Current status of insecticide susceptibility in the brown planthopper in Cambodia

Abstract

Little information exists about pesticide availability and its effect on pest control in rural regions of developing countries. The availability of different insecticidal ingredients in rural areas of Cambodia was determined by inspecting labels on products used by farmers and in retail shops. A large number of products available in markets and used by farmers contained abamectin, emamectin benzoate, cypermethrin, and chlorpyrifos. The effects on the brown planthopper (BPH; Nilaparvata lugens (Stål)) were investigated by comparing the susceptibility of three BPH populations in Cambodia to twelve active ingredients in 2015. All populations showed high susceptibility to abamectin and cypermethrin; however, regional differences in susceptibility were observed for the other ingredients. The implication was that farmers selected the most effective products based on sellers’ opinions. It is important to monitor insecticide use and BPH susceptibility in each region of Cambodia in order to minimize the risk of high BPH population densities.

Introduction

The use of chemical pesticides to control pests is widely practiced by farmers because it is convenient and easy to implement. Insecticides are conventionally used to control pest insect populations in paddy fields in many Asian countries.¹⁾ Following the Green Revolution, insecticide use dramatically increased in Southeast Asia,²⁾ such as on the Indochina Peninsula. In the recent years, a large amount of pesticides has been imported, for instance, 172,000 tons in Thailand in 2013, 116,582 tons in Vietnam in 2014, and 738,545 tons in Cambodia in 2015.^3–5)

In Cambodia, farmers experiencing severe crop damage from the brown planthopper (BPH; Nilaparvata lugens (Stål)), one of the most harmful insect pests on rice, tend to spray pesticides during the outbreak⁶⁾ and apply insecticide even when other insect pests were identified in the rice fields.⁷⁾ Pesticide use has therefore become one of the most important methods of pest control for rice farmers in Cambodia. After the establishment of regulations governing pesticide use in 2012, the sale of hazardous pesticides, such as those distributed in the 2000s, has decreased, as has labeling in foreign languages.^8–10) However, the status of insecticides available from markets and in use by farmers in rural areas is not well understood. Complexities arise because cross-border trade is still prevalent in areas close to international borders. Furthermore, most farmers depend on retailer opinion to select products and do not always remember the names of products they have used. It is therefore difficult to determine the current distribution of pesticides in Cambodia.

These factors are important in the context that frequent insecticide use has led to a rapid increase in BPH resistance to several insecticides in Asia; this has in turn led to a resurgence in BPH numbers, triggering its establishment as a major insect pest.^11,12) Recently, resistance to imidacloprid, a neonicotinoid insecticide, has increased in Asian countries such as Japan, China, Vietnam, and Thailand.^13–15) Although resistance to fipronil, a phenylpyrazole insecticide, is generally low in East and Southeast Asia, the 50% lethal dose (LD₅₀) values for fipronil are a little higher in the southern part than in the northern part of Vietnam.^14,16) BPH populations in Cambodia, which is located adjacent to southern Vietnam, might therefore also show a reduced susceptibility to these insecticides; however, this has not been previously investigated in Cambodia.

The aims of this study were therefore to (1) determine the availability of the active ingredients in insecticides by conducting a survey of retail shops and rice farmers in rural areas of Cambodia and (2) identify which ingredients were effective by testing the susceptibility of BPH specimens collected from three different locations in the main rice-producing provinces.

Materials and Methods

1. Survey

1.1. Markets

In order to determine the active ingredients available from retail shops in rural areas, insecticide package labels were reviewed in 20 shops from 10 of the main rice-producing provinces in Cambodia in 2014 (Fig. 1): northern areas (Battambang and Banteay Meanchey); central areas (Siem Reap, Kompong Thom, and Kompong Chhnang); and southern areas (Takeo, Kandal, Prey Veng, Svay Rieng, and Kompong Cham). The products were categorized by their active ingredients (AIs) according to the Mode of Action Classification of the Insecticide Resistance Action Committee (IRAC).¹⁷⁾ Products with the same label were counted as a single product in each area.

