Background

As per the World Malaria Report 2018, in 2017 80% of the global malaria burden was borne by 15 countries in sub-Saharan Africa and India. India (with 4% malaria cases) is one of the five countries that contributed 50% malaria cases worldwide. The other four countries are Nigeria (25%), Democratic Republic of the Congo (11%), Mozambique (5%), and Uganda (4%) [1]. Total malaria cases reported in 2017 in India were more than 9.5 million and this number is 3 million cases fewer than those reported in 2016. Plasmodium falciparum cases were 63.38% and the other major parasite species was Plasmodium vivax (about 33–34%); Plasmodium malariae and Plasmodium ovale continued to be about 2–3% of the total cases. Plasmodium knowlesi cases are so far reported only from Andaman and Nicobar Islands.

National framework for malaria elimination

In line with global developments in achieving the elimination of malaria in different countries, India has committed to eliminating malaria by 2030. The National Framework for Elimination of Malaria in India [2] was released on 16 February, 2016. Based on the epidemiological data of 2014, 15 states and Union territories (UTs) with annual parasite incidence (API) less than 1 are placed under Category 1, 11 with API less than 1 and 1 or more districts with API more than 1 under Category 2, and 10 with API more than 1 under Category 3. The national malaria control programme plans to achieve elimination in a phased and strategic manner, and prevent re-establishment of local transmission of malaria in areas where it has been eliminated and sustain a malaria-free status nationally by 2030. In order to reach the timely milestones and ultimately the elimination by 2030, key interventions recommended for the three categories are listed in detail in the document, and the vector control forms the major component of malaria control.

With the use of indoor residual spray (IRS) and long-lasting insecticidal nets (LLINs) in association with prompt case detection and treatment facilities, there has been a reduction in the incidence of malaria cases in the country. Epidemiological data of the country shows that certain areas still report intense malaria transmission [1].

Of the different technical and operational reasons identified for failure of control, lack of relevant knowledge on the behaviour of vectors could be one. There are reports from several countries [3,4,5,6] and from India [7, 8] showing drift in mosquito behaviour to rest outdoors owing to the use of insecticide interventions, especially LLINs. The concern is that the effectiveness of these tools would eventually be compromised. Reports on change in the behaviour of vectors are a warning and require attention to achieve elimination target. Hence, outdoor transmission and residual malaria are receiving renewed focus globally.

In the context of India’s commitment to malaria elimination by the year 2030, there is a need to revisit the existing knowledge on biology and bionomics of malaria vectors. The recently published monograph Guidelines for malaria vector control [9] states that “accurate species identification is crucial for all studies and surveillance activities on field populations of vectors. Many of the vectors belong to species complexes and require advanced molecular analyses for species identification, necessitating appropriate laboratory resources. Without accurate species identification, data collected on behaviour, distribution and infection rates for decision-making by control programmes will have limited use”.

Extensive studies have been carried out in different parts of the country and several books, articles, and reviews have been published (to mention a few: [10,11,12,13]). The objective of writing this review is to describe the biological characters and bionomics of major malaria vectors to highlight the changes that have occurred in the species prevalence and biological characters, as this information is important for planning vector control strategies. In this article, studies that need to be carried out to generate data to fill the gaps in existing knowledge in vector biology and bionomics, and generate data to quantify behavioural aspects to facilitate informed decisions in selecting tools/strategies to interrupt transmission effectively are suggested. Furthermore, mechanisms to integrate existing tools, additional vector control interventions to complement the existing ones, with a focus to address biological aspects of vectors are discussed in this review article.

