Topic: Distribution of Anopheles stephensi bioforms in selected districts of Rajasthan, India

Abstract

Introduction

Anopheles stephensi is a major urban malaria vector in Rajasthan, India, and is responsible for spreading persistent malaria throughout the year. In Rajasthan, An. stephensi is invariably distributed and has three bioforms discriminated based on the number of the ridge on the eggs viz; Type, Mysorensis, and Intermediate. The present study aimed to understand the distribution pattern of these bioforms in rural and urban setups as they also have differences in their malaria transmission capacity.

Methods

Gravid mosquitoes and the larvae were collected from different habitats of districts of Rajasthan. The gravid females An. stephensi were allowed to lay eggs. These eggs then were subjected to morphometric analysis and counted for the number of ridges for bioform identification.

Results

About 15000 ± 200 eggs were obtained from ~190 gravid An. stephensi collected from 45 localities (11 Urban & 34 Rural) of eight districts of Rajasthan. Out of which 3569 eggs were subjected to morphometric analysis. Mysorensis bioform (49.7%) was observed to have higher percent over Intermediate (25.5%) and Type (24.6%) bioforms. Mysorensis and Intermediate were found more in rural areas whereas the Type bioform dominated higher in urban areas.

Discussion

The Mysorensis bioform was found to be dominant throughout the year in all seasons in rural areas. Type bioform preferred indoor breeding places like underground tanks, cement tanks whereas other bioforms preferred outdoor breeding places. Egg size was found to be directly proportional to the number of ridges on the eggs (r = 0.55). No reproductive isolation was observed among these bioforms.

Conclusions

The Mysorensis bioform is more prominent than other bioforms. Subspecies level understanding helps to plan effective control measures according to the breeding site selection majorly by Type bioform, an efficient vector in this region.

Introduction

Malaria affects half of the world’s population and about 87 countries are at risk of infection. In 2022, approximately 249 million cases and more than six million deaths were reported [1]. According to the World Health Organization (WHO) Malaria Report 2023, the Southeast Asian Region contributes approximately 2% of the global burden of malaria, with India being the major contributor (65.2%) of this region [1]. In India, the five states i.e., Odisha, Chhattisgarh, Maharashtra West Bengal, and North-eastern states have contributed to about 85–90%, and Rajasthan accounted for 1–2% of the total malaria cases reported in India between 2012 and 2022 [2].

Anopheles is responsible for malaria in several countries across the world [1]. In India, out of 58 reported Anopheline species, only six are major vectors i.e. Anopheles culicifacies, Anopheles fluviatilis, Anopheles minimus, Anopheles baimaii, An. epiroticus and An. stephensi for malaria transmission [3,4]. Anopheles stephensi contributes to about 12% of malaria in India.

Although Anopheles culicifacies transmit malaria in rural parts of the country and contribute about 65% of cases annually and also maintain seasonality of malaria transmission and responsible for outbreaks [3]. Anopheles fluviatilis is responsible for the transmission of malaria (15%) in the plains and foothills [5]. Anopheles minimus, and An. baimaii causes malaria in the forest regions of the northeastern states of the country, and An. epiroticus transmits malaria in the Andaman and Nicobar Islands, which is a brackish water species [6]. The origin of An. stephensi Liston 1901 is Oriental region and in India, it is considered a major urban malaria vector. An. stephensi is responsible for persistent malaria transmission throughout the country except for Andaman and Nicobar Islands, however, scanty reports from North-East states of India [7]. The global presence of An. stephensi and its significant role in spreading malaria make it crucial for epidemiological study. Additionally, its surveillance and control are essential for comprehensive global disease control efforts [8,9].

Malaria vectors, other than An. stephensi have been characterized as species complexes with some morphologically indistinguishable sibling species which vary in their role in malaria transmission. Whereas An. stephensi still is not classified as sibling species but rather as bioforms. Although these are morphologically indistinguishable, there are no significant physiological and molecular distinct [6–8]. The distribution of An. stephensi is now global [10,11], and studies have shown that the species has expanded its geographic presence in various regions of the world such as Africa, the Middle East, and Sri Lanka, and has adapted to different urban environments [12,13]. In the decade between 2012 and 2023, An. stephensi invaded Djibouti (2012), Ethiopia (2016), Sudan (2016), Sri Lanka (2017), Somalia (2019), Nigeria (2020), Yemen (2021), Kenya (2022), Eritrea (2022), Ghana (2023) and these invasions are potentially linked with anthropogenic activities including construction, urbanization, transportation systems, and availability of suitable habitats (S1 Fig) [1,3,8,10,11,13–17].

In the Rajasthan state of India, An. stephensi has been reported as the primary malaria vector in both urban and rural areas [18–20]. In rural areas of western Rajasthan, “Tanka” and “Beri” are water-storing underground tanks, used due to scarcity of water [21,22]. These tankas and wells provide favorable breeding and resting environmental conditions to An. stephensi which likes to breed in them throughout the year. Moreover, An. stephensi finds favorable conditions for both breeding and resting within houses, which helps to skip the exposure to extreme environmental conditions such as heat and dryness.

Some studies in the past have highlighted that An. stephensi exists in more than one biological form and referred as bio-forms, variants, eco-variants, or sub-species [13,23,24]. The existence of two bioforms i.e. “Type” and “Mysorensis” was reported by Knowles and Basu (1934) [25]. Subsequently, Ramsay and McDonald (1936) and Mulligan and Baily (1936) expressed the possibility of the existence of two bioforms [26,27]. Sweet and Rao (1937) [12] exhibited significant differences in certain egg measurements of these two bioforms. Knight et. al. (1959) listed An. stephensi variety Mysorensis in ‘Catalog of the Mosquitoes of the World’ [15,28]. Subbarao et al [24] reported another bioform of this species with an egg ridge number 13–16 and designated it as an ‘Intermediate’ bioform. In India and Iran, this species exists in three biological forms viz. ‘Type’, ‘Intermediate’, and ‘Mysorensis’ [15,21,24,28]. Among these three bioforms, the Type bioform is reported to be an efficient vector of urban malaria, whereas, Mysorensis and Intermediate are considered to be rural and poor vectors [10,29].

