Mosquitoes are causing plenty of diseases including dengue fever, malaria, japanese encephalitis. Mosquitoes were collected from 1006 sties of six tehsils of district Sargodha. Different containers were inspected for mosquito sampling. RNA was extracted from female mosquitoes and used for cDNA synthesis followed by detection of arbovirus using primer. The site index (SI) and tehsil container index (TCI) of district Sargodha was 28.3 and 12.2 respectively. Overall, the percentage of Aedes spp, Culex spp. and Anopheles spp was 79, 20 and 0.4% respectively, in the collected population. Highest positive containers for Aedes mosquitoes were discarded tires. Overall, 12.2% of containers were found positive for mosquito population. Maximum number of larvae and adults were recorded from urban area of district Sargodha (SgdU). Arboviruses, Flavivviruses, were found in 5 pools (almost 3%) of the samples, while, other viruses such as Alphaviruses and Bunyaviruses were not detected in any of the samples. Man-made habitats are the main contributors to dengue fever mosquito population. There is a dire need of proper environment management to reduce the breeding places of mosquito and detect the virus in local population for environmental friendly control of dengue fever mosquito.
Article
Control of Aedes mosquito by targeting dengue fever virus in local mosquito population
https://doi.org/10.21203/rs.3.rs-1883195/v2
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Dengue Fever
Aedes mosquito
Arbovirus
Mosquito breeding sites
- Aedes mosquito are vector of dengue fever.
- Pakistan is facing dengue epidemic every year.
- Dengue fever mosquito breed in different container having fresh and rain water.
- Monitor the mosquito breeding site and sampling the dengue fever larvae is challenging.
- The detection of dengue fever virus in mosquito population emphasized the importance of mosquito breeding places management and their link to urbanization.
Mosquitoes are vector of various diseases like dengue fever, malaria, japanese encephalitis and lymphatic filariasis (Damapong et al. 2016). The major factor that plays an important role in transmission of diseases is the relative abundance of mosquitoes and fitness in the aquatic habitats (Alto et al. 2005). Dengue has the higher death rate than malaria (Chaiphongpachara et al. 2019). The development of mosquito life stages, larvae to pupa and then adult, depends greatly on temperature, nutrition and population density. The life cycle from larvae to adult could be as short as 7 days at 30°C with 65% humidity. Female mosquito mate within 2 to 6 days of development and start seeking swarms by 48 hours of emergence (Rueda et al. 1990). Larval stage counts for 135 hours in females and 118 hours in males and female emerge mostly in large numbers as compared to males. Culex and Anopheles mosquito breed in waste water and puddles respectively while, Aedes breed in fresh or rain water (Ulibarri et al. 2016). The population of dengue fever mosquito greatly depends on the man-made activities such as trade and urbanization. In highly populated areas, the more Aedes mosquito breeding container are present (Madzlan et al. 2016; Abílio et al. 2018). Different arboviruses are present in the mosquito population worldwide. Among the arbovirus, virus belonging to family flavivirus, bunyavirus and alphavirus are common in mosquito population around the world (Go et al. 2014; Bisimwa et al. 2016; Young 2018). The DENV has been endemic for many years in Pakistan because of the temperate climate of the country. Serotype (serotype 3) of dengue virus was reported in 2005 (Rasheed et al. 2013). Historically, only the presence of DENV subtypes 1 and 2 were detected in isolated outbreaks in Pakistan in the twentieth century(Khan et al. 2010). Since 2005, all four subtypes of DENV have spread throughout the country (Khan et al. 2008; Koo et al. 2013).
In spite of different control methods such as environmental source reduction, mechanical, biological, chemical and genetic (Baldacchino et al. 2015), people now-a-days largely depends upon chemical insecticides for vector control and reduction of breeding sites (Islam et al. 2020). A small number of insecticides are available which are persistently effective against target pest, eco-friendly and safe for human and other non-targeted organisms (Rezende-Teixeira et al. 2022). The extensive use of synthetic insecticide against selective species of an insect causes development of resistance in mosquito against that insecticide. The phenomenon of resistance in insects is actually the development of the ability in an insect to tolerate the approved dosage of the toxins. Generally, there are two types of resistance mechanisms, metabolic resistance and target site. Metabolic mechanism is the changes in the detoxification level of insecticide, while the target site involves the changes in sodium channel, GABA receptors and acetyl cholinesterase (Li et al. 2007).
