Vector abundance and behaviour in an area of low malaria endemicity in Bataan, the Philippines

Acta Tropica 63 (1997) 209 – 220

Vector abundance and behaviour in an area of low malaria endemicity in Bataan, the Philippines E.P. Torresa,*, N.P. Salazarb, V.Y. Belizarioc, A. Sauld a

Department of Parasitology and Medical Entomology, Research Institute for Tropical Medicine, Department of Health, Alabang, Muntinlupa, Metro Manila 1770, Philippines b SEAMEO TROPMED Central Office, 420 /6 Raj6ithi Road, Bangkok 10400, Thailand c College of Public Health, Uni6ersity of the Philippines Manila, Ermita, Manila, Philippines d Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Research, P.O. Royal Brisbane Hospital, Brisbane, Qld 4029, Australia Received 22 May 1996; revised 30 September 1996; accepted 7 October 1996

Abstract The vectorial importance of known and potential vectors in Morong, Bataan, Philippines was assessed based on human and animal baited collections of adult mosquitoes and on larval collections. Anopheles fla6irostris, the principal vector in the Philippines, was the most abundant among human landing catches, followed by An. maculatus sensu lato (s.l.). Both showed similar seasonal abundance with a peak during the early drier part of the year, which coincided with the peak in malaria cases. Both An. fla6irostris and An. maculatus s.l. fed throughout the night with the broad peak of capture from 00:00 to 04:00 and from 22:00 to 00:00, respectively. The two species had similar parous rates (0.76 and 0.72, respectively) giving an average life span equivalent to four feeding cycles. Neither vector was abundant with average human landing rates on collectors of 0.6 and 0.4 mosquitoes per person per night, respectively over the study period. An. maculatus s.l. showed a stronger preference for outdoor feeding compared to An. fla6irostris. An. maculatus s.l. was markedly zoophilic with a biting rate on water buffalo 50 times the human landing rate. An. fla6irostris was less zoophilic with a corresponding ratio of 7.5. It was concluded that in this area, An. fla6irostris is the principal vector. The combination of localised transmission, late night biting pattern * Corresponding author. Tel.: [email protected]

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and localised breeding sites of An. fla6irostris suggest that the use of bed nets and environmental management are relevant control measures that can be implemented through community participation. © 1997 Elsevier Science B.V. All rights reserved Keywords: Malaria; Anopheles fla6irostris; Philippines; Anopheles maculatus s.l.; Vectorial capacity

1. Introduction Malaria continues to be an important parasitic disease. It is transmitted by Anopheles mosquitoes. The behaviour of these vectors has profound effects on malaria transmission. Thus, there is a need to identify the mosquito species responsible for transmission in a given area and to determine their habits so that appropriate and effective control measures can be directed against them. Depending on location, An. fla6irostris is usually the principal vector. An. maculatus sensu lato (s.l.), An. litoralis, An. mangyanus and An. balabacensis may be secondary vectors (Salazar, 1989). An. fla6irostris prefers to breed in clear, partly shaded and slow flowing streams that abound in foothill areas (Smrkovski et al., 1982). Occasionally, it has the ability to adapt or utilise new habitats (Wooster and Rivera, 1985) such as irrigation ditches (Salazar et al., 1988), rice fields, pools and wells. In Palawan, it was observed to be mildly exophagic and zoophilic (Schultz, 1992). The maximum horizontal flight range of An. fla6irostris was reported to be about 1–2 km (Russell and Santiago, 1934). Although An. fla6irostris has been found to be a less efficient vector than An. balabacensis, a sylvatic species confined to deep forests of Palawan, still it is mainly responsible for the prevalence of malaria throughout the country. Keys for distinguishing An. fla6irostris from closely related non-vector species are not always reliable, but new genetic identification techniques using allozyme and rDNA markers should improve this situation (Foley et al., 1996). An. litoralis is associated with malaria transmission in the coastal areas of the southern provinces of Mindanao particularly Sulu (Cabrera et al., 1970; Catangui et al., 1972). Adult mosquitoes are mostly zoophilic and have been found 32 km inland from breeding sites (Baisas, 1975). An. maculatus s.l. co-exists with An. fla6irostris in the portion of the streams exposed to sunlight. Although members of this complex are strongly zoophilic, they appear to be responsible for malaria transmission at high altitudes with a flight range of about 2.5 km (Salazar, 1989). The vectorial status of An. maculatus s.l. in the Philippines needs to be investigated. Recent data suggests it is a complex containing 2 sibling species (Rattanarithikul and Harbach, 1990). Although An. mangyanus, has the same breeding habitats and seasonal prevalence as An. fla6irostris, the former seems to prefer habitats located near forest fringes. This limits its role as a vector. Indoor residual spraying remains the main vector control strategy utilised in all endemic areas by the Malaria Control Service. In areas such as Morong and

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Bataan, residual spraying is done bi-annually with Bendiocarb (Ficam) as the insecticide of choice. Despite these control efforts the disease is endemic in many parts of the country. This papers forms part of a multi-disciplinary study on factors influencing low endemicity of malaria in the Philippines (Belizario et al., 1997; Bustos et al., 1997; Espino et al., 1997; Lansang et al., 1997; Saul et al., 1997) with the aims of providing the basis for more effective integrated malaria control in low endemic areas.

