Impact of olfactory sensitivity preferences on midgut pathophysiology of Aedes mosquitoes towards different sugar variants

Impact of olfactory sensitivity preferences on midgut pathophysiology of Aedes mosquitoes towards different sugar variants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of olfactory sensitivity preferences on midgut pathophysiology of Aedes mosquitoes towards different sugar variants Ranjitha Sambanthan, Nur Faeza Abu Kassim, Sara Abdelrahman Abuelmaali, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7141836/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Feb, 2026 Read the published version in Parasitology Research → Version 1 posted 9 You are reading this latest preprint version Abstract The effective control of Aedes mosquitoes are increasingly challenging due to the presence of insecticide resistance in populations of mosquitoes. Hence, attractive targeted sugar baits (ATSBs) can be used as an alternative strategy to insecticides. The introduction of suitable attractant into baits is very crucial since it will determine the number of mosquitoes attracted to it. In this study, the ATSBs were prepared as a medium for liquid bait carriers in the development of mosquito baiting tools. The testing of non-choice and choice assay by Ae. aegypti and Ae. albopictus towards different group of ATSBs (mango, Chrysanthemum and mix) was performed. In addition, olfactory preference index by mosquitoes was calculated using preference index formula followed by independent t- test to determine the significant differences, (p <0.05) between ATSBs pair. Furthermore, the midgut tissue of the mosquitoes was observed by using histology method. The wild and lab strain Ae. aegypti illustrates a strong and significant attractancy towards mango ATSBs with a value of (p = 0.004) and ( p = 0.04) respectively. Mango attractant is the most preferred by the Aedes sp . However, the mix ATSBs is secondarily preferred while Chrysanthemum is the least preferred ATSBs. The histology results show that the midgut of mosquitoes feed on the treated ATSBs exhibit alteration and degeneration. In the prospect of integrated pest management, the ATSBs implementation in mosquito control may reduce the need for chemical insecticides since natural products have naturally occurring volatile emission properties that attract or repel the mosquitoes. Thus, sustain released of the natural attractant odour can be a major part of the attractive toxic sugar bait as an attractant and killing mechanism to overcome mosquito-borne diseases in future. Aedes baits histology midgut olfaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The Aedes mosquito is a disease vector of dengue, chikungunya, zika and yellow fever virus (Huang et al., 2020). The two Aedes species, commonly known for mosquito-borne diseases are Aedes aegypti and Aedes albopictus . Some of them are notable for biting nuisances and disease vector that is capable of carrying lethal pathogens to humans and other animals. The mosquito - borne diseases have been a serious global issue for the past 50 years, causing almost 390 million infections per year to humans (Bhatt et al., 2013). Many methods were introduced to reduce the risk of mosquito-borne disease, especially where no effective vaccines are available. However, the vector control requires a proper strategical method in term of understanding the biology and behaviour of the mosquitoes. Olfaction is very crucial for the survival and reproduction of mosquitoes because it enables the mosquitoes to locate resources such as food and oviposition localities. Thus, they have a complex sensory system. This system enables the mosquitoes to identify and respond to ecologically significant volatile organic chemicals and occasionally volatile inorganic compounds such as carbon dioxide and ammonia in their surroundings. Mosquitoes have evolved an intricate sensory system. Olfactory sensory neurons which are present in hair-like sensilla on the antennae, maxillary palps and proboscis are responsible for detecting odorants (Hill et al., 2021). The number and type on sensilla found on mosquito olfactory appendages differ depending on the species and stage of development. Odorants are presumed to diffuse through countless pores on the sensilla surface then entering an aqueous lymph to contact with the spectrum of molecular receptors found on dendrites of olfactory receptor neurons (Montell & Zwiebel, 2016). Three complementary methods of controlling mosquitoes can be implemented with odorants are as repellents that "push" mosquitoes away, "maskers" that restrict mosquito attraction to humans, and attractants that "pull" mosquitoes into traps that are set far from individuals (Mulatier et al., 2022). Originally, the purpose of attractive toxic sugar bait was to suppress the malaria vector, Anopheles gambiae at Mali, West Africa (Müller et al., 2010). Following the "attract and kill" approach, it has been recognized as an innovative technique that can effectively eradicate adult mosquito populations (Nur et al., 2020). The "attract and kill" approach used mosquito tendency to feed on sugar by attracting them to consume insecticidal sugar source. Commonly, attractive toxic sugar bait consists of one or more aromatic compounds as the attractant including fruit juice, floral scent, feeding stimulants and oral toxin (Nur et al., 2020). The well-known methods to deliver attractive toxic sugar bait solution is by using direct spraying on the vegetation surface or by fixing a bait station (Mangan et al., 2014). However, evaporation of the solution may become a major problem by altering the toxin concentration in the solution. Exposure to direct sunlight cause high evaporation rate (Cheng et al., 2011). Therefore, a bait matrix should be used to deliver the solution such as alginate hydrogel beads. The alginate hydrogel beads are made of sodium alginate where alginate is a naturally occurring anionic polysaccharide that can be extracted from marine brown algae such as Laminaria hyperborea, Laminaria digitate, Laminaria japonica, Ascophyllum nodosum and Macrocystis pyrifera (Singh et al., 2022). Usage of alginate hydrogel beads are highly recommended due to its biodegradable and non-toxic properties to the environment (Lee & Mooney, 2012). Additional advantageous features are high efficacy with low maintenance and it is cheap to produce (McCalla et al., 2020; Tay et al., 2020). The attracted targeted sugar baits (ATSBs) are mosquito control tools that utilize mosquitoes’ natural tendency to feed on plant sugars. By mixing sugar solutions with toxic agents or biological pesticides, ATSBs can attract mosquitoes that ingest the bait and are subsequently killed. Both male and female mosquitoes need sugar for energy, making ATSBs effective against a broad range of mosquito species. These baits are usually set in specific bait stations near human dwellings or mosquito breeding sites, where they can effectively reduce mosquito populations with minimal environmental impact (Fiorenzano et al., 2017). The infusion of proper attractant into the ATSBs is very crucial since the mosquitoes will detect the odour to reach the baits. Numerous materials have been used in recent years to actively study mosquito preferences. These include studies on mosquito attraction to synthetic flora-based attractants like phenylacetaldehyde, linalool oxide, phenylethyl alcohol, and acetophenone (Fikrig et al., 2017), plant tissue, ripe fruits, seedpods, floral and extra floral and host-derived chemicals including l-lactic and 1-octen-3-ol (Sissoko et al., 2019). The neurological and behavioral components of olfaction have been given a lot of attention meanwhile the physiological effects of these olfactory-mediated feeding decisions especially on the mosquito midgut is less understood. The midgut functions as the primary site of digestion, nutrient absorption, and microbial interaction. It plays a vital role in metabolic regulation and immune responses following sugar or blood intake (Barletta et al., 2017). The mosquito's physiological response to various dietary intake types and dosages can be observed through histological changes in midgut tissues, such as modifications in peritrophic matrix structure, cellular hypertrophy, or alterations in epithelial integrity (Wang et al., 2011). The mosquito olfactory-driven feeding preferences may indirectly influence these histological features since different sugars exhibit varying effects on tissue structure, enzyme activity, and gut microbes. However, the infusion of natural sugar extracts in polymer as attractant for mosquitoes are very limited. Thus, this study mainly focusing on preference index of adult Ae. albopictus and Ae. aegypti response towards fruit-based, floral-based and mix-based ATSBs. This study investigated the potential combination of hydrogel polymer application with ATSBs to control populations of Aedes mosquitoes and the findings provide an overview on the preference of mosquitoes exposed to different sugar variant. Examining the relationship between sugar feeding in relation to Aedes mosquito olfactory responses and its effect to the mosquito gut condition is a novel approach that may provide information on how various sources in environment influence their behavior and physiology. 2. Materials and methods 2.1 Mosquito colony maintenance The eggs of laboratory strain Ae. aegypti and Ae. albopictus were obtained from Vector Control Research Unit (VCRU), School of Biological Sciences (SBS), University of Science Malaysia. The rearing process was carried out at Insectary Building G025A – Medical Entomology Laboratory, SBS. The rearing process was conducted under the condition where the temperature was maintained at 27°C (± 2°C). Meanwhile, the relative humidity and a photoperiod were monitored at 75% (± 3%) and 12: 12 h (L:D) respectively. The eggs were hatched in dechlorinated water and routinely, the newly emerged larvae were transferred into a metallic tray filled with dechlorinated water (depth = 2 cm, diameter = 12 cm). The larvae were fed for two days once with a 2:1:1:1 ratio of fine powder formed by a mixture of cat food, beef liver, yeast and milk powder. Then, the pupae were placed in a 250mL capacity of plastic cup that were kept in mosquito rearing cages measuring 30 cm × 30 cm × 30 cm. The newly emerged adult was provided with a 10% sucrose solution as a food source. The eggs of wild strain Ae. aegypti and Ae. albopictus were collected around University of Science Malaysia and was reared in Insectary Building G025A – Medical Entomology Laboratory, SBS under same condition with laboratory strain. 2.2 ATSBs preparation The mango (Waterlily Thai Mango) and Chrysanthemum was purchased from Lotus Sungai Dua, Penang, Malaysia. Mango extract was obtained by peeling off the skin of mango and it was cut into small pieces. Then, distilled water added to the mango pieces with a dilution ratio of 2:1, distilled water: mango. It was blended together and the solution was filtered using filter paper. Extracted mango solution was used to make 30% v/v of mango (MG) ATSBs. Thus, 120mL of mango solution and 280mL of distilled water was mixed together. Then, to produce the ATSBs, 5g of sodium alginate powder was weighed and blended together with the mixture until homogenous suspension formed. It was then sonicated in an ultrasonic bath (BactoSonic®) at 40°C for 45 minutes. After 45 minutes, the emulsion was cooled to room temperature. Formation of the spherical ATSBs was carried out by dropping the emulsion into 0.2 M CaCl 2 solution with a rate of 300 rpm at 30°C. Finally, the ATSBs were filtered and washed using distilled water for three times to make sure excess chloride ion was removed from it. For 30% v/v Chrysanthemum (FL) ATSBs, the petals were detached and weighed. Control (C) ATSBs were produced by using sucrose solution. Similar steps were repeated for 30% v/v Chrysanthemum , 30% v/v mix mango & Chrysanthemum (MX) and sucrose ATSBs as in production of 30% v/v of mango ATSBs. Sucrose ATSBs were the control ATSBs. 2.3 Olfactometer design A modified olfactometer was used to determine the olfactory response of mosquitoes to different odors of ATSBs [Figure 1 ]. This modified olfactometer was designed based on the olfactometer designed by (Afify et al., 2014). The olfactometer consist of three main components which were holding chamber, flight chamber and olfactory bioassay chamber. The mosquito cage was covered by using transparent PVC cover with a thickness of 0.5 mm. Compartment for holding chamber was created by using a tube with a diameter and length, 4.4 cm and 12.5 cm respectively. One end of the tube was covered with mesh screen cap to enable airflow for the mosquito survival while the other end that was connected with flight chamber was covered with a sliding gate. Function of the holding chamber is to hold the mosquitoes for acclimation before it was introduced into flight chamber. The size of the flight chamber compartment measured as (30 cm × 30 cm × 30 cm) and it was integrated with three circular openings where each of the diameter were 4.4 cm. This chamber enabled the decision making of mosquitoes on flying direction to either one of the olfactory bioassay chambers. This olfactory bioassay chambers consisted of two tubes with a diameter and length, 4.4 cm and 12.5 cm respectively. One of the ends of this tube was covered with mesh screen cap while the other end was covered with a sliding gate which is connected to the flight chamber. Inner part of the olfactory bioassay chambers was covered with mesh screen too to ensure the mosquitoes were not attracted by visual attraction and also to prevent them from having direct contact with ATSBs. The door was designed to ease the cleaning process of this olfactometer. 