Background: Dengue fever has been responsible for around 12 countrywide large outbreaks in Pakistan, resulting in 286,262 morbidities and 1,108 deaths. Khyber Pakhtunkhwa (KP) is the most recently impacted province. This study aimed to investigate the molecular, epidemiological, and potential elements that contribute to increasing dengue transmission patterns, and knowledge, attitude, and practice (KAP) toward dengue in KP province.Method: This cross-sectional community-based study was conducted (June-December, 2021) in two phases. Phase I involved the epidemiological (n = 5,242) and molecular analysis of DENV in 500 randomly collected blood samples of the 2021 dengue outbreak in KP. Phase II focused on assessing dengue-KAP levels in healthy communities (n = 14,745, aged >18 years), adopting a cross-sectional clustered multistage sampling in eight districts (dengue-hotspot vs. non-hotspot) of KP. Chi-square tests and logistic regression analysis were applied.Results: Peshawar district had the highest dengue cases (60.0%) associated with the predominant co-circulation of DENV-2 (45.8%) and DENV-3 (50.4%) serotypes. A rise in cases was reported in October (41.8%) followed by September (27.9%) and August (14.4%; p < 0.001). Males (63.7%, p < 0.001) and individuals aged 16–30 years (37.0%, p < 0.001) were highly affected. General workers (18.0%), families with a monthly income of 10,000–20,000 Pak rupees (50.5%), unmarried (71.0%), uneducated (31%), families with higher human density (>10 individuals per household), and those (29.0%) who faced power outages for more than 7/24 h were the most affected. Moreover, co-morbidities like renal failure and bronchial asthma were associated with disease severity. A community survey on KAP revealed that an average of 74, 60, and 43% of the participants demonstrated good knowledge, attitudes, and dengue preventive practices, respectively.Conclusion: Multiple poor socioeconomic elements are influencing dengue fever transmission in the province. Higher KAP levels may explain the low frequency of dengue in non-hotspot districts. Our study emphasizes the need for effective and long-term public health education, strengthened vector surveillance, and expanded laboratory capacity for better diagnosis and management of dengue cases to better predict the burden and seasonality of disease in the country.

Mass egg production is an important component of Aedes albopictus mosquito control programs, such as the sterile insect technique and incompatible insect technique, which requires the releases of large number of sterile males. Developing standard operating procedures and optimized cages for adult maintenance of Ae. albopictus can improve the mass rearing efficiency. Three different sex ratios of females to males with a total number of 4,000 mosquitoes were tested by evaluating the insemination rate, egg production (total number of eggs per cage), female fecundity and egg hatch rate in small cage (30 × 30 × 30 cm). Blood meals with adenosine triphosphate (ATP, 0.05 g/ml), cage structures (Big cage A: 90 × 30 × 30 cm; Big cage B: 90 × 30 × 50 cm or 90 × 50 × 30 cm) and rearing densities (12,000, 16,000 and 20,000 mosquitoes, corresponding to 0.9 cm2/mosquito, 0.675 cm2/mosquito and 0.54 cm2/mosquito, respectively) were also tested and evaluated on the basis of egg production, female fecundity and egg hatch rate. An adult rearing unit holding 15 of Big cage A with optimal egg production was designed to produce 10 million eggs per rearing cycle in a 1.8 m2 space. Female to male ratios at 3:1 in small cages resulted in higher egg production but did not affect insemination rate, female fecundity and egg hatch rate. A concentration of 0.05 g/ml of ATP added to blood meals improved the blood-feeding frequency and thus increased the overall egg production per cage. Cage structures affected the egg production per cage, but not egg hatch rate. A medium rearing density at 0.675 cm2/mosquito (16,000 mosquitoes) resulted in higher egg production compared to both low and high densities. An adult rearing unit for Ae. albopictus on the basis of Big cage A has been developed with the capacity of producing 10 million eggs within 15 days. Our results have indicated that the adult rearing methods and adult maintenance unit are recommended for Ae. albopictus mass rearing in support of the establishment of a medium-sized mosquito factory.

