Influence of Temperature Change on the Growth and Susceptibility of the Common House Mosquito, Culex pipiens in Egypt to Some Insecticides

By transmitting major human diseases, mosquito species represent a serious threat worldwide in terms of public health. Most vector control programmes aiming to control life-threatening mosquitoes rely on the use of chemical insecticides. For the reason that only a few insecticides are used for public health, maintaining the efficacy of control programmes mostly relies on resistance management strategies. Development of such strategies requires understanding the factors influencing resistance together with characterizing the mechanisms involved. In this context, the present study aims to update current knowledge about the effect of temperature on the mosquito Culex pipiens population response to chemical insecticides. The results demonstrated that alteration of the temperature significantly affects Cx. pipiens populations. High temperature (25, 30°C) resulted in high survival rate (90, 95% respectively); while at temperature 20°C the survival rate was 80%. Egg hatching percentage was 95% after 24 h, at temperature 30°C and 50% after 24h, 50% after 48h at 25°C; however at 20°C egg hatching percentage was 100% after 48 h. In case of Cx. pipiens larvae that were reared under various temperatures pupated on day 5, 9 and 12 at 30°C, 25°C and 20°C, respectively. At high temperature 30°C, females emerged before males. On the other hand resistance of all Cx. pipiens populations to the selected chemical insecticides decreased with raising temperature. The obtained results also showed that there was significant change in acetylcholinesterase and glutathione -S-transferase level in both larvae and adult due to temperature changing. These results indicate that temperature is an important parameter that must be considered during the application of chemical assays or control of Cx. pipiens populations.


Introduction
Many of the most dangerous diseases such as malaria, filariasis, or dengue fever, are transmitted to humans by mosquitoes. Most vector control programmes largely based on the application of chemical insecticides. Today, insecticides resistance has been reported in all main mosquito vector species and geographical regions with high parasiterelated mortality and morbidity [1,2].
For Culicidae, as in other arthropods, temperature is one of the most important abiotic factors affecting the development and survival of the immature stages [3]. Mosquito growth and development rates are temperature dependent, with development times typically shortening [4], and body size either increasing [5] or decreasing [6] as temperature increases.
Insecticides are the most common strategy used for mosquito management [7,8]. However, insecticide toxicity does not only depend on the active ingredient. The efficacy of chemical insecticides against its target is also a function of the formulation, the biology of the insect, and the environment in which these interact [9]. Changes in ambient temperature can alter the toxicity of insecticides to ectothermic organisms [10].
Organophosphates are known to exhibit a positive correlation between ambient temperature and mortality for many insect species, while carbamates are known to exhibit a slightly negative correlation between ambient temperature and mortality for many insect species [11]. Environmental temperature has been shown to influence the outcome of insecticide exposure; temperature differences expected to occur naturally under field conditions can lead to notable variations in chemical efficacy [12].
This study aims to investigate the effect of temperature changing of mosquito larval habitats on subsequent development and relation of such factor with insecticides resistance.

Test Insects
Three Cx. pipiens populations were used in this study: resistant, susceptible and laboratory ones. All populations were reared in the Research Institute of Medical Entomology (Ministry of Health & Populations, Giza, Egypt) under standard conditions (26-28°C, 12 h:12 h light/dark period, 70-80% relative humidity) [13]. The susceptible and resistant populations were brought from The American Naval Medical Research 3 (NAMRU3) insectary and laboratory population was collected from field and reared in the laboratory for seven generation at the same conditions. The susceptible population was not exposed to any control agents since 1986 [14].
Mosquito eggs were transferred to different containers with different temperatures (20, 25 and 30°C), till emergence of the 4 th larval instar. Part of the 3 rd larval instars from each group was used for larval bioassays, while the remaining larvae were reared to the adult stage and used for adult bioassays. The larvae were fed on a fish food. The adults were fed on 10% sucrose solution [15]. The mosquitoes were reared and maintained in habitat free from insecticides and repellents. The tests were carried in incubators set at 20, 25 and 30°C.

Hatching Period
The newly laid eggs were placed in three water dishes for each population (resistant, susceptible and laboratory). For embryonation, a single raft of eggs (approximate 80-100 eggs) was placed in egg-hatching water dish contained dechlorinated water. Nine egg-hatching containers (three replicates for each population) were labeled with the kind of population, the temperature at which the test was set, and the date of the starting of the test and given an identifying number. Observations were reported at intervals of 24 hours and the numbers of larvae hatching from the eggs were recorded. A record of the number of larvae hatching was taken until maximum hatching was obtained. The hatching period was calculated according to the following equation [16].
The hatching period = The numbers of larvae X the number of days taken to hatch The maximum number of larvae hatched for that replicate