2.4 Evaluation of mosquito olfactory response The olfactory response evaluation of the mosquitoes towards 30% v/v of mango, Chrysanthemum , mix and sucrose ATSBs was carried out as two bioassay studies such as non-choice assay and choice assay. For the non-choice assay, the one of the olfactory bioassay chambers was filled with only attractant while the other one was filled with sucrose ATSBs. However, for the choice assay attractants were placed in both of the bioassay chamber. Experiment was started by aspirating 20 Ae. aegypti mosquitoes (10 male and 10 female) which was ranging from 5–7 days old. The aspirated mosquitoes were released into holding chamber and left it for 1 minute for acclimatization. Meanwhile, 30g of mango ATSBs was weighed and placed into one of the bioassay chambers while another chamber was filled with 30g of sucrose ATSBs as a negative control port. Then, the sliding gate for bioassay chambers were opened and two portable mini USB fans were placed at the end of each mesh cap. The lowest speed was set up to enable the scent of ATSBs flow into the flight chamber. After one minute, sliding gate of holding chamber was opened to release the mosquitoes into flight chamber and it was closed immediately once holding chamber was empty. After 2 minutes, bioassay chamber gate was closed and the number of mosquitoes on the mesh screen of the bioassay chambers were counted. The test was then repeated by placing two attractants in bioassay chambers for the choice assay. After completing the test for laboratory strain Ae. aegypti it was replaced with laboratory strain Ae. albopictus followed by wild strain with the same procedure according to Table 1 . The olfactometer was washed and dried before used it again to remove the lingering odour. New batch of mosquitoes that were not exposed to any of the odour was used in every test and each of the treatment was repeated for three times. Table 1 Attraction pairing for olfactory bioassay response of Aedes mosquitoes. Olfactory attraction bioassay Attraction pairs of ATSBs Pair labelling Chamber 1 Chamber 2 Non-choice assay Mango Chrysanthemum Mix Control Control Control MGC FLC MXC Choice assay Mango Mango Mix Chrysanthemum Mix Chrysanthemum MGFL MGMX MXFL *MGC refers to mango and control ATSBs; FLC refers to Chrysanthemum and control ATSBs; MXC refers to mix and control ATSBs; MGFL refers to mango and Chrysanthemum ATSBs; MGMX refers to mango and mix ATSBs and MXFL refers to mix and Chrysanthemum ATSBs. 2.5 Midgut histology analysis Both laboratory and wild strain female mosquitoes of Ae. aegypti and Ae. albopictus were fed with 30% w/v of mango, chrysanthemum, mix and control ATSBs. The mosquitoes were killed after three days and kept for overnight in 70% ethanol. After the dissection, the abdomens were stained for 15 minutes using eosin (0.5% of Alcoholic Eosin Y- Solution, Sigma-Aldrich). Then, the samples were dehydrated using gradual ethanol series such as 50% (30 minutes), 60% (15 minutes), 70% (15 minutes), 80% (30 minutes), 90% (30 minutes) and finally 100% (60 minutes). Subsequently, tissues were cleared in xylene for 15 minutes to enable paraffin embedding. Then, infiltration of samples was done by immersing in melted paraffin wax at 60°C in four stages (15 minutes each) which enable complete infiltration. Abdomens were embedded in paraffin wax using a tissue embedding center machine (Tissue Embedding & Cooling System – KEDEE) and cooled at -15°C overnight. The next day, specimen blocks were sectioned at 8 µm using a Leica BioCut Rotary Microtome. Then, the sectioned wax stripes were floated in a 40°C water bath and mounted onto glass slides. The glass slides were dried at 40°C overnight. Final staining process was performed by using histoclear, ethanol, hematoxylin, bluing agent and eosin. Finally, the samples were examined using a light microscope at 10X, 20X, and 40X magnification. 2.6 Statistical analysis The olfactory response for both Ae. aegypti and Ae. albopictus was obtained from Preference Index (PI), described by Nur et al., 2020. Preference Index was calculated using this formula: $$\:Preference\:Index\:\left(PI\right)=\frac{Number\:of\:mosquitoes\:in\:chamber\:1-Number\:ofmosquitoes\:in\:chamber\:2}{Number\:of\:mosquitoes\:in\:chamber\:1+Number\:ofmosquitoes\:in\:chamber\:2}$$ The PI value which ranging from − 1 to + 1 where negative value indicates mosquito repellence while positive value indicates mosquito attraction. Meanwhile, the value 0 refers to neutral response of mosquitoes. The normality of data for both non-choice and choice assay were checked by using Shapiro-Wilk test (p > 0.05). Moreover, equal variance across the sample was assumed when Levene’s test yield (p > 0.05). Then, parametric independent sample t-test was used to analyse the significances of the mosquito olfactory response expose to different attractant since the assumptions of Student’s t-test was met (p < 0.05). All statistical analyses were tested by using IBM SPSS statistic version 28.0. 3. Results 3.1 Response of Aedes sp in non-choice assay The response of Aedes sp. to three sets of attractant pairings was used to calculate their preference index (PI). Both the attractancy (positive response) and repellency (negative response) of mosquitoes to the specified attractants are determined by the set of bar graphs. Figure 2 shows the bar graph that represent mean preference index (PI) ± SE while Table 2 indicates the mean number of mosquitoes ± SE of laboratory and wild strain of Ae. aegypti and Ae. albopictus on non-choice assay with 3 pairs of treatment sets. For set MGC (mango and control), laboratory strain Ae. aegypti were significantly attracted to mango attractants compared to sugar (t ( 4 ) = 6.12, p = 0.04). Furthermore, for FLC ( Chrysanthemum and control) set, it shows a significant difference where (t ( 2 ) = − 4.01, p = 0.045). However, laboratory strain Ae. aegypti indicate that MXC (mix and control) has no significant different (t ( 3 ) = 2.00, p = 0.067) to attract mosquitoes. The mean PI ± SE in MGC displays a strong preference (0.72 ± 0.15) followed by MXC with least preference (0.29 ± 0.04). Nevertheless, FLC shows (-0.65 ± 0.07) moderate repellency by the mosquitoes (Fig. 2 ). The highest mean number of laboratory strain Ae. aegypti ± SE (6.00 ± 0.577) attracted to mango ATSBs compare with other treatment ATSBs. The Chrysanthemum ATSBs yield the lowest attraction (1.33 ± 0.333) while the control ATSBs recorded the highest (6.33 ± 1.202) number of mosquitoes when tested with FLC pair (Table 2 ). The mean PI ± SE for laboratory strain Ae. albopictus shows (0.79 ± 0.1), (-0.6 ± 0.22) and (0.21 ± 0.04) for the pair of MGC, FLC and MXC respectively. The high positive PI value indicates that this species was strongly preferred to MGC ATSBs and the lower PI value represents the least favourable response towards MXC. However, it repels towards FLC ATSBs since the PI value was negative (Fig. 2 ). When comparing the numbers of mosquitoes attracted to MG ATSBs, it displays relatively highest value (5.67 ± 0.333) than MX ATSBs (2.33 ± 0.333). Table 2 demonstrates the laboratory strain Ae. albopictus , obtained the most minimal value of mosquito attraction (1.67 ± 0.882) in FLC ATSBs compared to the other treatments used in this study. There was no statistically significant difference been observed for set of FLC (t ( 3 ) = -4.11, p = 0.20) and MXC (t ( 4 ) = 2.12, p = 0.101). In contrast, (t ( 4 ) = 10.61, p = 0.001) highlights that the preference of Ae. albopictus towards MGC shows a statistically significant value. The preference of wild strain Aedes sp . is distinct from the activity of laboratory strain. The wild strain Ae. aegypti illustrates a strong attractancy towards MGC with a value of 0.74 ± 0.13. In addition, a trend of weak attractancy (0.21 ± 0.21) was observed when the mosquitoes were exposed to the set of MXC ATSBs. Similar to laboratory strain Ae. aegypti (-0.65 ± 0.07), the wild strain also shows the same pattern where the value of PI is -0.57 ± 0.12. The negative value indicates that both of the mosquito strain repels moderately towards FLC ATSBs (Fig. 2 ). The highest number of mosquitoes attracted were towards mango (6.00 ± 0.577) followed by mix (3.00 ± 0.577) and Chrysanthemum (1.33 ± 0.333) ATSBs. By comparing the control ATSBs, maximum frequency of mosquitoes (5.00 ± 0.577) were attracted in the experiment set of FLC. The analysis of student t-test demonstrate that the MGC yield a significant difference where (t ( 4 ) = 6.12, p = 0.004) while FLC result was (t ( 3 ) = -5.50, p = 0.010). However, a non- significant result was obtained for MXC (t ( 4 ) = 1.23, p = 0.288) because the p-value is higher than 0.05. Next, the experiment carried out by using wild strain Ae. albopictus reveals that the mosquitoes are extremely attractive to mango ATSBs since the combination set of MGC gain PI of 0.84 ± 0.15. Conversely, the FLC set (-0.72 ± 0.18) shows that the mosquitoes are extremely repel to the Chrysanthemum ATSBs. A weak attractancy of mosquitoes were identified for the MXC set by considering PI value of 0.19 ± 0.18 (Fig. 2 ). Most prominent abundance of mosquitoes are attracted towards mango ATSBs (7.67 ± 0.333) compared to control ATSBs (0.67 ± 0.333). Additionally, mix ATSBs indicates the second highest in mosquito numbers (3.33 ± 0.333). According to Table 2 , least number was recorded for Chrysanthemum ATSBs (1.00 ± 0.577). The set of MGC and FLC reveals that there is a significant difference of (t ( 4 ) = 14.85, p = 0.001) and (t ( 3 ) = − 4.11, p = 0.020) respectively. In contrast, (t ( 2 ) = 0.54, p = 0.640) shows that MXC have non-significant impact on the mosquito attraction towards it. In conclusion, both laboratory and wild strain Aedes sp. are more likely to choose mango attractant compared to mix. However, the mosquitoes show a second interest to mix attractant compared to mango. Overall, Chrysanthemum repels both Ae. aegypti and Ae. albopictus from reaching the ATSBs in non-choice assay. Table 2 Mean number of mosquitoes ± SE of Aedes sp in non-choice assay. Set Mean number of mosquitoes ± SE Laboratory strain Wild strain Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus MGC MG C 6.00 ± 0.577 5.67 ± 0.333 6.00 ± 0.577 7.67 ± 0.333 1.00 ± 0.577 0.67 ± 0.333 1.00 ± 0.577 0.67 ± 0.333 FLC FL C 1.33 ± 0.333 1.67 ± 0.882 1.33 ± 0.333 1.00 ± 0.577 6.33 ± 1.202 6.00 ± 0.577 5.00 ± 0.577 5.33 ± 0.882 MXC MX C 3.00 ± 0.577 2.33 ± 0.333 3.00 ± 0.577 3.33 ± 0.333 1.67 ± 0.333 1.33 ± 0.333 2.00 ± 0.577 2.67 ± 1.202 *MG refers to mango ATSBs; FL refers to Chrysanthemum ATSBs; MX refers to mix ATSBs and C refers to control ATSBs. 3.2 Response of Aedes sp. in choice assay The preference index (PI) of Aedes sp . was determined by analysing their reaction towards three sets of attractant pairings (MGFL, MGMX, and MXFL). The set of bar graphs indicates the attractancy of mosquitoes (positive response) and repellency (negative response) to the designated attractants. The mean number of mosquitoes ± SE of laboratory and wild strains of Ae. aegypti and Ae. albopictus on choice assay experiment shown in Table 3 while Fig. 3 displays the bar graph representing mean PI ± SE. For set MGFL (mango and Chrysanthemum ), laboratory strain Ae. aegypti were not significantly attracted to mango attractants compared to Chrysanthemum (t ( 3 ) = 2.50, p = 0.82). However, for MGMX (mango and mix) set, it shows a significant difference where (t ( 3 ) = 3.50, p = 0.036). In addition, laboratory strain Ae. aegypti indicate that MXFL (mix and Chrysanthemum ) has significant different (t ( 4 ) = 4.24, p = 0.013) to attract mosquitoes. The mean PI ± SE in MGFL displays a strong preference (0.83 ± 0.17) followed by MGMX (0.70 ± 0.15) and MXFL (0.61 ± 0.20 ). The highest mean number of laboratory strain Ae. aegypti ± SE (3.00 ± 0.577) attracted to mango ATSBs compared to mix ATSBs (0.67 ± 0.333). The Chrysanthemum ATSBs yield the highest attraction (0.67 ± 0.333) when tested together with mix ATSBs while the lowest (6.33 ± 1.202) number of mosquitoes was recorded when tested using MGFL pair (Table 3 ). The choice assay study indicates that the PI value shows moderately positive results for laboratory strain Ae. albopictus . The PI value is the lowest for mango ATSBs (0.30 ± 0.09) when compared to mix ATSBs. However, when mango ATSBs tested together with Chrysanthemum ATSBs, the value rises to 0.49 ± 0.15. The highest PI obtained was 0.57 ± 0.23 in the testing of MXFL pair. Maximum number of laboratory strain Ae. albopictus attracted to mango ATSBs (4.33 ± 0.333) while (2.33 ± 0.333) was attracted to mix ATSBs in the assessment of the MGMX pair. For the MGFL set, a slight reduction in numbers occurred to mango ATSBs treatment (4.00 ± 0.577) compared to MGMX pair. Minimal number counts observed in MXFL, in which the mix yield (2.67 ± 0.333) whereas, (1.00 ± 0.577) was recorded in Chrysanthemum ATSBs. Hence, MXFL set displays a non-significant different value (t ( 3 ) = 2.50 p = 0.082). In contrast, MGFL (t ( 3 ) = 4.00, p = 0.025) and MGMX (t ( 4 ) = 4.00, p = 0.013) reveal that the pairs show significant different towards each other. The response of wild strain Ae. aegypti towards ATSBs are slightly different from laboratory strain. A strong attractancy response of mosquitoes were observed in PI of MGFL (0.70 ± 0.15) followed by MXFL (0.64 ± 0.18) and MGMX (0.45 ± 0.08) respectively. In order to the number of mosquitoes attracted to the ATSBs, the highest number was recorded in MGMX set as the mango ATSBs attracted (5.00 ± 0.577) while mix ATSBs drawn in (5.00 ± 0.577) mosquitoes. The least number observed in Chrysanthemum ATSBs (0.67 ± 0.333) in MGFL set. According to Table 3 , mix ATSBs attracted (4.00 ± 0.577) while Chrysanthemum ATSBs attracted only (1.00 ± 0.577) mosquitoes. The student t-test analysis reveals that all sets MGFL (t ( 4 ) = 5.66, p = 0.005), MGMX (t ( 4 ) = 3.67, p = 0.021) and MXFL (t ( 4 ) = 3.67, p = 0.021) in choice assay shows a significant different value. The response of wild strain Ae. albopictus shows a moderate attractancy in MGFL ( 0.44 ± 0.06) and MGMX (0.39 ± 0.06) except MXFL ( 0.30 ± 0.09) which indicates a weak attractancy value. A non-significant difference obtained for MGFL (t ( 3 ) = 2.83, p = 0.080) whereas, MGMX (t ( 3 ) = 4.03, p = 0.029) and MXFL (t ( 4 ) = 4.24, p = 0.013) shows significant difference. Counts of mosquitoes attracted to mango ATSBs (7.67 ± 0.333) is the highest value recorded for choice assay of wild strain Ae. albopictus . However, the least number was observed for Chrysanthemum ATSBs (0.67 ± 0.333) in MGFL pair. Finally, the number of mosquitoes attracted to mix ATSBs (5.33 ± 0.882) in MGMX and (3.33 ± 0.333) in MXFL are higher compared to their respective pair (Table 3 ). Overall, the results of choice assay show that the mango attractant is the most preferred by the Aedes sp . However, the mix ATSBs is secondarily preferred while Chrysanthemum is the least preferred ATSBs in choice assay. Table 3 Mean number of mosquitoes ± SE of Aedes sp in choice assay. Set Mean number of mosquitoes ± SE Laboratory strain Wild strain Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus MGFL MG FL 2.00± 0.577 4.00 ± 0.577 3.33 ± 0.333 7.67 ± 0.333 0.33 ± 0.333 1.33 ± 0.333 0.67 ± 0.333 0.67 ± 0.333 MGMX MG MX 3.00 ± 0.577 4.33 ± 0.333 5.00 ± 0.577 1.00 ± 0.577 0.67 ± 0.333 2.33 ± 0.333 2.00 ± 0.577 5.33 ± 0.882 MXFL MX FL 2.67 ± 0.333 2.67 ± 0.333 4.00 ± 0.577 3.33 ± 0.333 0.67 ± 0.333 1.00 ± 0.577 1.00 ± 0.577 2.67 ± 1.202 3.3 Histological observation The midgut condition of the mosquitoes exposed to different ATSBs illustrated through light microscopy image under scale bar of 20µm. The findings showed noticeable features in the midgut epithelial cells and overall midgut structure of lab and wild strain Ae. aegypti and Ae. albopictus that were fed with mango ATSBs (Fig. 4 ). Overall, the midgut structure was slightly degrading in all the samples. Thus, it causes minor breakage of the midgut. In addition, the arrangement of epithelial cells displays distinct changes. The epithelial cells in laboratory and wild strain Ae. albopictus (Fig. 4 (c) and (d)) were non-uniformly distributed and less visible. However, the epithelial cells in Ae. aegypti are clearly visible and it is closely packed. According to Fig. 5 , mosquitoes exposed to Chrysanthemum ATSBs show irregular shaped midgut which indicates it undergoes degradation and shrinkage. The density of epithelial cells still high and appeared to be slightly distanced or separated from midgut. Hence, it showed dispersed epithelial cells with several clearing regions. The Aedes sp. exposed to mix ATSBs had unique morphological patterns in their midgut and epithelial cells. The midgut shrinks which cause uneven size and shape of the midgut. Furthermore, dislocation of the midgut is observed in Fig. 6 (b) where the midgut of wild strain Ae. aegypti was pushed to a side. Degradation and minor instances of breakage midgut structure was observed. Epithelial cells surrounding the midgut are orderly arranged but it shows cell degeneration. The midgut structure of mosquitoes that exposed to control ATSBs were normal. The midgut is thicker and it has the even shape. There was no breakage or degradation of midgut is noticed. Additionally, the midgut is surrounded by the orderly arranged epithelial cells. All of the mosquito species fed on control ATSBs does not exhibit notable differences from one another (Fig. 7 ). 