Vector species prevalence in India

Six Anopheles species, Anopheles baimaii, Anopheles culicifacies, Anopheles fluviatilis, Anopheles minimus, Anopheles stephensi, and Anopheles sundaicus are implicated as primary vectors transmitting malaria in different eco-geographical regions of India. In addition, the secondary/local vectors Anopheles annularis, Anopheles nivipes, Anopheles philippinensis, and Anopheles varuna transmit malaria along with either one or two major vectors in different parts of the country. Anopheline vector fauna of this country is further enriched by the recognition of certain of these vector species as species complexes [11, 13]. The anopheline species that have been found as complexes and the members in each of these complexes found in India are: Culicifacies Complex (A, B, C, D, E), Dirus Complex (two-An. baimaii in the northeast and Anopheles elegans in the south), Fluviatilis Complex (S, T, U, V), Minimus Complex (An. minimus), Sundaicus Complex (species D), Annularis Complex (A and B) and Subpictus Complex (A, B, C, D) [11, 13]. These species, owing to their distinct biological characters and ecological preferences, show a specific distribution pattern. Malaria epidemiology in India is complex and the endemicity varies distinctly in diverse ecosystems of the country. The ecosystems vary in the proportions of two predominant malaria parasites P. falciparum and P. vivax, and the prevalence of the six major Anopheles vector species and their sibling species along with one or two vectors of local importance.

Eco-geographical distribution of major malaria vectors

The major ecosystems where malaria is endemic in the country are forests, rural plains, urban, coastal, and arid areas. There is a strong relationship between ecosystem and vector and parasite species prevalence to the malaria transmission in an area. Vector species have a distinct distribution pattern in the country and the pattern is governed by land use patterns and type of breeding sites available. For example, topography and climatic conditions in the forest eco-systems, in addition to influencing the prevalence of vector species, also affects longevity of the vectors. In laboratory studies at 27–28 °C and 70–80% relative humidity, P. falciparum takes longer to complete its sporogonic cycle than P. vivax in vectors [14] and there were similar observations in laboratory feeding experiments done at NIMR (unpublished). As all malaria vector species in India transmit both the major parasite species P. falciparum and P. vivax the differences observed in the prevalence of parasite species are due to variable climatic conditions in the ecosystems prevalent in the country.

Broadly, ecosystems and the major malaria vectors and their sibling species observed in different states are given in Table 1.

Table 1 Major malaria vector species prevalent in different ecosystems in India
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Biology and bionomics of major malaria vector species and their sibling species in different ecosystems

Anopheles culicifacies and An. fluviatilis are major vectors contributing to 75–80% malaria in India, and An. culicifacies alone is responsible for 60–70% of malaria. In a hilly-forested ecosystem, An. fluviatilis is the major vector species with An. culicifacies in the secondary role, and in plain and forest-fringe areas An. culicifacies is the predominant vector species. In certain plain areas, An. culicifacies is the only species transmitting malaria. Biology and bionomics studies carried out in India broadly reveal that An. culicifacies and An. fluviatilis predominantly rest indoors and bite indoors [10, 15,16,17]. Anopheles culicifacies is observed in high densities (per man-hour densities) and is predominantly a zoophilic species, and indoor-resting collections from cattle sheds are generally more than those from human dwellings [10, 17, 18]. In contrast, densities of An. fluviatilis are low with significantly higher numbers in human dwellings than in cattle sheds. This species has a high human blood index (HBI) ranging between 0.8 and 0.9 in areas where it has been implicated in malaria transmission [17, 19,20,21,22]. In Balaghat district of Madhya Pradesh, An. culicifacies tested positive for Plasmodium antigen, almost in equal number from cattle sheds and human dwellings. Furthermore, from outdoor light traps and indoor light traps positives were found. Of the total 67 An. fluviatilis collected, 3/13 positives were found from human dwelling pyrethrum spray collections [23]. In district Sundergarh (Odisha), in the forested areas, human biting rates (HBR) of An. culicifacies and An. fluviatilis were 0.6 and 6.47 per person per night, respectively, and in plain areas where only An. culicifacies was found, its HBR was the same as in forest areas [21]. In areas where both An. culicifacies and An. fluviatilis transmit malaria, as in hilly, forested villages of Odisha, low densities of An. fluviatilis from April to September are compensated by An. culicifacies, and from October to February–March An. fluviatilis, with its highest densities of the year, transmit malaria leading to perennial transmission [17, 21, 24].

