Al Thabiani Aziz, Jazem Abdullah Mahyoub, Hasibur Rehman, Shalini Saggu, Kadarkarai Murugan,Chellasamy Panneerselvam, Majed Salum S. Alrefaei, Marcello Nicoletti, Hui Wei, Angelo Canale,Giovanni BenelliDepartment of Biology，Faculty of Sciences，University of Tabuk，Tabuk，79，Saudi ArabiaDepartment of Biological Sciences，King Abdul Aziz University，Jeddah，Saudi ArabiaDivision of Entomology，Department of Zoology，School of Life Sciences，Bharathiar University，Coimbatore， 0，Tamil Nadu，IndiaDepartment of Environmental Biology，Sapienza University of Rome，Piazzale Aldo Moro ，008，Rome，ItalyInstitute of Plant Protection，Fujian Academy of Agricultural Sciences，Fuzhou，00，Fujian，People's Republic of ChinaDepartment of Agriculture，Food and Environment，University of Pisa，via del Borghetto 80，，Pisa，Italy



Insecticide susceptibility in larval populations of the West Nile vector Culex pipiens L. （Diptera：Culicidae）in Saudi Arabia

Al Thabiani Aziz1*, Jazem Abdullah Mahyoub2, Hasibur Rehman1, Shalini Saggu1, Kadarkarai Murugan3,
Chellasamy Panneerselvam1, Majed Salum S. Alrefaei2, Marcello Nicoletti4, Hui Wei5, Angelo Canale6,
Giovanni Benelli61Department of Biology，Faculty of Sciences，University of Tabuk，Tabuk，71491，Saudi Arabia
2Department of Biological Sciences，King Abdul Aziz University，Jeddah，Saudi Arabia
3Division of Entomology，Department of Zoology，School of Life Sciences，Bharathiar University，Coimbatore，641 046，Tamil Nadu，India
4Department of Environmental Biology，Sapienza University of Rome，Piazzale Aldo Moro 5，00185，Rome，Italy
5Institute of Plant Protection，Fujian Academy of Agricultural Sciences，Fuzhou，350013，Fujian，People's Republic of China6Department of Agriculture，Food and Environment，University of Pisa，via del Borghetto 80，56124，Pisa，Italy

ARTICLE INFO

Article history：

Received 19 Sep 2015

Received in revised form 17 Nov 2015

Accepted 25 Nov 2015

Available online 21 Mar 2016

Keywords：

Acute toxicity

Biosafety

Carbamates

Larvicides

Mosquito control programs

Pyrethroids

ABSTRACT

Objective：To investigate the susceptibility to some conventional and non-conventional insecticides in laboratory and field larval populations of the West Nile vector Culex pipiens L.（Cx. pipiens）, the dominant species in Jeddah Province, Saudi Arabia.

Methods：The tested conventional insecticides were Actikil and Pesgard, while the nonconventional ones were Bacilod, Dudim and Baycidal. Probit analysis and photomicroscopical observations were carried out to shed light on acute toxicity in laboratory and field Cx. pipiens strains.

Results：Cx. pipiens were more susceptible to Pesgard（LC50: 0.045 and 0.032 mg/L）than Actikil（0.052 and 0.038 mg/L）and Bacilod（0.129 and 0.104 mg/L）, for the field and laboratory strains, respectively. Results showed that treatments with the chitin synthesis inhibitor Dudim and Baycidal evoked morphological effects similar to those induced by other insect growth regulators. According to IC50values obtained（concentration which to inhibit the emergence of 50%of mosquito adults）, the compound Dudim （0.0003 and 0.0001 mg/L）was more effective against Cx. pipiens L. mosquitoes than Baycidal（0.0004 and 0.0003 mg/L）for both the field and laboratory strains, respectively. Conclusions：Our results provide baseline data to enhance control programs and orient publichealthdecisionsontheselectionofpesticidesagainstmosquitovectorsinSaudiArabia.

Original article http://dx.doi.org/10.1016/j.apjtb.2015.12.017

Tel: +966 556041263

E-mail: aalthbyani@ut.edu.sa

Foundation Project: Entomology and Toxicology Unit, University of Tabuk, Saudi Arabia with Grant No. 1/1/11/1435.

Peer review under responsibility of Hainan Medical University. The journal implements double-blind peer review practiced by specially invited international editorial board members.

