Anopheles minimus: its bionomics and role in the transmission of malaria in Assam, India V. Dev1 Indoor, day-resting collections of Anopheles minimus mosquitos from human dwellings in the study area in Assam, India, indicated that these insects were prevalent throughout the year and that their maximum abundance occurred from March to August. A. minimus was identified as a vector of malaria, and sporozoite infections were recorded every month of the year, with the highest rate occurring in October. The mosquito was highly anthropophilic and fed on human hosts (indoor) all through the night, but feeding was more pronounced between 01:00 and 04:00; the person-biting rate was 13.7 per night. Breeding occurred throughout the year in slow-flowing streams with grassy banks. A. fluviatilis was also identified as a vector of malaria in the study area but occurred in low density, and sporozoite infections were only seasonal.

Introduction

Materials and methods

Anopheles minimus used to be the most important vector of malaria in India along the foothills of the Himalayas from Uttar Pradesh (Terai region) to the north-eastern region of the country (1-5). However, following the application of DDT and other residual insecticides by the national malaria control/eradication programme, A. minimus disappeared from Uttar Pradesh (6, 7), and was believed to have also from the north-eastern region (8-10). Subsequently, the role played in malaria transmission by other vectors, i.e., A. philippinensis and A. balabacensis, was reported (9-11). However, because of persistent transmission of malaria in certain pockets in the north-eastern region, studies were initiated under the Plasmodium falciparum containment programme to identify the vectors and their relative importance. As a result, A. minimus was re-recorded in the foothills of the following states: Nagaland (12), Mizoram (13), Arunachal Pradesh (14), and Assam (15, 16). A. minimus was also recorded and identified as a malaria vector in Sonapur PHC, district of Kamrup, Assam (17). As a follow-up to these preliminary observations, additional data have been collected de novo on the seasonal prevalence, sporozoite rates, feeding behaviour, and breeding habitats of A. minimus in order to design appropriate malaria containment methods using an integrated disease vector control approach. At the same time the bionomics of A. minimus were studied and data were collected on malaria incidence.

Study area Sonapur PHC (altitude, 0-lSOm) is located 25km

I Senior Research Officer, Malaria Research Centre (ICMR), Field Station, P.O. Sonapur, Kamrup, Assam 782 402, India. Requests

for reprints should be sent to this address. Reprint No. 5673 Bulletin of the World Health Organization, 1996, 74 (1): 61-66

east of Gwahati on the south bank of the Brahmaputra river, adjacent to the border with Meghalaya State. The area is predominantly tribal, low-lying and prone to flooding, and most of the villages are located in the foothills. There are paddyfields in the plains interspersed with criss-crossing perennial streams. The primary occupation of the inhabitants is paddy-field cultivation (jhoom), and most of the people live under poor socioeconomic conditions. Typically, the houses consist of two or three rooms and are made of bamboo with thatched roofs; often there is a cattle shed close to each house. Rainfall in the area is heavy (1400-2200mm per annum) with premonsoon showers in April-May, and the maximum precipitation occurs in the period July-September. The relative humidity varies from 60% to 85%. Temperatures lie in the range 10-26°C in winter (November-February) and 23-33 °C throughout the rest of the year.

Methods Malaria incidence To determine the morbidity from malaria, we carried out active fever surveillance at weekly intervals in 50 villages within Sonapur PHC over the period 198991. Blood smears were taken from all fever cases and examined for malaria parasites. Cases found to be positive were given antimalarial drugs in accordance with the policy of the national malaria eradication programme. © World Health Organization 1996

61

V. Dev

Day-resting collections To identify the day-resting anopheline species and their relative proportions, collections were made in human dwellings (indoor) from 09:00 to 12:00 over the period October 1989 to September 1990 for a total of 311 man-hours. With the aid of a torch and a suction tube the insects were collected while they were resting on walls, clothes, and other articles within the houses. Subsequently, they were indentified using the pictorial keys produced by Wattal & Kalra (18) and graded for their abdominal condition and resting behaviour. The seasonal prevalences and man-hour densities of indoor resting A. minimus and A. fluviatilis were also

on an hourly basis and kept separate for identification purposes.