To cope with the dengue epidemics, there is a dire need to monitor the dengue fever mosquito and detect the arboviruses from the local population for vector management. The present research is aimed at monitoring the mosquito breeding sites and detection of arboviruses from the Aedes mosquito population before chemical control of Aedes mosquito. It is further hypothesized that the presence of Aedes mosquito is not correlated to the dengue fever in the human population.
I. Mosquito sampling
Sampling of dengue fever mosquito (larvae and adult) was performed at union council level from different part of district Sargodha, Punjab, Pakistan in 2021. The Site Index (SI) and Tehsil Container Index (TCI) were determined by using the following formula.
The breeding places such as discarded old tires outside, flower pots and trays, plastic containers, air coolers, water storage tank and mud pots were sampled. Aedes mosquito were brought to the Laboratory in collection vials for identification using a taxonomic key (Rueda 2004). Female Aedes mosquitoes were separated and pooled on the collection site bases. Each pool was having upto 20 female mosquitoes
II. Extraction of Viral RNA
RNA was extracted from pooled mosquito by using RNA extraction kit (Solarbio Life Sciences, China) according to the manufacturer’s recommendation. Briefly, 20 blood-fed female mosquito were ground in 1mL of lysate with tissue homogenizer and samples were placed at room temperature for 5 minutes. Chloroform (0.2 mL) was added to the homogenized samples followed by vigorous shaking for 15 seconds. The samples were centrifuges at 2-8°C, 12000 rpm, for 10 minutes and the supernatant was pipetted without touching the precipitate. The adsorption column was activated with column solution followed by centrifugation. The earlier collected supernatant was poured into the column after adding the 200 µl of absolute ethanol and let the sample stand for 5 minutes followed by centrifugation at 2-8°C at 12000 rpm for 2 minutes, and discarded the waste liquid. The column was rinsed twice with washing buffer and the flow-through was discarded. Then, the adsorption column was placed on a new tube and 80 µl RNase free ddH2O was added to the center of the column, left at room temperature for 5 minutes and centrifuged at 2-8°C, 12000 rpm for 2 minutes to obtain total RNA. The quantity and quality of extracted RNA was determined by using spectrophotometer (NanoDrop) by measuring absorbance at 260nm and 280nm. The ratio A260/A280 was used to determine the quality of RNA Ratios above 1.6 was considered good for downstream experiments.
III. cDNA synthesis
RNA was treated with DNase I (EN0521, Thermo Scientific) to remove trace amounts of DNA. One microliter of DNase I, RNase-free was used for 1 µg RNA. cDNA was synthesized by using RevertAid First Stand cDNA synthesis Kit (K1622, Thermo Scientific) according to the manufacturer’s recommendation. Briefly, a total of 500 µg RNA was used for cDNA synthesis.
RNA was mixed with 2 μL of random hexamer primer in a 0.2 ml PCR tube. The mixture was incubated in a thermocycler (T100, Biorad) for 10 min at 70 °C. The following components were added to the PCR tube after stopping the reaction: 4 μL of 5X Reaction Buffer (Thermo Scientific), 1 μl of 10 mM dNTPs, 1 μl of Ribolock RNAse Inhibitor (20U/μl) and 1 μl of RevertAid M-MuLV RT (200 U/μl). The mixture was then placed in a thermocycler at the following conditions: 25 °C for 15 min, 50 °C for 50 min, followed by 70 °C for 15 min in the thermocycler and hold at 4 °C. A total volume of reaction mixture was made to 20 μL. Negative control such as Reverse Transcriptase minus (RT minus) and No Template Control (NTC) and positive control with GADPH RNA (0.05 μg/ μL) were used to verify the results of the first strand cDNA synthesis steps.