All three bioforms of An. stephensi can only be differentiated based on egg morphological characters i.e., the number of ridges on the egg floats as there are no other morphological characteristics based on which the bioforms can be differentiated at the adult stage. The Type bioform has the highest egg ridges 16–22, whereas, the Mysorensis and Intermediate bioforms have 10–14 and 15–16 egg ridges respectively [12]. There is no comprehensive study on An. stephensi bioforms in the past 20 years by Nagpal et.al. (2003) [30].

Considering An. stephensi is a major malaria transmitting vector, there is a need to understand its distribution pattern and occurrence of different bioforms as they are reported to have different vectorial capacity. In the past taxonomic studies based on morphological characters and geographical distribution were done by Christopher in 1933 and documented in the records of "Fauna of British India"(6,7), the ‘Taxonomic Keys for Mosquitoes’ by Puri (1931) [31] and in ‘Anophelines of India’ by Rao 1984 [6]. Thereafter, no study on bioforms of An. stephensi taxonomy is comprehensively done. Therefore, the present study aims to gather information on the distribution of An. stephensi bioforms and their association with malaria transmission, focusing on their identification and distribution pattern in urban and rural areas of Rajasthan from 2020 to 2023.

Methodology

Study area

The Indian state of Rajasthan is located (27.0238° N and 72.2179° E) in the western part of the Indian subcontinent with spread of 342,239 km², that covers about 10.4% of India’s total geographical region. Rajasthan also shares its international border with Pakistan and with other Indian states i.e. Gujarat, Madhya Pradesh, Haryana, Punjab, and Uttar Pradesh. Rajasthan has about 69% of the "Thar" desert, also known as the Great Indian Desert, which is the easternmost extension of the vast Sahara Desert (measuring 2,85,680 km²). The state’s geography includes different ecological systems, including the desert in the west, the Aravalli hills, and the sub-humid region in the east. The arid part of Rajasthan receives less than 20 cm of rainfall per annum, with high-temperature extremes ranging from 0 degrees Celsius (November-January) to 50 degrees Celsius (May-June). On the other hand, the Aravalli hills receive between 50–100 cm of rainfall per annum, with relatively lower temperature extremes [32]. These extreme environmental conditions have a direct bearing on the prevalence of mosquitoes [33].

The study was carried out in urban and rural localities of 8 districts of Rajasthan situated in the western arid region (Jalore, Jodhpur, Barmer, Bikaner, Jaisalmer, and Pali) and Aravalli hilly region (Jaipur and Udaipur) (Fig 1).

[Figure omitted. See PDF.]

Adult mosquito collection

Anopheles habitats were surveyed 2–4 km around the villages and different locations for adults and larvae in different seasons for three years (2020–2023). Adult mosquitoes were collected at dawn and dusk (6:00–8:00 a.m.; 7:00–8:00 p.m.) from cattle sheds and houses [34]. Adult mosquitoes were collected using the hand catch method with aspirators and torches in separate collection cloth cages (9 x 9 x 9 cm size), and transported to the laboratory. Species identification of Anopheles was done using standard taxonomic keys, An. stephensi were separated and transferred to labelled cloth cages (12 x 12 x 12 cm size) for ovipositioning [5–8]. Individual field-collected gravid An. stephensi were allowed to lay eggs in separate bowls. About 10–15 eggs from each bowl were randomly picked up for morphometric observations under a compound microscope (40x) and after counting their ridges the egg laying female An. stephensi was classified into different subspecies. The collection sites of the mosquitoes were enumerated such as cattle sheds and human dwellings (such as bedrooms, living rooms, and bathrooms), and spaces like under stairs both in urban and rural settings. Information on the resting surface of the adult mosquitoes like cement walls, mud walls, wooden surfaces, hanging clothes, and roofs was gathered to understand the habitat preferences across different bioforms.

Larval collection

Larvae were searched and collected from all the habitats where the potential of larvae breeding could occur such as ponds, cement tanka, underground tanks, ditches, and plastic containers in the study sites. The collected larvae were reared into adults and the species were identified using the standard identification key [5–8]. To identify the bioform in the larval habitats these collected larvae obtained from different habitats were reared to adults separately and allowed to mate in different cages and allowed to lay eggs after blood meal in the laboratory condition i.e. 27±2°C and humidity 70%± 5%. The eggs were then morphologically identified based on ridge counts. Habitat preference was estimated on the basis of the F1 generation of the larvae collected from different habitats. We presented qualitative results of all bioform breeding preference in this communication.

Eggs ridge count and morphometric analysis

For morphometric analysis, the length, breadth, and number of ridges on the egg float were measured under a compound microscope (Zeiss stem 2000-c) at 10x and 40x magnification using an occulo-meter. The occulo-meter, was calibrated against a stage micrometer at 10x by superimposing the lines of the occulo-meter with a stage micrometer (10μm each division), and the number of ridges along one side of the egg float was counted. Each egg was measured three times under microscope to avoid focal bias out of three readings the reading that appeared tice was taken as correct reading whereas measurement of 10–12 eggs of isofemales were done to observe the mean values (Fig 2). The area of an egg was also calculated to understand any relation of an area with the egg arrangement on the water surface and the number of ridges. A formula for the calculation of area of elliptical area was used [35]. Area = π x transverse radius of egg x longitudinal radius of egg which is equal to the working formula (Considering the egg as an elliptical object.) as follows:

[Figure omitted. See PDF.]