4. Discussion Generally, mosquitoes need sugar meal as a source of carbohydrates, particularly to maintain population fitness. As a result, they conveniently consume any sugary food that is offered and might supply enough energy for them (Nur et al., 2020). The ecology of mosquitoes which includes their consumption of various forms of sugar, is described by phytophagy, which is notable for insects that ingest on plants. It includes extrude, plant sap, nectar (nectarivory), and fruit juices (frugivory) (Peach and Gries, 2020a). An excellent baiting tool could be created by developing bait attractants or repellents that imitate natural sugar sources, particularly when competing with the natural sugar sources found in their ecosystem. Species preferences such as exophagic and endophagic behavior may influence sugar selection (Tananchai et al., 2012 & 2019). Data gathered in this study regarding mosquito preference between mango and mix indicates significance attraction for both of the Aedes species. Although it was given a choice, still the mango ATSBs attracted mosquitoes the most. In the choice assay, the mosquitoes did not demonstrate the distinctive difference since the odour of the ATSBs were mixed together. They tend to confuse and their desire for consistent sugar consumption may be the cause that forcing them to consume on any readily available sugar sources (Nur et al., 2020). All of the laboratory and wild strain of Ae. aegypti and Ae. albopictus shows the similar pattern for non-choice assay, where they mostly attracted to mango but slightly attracted to mix. However, they show strong repellency towards Chrysanthemum ATSBs. Mango is selected because of their high sugar content and have been previously recorded for mosquito attraction. According to Bello-Pérez et al. (2007), ripe mango fruit contains a significant source of sugars (glucose, fructose, and sucrose) as well as other carbohydrates including starch and pectin. All of these substances are important from a flavour and nutritional perspective. About 15% of the total sugars in mature mangos are found in the fruit flesh. Sucrose is the main sugar in ripe mango fruit, but fructose is the main monosaccharide during the preclimatic period (Maldonado et al., 2019). Initial studies on mango fruit volatile compounds were primarily well-known for invention of ATSBs. The discovery regarding those chemical components satisfies the oral-toxic bait criteria for Aedes mosquito control. The identified volatile compounds that can attract the Aedes mosquitoes classified under alcohol, aldehyde, sesquiterpenes and monoterpenes groups. Sesquiterpenes and monoterpenes are the essential chemical compounds to enhance the flavour and fragrance of the plants. For instance, α-pinene, β-ocimene and Thujone are groups of monoterpenes while α-humule, Caryophyllene, and σ-cadinene are the sesquiterpene that are detachable for Aedes mosquitoes. Nevertheless, monoterpene is more volatile compared to sesquiterpenes. A groundbreaking study shown the ability of these volatile substances to attract the mosquitoes of both sexes. The chemical cues involved for plant-mosquito interactions are chemicals from the aldehyde class, such as hexanal and alcohol, more precisely (Z) 3-Hexen-1-ol (Nyasembe & Torto, 2014). According to previous study, a few floral semiochemicals were identified to guide the mosquitoes in locating the sugar sources such as ketones, terpenes, aldehydes, alcohols, fatty acids and fatty acid derivatives (Peach & Gries, 2019). There are many previous studies that discussed about the usage of fruits attractant to attract the mosquitoes. A prior study by Tenywa et al. (2017) noted that Anopheles spp. get attracted by the juice of subtropical fruits such bananas, mangoes, and guavas. Furthermore, Meza et al. (2020) have determined the essential components of mango juice that enhance the baiting attractant for Anopheles gambiae against malaria vectors. According to a research by Fiorenzano et al. (2017), mosquitoes responded to banana slices by piercing on them and sticking their proboscis into the fruit cavitation. It provided evidence for the potential benefit of these fruits in drawing mosquitoes. Mango fruit flesh is able to attract adult Ae. aegypti , however artificial mango fruit concentration appears to have little influence on the attraction factor for adult Ae. aegypti (Barbosa et al., 2019). In addition, both the aqueous and pellet forms of black pepper seed extraction were successful in drawing female Ae. aegypti to the study conducted by Briones et al. (2012). According to Sissoko et al. (2019), the fruit flesh of the Carica papaya (Papaya), C. melo (Honeydew melon), and M. indica (Mango) has been shown to be attractive to both male and female Ae. aegypti . In the experiment by Nur et al. (2020), the fruit peel of C. papaya was remarkably effective in drawing adult Ae. aegypti in both choice and no-choice experiments. The epithelial cells in the abdomen that was provided mango ATSBs showed a modified shape. Mango treatments resulted in a visible rupture of the epithelial cells. The natural biocides that are present in mangos may be the cause of this breakage. Compounds like tannins, alkaloids and polyphenols which have antimicrobial and biocidal qualities are known to be present in mangoes (Kim et al., 2021). The observed midgut breakage resulted from interactions between these biocides and the epithelial cells. The deterioration of the mosquito midgut following the consumption of mango may be linked to particular physiological and biochemical reactions driven on by the substances in mangos. These substances also cause degeneration by harming the midgut's lining cells. Additionally, these compounds also impair digestion and the absorption of nutrients by interfering with the action of the enzymes in the gut and damage gut tissues by producing reactive oxygen species which causes oxidative stress (Ribeiro & Schieber, 2009). Furthermore, due to their high sugar content, mangoes have the potential to cause cell damage by changing the osmotic balance in the mosquito gut (Kiani et al., 2022). Water inflow into the midgut due to this imbalance may result in distension and rupture. Despite their usual diet, mosquitoes might not be able to withstand such a high osmotic burden. This provides an atmosphere that encourages the growth of dangerous microorganisms which can destroy the lining of the gut. In order to test the mix baits attractancy effect, mango and Chrysanthemum were altered and improved using the blending method. This study conducted using a 1:1 combination of mango and Chrysanthemum as an attractant. During this study, the mosquitoes were exposed to the mixed attractant and either the mango or Chrysanthemum attractant at the same time. According to the observation, mosquitoes continue to be positively attracted to the combined attractants. This event confirms that the mango dominated the Chrysanthemum . As we know, Chrysanthemum are a powerful repellent that has been employed in the control approach, whereas mangoes are great attractants. Combined attractants improve the attractancy while combination of repellents enhance the repellency of the mosquitoes for a better application of ATSBs bait. Furthermore, enhancing the attractant component is necessary to increase the effectiveness of the ATSBs bait application since the bait must compete with the available sugar sources in the natural habitat. It clarifies how mangoes and Chrysanthemum provide a strong attraction for Aedes mosquitoes. It is clear from the results that the fruit extract is essential to the development of the ATSBs baiting tools since it demonstrates a positive attraction towards mango and Chrysanthemum in comparison to glucose (control). A significant difference structure of midgut and its epithelial cells was demonstrated by the mix ATSBs exposed mosquitoes. This is due to the toxic compounds present in mango and Chrysanthemum which cause adverse effects on it. The midgut of mosquitoes exposed to control ATSBs was seen to have closely packed columnar epithelial cells (Fig. 5.8). These cylindrical cells have a large and coarsely granular nucleus in the center of the cell. It is unclear whether if the various sugar varieties utilized in the feeding studies or the histology processing methods were accountable for this disruption. The fundamental reason of the observed cell destroying has to be investigated further. Plants provide mosquitoes with sugars, amino acids, and other resources through their nectars, honeydew, and decomposing fruits (Nyasembe et al., 2018). Mosquitoes employ a variety of sugars, including sucrose, fructose, and glucose, in the sugar-based meals they consume as their main energy source (Leyva et al., 2024). The types of sugar content in the attractant also influence the response of the mosquitoes towards the ATSBs. Carbohydrates are often divided into three primary types based on the number of sugar units such as monosaccharides, oligosaccharides, and polysaccharides. Monosaccharides or commonly termed as simple sugars are the most basic class of carbohydrates and the general formula is C n (H 2 O) n . They are known as aldoses when the functional group is -CHO (aldehyde), and ketoses when the functional group is -CO (keto). Glucose, galactose, and fructose are a few varieties of monosaccharides. Monosaccharides sugars have the reducing properties (Khadka, 2022). Around 3–10 monosaccharide molecules are present in oligosaccharides and it break down during hydrolysis. Depending on the quantity of monosaccharides, they can be either disaccharides or trisaccharides. Majority of the plants contain few oligosaccharides that occur naturally. Melibiose (galactose and glucose) and fructose combine to form raffinose, a trisaccharide that is present in many plants. For instance, maltose and sucrose. High molecular weight substances called polysaccharides are polymers of monosaccharides. They are non-sugary compounds because the sugary taste absence in it. Their primary focus is on two crucial functions such as energy and structural storage. Glycosidic bonds that develop at any one of a monosaccharide hydroxyl groups, causes branches to appear in polysaccharides. Example of polysaccharides including cellulose and starch. The sugar level in Chrysanthemum is minimal hence, they are not naturally sweet since they are predominantly made up of water, fibre, and trace nutrients. The approximate chemical compositions of Chrysanthemum were 12.8% in reducing sugar where fructose, glucose, and sucrose made up the majority of free sugars. Chrysanthemums like other flowering plants, store sugars and other carbohydrates for energy and to produce nectar that will draw pollinators. The main sugar that present include Chrysanthemum nectar and floral tissues contain sucrose, a common sugar that is essential for pollinators energy needs. The concentrations of these reducing sugars are lower than those of sucrose. They provide readily metabolizable energy and are produced when the enzyme invertase breaks down sucrose. The tissues of Chrysanthemum also contain polysaccharides, such as starch, which can be converted to simple sugars when the plant needs energy (Li et al., 2019). High glucose and fructose predominate in flowers visited by flies or beetles, while high sucrose proportion is frequently linked to flowers attracted for bees or butterflies (Wäckers, 2005). A damaged morphology of the midgut also appeared in the Chrysanthemum ATSBs treated mosquitoes. The loosely packed configuration of the epithelial cells demonstrated a clearing state. Some of the cells were also broken. Pyrethrin, a naturally occurring biocide frequently present in Chrysanthemums may have contributed to this fracture. According to Bowman et al. (2018), pyrethrin is well-known for its insecticidal qualities and application as a pesticide. Consequently, it is possible that the observed cell breakage was caused by the pyrethrin included in the treatment. The midgut of mosquitoes may shrink due to the toxic effects of pyrethrins. The pyrethrins causes cell damage, apoptosis and reduced the functionality of the midgut cells. Pyrethrins have the potential to directly harm the midgut's epithelial cells by rupturing cellular membranes or producing oxidative stress. Cellular shrinkage and tissue atrophy result from damage to midgut cells, which can also affect digestion and nutritional absorption (Nagai et al., 2022). Toxin exposure cause midgut tissues to