2. Methods

2.1. Study site The study site, the municipality of Morong, Luzon Island has been described in detail by Bustos et al. (1997). Briefly, it consists of a 20× 15 km area on the western side of the Bataan Peninsula facing the South China Sea (Fig. 1). The area has a narrow coastal plain along the west, then rises steeply through denuded hills to forested mountains in the east. It is traversed by many streams. Administratively, the area is divided into 5 regions called barangays (Bustos et al., 1997) of about

Fig. 1. Map of the study area showing sitios used for night biting catches and location of An. fla6irostris and An. maculatus s.l. breeding sites. The size of the shaded areas indicate the number of larvae collected per 20 dips in breeding sites close to the named sitio.

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4000 people. Within these areas are villages or sitios (Bustos et al., 1997) with an average of 35 houses.

2.2. Human landing catches Three sitios in 4 of the 5 barangay were selected and designated as collection sites (Fig. 1). These were chosen from sitios which from historical heath records had relatively high numbers of malaria cases, that were accessible throughout the year and which were well spaced within the barangay. No collection was done within the barangay of Poblacion, which is the main town in the area and where malaria transmission is very low (Belizario et al., 1997). Three houses per sitio were selected as stations for the indoor and outdoor night biting collections. All-night man landing catches were performed from 18:00 to 06:00 for 3 nights by 3 teams of collectors, three collectors staying inside and the other three staying outside of selected houses. Mosquitoes landing on the collectors’ bare legs were collected with an aspirator and transferred to a container. Each mosquito container was replaced with a new one every 2 h. The teams were replaced at 00:00. Collections were made for 18 person-nights per barangay. All anopheline mosquitoes in each container were identified by morphology and counted the following morning.

2.3. Carabao-baited trap (CBT) collections Carabao (water buffalo) collections were carried out in one sitio per barangay simultaneously with the human landing catches and whenever an animal was available. CBT was done for one night by keeping the animal overnight inside a mosquito net, one side of which was left open for mosquito entry. The net was closed prior to collection at 00:00 and 06:00. Mosquitoes were collected using a mechanical aspirator and identified in the laboratory.

2.4. Lar6al collections Anopheline larvae were collected by dipping technique (about 20 dips per collection site) from potential breeding habitats identified in a systematic survey of streams in the study area within 1 km of a sitio. Immatures were reared in the laboratory until the adults emerged, then identified.

2.5. Identification and parity determination Adult female anopheline specimens were identified based on morphology using the taxonomic keys of Cagampang-Ramos and Darsie (1970). All known malaria vectors were dissected to examine ovaries for parity by observation of tracheolar skeins (World Health Organization, 1975). Vectorial capacity (Garrett-Jones, 1964), the potential number of new human infections transmitted by mosquitoes from a single infectious human per day, was calculated from estimated human blood index and the measured survival based

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Fig. 2. Total number of potential vectors collected in human landing catches in Morong, Bataan. A: Jan–Feb 1992; B: Apr–May 1992; C: Jul – Aug 1992; D: Sep – Oct 1992; E: Jan – Feb 1993 F: May – Jun 1993.

using the formula of Saul et al. (1990). This avoids the mathematical error in the original vectorial capacity equations which underestimates vectorial capacity.

3. Results

3.1. Vector abundance and biting cycle An. fla6irostris was the most abundant mosquito among human landing catches through all collecting periods (Fig. 2). An. maculatus s.l. was the next most abundant. Both showed a similar seasonal variation, with the peak catches at the beginning of the year. Extensive surveys of breeding sites from February to April 1992 revealed widespread but low concentrations of An. fla6irostris and An. maculatus s.l. larvae in Morong. Both were found predominantly in pools in stream beds (Fig. 1), commonly within 100 m of houses. Very few adult An. litoralis or An. mangyanus were collected (2.6 and 4.6% of the An. fla6irostris catch, respectively) in landing catches. For both, the bulk of mosquitoes were caught in a few catches, precluding any determination of seasonal abundance (Fig. 2). Only two larvae of An. litoralis and one An. mangyanus larva were collected. A more detailed investigation of the distribution, seasonal abundance and biting time was carried out for An. fla6irostris. Seasonal abundance showed similar peaks in the early half of both 1992 and 1993 in all barangays sampled (Table 1). This