Wild gravid females were allowed to lay eggs, and subsequently, the egg ridges were counted and measured using an Occulo-meter. In this figure, 19 ridges were observed and counted under the microscope which is a ‘Type’ bioform.

Similarly, the arrangement of the eggs (ovipositional venation on the surface of the water) on the water surface was visualized first and then the number of ridge counts was done and recorded to get information related to egg arrangements and Type of bioform differentiation of different bioforms of An. stephensi.

All three bioforms of An. stephensi were categorized based on egg morphological characters i.e., the number of ridges on the egg floats. The eggs were categorized as Type bioform if egg ridges were between16-21, eggs bearing ridge count between 15–16 were categorized as Intermediate whereas, the eggs with ridge count between 10–14 considered as Mysorensis bioform respectively [12,29]. Iso-female lines of different bioforms were also reared in insectary conditions and eggs were obtained [34]. The eggs were counted and allowed to hatch and the percent hatching was calculated. Each gravid female was categorized based on the number of ridges on the eggs laid by them.

The crossbreeding experiments of bioforms, Mysorensis, and Type were conducted to understand reproductive isolation and to assess mating success in a controlled environmental condition in the laboratory at 27±2°C and humidity 70%± 5%. The mosquitoes collected from Isofemale lines of Type and Mysorensis were allowed to mate using different combinations, i.e., 4 male mysorensis and 2 female Type bioform and vice versa (4 males Type /2 females Mysorensis) (S1 Table). The experiment was conducted only once to evaluate the crossbreeding between two bioforms.

Data analysis

The distribution pattern analysis of field-collected An. stephensi was done using Microsoft Excel. The morphometric data of eggs was analyzed for length, ridges and area using one-way ANOVA (Analysis of Variance) in SPSS software (version 16) and x Chi-square and F test were used for test of significance for comparing egg morphometry of different bioforms. The map was prepared using Q GIS [36].

Ethics statement

This study was carried out as a part of an ongoing project (PHB/NIMR/EC/2020/199) with the approval of the Institute Ethics Committee ICMR- National Institute of Malaria Research, Sector 8, Dwarka, New Delhi.

Results

About 15,000 ± 200 eggs were obtained from ~362 gravid An. stephensi from 45 localities (11 Urban and 34 Rural) of 8 districts of Rajasthan, with 3569 eggs subjected to morphometric analysis. Mysorensis bioform was prominent (49.7%) followed by Intermediate (25.5%) and Type bioform (24.6%) (Table 1). The egg ridges exhibited considerable variation, encompassing a wide range extending from 9 to 23. The majority of the total egg sample (n = 3569), approximately 90%, fell within the range of 12 to 18 egg ridges very few eggs had low ridges from 9–11 and high ridges ranged from 22–23. It was observed that the egg ridges of the Mysorensis bioform were in the range between 9 and 14, whereas in the Intermediate bioform, this range was between 15 and 16, and the Type bioform has a range between 17 and 23. The most prevalent egg ridge range was found to be between 13 and 14, representing 38% of the sample, followed by 15 to 16, which accounted for 35.5% of the eggs (S2 Table).

[Figure omitted. See PDF.]

There was a distinct difference in the range of egg ridge counts between urban and rural areas in Rajasthan, with a total sample size of 3569 eggs. In urban settings, the range was notably constrained, spanning from 11 to 18 egg ridges. Conversely, in rural areas, the range extended from 9 to 23, indicating a broader variation in egg ridge counts within this context (Fig 3).

[Figure omitted. See PDF.]

As compared to Type and Intermediate bioforms, the presence of Mysorensis was significantly higher (50%) in both rural (48%) and urban (53%) settings. It was observed that An. stephensi Mysorensis (53.22%) and Intermediate (28.89%) were found more in rural areas (Fig 4). The Type bioform was significantly higher in urban areas (χ² = 47.69, p<0.00001, df = 5).

[Figure omitted. See PDF.]

The percentage of Mysorensis was more than double throughout the year except for a few months of winter (December to March) irrespective of their urban or rural habitats. In August and September, the composition reached about 85%. The increase in Type bioform composition was found to overlap with the malaria transmission season (October-December) in Rajasthan. It was also observed that the population size of Intermediate bioform was higher during January-July (Fig 5). Rainfall showed a positive correlation (r = 0.58) of a percent increase in the composition of Mysorensis. However, the correlation was not strong but the other bioforms showed a negative correlation with Intermediate (r = -0.401) and Type (r = -0.307) respectively. The population of Mysorensis was found to be significantly higher than the Type population of the region.

[Figure omitted. See PDF.]

The spikes in the graph not only explain the relationship between the mosquito composition and rainfall but also highlight the influence of the one-month lag period of rainfall on mosquito population dynamics.

F1 population obtained from the emergence of collected larvae from water bodies, house premises, and containers, indoors and outdoors, showed a preference for bioform breeding, it was observed that Type bioform preferred cemented tanks and underground tanks (Tankas) that are mostly indoors. On the other hand, bioform Mysorensis was found mostly in outdoor cattle tanks, pond ditches, outdoor cemented tanks, outdoor wells, and also in turbid water. Intermediate bioform was found in overhead tanks and cattle tanks that are outdoors similar to Mysorensis bioform (Table 2).

[Figure omitted. See PDF.]

The eggs laid on water surface were observed to be laid in some pattern. The eggs laid in a linear arrangement were from the Mysorensis and Intermediate bioforms (Fig 6A and 6B) having lower ridge counts. Whereas, the eggs with high ridge counts (Type bioform) were laid in a rosette formation (Fig 6C).

