Efficacy of Phosphine Gas Against the Darkling Beetle (Alphitobius Diaperinus)

F.L. Gazoni, C.G. Wilsmann, Fernanda Flores,,, et al... 2011. Efficacy of Phosphine Gas Against the Darkling Beetle (Alphitobius diaperinus). Acta Scientiae Veterinariae. 39(2): 965. Acta Scientiae Veterinariae, 2011. 39(2): 965.

ORIGINAL ARTICLE ISSN 1679-9216 (Online) Pub. 965

Efficacy of Phosphine Gas Against the Darkling Beetle (Alphitobius diaperinus)

Fabio Luis Gazoni1,,, César Gustavo Wilsmann1,,, Fernanda Flores1,,, Flávio Silveira 1,,, Rafael Azambuja Bampi1, Roger Boufleur2 & Maristela Lovato1

ABSTRACT

Background: The darkling beetle, Alphitobius diaperinus is the most commonly beetle found in poultry sheds and causes economic and sanitary impact in the poultry industry. The life cycle of the mealworm can vary from one to three months depending on environmental conditions, and adults can survive for up to one year. The insect lives in the poultry litter where it eats feed and organic waste. The temperature in the poultry house and the accumulation of feed and organic matter promote ideal conditions for beetle infestation. The consumption of beetles affects feed conversion in poultry, especially in the first days of life and it is often cited as a vector of viral, bacterial and parasitic pathogens. The control of its populations is generally achieved by insecticide application on the walls and floor, but resistant populations of beetles are often reported. Phosphine gas is used as a fumigant to control insects in stored grain. In this study the efficacy of phosphine gas against this beetle was evaluated. Materials, Methods & Results: Two experiments were conducted: one in vitro trial, and a trial simulating field conditions. The in vitro trial aimed to evaluate the exposure time required (ETR) to obtain 100% insect mortality, in the presence and absence of wood shavings. Adults and larvae were tested separately. In treatment T1, 100 adult beetles were placed in a petri dish without poultry litter; treatment T2, had 100 adult beetles per plate and filled with sterilized poultry litter. Treatments T3 and T4 had 100 A. diaperinus larvae per plate, in absence and presence of poultry litter, respectively. Three repetitions were performed for each treatment. Insect mortality in plates was monitored at 5 min intervals. The absence of beetle movements after shaking the plate was considered an indicator of insect mortality. The field evaluation was carried out in a poultry housNe with litter infested with A. diaperinus. The evaluation was made in areas of 1 m² where 2 gm-3 of aluminum phosphide was applied, according to manufactures recommendation. The area of product application was covered with a plastic cover to seal the litter. Under in vitro conditions the ETR for total mortality of insects in plates with presence of litter was 15 min for larvae and 20 min for adults. In plates without poultry litter the ETR for A. diaperinus mortality was 25 and 35 min, for larvae and adults, respectively. It was observed that the presence of poultry litter in the plates reduced the volume and so increased the phosphine gas concentration, causing more rapid inset death. The mortality of adults and larvae insects occurred approximately 30 s after the exposure to the product. All the insects exposed to the phosphine gas died, resistant insects were not observed. In the field test the ETR to obtain 100% of mortality was 90 min for larvae and adults. Discussion: The phosphine gas showed good potential to be used in strategic control of A. diaperinus. The product was effective in the killing adults and larvae beetles on in vitro and in field trials. However, more studies are needed to establish the issues related to safe use of PH3 in poultry facilities. Keywords: Darkling Beetle, Pesticide, Phosphine Gas, Poultry.

Received: November 2010 www.ufrgs.br/actavet Accepted: February 2011 1Laboratório Central de Diagnóstico de Patologias Aviárias, Departamento de Medicina Veterinária Preventiva (DMVP), Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil. 2Centro de Diagnóstico e Pesquisa em Patologia Aviária (CDPA), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. CORRESPONDENCE: F.L. Gazoni [[email protected] - Fone: + 55 (55) 3220- 8072]. Campus Universitário, Camobi, CEP 97105-900 Santa Maria, RS, Brazil.

1 F.L. Gazoni, C.G. Wilsmann, Fernanda Flores,,, et al... 2011. Efficacy of Phosphine Gas Against the Darkling Beetle (Alphitobius diaperinus). Acta Scientiae Veterinariae. 39(2): 965.

