The Potency of Aedes species in transmitting dengue fever virus with evaluating the susceptibility of vector larval stages to some insecticides

The present study was planned to use the technique of Polymerase chain reaction (PCR) to detect the virus of dengue fever in mosquito females of Aedes aegypti (L.) and Ae. caspius (Pallas). The results indicated that all samples tested for virus of dengue were found negative (-) except one sample of Ae. aegypti which was positive(+). On the other hand, susceptibility levels of mosquito larvae of field strains of Ae. aegypti, which is the vector of dengue fever, to some insecticides currently in use in Jeddah governorate were determined. The LC50 values (concentration which kills 50% of mosquito larvae) were taken in consideration. Mosquito larvae of Ae. aegypti proved to be more susceptible to Bactilarvae (0.012 ppm) than Safroten (0.020 ppm), Temephos (0.032ppm), Solfac (.039 ppm) and Icon (0.047ppm), respectively.


INTRODUCTION
Dengue fever is an acute viral disease of the tropical and sub-tropical regions around the world, especially in urban and semi-urban areas (Halsted et al., 2001).A recent estimate shows that more than 50 million people are at risk of dengue virus exposure worldwide.Annually, there are 2 million infections, 500,000 cases of dengue hemorrhagic fever, and 12,000 deaths (Guha-Sapir and Schimme, 2005;WHO, 2005).
In Jeddah city, Saudi Arabia, dengue fever made its first appearance in 1994; and by the end of the year, approximately 300 cases were diagnosed and three dengue serotypes (Den1, Den2, and Den3) of viruses were reported (Fakeeh and Zaki, 2003).In 2006, dengue fever reported cases had risen drastically compared to the earlier recorded numbers.The government of Saudi Arabia has responded to the problem immediately by allocating more than one billion Saudi Riyals (more than two hundred fifty US dollars) to combat the disease and to bring down the number of confirmed cases drastically.The government decision has identified roles and responsibilities among different government agencies and ministries including the Ministry of Municipality and Rural Affairs; Ministry of Health; Ministry of Agriculture, and Ministry of Water and Electricity (Aburas, 2007).On the other hand, dengue fever is a mosquito-borne viral illness that caused by one of the four serotypes of dengue virus belonging to the family Flaviviridae and predominantly transmitted by many members of Aedes species.Lanciott (1992) was the first to describe the technique of polymerase chain reaction (PCR) to detect dengue virus in the body of the mosquito vector, and isolated the four dengue virus patterns from mosquito A. aegypti.Fouque et al. (2004), and David et al. (2003) identified three types of mosquitoes, Ae.Aegypti, Ae. hensilli, Ae.albopictus as vectors of dengue virus.
Currently there is no single vaccine with an economic value is available against serotype of dengue viruses, and therefore the primary protection against infection with the virus depends on the control of mosquitoes.The spraying of pesticides in the hotbeds of breeding to reduce the source of infection is a modern method which is used globally to reduce the reproduction and multiplication of mosquito vectors of disease and restrict their number in the environment to the extent that they do not spread the disease (WHO, 1990).This study aims to use the new approach to mosquitoes control through the identification of the carrier type of the virus that causes dengue fever with an evaluation of the effectiveness of some insecticides commonly used in control programs in the province of Jeddah against the larvae of the carrier type.

MATERIALS AND METHODS Molocular studies of dengue virus Preparation of mosquitoes samples
Light traps of Black-Hole was used to collect samples of adult mosquitoes from various locations of the Jeddah province, samples after collection from the field brought to the laboratory and kept in the low temperature (-86 ° C) to kill insects by freezing while preserving the genetic material.The female mosquito of genus Aedes species were isolated by using anatomical microscope and the identification of the species was done by using taxonomic keys (Harbach, 1988;Al-Tubiakh, 1995).Each type of insects has been distributed at each location within the vial tubes and a special code number was given to each sample.

Isolate the DNA of the virus
The viral RNA was extracted from the complex of mosquito sample and 140 ml of it was added to Kit Qiagen (QIA amp-Viral RNA Mini Kit Cat.NO.5294) according to the proposed kit protocol (MWG-Biotech, Germany).

