The Assessment of Morphometric Variation of Aedes aegypti Larvae with the Seasonal Environment Temperature Inversion in Selected Areas of Lahore, Pakistan

Mosquitoes are an important group of insects because they spread pathogens that cause disease in human and animal (Giesen et al., 2020). The primary vector of various human arboviral diseases, including dengue, Zika, and chikungunya fever, is a mosquito of the genus Aedes, namely Aedes aegypti (Rocklov and Dubrow, 2020).

As a poikilotherm, the internal temperature of insects, including mosquitoes, fluctuates and rely on the temperature of the nearby environment (Reinhold et al. 2018). Temperature changes affect physiology, behaviour, ecology, and insect survival (Wimalasiri-Yapa et al. 2021). Because of seasonal thermal fluctuations, insect development faces the risks, such as desiccation, metabolic changes, and loss of mobility (Caminade et al., 2019).

The development of insects primarily depends on temperature and can be delayed or accelerated by altering the temperature (Beck-Johnson et al., 2013). The Aedes mosquitoes have 4 life stages: egg, larva, pupa and adult. The entire life cycle, from an egg to an adult, takes approximately 8-10 days. In Ae. aegypti, there is a direct association between the mosquito’s immature development stage and the temperature (Farjana et al., 2012). The development rate improved linearly with temperatures (22°C to 28°C) (Farjana et al., 2012; Barreaux et al., 2018; Sasmita et al., 2019). It is critical to follow on for about ten days the advancement from the egg to the adult stages (Bayoh and Lindsay, 2003) and Christiansen (2015) described that no adults appeared at temperatures below 18°C or above 34°C after pupation, but at 35°C, all larvae died before emergence (Christiansen-Jucht et al., 2015).

Since mosquitoes are also poikilotherms, almost all biological activity is affected by ambient environmental conditions, such as humidity and temperature (Wimalasiri-Yapa et al., 2021). Given the increasing phenomenon of climate change, it is important to understand how mosquitoes respond to changes in body characteristics and critical environmental parameters needed to predict survival rates. The present study assessed the changes in body sizes of Ae. aegypti larvae in two different seasons of the year, i.e., Southwest Monsoon (June through September) and Retreating Monsoon (October and November), to better understand the fluctuation in the development rate of Ae. Aegypti larvae in different environmental temperatures.

A total of 216 (n= 36/month) Ae. aegypti larvae were collected during the southwest monsoon (June through September) and retreating monsoon (October and November). The larvae were collected from selected areas of Wahga town and Allama Iqbal town of the district Lahore. The collected samples were then transported to the Department of Environmental Science and Engineering at the Government College University Faisalabad for further investigations. The larval samples were preserved in 70% formalin according to (Khan et al., 2018) method. The collected larvae were identified to species level with the aid of a microscope by (Rueda, 2004) identification key. For the control, larvae were reared at laboratory maintained optimum temperature and relative humidity i.e., 28±1°C and 80%±5, respectively.

After mounting with Hoyer’s medium for morphometric parameters assessment, the dead larvae were scrutinized. The head length, head width, thoracic length, thoracic width, abdominal length, abdominal width, and total length of larvae were determined with the aid of a stereomicroscope (BOECO BST-606, Germany) and method described by (Gunathilaka et al., 2019; Sutiningsih et al., 2019).

The statistical determination of the length and width of variations in body segments of the larva was analyzed with Student’s t-test using Prism v.7 (GraphPad Software, San Diego, CA, USA). The P-value (p<0.05) was considered significant.

