ABSTRACT

This study examined the effectiveness of sodium chloride (NaCl) as an oviposition repellent for Aedes albopictus females. Oviposition responses to 0.5%, 0.75%, 1.00%, 1.25%, and 1.5% solutions of pure NaCl were evaluated over 8 days using ovitraps. Gravid Ae. albopictus females showed a reduction in oviposition at all NaCl concentrations. Compared with controls, the inhibition of oviposition ranged from 84.4% to 97.0% at concentrations above 0.5% NaCl. We also show that NaCl is effective for oviposition control of gravid females when laying their overwintering eggs. Our results showed that a 0.5% NaCl solution is effective for use as an oviposition repellent against Ae. albopictus females.

In Asia, the congeneric mosquitoes Aedes aegypti (L.) and Ae. albopictus (Skuse) are the main vectors of dengue hemorrhagic fever, a prostrating, sometimes epidemic disease that affects humans and is often fatal (Simmons et al. 2012). Dengue outbreaks occurred in Japan during World War II, and Ae. albopictus was identified as the vector (Hotta 1998). Although the disease is no longer considered to be endemic to Japan, a total of 162 people contracted dengue fever via Ae. albopictus in 2014 (Shimada et al. 2016). There has also recently been a marked increase in the number of travelers infected with the dengue virus in Japan, and a total of 868 cases were reported between 1999 and 2010 (Takasaki 2011). In 2019, the Ministry of Health, Labor and Welfare in Japan reported that there were 2 autochthonous cases of dengue fever in Japan (MHLW 2019). This increase in the prevalence of dengue fever implies that climate change and globalization have increased the risk of contracting dengue fever in temperate regions (Gubler 2011, Ebi and Nealson 2016).

To date, prevention measures have focused on prophylaxis and/or decreasing the chance of being bitten by a carrier mosquito (WHO 2009). Given the problems associated with insecticide resistance (Vontas et al. 2012) and the effects of chemicals on nontarget species (Milam et al. 2000), more attention is being paid to alternative or combined control methods. For example, source reduction is considered to be the single most effective method for controlling container-inhabiting Aedes species (Faraji and Unlu 2016). Furthermore, the addition of salt to used tires and other potential habitats is recommended in Japan (MHLW 2015). The larvae of some Aedes species, such as Ae. albopictus and Ae. aegypti, cannot tolerate salty water (Mukhopadhyay et al. 2010, Ramasamy et al. 2011, Brito-Arduino et al. 2015). We hypothesized that Ae. albopictus adults avoid salty water when selecting oviposition sites. An understanding of mosquito oviposition behavior is necessary for the development of surveillance and control programs directed against Aedes-specific disease vectors (Day 2016). We therefore assessed the effectiveness of using sodium chloride (NaCl) as an oviposition repellent in Ae. albopictus females.

All of the mosquitoes used in our experiments were Ae. albopictus, which were derived from 6 colonies collected in Sendai city, Miyagi Prefecture, Japan, during the summer of 2019. The sampling locality was approximately 13 km from the coast. Adults were kept in cages (320 × 220 × 220 mm) covered with surgical cotton stocking. The mosquitoes were provided 10% sucrose solution ad libitum and maintained at high humidity (>60% RH) and at 25–28°C under natural photoperiod conditions. Blood meals from a human were provided for at least 15 min daily. This research was approved by the Research Ethics Committee at Miyagi University, Japan.

Ovitraps used in the study were plastic cylinders measuring 85 mm high and 75 mm wide, which were lined with black paper on the inside. Gravid solution was made by incubating rice straw in filtered water for 10 days at 25°C. Aedes albopictus females laid their eggs on the black paper in the ovitraps containing the gravid solution in the cage. We then collected eggs and reared larvae in an enamel tray (200 × 250 × 50 mm) containing filtered water at room temperature. The number of larvae kept in each enamel tray was maintained at approximately 200 individuals. Larvae were supplied continuously with yeast-based rat feed.

