Malaria is a parasitic infection transmitted by Anopheles mosquitoes, affecting approximately 249 million individuals worldwide and leading to over 608,000 deaths per year [1]. India is making significant progress toward malaria elimination, having exited the WHO's  High Burden to High Impact (HBHI) group in 2024 with goal is to achieve zero indigenous cases by 2027[2]. The spread of vector-borne diseases is influenced by various demographic, environmental, and social factors, including global travel, unplanned urbanization, climate change, and the adaptation of vectors. [3]. Understanding the spatial distribution of disease vectors, knowledge about the mosquito species and their bionomics, size and peaks of activities, seasonal changes in vector population, etc. are very important aspects in planning effective intervention and prevention strategies [4]. The Anopheles genus is the primary vector for malaria transmission in India, An. culicifacies, An. stephensi, An. fluviatilis accounting for 60-70% of all reported malaria cases annually in the country [5]. A species complex is a well-defined taxonomic group comprising morphologically identical and closely related species. Beyond the variations in vector status among these species, sibling species exhibit significant differences in their geographical distributions [6]. An. culicifacies stands out as a notable species complex, consisting of five isomorphic species (A, B, C, D, and E) that, while challenging to differentiate morphologically, show distinct behavioral characteristics. Each sibling species possesses unique qualities regarding vector capacity, biting preferences, and vulnerability to malaria parasites [7]. The presence of multiple sibling species contributes to challenges in vector control because different species can develop varying levels of resistance to insecticides [8]. Continuous observation of vector populations, their vulnerability to insecticides, and the extent of intervention coverage is essential for efficient and sustainable malaria management [9]. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are proven malaria vector control measures implemented in India. However, the emergence of insecticide resistance presents significant challenges for the effectiveness of these vector control strategies.  [10].

In this context, study was planned with mentioned objectives:

1. To generate data on resting and feeding habits of An. culicifacies in areas with IRS/LLIN.
2. To identify the sibling species composition and the vectors susceptibility to insecticides.
3. Stratification of the areas based on distribution of the prevalent vector species and epidemiological indices for suggestive appropriate situation specific vector control strategy.

Methodology:

Study site and population:

Based on the malaria incidence six villages of two PHCs in each district Surendranagar and Dahod included in the study. Surendranagar district (22.730 N 71.510 E) is canal irrigated area of Saurashtra, Gujarat. Three villages, namely Navalgarh, Pratappur, Rajchardi of Methan PHC and Ramparda, Davapara and Tidana villages of Vagheriya PHC in Surendranagar were selected. Dahod district (20.50 “2” N 740 15’ 28’’ E) is forested area with more than 75 % tribal population. Dahod is adjacent to Rajasthan and Madhya Pardesh states on its Eastern border.  Three villages Dungra, Doki, Khurad (PHC: Rentiya) and Dageriya, Dharadungar and Inami villages (PHC: Mirakhedi) were included in the study (Figure.1). In both the districts IRS and LLINs strategy were used for vector control. In Surendranagar district, all the three villages of Methan PHC were covered by IRS and LLINs both interventions. Vaghedya PHC of Surendranagar district, Ramparda village covered by IRS not by LLINs. Tidana village covered by only LLINs not by IRS intervention. In Dahod district, Doki village covered by only IRS.  In daharadungar village LLINs were distributed.  Dungra, Khurod, Dageriya and inami village were covered by both LLINs and IRS intervention. The flow chart of overall study is shown in Fig.2.

Figure.1: Study area, district wise in MAP of Gujarat

Figure.2.0: Flow Chart of Overall Study

Sampling of Mosquito Population

The indoor and outdoor resting, bait, and trap collections were conducted monthly in each district across sentinel villages, adhering to the established WHO protocols. In each village, we strategically selected 4 human dwellings (HD) and 4 cattle sheds (CS) for indoor resting mosquito collections. 

Indoor resting mosquito collection from human dwellings (by handcatch or Mechanized aspirtor)

Adult female Anopheles mosquitoes were successfully collected from indoor locations. Sampling took place in four fixed human dwellings, utilizing flashlights and mouth aspirators during the early morning hours from 0600 to 0800 hrs, with each collection lasting 15 minutes. Collections made from four randomly selected human dwellings. Following the initial hand captures, a pyrethrum spray collection (PSC) was conducted in the same rooms. The mosquitoes obtained through hand catching were stored in paper cups, while those collected via PSC were preserved in Petri dishes lined with wet cotton and filter paper. and transported to the laboratory for processing The total number of mosquitoes/species resting per structure were calculated as density (per man per hour density for HC) or numbers of mosquitoes/room (PSC).

Cattle shed Collections dwellings (by handcatch or Mechanised aspirtor)

Mosquitoes resting in four fixed and four random cattle sheds (from the roof and walls) were collected. The total number of mosquitoes/species resting per structure were calculated as density (per man per hour density). 

