Larvicidal and Adult Mosquito Vector Attractant Activity of Tremella fuciformis Berk Mushroom Extract on Aedes aegypti ( L . ) and Culex sitiens Wiedemann ( Diptera : Culicidae )

© 2018 Tanawat Chaiphongpachara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Tanawat Chaiphongpachara, College of Allied Health Science, Suan Sunandha Rajabhat University, Samut Songkhram 75000, Thailand. E-mail: tanawat.ch @ ssru.ac.th Larvicidal and Adult Mosquito Vector Attractant Activity of Tremella fuciformis Berk Mushroom Extract on Aedes aegypti (L.) and Culex sitiens Wiedemann (Diptera: Culicidae)


INTRODUCTION
Mosquito-borne diseases are a major public health issue, especially in tropical and subtropical countries. They include Zika virus disease, malaria, dengue fever, Japanese encephalitis, chikungunya, and filariasis (Service, 2008;World Health Organization, 2016). Globally, more than 1 million people die from these diseases every year (World Health Organization, 2014). In Thailand, mosquito-borne diseases are one of the most important health concerns, requiring urgent resolution. According to Ministry of Public Health of Thailand reports, in 2016 dengue fever had the highest incidence rate at 58.79 cases per one hundred thousand people, followed by malaria, chikungunya, Japanese encephalitis, and filariasis (Ministry of Public Health, 2016). For control of mosquito borne diseases to reduce the risk of infection will focus mosquito population in the endemic area (Roiz et al., 2012) in two ways including reducing the number of both the larval and adult stages of mosquitoes.
Currently, the most common and popular method for controlling mosquitoes is the use of chemical insecticides, but these have harmful effects on humans, animals, and the environment (Nkya et al., 2013). Furthermore, mosquitoes have become resistant to some insecticides, for example, the larvicide temephos . Thailand has reported mosquito resistance in many areas, resulting in difficulties in controlling the dengue vector population (Pimsamarn et al., 2009).
Mosquito traps are useful tools to reduce mosquito populations (Okumu et al., 2010). Currently, various odors are used for trap optimization, including octenol, carbon dioxide, and lactic acid. These are based on the olfactory attraction of female mosquitoes, which require proteins from the blood of humans or animals for egg production (Takken and Kline, 1989).
Octenol (1-octen-3-ol) is a volatile organic compound found in the perspiration and breath of humans or animals. It has also been reported that octenol is found in some mushrooms and that it is toxic to insects (Inamdar and Bennett, 2014). Recently, studies have reported that some mushrooms have larvicidal activity including Aspergillus flavus, Chrysosporium lobatum, Penicillium spp., Podospora spp., Xylaria nigripes, Chlorophyllum spp., Steccherinum spp. and Thaeogyroporus porentosus (Matasyoh et al., 2011;Mohanty and Prakash, 2009;Govindarajan et al., 2005;Thongwat et al., 2015). It is possible, therefore, that certain fungi could have both adult attractant and larvicidal properties, with potential as an alternative method of mosquito control.
We selected Tremella fuciformis Berk, an edible fungus that is popular with consumers in Thailand, for assay of larvicidal and adult mosquito attractant activity, and we tested this on Aedes aegypti (L.) (a dengue fever vector) and Culex sitiens Wiedemann (a filariasis and Japanese encephalitis vector), both major vectors in coastal habitats (Chaiphongpachara and Sumruayphol, 2017).

MATERIALS AND METHODS Mushroom collection and extract preparation
T. fuciformis mushrooms were purchased during September 2016 from Talat Thai market, Pathum Thani province in Thailand (14°4'54.51"N, 100°37'53.06"E), and then taken to the College of Allied Health Sciences, Suan Sunandha University, Samut Songkhram province education center. They were then washed thoroughly and their identity confirmed by morphological characteristics using taxonomic identification keys (Largent and Thiers, 1977).
The mushrooms were dried in the shade at ambient temperature (~37°C), ground into powder using a blender, and infused in 95% ethanol at room temperature for 48 hours. The mushroom extract was filtrated through Whatman filter paper and dried in a rotary evaporator. The yield of crude extract was recorded, dissolved in methanol for adult mosquito bioassay and distilled water for larvicidal bioassay, and stored at −20°C before investigations in the laboratory.

Mosquito collection and rearing
Eggs of Ae. aegypti (WHO susceptible strain Bora-Bora) were supplied by the Faculty of Tropical Medicine, Mahidol University, while Cx. sitiens larvae, a coastal vector, and were collected using a standard mosquito dipper from water with a salinity level of more than 0.05 ppt in coastal areas of Samut Songkhram province, Thailand, 200 m inland from the sea. The eggs and larvae were placed separately in trays (25 × 30 × 5 cm) containing filtered water and rearing were carried out at 25 ± 2°C with 10:14 h light: dark cycle with 0.1 g food provided daily. The pupae were transferred to cages (30 × 30 × 30 cm) for adult mosquito emergence.