Oviposition responses to water containing NaCl were evaluated using 2 methods. In the 1st method, 0.5%, 0.75%, 1.00%, 1.25%, and 1.5% (w/v) solutions of pure NaCl were prepared by dissolving 5 g, 7.5 g, 1.0 g, 12.5 g, and 15.0 g of NaCl in 1,000 ml of ion-exchange water. The 5 solutions were added to polypropylene cups (35 mm diam, 75 mm high, 120 ml capacity). Cups containing ion-exchange water were also prepared as a control. Each cup contained 35 ml of test solution and was lined with black paper to act as ovitrap. Three cups of each test solution (3 × 5 = 15 cups) and 3 control cups were placed randomly into each oviposition cage (350 × 220 × 220 mm), and rotated in a clockwise manner every day to eliminate the effect of ovitrap placement.

Batches of 36 to 46 adults from F1 generations—18 to 23 males and females—were released into cages. After 8 days, the cups were removed and the number of eggs in the control and experimental treatments were counted to assess the oviposition preference of Ae. albopictus. The oviposition repellent effect of the NaCl concentration was estimated using an oviposition inhibition (OI) score, which was calculated using the following equation and in accordance with inhibition test methods for testing chemicals (Jinguji et al. 2018):
formula
where Otox denotes the mean number of eggs laid in the NaCl solution and Ocon denotes the mean number of eggs laid in the control. The experiment was conducted from August 8 to August 15 and from August 31 to September 7. Mosquitoes were supplied continuously with a 10% sucrose solution. Photophase light was provided by natural sunlight in the laboratory (photoperiod of approximately 14 h light and 10 h dark) under high humidity (>70% RH) at 26–29°C. In addition, blood meals from a human were provided for at least 15 min daily.

In the 2nd method, 0.1%, 0.5%, 1.0%, and 1.5% (w/v) solutions of pure NaCl were prepared by dissolving 1.0 g, 5.0 g, 10.0 g, and 15.0 g of NaCl in 1,000 ml of ion-exchange water. The 4 solutions were added to polypropylene cups (35 mm diam, 75 mm high, 120 ml capacity). Cups containing ion-exchange water were also prepared as a control. Each cup contained 35 ml of test solution and was lined with black paper to act as ovitrap. Into an oviposition cage (350 × 220 × 220 mm) 3 cups of test solution and 3 control cups were inserted separately (3 cups × 5 cages = 15 cups). Batches of 11 males and 11 females adults of Ae. albopictus were released into each cage. The experiment was conducted from September 15 to October 8 to clarify the effectiveness of controlling the oviposition of gravid females seeking to lay their overwintering eggs. The salt medium was replenished after 8 days and 16 days, and mosquitoes were supplied continuously with a 10% sucrose solution. Photophase light was provided by natural sunlight in the laboratory (photoperiod of approximately 12 h light and 12 h dark). In addition, human blood meals were provided for at least 15 min daily. After 24 days, the ovitraps were removed and the eggs deposited on black paper were counted. In both methods, eggs floating on the water surface and submerged in the cups were counted and added to the total number of eggs that were laid on the oviposition substrates. In the event that any dead adults were observed during the course of the experiment, new adult mosquitoes were introduced into the cage. In the 2nd oviposition bioassay, to evaluate which NaCl solution was significantly different from the control, the number of eggs in each NaCl solution was analyzed using the Dunnett's test.

In the 1st method, gravid Ae. albopictus females showed a reduction in oviposition at all NaCl concentrations (Table 1). The OI ranged from 90.3% to 97.0% and 84.4% to 96.8% in the 2 experimental periods. In the 2nd method, the number of eggs in 0.5%, 1.0%, and 1.5% NaCl solutions was significantly lower than that in the control treatment (Dunnett's test; 0.5%: t-value = 3.404, df = 4, P = 0.021; 1.0%: t-value = 4.716, df = 4, P = 0.0027; 1.5%: t-value = 5.618, df = 4, P < 0.001; Fig. 1).