Polymerase chain reaction (PCR)
During Reverse transcription polymerase chain reaction (RT-PCR) two primers (D1, D2) were used to identify the virus that causes dengue fever in addition to the DNA extracted with the rest of the chemicals for interaction, (Table 1).The overall mix has been developed (25 micro liter of reaction mixture) inside the PCR tube, which is specially designed to withstand the heat of polymerization chain reactions.(The device, named Ependroff Master cycler® gradient thermal cycler from Germany.).
7.5 Water treatment free of RNA ase 5 RNA extracted 25 Total * The polymerase enzyme solution and the regulator in the presence of magnesium salts stimulate the prefixes on the link DNA to be doubled and reduce the link prefixes to other sites on the DNA similar to the gene to be isolated.Amplification of DNA sequence which requires three temperature shifts was done in the thermal cycler.The polymerase chain reaction was done in three steps.
The Potency of Aedes species in transmitting dengue fever virus with evaluating 111 1.In the first step the temperature was raised to 95 0 C for 15 minutes to denature the double stranded DNA (dsDNA) which unzip the DNA strand.2. In the second step the temperature is reduced to 55 0 C for one minute for annealing of the primers to single strand DNA (ssDNA).(High temperature do not allow primers to anneal efficiently).This allows the link between two strings in 3' end of the one primer and 5' end of other primer.3. The third step is to extend the DNA by raising the temperature to 72 0 C for two minutes to construct a DNA chain by Taq polymerase enzyme (isolated from different thermophilic bacteria) which attaches at each primers site and extend a new DNA strand.After this extension step the temperature is raised to 72 0 C for ten minutes.This stage is called final extension.Millions of DNA copies will be formed after 45 cycles.At the end of PCR session the out puts of RT-PCR was subjected to electrophoresis on 1.5% Agarose gel. 3 ml (it is not a true volume) of the sample was mixed with 2 ml (it is not a true volume) of Ethidium Bromide dye (Et-Br) after the integration with barcode (marker) DNA, then the sample was loaded on the gel and a current of 450 volts (it is not a true volts) for a period of eighteen hours (it is not a true time) was passed to get a length of 100pairs baseline for comparison with a sample of DNA of the virus and backwater from the tissues of insects by using electrophoresis of nucleic acids and products of PCR samples.

Mosquito strain rearing
Tests were performed on a field strain of Aedes aegypti (L.) raised from wild larvae, collected from Jeddah.The larvae were reared until pupation and adult emergence took place for maintaining the stock colony.This strain was maintained at a room temperature of (27 ± 1C • ) and (70 ± 5% R. H.) with a 14: 10 (L: D) photoperiod throughout this study.

Larval bioassay
Susceptibility tests of A. aegypti larvae were conducted according to the procedure recommended by WHO (1981).Treatments were carried out by using early fourth instar larvae to serial concentrations of the tested insecticides for 24 hrs in groups of glass beakers containing 100 ml of distilled water.Five replicates of 20 larvae for each concentration and for control trials were set up. Larval mortalities were record at 24hrs post-treatment (WHO, 2005).

Statistical analysis
The average larval mortality data were subjected to probit analysis for calculating the LC 50 and LC 95 , values which calculated by using the Finney (1971) method.GW BASIC probit1 Statistical software was used.