Outdoor resting mosquito collection (Hand catch method or mechanized aspirator)

Collections were made in possible mosquito resting places outdoors in village such temples, underneath culverts, abandoned houses and structures, bushes were searched for outdoor resting malaria vectors. The mosquito collection from above mentioned places carried out with utilizing flashlights and mouth aspirators during the early morning hours from 0600 to 0800 hrs, The female’s mosquito caught from different habitats were kept in separate paper cups, marked with habitat code and transported safely to the laboratory for processing. The number of mosquitoes/species calculated as the per man hour outdoor mosquito density (nos. Mosquito/ man/ hour).

Light-trap collection

Light-traps collection from indoors and outdoors were done once in each village at monthly interval. One battery operated CDC light trap was hung 1.8 meter above the floor, where people rest and spend time, or in the open spaces away from the habitation in the village. One trap was kept indoor and another outdoors. Trapped mosquitoes targeted during an interval from 6:00 PM to 6:00 AM   Mosquitoes density were calculated as nos. of mosquitoes/light trap.

Blood meal analysis:

Blood meals from An. culicifacies were collected on Whatman no. 1 filter paper, enabling PCR methods to accurately determine the human blood index and assess feeding preferences.

Human landing collection (HLC)

Conducting an overnight collection of mosquitoes by utilizing a human volunteer(mosquitoes landing on human bait), Indoor and outdoor were carried from dusk to dawn (18:00 to 06:00 h). Monthly human landing collection were done in each district indoors and outdoors.

A systematic approach was employed to collect mosquitoes immediately upon landing on the host, thereby minimizing the likelihood of bites on volunteers. Each hour of collection utilized a paper cup secured with netting to ensure effective capture. The collected mosquitoes were promptly transported to the field laboratory for species identification and vector analysis. All female An. culicifacies specimens were processed for parity, with their heads and thoraxes preserved for subsequent PCR assays.

Processing of mosquito samples

Anopheline mosquitoes were identified to species using a standard identification key based on their morphological characteristics (11-12). The physiological conditions of their stomachs were clearly classified into categories: unfed (UF), fully fed (FF), half gravid (HG), and gravid (G). Blood meals from An. culicifacies were expertly collected on Whatman no. 1 filter paper for host source analysis. PCR methods were used to accurately determine the feeding preferences (the Human Blood Index).

Sibling species identification

Morphologically identified specimens stored in isopropanol were processed to identify the An. culicifacies sibling species (13). Five set of PCR primers were used for sibling species identification. PCR conditions were: one cycle of denaturation at 95°C for 5 min followed by 35 cycles of each of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 60 s, and final extension at 72°C for 7 min. The product was electrophoresed on 2.5% agarose gel to detect the diagnostic bands to identify the species.

Wild caught An. culicifacies were collected in the morning hours from cattle sheds, human dwellings and outdoor shelters by hand catch method. Ovaries pooled from half gravid females and preserved in isopropanol. Sibling species identified and proportion of each sub-species determined.

 Susceptibility status of vectors to different insecticides

The susceptibility of An. culicifacies to pyrethroids was assessed using the standard WHO method (14). Field-collected mixed age population of An. culicifacies mosquitoes from an unsprayed village were exposed to WHO impregnated papers with malathion, deltamethrin, and alpha-cypermethrin. Each insecticide was tested with a minimum of 100 mosquitoes (25 per replicate), alongside a control group of 50 mosquitoes (25 per replicate). Tests were conducted in the NIMR field laboratory at 27±2ºC and 60–70% humidity. Mortality was recorded 24 hours post-exposure. If control mortality was between 5% and 20%, Abbott's formula was applied to correct the mortality values. (15).

Vector incrimination

The head and thorax of An. culicifacies were effectively utilized to detect sporozoites through robust PCR-based methods (16). The specimens were rigorously tested for species-specific circumsporozoite antigens (CSP) of Pf, Pv210, and Pv247, following the well-established protocol outlined by Akhatar et al. (16). 

Stratification of the areas based on distribution of the prevalent vector species and epidemiological indices for suggestive appropriate situation specific vector control strategy.

WHO insecticide susceptibility assay was conducted to access the insecticide susceptibility towards An. culicifacies. LLINs were distributed by Govt of Gujarat in venerable population. Immature and important Vector were collected form the study site.

Results:

Cattle shed, (CS), Human dwelling (HD), pyrethrum spray sheet collection (PSC) and Outdoor collection (OD):  

Month wise, Cattle shed (CS), Human dwelling (HD), pyrethrum spray sheet collection (PSC) and Outdoor collection (OD) densities were shown in Dahod district Table.1 and Fig.3 and Surendranagar Table.2 & Fig.4. In all the collection, Cattle shed, (CS), Human dwelling (HD), pyrethrum spray sheet collection (PSC) and Outdoor collection (OD) shown higher densities in post monsoon (August to Sept month) and in the month of March and April. In the cattle shed collection highest densities were observed as compare to Human dwelling (HD), pyrethrum spray sheet collection (PSC) and Outdoor collection (OD). Lowest density was observed in the Outdoor collection. The vector density was higher in the pyrethrum spray sheet collection (PSC) as compare the Human dwelling (HD).  During Monsoon, season more breeding site were generated. Vector densities were increased after monsoon season.