IV. PCR amplification of first strand cDNA
The PCR amplification of targeted viral sequences in the cDNA was performed in a 50-μL reaction containing: 0.5 μl of Taq DNA polymerase 5 U/ µL, 0.3 µM s each of forward and reverse primer, 2 μl of the cDNA, 5 µl of 10X PCR buffer, 1 µl of 10mM dNTP mix, 3 µl of 25 mM MgCl2 and 35.5 μl of nuclease free water to top up to 50 μl. The primers with sequence and amplification condition used for the amplification of viral cDNA were presented in Table 1 .
V Detection of Arbovirus
PCR products were separated on 1.5% agarose gel of 4mm thickness using electrophoresis apparatus. One microliter of loading dye was mixed with 5 μL of PCR product. All samples including positive and negative control were loaded on the gel. The image of gel was visualized with Gel Doc System.
VI. Patient and Public Involvement statement
Patients were not involved in the current study. Data of mosquito was collected from mosquito breeding sites and arbovirus was detected in the population of mosquito, not in human, so patients were not involved.
Collection of Aedes mosquito from their breeding sites
A total of 1006 sites were surveyed from six tehsil such as Bhalwal, Kotmomin, Sahiwal, Sargodha, Shahpur, Silanwali of district Sargodha to collect the mosquito adult and larvae for the detection of dengue fever virus from the mosquito adult population. Collected adults and larvae were used for arbovirus detection and bioassays respectively. The site index (SI) was 23.4, 20.8, 45.2, 17, 21.7, 66.4 and 20.7 for the tehsil Bhalwal, Kotmomin, Sahiwal, Sargodha, Shahpur, Silanwali respectively. Overall, SI of district Sargodha was 28.3. Similarly, out of 5508 inspected container, 673 container was found positive for mosquito larvae and adults. The tehsil container index (TCI) was 7.9, 7.8, 12, 19.8, 14.4, 12.5 and 11.3 for tehsil Bhalwal, Kotmomin, Sahiwal, Sargodha, Shahpur, Silanwali, respectively with an overall TCI of 12.2. Largely, the percentage of Aedes spp, Culex spp. and Anopheles spp was 79, 20 and 0.4 % respectively, in the collected population (Table 2). Discarded tires, flower pots, plastic container, air cooler, water storage tanks and mud pots showed 35.5, 1.6, 6.1, 14.5, 18.1 and 7.6 % positive container for mosquito (larvae and adult) respectively. Highest positive containers were discarded tires. Overall, 12.2 % of containers were found positive for mosquito population Figure 1. Larval and adult population of Aedes mosquito started increasing after June 2021. The population gradually increased from July, August, September, October and November 2021 in almost all tehsils of district Sargodha. Maximum number of larvae and adults were recorded from urban area of tehsil Sargodha, district Sargodha (SgdU) followed by Silanwali, rural area of tehsil Sargodha (SgdR), Shahpur, Sahiwal, Bhalwal and Kotmomin Figure 2 and Figure 3. Detailed population of Aedes larvae and adults from different tehsils at union council level was presented in supplementary Figure 1 to Figure 7.