[Figure omitted. See PDF.]

In Mysorensis (A) and the Intermediate bioform (B), eggs are arranged linearly, whereas in the Type bioform (C), eggs are arranged in a rosette pattern.

The Crossbreeding experiments of bioforms Mysorensis and Type showed no reproductive isolation. The isofemale of Mysorensis laid 75.5 (average of 2 females) with variable egg ridges ranging from 14 to 18 when allowed to mate with males from Type iso-female line with 90.7% emergence and 82% survival till adult formation. Similar results were obtained when isofemale of Type bioform were allowed to mate with males obtained from the isofemale line of Mysorensis bioform (Average eggs 88.5; ridge range 13–18; and 80% pupation).

Gravid females of both bioforms (n = 20 each) were allowed to lay eggs individually, revealing that the Type bioform lays significantly more eggs (75±5.16) than the Mysorensis (65±2.79), with the egg-laying capacity of the Type bioform being significantly higher (χ² = 7.39, p≥0.025).

Average areas of eggs were compared between, bioforms, and Type was found to be significantly bigger area (p< 0.001) than other bioforms. The size of eggs was larger collected from rural areas than in urban localities although the difference was not statistically significant. This difference in size was found in all bioforms (Fig 7).

[Figure omitted. See PDF.]

On the other hand, hatchability was highest in Mysorensis bioform followed by Type (76%) and Intermediate (72%) Type, the difference was not significant (χ² = 2.64 at df = 2 for Mysorensis) (Table 3).

[Figure omitted. See PDF.]

Overall parameters among the eggs from the gravid mosquitoes collected from rural locations were larger than those collected from urban areas. The average egg length of Type bioform was larger (550.8±0.028μm) than Intermediate (533.0±0.023μm) and Mysorensis (515.0±0.025μm). This consistent trend of size differences was also observed in the surface area of the eggs, with the bioform Type demonstrating significantly larger surface areas. (Table 4).

[Figure omitted. See PDF.]

Table 4 shows the egg morphometry, including the average length, width, and area of bioforms. The average length of bioform Type was found to be significantly higher than other bioforms (F = 576.8; P≤0.001. Similarly, the mean egg areas of the bioforms also showed significant differences (F = 124.7, P≤0.001).

Discussion

Malaria is more prominent in rural areas than in urban areas of Rajasthan [37]. Mysorensis bioform is said to be prominent in rural which our study also confirms. However, some studies suggest that Mysorensis is a zoophilic and a poor vector [38]. Intermediate and Mysorensis bioforms were mainly found in rural settings in and around Delhi [23]. Our study also revealed similar findings for the distribution of Mysorensis. The Intermediate bioform was comparatively higher in the rural areas, while the Type bioform was dominant in urban areas. However, the difference was not significant, suggesting that a mixed population of bioforms occurs in both urban and rural areas depending on the availability of breeding sites. A similar distribution was observed by other studies conducted in the extreme western regions of Baluchistan, and Iran [16,39].

Anopheles stephensi, mysorensis bioform was reported to be dominant in Sri Lanka [40]. Our field collections also showed that there are seasonal variations in the distribution of different bioforms, Mysorensis bioform remained the predominant species during the post-monsoon season where outdoor breeding was predominant this also coincides with transmission season in Rajasthan. Additionally, during the monsoon and post-monsoon season (July to November), Mysorensis bioform co-exists with the seasonal An. culicifacies. All bioforms occur in both rural and urban locations of Rajasthan similar to the other studies conducted earlier [41]. Interestingly, although the breeding sites were not isolated to any specific bioform, the Mysorensis bioform was primarily found in outdoor shallow water bodies (Table 4). The selection of breeding habitat also depends upon larvivorous predators. Studies have reported that the presence of predators like Gambusia/Copepods/ Tadpoles deter oviposition by airborne mosquitoes as these predators regulate mosquito populations and even influence their oviposition and habitat-selection behavior [42].

The egg of An. stephensi is shiny jet-black in color and has a ‘skiff‐shaped’ appearance with its anterior end slightly broader than the posterior end [43]. Type bioform eggs were found significantly larger than other bioforms (Table 4). The largest average egg length was observed in a study conducted in Sri Lanka as mentioned in Table 5.

[Figure omitted. See PDF.]

The average length of Mysorensis eggs was found to be larger than the average egg length of Mysorensis studied in the rest of India, Pakistan, and Iran.

The length of eggs corresponds to the number of ridges on the eggs and showed positively correlated (r = 0.55) (Table 4) i.e., higher the ridge number means larger the length of eggs.

Type species distribution was restricted and contributed about 23.62% of composition in the overall distribution of the bioforms in Rajasthan. The percent composition of Type and Mysorensis populations was observed dynamic and kept on changing during different seasons during summers (April–June) the percent composition of Type bioform was increased in all the urban and rural settings (Fig 4). This may be attributed to the fewer outdoor breeding sites available in the dry season to Mysorensis and the Type bioform is adapted to underground tanks and indoor habitats. (Table 4)

The transmission capacity of mosquitoes is also dependent on their anthropophilic behavior and the presence of the cattle population which may provide zooprophylaxsis. A study on An. stephensi in southern Iran by Vatandoost et. al. (2006) [16] also concluded that the transmission capacity depends on the availability of cattle in the rural areas due to the presence of bioform Mysorensis which is known as zoophilic and considered as a poor vector [24]. However, in western Rajasthan rural areas, our study showed the presence of Type bioform which is considered an efficient vector due to its high anthropophilic index and high transmission rates despite the cattle population in rural areas. A recent study in Iran suggests that Mysorensis has the potential to survive long enough to be re-infected and transmit Plasmodium vivax malaria several times and has transmission capacity at par with other vector species [45]. which is evident in western Rajasthan as well where Plasmodium vivax malaria is prominent. The distribution of Type bioform in rural areas needs more investigation, especially during hot summers.