INTRODUCTION cypermethrin use. The insects were extracted from samples of broiler-house litter by sieving through 2- Alphitobius diaperinus (Coleoptera: and-3mm screens, and then they were transported to Tenebrionidae) is an abundantly found beetle in the laboratory. They were held at 25ºC and 55% RH poultry sheds where the temperature, accumulation for at least 1wk and then tested [11]. of waste feed, organic material and adequate moisture promotes conditions for the development of darkling In vitro test beetle, making it the most abundant insect in the litter The in vitro tests were performed in sterile in poultry houses in Brazil [1]. In the first day of life Petri dishes (95x15mm) and the beetles were broilers can consume over than 450 larvae per day separated into four treatment groups in three replicates even if they have food at their disposal [2]. This as follows: Treatment 1 (T1) with 100 adult of lesser consumption affects feed conversion in poultry, mealworm per plate without litter and Treatment 2 especially in the first days of life [3] and they are (T2) containing 100 adult beetles per plate with sterile often implicated as a vector of viral, bacterial and litter. Treatments 3 (T3) and 4 (T4) were composed parasitic pathogens [4-6]. In addition, this insect can of 100 larvae per plate in the absence and presence cause significant damage to broiler houses by of litter, respectively, and a control treatment (TC) tunneling into the structural components of the sheds without exposure to PH3. The plates were exposed and into clay floors [6]. to 2 mg-3 of aluminum phosphide, according to the The control of its populations is generally manufacturer prescriptions for other insecticide uses achieved by spraying insecticide on the walls and [10]. The plates were monitored in five min intervals floor, after litter removal and before placing new in order to observe the exposure time required (ETR) chicks. Resistant populations are often reported [7- to obtaining 100% insect mortality. The beetle’s 9], and this concern has prompted a search for new mortality was assessed according to the literature [12] alternatives to minimize the resistance problem. and a correction for the control of mortality was made

Phosphine gas (PH3) is extremely toxic to by the use of Abbott’s formula [13]. insects and is the most used fumigation agent in stored In situ test grain for controlling pests. Its mechanism of action is based on partial inhibition of the electron transport The tests were made at a poultry house with chain that increases the generation of superoxide 8,000 birds in Garibaldi, RS, Brazil. Three sites of 1 dismutase and hydroxyl radicals. These radicals m² for each treatment in random locations were initiate lipid peroxidation, which results in cell damage separated into the shed from a control treatment, followed by insect’s death [10]. immediately after removing the chicken flock. The This study aimed to evaluate the efficacy of litter was excavated in order to check its depth to phosphine gas againts the darkling beetle (Alphi- determine what volume of litter would be exposed to PH . Then the dose required for the volume was tobius diaperinus) in vitro and in a poultry facility. 3 calculated. Thus, the dosage of 2 mg-3 of aluminum MATERIALS AND METHODS phosphide was used. After determining the dosage,

PH3 was inserted into the litter in a depth of about 15 Chemical cm, covered with plastic and sealed the edges. There The Gastoxin® is composed of aluminum were three treatments with 30, 60 and 90 min of phosphide (57% m/m), aggregates and adjuvants. It exposure to the product before the plastic was is presented in 3 g fumigation pellets. The product is removed and the insect mortality was assessed. manufactured by Bernardo Quimica SA1. Statistical analysis Insects Statistical analysis of data obtained was Beetles were collected from two Brazilian performed to test mean comparison by Tuckey, with broiler farms in the south of Brazil, a traditional a significance level of 95%. The test was performed poultry producing center, with varying histories of using the SAS statistical program 9.1 version [14].

2 F.L. Gazoni, C.G. Wilsmann, Fernanda Flores,,, et al... 2011. Efficacy of Phosphine Gas Against the Darkling Beetle (Alphitobius diaperinus). Acta Scientiae Veterinariae. 39(2): 965.

RESULTS the insects gradually until 100% of mortality. All

samples tested were 100% susceptible to PH3, In vitro test showing no signs of resistance. Data of in vitro tests performed in this Field trial experiment are shown in Table 1. The data shows The field trial obtained 100% mortality of that the presence of litter reduced the ETR when insects after an exposure period of 90 min (Table compared with T1 (P <0.05) occurring 100% of 2). At 30 and 60 min of exposure, live insects were mortality in 35min. It was also observed that larvae still observed in the treated area. At these points we were more susceptible to PH exposure and mortality 3 observed the insect mortality was concentrate around occurred in less time than adults (15min) (P the area of the PH application, large numbers of live <0.05). The early mortality of both adults and larvae 3 insects were present at the edges of the treatment occurred in about 30 s after exposure to PH , reaching 3 area. There was no mortality in the control treatment.

Table 1. Time required to obtain 100% mortality of Alphitobius diaperinus, -3 in larval and adult stages after exposure to PH3 (2 mg ) in the in vitro test. Treatments Adultsa Larvaea

T1 Litter 20' 15'

T2 No litter 35' 25'

TC - N N

N: No mortality after 180min observation. aAll times reveal a significant difference (P < 0,05). N Table 2. Time required to obtain 100% mortality of -3 Alphitobius diaperinus after exposure to PH3 (2 mg ) in the in situ test. Treatments Mortality Time

S1 - 30'

S2 - 60'

S3 100% 90'

S = Site (1m2)

DISCUSSION to larvae and adults of a field population of A. A recent study [15], in the United Kingdom, diaperinus [17]. Their results indicated that for most used the FAO impregnated filter-paper method [16] of the insecticides tested, larvae were more susceptible was used to test adult beetles and to develop than adult beetles. Although recent research has shown discriminating concentrations for iodofenphos and that resistance to the current registered insecticides malathion. They found that almost one half of the (fenitrothion and cyfluthrin) exists in broiler and field populations they tested were resistant to one or caged layer populations in Australia and in the USA both insecticides. A topical dosing method was used, [8,18]. In this study, we used modifications of the FAO in the USA, to test the toxicity of seven insecticides recommended methods for measurement of pest