Detection of arbovirus from mosquito adults
The result of arbovirus detection from mosquito (cDNA) collected from the 186 Union Councils showed the PCR product of 296bp amplified with a primer of dengue virus. These results clearly indicated that the five union councils, one UC from each tehsil Silanwali (UC No 118), Sahiwal (UC No. 142), Shahpur (UC No. 157) and two UC from Sargodha (UC No. 18 and 67), were positive for dengue virus Figure 4. Arboviruses, Flavivviruses, were found in 5 pools (almost 3 %) of the samples, while, other viruses such as Alphaviruses and Bunyaviruses were not found in any of the samples. It is likely that this family of arboviruses is very common in the mosquito population
Mosquitoes are vector of various diseases. Dengue fever has the highest death rate than malaria (Chaiphongpachara et al. 2019). Sargodha, the province of Punjab, is one the populated districts of Pakistan with a population of 3.7 million. The current study aimed at recording the breeding sites and population of dengue fever mosquito (Aedes spp.) from district Sargodha across three consecutive seasons (from summer, monsoon and winter season) and detecting the arbovirus responsible for dengue fever. The larvae and adults were recorded from all tehsils of district Sargodha. Tehsil Shahpur exhibited the highest number of mosquito infested sites as compared to tehsil Sargodha. While, the earlier mentioned tehsil is less populated than Sargodha. The increased infested sites could be due to the lack of mosquito control practices in less populated or neglected areas by district council. However, the urban area of tehsil Sargodha showed the highest number of mosquito infested container. It is likely that the more mosquito breeding containers including tires and water storage tanks are discarded or used in the populated areas. Hence, more mosquito infested containers are present in populated areas (Paul et al. 2018). Tires are commonly found in outdoor areas where rainwater remains stagnant, making them ideal for Aedes oviposition. Overall, tires and water storage tanks were found as favorite breeding place for dengue fever mosquito (Ulibarri et al. 2016). Conclusively, dengue fever mosquito has established its population in almost all tehsils of district Sargodha. In addition to Aedes, the larvae of other medically important mosquito including Culex and Anopheles were recorded from the breeding sites of dengue fever mosquito. Dengue fever mosquitoes breed in fresh or rain water, while, other mosquitoes such as malarial mosquitoes (Anopheles) breed in puddles (Mattah et al. 2017) and west nile fever (Culex) preferable breed in sewage water (Calhoun et al. 2007). The highest population of Culex mosquito was found in the highly populated tehsil of district Sargodha. The increased population of Culex could be linked to urbanization. Similarly, the population of malarial mosquito was recorded either from tehsil Bhalwal or Sargodha. Generally, the population of west nile fever and malarial mosquito was very less in the breeding places of dengue fever mosquito. The population gradually increased from July and reached to maximum in November. In Sargodha, monsoon season sets in July and temperature starts decreasing from July to December. It is already known that the population of mosquito is weather dependent. Rainfall provides water to breeding places while, temperature is important for life cycle (Polwiang 2015). Mosquitoes, Ae. aegypti and Ae. albopictus, have ability to transmit virus when incubated at 28°C (Chouin-Carneiro et al. 2020). Taken together, the data suggest that man-made habitats in the urban and rural areas lead to the development of mosquito breeding places.
The results of arbovirus detection from adult mosquitoes collected from different tehsils of district Sargodha exhibited the presence of virus, Flaviviruses, from five pools of the samples, whiles, viruses from other family such as Alphaviruses and Bunyaviruses were not found. It is likely that this family of arboviruses is very common in the mosquito population. These results correlate with the previous study (Weller et al. 2014; Bisimwa et al. 2016). It is worth mentioning that arbovirus was not detected in all mosquito populations collected from different tehsils and UC. It is suggested to detect or monitor the presence of dengue fever virus in the local mosquito population before the fogging of chemical insecticides in a spot, positive for Aedes mosquito to avoid harmful impact on environment and non-target organism. The dengue fever cases are likely to increase in the district if the dengue fever virus is not monitored in the local population of Aedes mosquito.
A total of 1006 sites were surveyed for mosquito collection from district Sargodha. Highest positive containers for Aedes mosquitoes were discarded tires. Maximum number of larvae and adults were recorded from urban area of tehsil Sargodha, district Sargodha (SgdU) Arboviruses, Flavivviruses, were found in 5 pools of the samples. Man-made habitats are the main contributors to dengue fever mosquito population. There is a dire need of proper monitoring of dengue virus in the local mosquito population and environment management to reduce the breeding places of mosquito, hence, to control the dengue fever mosquito.
