Many insect vector species of medical and veterinary importance are frequently found in animal sheds [1
]. Moreover, they may acquire pathogens, such as viruses or protozoa, from animals and transfer these to humans during subsequent blood feedings [3
]. Animal shelters are therefore important not only for sustaining insect vector populations, however also serve as potential sources of pathogens. Use of zooprophylaxis, which can reduce contact rates between vectors and humans by diverting bites towards non-human animals, can be integrated into vector control with chemical insecticides used in public health [5
Many mosquito vector species readily feed on both humans and other animals, however some species are primarily zoophilic. For example, Anopheles culicifacies
s.l., a malaria vector in India, feeds mainly on cattle [6
]. Livestock serve as a plentiful source of blood which contributes to sustaining vector populations such as An. arabiensis
and An. funestus
s.l., the malaria vectors in Africa [7
]. Many Culex
species are vectors of arboviral diseases and filariasis [9
]. Members of the Culex vishnui
subgroup, such as Cx. tritaeniorhynchus
and Cx. vishnui
, are important vectors of Japanese encephalitis virus (JEV) and feed primarily on cattle and pigs [10
]. Culex quinquefasciatus
, a vector of bancroftian filariasis, feeds on a variety of hosts including birds, cattle, dogs, cats, and humans [11
]. It is also abundant in pig farms and has been found to be infected with JEV [12
], although its role in the transmission of the virus is not established.
Despite the risk of disease transmission, many prefer not to use neurotoxic insecticides to control insect vectors in animal sheds. Single dermal applications of recommended doses of pyrethroids resulted in the presence of residual insecticides in milk [13
]. In addition, problems of insecticide resistance occurring in many mosquito species hampers the efficacy of insecticide usage [14
]. To cope with these problems, the use of alternative insecticides that are environmentally friendly while also utilizing a different mode of action have been recommended for mosquito control [15
Pyriproxyfen (PPF) is an insect growth regulator that has been used effectively to control mosquitoes in the aquatic stages [16
]. Tarsal contact with high doses of PPF affects egg maturation in females and the hatchability of laid eggs [18
]. In a semi-field study using small microcosms containing breeding habitats and a PPF-treated polyethylene bed net (350 mg/m2
) with holes and a confined mouse inside, the population of Ae. albopictus
in the microcosms was significantly reduced [23
]. This reduction occurred because the female mosquitoes were exposed to PPF on the nets both before and after blood feeding during their attempts to enter and exit through the holes. Moreover, in an experimental hut, Ngufor et al. [24
] demonstrated that bed nets treated with PPF and PPF + permethrin significantly reduced the fecundity and fertility of surviving blood-fed An. gambiae
Artificial resting boxes of various sizes and shapes were used to collect mosquitoes that naturally prefer to rest in the shade or protected locations [1
]. They have proved to be useful for quickly sampling Ae. aegypti
inside houses and Anopheles
populations outdoors [25
]. Since PPF does not have a repellency effect [27
], PPF-treated resting devices are effective against Ae. aegypti
In animal shelters, we often observed that female mosquitoes may rest for a period of time on objects around animals. These resting periods occur both before and after blood feeding and may last for several hours. Some species, particularly after blood-feeding, may be found resting around animal sheds. This behavior allows us to develop new strategies for controlling zoophagic mosquito species.
2. Materials and Methods
2.1. Reproductive Effect of PPF on Mosquitoes Determined by Cone Bioassay
Since little is known about the effective dosage of PPF against Culex
adult mosquitoes, our preliminary study was carried out by using CDC bottle bioassays [30
] with 250 mL Duran bottles with inner surfaces of approximately 300 cm2
. Fully blood-fed Cx. quinquefasciatus
females (NIH strain, Bangkok, Thailand) were exposed for 30 min to varying doses of PPF. The results revealed that the oviposition of mosquitoes was totally inhibited at 333 mg/m2
. However, the treatment of glass bottles with PPF is not directly applicable to surface treatment within animal shelters. In this experiment, we used a simple spraying method to coat a surface and then evaluated reductions in reproductivity of Cx. quinquefasciatus
(NIH strain) and wild Anopheles
females using cone bioassays according to WHO protocol [31
]. For the laboratory Cx. quinquefasciatus
, bioassays were performed on black polypropylene sheets (35 × 25 cm), while gray polypropylene sheets (65 × 122 cm) were used for bioassays with wild mosquitoes. The sheets were rubbed with sand paper and then sprayed with a PPF solution. This solution consisted of technical grade PPF dissolved in acetone at dosages of 333 mg/m2
and 666 mg/m2
and was administered using a foggy sprayer for ironing (1 L) (Moong Pattana PCL., Bangkok, Thailand). In a previous study, a dosage of 1.3% PPF (or about 455 mg/m2
) was applied for the treatment of a barrier against Ae. albopictus
]. An amount of 166 mL acetone was found to be sufficient to cover a 1 m2
area. Control sheets were sprayed with acetone alone. The sprayed sheets were allowed to dry and were then stored in a dark place until use.