These studies establish the distinct difference in intensity of malaria transmission in plain and forest areas in the same district, and also show distinct difference in transmission efficiencies of these two vector species. Similar observations were made in Madhya Pradesh in areas of Mandla and Dindori districts, which are different in terrain and forest cover [25]. Anopheles culicifacies was incriminated from both the districts, while only An. fluviatilis from evergreen forests of Dindori. In Madhya Pradesh, in whole night collections using light traps, An. culicifacies and An. fluviatilis were collected in outdoor traps [23, 25]. There was no difference in the number of An. culicifacies in indoor and outdoor light traps, while a higher number of An. fluviatilis was collected in outdoor traps than in indoor resting collections, suggesting the preferential exophilic nature of this species. In Panna district (Madhya Pradesh), families that spent about 3 weeks in the forest for the collection of mahua flowers (Madhuca indica used for making liquor) returned with falciparum malaria infection [26]. These studies suggest the occurrence of outdoor transmission of malaria in forest areas of central India. In Assam, Meghalaya, Manipur and Sikkim States in the northeast where deforestation was done for agricultural purpose and rice cultivation is in practice, An. culicifacies was found with higher sporozoite rates than An. minimus. Irrigation channels for rice cultivation were one of the important breeding sites for An. culicifacies, and seen to be responsible for the increased presence of this vector in these States [27]. Similarly, in Thar Desert area of northwestern Rajasthan, with the development of canal-irrigation system, An. culicifacies established itself as a vector [28]. However, An. stephensi continues to be the major vector in irrigated and non-irrigated villages in these areas of Rajasthan [29].

Of the five sibling species identified in the Culicifacies Complex, except species B (which is either a poor or non-vector), all other species (A, C, D, E) transmit malaria in different parts of the country [23, 30,31,32,33,34]. Epidemiological and laboratory susceptibility studies support the poor vector status of species B [35, 36]. All these sibling species have a distinct distribution pattern in the country with species B prevalent in all the areas surveyed either exclusively or in sympatric association with other sibling species [11, 13]. Host feeding preference studies of species A, B, C, and D showed them to have low HBI ranging between 0 and 0.05 [13, 37]. Distinct seasonal variations in prevalence were observed among the sibling species [11, 13, 18]. Among the four sibling species examined for the biting rhythm, species A and B showed peak biting activity in the second quarter of the night, between 22:00 and 24:00 h, in all the seasons. Species C and D showed a different biting rhythm with peak biting in the first quarter between 18:00 and 21:00 h in April [13, 38]. In Chhattisgarh, Madhya Pradesh and Odisha, species C is the predominant sibling species [17, 21, 31]. In Madhya Pradesh, biting in early hours (first quarter of the night) was 60% for species C and 30 to 40% for the next predominant vector species, species D [38]. For species A, B, C, and D, the proportion of biting in the first quarter was highest in April (biting rhythms of species E has not been studied so far). In Madhya Pradesh, in April, active malaria transmission of P. vivax and P. falciparum cases was observed where species C was predominant [39], and in Jharkhand, transmission was reported in April, although An. culicifacies was in low densities [40].

Among the four members S, T, U, and V of the Fluviatilis Complex, species S was found with very high anthropophagy (90–98%) and positivity to P. vivax and P. falciparum infections [11, 17, 19]. Species T is predominantly zoophilic [19], and a few specimens positive for P. vivax and P. falciparum sporozoite antigen were found in forest villages of Madhya Pradesh [23]. Sporozoite antigen-positive specimens of species U have not been found so far. Species V was identified in district Hardwar in Uttarakhand State in sympatric association with species T and U. In the indoor collections, 70% specimens of species V were collected from human and mixed dwellings and its HBI was 0.04, while in the same sample collection, species T and U were totally zoophilic [41]. Because of distinct differences in the distribution of these sibling species and their feeding preference, great variation in the role of An. fluviatilis sensu lato in malaria transmission was observed in the country. Anopheles fluviatilis species S is a highly efficient sibling species of this complex and the major vector in hilly, forested villages of Chhattisgarh [20] and Odisha [8, 17, 21, 24, 33]. However, in recent studies in Keonjhar and Sundergarh districts in Odisha State in contrast to earlier observations, species T was predominant and along with species S it was found in higher numbers in cattle sheds than in human dwellings [7, 8]. In the Singhbhum hill area in Keonjhar district, An. minimus was reported along with An. fluviatilis species S (90%) and species T (9.1%) in indoor and outdoor human landing catches for the first time outside of the northeast [42, 43]. Anopheles minimus and An. fluviatilis S were observed throughout the year and were highly anthropophilic with 92 and 90.2% human blood positivity, respectively. Recently, An. minimus has been found in other districts in India, West Singhbhum district, Jharkhand (MK Das, pers. comm.) and Kalahandi district, Odisha (RK Hazara, pers. comm.). Anopheles annularis is a secondary vector in Odisha. This taxon is a complex of two sibling species, A and B [44].