Larval breeding surveys To determine the species-specific breeding sites of the mosquitos, larval surveys were carried out over the period April 1988 to March 1989. Larvae were collected from different habitats including ponds, wells, ditches, pits, bamboo holes (cut bamboos), paddy-fields, and streams. Immature larvae were reared in the laboratory until emergence, for identification purposes.

determined.

Results

Vector identification and host preferences The anophelines collected indoors from human dwellings during day-resting catches were dissected in normal saline solution (0.9%) to detect any sporozoites. Data were collected for the period July 1989 to September 1990 to determine the seasonal infection rates. Host blood-meal analyses were carried out for A. minimus using agar gel diffusion (19).

Parasitological observations Individuals who were positive for malaria were detected at the weekly fever surveillance visits during all months in 1989-91 (Table 1). Nevertheless, there was a notable increase in the number of fever cases, with a corresponding increase in the number of malaria positives from May to September/October. This increase was largely due to P. falciparum infections and coincided with the wet season (monsoon). For the remainder of the year (winter), transmission continued but there was a marked decrease in the number of cases. Over the study period the slide positivity rate ranged from 22.9% to 36.5%, with the

Human biting collections To study the feeding behaviour and nocturnal movements of the mosquitos, a total of nine whole night catches (18:00 to 5:00) were made inside the dwellings over the period June 1988 to October 1989 (the active malaria transmission season). The collections were made on the exposed arms and legs of human volunteers by an experienced insect collector using a torch and a suction tube; the collections were made

majority (>74%) being P. falciparum infections.

Entomological observations Indoor resting collections. From the day-resting collections 14 anopheline species were recorded, of

Table 1: Malaria incidence in the study villages, Sonapur PHC, Assam, India, 1989-91 1989 (n = 21 547)a

No. of fever Month

January February March

Aprl May June July August

September October November December Total

cases

No. +ve for malaria

1990 (n =

No. +ve, P. falciparum

491 704 479 489 1 028 1 324 1131 1 081 975 973 893 582

99 44 26 59 276 438 403 407 293 243 249 176

76 32 17 47 208 396 379 367 263 199 200 131

10150

2713

2315

No. of fever cases

No. +ve for malaria

638 627 862 394 705 905

152 74 146 41 214 211

1 274 830

327 244

618 529 434 432 8248

22262)8

1991 (n = 22452)8

No. +ve,

No. of fever

P. falciparum

cases

No. +ve for malaria

No. +ve, P. falciparum

133 95 122 132

90 40 90 17 144 174 298 213 108 72 96 100

314 403 373 922 833 993 1 366 1 381 987 843 802 651

96 51 50 242 281 347 619 688 391 324 307 202

69 37 36 183 241 298 437 500 336 221 186 136

1 891

1442

9868

3598

2680

a n = population. 62

WHO Bulletin OMS. Vol 74 1996

Bionomics of Anopheles minimus in Assam

which A. minimus (40.4%) and A. vagus (43.6%) were the most frequent. A. varuna and A. fluviatilis accounted for 6.4% and 4.6%, while remaining species comprised less than 2% of the total collected (Table 2). All the insects were found resting on walls, hanging clothes and other articles, and on the underside of beds. Most of the A. minimus were either fully fed, semigravid or gravid in almost equal proportions. However, the distribution of A. minimus was patchy in the villages, some houses regularly yielding large numbers of this species while others did not. A. minimus mosquitos were found throughout the year; however, their density (No. caught per man-hour) was highest from March to August with a dip in July, and thereafter remained low until January (Fig. 1). A. fluviatilis mosquitos were encountered from January to July but in low numbers, and their density varied from 0.10 to 3.15, with the maximum in March. Other known malaria vectors, e.g., A. dirus and A. culicifacies, were also recorded but in very low numbers.

Fig. 1. Seasonal prevalence and density (No. caught per man-hour) of Anopheles minimus, Sonapur PHC, Assam, India, October 1989 to September 1990.