The higher percentage of hatching of Mysorensis eggs may be attributed to the high competition in outdoor breeding species than mostly container breeder-like Type bioforms more study is needed to explain the egg viability and hatching. The Type showed less hatching ability than other bioforms, despite breeding in safe places like tanks drums and underground tankas.

In our controlled laboratory study it was found that Mysorensis and Type bioform mate easily in caged conditions (under laboratory conditions of 27±2°C and humidity 70%± 5%) we put both conditions using male Mysorensis versus female Type An. stephensi (4 males ‘Mysorensis’ /2 females ‘Type’) and vice versa and found successful mating and emergence, similar findings were shown by Rutledge et al(1970) [23] who did not find any hybrid sterility in the crosses between bioforms laboratory strains derived from Iran. Our results of the cross-breeding experiment showed that there is no reproductive isolation between Anopheles stephensi bioforms however, this was observed that the F1 generation obtained by these experiments suggests that the egg ridges also change with cross-breeding i.e. if the Type female was crossed with Mysorensis male they tend to lay eggs with lower egg ridges than the Type bioform. The adults obtained from the cross did not show any sterility and reduction in egg numbers (egg laid /female) when they were allowed to mate among themselves. Our study showed Mysorensis prefers larger ponds, and wetlands of fresh water more a peridomestic habitat with open locations whereas the Type habitat is more in small cemented containers, underground tanks (tankas) and they prefer peridomestic to indoor breeding as well in coolers cattle tanks and underground tanks (Table 2). The Type was found to lay more eggs than Mysorensis which may be attributed to the indoor breeding habit where competition with other mosquito species is comparatively less and the survival of mosquitos is higher in indoor climatic conditions than those breeds outdoors. However, more studies need to be done to find the egg-laying capacity of these bioforms.

Based on egg length correlation with the number of ridges on eggs it was seen that if the length of an egg increases there is an increase in ridge count (r = 0.5570, weak correlation), in rural (r = 0.565) the correlation was lower than in urban (r = 0.6347). The high number of ridges supports larger eggs which may be similar to the larger boats requiring more water displacement for floating on water. There is a need to have an in-depth study of the environmental conditions of An. stephensi of bioforms as it is observed that there is a change in ridge numbers in laboratory conditions after a few generations of captivity this might be an environmental alteration and may be the larval food, competition, blood feeding to adult females, etc. Some molecular studies and Spiracular indices [30] as identifying tools could not find a significant difference in the bioforms although these two races of An. stephensi were given sub-species status by Puri [13] and this was accepted by Stone et al [28], which needs an in-depth study of ecological variation whether to keep species biotype or ecotype only. Studies conducted by Alam et.al. (2008) & Mishra et.al. (2021), found no significant difference between ribosomal DNA ITS2 and D-3 domains and the An. stephensi odorant binding protein (AsteObp1), in the two sub-variants of Mysorensis and Type, as observed by Gholizadeh et. al. [46–49].

Although there was no clear-cut isolation in the breeding habitats of bioforms but Type bioform was exclusively found in indoors and underground tanks called “Tankas”. They were also adapted to low light conditions, on the other hand, Mysorensis was prevalent in seepage water and distributed away from human dwellings. These breeding area selections might be responsible for the slight difference in bioform isolation and making them reproductively isolated in sympatric conditions. Further studies on niche selection and isolation of ecotypes of An. stephensi are necessary to evaluate the differential distribution of bioforms in the region. The mating experiment suggested that, given the opportunity, they can interbreed, an in-depth study on breeding isolation on these bioforms is required. Studies have also shown that the distribution of Anopheles stephensi is increasing in different continents along with all its bioforms, suggesting co-habitation of all bioforms indoor and outdoor habitats (Sri Lanka, Africa [1,3,8,10,11,13–17]. Studies on the distribution of bioforms may provide evidence of uneven malaria incidences in similar mosquitogenic systems. However, this study has certain limitations i.e. Scanning Electron Microscope for surface structure studies of eggs and molecular identification on the eggs for bioform identification was not conducted. Additionaly, the cross-breeding experiment was conducted only once.

Conclusion

Mysorensis bioform was dominantly found in both urban and rural areas. The percent composition of Type bioform was more in the rural than in the urban areas. The distribution of An. stephensi bioforms depends on the availability of preferred breeding habitat i.e. Type bioform was highly associated with indoor and large container breeding, while the Mysorensis bioform was associated with natural water bodies such as ditches and ponds.

This study revealed that bioforms are not reproductively isolated. While the number of egg ridges, the key for bioform identification, is correlated with the size of the egg. This finding can be used for the selection and implementation of control interventions efficiently. Further study is warranted to understate the epidemiological impact of different bioforms and the impact of habitat availability on the distribution of bioforms. The Type bioform of An. stephensi in both rural and urban might be playing a crucial role in the persistent transmission of malaria in the region. The findings of this study can be used by the state control program and the National Vector-borne Diseases Control Program of the country.

Supporting information

S1 Fig. The timeline of the distribution and invasion into different countries illustrates the spread of Anopheles stephensi from its native countries to various countries from 2012 in Asia, the Middle East, and Africa.