undergo apoptosis, or programmed cell death which reduces tissue size and cell volume (Elmore et al, 2007). In contrast, the odour of sweet alyssum ( Lobularia maritima ) recorded to attract Ae. aegypti (Von Oppen et al. , 2015). The attractiveness of the adult Ae. aegypti of several blooming plant species was evaluated using their blossoming branches in the Sissoko et al. (2019) study. The Ae. aegypti males and females were attracted to the flowering branches of Acacia macrostachya , A. salicina , Galphimia gracilis , and Prosopis juliflora among the treatments. Since the concentration of attractants influence the study, further studies on the different combinations of attractants concentration are highly suggested. Others despite than mango and Chrysanthemum as the attractant and repellent component, different fruits and flowers are also suggested to expand the choice selection in preparing the mosquito baits. Since the ATSBs needs to compete with the other available sugar sources in the environment, it is fundamental to boost up their attractiveness. Additionally, further studies on a synthetic chemical compound for attractant components also suggested reducing the production cost. 5. Conclusion In conclusion, the study revealed that the high sugar content in mango attracting the maximum number of mosquitoes when infused in ATSBs. However, the low sugar content in Chrysanthemum ATSBs repel the mosquitoes but the mixture of mango of Chrysanthemum shows the mosquitoes attracted towards it. Thus, the sugar content of mango dominating the mix ATSBs. The attractants increase the ATSBs attractiveness, however glucose alone can be used as a phagostimulant for the bait. Histological analysis of the mosquito midgut revealed structural changes in response to different ATSBs. Notable alterations included epithelial disruption and damage to the peritrophic membrane, particularly in mosquitoes exposed to the mixed and floral-based baits. These findings suggest that ATSBs can significantly affect midgut integrity, potentially impairing digestion and pathogen transmission, and support their use as effective vector control strategies. An excellent baiting tool could be created by developing bait attractants or repellents that imitate natural sugar sources, particularly when competing with the natural sugar sources found in their ecosystem. Declarations Acknowledgment We would like to thank the Vector Control Research Units (VCRU) for egg supplies, BioG Expert Sdn. Bhd. and School of Biological Sciences for hydrogel beads preparation facilities and samples. This work was supported by Ministry of Higher Education Malaysia, Fundamental Research Grant Scheme (FRGS) with Reference No: FRGS/1/2022/STG03/USM/02/8. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nur Faeza Abu Kassim, Ranjitha Sambanthan and Wan Maryam Wan Ahmad Kamil. The first draft of the manuscript was written by Ranjitha Sambanthan and Wan Maryam Wan Ahmad Kamil edited the full draft of the manuscript. Sumiyyah Sabar, Kamarul Zaman Zarkasi, Wan Rosli Wan Ishak, Sara A. Abuelmaali and Cameron E. Webb validated the related results. All authors read and approved the final manuscript. Funding This work was supported by Ministry of Higher Education Malaysia, Fundamental Research Grant Scheme (FRGS) with Reference: FRGS/1/2022/STG03/USM/02/8. Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Ethics approval USM/IACUC/2022/(137)(1223) has specifically approved this study. Consent to participate Not applicable. Consent to publish Not applicable. Conflict of interest The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7141836","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491317106,"identity":"388d4a54-683e-40f2-9d58-3cb827150bf9","order_by":0,"name":"Ranjitha Sambanthan","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Ranjitha","middleName":"","lastName":"Sambanthan","suffix":""},{"id":491317107,"identity":"13739999-2133-4fb4-b134-456e67e0ae34","order_by":1,"name":"Nur Faeza Abu Kassim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqUlEQVRIiWNgGAWjYJCCDyCCnxQdjDNApGQDyVoMDhCr3lwi92HDj4o6eeMb6U83MOYcJqzFcka6YWPPGTbDbTdyzG4wbiNCi8GNNPYHvG08jEAtbERrYWz8+0/CfvOM9GfEa2nmbTBI3CCRQKzDzjxjbJY5lpA848wbsxuJ29KJ0HIc6LA3NXW2/e1Ah33cZk1YCypIYGgmVQsDQx3pWkbBKBgFo2DYAwBfUD6xjjMUWQAAAABJRU5ErkJggg==","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Nur","middleName":"Faeza Abu","lastName":"Kassim","suffix":""},{"id":491317108,"identity":"86ec7f76-26ca-426f-9cb9-dc334d371170","order_by":2,"name":"Sara Abdelrahman Abuelmaali","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"Abdelrahman","lastName":"Abuelmaali","suffix":""},{"id":491317110,"identity":"4d2da8c8-82d0-4652-8b14-89f67ae7570f","order_by":3,"name":"Wan Maryam Wan Ahmad Kamil","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Wan","middleName":"Maryam Wan Ahmad","lastName":"Kamil","suffix":""},{"id":491317111,"identity":"0655e4c4-4dc2-468f-a892-a34a176f4cf4","order_by":4,"name":"Sumiyyah Sabar","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Sumiyyah","middleName":"","lastName":"Sabar","suffix":""},{"id":491317114,"identity":"02d8f4ce-700e-4dbd-a74d-efb15dd57d30","order_by":5,"name":"Kamarul Zaman Zarkasi","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Kamarul","middleName":"Zaman","lastName":"Zarkasi","suffix":""},{"id":491317115,"identity":"8e65482d-ca2d-47cc-814c-40e53dae1121","order_by":6,"name":"Wan Rosli Wan Ishak","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Wan","middleName":"Rosli Wan","lastName":"Ishak","suffix":""},{"id":491317118,"identity":"0852b1b4-9a8b-4902-8fb7-f3962181abed","order_by":7,"name":"Cameron E. Webb","email":"","orcid":"","institution":"Westmead Hospital","correspondingAuthor":false,"prefix":"","firstName":"Cameron","middleName":"E.","lastName":"Webb","suffix":""}],"badges":[],"createdAt":"2025-07-16 15:53:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7141836/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7141836/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00436-025-08624-8","type":"published","date":"2026-02-09T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87748863,"identity":"31af7c52-55e7-4648-a7a7-1581c40ecff0","added_by":"auto","created_at":"2025-07-28 14:39:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33112,"visible":true,"origin":"","legend":"\u003cp\u003eDesign of modified olfactometer\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141836/v1/9493da7b827643db0cfd2134.jpg"},{"id":87748102,"identity":"fdaae705-2db1-4c13-8483-5b20cbf4d863","added_by":"auto","created_at":"2025-07-28 14:31:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45621,"visible":true,"origin":"","legend":"\u003cp\u003eMean preference index (PI) ± SE of laboratory and wild strain of \u003cem\u003eAe. aegypti \u003c/em\u003eand \u003cem\u003eAe. albopictus\u003c/em\u003e on non-choice assay with 3 pairs of attractant sets (MGC, FLC and MXC). *MGC refers to mango and control ATSBs; FLC refers to \u003cem\u003eChrysanthemum\u003c/em\u003e and control ATSBs and MXC refers to mix and control ATSBs.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141836/v1/49c2d39c035db248fd54181f.jpg"},{"id":87749462,"identity":"8e61dad3-7084-47b8-bf37-4c06082e1747","added_by":"auto","created_at":"2025-07-28 14:47:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50163,"visible":true,"origin":"","legend":"\u003cp\u003eThe bar graph represents the mean preference index (PI) ± SE of laboratory and wild strain of \u003cem\u003eAe. aegypti \u003c/em\u003eand \u003cem\u003eAe. albopictus\u003c/em\u003e on choice assay with 3 pairs of attractant sets (MGFL, MGMX and MXFL).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141836/v1/c97ab495fd5d7a6b1f26c448.jpg"},{"id":87748865,"identity":"9133b43b-cc88-4544-80c0-f68c460b6245","added_by":"auto","created_at":"2025-07-28 14:39:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51405,"visible":true,"origin":"","legend":"\u003cp\u003eHistological section of \u003cem\u003eAedes sp \u003c/em\u003emidgut exposed to mango ATSBs in cross-section position under magnification of 20x. (a) \u003cem\u003eAe. aegypti \u003c/em\u003e(Laboratory strain) (b) \u003cem\u003eAe. aegypti \u003c/em\u003e(Wild strain) (c) \u003cem\u003eAe. albopictus \u003c/em\u003e(Laboratory strain) (d) \u003cem\u003eAe. albopictus \u003c/em\u003e(Wild strain). EC, epithelial cell and MG, midgut. Scale bars, 20mm.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141836/v1/49241e923c37d0a82771cbc1.jpg"},{"id":87748106,"identity":"204e1fd2-34a8-4362-8b8e-180b00d76923","added_by":"auto","created_at":"2025-07-28 14:31:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":65763,"visible":true,"origin":"","legend":"\u003cp\u003eHistological section of \u003cem\u003eAedes sp \u003c/em\u003emidgut exposed to \u003cem\u003eChrysanthemum \u003c/em\u003eATSBs in cross-section position under magnification of 20x. (a) \u003cem\u003eAe. aegypti \u003c/em\u003e(Laboratory strain) (b) \u003cem\u003eAe. aegypti \u003c/em\u003e(Wild strain) (c) \u003cem\u003eAe. albopictus \u003c/em\u003e(Laboratory strain) (d) \u003cem\u003eAe. albopictus \u003c/em\u003e(Wild strain). EC, epithelial cell and MG, midgut. Scale bars, 20mm.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141836/v1/ee9bb6ed1aaa43f788bd1e00.jpg"},{"id":87748110,"identity":"424cc851-0d78-41f1-b566-a02531a0359f","added_by":"auto","created_at":"2025-07-28 14:31:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":57535,"visible":true,"origin":"","legend":"\u003cp\u003eHistological section of \u003cem\u003eAedes sp \u003c/em\u003emidgut exposed to mix ATSBs in cross-section position under magnification of 20x. (a) \u003cem\u003eAe. aegypti \u003c/em\u003e(Laboratory strain) (b) \u003cem\u003eAe. aegypti \u003c/em\u003e(Wild strain) (c) \u003cem\u003eAe. albopictus \u003c/em\u003e(Laboratory strain) (d) \u003cem\u003eAe. albopictus \u003c/em\u003e(Wild strain). EC, epithelial cell and MG, midgut. Scale bars, 20mm.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141836/v1/4b68cbb45d8c334ea67ced26.jpg"},{"id":87748114,"identity":"6c8aa55b-4526-4480-890c-93a48965a2cf","added_by":"auto","created_at":"2025-07-28 14:31:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":72460,"visible":true,"origin":"","legend":"\u003cp\u003eHistological section of \u003cem\u003eAedes sp \u003c/em\u003emidgut exposed to control ATSBs in cross-section position under magnification of 20x. (a) \u003cem\u003eAe. aegypti \u003c/em\u003e(Laboratory strain) (b) \u003cem\u003eAe. aegypti \u003c/em\u003e(Wild strain) (c) \u003cem\u003eAe. albopictus \u003c/em\u003e(Laboratory strain) (d) \u003cem\u003eAe. albopictus \u003c/em\u003e(Wild strain). EC, epithelial cell and MG, midgut. Scale bars, 20mm.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7141836/v1/d3381442ef5869465eb86069.jpg"},{"id":102785383,"identity":"64f0d876-e765-416f-b76c-5d196ec0826a","added_by":"auto","created_at":"2026-02-16 16:06:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1281888,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7141836/v1/355c24eb-3a85-46b7-9ec3-a30dfa2b5406.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of olfactory sensitivity preferences on midgut pathophysiology of Aedes mosquitoes towards different sugar variants","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe \u003cem\u003eAedes\u003c/em\u003e mosquito is a disease vector of dengue, chikungunya, zika and yellow fever virus (Huang et al., 2020). The two \u003cem\u003eAedes\u003c/em\u003e species, commonly known for mosquito-borne diseases are \u003cem\u003eAedes aegypti\u003c/em\u003e and \u003cem\u003eAedes albopictus\u003c/em\u003e. Some of them are notable for biting nuisances and disease vector that is capable of carrying lethal pathogens to humans and other animals. The mosquito\u003cem\u003e-\u003c/em\u003eborne diseases have been a serious global issue for the past 50 years, causing almost 390\u0026nbsp;million infections per year to humans (Bhatt et al., 2013). Many methods were introduced to reduce the risk of mosquito-borne disease, especially where no effective vaccines are available. However, the vector control requires a proper strategical method in term of understanding the biology and behaviour of the mosquitoes.\u003c/p\u003e\u003cp\u003eOlfaction is very crucial for the survival and reproduction of mosquitoes because it enables the mosquitoes to locate resources such as food and oviposition localities. Thus, they have a complex sensory system. This system enables the mosquitoes to identify and respond to ecologically significant volatile organic chemicals and occasionally volatile inorganic compounds such as carbon dioxide and ammonia in their surroundings. Mosquitoes have evolved an intricate sensory system. Olfactory sensory neurons which are present in hair-like sensilla on the antennae, maxillary palps and proboscis are responsible for detecting odorants (Hill et al., 2021). The number and type on sensilla found on mosquito olfactory appendages differ depending on the species and stage of development. Odorants are presumed to diffuse through countless pores on the sensilla surface then entering an aqueous lymph to contact with the spectrum of molecular receptors found on dendrites of olfactory receptor neurons (Montell \u0026amp; Zwiebel, 2016). Three complementary methods of controlling mosquitoes can be implemented with odorants are as repellents that \"push\" mosquitoes away, \"maskers\" that restrict mosquito attraction to humans, and attractants that \"pull\" mosquitoes into traps that are set far from individuals (Mulatier et al., 2022).\u003c/p\u003e\u003cp\u003eOriginally, the purpose of attractive toxic sugar bait was to suppress the malaria vector, \u003cem\u003eAnopheles gambiae\u003c/em\u003e at Mali, West Africa (M\u0026uuml;ller et al., 2010). Following the \"attract and kill\" approach, it has been recognized as an innovative technique that can effectively eradicate adult mosquito populations (Nur et al., 2020). The \"attract and kill\" approach used mosquito tendency to feed on sugar by attracting them to consume insecticidal sugar source. Commonly, attractive toxic sugar bait consists of one or more aromatic compounds as the attractant including fruit juice, floral scent, feeding stimulants and oral toxin (Nur et al., 2020). The well-known methods to deliver attractive toxic sugar bait solution is by using direct spraying on the vegetation surface or by fixing a bait station (Mangan et al., 2014). However, evaporation of the solution may become a major problem by altering the toxin concentration in the solution. Exposure to direct sunlight cause high evaporation rate (Cheng et al., 2011). Therefore, a bait matrix should be used to deliver the solution such as alginate hydrogel beads. The alginate hydrogel beads are made of sodium alginate where alginate is a naturally occurring anionic polysaccharide that can be extracted from marine brown algae such as \u003cem\u003eLaminaria hyperborea, Laminaria digitate, Laminaria japonica, Ascophyllum nodosum\u003c/em\u003e and \u003cem\u003eMacrocystis pyrifera\u003c/em\u003e (Singh et al., 2022). Usage of alginate hydrogel beads are highly recommended due to its biodegradable and non-toxic properties to the environment (Lee \u0026amp; Mooney, 2012). Additional advantageous features are high efficacy with low maintenance and it is cheap to produce (McCalla et al., 2020; Tay et al., 2020).