Effectiveness of Pleurotus eryngii (King Oyster Mushroom) Extract for Killing Larvae and Attracting Adult Mosquito Vectors in Samut Songkhram Province of Thailand.
Mosquitoes carry many human diseases that are major public health issues around the world, especially in tropical and sub-tropical countries . Mosquito-borne diseases include dengue fever, chikungunya, malaria, filariasis, West Nile virus, yellow fever, Zika virus, and Japanese encephalitis, and according to World Health Organization (WHO) estimates, more than one million people die from these diseases every year [2,3]. Thailand has epidemic areas of mosquito-borne diseases, primarily because it is located in a tropical area. In 2017, the Thai Ministry of Public Health reported a total of 65,000 patients suffered from mosquito-borne diseases . This number indicates that these disease are a major problem and should be resolved urgently.
There are two ways to control mosquito vectors, namely reducing the number of larvae or adult mosquitoes. Temephos, a chemical that is not toxic to humans or animals, is widely used on mosquito larvae in all parts of Thailand . However, long-term use of these chemical causes vector resistance. Indeed, temephos resistance has been reported in many areas of Thailand, and this phenomenon makes it difficult to control the mosquito population . Further, temephos use is usually successful only for Aedes spp., especially Ae. aegypti, since its spawning habits are in household water containers . Other mosquito species, such as Culex spp. and Anopheles spp., spawn in large water resources in nature. It is unlikely that temephos can or will be used for larvae control.
Octenol (1-octen-3-ol) is a volatile substance that emanates from human sweat and breath. Female mosquitoes use this scent to find bait to suck blood to obtain the blood protein that is required for egg development . Currently, many types of mosquito traps use octenol to increase the efficiency of mosquito attraction. One such trap, the mosquito magnet , exhibits high performance, but its expense makes it unpopular in Thailand. It has been reported that octenol can found in mushrooms and it is reportedly toxic to insect larvae . Therefore, it is possible that edible mushrooms in Thailand could be developed for use as larvicides and as substances that attract mosquitoes to replace the expensive synthetic substances on the market.
In this study, we examined the popular and edible king oyster mushroom (Pleurotus eryngii), reported to contain octenol as a component , to study its efficacy and efficiency for killing larvae and attracting adult mosquito vectors in the laboratory. This study was conducted using prominent mosquito vectors in coastal areas of Samut Songkhram province, including Ae. aegypti, a dengue fever vector, and Cx. sitiens, a filariasis and Japanese encephalitis vector . We hypothesised that this extract could reduce mosquito numbers in this area and consequently further reduce the number of new patients with mosquito-borne diseases.
Materials and Methods
P. eryngii was collected from the Talat Thai market, Khlong Luang district, Pathum Thani province, the center of trade in agricultural products in Thailand (14[degrees]4'54.51"N 100[degrees]37'53.06"E) in September 2016 (Figure 1). Mushroom samples were transported to the College of Allied Health Sciences (Suan Sunandha University, Samut Songkhram province). Specimens were identified morphologically using the mushroom taxonomic keys [13-16].
P. eryngii samples were cut into small pieces and fermented with 95% ethanol at room temperature for 48 h. The mushroom extract was filtered and evaporated under reduced pressure at 50[degrees]C using a rotary evaporator to obtain the crude extract. The crude extract was then dried by freeze dehydration at -85[degrees]C for approximately 24 h. The yields of P. eryngii extract were weighed, recorded, dissolved in ethanol and stored at -20[degrees]C before laboratory testing.
Ae. aegypti (Bola Bola strain, F171) were obtained from the Faculty of Tropical Medicine (Mahidol University). Cx. sitiens were collected from the coastal area of Samut Songkhram province, which is 200 m away from the sea (13[degrees]23'31.57"N 100[degrees]1'59.36"E), using a standard mosquito dipper in a water source with a salinity level of more than 0.05 parts per thousand (ppt). Ae. aegypti eggs and Cx. sitiens larvae were placed in separate trays (25 x 30 x 5 cm) that contained filtered water; 0.1 g dog food was provided daily. When eggs or larvae reached the pupae stage, they were transferred to cages (30 x 30 x 30 cm) to facilitate adult emergence.
Bioassay for P. eryngii extract larvicidal effect
Larvicidal tests were modified according to WHO . Five concentrations of extract were used (120, 12, 1.2, 0.12 and 0.012 mg/L). Filtered water containing the appropriate concentration of the substance was added to 6 ounce glasses and 20 late third instar or early forth instar larvae were added. After 24 h, the number of dead larvae was recorded. For the control group, only solvent was added to the filtered water in the test glass. Each concentration was repeated 3 times.
Bioassay to test P. eryngii extract adult mosquito attraction
Three concentrations of mushroom extract were used to test adult mosquito attraction (100, 10, and 1 mg/L). We conducted this bioassay using a modified Y tube, according to Geier et al. , using 20 mosquitoes per concentration. Mosquitoes were released into the tube, which has 2 sides: the left contained P. eryngii extract and the right contained solvent. The number of mosquitoes that flew to each end was counted and recorded. This experiment was repeated 3 times for each concentration.
The number of dead larvae or attracted mosquitoes are expressed as the mean [+ or -] standard deviation (S.D.). Statistical comparison for adult attraction between P. eryngii extract and octenol was performed using a two-tailed t-test. p <0.05 was considered statistically significant.