1. Introduction

The development and use of insecticides have produced immeasurable benefits for humankind as they kill unwanted insect pests by disruption of their vital processes through chemical action. Therefore, they have been a major contributor to the upsurge in agricultural productivity over the past three decades. Their use has not only resulted in foodstuffs of the highest quality but also has saved millions of lives through eradication of disease-carrying insects［1］.

Mosquitoes are considered as the most important group of insect which have ability to spread the disease in the world［2–4］. Chemical control is one of the main ways to combat mosquito vectors of medical and veterinary importance. In the beginning of the 19th century, the discovery of a group of chlorinated hydrocarbons such as DDT and its derivatives boosted up pest control. Later on, many synthetic chemical insecticides［i.e., organophosphates, carbamates and insect growth regulators （IGRs）］were successfully used to control mosquitoes［5,6］. Over the last century, natural and synthetic insecticides usagehas increased in the agricultural and public health sectors［7,8］. This has saved millions of tons of agricultural food resources and human lives. However, the introduction of synthetic insecticides has arisen the problem of resistance in vector-borne diseases［9］. Resistance is the developed ability in a strain of insects to tolerate the doses of insecticides, which may be lethal to the majority of individuals in a normal population of the same species. This is reflected in repeated failure of an insecticide to achieve the expected level of control of insects when used according to the product label recommendations and where problems of product storage, application and unusual climatic or environmental conditions can be eliminated as causes of the failure［10］. To overcome these problems, scientists discovered other types of chemicals known as IGRs. IGR is a chemical group of pesticides, which may be used as mosquito larvicides and divided into two families；the juvenoid family, which affects the growth process of the insects, and the chitin synthesis inhibitors, which disrupt the transformation processes. In general, IGRs are safer to fishes, amphibians, mammals and birds, if compared with other pesticides［11］. Pyriproxyfen, belonging to this family, is an IGR that affects the physiology of morphogenesis, reproduction and embryogenesis of insects. It exhibits a high level of activity against mosquito larvae inhibiting adult emergence at low dose ［12,13］. Furthermore, Vythilingam et al.［14］showed the longterm effectiveness of pyriproxyfen against dengue vectors in Asia. Similarly, Sihunincha et al.［15］showed that, pyriproxyfen prevented adult emergence at extremely low concentrations （LC50= 0.012 mg/L）when applied to late mosquito instars. In this research, we determine the susceptibility levels on the West Nile vector Culex pipiens L.（Cx. pipiens）testing conventional and non-conventional insecticides in Jeddah province of Saudi Arabia. Results provide baseline data to enhance control programs and orient public health decisions on the selection of pesticides in Saudi Arabia.

2. Materials and methods

2.1. Collection sites

Mosquito larvae were collected from domestic and outside containers around homes throughout Jeddah City, Saudi Arabia, located between latitude 21°29′31′′N and longitude 39°11′24′′E.

All applicable international and national guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

2.2. Mosquito collection and colonization

Cx. pipiens larvae were maintained at the laboratory of University of Tabuk under conditions of controlled temperature［（27±1）°C］and relative humidity［（70±5）%］with a constant photoperiod（light: dark = 14 h:10 h）. Pupae were transferred from water medium to standard mosquito rearing cages （30 cm×30 cm×30 cm）. Subsequently, adults were kept in cages and provided with a cotton wick soaked with 10%glucose solution for post-emergence. After a period of 4 days, sugar-fed females were starved for 24 h prior to feed on artificial blood suppliers. Blood-fed females were allowed to assimilate the blood meals for 48 h. Gravid females were given access to oviposition sites consisting of small glass containers （23 cm×17 cm×8 cm）lined with filter paper as egg deposition sites. Eggs were dried under laboratory conditions. Samples of eggs from filial generation 13 were hatched in cool sterilized water. Newly enclosed larvae were reared in plastic trays and fed every two days with a powdered mixture of biscuits, dried yeast, and fat-free milk（1:1:1）. Late 3rd or early 4th instar larvae of generation 12 were used for larval bioassays. Adult experiments were conducted using sugar-fed（10%glucose solution）3–5 day-old adults derived from wild larvae after one generation under laboratory conditions.

2.3. Insecticides

Conventional insecticides: the Actikil 50%（active ingredient: pirimiphos-methyl 5%）, and Pesguard Fg161（active ingredient: D-tetramethrin 4%；cyphenothrin 6%）.

Non-conventional insecticides: Bacilod 5000 ITU（active ingredient: Bacillus thuringiensis var. israelensis）, Dudim 4G （active ingredient: diflubenzuron 4%）, and Baycidal（active ingredient: triflumuron 25%）.