\u003c/p\u003e\u003cp\u003eThe attracted targeted sugar baits (ATSBs) are mosquito control tools that utilize mosquitoes\u0026rsquo; natural tendency to feed on plant sugars. By mixing sugar solutions with toxic agents or biological pesticides, ATSBs can attract mosquitoes that ingest the bait and are subsequently killed. Both male and female mosquitoes need sugar for energy, making ATSBs effective against a broad range of mosquito species. These baits are usually set in specific bait stations near human dwellings or mosquito breeding sites, where they can effectively reduce mosquito populations with minimal environmental impact (Fiorenzano et al., 2017). The infusion of proper attractant into the ATSBs is very crucial since the mosquitoes will detect the odour to reach the baits. Numerous materials have been used in recent years to actively study mosquito preferences. These include studies on mosquito attraction to synthetic flora-based attractants like phenylacetaldehyde, linalool oxide, phenylethyl alcohol, and acetophenone (Fikrig et al., 2017), plant tissue, ripe fruits, seedpods, floral and extra floral and host-derived chemicals including l-lactic and 1-octen-3-ol (Sissoko et al., 2019).\u003c/p\u003e\u003cp\u003eThe neurological and behavioral components of olfaction have been given a lot of attention meanwhile the physiological effects of these olfactory-mediated feeding decisions especially on the mosquito midgut is less understood. The midgut functions as the primary site of digestion, nutrient absorption, and microbial interaction. It plays a vital role in metabolic regulation and immune responses following sugar or blood intake (Barletta et al., 2017). The mosquito's physiological response to various dietary intake types and dosages can be observed through histological changes in midgut tissues, such as modifications in peritrophic matrix structure, cellular hypertrophy, or alterations in epithelial integrity (Wang et al., 2011). The mosquito olfactory-driven feeding preferences may indirectly influence these histological features since different sugars exhibit varying effects on tissue structure, enzyme activity, and gut microbes.\u003c/p\u003e\u003cp\u003eHowever, the infusion of natural sugar extracts in polymer as attractant for mosquitoes are very limited. Thus, this study mainly focusing on preference index of adult \u003cem\u003eAe. albopictus\u003c/em\u003e and \u003cem\u003eAe. aegypti\u003c/em\u003e response towards fruit-based, floral-based and mix-based ATSBs. This study investigated the potential combination of hydrogel polymer application with ATSBs to control populations of \u003cem\u003eAedes\u003c/em\u003e mosquitoes and the findings provide an overview on the preference of mosquitoes exposed to different sugar variant. Examining the relationship between sugar feeding in relation to \u003cem\u003eAedes\u003c/em\u003e mosquito olfactory responses and its effect to the mosquito gut condition is a novel approach that may provide information on how various sources in environment influence their behavior and physiology.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Mosquito colony maintenance\u003c/h2\u003e\u003cp\u003eThe eggs of laboratory strain \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e were obtained from Vector Control Research Unit (VCRU), School of Biological Sciences (SBS), University of Science Malaysia. The rearing process was carried out at Insectary Building G025A \u0026ndash; Medical Entomology Laboratory, SBS. The rearing process was conducted under the condition where the temperature was maintained at 27\u0026deg;C (\u0026plusmn; 2\u0026deg;C). Meanwhile, the relative humidity and a photoperiod were monitored at 75% (\u0026plusmn; 3%) and 12: 12 h (L:D) respectively. The eggs were hatched in dechlorinated water and routinely, the newly emerged larvae were transferred into a metallic tray filled with dechlorinated water (depth\u0026thinsp;=\u0026thinsp;2 cm, diameter\u0026thinsp;=\u0026thinsp;12 cm). The larvae were fed for two days once with a 2:1:1:1 ratio of fine powder formed by a mixture of cat food, beef liver, yeast and milk powder. Then, the pupae were placed in a 250mL capacity of plastic cup that were kept in mosquito rearing cages measuring 30 cm \u0026times; 30 cm \u0026times; 30 cm. The newly emerged adult was provided with a 10% sucrose solution as a food source. The eggs of wild strain \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e were collected around University of Science Malaysia and was reared in Insectary Building G025A \u0026ndash; Medical Entomology Laboratory, SBS under same condition with laboratory strain.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 ATSBs preparation\u003c/h2\u003e\u003cp\u003eThe mango (Waterlily Thai Mango) and \u003cem\u003eChrysanthemum\u003c/em\u003e was purchased from Lotus Sungai Dua, Penang, Malaysia. Mango extract was obtained by peeling off the skin of mango and it was cut into small pieces. Then, distilled water added to the mango pieces with a dilution ratio of 2:1, distilled water: mango. It was blended together and the solution was filtered using filter paper. Extracted mango solution was used to make 30% v/v of mango (MG) ATSBs. Thus, 120mL of mango solution and 280mL of distilled water was mixed together. Then, to produce the ATSBs, 5g of sodium alginate powder was weighed and blended together with the mixture until homogenous suspension formed. It was then sonicated in an ultrasonic bath (BactoSonic\u0026reg;) at 40\u0026deg;C for 45 minutes. After 45 minutes, the emulsion was cooled to room temperature. Formation of the spherical ATSBs was carried out by dropping the emulsion into 0.2 M CaCl\u003csub\u003e2\u003c/sub\u003e solution with a rate of 300 rpm at 30\u0026deg;C. Finally, the ATSBs were filtered and washed using distilled water for three times to make sure excess chloride ion was removed from it. For 30% v/v \u003cem\u003eChrysanthemum\u003c/em\u003e (FL) ATSBs, the petals were detached and weighed. Control (C) ATSBs were produced by using sucrose solution. Similar steps were repeated for 30% v/v \u003cem\u003eChrysanthemum\u003c/em\u003e, 30% v/v mix mango \u0026amp; \u003cem\u003eChrysanthemum\u003c/em\u003e (MX) and sucrose ATSBs as in production of 30% v/v of mango ATSBs. Sucrose ATSBs were the control ATSBs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Olfactometer design\u003c/h2\u003e\u003cp\u003eA modified olfactometer was used to determine the olfactory response of mosquitoes to different odors of ATSBs [Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e]. This modified olfactometer was designed based on the olfactometer designed by (Afify et al., 2014). The olfactometer consist of three main components which were holding chamber, flight chamber and olfactory bioassay chamber. The mosquito cage was covered by using transparent PVC cover with a thickness of 0.5 mm. Compartment for holding chamber was created by using a tube with a diameter and length, 4.4 cm and 12.5 cm respectively. One end of the tube was covered with mesh screen cap to enable airflow for the mosquito survival while the other end that was connected with flight chamber was covered with a sliding gate. Function of the holding chamber is to hold the mosquitoes for acclimation before it was introduced into flight chamber. The size of the flight chamber compartment measured as (30 cm \u0026times; 30 cm \u0026times; 30 cm) and it was integrated with three circular openings where each of the diameter were 4.4 cm. This chamber enabled the decision making of mosquitoes on flying direction to either one of the olfactory bioassay chambers. This olfactory bioassay chambers consisted of two tubes with a diameter and length, 4.4 cm and 12.5 cm respectively. One of the ends of this tube was covered with mesh screen cap while the other end was covered with a sliding gate which is connected to the flight chamber. Inner part of the olfactory bioassay chambers was covered with mesh screen too to ensure the mosquitoes were not attracted by visual attraction and also to prevent them from having direct contact with ATSBs. The door was designed to ease the cleaning process of this olfactometer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Evaluation of mosquito olfactory response\u003c/h2\u003e\u003cp\u003eThe olfactory response evaluation of the mosquitoes towards 30% v/v of mango, \u003cem\u003eChrysanthemum\u003c/em\u003e, mix and sucrose ATSBs was carried out as two bioassay studies such as non-choice assay and choice assay. For the non-choice assay, the one of the olfactory bioassay chambers was filled with only attractant while the other one was filled with sucrose ATSBs. However, for the choice assay attractants were placed in both of the bioassay chamber. Experiment was started by aspirating 20 \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes (10 male and 10 female) which was ranging from 5\u0026ndash;7 days old. The aspirated mosquitoes were released into holding chamber and left it for 1 minute for acclimatization. Meanwhile, 30g of mango ATSBs was weighed and placed into one of the bioassay chambers while another chamber was filled with 30g of sucrose ATSBs as a negative control port. Then, the sliding gate for bioassay chambers were opened and two portable mini USB fans were placed at the end of each mesh cap. The lowest speed was set up to enable the scent of ATSBs flow into the flight chamber. After one minute, sliding gate of holding chamber was opened to release the mosquitoes into flight chamber and it was closed immediately once holding chamber was empty. After 2 minutes, bioassay chamber gate was closed and the number of mosquitoes on the mesh screen of the bioassay chambers were counted. The test was then repeated by placing two attractants in bioassay chambers for the choice assay. After completing the test for laboratory strain \u003cem\u003eAe. aegypti\u003c/em\u003e it was replaced with laboratory strain \u003cem\u003eAe. albopictus\u003c/em\u003e followed by wild strain with the same procedure according to Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The olfactometer was washed and dried before used it again to remove the lingering odour. New batch of mosquitoes that were not exposed to any of the odour was used in every test and each of the treatment was repeated for three times.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAttraction pairing for olfactory bioassay response of \u003cem\u003eAedes\u003c/em\u003e mosquitoes.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eOlfactory attraction bioassay\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eAttraction pairs of ATSBs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePair labelling\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChamber 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChamber 2\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNon-choice assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMango\u003c/p\u003e\u003cp\u003e\u003cem\u003eChrysanthemum\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMix\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003cp\u003eControl\u003c/p\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMGC\u003c/p\u003e\u003cp\u003eFLC\u003c/p\u003e\u003cp\u003eMXC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChoice assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMango\u003c/p\u003e\u003cp\u003eMango\u003c/p\u003e\u003cp\u003eMix\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eChrysanthemum\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMix\u003c/p\u003e\u003cp\u003e\u003cem\u003eChrysanthemum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMGFL\u003c/p\u003e\u003cp\u003eMGMX\u003c/p\u003e\u003cp\u003eMXFL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e*MGC refers to mango and control ATSBs; FLC refers to \u003cem\u003eChrysanthemum\u003c/em\u003e and control ATSBs; MXC refers to mix and control ATSBs; MGFL refers to mango and \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs; MGMX refers to mango and mix ATSBs and MXFL refers to mix and \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Midgut histology analysis\u003c/h2\u003e\u003cp\u003eBoth laboratory and wild strain female mosquitoes of \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e were fed with 30% w/v of mango, chrysanthemum, mix and control ATSBs. The mosquitoes were killed after three days and kept for overnight in 70% ethanol. After the dissection, the abdomens were stained for 15 minutes using eosin (0.5% of Alcoholic Eosin Y- Solution, Sigma-Aldrich). Then, the samples were dehydrated using gradual ethanol series such as 50% (30 minutes), 60% (15 minutes), 70% (15 minutes), 80% (30 minutes), 90% (30 minutes) and finally 100% (60 minutes). Subsequently, tissues were cleared in xylene for 15 minutes to enable paraffin embedding. Then, infiltration of samples was done by immersing in melted paraffin wax at 60\u0026deg;C in four stages (15 minutes each) which enable complete infiltration.