Fig. 1. Sampling locations in Cambodia. The market survey was conducted in the main rice-producing provinces (shown enclosed by the bold line): northern (Battambong, and Banteay Meanchey) (unshaded), central (Siem Reap, Kompong Thom, and Kompong Chhnang) (dots), and southern areas of Cambodia (Takeo, Kandal, Prey Veng, Svay Rieng, and Kompong Cham) (diagonal lines). The survey of farmers was conducted in an area in the southern region (white circles). For the susceptibility tests, BPH specimens were collected from three locations (black circles): one in the north (BB), and two in the south (TK and SR).

1.2. Rice farmers

Farmers were surveyed in 2014 to determine which insecticides they used in the rainy season. In the southern region of Cambodia (Fig. 1), Takeo, Prey Veng and Svay Rieng Province, where severe damage by the BPH has been experienced,¹⁸⁾ 53 rice farmers were surveyed and asked to retain insecticide packaging after use. From this, we verified product names and ingredients. The products were categorized by type of AI, and products with the same label were counted as a single product.

2. Bioassay

2.1. Insects

In August 2015, BPH specimens were collected from three locations in the main rice-producing area: TK, Takeo Province (N10°41′10.6, E104°51′01.7); SR, Svay Rieng Province (N11°05′64.3, E105°81′67.6); and BB, Battambang Province (N13°04′28.0, E103°12′86.8) (Fig. 1). Under the Material Transfer Agreement between Nagoya University and the Royal University of Agriculture, Cambodia, the BPH specimens were imported and then reared in a chamber in an isolated insect-breeding room at Nagoya University (26°C, 80% humidity, 16 hr light : 8 hr dark (16L : 8D)). They were fed with rice seedlings of the variety ‘Nipponbare.’

2.2. Insecticides

Twelve commercial insecticides were used to conduct a seedling dipping test. Each insecticide contained one of 12 active ingredients, representative of six insecticide types: BPMC (carbamates); malathion and MEP (organophosphates); cypermethrin and etofenprox (pyrethroids); dinotefuran, nitenpyram, imidacloprid, and clothianidin (neonicotinoids); abamectin and emamectin benzoate (avermectins); and fipronil (phenylpyrazoles). Eight of the 12 AIs were available in retail shops and used on farmers' fields in Cambodia; the others were applied to rice fields in Japan (Table 1).

Table 1. Insecticides used in the susceptibility test

Typea)	Trade name	Active Ingredient (%)	Recommended concentration (ppm)	Application to rice in Japanb)	Available in Cambodiac)
Carbamates	Bassa	BPMC (50)	250–500	○	○
Organophosphates	Malathion	Malathion (50)	250	○
	Sumithion	MEP (50)	500	○
Pyrethroids	Agrosrin	Cypermethrin (6)	30–60		○
	Trebon	Etofenprox (20)	100	○
Neonicotinoids	Arubarin	Dinotefuran (20)	66.7	○	○
	Bestguard	Nitenpyram (10)	33.3	○	○
	Adomaiya	Imidacloprid (1)	5.0	○	○
	Dantotsu	Clothianidin (16)	160	○
Avermectins	Agrimec	Abamectin (1.8)	18.0		○
	Affirm	Emamectin benzoate (1)	5.0		○
Phenylpyrazoles	Prince	Fipronil (5)	16.7	○	○

^a) Active ingredients are categorized according to the Mode of Action IRAC classification. ^b) Active ingredients included in the list registered for application to rice by the Food and Agricultural Materials Inspection Center. ^c) Active ingredients applied by farmers in southern Cambodia.