Anopheles baimaii and An. minimus are vectors in the northeastern states. Anopheles baimaii is reported from all the states in the northeast (Sikkim, unpublished) [45]. Anopheles baimaii which is predominantly exophilic rests during day time on tree trunks/creepers in forests. It bites indoors and outdoors, and it briefly rests indoors on walls for about 20–30 min before biting [46]. Recently it has been collected indoors in large numbers in the State of Tripura [47, 48]. It is highly anthropophilic species and in Assam its HBI ranged from 0.667 to 1.0 during different months (average 0.923) [46]. Biting was observed in all the four quarters of the night with about 6–7% in the first quarter, 75% of biting in the second and third quarters, and 20% in the fourth quarter. In this study in Assam, 21% of overall effective entomological inoculation rate (EIR) was seen in the first quarter of the night [49]. Anopheles elegans, the second member in the Dirus Complex reported from southern India, has not been incriminated as vector so far. Presently only one sibling species of the Minimus Complex, An. minimus is reported from India. This species is reportedly endophilic, endophagic and highly anthropophagic and is found in low densities ranging from < 1 to 7 mosquitoes per man-hour in indoor resting collections [50]. Preference to bite humans was very high (93%) and sporozoite rate was 3.3% with sporozoite positives found during all months of the year. Biting activity was observed throughout the night with peak biting after midnight, between 01:00 and 04:00 h. In the northeastern states, in addition to An. baimaii and An. minimus, An. nivipes and An. philippinensis play a secondary role in the transmission of malaria. Anopheles nivipes was observed in Arunachal Pradesh, Assam, Manipur, Meghalaya, and Nagaland [51]. Between these two closely related mosquito species under the Annularis Group, An. nivipes was predominant in Assam and Nagaland, while An. philippinensis was more prevalent in the states of Mizoram and Arunachal Pradesh. Anopheles nivipes was incriminated as a vector of P. falciparum in Nagaland bordering Assam, and both these species were reported exophilic in behaviour and predominantly zoophilic [52].

Anopheles sundaicus is now found only in Andaman and Nicobar Islands and was not reported from the mainland after its last report in 1974–75 from South 24 Parganas district in West Bengal [10]. Prior to its disappearance from mainland, this species was reported from Andhra Pradesh, Odisha and West Bengal [10]. This is a species complex, and only one species, species D (cytotype D) was found in the islands [53, 54]. It has a low preference to bite humans, with HBI of 0.025, but in exclusive human dwelling collections, HBI was 0.18 [55]. It was collected indoors from both human dwellings and cattle sheds, and also from outdoors. Exophagy and bimodal biting activity with peak biting around 23.00 h and the second peak around 02.00 h were observed [56]. In a recent study conducted in the Andaman and Nicobar Islands, An. sundaicus was found positive for P. knowlesi [57].