The results showed that seasonal thermal fluctuations affected the total body size, head length and width, abdominal length and width, and thoracic length and width of Ae. aegypti larvae (Figure 1). It was found that the average head length (0.51±0.01mm) during southwest monsoon significantly increased (P ≤ 0.01) (0.62 ±0.02mm) during the retreating monsoon season (Figure 1a). Similarly, the average head width in southwest monsoon was 0.54±0.01mm, which significantly increased (P ≤ 0.05) in retreating monsoon (0.63±0.02mm) (Figure 1b). The abdominal length in southwest monsoon (3.57 ±0.07mm) got significantly increased (P ≤ 0.05) in retreating monsoon (3.91±0.09mm) (Figure 1c). At the same time the abdominal width in southwest monsoon (0.68±0.02mm) was also significantly increased (P ≤ 0.05) in retreating monsoon (0.74 ±0.02mm) (Figure 1d). Furthermore, the thoracic length significantly increased (P ≤ 0.05) during retreating monsoon from 0.83±0.01mm in southwest monsoon to 0.90±0.03mm (Figure 1e). The average thoracic width during southwest monsoon was recorded at 0.95±0.02mm which also significantly increased (P ≤ 0.05) in retreating monsoon (1.07±0.04mm) (Figure 1f). The overall size of Ae. aegypti larvae in Southwest Monsoon was recorded at 4.91±0.05mm that increased highly significantly (P≤0.001) in retreating monsoon (5.43±0.20mm) (Figure 1g). While the average overall size of the larvae under laboratory temperature were recorded as 4.343±0.40 mm. The temperature variation during Southwest Monsoon and Retreating Monsoon are shown in (Figure 2).

Changing environmental temperatures affect the development stages of mosquitoes and have a significant impact on their population dynamics (Couret and Benedict, 2014). The temperatures between 16°C-34°C are suitable for Aedes aegypti development, and at water temperatures below 8°C, the larvae become immobile and die within a few weeks (Cristophers, 1960). In Pakistan, dengue control field staff identify Aedes mosquitoes, on the bases of larval and siphon tube size. So, the information about the size is important to minimize the chances of wrong identification of the dengue vectors at the initial stage. Therefore, we conducted this study to observe the difference in body size and different body structures of Ae. aegypti larvae in two seasons, i.e., southwest monsoon (June through September) and retreating monsoon (October and November). To our knowledge, this is the first study to assess the morphometric variation of Aedes aegypti larvae with the seasonal environment temperature inversion in selected areas of Lahore, Pakistan.

Recent studies also showed that at lower temperatures, the survival of larvae is correlated with food availability in water and intraspecific density (Couret and Benedict, 2014; Couret et al., 2014). Aedes aegypti larvae with sufficient nutrient supply at cool ambient temperatures (15°C) can remain at a particular age for several months (Andrew and Bar, 2013; Brady et al., 2014; Foster and Walker, 2019).

It has been established that a temperature between 31°C-32°C is optimal for larvae to complete their development, and mortality threshold temperature ranges between 14°C and 38°C (Bar-Zeev, 1957, 1958). The survival of mosquitoes during the development stages depends on the regional temperature and their tolerance to cold and heat (Teng and Apperson, 2000).

Previously the pattern of the development time and size of the A. aegypti and A. albopictus mosquito is affected by changes in the temperature of the surrounding environment when it exceeds the lower critical development threshold (Yang et al., 2009). The findings of (Couret et al., 2014) showed that changes in the temperature could alter the stage and size of development starting from the larval stage. In a study conducted by (Farjana et al., 2012) the rate of development slowed and size increased as temperature decreased.

The study concluded that changes in meteorological conditions during the development of Aedes aegypti have a significant effect on the larval size and low temperatures of breeding water significantly increase (P≤0.05) the body size, head, thorax and abdomen of larvae. Climate change affects the immature stages of larval development. The findings might help in improving Aedes aegypti control measures and monitoring strategies by better understanding variation in larval development rates under different environmental temperatures. However, the study is limited in scope due to the smaller sample sizes and was carried out in two towns of one district only i.e., district Lahore. We suggest further studies with larger sample size and different settings across the dengue-endemic areas of the country.

The authors thank the Department of Health, Government of Punjab for the permission for and support for this worthy study.

The authors have declared no conflict of interest.