Standardized larval rearing units for mosquito production are essential for the establishment of a mass-rearing facility. Two larval rearing units, developed respectively by the Guangzhou Wolbaki Biotech Co. Ltd. (Wolbaki) and Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture (FAO/IAEA-IPCL), are tested to assess their potential uses to mass-rear the larval stages of Aedes albopictus in support of the establishment of a medium-scale mosquito facility for the application of mosquito genetic control strategies. The triple Wolbachia-infected Ae. albopictus strain (HC strain) was used in this study. The effects of larval densities of two larval rearing trays (corresponding to 2.4, 3.0 and 3.6 larvae/cm2) and tray size/position (top, middle and bottom layers) on the pupae production and larval survival were assessed when trays were stacked within the larval rearing units. The male pupae production, female pupae contamination after sex separation, and male mating competitiveness were also studied by using both larval rearing units in their entirety. The optimal larval rearing density for Wolbaki-tray (Wol-tray) was 6,600 larvae (equal to 3.0 larvae/cm2) and 18,000 larvae (3.6 larvae/cm2) for the FAO/IAEA-IPCL tray (IAEA-tray). No significant difference in pupae production was observed when trays were stacked within top, middle or bottom layers for both units. At thirty-four hours after the first pupation, the average male pupae production was (0.89 × 105) for the Wol-unit and (3.16 × 105) for the IAEA-unit. No significant difference was observed in female pupae contamination between these two units. The HC males showed equal male mating competitiveness to wild type males for mating with wild type females in large cages, regardless of whether they were reared in the Wol-unit or IAEA-unit. The current study has indicated that both the Wol-unit and IAEA-unit are suitable for larvae mass-rearing for Ae. albopictus. However, the IAEA-unit, with higher male production and less space required compared to the Wol-unit, is recommended to be used in support of the establishment of a medium-sized mosquito facility.

Combined incompatible and sterile insect technique (IIT-SIT) has been considered to be an effective and safe approach to control mosquito populations. Immobilization of male adults by chilling is a crucial process required for the packing, transportation and release of the mosquitoes during the implementation of IIT-SIT for mosquito control. In this study, effects of chilling on the Aedes albopictus males with triple Wolbachia infections (HC line), a powerful weapon to fight against the wild type Ae. albopictus population via IIT-SIT, were evaluated under both laboratory and field conditions. Irradiated HC (IHC) males were exposed to 1, 5 and 10°C for 1, 2, 3, 6 and 24 h. The survival rate of the post-chilled IHC males was then monitored. Longevity of post-chilled IHC males was compared to non-chilled males under laboratory and semi-field conditions. Mating competitiveness of IHC/HC males after exposure to 5 or 10°C for 0, 3 and 24 h was then evaluated. Effects of compaction and transportation under chilled conditions on the survival rate of IHC males were also monitored. The optimal chilling conditions for handling IHC males were temperatures between 5 and 10°C for a duration of less than 3 h with no negative impacts on survival rate, longevity and mating competitiveness when compared to non-chilled males. However, the overall quality of post-chilled IHC/HC males decreased when exposed to low temperatures for 24 h. Reduced survival was observed when IHC males were stored at 5°C under a compaction height of 8 cm. Transportation with chilling temperatures fluctuating from 8 to 12°C has no negative impact on the survival of IHC males. This study identified the optimal chilling temperature and duration for the handling and transportation of Ae. albopictus IHC male adults without any detrimental effect on their survival, longevity and mating competitiveness. Further studies are required to develop drone release systems specific for chilled mosquitoes to improve release efficiency, as well as to compare the population suppression efficiency between release of post-chilled and non-chilled males in the field. Combining the incompatible insect technique (IIT) and sterile insect technique (SIT) is considered to be a promising approach to control Aedes albopictus, the world’s most invasive mosquito species which can transmit dengue and zika. The combined IIT-SIT technique depends on the release of overflooding numbers of mass-reared male mosquitoes to sterilize female mosquitoes through mating, resulting in the reduction of the wild mosquito population. In existing SIT programs against agricultural pests, male insects are often immobilized by chilling to pack, transport and release them and thereby increase handling efficacy. Chilling for the immobilization of male mosquitoes may also improve program efficiency in the IIT-SIT against mosquitoes. Thus, it is essential to ascertain the optimal chilling conditions for handling Ae. albopictus male mosquitoes. In this study, we found that Ae. albopictus males could be chilled at 10°C for 3 h without negative impacts on the biological qualities such as survival, longevity and mating competitiveness. Our results show that male mosquitoes could preserve a high level of quality under optimal chilling conditions, which indicates that immobilization by chilling is an important element for the combined IIT-SIT for mosquito control.