Table 1.

Oviposition inhibition of Aedes albopictus females at different sodium chloride (NaCl) concentrations.

Oviposition inhibition of Aedes albopictus females at different sodium chloride (NaCl) concentrations.
Oviposition inhibition of Aedes albopictus females at different sodium chloride (NaCl) concentrations.
Fig. 1.

Mean number of eggs oviposited in ovitraps containing sodium chloride (NaCl) solutions with batches of 11 males and 11 females. Characters denote significant differences compared with the control treatment (* P < 0.05, ** P < 0.01, *** P < 0.001). Error bars indicate standard deviation.

Fig. 1.

Mean number of eggs oviposited in ovitraps containing sodium chloride (NaCl) solutions with batches of 11 males and 11 females. Characters denote significant differences compared with the control treatment (* P < 0.05, ** P < 0.01, *** P < 0.001). Error bars indicate standard deviation.

In this study, the number of eggs decreased gradually with increasing salinity in ovitraps for the 24 days of the experiment (Fig. 1). Similar results have been confirmed in the laboratory and field experiments. In the study conducted for 24 h in the laboratory, Ae. albopictus showed strong negative correlations among the number of eggs deposited and the NaCl concentration of the ovitraps (Panigrahi et al. 2014, Gunathilaka et al. 2017). In an oviposition experiment performed in the field with ovitraps having salt gradients ranging from 2% to 20%, the number of larvae collected decreased with increasing salinity of the ovitraps (Ramasamy et al. 2011). According to the results of the present study, saline water with a concentration of 0.5% NaCl, which is the lowest concentrated solution used, had a strong repellent effect (90.3%) on the oviposition of overwintering eggs (Table 1). These findings showed that Ae. albopictus females typically avoid laying eggs in saline oviposition medium for short or long periods. However, Gunathilaka et al. (2017) showed that 0.2% NaCl solution was preferred over distilled water for oviposition by Ae. albopictus, implying that Ae. albopictus may exhibit a wide preference range for the salinity of the oviposition medium. Future research should therefore focus on this wider preference range, especially in oviposition medium with low NaCl concentrations.

Aedes albopictus has adapted well to urban environments where larvae are found in artificial containers, such as discarded tires, drains, cemetery urns, plastic buckets, flowerpots, pans, corrugated extension spouts, and water storage containers (Rochlin et al. 2013). Some studies have demonstrated that source reduction is labor intensive, time consuming, and costly (Fonseca et al. 2013), and might not be effective if it is not combined with other control methods (Wheeler et al. 2009). Thus, adding NaCl or salt to sites that are either inhabited by mosquito larvae or that are considered optimal for oviposition could potentially be employed as a measure for controlling Ae. albopictus. For example, adding 5 g of salt per 1,000 g water to an artificial container like a drain could potentially be employed as a measure for controlling Ae. albopictus. However, the application of salt to these environments needs to be carefully considered and factors such as the corrosion of metal containers and salt damage to crops need to be considered. It has been shown that Ae. albopictus can adapt to brackish water conditions and can oviposit and undergo preimaginal development in unused wells and discarded artificial containers (Ramasamy et al. 2011). We consider that Ae. albopictus has the ability to lay eggs that develop into adults in saline water. We therefore need to continuously monitor Ae. albopictus populations after adding salt to their habitat.

We thank Kyoko Jinguji for assistance in the laboratory. This work was supported in part by a grant from JSPS KAKENHI (Grant No. JP 15K07651).

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Author notes

1

Faculty of Food and Agricultural Sciences, Fukushima University, Japan.

2

School of Food, Agricultural and Environmental Sciences, Miyagi University, Sendai, Miyagi, Japan.

3

Institute for Agro-Environmental Sciences Division of Biodiversity, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.