Larvicidal Effect of Some Selected Salts Against the Dengue Vector Mosquito, Aedes aegypti (Diptera: Culicidae) in Bangladesh CURRENT STATUS: POSTED

Background The container breeding mosquito, Aedes aegypti is the major universal vector of dengue viruses capable of causing fever (DF), fever and dengue shock syndrome (DSS). Vector control may have a potential role in reducing the morbidity and mortality of human by DHF/DSS. The purposes of this work were to evaluate the larvicidal effect of some salt solutions against the larvae of Ae. aegypti . Methods Freshly collected l arvae were transferred to the laboratory and reared using rainwater as a rearing medium with yeast granules as larval food. Five salts i.e. AgNO 3 , HgCl 2 , CdCl 2 , CuSO 4, and CuCl 2 were tested to assess the larvicidal effect on both the 1st and 3rd instars larvae of Ae. aegypti . Serial concentrations (0, 1, 3, 5, 7 &10 ppm) of each salt were prepared by using distilled water as the solvent. Results Silver nitrate (AgNO 3 ) was noted as the most effective larvicide followed by HgCl 2 , CdCl 2 , CuSO 4 and CuCl 2 against Ae. aegypti larvae. The LC 50 of AgNO 3 against 1st and 3rd instars larvae were 0.118 and 1.659 ppm, and the LC 90 were 2 and 3.347 ppm, respectively. The LT 50 of AgNO 3 (1 ppm) against 1st and 3rd instars larvae were 0.575 and 30.42hrs, and the LT 90 was 6 and 67.49 hrs, respectively. All of the first instar larvae were killed and failed to pupate with every salt of each concentration (1–10 ppm) within 7 days. Whereas 3rd instar larvae were also unable to pupate entirely in AgNO 3 and HgCl 2 solutions but very few (5-36.66%) pupation was found at 7 ppm & higher concentrations of CdCl 2 , CuSo 4, and CuCl 2 salts. CdCl 2 , CuSo 4 and CuCl 2 prevented 66.66%, 57.14% and 50% adult emergences from pupae at 5 ppm concentration respectively. The


Background
Aedes aegypti (L.) is the predominant vector of dengue; a mosquito-borne arbovirus. It is also a recognized vector of Zika, Chikungunya, and Yellow fever viruses. Like malaria, dengue virus had become a global threat [1][2]. Dengue fever (DF) is an acute and painful disease that is often not lethal but dengue hemorrhagic fever (DHF) may lead to Dengue shock syndrome (DSS), i.e. circulatory failure which is often lethal within 12 to 24 hrs. Dengue virus is transmitted almost wholly from man to mosquito and mosquito to man [3]. The dengue virus spread rapidly and the disease develops into pandemic proportion [4]. A recent study estimated that there were more people at risk of dengue infection calculating up to 3.97 billion those living in 128 countries [5]. Both the species of Aedes mosquito breed frequently in artificial containers such as water-storage vessels, flower vases, water accumulations, discarded tins, automobile tires, and blocked gutters. These also provide excellent larval habitat and adult resting sites [6]. Though dengue has been reported as an urban disease [7] in the recent past dengue infection and the vector mosquitoes have been detected in rural areas of Thailand and India [8][9]. Unplanned urbanization in cities and technological advancement in villages might have played a significant role in dengue transmission [10,11].
Mosquito control is a worldwide problem due to their vector nature and the resurgence of much infectious disease [12]. The best way to keep a check on the larval population because breeding grounds are confined to limited stagnant water bodies. The major existing means of mosquito control all over the world is the employment of synthetic insecticides [13]. Several insecticides have been indiscriminately used against the mosquito larvae and as a result over the time they have developed resistance to a wide variety of chemicals used against them i.e. Ae. aegypti resistant to Malathion, Temephos, Permethrin, Propoxur [14][15]and Fenitrothion [16]. Though chemical controls require a short time and work quickly but have some adverse effects on man and the environment. Burdick et al. [13] observed that residual toxicity may be magnified biologically in the food chain. On the other hand, salts are generally safer, less hazardous and environmentally friendly than chemical pesticides.
Therefore the present study was conducted to determine the effects of salt solutions on the larvae of Ae. aegypti.