For the bioassays, plastic cones were attached to each of polypropylene sheets and placed vertically (Figure 1
). Then, 10 newly blood-fed Cx. quinquefasciatus
females were released into each of the plastic cones and were exposed for 30 min. After exposure, the mosquitoes were transferred individually to plastic cups (236 mL), hereafter called ovicups, and were provided with a 10% sugar solution. For wild mosquitoes, fully blood-fed Anopheles
and culicine mosquitoes were collected by the aid of an aspirator and touch from a cow shed at Ban Thung Kor Laan, Doi Saket District, Chiang Mai Province, Thailand. The collected mosquitoes were released into each cone (maximum 10 females) and were left for 30 min exposure. Thereafter, they were placed in cups and transferred to the laboratory. In the morning, all of the treated and control mosquitoes were identified morphologically using the keys of Rattanarithikul et al. [33
] and were transferred individually to ovicups.
The exposed and control mosquitoes were reared for four days to ensure egg maturation. Three days after exposure, each cup was filled with 70 mL of hay infused water (10 g of dry grass submerged in 1 L of water for one week and diluted with 10 parts of water before use) as an oviposition medium. The females were allowed to lay eggs for a maximum of one week. The laid eggs were then allowed to hatch for three days. The hatched larvae in each ovicup were reared with rabbit food (Tops RabbitTM, Nakhon Pathom, Thailand) until the emergence of adults. The oviposition rates (number of oviposited females per total females), numbers of eggs laid, eggs hatched, pupation rates, and adult emergence rates were recorded. The parental females were examined for insemination status and for the presence of retained eggs in the ovaries. The experiment was performed in triplicate for a total of 30 females in each group.
2.2. Reproductive Effects with Different Exposure Times
This experiment evaluated the effects on reproductivity in free-flying female mosquitoes that had rested continuously on PPF-treated sheets for various amounts of time. Four rubbed polypropylene sheets (65 × 122 cm, gray color) were sprayed with PPF at the dosage of 666 mg/m2
, as mentioned previously. Control sheets were sprayed with acetone only. Thereafter, they were marked with grid lines and labeled (Figure 2
) to facilitate locating the mosquitoes. The treated and control devices (four sheets each) were set up at dusk and allowed to stand for 3 h (6.00 to 9.00 pm) during which fully blood-fed females that remained continuously on the sheets for 5, 10, and 20 min were collected with the aid of an aspirator, torch, and timer. They were then transferred to the laboratory for rearing. In the laboratory, engorged female Anopheles
were sorted out and morphologically identified to species. They were reared individually to determine the reproductive parameters as described previously.
2.3. Residual Activity of PPF-Treated Polypropylene Sheets
The above gray PPF-treated sheets (666 mg/m2
) in Section 2.1
were kept in a dark place in the laboratory and their residual activity was evaluated 3 and 5 months after treatment. Engorged female mosquitoes were collected from the cowshed and transferred to the laboratory. After morphological identification, five blood-fed females of each mosquito species were released into the plastic cone which was attached to the PPF-treated polypropylene sheets for 30 min. Three replicates were conducted for a total of 15 females per species. After exposure, mosquitoes were kept individually in ovicups and were reared to determine the effects of PPF exposure on reproductivity, as mentioned previously.
2.4. PPF-Treated Resting Boxes for Controlling Cx. quinquefasciatus
This experiment was meant to evaluate the efficacy of PPF-treated resting boxes on the reproductivity of Cx. quinquefasciatus females. The experiment was conducted at a chicken farm (housing approximately 50 chickens) in Chiang Mai city. Mosquito resting boxes (35 × 35 × 55 cm) were constructed using black polypropylene sheets and had a circular hole (20 cm diameter) on top. The inner surface of the box was sprayed with technical grade PPF dissolved in acetone at a concentration of 666 mg/m2. The control box was sprayed with acetone alone.
One control and one PPF-treated resting box were placed at the same site on alternative nights for six consecutive nights, starting at 6 pm and collected at 8 am, in December 2016. All mosquitoes that were found in the box were removed and taken to the laboratory. Mosquitoes were identified to species according to Rattanarithikul et al. [33
]. Fully engorged Cx. quinquefasciatus
females were selected at random and reared individually in ovicups. Three days after, each cup was filled with the oviposition medium and mosquitoes were allowed to lay eggs for a maximum of one week. The oviposition rate, number of laid eggs, hatched larvae, pupae, and adults were recorded. Ovaries were dissected and examined for insemination status and the presence of retained eggs.
The PPF-treated and control boxed were stored in a dark room in the laboratory and reused on months 3 and 5 to observe the residual activity. Collection, rearing, and the determination of reproductive parameters were as described previously.
The potential attraction of adult Cx. quinquefasciatus to the resting boxes was evaluated in February 2017. In this study, we wanted to compare the number of mosquitoes collected by one box and two boxes which were placed on alternative nights for six consecutive nights. The station of one box was fixed and the second box was placed approximately 15 m apart. All Cx. quinquefasciatus found in the boxes were separated, counted, and scored as non-blood fed, blood-fed, half-gravid, and gravid. Male mosquitoes were also counted.