Table 1: Cattle shed, (CS), Human dwelling (HD), pyrethrum spray sheet collection (PSC) and Outdoor collection (OD) in Dahod: 

Fig 3: CS, HD, PSC and Outdoor collection in Dahod

Table 2: Cattle shed, (CS), Human dwelling (HD), pyrethrum spray sheet collection (PSC) and Outdoor collection (OD) in Surendranagar: 

Fig 4: CS, HD, PSC and Outdoor collection in Surendranagar

Light Trap Collection Indoor & Outdoor:

The light traps were fixed indoor and outdoor of the houses. Month wise collection was made from each district.  An. culicifacies were observed in high density in the month of August and Sept in in Dahod Table.3 and Fig.4.  In Surendranagar district, high density An. culicifacies were observed during Feb, August and Sept Table.4 and Fig.6.  Both the district, number of Anopheles culicifacies was higher indoor as compare to outdoor collection. Mosquitoes were collected once in the morning to determine the proportion of species in the area, measured as the number of mosquitoes per trap-night (no. mosquitoes/trap).

Table 3. Light Trap Collection Indoor & Outdoor Dahod :

Fig. 5. Light Trap Collection Indoor & Outdoor Dahod:

Table 4. Light trap colletion Indoor & Outdoor

Fig.6.0.  Light trap colletion Indoor & Outdoor Surendranagar:

Species Composition:

In Dahod district, during indoor resting collection, 6 anopheline species were collected namely An. culicifacies, An. stephensi, An. fluviatilis, An. subpictus, An. annularis and An. tessellatus.  Culex was also collected. Indoor collection, An. culicifacies is the primary vector.   Indoor collection, An. culicifacies is the highest number and followed by An. subpictus and An. annularis. In Surendranagar, during indoor resting collection, 5 anopheline species were collected namely An. culicifacies,  An. stephensi, An. subpictus, An. annularis and An. fluviatillis. (Table 5). An. culicifacies density were high in tribal and rural villages of Dahod district as compare the Surendranagar. An. stephensi is an urban malaria vector but still found in higher density of tribal and rural area of  both the districts. An. stephensi density were 10 time higher in Surendranagar as compare the Dahod district.  In the outdoor collection, An. annularis was the dominant species among anopheline followed by An. subpictus and An. culicifacies.  Outdoor collection, only Anopheline species and Culex were found.  In outdoor collection, Anopheline density were higher in the Surendranagar district as compare the Dahod district.  Outdoor collection was shown in Table.6.

Table 5. Indoor Mosquito species composition

Table 6. Outdoor Mosquito species composition

Both the districts,  parity status were shown in Fig.7. Most of the vectors found Monoparous in the transmission season. The parous rate of An. culicifacies was nearly 47.46% in Dahod district which was high in transmission season (July-Oct). It was low in summer season. This coincides with the malaria cases reported in the districts. The parous rate of An. culicifacies in both the districts varied among months ranging from 14% in Feb to 47% in August. Most of the mosquitoes were found as nulli parous followed by monoparous. Di and Tri parous were absent.

Fig 7. Parity status of An. culicifacies in Dahod & Surendranagar District

Human Landing Collection:

Mosquitoes were collected throughout the night, on human bait (volunteers)carried out from dusk to dawn. (1800 hrs to 0600 hrs) Landing collection was made hourly. Attempt made to collect mosquitoes as soon as they land on the host to prevent  the actual biting. The biting rate of An. culicifacies was highest in the time of retreating monsoon (Aug to Oct) period. An. culicifacies was captured in all the month indoor and outdoor collection. Anopheles culicifacies density shown as  per bait/night.  Month wise Human landing collection of Dahod & Surendranagar were shown in  Fig.8 & 9.

Fig.8 Biting rate of An.culicifacies in Dahod

An. culicifacies were biting mainly two times (1) 20:00 to 21:00 pm and (2) 2:00 am to 4:00am In Indoor and Outdoor HLC. LLIN can be used for protection whole night.

Fig.9 Biting rate of An.culicifacies in Surendranagar

An. culicifacies Gonotrophic Condition:

The analysis of physiological condition of abdomen of An. culicifacies were shown in Dahod & Surendranagar district Figure. 10 & 11.  In the monsoon and post monsoon season higher proportion of freshly fed (FF) as compare to half-gravid (HG) and gravid (G) were observed.

Fig. 10.0. An. culicifacies Gonotrophic Condition of Dahod

During monsoon season most of the new breeding sites were appeared . Mosquitoes density were increased due to monsson / post monsoon season. These increased mosquitoes density were taken the blood from 8-9 pm and 2-4 am. These blood fed mosquitoes were collected in dark hours.  

Fig 11. An. culicifacies Gonotrophic Condition of Surendranagar

UF:Unfed, FF: Freshly Fed, HG: Half Gravid, G: Gravid