P. eryngii extract had no effect on Ae. aegypti larvae, while the extract minimally killed Cx. sitiens larvae at all concentrations except 1.2 mg/L (Table 1). The highest octenol concentration (120 mg/L) killed less Ae. aegypti than Cx. sitiens larvae (9.33 [+ or -] 4.93 compared to 19.67 [+ or -] 0.58, respectively). For the P. eryngii control group, no larval death was observed, while the octenol control group caused slight larval death for Ae. aegypti (0.33 [+ or -] 0.58; Table 1) and Cx. sitiens (1.33 [+ or -] 0.58; Table 1).
The 10 g/mL concentration of P. eryngii extract attracted the most adult Ae. aegypti and Cx. sitiens mosquitoes, followed by 1 and 100 g/mL concentrations, respectively. The 10 g/mL octenol concentration also attracted the most adults for both species (Figure 2). At all concentrations, P. eryngii extract attracted more Ae. aegypti than Cx. sitiens adults (Figure 2). However, compared to all octenol concentrations, P. eryngii extract attracted significantly fewer adult mosquitoes for both species (Figure 2).
In this study, we investigated the effectiveness of P. eryngii mushroom extract for killing larvae and attracting adult mosquitoes. We found that P. eryngii extract was ineffective in killing Ae. aegypti larvae, but it appeared mildly larvicidal toward Cx. sitiens at certain concentrations. However, the minimal Cx. sitiens larval death may be due to our use of larvae from nature (rather than a laboratory strain), since the number of larval deaths in the extract and control groups were similar. Thus, P. eryngii extract is not suitable to kill mosquito larvae in the field. This finding is consistent with previous research from Thongwat et al.  who screened the ability of 143 mushroom species in Thailand to kill mosquito larvae and found them to be almost entirely ineffective. There were only 6 mushroom species with potential larvicidal activity, all of which were wild, expensive and seasonal. Thus, it appears quite difficult to further develop a product to control mosquito larvae based on mushroom extracts.
For adult mosquito control, mosquito traps are an effective control option with substantial interest worldwide. Currently, mosquito trap development focuses on optimising traps to avoid polluting the environment . The use of odor in traps is one of the most important tools used to increase the effectiveness of mosquito control. Octenol is a powerful mosquito attractant , but it is expensive. It attracts mosquitoes because it emanates from humans and animals that would serve as prey for blood-feeding female mosquitoes . Several studies have documented that mushrooms, including P. eryngii, contain octenol . The results in this study showed that 10 g/mL P. eryngii extract most effectively attracted adult mosquitoes. This finding is consistent with other octenol research that demonstrated higher octenol concentrations did not necessarily attract mosquitoes more efficaciously than lower concentrations . Since the insect odor system has a specific odor range, it varies according to mosquito species.
Our research found that P. eryngii extract attracted Ae. aegypti better than Cx. sitiens at all concentrations. This finding is similar to previous research by Cilek et al.  that reported that octanol attracted Ae. albopictus better than Cx. quinquefasciatus. However, at all concentrations P. eryngii extract attracted significantly fewer adult mosquitoes than octenol. Nevertheless, at 10 g/mL, the extract attracted more than half of all Ae. aegypti mosquitoes (58.33%).
These finding is the first to demonstrate that P. eryngii extract could possibly be developed to further enhance mosquito lure efficiency. Although the performance of P. eryngii extract is different with octanol but extract was effective in attracting more than half of all mosquitoes in the laboratory. The advantages of this extract are an inexpensive and eco-friendly way to increase the efficiency of mosquito traps.
We would like to thank the College of Allied Health Science, Suan Sunandha Rajabhat University, Thailand, for their kind support of our research.
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Tanawat Chaiphongpachara (*), Aegkapun Bumrungsuk, Chichanok Chitsawaeng, Kantima Sumchung and Kitthisak Khlaeo Chansukh
College of Allied Health Sciences, Suan Sunandha Rajabhat University, Thailand
(*) Corresponding author: Tanawat Chaiphongpachara, College of Allied Health Sciences, Suan Sunandha Rajabhat University, Thailand, Tel: +66835865775; E-mail: firstname.lastname@example.org
Received date: June 27, 2018; Accepted date: July 10, 2018; Published date: July 17, 2018
Table 1: Mean number of dead Ae. aegypti and Cx. sitiens larvae. Concentration n Mean [+ or -] S.D. number of dead larvae (mg/L) P. eryngii Ae. aegypti Cx. sitiens 120 20 0.00 [+ or -] 0.00 1.33 [+ or -] 1.53 12 20 0.00 [+ or -] 0.00 1.67 [+ or -] 0.58 1.2 20 0.00 [+ or -] 0.00 0.00 [+ or -] 0.00 0.12 20 0.00 [+ or -] 0.00 0.67 [+ or -] 0.58 0.012 20 0.00 [+ or -] 0.00 0.67 [+ or -] 0.58 Concentration Mean [+ or -] S.D. number of dead larvae (mg/L) Octenol Ae. Aegypti Cx. sitiens 120 9.33 [+ or -] 4.93 19.67 [+ or -] 0.58 12 0.33 [+ or -] 0.58 9.67 [+ or -] 3.06 1.2 0.00 [+ or -] 0.00 0.00 [+ or -] 0.00 0.12 1.00 [+ or -] 1.00 5.00 [+ or -] 2.65 0.012 0.33 [+ or -] 0.58 5.00 [+ or -] 2.65
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|Title Annotation:||Research Article|
|Author:||Chaiphongpachara, Tanawat; Bumrungsuk, Aegkapun; Chitsawaeng, Chichanok; Sumchung, Kantima; Chansukh|
|Publication:||Biology and Medicine|
|Date:||Jul 1, 2018|
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