The three forms of An. stephensi distinguished on the basis of ridge number on floats of eggs are type form predominant in urban areas, intermediate in semi-urban areas, and var. mysorensis in rural areas [58]. As no mating barrier was observed in laboratory crosses between the three forms, and that egg morphological and chromosomal inversion polymorphism studies in rural and urban areas suggested the three forms to be differentially found in different ecosystems, they were referred as ecological forms [58,59,60]. Anopheles stephensi is the major malaria vector in urban areas and transmits at low densities [61]. While this species is the major vector in arid zones of rural Rajasthan [28, 29], it is considered as a poor/non vector in the rural areas of other parts of India [58]. In certain parts of Iran An. stephensi var. mysorensis was found as the only vector transmitting malaria [62]. In these areas, animal hosts were very low in number or were totally absent. Recently this species is speculated to be a complex based on examination of odorant binding protein 1 intron I sequence in An. stephensi specimens collected from Iran and Afghanistan [63]. The three biological species recognized as species A, B and C correspond to type form, intermediate form and var. mysorensis, respectively. The main strategy to interrupt malaria transmitted by An. stephensi in urban areas by the National Vector Borne Disease Control Programme (NVBDCP) under the urban malaria scheme is larval control, and in rural areas of Rajasthan where this species is reported resting and biting indoors, indoor residual spraying is used. In Rajasthan, in pre-DDT era this species was found resting on the walls of the houses, but recently in Jodhpur district of Rajasthan this species was found resting on household objects (hanging clothes, furniture, stacked clothes, etc.) avoiding walls both in insecticide sprayed and unsprayed villages, suggesting change in its resting behaviour [64]. Furthermore, An. stephensi biting was observed outside the houses in courtyards during dusk, and it was found entering the houses after 23.00 h and most of the entry was between 01.00 and 04.00 h. In Goa State, An. stephensi is the major vector. In this state An. stephensi could not be collected from well-built houses, and large collections were made from huts near construction sites [65]. In human landing catches inside the houses, seasonal variations were observed in biting times [66]. In Chennai city, An. stephensi is the vector and this city contributes 60–70% of malaria cases of Tamil Nadu State [67]. In one of the high malaria-endemic areas of Chennai, higher densities of An. stephensi were observed in cattle sheds in the vicinity of human dwellings than in human dwellings [68]. Maximum mosquito collections were from houses with thatched roofs and only about 5% were from houses with asbestos and tiled roofs. In addition to An. stephensi, An. subpictus was found positive for sporozoites in coastal areas of Goa [69] and in Chennai [68]. In Goa, sporozoite positive specimens were identified as species B of An. subpictus. Anopheles subpictus is reported to be a complex of 4 sibling species [70], and species B, which is a coastal species, was earlier incriminated in Puducherry [71].

Vector control strategies in use

Under NVBDCP, India vector control has been playing an important role in disease management. The two main vector control strategies that are being used are indoor spraying with residual insecticides (IRS) and LLINs targeting adult mosquitoes in rural areas of the country. In urban areas, where vector breeding is in defined and confined habitats, larval control using chemical insecticides, bacterial pesticides and larvivorous fish is the applied strategy. In the northeastern states and in forested areas of the states in Central India, LLINs are being distributed to saturation.

Current situation on responses of major vectors to vector control tools

For indoor residual spraying, DDT, malathion and pyrethroids have been introduced in the malaria control programme in a sequential order. In 1959 An. culicifacies was reported resistant to DDT [72], to hexachlorocyclohexane [73] and in 1973, to malathion [74]. The differential development of resistance among the sympatric sibling species under similar selection pressure was observed [11, 15, 75,76,77]. Now that IRS has been in practice for more than six decades, vector species have developed increased levels of resistance to one or more insecticides in a given area depending on the use of insecticides and the selection pressure exerted on the vector species. In a recent review of the resistance/susceptible status of vector species to different insecticides, it is mentioned that An. culicifacies in the rural plains have exhibited widespread resistance [78]. In 70% of the districts examined, this species has shown resistance to at least one insecticide, while in some to two and in some other districts to all three classes of insecticides. Resistance to pyrethroids has been found widespread in Chhattisgarh, Madhya Pradesh and Odisha while in other states it was sporadic. With reference to other vector species: An. fluviatilis in hilly, forested and foothill areas, where it is the major vector, was mostly susceptible to DDT, in one district even to malathion and fully susceptible to pyrethroid; An. baimaii and An. minimus (except in one district resistant to DDT), which are major vectors in the northeastern states, were fully susceptible to all the three insecticides; An. sundaicus was reported to be resistant to DDT and malathion in Car Nicobar; and, An. stephensi, a vector in urban areas, is susceptible to Temephos, used for larval control, and is also susceptible to bacterial pesticide Bacillus thuringiensis israelensis (Bti) [78]. Scaling up of LLIN intervention has brought about changes in sibling species composition, resting behaviour and feeding preferences of An. fluviatilis in Odisha [7, 8].