2.3. Testing and analysis

We followed a slightly modified version of the leaf-dipping method recommended by the IRAC. Five concentrations of insecticide solutions were prepared, including water as a negative control, and 0.02% of an agrochemical spreader was added to all concentrations (Dain; Takemoto Yushi Co., Ltd.). Rice seedlings (∼10 days old) were dipped in the solutions for 10 sec and then left to dry completely. Treated seedlings were placed in a plastic container (6 cm diameter×10 cm height) with approximately 10 BPH adults and left for 48 hr (carbamate, organophosphates, pyrethroid, and abarmectin treatments) or 72 hr (neonicotinoids, fipronil, and emamectin benzoate treatments) at room temperature (25±2°C). Three replications of each concentration were prepared. Following testing, BPH individuals were classified as either dead or alive and then counted to calculate the median lethal concentration (LC₅₀) using probit analysis.¹⁹⁾

Results

1. Active ingredients available from retail shops and farmers

In the 20 shops investigated, a total of 25 AIs of 12 types were identified in the form of single-AI products, while 28 AIs of 11 types were found in the form of composite insecticides (Fig. 2). A larger number of products were found to contain organophosphates, pyrethroids, and avermectins, particularly abamectin, emamectin benzoate, cypermethrin, and chlorpyrifos, when compared to products containing other insecticides. We did not find any regional differences in the number of products based on each AI; however, product labeling was not the same among the regions. Not many neonicotinoid products were available, although five neonicotinoid AIs (acetamiprid, dinotefuran, imidacloprid, thiamethoxam, and nitenpyram) were found. Products containing organophosphates and neonicotinoids were mainly found in composite insecticides; avermectins were in single-AI products; and cypermethrin was found in both single and composite products. Furthermore, the labels of insecticides used by farmers listed a total of 13 AIs of eight types as single AI products and 22 AIs of 10 types as composite insecticides (Fig. 3). A similar pattern was detected between products surveyed in retail shops and those in use by farmers. The major AIs identified were cypermethrin, abamectin, emamectin benzoate, and chlorpyrifos. These results show that a large variety of insecticides are available to farmers from retail shops in rural areas. We tested the susceptibility of the BPH to eight of these available AIs (Table 1).

Fig. 2. Commercial products found in retail shops, grouped by active ingredients. The numbers in parentheses are the number of retail shops in which we conducted the survey in the northern, central, and southern regions of Cambodia. The names of group: ¹⁾ pymetrozine, pyridine azomethine derivatives and ²⁾ nereistoxin, nereistoxin analogues.

Fig. 3. Commercial products confirmed from the insecticide packaging used by farmers, grouped by active ingredients. The numbers in parentheses indicate the number of farmers. The name of group: ¹⁾ pymetrozine, pyridine azomethine derivatives.

2. Susceptibility to insecticides

Although knowledge of the field history of spraying by local farmers in each location site is necessary to fully understand insecticidal susceptibility, due to current conditions in Cambodia, we were unable to collect the exact information from each farmer; therefore, we used colonies multiplied under laboratory conditions to examine BPH susceptibility to the most commonly used insecticides. Susceptibility to both the organophosphates insecticides, malathion and MEP, was tested, although neither was found in the products highlighted in this study (Table 1). Malathion was determined to have an LC₅₀ value that was approximately two and five times higher than the concentration recommended for application in the BB and TK populations, respectively. Conversely, MEP had an LC₅₀ value within recommended application concentrations for all three populations, indicating that MEP is lethal to Cambodian BPH populations (Table 2). The LC₅₀ values of BPMC were higher than the recommended concentration for TK and SR populations (Table 2).

Table 2. LC₅₀ values of different types of insecticides to BPH from three populations in Cambodia