The Larval Duration
Once hatching had taken place, the larvae were observed every 24h and the number of pupae formed in each egghatching dish was recorded.
The larval period was calculated according to the following equation [16].
The larval duration = The number of pupae at each observation X the number of days since the larvae were hatched / The total number of pupa for that replicate

The Pupal Duration
The pupae were collected with a little water using a large pipette and moved into plastic cup and put in special mosquito cage. Observations were reported every 24h hours and the number of adults emerging recorded until all adult emerged. The duration of the pupal period was calculated according to the following equation [16].
The duration of the pupal period = The total number of adults emerged X the number of days The total period in days in which all the adults emerged

The Survival Rate and Emergence of Male and Female
Surviving of all stages was monitored and recorded daily. The emerged adult males and females were counted and observed to determine which of them emerged first.

Larvicides
Three recommended organophosphorus insecticides by the World Health Organization's Pesticide Evaluation Scheme (WHOPES) for control mosquito larvae [17]; temephos, chloropyrifos and pirimiphos methyl were used in the study.
To perform bioassays, single-concentration diagnostic tests were conducted as proposed by [18] to monitor insecticide resistance. The diagnostic concentrations were; 0.02 ppm for temephos, and 0.01 ppm for chloropyrifos, while the diagnostic concentration for pirimiphos methyl was calculated by measuring LC 99 of susceptible population and then duplicate (0.1 ppm).
Larval bioassay was performed according to the WHO standard method [19]. For bioassay, four replicates for each selected insecticide were conducted against newly 3 rd larval instar. Test beakers of 500 ml capacity, each containing 249 ml tap water were prepared. In each, 1 ml of each concentration was infiltrated under the water surface with a pipette. After 30 min. of preparing the insecticide solution a group of 25 larvae were placed in each beaker. The test was run at the same three temperature degrees (20, 25 and 30°C) at which the larvae were previously reared. Larvae were left for 24 h and mortality was then recorded. Moribund larvae were considered as dead ones. Another group of larvae was used as control. The control tests were set up by adding 1 ml of ethanol (the solvent of used chemical insecticides) into water and mortality never exceeded 4%.
Twenty sucrose-fed 4 day adult female mosquitoes per replicate were used for this bioassay. The principle of this test is to expose of females of each different larval breeding habitat (temperature: 20, 25, 30°C) for one hour in a specially designed plastic tube lined with a filter paper impregnated with diagnostic concentration of the insecticide. After the exposure the mosquitoes were transferred to another tube lined with clean filter paper and mosquito adult mortality is observed after 24 hours. For the control, impregnated-papers containing 1 ml ethanol were used.

Biochemical Assay
To determine the effect of temperature on enzymes of the mosquitos, adults and larvae of the three tested C. pipiens populations that reared at different temperature degrees (20, 25, 30°C) were submitted to biochemical assays for general acetylcholinesterase AChE, glutathione S-transferase GST and total protein.

Preparation of Insects for Analysis
The survived larvae and adults of C. pipiens were collected after insecticide application for each different temperature then weighted and mechanically homogenized in distilled water by a chilled glass telfon homogenizer (ST-Mechanic-Preczyina, Poland). Homogenates were centrifuged by refrigerated centrifuge (6 MR, USA) at 8000 r.p.m. for 15 min at 5°C. The deposits were discarded and the supernatant was kept in a deep freezer till use.

Determination of Total Protein Content
Total protein quantification of mosquito homogenates was performed using Bradford reagent with bovine serum albumin as the standard protein [20] to normalize enzyme activity levels by protein content.

Determination of Acetylcholinesterase (AChE)
Activity Acetylcholinesterase (AChE) activity was measured by using acetylcholine bromide (AChBr) as substrate, the produced color was measured calorimetrically using double beam ultraviolet/visible spectrophotometer (Sectronic 1201, Milton Roy Co., USA) used to measure absorbance of colored substances at 515 nm [21].

Determination of GST (Glutathione S-Tranferases)
Activity Glutathione S-transferase (GST) catalyzes the conjugation of reduced glutathione (GSH) with 1-chloro 2,4dinitrobenzene (CDNB) via the -SH group of glutathione. Enzyme activity was measured by a reaction mixture of 10 µL of the insect homogenate plus 200 µL of reduced glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB) solution (10 mM reduced glutathione dissolved in 0.1 M phosphate buffer pH 6.5 and 3 mM CDNB originally dissolved in 1 mL methanol) [22]. The mixture was incubated at 30°C for 5 min then the enzyme activity was measured at 340 nm with spectrophotometer. The enzyme activity was reported as µΜ/min/mg protein using the extinction co-efficient corrected for the path length of the solution in the microtitre plate well.