\u003c/p\u003e\u003cp\u003eAbdomens were embedded in paraffin wax using a tissue embedding center machine (Tissue Embedding \u0026amp; Cooling System \u0026ndash; KEDEE) and cooled at -15\u0026deg;C overnight. The next day, specimen blocks were sectioned at 8 \u0026micro;m using a Leica BioCut Rotary Microtome. Then, the sectioned wax stripes were floated in a 40\u0026deg;C water bath and mounted onto glass slides. The glass slides were dried at 40\u0026deg;C overnight. Final staining process was performed by using histoclear, ethanol, hematoxylin, bluing agent and eosin. Finally, the samples were examined using a light microscope at 10X, 20X, and 40X magnification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe olfactory response for both \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e was obtained from Preference Index (PI), described by Nur et al., 2020. Preference Index was calculated using this formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Preference\\:Index\\:\\left(PI\\right)=\\frac{Number\\:of\\:mosquitoes\\:in\\:chamber\\:1-Number\\:ofmosquitoes\\:in\\:chamber\\:2}{Number\\:of\\:mosquitoes\\:in\\:chamber\\:1+Number\\:ofmosquitoes\\:in\\:chamber\\:2}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe PI value which ranging from \u0026minus;\u0026thinsp;1 to +\u0026thinsp;1 where negative value indicates mosquito repellence while positive value indicates mosquito attraction. Meanwhile, the value 0 refers to neutral response of mosquitoes. The normality of data for both non-choice and choice assay were checked by using Shapiro-Wilk test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Moreover, equal variance across the sample was assumed when Levene\u0026rsquo;s test yield (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Then, parametric independent sample t-test was used to analyse the significances of the mosquito olfactory response expose to different attractant since the assumptions of Student\u0026rsquo;s t-test was met (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All statistical analyses were tested by using IBM SPSS statistic version 28.0.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Response of \u003cem\u003eAedes sp\u003c/em\u003e in non-choice assay\u003c/h2\u003e\u003cp\u003eThe response of \u003cem\u003eAedes sp.\u003c/em\u003e to three sets of attractant pairings was used to calculate their preference index (PI). Both the attractancy (positive response) and repellency (negative response) of mosquitoes to the specified attractants are determined by the set of bar graphs. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the bar graph that represent mean preference index (PI)\u0026thinsp;\u0026plusmn;\u0026thinsp;SE while Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicates the mean number of mosquitoes \u0026plusmn; SE of laboratory and wild strain of \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e on non-choice assay with 3 pairs of treatment sets.\u003c/p\u003e\u003cp\u003eFor set MGC (mango and control), laboratory strain \u003cem\u003eAe. aegypti\u003c/em\u003e were significantly attracted to mango attractants compared to sugar (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;6.12, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). Furthermore, for FLC (\u003cem\u003eChrysanthemum\u003c/em\u003e and control) set, it shows a significant difference where (t (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;4.01, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045). However, laboratory strain \u003cem\u003eAe. aegypti\u003c/em\u003e indicate that MXC (mix and control) has no significant different (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.00, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.067) to attract mosquitoes. The mean PI\u0026thinsp;\u0026plusmn;\u0026thinsp;SE in MGC displays a strong preference (0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15) followed by MXC with least preference (0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04). Nevertheless, FLC shows (-0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) moderate repellency by the mosquitoes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe highest mean number of laboratory strain \u003cem\u003eAe. aegypti\u003c/em\u003e \u0026plusmn; SE (6.00 \u0026plusmn; 0.577) attracted to mango ATSBs compare with other treatment ATSBs. The \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs yield the lowest attraction (1.33 \u0026plusmn; 0.333) while the control ATSBs recorded the highest (6.33 \u0026plusmn; 1.202) number of mosquitoes when tested with FLC pair (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe mean PI\u0026thinsp;\u0026plusmn;\u0026thinsp;SE for laboratory strain \u003cem\u003eAe. albopictus\u003c/em\u003e shows (0.79 \u0026plusmn; 0.1), (-0.6 \u0026plusmn; 0.22) and (0.21 \u0026plusmn; 0.04) for the pair of MGC, FLC and MXC respectively. The high positive PI value indicates that this species was strongly preferred to MGC ATSBs and the lower PI value represents the least favourable response towards MXC. However, it repels towards FLC ATSBs since the PI value was negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). When comparing the numbers of mosquitoes attracted to MG ATSBs, it displays relatively highest value (5.67 \u0026plusmn; 0.333) than MX ATSBs (2.33 \u0026plusmn; 0.333). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrates the laboratory strain \u003cem\u003eAe. albopictus\u003c/em\u003e, obtained the most minimal value of mosquito attraction (1.67 \u0026plusmn; 0.882) in FLC ATSBs compared to the other treatments used in this study. There was no statistically significant difference been observed for set of FLC (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) = -4.11, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.20) and MXC (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.12, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.101). In contrast, (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;10.61, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) highlights that the preference of \u003cem\u003eAe. albopictus\u003c/em\u003e towards MGC shows a statistically significant value.\u003c/p\u003e\u003cp\u003eThe preference of wild strain \u003cem\u003eAedes sp\u003c/em\u003e. is distinct from the activity of laboratory strain. The wild strain \u003cem\u003eAe. aegypti\u003c/em\u003e illustrates a strong attractancy towards MGC with a value of 0.74 \u0026plusmn; 0.13. In addition, a trend of weak attractancy (0.21 \u0026plusmn; 0.21) was observed when the mosquitoes were exposed to the set of MXC ATSBs. Similar to laboratory strain \u003cem\u003eAe. aegypti\u003c/em\u003e (-0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07), the wild strain also shows the same pattern where the value of PI is -0.57 \u0026plusmn; 0.12. The negative value indicates that both of the mosquito strain repels moderately towards FLC ATSBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe highest number of mosquitoes attracted were towards mango (6.00 \u0026plusmn; 0.577) followed by mix (3.00 \u0026plusmn; 0.577) and \u003cem\u003eChrysanthemum\u003c/em\u003e (1.33 \u0026plusmn; 0.333) ATSBs. By comparing the control ATSBs, maximum frequency of mosquitoes (5.00 \u0026plusmn; 0.577) were attracted in the experiment set of FLC. The analysis of student t-test demonstrate that the MGC yield a significant difference where (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;6.12, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004) while FLC result was (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) = -5.50, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010). However, a non- significant result was obtained for MXC (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;1.23, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.288) because the p-value is higher than 0.05.\u003c/p\u003e\u003cp\u003eNext, the experiment carried out by using wild strain \u003cem\u003eAe. albopictus\u003c/em\u003e reveals that the mosquitoes are extremely attractive to mango ATSBs since the combination set of MGC gain PI of 0.84 \u0026plusmn; 0.15. Conversely, the FLC set (-0.72 \u0026plusmn; 0.18) shows that the mosquitoes are extremely repel to the \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs. A weak attractancy of mosquitoes were identified for the MXC set by considering PI value of 0.19 \u0026plusmn; 0.18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Most prominent abundance of mosquitoes are attracted towards mango ATSBs (7.67 \u0026plusmn; 0.333) compared to control ATSBs (0.67 \u0026plusmn; 0.333). Additionally, mix ATSBs indicates the second highest in mosquito numbers (3.33 \u0026plusmn; 0.333). According to Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, least number was recorded for \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs (1.00 \u0026plusmn; 0.577). The set of MGC and FLC reveals that there is a significant difference of (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;14.85, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;4.11, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020) respectively. In contrast, (t (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.54, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.640) shows that MXC have non-significant impact on the mosquito attraction towards it.\u003c/p\u003e\u003cp\u003eIn conclusion, both laboratory and wild strain \u003cem\u003eAedes sp.\u003c/em\u003e are more likely to choose mango attractant compared to mix. However, the mosquitoes show a second interest to mix attractant compared to mango. Overall, \u003cem\u003eChrysanthemum\u003c/em\u003e repels both \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e from reaching the ATSBs in non-choice assay.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean number of mosquitoes \u0026plusmn; SE of \u003cem\u003eAedes sp\u003c/em\u003e in non-choice assay.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c2\" namest=\"c1\" rowspan=\"3\"\u003e\u003cp\u003eSet\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eMean number of mosquitoes \u0026plusmn; SE\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eLaboratory strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eWild strain\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eAe. albopictus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eAe. albopictus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMG\u003c/p\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFLC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFL\u003c/p\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.67 \u0026plusmn; 0.882\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.33 \u0026plusmn; 1.202\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.33 \u0026plusmn; 0.882\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMXC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMX\u003c/p\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.67 \u0026plusmn; 1.202\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e*MG refers to mango ATSBs; FL refers to \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs; MX refers to mix ATSBs and C refers to control ATSBs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Response of \u003cem\u003eAedes sp.\u003c/em\u003e in choice assay\u003c/h2\u003e\u003cp\u003eThe preference index (PI) of \u003cem\u003eAedes sp\u003c/em\u003e. was determined by analysing their reaction towards three sets of attractant pairings (MGFL, MGMX, and MXFL). The set of bar graphs indicates the attractancy of mosquitoes (positive response) and repellency (negative response) to the designated attractants. The mean number of mosquitoes \u0026plusmn; SE of laboratory and wild strains of \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e on choice assay experiment shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e while Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e displays the bar graph representing mean PI\u0026thinsp;\u0026plusmn;\u0026thinsp;SE.\u003c/p\u003e\u003cp\u003eFor set MGFL (mango and \u003cem\u003eChrysanthemum\u003c/em\u003e), laboratory strain \u003cem\u003eAe. aegypti\u003c/em\u003e were not significantly attracted to mango attractants compared to \u003cem\u003eChrysanthemum\u003c/em\u003e (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.50, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.82). However, for MGMX (mango and mix) set, it shows a significant difference where (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.50, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036). In addition, laboratory strain \u003cem\u003eAe. aegypti\u003c/em\u003e indicate that MXFL (mix and \u003cem\u003eChrysanthemum\u003c/em\u003e) has significant different (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.24, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) to attract mosquitoes. The mean PI\u0026thinsp;\u0026plusmn;\u0026thinsp;SE in MGFL displays a strong preference (0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17) followed by MGMX (0.70 \u0026plusmn; 0.15) and MXFL (0.61 \u0026plusmn; 0.20\u003cb\u003e).\u003c/b\u003e The highest mean number of laboratory strain \u003cem\u003eAe. aegypti\u003c/em\u003e \u0026plusmn; SE (3.00 \u0026plusmn; 0.577) attracted to mango ATSBs compared to mix ATSBs (0.67 \u0026plusmn; 0.333). The \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs yield the highest attraction (0.67 \u0026plusmn; 0.333) when tested together with mix ATSBs while the lowest (6.33 \u0026plusmn; 1.202) number of mosquitoes was recorded when tested using MGFL pair (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe choice assay study indicates that the PI value shows moderately positive results for laboratory strain \u003cem\u003eAe. albopictus\u003c/em\u003e. The PI value is the lowest for mango ATSBs (0.30 \u0026plusmn; 0.09) when compared to mix ATSBs. However, when mango ATSBs tested together with \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs, the value rises to 0.49 \u0026plusmn; 0.15. The highest PI obtained was 0.57 \u0026plusmn; 0.23 in the testing of MXFL pair. Maximum number of laboratory strain \u003cem\u003eAe. albopictus\u003c/em\u003e attracted to mango ATSBs (4.33 \u0026plusmn; 0.333) while (2.33 \u0026plusmn; 0.333) was attracted to mix ATSBs in the assessment of the MGMX pair. For the MGFL set, a slight reduction in numbers occurred to mango ATSBs treatment (4.00 \u0026plusmn; 0.577) compared to MGMX pair. Minimal number counts observed in MXFL, in which the mix yield (2.67 \u0026plusmn; 0.333) whereas, (1.00 \u0026plusmn; 0.577) was recorded in \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs. Hence, MXFL set displays a non-significant different value (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.50 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.082). In contrast, MGFL (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.00, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025) and MGMX (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.00, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) reveal that the pairs show significant different towards each other.