3 F.L. Gazoni, C.G. Wilsmann, Fernanda Flores,,, et al... 2011. Efficacy of Phosphine Gas Against the Darkling Beetle (Alphitobius diaperinus). Acta Scientiae Veterinariae. 39(2): 965. resistance to evaluate the efficacy of PH3 againts the of wood shavings, the space inside plates is occupied; lesser mealworm. There is no data about the efficacy therefore the concentration of PH3 becomes larger of this agent againts A. diaperinus. and accelerates the time of insect’s death. Another The issue of resistance in populations of observation in the field trial was the insects that moved A. diaperinus to insecticides in Brazil has not yet been to the margins of the covered area were probably reported with due merit. It has only been discussed demonstrating their instinct for escaping, and a large without further scientific evidence, between farmers number of dead insects were found on the periphery and the poultry industry. The most commonly used of the covered area. insecticide in Brazil contains pyrethroid compounds Another factor that should be considered for such as cypermethrin and it is well documented that PH3 use is related to its release curve from aluminum beetles in the field can develop high levels of phosphide. This curve shows that PH3 needs a period resistance against pyrethroid agents. of 36 h in order to a complete gas release by The history of insecticides use at this facility aluminum phosphide [20]. The aluminum phosphide included previous applications of cypermethrin for wafer used in in vitro tests was crushed for weighing; at least five years. In addiction it was observed that this increased at the contact surface area of aluminum insect population were high even with these phosphide and accelerated the gas release. In the field applications. So it is possible that the resistance for trial mortality occurred in a short time compared to cypermethrin was present in those beetle fields. the gas release curve. Thus, given the time to release The success of any fumigation operation the gas, the compound demonstrated a great depends largely on the standard of gas-tightness of effectiveness in controlling this pest. In addition, the the space to be fumigated. The fumigant gas must be chemical residue in the litter is low according to the retained for a minimum period to be effective against release curve, the total liberation of PH3 takes place insects; at temperatures >20ºC, a three-to-five-day within 26 h with a good safety margin. exposure of grains to an effective concentration of Taking into account that A. diaperinus has

PH3 is generally sufficient to control most of the insect developed resistance to the currently registered broiler pests and their developmental stages, but the com- house insecticides, and that PH3 resistance has plete control of some insects, such as Sitophilus emerged in other pest species [21] a good integrated species, requires exposure for 12 days or longer [16]. management approach to the control of lesser The standard of gas-tightness required for effective mealworm and well planned resistance management fumigation is, however, rarely met under practical strategies will be needed to minimize the likely field conditions found in many countries. An early development of PH3 resistance. An integrated pest fall in the concentration of PH3 generally results in management was proposed for this beetle [22] and the failure of fumigation because surviving insects an insecticide rotation program would be developed rapidly rebuild their population in the absence of any by the broiler industry [23]. However, such a program residual insect toxicant. Frequent repeated exposures has not been developed due to the limited number of to sublethal levels of the toxicant could also lead to compounds from different insecticide classes the selection of resistant insects. Repeated applications registered for such use. This issue is still a major of PH3 in structures resulted in leakage and sub lethal concern today and this study demonstrates that PH3 dosage leading to the development of resistance by could be a chemical alternative to control this pest, various insects [18]. Resistance to PH3 has been which makes the development of such program reported by several authors [19], however it has not possible. Future studies are needed to take the first observed for this beetle. steps toward the use of PH3 as an efficient and safe

Therefore, PH3 must be applied uniformly alternative for controlling this beetle in the poultry covering the sides of the treated area, so that there industry. should not be any escape of insects after the CONCLUSION fumigation. This fact allows us to conclude that PH3 requires a longer exposure in larger volumes of treated Alphitobius diperinus in both adult and larval litter so that PH can penetrate and spread throughout 3 forms is susceptible to PH3 in a concentration of 2 the area. In vitro results indicate that in the presence -3 mg and PH3 demonstrated high efficacy both in vitro

4 F.L. Gazoni, C.G. Wilsmann, Fernanda Flores,,, et al... 2011. Efficacy of Phosphine Gas Against the Darkling Beetle (Alphitobius diaperinus). Acta Scientiae Veterinariae. 39(2): 965. and in field conditions. It could become another Ethical approval. This research was approved by the Ethics chemical alternative to control this pest. Additional and Experiment Committee in the Federal University of Santa Maria (UFSM) under the following process number: studies in poultry houses are needed to evaluate its 23081.007229/2009-64. action against a large population and other experiments to determine its toxicity in chickens are necessary for Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content its use in poultry facilities. and writing of the paper.

SOURCES AND MANUFACTURERS 1GASTOXIN ®, BERNARDO CHEMICALS SA, São Vicente, São Paulo, Brazil. Technical Manual. Available at: http://www.bequisa.com.br/download/outros/ manual_tecnico_gastoxin.pdf

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