Statistical Analysis
Mortality data of toxicological tests and values of biochemical assay were calculated by using Microsoft Excel (Microsoft Corporation, 2007). Data of the biological parameters and biochemical results were statistically analyzed using t-tests and ANOVA using SPSS (20) [23] for Windows to find out if there were significant differences in all tested result.

Biology
The obtained data demonstrated that changing temperature had the same effect on the survival rate, growth duration of the three tested mosquito populations (resistant, susceptible and laboratory): Table 1 showed that temperature has an effect on egg hatching, larval and pupal periods as well as survival rate of mosquitoes. There were significant differences in the embryonation periods under each of the three experimental temperatures at (p <0.05). At 20°C, the eggs of Cx. pipiens took an average of 2.4 days for embryonation while at25°C; the eggs need an average of 1.7 and at 30 o C, the eggs hatched after 1.13 days.
There was inverse relationship between the temperature and larval duration as well as pupal duration, that as the temperature rose, the duration of these immature stages reduced. With rising temperature, the survival rate of the mosquitoes increased, that at 30°C the survival rate was 95%. Also it was observed that, the female emerged before male at temperature 30°C.  20 2.4 ± 0.15 a 12.5 ± 0.25 a 3.6 ± 0.15 a 80 25 1.7 ± 0.15 a 9 ± 0.2 a 2.13 ± 0.08 a 90 30 1.13 ± 0.08 a 5.1 ± 0.2 a 2 ± 0.12 a 95 a: means that there is significant difference between three groups(p <0.05). Table 2 demonstrated the mortality percentage of the three mosquito populations due to the exposure to the selected larvicides at different temperature.

Larvae
The result showed that all tested populations were more sensitive to temephos at all temperature degrees with mortality percent 100% followed by pirimiphos methyl and the lowest effective insecticide was chloropyrifos. On the other hand, all populations were more susceptible at low temperature than at high temperature, that the minimum mortality percent occurred at 30°C followed by 25°C and maximum mortality percent occurred at 20°C. As expected, susceptible population exhibited the lowest resistance to larval insecticides then laboratory population followed by resistant population which has the highest degree of resistant especially at temperature 30°C.

Adults
From the tabulated data in table 3 it could be observed that all populations were more susceptible at low temperature than at high ones. The minimum mortality percent occurred at 30°C followed by 25°C and highest mortality occurred at 20°C. Lambda-cyhalothrin was the most effective insecticide followed by deltamethrin, while permethrin was the least effective insecticide. Also, the susceptible population recorded the highest mortality percentage, followed by the laboratory one then the resistant population.

Larval Biochemical Assay
The biochemical assay results were represented in figures  (1 and 2). The resistant population had highest level of the enzymes especially at temperature 30 °C, followed by laboratory population, while susceptible population recorded the lowest enzyme activity levels. In all populations, by increasing the temperature degrees the levels of AChE and GST increased. Figure 1 showed that the lowest levels of AChE observed at 20°C (1.9, 2.3 and 4.11µg AChBr/min/mg protein) for susceptible, laboratory and resistant population, respectively. Temperature 25°C showed moderate level of AChE (1.97, 2.7 and 4.23µg AChBr/min/mg protein) for susceptible, laboratory and resistant population, respectively. On other hand temperature 30°C resulted appearance of the highest level of AChE (3.6, 4.1 and 7 µg AChBr/min/mg protein) for susceptible, laboratory and resistant population, respectively. According to fig. 2, the enzyme GST level of susceptible population was (1004, 1214 and 1659 n mole sub conjugated/min/mg protein) at temperature 20, 25 and 30°C, respectively, while GST level of laboratory population was recorded (1421, 1655 and 1796 n mole sub conjugated/min/mg protein) at temperature 20, 25 and 30°C, respectively. The resistant ones had GST levels (1609, 1974 and 2094 n mole sub conjugated/min/mg protein) at temperature 20, 25 and 30°C, respectively.

Adult Biochemical Assay
The biochemical assay results of the adult mosquitoes were plotted in figures 3, 4. The resistant population recorded high levels of the enzymes elevation followed by laboratory population, while susceptible population had the lowest enzyme activity. Temperature 30°C elevated the enzymes activity in all tested populations if compared with the two other degrees. Figure 3 showed that the least levels of AChE observed in temp. 20°C (5, 5.2 and 6 (µg AChBr/min/mg protein) for susceptible, laboratory and resistant population, respectively. Temperature 25°C showed moderate level AChE (4.9, 4.8 and 5.9 µg AChBr/min/mg protein) for susceptible, laboratory and resistant population, respectively. On other hand, temperature 30°C showed the highest level of AChE (6.8, 7.4 and 7.9 µg AChBr/min/mg protein) for susceptible, laboratory and resistant population, respectively. GST activity for susceptible population was (1254, 1177 and1745 n mole sub conjugated/min/mg protein) at temperature 20, 25 and 30°C, respectively, while GST activity for laboratory population was (1284, 1493and1937 n mole sub conjugated/min/mg protein) for temperature 20, 25 and 30°C, respectively. While the resistant strain exhibited elevation in activity of GST (1425, 1377 and 2000 n mole sub conjugated/min/mg protein) for temperature 20, 25 and 30°C, respectively.