Typea)	Active ingredient	Recommended concentration (ppm)	LC50 (95% CL) (ppm)
			TK	SR	BB
Carbamates	BPMC	250–500	1019.8	1609.4	590.0
			(918.9–1125.3)	(1465.9–1751.5)	(523.0–645.1)
Organophosphates	Malathion	250	1401.3	300.4	513.8
			(1294.3–1527.2)	(202.3–484.8)	(311.2–1018.7)
	MEP	500	120.6	165.3	288.2
			(104.3–140.6)	(148.7–182.2)	(242.5–345.7)
Pyrethroids	Cypermethrin	30–60	22.0	52.3	14.7
			(16.3–28.2)	(37.4–87.0)	(8.0–21.7)
	Etofenprox	100	639.1	1351.4	744.7
			(599.0–682.7)	(1198.3–1596.2)	(675.0–834.2)
Neonicotinoids	Dinotefuran	66.7	268.3	230.0	50.3
			(227.7–314.9)	(186.6–321.2)	(19.4–75.7)
	Nitenpyram	33.3	39.8	205.3	14.1
			(25.8–59.4)	(139.3–339.3)	(7.6–23.1)
	Imidacloprid	5.0	>2500b)	>2500b)	1265.8
			(1/26)	(1/27)	(721.3–4387.9)
	Clothianidin	160	620.6	1482.7	303.0
			(533.3–732.9)	(1326.0–1783.3)	(193.2–595.2)
Avermectins	Abamectin	18.0	0.6	0.1	0.3
			(0.4–1.0)	(0.0–0.3)	(0.0–0.8)
	Emamectin benzoate	5.0	57.0	40.9	45.8
			(48.6–80.8)	(35.6–50.7)	(36.8–65.8)
Phenylpyrazoles	Fipronil	16.7	119.2	83.2	13.8
			(88.4–214.2)	(56.2–143.9)	(10.8–17.2)

^a) Active ingredients are categorized according to the Mode of Action IRAC classification. ^b) The highest concentration used to treat BPH, and the dead/treated number of BPH, are shown in parentheses.

Cypermethrin, which is classified as a pyrethroid insecticide, was commonly available for purchase, and farmers confirmed it to be effective in controlling all BPH populations (LC₅₀=22.0 ppm in TK, 52.3 ppm in SR, 14.7 ppm in BB). The LC₅₀ value of etofenprox was 6.3–13.5 times higher than the recommended concentration for all three populations; the SR population showed the lowest susceptibility. These results indicated that BPH populations had different responses to each of the AIs, even to AIs in the same class of insecticide.

Neonicotinoid insecticides, which were developed more recently than the organophosphates and pyrethroid insecticides, were also found in use in rural areas. Susceptibility to three neonicotinoid AIs was lowest in the SR population, followed by the TK and BB populations. Dinotefuran was lethal to BPH populations from BB when applied at the recommended concentration (LC₅₀=50.3 ppm), and nitenpyram was lethal at recommended concentrations to both the BB and TK populations (LC₅₀=14.1 and 39.8 ppm, respectively). Furthermore, all of the populations exhibited low susceptibility to imidacloprid; the LC₅₀ value for the BB population was higher than 1000 ppm, and only 4% of imidacloprid-treated BPHs in the SR and TK populations died after treatment with a concentration of 2500 ppm, indicating that these two populations had a lower susceptibility to imidacloprid than did the BB population.

All three BPH populations exhibited high susceptibility to abamectin, a insecticide frequently found in Cambodia, with LC₅₀ values of 0.6, 0.1, and 0.3 ppm for the TK, SR, and BB populations, respectively. In contrast, the LC₅₀ values for emamectin benzoate tended to be higher than the recommended application concentrations for all three populations.

The LC₅₀ values of fipronil for the TK and SR populations were approximately five to seven times higher than the recommended concentration (LC₅₀=119.2 ppm in TK, 83.2 ppm in SR), whereas the value for the BB population was effective at the recommended concentration (LC₅₀=13.8 ppm).