Mosquito rearing
The period from July to September has been reported as the peak dengue season in Bangladesh.
Freshly collected Ae.aegypti larvae were brought to the laboratory and cleaned with distilled water.
These larvae were reared using rainwater (previously stored) as a rearing medium at the IRES (Insect Rearing and Experimental Station), Department of Zoology, Jahangirnagar University, Savar, Dhaka at ambient temperature and relative humidity (28 ± 2 °C and 70 ± 5% RH). In each rearing container (10 × 10 × 5 cm 3 ) about 500 ml rainwater was taken and 500 healthy larvae were released. Larval food (0.10 gm yeast granules) was regularly added to each rearing container. Each container along with the larvae was placed inside a rearing cage (30 × 30 × 30 cm 3 ) made of iron wire and fine mesh size mosquito net to prevent egg-laying by other mosquitoes. To avoid fungal contamination the larvae were transferred from the old container to another at regular intervals. Inspections were made 6 hourly to measure water temperature, relative humidity, larval molting and wastage of food if any. Sometimes a surface film or scum food was removed with a spuit or a brush and yeast granules (˂0.02 gm/100 ml) were newly added to the rearing medium, if required. As the larvae developed, the pupae were picked with the help of a pipette and transferred to a glass jar (covered with gauze net).
About 500 pupae were transferred to a new plastic bowl containing rainwater for adult emergence. No food was supplied for this non-feeding stage. Adult mosquitoes were identified morphologically under stereomicroscopes using taxonomic keys [17][18][19][20][21], within a few hours after sampling.
A ratio of 3 females to 1 male is preferable for mating in a cage. Therefore a batch of 100 males and 300 females were housed together in a cage (30 × 30 × 30 cm 3 ) for about 5-6 days to mate. Cotton pads soaked with 10% glucose solutions were placed inside the cage for food supplement of the adults. Gravid females were then removed to another cage and a pigeon (Columba livia) was kept tight for about an hour on the roof of rearing cage for sucking blood meal by the adult mosquitoes.
Five plastic cups (125 ml) filled with distilled water (2.5 cm depth) were lined with a 3̎ wide strip of filter paper were placed inside each cage for laying eggs. Eggs were removed at regular intervals and kept in air-dried condition for subsequent use. When required, those eggs were released in the rainwater of a plastic bowl for hatching. During the period of hatching 0.02 gm, glucose or yeast granules were added in each 100 ml distilled water as larval food.

Preparation of stock solutions
Salt solutions of different concentrations were prepared through the process of serial dilution. Serial dilution is a method used to stepwise dilute substance into solution with constant dilution factor in each step. In this method, 0.1 gm salt was mixed with 100 ml water for preparing a stock solution of 1000 ppm. After that 1 ml, 0.7 ml, 0.5 ml, 0.3 ml and 0.1 ml of stock solution were mixed with 99, 99.3, 99.5, 99.7 and 99.9 ml of water respectively for preparing 10, 7, 5, 3 and 1 ppm salt solutions.

Larvicidal testing procedure
For experimenting with each of the salt solutions, thirty-six plastic cups (125 ml) were taken and cleaned with distilled water. They were air-dried and arranged in groups 1, 2, 3, 4, 5 and 6, where each of the groups having six cups. Each cup of the group 2, 3, 4, 5 and 6 was filled with 99.9, 99.7, 99.5 ml, 99.3 and 99 ml of rainwater respectively. Afterward, 0.1, 0.3, 0.5, 0.7 and 1 ml of the stock solutions of salt were added to each cup of groups 2, 3, 4, 5 and 6 respectively. Cups of group 1 were used as control i.e. no salt solutions were added. The first and third instars larvae were selected for the study, because of the easy differentiation of these two instars and separate sets of experiments were carried out for each of the instars. The fourth instar larvae were avoided for the possibility of immediate pupation. Thereby a batch of ten larvae of a particular instar was collected from the rearing container and released into each cup of the group 2, 3, 4, 5 and 6. An amount of 0.2 gm yeast granules were added regularly to each cup as larval food. All of the plastic cups along with larvae were placed inside the rearing cage to prevent any contamination and egg-laying by other mosquitoes. The cups of the experiment with a particular type of salt were kept in a separate cage.
The larvae which showed no signs of motion were regarded as dead. Their number was counted and recorded at the time of each inspection. Inspections were made at 6 hours interval. To eliminate the impact of the decomposing larval food, it was removed with a spuit from each cup during the last inspection of the day. A little amount (< 0.02 gm/100 ml) of larval food was newly added to each cup if required. The mortality of larvae (after 24 hours), duration of larval instars, number of pupae, the mortality of pupae and adult emergence were recorded. Statistical Analysis LC 50 , LC 90 , LT 50 , LT 90 , 95% confidence interval were generated through probit analysis using Statistical Package for Social Sciences (SPSS®) version 20 [22]. Mean mortality of larvae, standard error, % pupation, % pupal mortality and % adult emergence of mosquitoes were calculated using MS Excel, 2013.