Discussion
Climate change is expected to lead to global and regional changes in environmental temperature and other climatic variables [24], which are likely to have an impact on different disease vectors distribution [25,26]. It is thought that global warming may make currently in hospitable regions amenable to vector expansion along altitudinal gradients [27]. In order to generate useful predictions of any disease transmission and the impact of intervention programmes, the full impact of environmental conditions on the life history parameters and population dynamics of disease vectors needs to be taken into account when forecasting transmission [28].
The results demonstrate that alteration of the temperature affects egg hatching, duration of the larval and pupal stages of the mosquito, emergence of adult in all studied populations of mosquito, Cx. pipiens. At high temperature Cx. pipiens life cycle became shorter (mosquito development occurred faster) while at low temperature mosquito took long time for complete development. The present study proved that temperature had great effects on biology as well as susceptibility of Cx pipiens mosquitoes.
These findings agree with [29,30] who found that temperature has effects on the emergence, survival and the subsequent behavior of the adult Cx. pipiens mosquitoes and Ochlerotatus albifasciatus mosquitoes. Also [16,31] observed that in higher temperatures the mosquitoes complete their life cycle faster but also died earlier.
Generally, the survival rate in this study was high at temperature between 25-30°C and this finding agreed with [32,33] who found that the rate of larval survive decreased at temperatures below 18°C and above 32°C in Anopheles gambiae and Cx tarsalis respectively. Also [31] established that when the larvae of Aedes aegypti were exposed to a range of varying temperatures, mortality was observed to increase with increase temperatures above certain levels with the temperature being completely lethal at 43°C. Changes in ambient temperature can alter the toxicity of insecticides to ectothermic organisms [10].
The present data showed that larval breeding conditions considerably influence the susceptibility of Culex mosquitoes to different insecticides. The susceptibility of Cx. pipiens increased as the temperature decreased. This findings agreed with [11,34,35,36,37] who found that the temperature increases often associated with decrease in toxicity of different insecticides against different mosquito species. Also, the results agreed with [38] who estimated that pre-exposing larvae to high temperatures resulted in increased tolerance to the insecticide.
On other hand the present results are disagreement with [39,40], who observed that organophosphorus insecticides toxicity increases with increasing temperatures.
To gain satisfactory explanation for changing the response of mosquitoes to the used insecticides with temperature changing, a biochemical assay was conducted for certain two enzymes which are known to be implicated in insect resistance. The enzymes are acetylcholine esterase AChE and glutathione-s-transferase GST. AChE is an enzyme that found in the center nervous system and its functions is removing acetylcholine from its post synaptic receptors. The result of this action is the hydrolysis of acetylcholine into acetate and prolonged neuron excitation [41]. Inhibition of AChE by bio-insecticides causes a desensitization of acetylcholine receptors and leads to eventual death of the organism. The three gene families, GSTs, cytochrome P450 monooxygenases (cytochrome P450s) and hydrolases are mainly involved in the detoxification of insecticides, whereas, three proteins, including acetylcholinesterase, sodium channels and GABA receptor, have been mainly implicated in decreased target site sensitivity in insect nervous system [42].
At high temperature Cx. pipiens showed more resistance to the used insecticides and elevated activity of AChE as well as GST. The activation of GST and AChE may explain increase of resistance in the mosquito population.
Temperature affects not only insecticide ingredients but also enzymes and proteins, which play an important role in the survival of many organisms [43]. The biochemical studies agreed with [44] who found that decreased temperature may also increase the susceptibility of neurons of crayfish to pyrethroid insecticides making them more sensitive to excitation.
The current findings suggested that high larval rearing temperatures cause changes in the activity levels of AChE and GST enzymes which lead to make larvae being more resistant to insecticides. This means that any modifications in the levels of resistance of the mosquitoes to any applied chemical control may be due to the impact of the temperature, so the environmental conditions at which the insecticides are used must be took in consideration.

Conclusion
The obtained results suggested that, high larval rearing temperatures cause changes in the activity levels of AChE and GST enzymes which lead to make larvae being more resistant to insecticides. It could be concluded that, water temperature plays a fundamental role not only on development and biodiversity of mosquito larvae but also on the susceptibility to some insecticides by affecting the level of detoxified enzymes which interfere with insecticide resistance and their assays. Mosquito larval mortality was observed to increase with increase temperatures.