This chronology provides specific details about the expansion of Anopheles stephensi, tracing its journey from its origin and highlighting its invasion into different countries in Asia, the Middle East, and Africa.

https://doi.org/10.1371/journal.pone.0313227.s001

(DOCX)

S1 Table. Crossbreeding Experiment between Anopheles stephensi bioforms.

https://doi.org/10.1371/journal.pone.0313227.s002

(DOCX)

S2 Table. Morphometric analysis of An. stephensi bioform egg ridges from different locations.

https://doi.org/10.1371/journal.pone.0313227.s003

(DOCX)

S3 Table. Excel file of raw data of the study.

https://doi.org/10.1371/journal.pone.0313227.s004

(XLS)

Acknowledgments

The authors express their sincere gratitude to the Director of ICMR-NIMR for their unwavering support, and to the NIMR staff for their invaluable assistance during the field collections.

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10. 10. Surendran SN, Sivabalakrishnan K, Sivasingham A, Jayadas TTP, Karvannan K, Santhirasegaram S, et al. Anthropogenic factors driving recent range expansion of the malaria vector Anopheles stephensi. Front Public Health. 2019;7:53.

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* View Article

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* View Article

* Google Scholar

20. 20. Tyagi BK. Malaria in the Thar Desert: a critical review. ICMR Bull. 1995;25(9).

* View Article

* Google Scholar

21. 21. Tyagi BK, Yadav SP. Malariological and sociological significance of ‘tanka’ and ‘beri’ in the Thar Desert, Western Rajasthan, India. J Arid Environ. 1996;33(4):497–501.

* View Article

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22. 22. Singh H, Gupta SK, Vikram K, Saxena R, Sharma A. The impact of mosquito proof lids of underground tanks “tanka” on the breeding of Anopheles stephensi in a village in western Rajasthan, India. Malar J. 2021 Dec 1;20(1).

* View Article

* Google Scholar

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* View Article

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24. 24. Subbarao SK, Vasantha K, Adak T, Sharma VP, Curtis CF. Egg‐float ridge number in Anopheles stephensi: ecological variation and genetic analysis. Med Vet Entomol. 1987;1(3):265–71.

* View Article

* Google Scholar

25. 25. Knowles R, Basu BC. Mosquito Prevalence and Mosquito-borne Diseases in Calcutta City. Records of the Malaria Survey of India. 1934;4(3).

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26. 26. GC Ramsay OBE, Macdonald G. The Species Control of Anophe Lines in India. Ind Med Gaz. 1936;71(12):699. pmid:29012618

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27. 27. Mulligan HW, Baily JD. Malaria in Quetta, Baluchistan. Records of the Malaria Survey of India. 1936;6(2): 289–385.

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28. 28. Stone A, Knight KL, Starcke H. A synoptic catalog of the mosquitoes of the world (Diptera, Culicidae). A Synoptic Catalog of the Mosquitoes of the World (Díptera, Culicidae). 1959: 358-pp.

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29. 29. Hinton HE. Observations on the biology and taxonomy of the eggs of Anopheles mosquitos. Bull Entomol Res. 1968;57(4):495–508. pmid:5659158

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* Google Scholar

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* Google Scholar

31. 31. Puri IM. Larvae of anopheline mosquitoes, with full description of those of the Indian species. Larvae of Anopheline Mosquitoes, with Full Description of those of the Indian Species. 1931;(Memoir No. 21).

32. 32. Pingale SM, Khare D, Jat MK, Adamowski J. Spatial and temporal trends of mean and extreme rainfall and temperature for the 33 urban centers of the arid and semi-arid state of Rajasthan, India. Atmos Res. 2014;138:73–90.

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33. 33. Bansal SK, Singh K V. Prevalence and seasonal distribution of anopheline fauna in district Bikaner (Rajasthan). Indian J Malariol. 1993;30(3):119–25. pmid:8131880

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* Google Scholar

34. 34. World Health Organization. Manual on practical entomology in malaria. World Health Organization; 1995.

35. 35. Tiznado-Hernandez ME, Fortiz-Hernandez J, Ojeda-Contreras AJ, Rodriguez-Felix A. Use of the elliptical mathematical formula to estimate the surface area of cladodes in four varieties of Opuntia ficus-indica. Journal of the Professional Association for Cactus Development. 2010;12:98–109.

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36. 36. QGIS DT. Geographic Information System. Open Source Geospatial Foundation Project. 2018;3(4):10.

37. 37. Urban Malaria Scheme (UMS). [Internet]. [cited 2024 Sep 30] Available from: https://ncvbdc.mohfw.gov.in/index4.php?lang=1&level=0&linkid=529&lid=3822.

38. 38. Subbarao SK, Nanda N, Rahi M, Raghavendra K. Biology and bionomics of malaria vectors in India: existing information and what more needs to be known for strategizing elimination of malaria. Malar J [Internet]. 2019 [cited 2023 Nov 23];18:396. Available from: pmid:31796010

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* View Article

* Google Scholar

40. 40. Jude J, Gunathilaka N, Udayanaga L, Fernando D, Premarathne P, Wickremasinghe R, et al. Biology, bionomics and life-table studies of Anopheles stephensi (Diptera: Culicidae) in Sri Lanka and estimating the vectorial potential using mathematical approximations. Parasitol Int. 2023;93:102715.

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41. 41. Oshaghi MA, Yaaghoobi F, Vatandoost H, Abaei MR, Akbarzadeh K. Anopheles stephensi biological forms; geographical distribution and malaria transmission in malarious regions of Iran. Pakistan Journal of Biological Sciences. 2006;9(2):294–8.

* View Article

* Google Scholar

42. 42. Kumar R, Dahms H-U, Muhid P, Tseng L-C, Hwang J-S. Potential of three aquatic predators to control mosquitoes in the presence of alternative prey: a comparative experimental assessment. Mar Freshw Res. 59:817–835.

* View Article

* Google Scholar

43. 43. Malhotra PR, Jatav PC, Chauhan RS. Surface morphology of the egg of Anopheles stephensi sensu stricto (Diptera, Culicidae). Italian Journal of Zoology. 2000;67(2):147–51.