Challenges and possible solutions to effective vector control

To achieve effective vector control towards elimination, among various confounding factors it is important to identify the challenges pertaining to the biological and bionomic characters of vectors and related operational issues. Those identified and the few more that require attention are listed in Table 2, in order to facilitate taking informed decisions on strategies to be used to limit transmission.

Table 2 Challenges identified and possible solutions for effective vector control
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Conclusions

The use of indoor spray with residual insecticides and LLINs to target adults is the cornerstone of the national malaria control programme. The efficacy of these interventions depends on the biological characters of the vector species, such as resting and feeding behaviour. The success of IRS depends exclusively on the indoor resting (endophilic) behaviour of vector species irrespective where they feed, while for LLINs it depends on site of use (indoor/outdoor) and on feeding time of biting. Considering that the major vectors are mainly indoor resting and endophagic, these strategies are being implemented against all the vector species in the country. In the light of widespread resistance in An. culicifacies to the three classes of insecticides in use for interventions, there is an urgent need to implement novel strategies to overcome resistance [79], which includes use of insecticide molecules with novel modes of action for the management of resistance and for the effective control of vector species. Soon interventions using combinations of synergists with insecticides and mixtures containing new class of insecticides for IRS and LLINs will be available. The data presented in the section on biology and bionomics points out the presence of populations of vector species that are exophilic and early biting. Cultural and agricultural practices in certain endemic areas make people vulnerable to biting by vector species that are exophilic and early biters. To address outdoor and early biting of vector species there is an urgent need for new tools and to evaluate them for their efficacy and feasibility to use in different ecosystems. In recent years in different endemic countries newer tools are being tested for their efficacy, such as spatial dispensers using volatile pyrethroids [80] and for treating eve ribbons and odour-baited traps [81], eave tubes [8], totally mosquito-proof portable huts for the protection of rice cultivators [82], attractive toxic sugar baits [83, 84], etc., are being evaluated in malaria-endemic countries. Insecticide-impregnated sheets, blankets, personal clothes, and hammocks, etc., can be used to protect people who stay in forests for specific occupations. Novel and emerging tools for species-specific control include Wolbachia-based disease control strategy, sterile insect technique (SIT), incompatible insect technique (IIT), gene drive technology, etc. While SIT and IIT are used for population suppression, Wolbachia transinfected mosquitoes and those that are modified using gene drive/editing technology can be used for population replacement to control the disease they transmit. A strain of An. stephensi transinfected with Wolbachia from Aedes albopictus showed refractoriness to P. falciparum [85]. Another strain of An. stephensi that was genetically engineered to express genes targeted against the malaria parasite P. falciparum using CRISPR–Cas9 system interrupted the development of P. falciparum [86]. Both these strains are yet to be field-tested. Species-specific tools, although very effective, have a limitation: in many areas more than one vector transmits malaria and many of the major malaria vectors are species complexes. This necessitates the need for the release of more than one species strain in an area. These techniques are advantageous in areas where only one species is responsible for the transmission, and because they could provide protection from disease while not attempting species elimination.

With intensive control activities to reach the elimination target, regular surveillance of vectors for changes in prevalence of vector species and their behavioural aspects, and regular monitoring of insecticide resistance should be made routine activities by the programme. The need of the hour is to identify the knowledge gap and to generate data to fill it. Equally important is to test new tools for their efficacy and their suitability in different ecosystems the vector species are occupying.