\u003c/p\u003e\u003cp\u003eThe response of wild strain \u003cem\u003eAe. aegypti\u003c/em\u003e towards ATSBs are slightly different from laboratory strain. A strong attractancy response of mosquitoes were observed in PI of MGFL (0.70 \u0026plusmn; 0.15) followed by MXFL (0.64 \u0026plusmn; 0.18) and MGMX (0.45 \u0026plusmn; 0.08) respectively. In order to the number of mosquitoes attracted to the ATSBs, the highest number was recorded in MGMX set as the mango ATSBs attracted (5.00 \u0026plusmn; 0.577) while mix ATSBs drawn in (5.00 \u0026plusmn; 0.577) mosquitoes. The least number observed in \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs (0.67 \u0026plusmn; 0.333) in MGFL set. According to Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, mix ATSBs attracted (4.00 \u0026plusmn; 0.577) while \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs attracted only (1.00 \u0026plusmn; 0.577) mosquitoes. The student t-test analysis reveals that all sets MGFL (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;5.66, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005), MGMX (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.67, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021) and MXFL (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.67, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021) in choice assay shows a significant different value.\u003c/p\u003e\u003cp\u003eThe response of wild strain \u003cem\u003eAe. albopictus\u003c/em\u003e shows a moderate attractancy in MGFL \u003cb\u003e(\u003c/b\u003e0.44 \u0026plusmn; 0.06) and MGMX (0.39 \u0026plusmn; 0.06) except MXFL \u003cb\u003e(\u003c/b\u003e0.30 \u0026plusmn; 0.09) which indicates a weak attractancy value. A non-significant difference obtained for MGFL (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.83, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.080) whereas, MGMX (t (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.03, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029) and MXFL (t (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.24, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) shows significant difference. Counts of mosquitoes attracted to mango ATSBs (7.67 \u0026plusmn; 0.333) is the highest value recorded for choice assay of wild strain \u003cem\u003eAe. albopictus\u003c/em\u003e. However, the least number was observed for \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs (0.67 \u0026plusmn; 0.333) in MGFL pair. Finally, the number of mosquitoes attracted to mix ATSBs (5.33 \u0026plusmn; 0.882) in MGMX and (3.33 \u0026plusmn; 0.333) in MXFL are higher compared to their respective pair (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOverall, the results of choice assay show that the mango attractant is the most preferred by the \u003cem\u003eAedes sp\u003c/em\u003e. However, the mix ATSBs is secondarily preferred while Chrysanthemum is the least preferred ATSBs in choice assay.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean number of mosquitoes \u0026plusmn; SE of \u003cem\u003eAedes sp\u003c/em\u003e in choice assay.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c2\" namest=\"c1\" rowspan=\"3\"\u003e\u003cp\u003eSet\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eMean number of mosquitoes \u0026plusmn; SE\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eLaboratory strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eWild strain\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eAe. albopictus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eAe. albopictus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMGFL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMG\u003c/p\u003e\u003cp\u003eFL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.00\u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMGMX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMG\u003c/p\u003e\u003cp\u003eMX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.33 \u0026plusmn; 0.882\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMXFL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMX\u003c/p\u003e\u003cp\u003eFL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.33 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.67 \u0026plusmn; 0.333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.00 \u0026plusmn; 0.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.67 \u0026plusmn; 1.202\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Histological observation\u003c/h2\u003e\u003cp\u003eThe midgut condition of the mosquitoes exposed to different ATSBs illustrated through light microscopy image under scale bar of 20\u0026micro;m. The findings showed noticeable features in the midgut epithelial cells and overall midgut structure of lab and wild strain \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e that were fed with mango ATSBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall, the midgut structure was slightly degrading in all the samples. Thus, it causes minor breakage of the midgut. In addition, the arrangement of epithelial cells displays distinct changes. The epithelial cells in laboratory and wild strain \u003cem\u003eAe. albopictus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (c) and (d)) were non-uniformly distributed and less visible. However, the epithelial cells in \u003cem\u003eAe. aegypti\u003c/em\u003e are clearly visible and it is closely packed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAccording to Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, mosquitoes exposed to \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs show irregular shaped midgut which indicates it undergoes degradation and shrinkage. The density of epithelial cells still high and appeared to be slightly distanced or separated from midgut. Hence, it showed dispersed epithelial cells with several clearing regions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eAedes sp.\u003c/em\u003e exposed to mix ATSBs had unique morphological patterns in their midgut and epithelial cells. The midgut shrinks which cause uneven size and shape of the midgut. Furthermore, dislocation of the midgut is observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(b) where the midgut of wild strain \u003cem\u003eAe. aegypti\u003c/em\u003e was pushed to a side. Degradation and minor instances of breakage midgut structure was observed. Epithelial cells surrounding the midgut are orderly arranged but it shows cell degeneration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe midgut structure of mosquitoes that exposed to control ATSBs were normal. The midgut is thicker and it has the even shape. There was no breakage or degradation of midgut is noticed. Additionally, the midgut is surrounded by the orderly arranged epithelial cells. All of the mosquito species fed on control ATSBs does not exhibit notable differences from one another (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eGenerally, mosquitoes need sugar meal as a source of carbohydrates, particularly to maintain population fitness. As a result, they conveniently consume any sugary food that is offered and might supply enough energy for them (Nur et al., 2020). The ecology of mosquitoes which includes their consumption of various forms of sugar, is described by phytophagy, which is notable for insects that ingest on plants. It includes extrude, plant sap, nectar (nectarivory), and fruit juices (frugivory) (Peach and Gries, 2020a). An excellent baiting tool could be created by developing bait attractants or repellents that imitate natural sugar sources, particularly when competing with the natural sugar sources found in their ecosystem.\u003c/p\u003e\u003cp\u003eSpecies preferences such as exophagic and endophagic behavior may influence sugar selection (Tananchai et al., 2012 \u0026amp; 2019). Data gathered in this study regarding mosquito preference between mango and mix indicates significance attraction for both of the \u003cem\u003eAedes\u003c/em\u003e species. Although it was given a choice, still the mango ATSBs attracted mosquitoes the most. In the choice assay, the mosquitoes did not demonstrate the distinctive difference since the odour of the ATSBs were mixed together. They tend to confuse and their desire for consistent sugar consumption may be the cause that forcing them to consume on any readily available sugar sources (Nur et al., 2020). All of the laboratory and wild strain of \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e shows the similar pattern for non-choice assay, where they mostly attracted to mango but slightly attracted to mix. However, they show strong repellency towards \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs.\u003c/p\u003e\u003cp\u003eMango is selected because of their high sugar content and have been previously recorded for mosquito attraction. According to Bello-P\u0026eacute;rez et al. (2007), ripe mango fruit contains a significant source of sugars (glucose, fructose, and sucrose) as well as other carbohydrates including starch and pectin. All of these substances are important from a flavour and nutritional perspective. About 15% of the total sugars in mature mangos are found in the fruit flesh. Sucrose is the main sugar in ripe mango fruit, but fructose is the main monosaccharide during the preclimatic period (Maldonado et al., 2019). Initial studies on mango fruit volatile compounds were primarily well-known for invention of ATSBs. The discovery regarding those chemical components satisfies the oral-toxic bait criteria for \u003cem\u003eAedes\u003c/em\u003e mosquito control. The identified volatile compounds that can attract the \u003cem\u003eAedes\u003c/em\u003e mosquitoes classified under alcohol, aldehyde, sesquiterpenes and monoterpenes groups. Sesquiterpenes and monoterpenes are the essential chemical compounds to enhance the flavour and fragrance of the plants. For instance, α-pinene, β-ocimene and Thujone are groups of monoterpenes while α-humule, Caryophyllene, and σ-cadinene are the sesquiterpene that are detachable for \u003cem\u003eAedes\u003c/em\u003e mosquitoes. Nevertheless, monoterpene is more volatile compared to sesquiterpenes. A groundbreaking study shown the ability of these volatile substances to attract the mosquitoes of both sexes. The chemical cues involved for plant-mosquito interactions are chemicals from the aldehyde class, such as hexanal and alcohol, more precisely (Z) 3-Hexen-1-ol (Nyasembe \u0026amp; Torto, 2014).\u003c/p\u003e\u003cp\u003eAccording to previous study, a few floral semiochemicals were identified to guide the mosquitoes in locating the sugar sources such as ketones, terpenes, aldehydes, alcohols, fatty acids and fatty acid derivatives (Peach \u0026amp; Gries, 2019). There are many previous studies that discussed about the usage of fruits attractant to attract the mosquitoes. A prior study by Tenywa et al. (2017) noted that \u003cem\u003eAnopheles\u003c/em\u003e spp. get attracted by the juice of subtropical fruits such bananas, mangoes, and guavas. Furthermore, Meza et al. (2020) have determined the essential components of mango juice that enhance the baiting attractant for \u003cem\u003eAnopheles gambiae\u003c/em\u003e against malaria vectors. According to a research by Fiorenzano et al. (2017), mosquitoes responded to banana slices by piercing on them and sticking their proboscis into the fruit cavitation. It provided evidence for the potential benefit of these fruits in drawing mosquitoes. Mango fruit flesh is able to attract adult \u003cem\u003eAe. aegypti\u003c/em\u003e, however artificial mango fruit concentration appears to have little influence on the attraction factor for adult \u003cem\u003eAe. aegypti\u003c/em\u003e (Barbosa et al., 2019). In addition, both the aqueous and pellet forms of black pepper seed extraction were successful in drawing female \u003cem\u003eAe. aegypti\u003c/em\u003e to the study conducted by Briones et al. (2012). According to Sissoko et al. (2019), the fruit flesh of the \u003cem\u003eCarica papaya\u003c/em\u003e (Papaya), \u003cem\u003eC. melo\u003c/em\u003e (Honeydew melon), and \u003cem\u003eM. indica\u003c/em\u003e (Mango) has been shown to be attractive to both male and female \u003cem\u003eAe. aegypti\u003c/em\u003e. In the experiment by Nur et al. (2020), the fruit peel of \u003cem\u003eC. papaya\u003c/em\u003e was remarkably effective in drawing adult \u003cem\u003eAe. aegypti\u003c/em\u003e in both choice and no-choice experiments.\u003c/p\u003e\u003cp\u003eThe epithelial cells in the abdomen that was provided mango ATSBs showed a modified shape. Mango treatments resulted in a visible rupture of the epithelial cells. The natural biocides that are present in mangos may be the cause of this breakage. Compounds like tannins, alkaloids and polyphenols which have antimicrobial and biocidal qualities are known to be present in mangoes (Kim et al., 2021). The observed midgut breakage resulted from interactions between these biocides and the epithelial cells. The deterioration of the mosquito midgut following the consumption of mango may be linked to particular physiological and biochemical reactions driven on by the substances in mangos. These substances also cause degeneration by harming the midgut's lining cells.\u003c/p\u003e\u003cp\u003eAdditionally, these compounds also impair digestion and the absorption of nutrients by interfering with the action of the enzymes in the gut and damage gut tissues by producing reactive oxygen species which causes oxidative stress (Ribeiro \u0026amp; Schieber, 2009). Furthermore, due to their high sugar content, mangoes have the potential to cause cell damage by changing the osmotic balance in the mosquito gut (Kiani et al., 2022). Water inflow into the midgut due to this imbalance may result in distension and rupture. Despite their usual diet, mosquitoes might not be able to withstand such a high osmotic burden. This provides an atmosphere that encourages the growth of dangerous microorganisms which can destroy the lining of the gut.