* View Article

* Google Scholar

44. 44. Afridi MK, Talibi SA, Rashid SA, Husain MZY. Identification of Races of A. stephensi prevalent in the Federal Karachi Area by Measurement of their Ova. Pak J Health. 1958;8(2):71–6.

* View Article

* Google Scholar

45. 45. Mesghali A. Detailed report on the distribution of the mosquitoes of the genus Anopheles in Iran. 1961.

* View Article

* Google Scholar

46. 46. Alam MT, Bora H, Das MK, Sharma YD. The type and mysorensis forms of the Anopheles stephensi (Diptera: Culicidae) in India exhibit identical ribosomal DNA ITS2 and domain-3 sequences. Parasitol Res. 2008;103:75–80. pmid:18309520

* View Article

* PubMed/NCBI

* Google Scholar

47. 47. Gholizadeh S, Firooziyan S, Ladonni H, Hajipirloo HM, Djadid ND, Hosseini A, et al. The Anopheles stephensi odorant binding protein 1 (AsteObp1) gene: a new molecular marker for biological forms diagnosis. Acta Trop. 2015;146:101–13. pmid:25795618

* View Article

* PubMed/NCBI

* Google Scholar

48. 48. Mishra S, Sharma G, Das MK, Pande V, Singh OP. Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vector Anopheles stephensi. PLoS One. 2021;16(6):e0253173. pmid:34125861

* View Article

* PubMed/NCBI

* Google Scholar

49. 49. Khan J, Gholizadeh S, Zhang D, Wang G, Guo Y, Zheng X, et al. Identification of a biological form in the Anopheles stephensi laboratory colony using the odorant-binding protein 1 intron I sequence. PLoS One. 2022;17(2):e0263836. pmid:35192647

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Citation: Singh S, Marwal R, Lata S, Saroha P, Gupta SK, Singh H (2025) Topic: Distribution of Anopheles stephensi bioforms in selected districts of Rajasthan, India. PLoS ONE 20(2): e0313227. https://doi.org/10.1371/journal.pone.0313227

About the Authors:

Sangeeta Singh

Contributed equally to this work with: Sangeeta Singh, Robin Marwal

Roles: Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft

Affiliations: ICMR-National Institute of Malaria Research, New Delhi, India, Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

ORICD: https://orcid.org/0009-0002-7526-6586

Robin Marwal

Contributed equally to this work with: Sangeeta Singh, Robin Marwal

Roles: Conceptualization, Data curation, Formal analysis, Methodology, Software, Writing – original draft, Writing – review & editing

Affiliation: National Centre for Diseases Control, New Delhi, India

ORICD: https://orcid.org/0000-0002-8314-1743

Suman Lata

Roles: Formal analysis, Investigation, Methodology, Resources, Writing – review & editing

Affiliations: ICMR-National Institute of Malaria Research, New Delhi, India, Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

Poonam Saroha

Roles: Data curation, Methodology, Software, Writing – review & editing

Affiliations: ICMR-National Institute of Malaria Research, New Delhi, India, Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

Sanjeev Kumar Gupta

Roles: Resources, Software, Supervision, Writing – review & editing

Affiliation: ICMR-National Institute of Malaria Research, New Delhi, India

ORICD: https://orcid.org/0000-0001-7583-2301

Himmat Singh

Roles: Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft

E-mail: [email protected]

Affiliations: ICMR-National Institute of Malaria Research, New Delhi, India, Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

ORICD: https://orcid.org/0000-0001-7682-651X

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14. Gayan Dharmasiri AG, Perera AY, Harishchandra J, Herath H, Aravindan K, Jayasooriya HTR, et al. First record of Anopheles stephensi in Sri Lanka: a potential challenge for prevention of malaria reintroduction. Malar J. 2017;16(1):1–9.

15. Knight KL. A catalog of the mosquitoes of the world (Diptera: Culicidae). Thomas Say Found. Entomol Soc Am. 1977;6:1–611.

16. Vatandoost H, Oshaghi MA, Abaie MR, Shahi M, Yaaghoobi F, Baghaii M, et al. Bionomics of Anopheles stephensi Liston in the malarious area of Hormozgan province, southern Iran, 2002. Acta Trop. 2006;97(2):196–203.

17. Vector alert: Anopheles stephensi invasion and spread in Africa and Sri Lanka [Internet]. [cited 2023 Oct 7]. Available from: https://www.who.int/publications/i/item/9789240067714.

18. Tyagi BK, Singh K V, Bansal SK, Yadav SP. Malaria epidemic in some villages of north-western desert Rajasthan. J App Zool Res. 1994;5:152–5.

19. Tyagi BK, Yadav SP. Correlation of irrigation and flood water management with malaria in the Thar desert. 1997.

20. Tyagi BK. Malaria in the Thar Desert: a critical review. ICMR Bull. 1995;25(9).

21. Tyagi BK, Yadav SP. Malariological and sociological significance of ‘tanka’ and ‘beri’ in the Thar Desert, Western Rajasthan, India. J Arid Environ. 1996;33(4):497–501.

22. Singh H, Gupta SK, Vikram K, Saxena R, Sharma A. The impact of mosquito proof lids of underground tanks “tanka” on the breeding of Anopheles stephensi in a village in western Rajasthan, India. Malar J. 2021 Dec 1;20(1).

23. Rutledge LC, Ward RA, Bickley WE. Experimental hybridization of geographic strains of Anopheles stephensi (Diptera: Culicidae). Ann Entomol Soc Am. 1970;63(4):1024–30.