Results
Effects on 1st and 3rd instar larvae Results on the LT 50 and LT 90 values for 1st instar larvae at 1 ppm concentration of all salts were shown in Table   2. Among all salts lowest LT 50 (0.575 hrs) and LT 90 (6 hrs) values were found at 1 ppm concentration of AgNO 3   Effects on pupation and adult emergenceÌ t was noted that 100% 1st instar larvae died at each concentration (1-10 ppm) of every salt within 7 days. All larvae have died within 9, 6 and 3 hours when exposed to 1 ppm, 3 ppm and 5 ppm solutions of AgNO 3 respectively. On the other hand, 46%, 93% and 100% 3rd instar larvae were killed within 24, 24 and 9 hours when exposed to 1, 3 and 10 ppm solutions of AgNO 3 respectively. No pupation and adult emergence was also observed from 3rd instar larvae even at 7 and 10 ppm concentrations of CdCl 2 , CuSO 4 and CuCl 2 salt solutions (Table 3). Table 3 Effect of salts on pupation, pupal mortality and adult emergence from the 3rd instar larvae of Ae.

Discussion
The killing efficiency of salt solutions on the larvae of mosquito might be due to its impact on the cells of the anal gill. Similar larvicidal efficacy was reported by Wigglesworth [23] that the cells of anal gills in Ae. argenteus (Poir.) become swollen, perhaps due to the diffusion of the hypertonic NaCl into the cells which were caused by the difference of the concentration between hemolymph and external medium. He also stated that the concentration of NaCl in the cells rises above that in the hemolymph and water from the latter moves into the cells by osmosis.
This difference of osmotic pressure between body fluids and the external medium is the main factor to swell up of cells. MacFie [24] also found that the destructive action of a salt solution of 2% or higher on the larvae of Stegomyia fasciata (= Ae. aegypti) is due to the hypertonicity of the solution. Bradley [25] stated that only 5% of all extant species of the family Culicidae are capable of surviving in saltwater and those species of Aedes do not possess an additional segment of the rectum cannot survive for not eliminating excess ions. During the present study, it was also observed that anal gills of Aedes larvae become swollen by absorbing salts solution and then larvae have died. It was noted that all salt solutions were able to kill 1st instar larvae of Ae. aegypti at a lower concentration. It is pertinent to mention here the findings of Suzuki [26] who found that LC 50 of AgNO 3 and HgCl 2 were 1.7 ppm (0.00017%) and 1 ppm (0.0001%) respectively against fourth instar larvae of Culex pipiens pallens.
In the present study, it was noted that AgNO 3 kills 60% of 3rd instar larvae at 1 ppm (0.0001%) concentration and when concentration increased to 3 ppm (.0003%) mortality also increased to 98.3%. Riaz et al. [27] reported that a 10% NaCl solution killed the majority of larvae of Ae. aegypti in the laboratory and 100% mortality were achieved within the minimum time when exposed to a 20% salt solution. MacFie [28] reported that undiluted seawater killed larvae of Stegomyia fasciata within 2 to 4 hrs and 50% or more diluted seawater caused death after 24 hrs. Wigglesworth [29] conducted experiments with sodium chloride and found good results at 1.25% solution against Aedes species within 4-7days. Wigglesworth [30] also stated that 1% of NaCl is effective to kill larvae within a week and mortality increased with the increase of salt concentrations. Suzuki [26] studied the relation of required time to death of 50% of larvae (TD 50 ) to the concentration of the salts and calculated the order of decreasing toxicity of the heavy metal salts. He also stated that TD 50 increases in parallel to the decreases in salt concentration. High larval toxicity of AgNO 3 against Aedes aegypti larvae was found in the present study. Nearly similar result were found by Suzuki [26] in Japan.
There were no pupation and adult emergence from 3rd instar larvae in the solution of AgNO 3 and HgCl 2 . Though very few pupations (5-36.66%) were found in other three salt solutions they could not develop into adult or died or might be lengthened its time of emergence. Our results support the Pappas et al. [31] from Peru who reported that less than 50% of the larvae of Culiseta inornata reached the pupal stage at sodium chloride concentration above 0.01 m.

Conclusion
During the present study, it was recorded that all of the tested salt solutions were effective in killing off both the The lowest concentration (1 ppm) of low effective salt CuCl 2 also killed 90% larvae within 220.628 hrs ( Table 2).
The LC 50 and LC 90 values of each salt were lower for 1st instar than 3rd instar. Among five salts AgNO 3 has the lowest LT 50 and LT 90 values were 0.575 and 6 hrs for 1st instar whereas 30.421and 67.492 hrs for 3rd instar respectively. AgNO 3 and HgCl 2 successfully prevented pupation and adult emergence of both instars (1st & 3rd ) larvae. These promising results could be useful in the search for a more effective and eco-friendly larvicidal product against Ae. aegypti, especially in the areas where mosquitoes are highly resistant to chemical insecticides. However, the findings of this laboratory-based study need to be evaluated in the field conditions.
Besides, further investigation regarding the residual toxicity on non-target organisms' extremely important and requiring attention soon.

Ethics approval and consent to participate
Not applicable

Consent for publication
Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.