2.4. Larval bioassays

Experiments were conducted following the method by Aziz et al.［16］. Batches of 20 larvae were added to glass beakers filled with 200 mL of water containing different concentrations of the five insecticides: i.e., Actikil, Pesguard, Bacilod, Dudim and Baycidal. When larvae were introduced into the beakers, 0.02 g of the powdered mixture was added to avoid death by starvation.

The concentrations applied for conventional insecticides Actikil and Pesguard were 0.02–0.15 and 0.02–0.20. The concentrations applied for non-conventional insecticides, Bacilod, Dudim and Baycidal were 0.05–0.5, 0.0001–0.0040, and 0.0002–0.0020, respectively. These concentrations of each insecticide were tested on fourth instar larvae in five replications, forbothlaboratoryandfield-strains.Ineachcase,thesamenumber of glass beakers with the same treatment but without insecticide served as controls. Beakers were inspected 24 h after introduction of larvae and the numbers of dead larvae were recorded.

2.5. Data analysis

In the Actikil, Pesguard and Bacilod insecticides bioassay experiment, the numbers of dead larvae were determined after 24 h. Following［17］, larvae incapable of reaching the water surface for oxygen and those showing no diving reaction characteristics when the water was disturbed were considered dead. For IGR insecticides Dudim and Baycidal, daily inspections were carried out until adult emergence, dead larvae were recorded. The distortion was calculated. Results were excluded from analysis if mortality rate was above 20%. In addition, if the percentage ranged between 5%and 20%, the mortality was corrected using the Abbott's formula［18］. Data from larval bioassays were subjected to probit analysis［19］. The concentrations of agents that killed 50%and 90%of mosquito larvae in 24 h（LC50and LC90, respectively）were used to judge the larvicidal activity of the tested products［20］. The resistance status was determined according to World Health Organization［21］.The following formula was used to evaluate resistance ac

cording to Aziz et al. and Pungasem et al.［16,22］.

Resistant ratio（RR）=

where, RR＜2 is susceptible strain, 10＞RR＞2 is tolerant strain, RR＞10 is resistant strain.

3. Results

3.1. Susceptibility of Cx. pipiens larvae

The different patterns of larval susceptibility were reported in Table 1. The lowest percentage mortality（12%）was recorded among wild larvae exposed to Actikil, whereas the highest mortality rate（98%）was observed for laboratory strain larvae exposed to Actikil and Pesguard. The percentage mortality rates of Cx. pipiens exposed to larvicides were lower in the field strain than in the laboratory strain. The LC50of Actikil for the laboratory strain was 0.038 mg/L, which was 1.37 fold lower than that of the field strain. The LC50of Pesguard for the laboratory population was 0.032mg/L, which was 1.41fold lower thanthatofthe field strain. The LC50of Bacilod for the laboratory strain was 0.104 mg/L, which was 1.24 fold lower than that of the field strain. Pesguard wasthemosteffectivelarvicideinbothfieldandlaboratorystrains, followedby Actikil（Figure1A–C）.Theslopesweregreaterforthe laboratory strain than the wild strain for Actikil（3.11 vs. 2.97, respectively）,Pesguard（2.449vs.2.098,respectively）,and Bacilod （2.435vs.1.827,respectively）,indicatinghomogeneityofresponse tothetestedlarvicides.Overall,fieldlarvaewerelesssusceptibleto the larvicides than the laboratory-adapted larvae.

3.2. Impact of IGR Dudim and Baycidal on young instars

Table 1 Susceptibility of the 4th larval stage of Cx. pipiens L. to different insecticides.

Figure 1. The effect of larval treatments with Actikil（A）, Pesguard（B）and Bacilod（C）on the 4th larval stage of Cx. pipiens.

Table 2 Impact of the IGR Dudim on the developmental stages of laboratory and field strains of Cx. pipiens.

Treated 4th instar larvae of Cx. pipiens field and laboratory strain with Dudim delayed effects insecticides at 0.0001 and 0.0040 concentrations showed percentage mortalities from 2%to 30%and 5%to 21%, respectively（Table 2）. Whereas the percentage mortality was 3%–31%for field strain and 4%–23% for laboratory-reared larvae exposed to Baycidal at 0.0002–0.0020 concentrations（Table 3）. The percentage mortality of larvae exposed to cuticle synthesis inhibitors was lower in the field strain than in the laboratory strain.