24. Subbarao SK, Vasantha K, Adak T, Sharma VP, Curtis CF. Egg‐float ridge number in Anopheles stephensi: ecological variation and genetic analysis. Med Vet Entomol. 1987;1(3):265–71.

25. Knowles R, Basu BC. Mosquito Prevalence and Mosquito-borne Diseases in Calcutta City. Records of the Malaria Survey of India. 1934;4(3).

26. GC Ramsay OBE, Macdonald G. The Species Control of Anophe Lines in India. Ind Med Gaz. 1936;71(12):699. pmid:29012618

27. Mulligan HW, Baily JD. Malaria in Quetta, Baluchistan. Records of the Malaria Survey of India. 1936;6(2): 289–385.

28. Stone A, Knight KL, Starcke H. A synoptic catalog of the mosquitoes of the world (Diptera, Culicidae). A Synoptic Catalog of the Mosquitoes of the World (Díptera, Culicidae). 1959: 358-pp.

29. Hinton HE. Observations on the biology and taxonomy of the eggs of Anopheles mosquitos. Bull Entomol Res. 1968;57(4):495–508. pmid:5659158

30. Nagpal BN, Srivastava A, Kalra NL, Subbarao SK. Spiracular indices in Anopheles stephensi: a taxonomic tool to identify ecological variants. J Med Entomol. 2003;40(6):747–9. pmid:14765648

31. Puri IM. Larvae of anopheline mosquitoes, with full description of those of the Indian species. Larvae of Anopheline Mosquitoes, with Full Description of those of the Indian Species. 1931;(Memoir No. 21).

32. Pingale SM, Khare D, Jat MK, Adamowski J. Spatial and temporal trends of mean and extreme rainfall and temperature for the 33 urban centers of the arid and semi-arid state of Rajasthan, India. Atmos Res. 2014;138:73–90.

33. Bansal SK, Singh K V. Prevalence and seasonal distribution of anopheline fauna in district Bikaner (Rajasthan). Indian J Malariol. 1993;30(3):119–25. pmid:8131880

34. World Health Organization. Manual on practical entomology in malaria. World Health Organization; 1995.

35. Tiznado-Hernandez ME, Fortiz-Hernandez J, Ojeda-Contreras AJ, Rodriguez-Felix A. Use of the elliptical mathematical formula to estimate the surface area of cladodes in four varieties of Opuntia ficus-indica. Journal of the Professional Association for Cactus Development. 2010;12:98–109.

36. QGIS DT. Geographic Information System. Open Source Geospatial Foundation Project. 2018;3(4):10.

37. Urban Malaria Scheme (UMS). [Internet]. [cited 2024 Sep 30] Available from: https://ncvbdc.mohfw.gov.in/index4.php?lang=1&level=0&linkid=529&lid=3822.

38. Subbarao SK, Nanda N, Rahi M, Raghavendra K. Biology and bionomics of malaria vectors in India: existing information and what more needs to be known for strategizing elimination of malaria. Malar J [Internet]. 2019 [cited 2023 Nov 23];18:396. Available from: pmid:31796010

39. Oshaghi MA, Yaaghoobi F, Abaie MR. Pattern of mitochondrial DNA variation between and within Anopheles stephensi (Diptera: Culicidae) biological forms suggests extensive gene flow. Acta Trop. 2006;99(2–3):226–33.

40. Jude J, Gunathilaka N, Udayanaga L, Fernando D, Premarathne P, Wickremasinghe R, et al. Biology, bionomics and life-table studies of Anopheles stephensi (Diptera: Culicidae) in Sri Lanka and estimating the vectorial potential using mathematical approximations. Parasitol Int. 2023;93:102715.

41. Oshaghi MA, Yaaghoobi F, Vatandoost H, Abaei MR, Akbarzadeh K. Anopheles stephensi biological forms; geographical distribution and malaria transmission in malarious regions of Iran. Pakistan Journal of Biological Sciences. 2006;9(2):294–8.

42. Kumar R, Dahms H-U, Muhid P, Tseng L-C, Hwang J-S. Potential of three aquatic predators to control mosquitoes in the presence of alternative prey: a comparative experimental assessment. Mar Freshw Res. 59:817–835.

43. Malhotra PR, Jatav PC, Chauhan RS. Surface morphology of the egg of Anopheles stephensi sensu stricto (Diptera, Culicidae). Italian Journal of Zoology. 2000;67(2):147–51.

44. Afridi MK, Talibi SA, Rashid SA, Husain MZY. Identification of Races of A. stephensi prevalent in the Federal Karachi Area by Measurement of their Ova. Pak J Health. 1958;8(2):71–6.

45. Mesghali A. Detailed report on the distribution of the mosquitoes of the genus Anopheles in Iran. 1961.

46. Alam MT, Bora H, Das MK, Sharma YD. The type and mysorensis forms of the Anopheles stephensi (Diptera: Culicidae) in India exhibit identical ribosomal DNA ITS2 and domain-3 sequences. Parasitol Res. 2008;103:75–80. pmid:18309520

47. Gholizadeh S, Firooziyan S, Ladonni H, Hajipirloo HM, Djadid ND, Hosseini A, et al. The Anopheles stephensi odorant binding protein 1 (AsteObp1) gene: a new molecular marker for biological forms diagnosis. Acta Trop. 2015;146:101–13. pmid:25795618

48. Mishra S, Sharma G, Das MK, Pande V, Singh OP. Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vector Anopheles stephensi. PLoS One. 2021;16(6):e0253173. pmid:34125861

49. Khan J, Gholizadeh S, Zhang D, Wang G, Guo Y, Zheng X, et al. Identification of a biological form in the Anopheles stephensi laboratory colony using the odorant-binding protein 1 intron I sequence. PLoS One. 2022;17(2):e0263836. pmid:35192647