20 3 18 '516

Q.14

c

oco 12 10 a" 86 4 -

-

I.'R

,

month of the year, with the maximum infection rate in October. Interestingly, no infections were recorded in November and December 1989 but during these months in 1990, infections occurred. Similarly

minimus and A. fluviatilis were identified as vectors of malaria. Over the period July 1989 to September 1991, a total of 4680 A. minimus were dissected; 155 of which were sporozoite-positive, corresponding to an overall infection rate of 3.3% (Table 3). This species was positive for sporozoites practically every Table 2: Distribution of anopheline species and their abdominal condition in day-resting collections in indoor human dwellings, Assam, India, October 1989September 1990 Abdominal conditiona Anopheles species

UF

FF

SG

A. aconitus A. annularis A. culicifacies A. dirus A. fluviatilis A. jeyporiensis A. kochi A. maculatus A. minimus A. nigerrimus A. philippinensis A. splendidus A. vagus A. varuna

-

-

-

-

8 0 0 9 11

47 1 1 107 51

21 3 0 46 8

8 1 1 93 12

-

-

-

-

1 39

22 665

0 767

1 758

-

-

-

-

b

1990

-4-1989

From the results of the day-resting collections, A.

a

cz

Table 3: Seasonal sporozoite infection in A. minimus, Sonapur PHC, Assam, India, 1989-91

Sporozoite rates

Total

a

0c2-

c]nu

G

Total

(1.0)b (1.5) (0.09) (0.04) (4.6) (1.5) (0.05) (0.4)

1

4

4

3

-

-

-

-

10

184

82

76

55 84 5 2 255 82 3 24 2229 4 12 6 2406 352

-

-

-

-

5519 (100.0)

-

UF unfed; FF fully fed; SG semigravid; G Figures in parentheses are percentages. =

WHO Bulletin OMS. Vol 74 1996

=

(40.4) (0.07) (0.2) (0.1) (43.6)

(6.4)

gravid.

No. dissected

No. gland-positive

Infection rate (%)

802 284 386 114 38 28

39 12 11 8 0 0

4.86 4.22 2.85 7.02 0.00 0.00

106 223 282 217 328 172 52 25 5 94 227 182

1 3 2 2 11 7 1 0 0 8 7 3

0.94 1.35 0.71 0.92 3.35 4.07 1.92 0.00 0.00 8.51 3.08 1.65

112 38 96 245 15 30

0 1 0 7 13 17 1 1

0.00 2.63 0.00 2.86 3.89 6.94 6.67 3.33

4680

155

3.31

1989

July August September October November December 1990

January February March April May June July August September October November December 1991 January February March April May June August

September Total

245 334

63

V. Dev Table 4: Results of hourly collections of malaria vectors on human bait, Sonapur PHC, Assam, India, JuneOctober 1988 No. collected per person during the hours of:

Anopheles species 18-19 A. minimus A. philippinensis A. dirus a

b

0.25 0.50 0.25

19-20

20-21

21-22

22-23

23-24

0-1

1-2

2-3

3-4

4-5

Total

Biting ratea

0.25 0.50 0.25

1 7 1

12 1 0

14 3 0

16 10 0

7 6 0

25 2 1

25 2 0

18 1 1

5 0.25 0

123.50 (9)b 33.25 (9) 3.50 (9)

13.72 3.69 0.39

Biting rate per person per night. Figures in parentheses are the number of nights when collections were made.

in August and September 1990 no infections were recorded (perhaps because of the low sample sizes) but were found during these months in 1989 and 1991. An analogous situation applied to January and March in 1991 and 1990. Also, A. fluviatilis mosquitos were positive for sporozoites: between January and July, 207 specimens were dissected, four of which were positive (one each in February, March, April and July). All other known malaria vector species in the study area, i.e., A. dirus, A. philippinensis, A. culicifacies, A. maculatus, and A. annularis, were sporozoite-negative.