Table 3 Impact of the IGR Baycidal on the developmental stages of laboratory and field strains of Cx. pipiens.

Figure 2. Morphological abnormalities in the developmental stages of Cx. pipiens after treatment with Baycidal or Dudim.

Generally, the mortality rates were associated mainly with failure to pupate. The post-effect of Dudim and Baycidal on the adult stage of Cx. pipiens was evaluated studying percentage of adult emergence. Some Cx. pipiens individuals that have succeeded to reach to the adult stage have folded wings（Figure 2）. We used IC50which is a measure of 50%inhibition of adultemergence following insecticide exposure. The percentage mortality rates of Cx. pipiens exposed to Dudim were 22.6%–92.6%and 35.48%–97.88%for field strain and laboratory strains respectively, at concentrations ranging from 0.0001 to 0.0040 mg/L, respectively. The value of IC50was 0.0003 and 0.0001 mg/L for field and laboratory strains, respectively （Figure 3）.

The effective concentration of Baycidal was between 0.0002 and 0.002 mg/L, and the given adult emergence inhibition was 25%–90%and 32%–95%for field population and laboratory population respectively. Whereas, the IC50was 0.0004 and 0.0003 for field strain and laboratory strains（Table 4）. According to the resistance ratio obtained the 4th larvae stage of Cx. pipiens, the field strain was susceptible to Baycidal compound（Resistant ratio = 1.33）, as compared from the tolerant strain for Dudim（Resistant ratio = 3）（Table 4）.

Figure 3. Effect of larval treatment with the IGR insecticides on survived adults of Cx. pipiens.

Table 4 Susceptibility of the 4th larval stage of Cx. pipiens to delayed effects of different insecticides.

4. Discussion

The present study was performed to determine the susceptibility of the West Nile vector Cx. pipiens to some conventional and non-conventional insecticides in Saudi Arabia. Larvae of Cx. pipiens were more susceptible to Pesgard（0.045 and 0.032 mg/L）over Actikil（0.052 and 0.038 mg/L）and Bacilod （0.129 and 0.104 mg/L）, both for field and laboratory strains. On the other hand, the response of 4th instar of Cx. pipiens to tested insecticides（Actikil 50%, Pesguard and Bacilod）depends on the mode of action and the concentration of the active ingredient. According to IC50values obtained, the compound Dudim was more effective over Baycidal both for lab and field strains. In agreement with our findings, Saleh et al.［23,24］reported that the differences in the percentages of larval mortality increased proportionally with the increase in the use of concentration. Furthermore, mortality in the pupal stage and damage to adult emergence was also observed. Results revealed that larvae exposed to Dudim and Baycidal showed abnormalities in developmental stages such as larval siphon, pupal trumpets, unmelanized pupae（albino pupae）, and/or adults failure to emerge from the pupae［25,26］. The resistance ratio of Cx. pipiens treated with Actikil, Pesguard, Bacilod and Baycidal showed that this mosquito species was susceptible, whereas it was tolerant against Dudim, even if it did not reach the resistance level. This may be linked to the fact that the authority of Jeddah Province recently used a successful vector control programs against Cx. pipiens larvae, with proper rotation between the insecticidal groups, to avoid resistance development. The slightly difference in the effective concentration range among these studies could be linked to the differences between strains, biological response of the tested larvae, formulation of compounds and experimental conditions. Long-term follow-up studies are needed to determine how the environmental conditions affect the larvicidal effectiveness of these pesticides in field control programs. The reason may be due to the difference susceptibility of Cx. pipiens to different active ingredients［27］. This result showed that the larval mortality increased with concentration raised. In addition, the findings of this study were in agreement with several studies conducted in different parts of the world about the effect of insecticide against the mosquitoes［28–30］. Our findings revealed that the larvae treated with IGRs showed deformed abnormalities in developmental stages of Cx. pipiens after treatment and other intermediate stages including larval siphon, pupal trumpets, unmelanized pupa and failure of adults to emerge from the pupal skins. These observed abnormalities on developmental stages could be due tomorphological aberrations leading to the failure of successful emergence from exuviae of pupal stages［31］.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

Wearegratefultotheteamofthe Entomologyand Toxicology. The research was funded by Entomology and Toxicology Unit, University of Tabuk, Saudi Arabia with Grant no. 1/1/11/1435.

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*Corresponding author:Al Thabiani Aziz, Department of Biology, Faculty of Sciences, University of Tabuk, Tabuk, 71491, Saudi Arabia.