Acknowledgement
I, Dr. M. Asam Riaz, am very thankful to Higher Education Commission (HEC) of Pakistan for providing funding for this project (No. 20-11326/NRPU/R&D/HEC/2020). We are grateful to Dr. Abu Bakar Muhammad Raza for his valuable comments and suggestions.
Author Contribution Statement
MAR contributed to study design, conceived and coordinated the study, participated in sample preparation and data analysis and wrote the manuscript. IS, MBB, ZA and SI performed mosquito sampling and statistical analysis and help to draft the manuscript. MIU contributed to data analysis and help to draft the manuscript. MZM and MA contributed to sample preparation, data analysis and helped to draft the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by Higher Education Commission (HEC) of Pakistan grant number 20-11326/NRPU/R&D/HEC/2020.
Competing interests
The authors have no relevant financial or non-financial interests to disclose.
Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request
Ethical Approval Statement
The data is not collected from human. Research Ethics Committee has confirmed that no ethical approval is required.
Consent to participate
Not applicable
Consent to publish
Not applicable
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Table 1: Sequence of primer with targeted gene or protein of arbovirus (Eastwood et al. 2017).
|
Virus |
Target gene/ protein |
Primer |
Sequence (5⸍----3⸍) |
Cycling conditions |
|||
|
Alphavirus
|
N-Protein |
BCS82C
BCS332V |
ATC ACT GAG TTG GAG TTT CAT GAT GTC GCTGT TCC TGT TGC CAG GAA AAT |
95ºC (10 min) |
95ºC (30 sec) 49ºC (30 sec) 72ºC (30 sec) For 35 cycles |
72ºC (7 min) |
4ºC (Hold) |
|
Flavivirus |
NS5 |
FU1
CFD2 |
TAC AAC ATG ATG GGA AAG AGA GAG AA GTG TCC CAG CCG GCG GTG TCA TTCA GC |
95ºC (10 min) |
95ºC (30 sec) 55ºC (30 sec) 68ºC (30 sec) For 35 cycles |
72ºC (10 min) |
4ºC (Hold) |
|
Bunyavirus |
NSP4 |
VIR2052F
VIR2052R |
TGG CGC TAT GAT GAA ATC TGG AAT GTT TAC GAT GTT GTC GTC GCC GAT GAA |
||||
Table 2: Population of mosquito found in different containers from six tehsils of district Sargodha.
|
Sr. No. |
Tehsil |
Bhalwal |
Kotmomin |
Sahiwal |
SgdR* |
SgdU** |
Shahpur |
Silanwali |
Total |
|
1 |
No. of inspected sites |
94 |
106 |
115 |
247 |
212 |
116 |
116 |
1006 |
|
2 |
No. of infested sites |
22 |
22 |
52 |
42 |
46 |
77 |
24 |
285 |
|
|
Site Index (SI) |
23.4 |
20.8 |
45.2 |
17.0 |
21.7 |
66.4 |
20.7 |
28.3 |
|
|
|
|
|
|
|
|
|
|
|
|
3 |
No. of inspected container |
784 |
731 |
852 |
731 |
794 |
821 |
795 |
5508 |
|
4 |
No. of infested container |
62 |
57 |
102 |
145 |
114 |
103 |
90 |
673 |
|
|
Tehsil Container Index (TCI) |
7.9 |
7.8 |
12.0 |
19.8 |
14.4 |
12.5 |
11.3 |
12.2 |
|
|
|
|
|
|
|
|
|
|
|
|
5 |
Total No. of mosquito |
506 |
385 |
875 |
1508 |
2333 |
1827 |
812 |
8246 |
|
|
Aedes spp. |
356 |
258 |
785 |
945 |
1876 |
1543 |
756 |
6519 (79%) |
|
|
Culex spp. |
124 |
127 |
90 |
557 |
449 |
284 |
56 |
1687 (20%) |
|
|
Anopheles spp. |
26 |
0 |
0 |
6 |
8 |
0 |
0 |
40 (0.4%) |
*SgdR = Rural area of tehsil Sargodha, **SgdU = Urban area of tehsil Sargodha
No competing interests reported.