Larvicidal bioassay
Larvicidal bioassay of the T. fuciformis extract was conducted following WHO protocols (WHO, 2016). Five concentrations of mushroom extract were prepared (120, 12, 1.2, 0.12, and 0.012 mg/L) using filtered water in six-ounce glass containers, to which were added 20 late third instar or early fourth instar larvae. After 24 h the number of dead larvae was counted and recorded. All experiments were done in triplicate. For the control group, we used a mixture of filtered water and extraction solvent. Octenol (1-Octanol EMPLURA ® from Merck KGaA COMPANY, Darmstadt, Germany) at the same concentrations was used as a benchmark for testing against larvae and ault mosquitoes.

Adult mosquitoes attractant bioassay
Mushroom extract at concentrations of 100, 10, and 1 mg/L following Cilek et al. (2011) were used in adult mosquito attraction tests, conducted using a modified Y-tube, according to the method of Geier et al. (Geier and Boeckh, 1999). At each concentration level, 20 healthy adult female mosquitoes were tested, with four replicates at each concentration. In this bioassay, one arm of the Y-tube provides a flow of the test substance (the T. fuciformis extract) and the other is the control; when all 20 mosquitoes had flown down either one arm or the other, we counted and recorded the results.

Statistical analyses
Numbers of dead larvae and numbers of mosquito attracted were expressed as mean ± S.D. (standard deviation).
The T-test was used for comparisons, with differences regarded as significant at p ≤ 0.05.

Efficacy of T. fuciformis extract as a larvicide
At all concentrations, T. fuciformis extract showed no larvicidal activity against Ae. aegypti, and only a slight (nonsignificant) effect against Cx. sitiens. (Table 1), while Octenol at the highest concentration (120 mg/L) killed nearly all the Cx sitiens larvae (19.00 ± 1.00 of 20) and around half the Ae. aegypti larvae (9.00 ± 1.00 of 20). In the control groups, none of the Ae. aegypti larvae died, and only very few of the Cx. sitiens larvae died.

Efficacy of T. fuciformis extract for attracting adult mosquitoes
At all concentrations, octenol was a significantly (p ≤ 0.05) better attractant for Ae. aegypti and Cx. sitiens than T. fuciformis extract (Table 2). Of the three concentrations of attractants tested, the intermediate concentration (10 mg/L) was most effective for both species, followed by 100 and 1 mg/L. Ae. aegypti adults were more attracted than Cx. sitiens in all tests.
We studied the larvicidal effect and adult female mosquito attractant activity of T. fuciformis mushroom extract on Ae. aegypti and Cx. sitiens. There are reports of the toxicity of octenol to small insects (Inamdar and Bennett, 2014) consistent with the results of this study; here, the highest concentration (120 mg/L) killed almost all Cx. sitiens larvae (19.00 ± 1.00 of 20) and about half the Ae. aegypti larvae (9.00 ± 1.00 of 20). However, the T. fuciformis mushroom extract was not effective against Ae. aegypti larvae and only very slightly effective on Cx. sitiens larvae and our results indicate that it is unlikely that T. fuciformis extracts as prepared in this study could be used to control mosquito larvae. This result is consistent with that of Thongwat et al. (2015), who screened 143 species of mushroom against Ae. aegypti larvae in the laboratory and found larvicidal activity in only Thaeogyroporus porentosus, Xylaria nigripes, Chlorophyllum spp., Steccherinum spp., and two unidentified species.  The same letter in the row between T. fuciformis extract and octenol in each concentration is not significantly different at p ≤ 0.05.
Apart from octanol's toxicity to aquatic insects, it is a substance that can be used to attract mosquitoes. Octenol is a powerful attractant for female mosquitoes looking for a blood meal, and several studies have reported that octenol is contained in mushrooms, including T. fuciformis (Mau et al., 1997). The T. fuciformis extract used in our study attracted more than 60% of Ae. aegypti at a concentration of 10 mg/L, though at the same concentration the rate of attraction for Cx. sitiens was poorer at around 35%. These results are consistent with previous research that found that octenol at a concentration of 10 mg/L was best for attracting mosquitoes, with Aedes spp. more attracted than Culex spp. (Cilek et al., 2011). In this study, the T. fuciformis extract produced a significantly lower attraction response than octenol at all concentrations (p ≤ 0.05), a difference that may arise from the concentration of bioactive substances in the T. fuciformis mushroom.

CONCLUSION
This experiment is the first to reveal the ability of T. fuciformis mushroom extract to control mosquitoes. Although the performance of T. fuciformis extract is not equal to that of octanol, it was effective in attracting more than half of all mosquitoes in the laboratory tests and could be an eco-friendly way to increase the efficiency of mosquito traps.