Discussion

In Cambodia, products containing chlorpyrifos, cypermethrin, abamectin, and emamectin benzoate were abundant in retail shops and in use on agricultural fields. Chlorpyrifos and cypermethrin were the products primarily used; abamectin, methomyl, and endosulfan were also used by small-scale farmers in Thailand.²⁰⁾ In Vietnam, farmers employed several AIs: BPMC, thiamethoxam, buprofezin, cartap hydrochloride, fipronil, α-cypermethrin, isoprocarb, chlorpyrifos ethyl, cypermethrin, etofenprox, quinalphos, phenthoate, and chlorpyrifos. There is evidence that these insecticides pollute drinking water and pose a risk to farmers’ health.^21,22) Given the availability of various insecticides in these countries, controlling their distribution and use in Cambodia poses challenges. Farmers can easily purchase pesticides, not only in their own country, but also in neighboring countries,⁸⁾ where the products available for purchase are mostly dependent on import from other countries, including China and Singapore,²³⁾ thus promoting secondary import. Product selection should not rely only on seller opinion. Farmers and farm workers need a sufficient understanding of the large variety of available insecticide products to enable them to make informed choices.

BPH populations collected from three locations in Cambodia were tested to determine their susceptibility to AIs available in rural areas. All three BPH populations revealed a high susceptibility to abamectin and cypermethrin; these products were applied by farmers across a large area,⁷⁾ indicating that the effective ingredients are widely available in rural locations. Many products based on the same AIs were, however, labeled differently (Figs. 2 and 3), and one finding was that farmers are generally unaware that these products will have the same efficacy. This is important to note, because intensive and extensive use of the same AIs in adjacent regions and countries might result in decreased BPH susceptibility, even though these AIs still currently produce lethal effects. Cypermethrin is, in fact, already in frequent use in both Vietnam and Thailand.^20,21)

The development of resistance to imidacloprid has been reported in both East and Southeast Asia.¹³⁾ This has probably been induced by its intensive and dominant use across Asia, as well as the origins and destinations of BPH migration.²⁴⁾ In Cambodia, the LC₅₀ values of imidacloprid, fipronil, and BPMC in TK and SR populations were higher than those in the BB population. The sites of TK and SR population are located on the southernmost parts of Cambodia, neighboring on the southern part of Vietnam. Matsumura and Sanada (2010) reported that the LD₅₀ values of these three insecticides were higher in the southern part of Vietnam than in the northern parts of Vietnam. This may suggest that the BPH populations in southern Cambodia show similar characteristics in terms of insecticide susceptibility to southern Vietnam populations, caused by BPH migration between these neighboring areas.

This is the first report of BPH susceptibility to various insecticidal AIs based on information about AIs available in rural areas of Cambodia. We found that farmers tended to use the AIs they found most effective. However, the “cocktail” approach and the extensive application of insecticide creates the risk of increased BPH resistance, thus triggering pest insect outbreaks. Although the LC₅₀ values from three Cambodian field populations are not sufficient data to compare insecticide resistance level in Cambodia due to the lack of data on the Cambodian BPH susceptible level, regional differences in susceptibility between three populations indicate the importance of monitoring insecticide use at the level of AI content and of considering BPH susceptibility on a regional basis between the south and north of Cambodia. Also of importance will be the sharing of information on susceptibility and resistance between neighboring countries.

Acknowledgment

This study was conducted under the terms of the material transfer agreement between the Royal University of Agriculture, Cambodia, and the Graduate School of Bioagricultural sciences, Nagoya University, Japan. We would like to express our gratitude to Dr. Ngo Bunthan, Dr. Seng Mom, and Dr. Chay Chim from the Royal University of Agriculture, Cambodia, for their understanding and support of this study. We also thank Dr. Ouk Makara, Dr. Khay Sathya, Ms. Yin Theavy, and the staff of the Plant Protection Office of the Cambodian Agriculture Research Development Institute for conducting the preliminary tests to check BPH susceptibility in Cambodia. We are also grateful to Dr. Pheap Visarto and Mr. Ning Chhay in the General Directorate of Agriculture in Cambodia for providing information about pesticide regulations, and to Dr. Daigo Takemoto, Dr. Ken Miura, and Dr. Chieka Minakuchi for rearing BPH at Nagoya University. Finally, this study was partially funded by the Japan Society for the Promotion of Science (KAKENHI Grant Number 14J03722).

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