\u003c/p\u003e\u003cp\u003eIn order to test the mix baits attractancy effect, mango and \u003cem\u003eChrysanthemum\u003c/em\u003e were altered and improved using the blending method. This study conducted using a 1:1 combination of mango and \u003cem\u003eChrysanthemum\u003c/em\u003e as an attractant. During this study, the mosquitoes were exposed to the mixed attractant and either the mango or \u003cem\u003eChrysanthemum\u003c/em\u003e attractant at the same time. According to the observation, mosquitoes continue to be positively attracted to the combined attractants. This event confirms that the mango dominated the \u003cem\u003eChrysanthemum\u003c/em\u003e. As we know, \u003cem\u003eChrysanthemum\u003c/em\u003e are a powerful repellent that has been employed in the control approach, whereas mangoes are great attractants. Combined attractants improve the attractancy while combination of repellents enhance the repellency of the mosquitoes for a better application of ATSBs bait. Furthermore, enhancing the attractant component is necessary to increase the effectiveness of the ATSBs bait application since the bait must compete with the available sugar sources in the natural habitat. It clarifies how mangoes and \u003cem\u003eChrysanthemum\u003c/em\u003e provide a strong attraction for Aedes mosquitoes. It is clear from the results that the fruit extract is essential to the development of the ATSBs baiting tools since it demonstrates a positive attraction towards mango and \u003cem\u003eChrysanthemum\u003c/em\u003e in comparison to glucose (control).\u003c/p\u003e\u003cp\u003eA significant difference structure of midgut and its epithelial cells was demonstrated by the mix ATSBs exposed mosquitoes. This is due to the toxic compounds present in mango and \u003cem\u003eChrysanthemum\u003c/em\u003e which cause adverse effects on it. The midgut of mosquitoes exposed to control ATSBs was seen to have closely packed columnar epithelial cells (Fig.\u0026nbsp;5.8). These cylindrical cells have a large and coarsely granular nucleus in the center of the cell. It is unclear whether if the various sugar varieties utilized in the feeding studies or the histology processing methods were accountable for this disruption. The fundamental reason of the observed cell destroying has to be investigated further.\u003c/p\u003e\u003cp\u003ePlants provide mosquitoes with sugars, amino acids, and other resources through their nectars, honeydew, and decomposing fruits (Nyasembe et al., 2018). Mosquitoes employ a variety of sugars, including sucrose, fructose, and glucose, in the sugar-based meals they consume as their main energy source (Leyva et al., 2024). The types of sugar content in the attractant also influence the response of the mosquitoes towards the ATSBs. Carbohydrates are often divided into three primary types based on the number of sugar units such as monosaccharides, oligosaccharides, and polysaccharides. Monosaccharides or commonly termed as simple sugars are the most basic class of carbohydrates and the general formula is C\u003csub\u003en\u003c/sub\u003e(H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003en\u003c/sub\u003e. They are known as aldoses when the functional group is -CHO (aldehyde), and ketoses when the functional group is -CO (keto). Glucose, galactose, and fructose are a few varieties of monosaccharides. Monosaccharides sugars have the reducing properties (Khadka, 2022). Around 3\u0026ndash;10 monosaccharide molecules are present in oligosaccharides and it break down during hydrolysis. Depending on the quantity of monosaccharides, they can be either disaccharides or trisaccharides. Majority of the plants contain few oligosaccharides that occur naturally. Melibiose (galactose and glucose) and fructose combine to form raffinose, a trisaccharide that is present in many plants. For instance, maltose and sucrose. High molecular weight substances called polysaccharides are polymers of monosaccharides. They are non-sugary compounds because the sugary taste absence in it. Their primary focus is on two crucial functions such as energy and structural storage. Glycosidic bonds that develop at any one of a monosaccharide hydroxyl groups, causes branches to appear in polysaccharides. Example of polysaccharides including cellulose and starch.\u003c/p\u003e\u003cp\u003eThe sugar level in \u003cem\u003eChrysanthemum\u003c/em\u003e is minimal hence, they are not naturally sweet since they are predominantly made up of water, fibre, and trace nutrients. The approximate chemical compositions of \u003cem\u003eChrysanthemum\u003c/em\u003e were 12.8% in reducing sugar where fructose, glucose, and sucrose made up the majority of free sugars. Chrysanthemums like other flowering plants, store sugars and other carbohydrates for energy and to produce nectar that will draw pollinators. The main sugar that present include Chrysanthemum nectar and floral tissues contain sucrose, a common sugar that is essential for pollinators energy needs. The concentrations of these reducing sugars are lower than those of sucrose. They provide readily metabolizable energy and are produced when the enzyme invertase breaks down sucrose. The tissues of \u003cem\u003eChrysanthemum\u003c/em\u003e also contain polysaccharides, such as starch, which can be converted to simple sugars when the plant needs energy (Li et al., 2019). High glucose and fructose predominate in flowers visited by flies or beetles, while high sucrose proportion is frequently linked to flowers attracted for bees or butterflies (W\u0026auml;ckers, 2005). A damaged morphology of the midgut also appeared in the \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs treated mosquitoes. The loosely packed configuration of the epithelial cells demonstrated a clearing state. Some of the cells were also broken. Pyrethrin, a naturally occurring biocide frequently present in \u003cem\u003eChrysanthemums\u003c/em\u003e may have contributed to this fracture. According to Bowman et al. (2018), pyrethrin is well-known for its insecticidal qualities and application as a pesticide. Consequently, it is possible that the observed cell breakage was caused by the pyrethrin included in the treatment. The midgut of mosquitoes may shrink due to the toxic effects of pyrethrins. The pyrethrins causes cell damage, apoptosis and reduced the functionality of the midgut cells. Pyrethrins have the potential to directly harm the midgut's epithelial cells by rupturing cellular membranes or producing oxidative stress. Cellular shrinkage and tissue atrophy result from damage to midgut cells, which can also affect digestion and nutritional absorption (Nagai et al., 2022). Toxin exposure cause midgut tissues to undergo apoptosis, or programmed cell death which reduces tissue size and cell volume (Elmore et al, 2007).\u003c/p\u003e\u003cp\u003eIn contrast, the odour of sweet alyssum (\u003cem\u003eLobularia maritima\u003c/em\u003e) recorded to attract \u003cem\u003eAe. aegypti\u003c/em\u003e (Von Oppen \u003cem\u003eet al.\u003c/em\u003e, 2015). The attractiveness of the adult \u003cem\u003eAe. aegypti\u003c/em\u003e of several blooming plant species was evaluated using their blossoming branches in the Sissoko et al. (2019) study. The \u003cem\u003eAe. aegypti\u003c/em\u003e males and females were attracted to the flowering branches of \u003cem\u003eAcacia macrostachya\u003c/em\u003e, \u003cem\u003eA. salicina\u003c/em\u003e, \u003cem\u003eGalphimia gracilis\u003c/em\u003e, and \u003cem\u003eProsopis juliflora\u003c/em\u003e among the treatments.\u003c/p\u003e\u003cp\u003eSince the concentration of attractants influence the study, further studies on the different combinations of attractants concentration are highly suggested. Others despite than mango and \u003cem\u003eChrysanthemum\u003c/em\u003e as the attractant and repellent component, different fruits and flowers are also suggested to expand the choice selection in preparing the mosquito baits. Since the ATSBs needs to compete with the other available sugar sources in the environment, it is fundamental to boost up their attractiveness. Additionally, further studies on a synthetic chemical compound for attractant components also suggested reducing the production cost.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, the study revealed that the high sugar content in mango attracting the maximum number of mosquitoes when infused in ATSBs. However, the low sugar content in \u003cem\u003eChrysanthemum\u003c/em\u003e ATSBs repel the mosquitoes but the mixture of mango of \u003cem\u003eChrysanthemum\u003c/em\u003e shows the mosquitoes attracted towards it. Thus, the sugar content of mango dominating the mix ATSBs. The attractants increase the ATSBs attractiveness, however glucose alone can be used as a phagostimulant for the bait. Histological analysis of the mosquito midgut revealed structural changes in response to different ATSBs. Notable alterations included epithelial disruption and damage to the peritrophic membrane, particularly in mosquitoes exposed to the mixed and floral-based baits. These findings suggest that ATSBs can significantly affect midgut integrity, potentially impairing digestion and pathogen transmission, and support their use as effective vector control strategies. An excellent baiting tool could be created by developing bait attractants or repellents that imitate natural sugar sources, particularly when competing with the natural sugar sources found in their ecosystem.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Vector Control Research Units (VCRU) for egg supplies, BioG Expert Sdn. Bhd. and School of Biological Sciences for hydrogel beads preparation facilities and samples. This work was supported by\u0026nbsp;Ministry of Higher Education Malaysia, Fundamental Research Grant Scheme (FRGS) with Reference No: FRGS/1/2022/STG03/USM/02/8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nur Faeza Abu Kassim, Ranjitha Sambanthan and Wan Maryam Wan Ahmad Kamil. The first draft of the manuscript was written by Ranjitha Sambanthan and Wan Maryam Wan Ahmad Kamil edited the full draft of the manuscript.\u0026nbsp;Sumiyyah Sabar, Kamarul Zaman Zarkasi, Wan Rosli Wan Ishak, Sara A. Abuelmaali and Cameron E. Webb validated the related results.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Ministry of Higher Education Malaysia, Fundamental Research Grant Scheme (FRGS) with Reference: FRGS/1/2022/STG03/USM/02/8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUSM/IACUC/2022/(137)(1223) has specifically approved this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHuang, H., Ren, Z., Gao, X., Hu, X., Zhou, Y., Jiang, J., \u0026amp; Zheng, S. 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(2023, May 12). \u003cem\u003eChrysanthemum\u003c/em\u003e. https://www.britannica.com/plant/chrysanthemum\u003c/li\u003e\n\u003cli\u003eVonOppen, S., Masuh, H., Licastro, S., Zerba, E., \u0026amp; Gonzalez-Audino, P. (2015). A floral-derived attractant for \u003cem\u003eAedes aegypti \u003c/em\u003emosquitoes. Entomologia Experimentalis et Applicata, 155(3), 184\u0026ndash;192. https://doi.org/10.1111/eea.12297\u003cu\u003e\u003c/u\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aedes, baits, histology, midgut, olfaction","lastPublishedDoi":"10.21203/rs.3.rs-7141836/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7141836/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe effective control of \u003cem\u003eAedes\u003c/em\u003e mosquitoes are increasingly challenging due to the presence of insecticide resistance in populations of mosquitoes. Hence, attractive targeted sugar baits (ATSBs) can be used as an alternative strategy to insecticides. The introduction of suitable attractant into baits is very crucial since it will determine the number of mosquitoes attracted to it. In this study, the ATSBs were prepared as a medium for liquid bait carriers in the development of mosquito baiting tools. The testing of non-choice and choice assay by \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003etowards different group of ATSBs (mango, \u003cem\u003eChrysanthemum\u003c/em\u003eand mix) was performed. In addition, olfactory preference index by mosquitoes was calculated using preference index formula followed by independent t- test to determine the significant differences, (p \u0026lt;0.05) between ATSBs pair. Furthermore, the midgut tissue of the mosquitoes was observed by using histology method. The wild and lab strain \u003cem\u003eAe. aegypti\u003c/em\u003eillustrates a strong and significant attractancy towards mango ATSBs with a value of (p = 0.004) and (\u003cem\u003ep \u003c/em\u003e= 0.04) respectively. Mango attractant is the most preferred by the \u003cem\u003eAedes sp\u003c/em\u003e. However, the mix ATSBs is secondarily preferred while \u003cem\u003eChrysanthemum\u003c/em\u003e is the least preferred ATSBs. The histology results show that the midgut of mosquitoes feed on the treated ATSBs exhibit alteration and degeneration. In the prospect of integrated pest management, the ATSBs implementation in mosquito control may reduce the need for chemical insecticides since natural products have naturally occurring volatile emission properties that attract or repel the mosquitoes. Thus, sustain released of the natural attractant odour can be a major part of the attractive toxic sugar bait as an attractant and killing mechanism to overcome mosquito-borne diseases in future.\u003c/p\u003e","manuscriptTitle":"Impact of olfactory sensitivity preferences on midgut pathophysiology of Aedes mosquitoes towards different sugar variants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 14:30:55","doi":"10.21203/rs.3.rs-7141836/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-15T17:34:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-14T14:21:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"57050342727351338178899415023746865031","date":"2025-08-11T13:19:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-30T17:16:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330506704503333416847707812756841058992","date":"2025-07-30T15:04:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-24T08:32:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-24T07:38:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-24T06:16:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2025-07-16T15:45:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1a61097f-913d-4c58-ad28-5999782d1d54","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:03:12+00:00","versionOfRecord":{"articleIdentity":"rs-7141836","link":"https://doi.org/10.1007/s00436-025-08624-8","journal":{"identity":"parasitology-research","isVorOnly":false,"title":"Parasitology Research"},"publishedOn":"2026-02-09 15:57:46","publishedOnDateReadable":"February 9th, 2026"},"versionCreatedAt":"2025-07-28 14:30:55","video":"","vorDoi":"10.1007/s00436-025-08624-8","vorDoiUrl":"https://doi.org/10.1007/s00436-025-08624-8","workflowStages":[]},"version":"v1","identity":"rs-7141836","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7141836","identity":"rs-7141836","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}