2.5. Statistical Analysis
The oviposition rates were analyzed by Pearson’s chi-squared test or Fisher’s Exact Test. Mean numbers of eggs laid, hatched larvae, pupae, and emergent adults were analyzed by negative binomial regression using SPSS version 22.0 (IBM Corp., Armonk, NY, USA).
Most studies on the use of PPF-treated surfaces for mosquito control have focused on the development of PPF-treated ovitraps which are suitable for container-breeding mosquitoes such as Aedes aegypti
and Ae. albopictus
]. However, ovitraps are generally not attractive for most Culex
]. Cattle, pigs, and chicken are commonly found throughout rural Thailand. These animals serve as important blood sources for both vector and non-vector mosquitoes. To our knowledge, our study appears to be the first to demonstrate that residual PPF spraying on a small surface area is a potential method for controlling Culex
mosquitoes when they are seeking blood meals in animal shelters.
In the current study, exposure of Culex
mosquitoes for 30 min on the PPF-sprayed polypropylene sheets at a dosage of 666 mg/m2
totally disrupted the fecundity of these mosquitoes. This concentration is higher than that tested in our previous study using the CDC bottle assay in which the oviposition of Cx. quinquefasciatus
mosquitoes was totally inhibited at 333 mg/m2
. This difference may be attributed to there being a small loss of chemicals during spraying and the fact that the PPF concentration over the sprayed sheet was not homogenized. Nonetheless, our spraying method is a simple and inexpensive method for applying PPF to surfaces and could be repeated easily. Therefore, this concentration (666 mg/m2
) is recommended for residual spraying. This concentration is higher than the dose of 1% PPF (350 mg/m2
) that is normally used for coating nets, including commercially available treated nets (e.g., Olyset Duo®
, Sumitomo) [22
]. Higher doses of PPF (≥ 1000 mg/m2
) have also been evaluated [18
], however such doses may be too high and not economical for large scale use.
The reproductive effect of PPF varies depending on mosquito species. In pyrethroid-resistant Ae. aegypti
strains, exposure for 10 min on PPF-sprayed sheets at a dosage of 333 mg/m2
completely inhibited fecundity [28
]. In the current study, Culex
species appeared to be more sensitive than Anopheles
species studied, particularly when the exposure time was short (5 min). Such differences are probably due to differences in PPF absorption, resistance levels, and/or resistance mechanisms. Insecticide resistance with various mechanisms has been detected in several species of Culex
, and Anopheles
in Thailand [48
]. A recent study demonstrated that a subset of cytochrome P450 enzymes can metabolize PPF in An. gambiae
]. However, the current study suggests that the effects of long PPF exposure (20 min or longer) are not mitigated by currently known resistance mechanisms.
The residual effect of PPF-sprayed sheets or resting boxes, which was effective over three months, is based on 30 min of exposure time. This may underestimate what would occur in natural conditions because it was observed that newly blood-fed females usually rest for several hours on surrounding surfaces in cowsheds before they disappeared in the morning. Newly blood-fed females are likely to rest in their preferable resting places, including the resting box, one day or longer during egg maturation. Longer exposures to PPF could increase the chances of chemical uptake. However, PPF is light-sensitive and this property may be disadvantageous for outdoor residual spraying applications. In the current study, the PPF-treated sheets were kept in a dark place during the evaluation of the residual effect. Hence, shorter periods of residual activity is expected if it is used under real conditions. This would need to be evaluated in field conditions.
Besides the polypropylene sheets that were used in the current study, PPF may be applied to a variety of surface substrates. In general, insecticidal residues are more persistent on nonporous surfaces such as metal and tile compared with more porous surfaces such as concrete or wood [51
]. Little is known about the persistence of PPF on different surfaces under natural conditions and, as such, warrants further study. In addition, some cattle owners protect their animals from insect bites by using nets. Treatment of nets with PPF may be an alternative for controlling insect populations.
The current study demonstrated that the artificial resting boxes are attractive to various physiological stages of Cx. quinquefasciatus
females, mainly blood-fed, as well as males. This species is active at nighttime and at dawn seeks rest in dark corners in and around homes. Hence, treatment with residual PPF could be useful for controlling this vector species, especially in areas where they are found in abundance, such as chicken farms or animal sheds. PPF affects the fecundity of female mosquitoes, including those exposed before or after blood-feeding or while gravid [18
]. In addition, our device could also contaminate male mosquitoes and may help to transfer PPF to virgin females via mating, as observed in Ae. albopictus
and Ae. aegypti
Both the PPF-treated sheets and PPF-treated resting boxes represent a “passive” approach to the control of mosquitoes in livestock. Such methods do not require much attention yet continue to affect mosquitoes over time. Retreatment may be applied every three months to maintain control. The portable nature of the polypropylene sheets and the resting boxes as well as their low cost per unit suggests that they could be efficiently deployed in high numbers with minimal disruption to animals or local residents. The results from this study provide important information on the use of PPF which could be implemented in the future for controlling mosquito-borne diseases.