Feeding behaviour Of 151 blood meals collected in October and November from A. minimus, 140 were of human blood, with the remainder containing blood from animals other than cattle. Thus, A. minimus were attracted to human hosts, with an anthropophilic index as high as 93%. This was confirmed by the results of the wholenight human-bait catches (Table 4). A. minimus fed throughout the night but feeding was more pronounced between 01:00 and 04:00. This species was the most predominant in the whole-night catches and its biting rate was 13.72 per person per night. Other vector species, i.e., A. philippinensis and A. dirus, were also collected on human bait but in low numbers.

Breeding habitats A. minimus bred throughout the year in slow-flowing streams with grassy banks and sometimes also in rice fields that had streams with perceptible flow of water. Other potential vector species that bred in streams included A. culicifacies and A. maculatus.

Discussion With the introduction of residual insecticides for vector/malaria control in India, there have been 64

records of changes in the composition of mosquito species and their bionomics, e.g., A. fluviatilis in the Western Ghats (5), A. culicifacies in Tamil Nadu (20), A. sundaicus along the eastern coast (21), and A. philippinensis in West Bengal (5). It is therefore all the more important to study these changes and to formulate appropriate control strategies. Data are available on the bionomics of A. minimus in the preDDT era (1-5). The data on its prevalence, resting habits, feeding behaviour, breeding habitats and sporozoite rates presented here (Tables 2-4), indicate that it has staged a comeback with all its bioecological characteristics intact. A. minimus was prevalent throughout the year in the study area and is endophilic, endophagic, and highly anthropophilic. Also the sporozoite infectivity rates show that it lives long enough to actively transmit malaria through most of the year. The entomological observations closely paralleled the incidence of malaria in the study area (Table 1). Malaria was detected every month of the

year, the majority of infections being P. falciparum. From the monthly parasite prevalence data, it was apparent that transmission was persistent, with May to September/October representing the peak period (monsoon season). This peak transmission period preceded the peak density for A. minimus (Fig. 1), perhaps because of the extrinsic development of the parasite in the vector. During the winter, malaria transmission was supplemented by

A. fluviatilis, thus accounting for the perennial pattern of the disease in the study area. Since A. minimus is highly sensitive to DDT (22), it is plausible that it was reduced in numbers as a consequence of spraying, but that it remained in certain remote foci. Nevertheless, like many other anopheline complexes (23), the existence of A. minimus sibling species cannot be overlooked. The fact that A. minimus disappeared from the Terai area of Uttar Pradesh (7), but reappeared in the north-east part of the state suggests the existence of sibling species within its domain of distribution. Indeed, based on population genetic evidence (24) and behavioural studies (25), A. minimus sibling species WHO Bulletin OMS. Vol 74 1996

Bionomics of Anopheles minimus in Assam

have been documented in Thailand. A. minimus is also known to have disappeared from Nepal (26), while in Thailand it is found only in the forested foothills, having disappeared from the plains (25). Since A. minimus is highly anthropophilic and is sporozoite-positive practically every month of the year, it can be taken to be the principal malaria vector in the study area. It has also been found to be prevalent and identified as a vector in malariaendemic tea estates and other endemic pockets in Assam (27). A. fluviatilis played only a supplementary role as a malaria vector in the study area, its density was low, and its sporozoite infection was seasonal. The influence of other vectors, i.e., A. philippinensis and A. dirus, could not clearly be established. The indoor resting characteristics and breeding habitats of A. minimus made it amenable to control by residual insecticides and larvicides. Since its peak biting activity occurred from midnight onwards, the use of insecticide-impregnated bednets in conjunction with larvicides would be a more appropriate strategy for the containment of malaria than the large-scale use of insecticides (28).

Acknowledgements am indebted to Dr V.P. Sharma for providing research facilities, and to Mr N.L. Kalra for his constructive criticism and extensive revision of the manuscript. Dr D.S. Choudhury, Dr T. Adak, Dr B.N. Nagpal, and Dr A. Srivastava are thanked for their active interest and suggestions. Thanks are also due to Dr A. Roy and Dr H. Joshi for carrying out the host blood-meal analysis, and to Dr S.K. Subbarao for critically reviewing the manuscript. The technical assistance provided by the field staff is gratefully acknowledged.