RESULTS AND DISCUSSION Molecular studies of dengue virus
Fig. 1 Shows radiological picture of the agarose gel and bundles of DNA dyed textured Eithidium bromide (Et Br).As shown in the figure contains the first slot on the DNA scale.DNA Ladder is a cut or packages of different lengths of DNA known to contain holes from 2 to 9 DNA samples isolated from female mosquitoes Ae. aegypti to be detected for their ability to carry the virus that causes dengue fever and take Code numbers of 494 to 502 and contains openings from 10 to 14 DNA samples isolated from female mosquitoes Ae. caspiusbe detected for their ability to carry the virus that causes dengue fever and take code numbers from MP65 to MP69 and contains the slot 15 on DNA isolated from virus positive samples for comparison.It contains the slot 16 of a sample free of DNA solution as a negative control.The results showed that the sample with code number 501 were carriers of the virus as it appears on the gel packs stacked on top of each other was almost identical to the positive control treatment.This confirms that the sample with code number 501 isolated from female Ae.aegypti, which was collected from the area of the country to examine the positive polymerase chain, was getting package DNA with a length of 511 base pairs.Overall this study showed the ability of female Ae.Aegypti in transmitting the virus that causes dengue fever in Jeddah.It was only in one sample which carriers the virus, while the rest of the samples were negative.These results are consistent with many previous studies and demonstrated the ability of Ae. aegypti in the transfer of dengue virus using PCR technique in different regions of the world Feres et al. 2006;Pinheiro et al., 2005) ; (Urdaneta et al., 2005).Evaluation of some insecticides against larvae of Aedes aegypti mosquitoes of dengue virus in Jeddah City.
Results of susceptibility levels of fourth instar larvae of A. aegypti after treatments with different concentrations of the chemical insecticides Solfac, Icon, Temephos and Safroten as well as the bacterial insecticide Bactilarvae were recorded in Table 3.The effective concentrations of the chemical insecticide ranged from 0.01 -0.12 ppm for the Ae.aegypti mosquito larvae.The corresponding larval mortalities were in respect 17-98% (Table 3).Records of susceptibility levels of A. aegypti mosquito larvae against the bacterial insecticide Bactilarvae are indicated that the range of effective concentrations of the test biocide was 0.01-0.08ppm.Results of larval mortalities varied from 38-96%.According to the LC 50 values, the results confirms that the bacterial insecticide Bactilarvae was more effective against the larvae of Ae. aegypti larvae than chemical insecticides, Safroten, Temephos, Solfac and Iconat about 1.6,2.66,3.25 and 3.92 Folds,respectively.Table 1: Larvicidal activity of tested compounds against A. aegypti.a: Five replicates, 20 larvae each.(Nil mortality in control samples) b: Tabulated value of χ 2 = 7.81 when the (df) degrees of freedom=3,The level of significance (0.05) to which is larger than the calculated value of χ 2 and this confirms data homogeneity.c: LFL -lower fiducial limit.d: UFL -upper fiducial limit, χ 2 -Chi-square value.
Generally, comparison of larvicidal activity of the tested insecticides against A. aegypti larvae indicates that the bacterial insecticide Bactilarvae proved to be the most effective compound followed by the OP Safroten, Temephos, and Solfac while the Icon was the least effective one as shown in Fig, 2. In general, the records indicated that mosquito larvae were more susceptible to the tested bacterial insecticide when compared with the chemical insecticides.The increase in the tolerance level of chemical insecticides may be due to the extensive local application of different chemical insecticides during control measures against present immature and adult mosquito species vectors in Jeddah.However, laboratory and field studies in this respect were carried out by several authors against a wide spectrum of mosquitoes (Saleh and Kassem, 1982;Hemingway and Ranson, 2008;Liu et al., 2004;Fansiri et al. 2006;Al-Ghamdi et al. 2008;Qusti et al. 2010;Al-Ghamdi and Mahyoub, 2010;Saleh and Aly, 1987;Canyon and Hii, 1999;Nazni et al., 2005;Lawler et al., 2007).

ACKNOWLEDGEMENTS
We extend our sincere thanks and gratitude to His Excellency Dr / Issam

Fig. 1 :
Fig. 1: The radiological image of agarose gel (1.5%) and stained by the bundles of DNA after duplicate serial by polymerization reactions.The length of the virus band is 511 base pairs.Column A: DNA marker ladder in 100-base pairs.Column B: Tested samples for carrying the virus that causes dengue fever.Column C: Positive control for the virus length of 511 base pairs.Column D: Negative control (solution is free of DNA).

Fig. 2 :
Fig. 2: lethal concentrations for 50% of fourth instar larvae of the mosquito Ae. aegypti after 24 hours of treatment with pesticides commonly used in mosquito control programs in the province of Jeddah.

Table 1 :
Diagnostic materials used in the detection of dengue virus in the body of female Aedes mosquitoes collected from different locations in Jeddah city.
Bin Ibrahim Al-Azhar, head of infectious organisms at King Fahd Medical-King Abdulaziz University to help us