Resume Bionomie et r6le d'Anopheles minimus dans la transmission du paludisme dans l'Assam, Inde Les captures d'Anopheles minimus effectuees dans leurs refuges diurnes a l'interieur d'habitations humaines ont indique que ces insectes y sont pr6sents tout au long de l'annee avec un pic d'abondance entre mars et aout. A. minimus s'est avere etre un vecteur du paludisme et l'on a enregistre des infestations sporozo itaires toute l'ann6e, les taux d'infestation les plus 6lev6s survenant en octobre. Ce moustique est tres anthropophile et prend ses repas de sang sur I'homme (a l'interieur des habitations) durant toute la nuit avec une frequence augment6e entre 1 WHO Bulletin OMS. Vol 74 1996

heure et 4 heures du matin; le taux de piqOres est de 13,72 par homme par nuit. Les gites larvaires de cette espece, qui se reproduit en continu, sont des cours d'eau lents aux berges herbeuses. Dans la zone d'6tude, A. fluviatilis s'est av6r6 etre un autre vecteur du paludisme, mais avec une densite de population moindre, et les infestations sporozoitaires dues a cette espece sont saisonnieres. La pr6valence de ces vecteurs correspond a l'incidence du paludisme dans la zone

d'6tude.

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V. Dev 14. Dutta P, Baruah BD. Incrimination of Anopheles minimus Theobald as vector of malaria in Arunachal Pradesh. Indian journal of malariology, 1987, 24: 159-1 62. 15. Kareem MA et al. A preliminary report on some entomological observations in malaria endemic area of Kamrup district, Assam. Journal of communicable diseases, 1985, 17: 29-35. 16. Nagpal BN, Sharma VP. Survey of mosquito fauna of northeastern region of India. Indian joumal of malariology, 1987, 24: 143-149. 17. Wajihullah et al. Anopheles minimus in Assam. Current science, 1992, 63: 7-9. 18. Wattal BL, Kaira NL. Regionwise pictorial keys to the female Indian Anopheles. Bulletin of the National Society of India for Malaria and other Mosquitoborne Diseases, 1961, 9: 85-138. 19. Collins RT et al. An adaptation of the gel diffusion technique for identifying the source of mosquito blood meals. Indian joumal of malariology, 1986, 23: 81-89. 20. Chandrahas RK et al. On density measurements of A. culicifacies (Giles) during malaria transmission season in Pattukkottai, Thanjavur district, Tamil Nadu. Indian joumal of medical research, 1979, 70: 429434. 21. Kalra NL. Emergence of malaria zoonosis of simian origin as natural phenomenon in Greater Nicobars, Andaman and Nicobar Islands. Joumal of communicable diseases, 1980, 12: 40-44.

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22. Gilroy AB. Malaria control on tea estates in Assam, 1947-1956. Indian journal of malariology, 1958, 12: 157-164. 23. Subbarao SK et al. Cytotaxonomy of certain malaria vectors in India. In: Service MW, ed. Biosystematics of haematophagous insects. Oxford, Clarendon Press, 1988: 25-37 (Systematics Association Special Vol.

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24. Green CA et al. Population-genetic evidence for two species in Anopheles minimus in Thailand. Medical and veterinary entomology, 1990, 4: 25-34. 25. Nutsathapana SP et al. The behaviour of Anopheles minimus Theobald (Diptera: Culicidae) subjected to differing levels of DDT selection pressure in Northern Thailand. Bulletin of entomological research, 1986, 76: 303-312. 26. Parajuli MB et al. Nationwide disappearance of Anopheles minimus Theobald 1901, previously the principal malaria vector in Nepal. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1981, 75: 603. 27. Dev V, Sharma VP. Morbidity due to malaria in Assam: an investigation. In: Abstracts of the Xl National Congress on Parasitology, Udaipur, Rajasthan, 22-24 February 1994. 1994: 29. 28. Dev V et al. Field trials with insecticide impregnated bednets for malaria control in Assam. In: Kumar S et al., eds. Tropical diseases: molecular biology and control strategies. New Delhi Publication and Information Directorate, Council of Scientific and Industrial Research, 1994: 387-396.

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