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Table 1 Human landing rate of An. fla6irostris in barangays by period of collection Morong, Bataan, Philippines, 1992–1993 Barangay

Mabayo Binaritan Sabang Nagbalayong a

Human landing ratea Jan–Feb 1992

Apr–May 1992

Jul – Aug 1992

Sep – Oct 1992

Jan – Feb 1993

May – Jun 1993

0.6(0.3) 3.8(0.2) 0.7(0.2) 7.3(0.6)

7.4(0.6) 1.5(0.3) 2.0(0.3) 3.1(0.5)

0.2(0.1) 1.7(0.5) 0.4(0.1) 0.8(0.2)

0.2(0.1) 0.6(0.3) 1.5(0.3) 0

5.4(0.4) 3.8(0.2) 1.2(0.2) 0.6(0.1)

0.3(0.1) 0.6(0.1) 0.8(0.2) 2.4(0.4)

Number of mosquitoes per person per night (S.E.).

corresponds to the dry season in Morong. and is also the time when the majority of malaria cases are recorded at the two clinics serving the area (Belizario et al., 1997) (Fig. 3). As expected, collections from individual sitios showed more variation, but most gave peak values during the high malaria period (Table 2). However, there was some variation in the time of peak catches. Two sitios in barangay Sabang, Anahao and Matico, gave peak catches in Sep–Oct 1992, a period when the average human landing rate for all collections was at its minimum. An. fla6irostris showed a broad feeding time, with the mean time of capture at 01:00, but extending into the early morning (Table 3). There was a slight difference in the mean feeding time through the year, with feeding in the peak seasons tending

Fig. 3. Comparison of parasitological, entomological and meteorological data in Morong, Bataan, Philippines, Jan 1992–Feb 1993. Malaria incidence ( – – ) is the number of cases of malaria diagnosed in the two clinics per month per 1000 population. An. fla6irostris abundance ( – ’ – ) was measured as the number caught per person-night in human landing catches. Rainfall (white bars) is in mm per month.

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Table 2 Average and peak human landing rate of An. fla6irostiris by sitio indicating the time of peak catches Sitio

Average human landing rate (S.E.)

Peak human landing ratea

Mabayo Minanga Dose Ginian

1.1 (0.2) 3.9 (0.5) 2.1 (0.4)

3.5c 15.7c 8.5f

Binaritan Canawan Nagbaytu Timak

1.4 (0.3) 2.1 (0.2) 1.2 (0.3)

5.5c 5.0c 5.5f

Sabang Anahao Lalayan Matiko

2.0 (0.1) 1.2 (0.2) 0.2 (0.1)

3.3e 3.2c 0.8e

Nagbalayong Marucdoc Sampalok Gantuan

4.4 (0.4) 2.5 (0.4) 0.3 (0.1)

11.0b 10.8b 1.0d

a

Time of peak catches. 1993.

b

Jan–Feb 1992. c Apr – May 1992.

d

Jul – Aug 1992. e Sep – Oct 1992. f Jan – Feb

to be earlier than in the low transmission season (mean feeding times 01:29, 00:47, 00:41, 00:31, 01:13 and 01:11 for collections made in Jan–Feb 1992, Apr–May 1992, Jul – Aug 1992, Sep – Oct 1992, Jan–Feb 1993 and May–Jun 1993, respecTable 3 Collections by location and time of collection in Morong, Bataan, Philippines Location

Time of collection 18:00

20:00

22:00

00:00

02:00

04:00

–20:00

–22:00

– 00:00

– 02:00

– 04:00

– 06:00

27 65 92 (10.8)

81 100 181 (21.2)

83 126 209 (24.5)

112 113 225 (26.4)

70 50 120 (14.1)

375 (44.0) 478 (56.0) 853

10 36 46 (18.5)

14 50 64 (25.8)

10 37 47 (19.0)

16 21 37 (14.9)

11 13 24 (9.7)

68 (28.0) 180 (72.0) 248

An. fla6irostris a Indoor 2 Outdoor 24 Total 26 (3.05) An. maculatus b Indoor 7 Outdoor 23 Total 30 (12.1)

Total

Numbers in parenthesis are percentages. Significant difference in time of indoor and outdoor catches x 2 =36.6, df = 5, PB0.001 by x 2-square test for homogeneity. c Significant difference in time of indoor and outdoor catches x 2 =11.7, df= 5, PB0.05. a

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Table 4 Human-biting rate versus carabao biting rate for potential malaria vectors Species

Man biting rate

Carabao biting rate

An. An. An. An.

0.6 (0.1) 0.4 (0.2) 0.02 (0.04) 0.3 (0.05)

4.5 2.3 0.4 14.4

fla6irostris mangyanus litoralis maculatus s.l.

(0.3) (0.5) (0.3) (0.5)

Number of mosquitoes/host/night (S.E.).

tively). An. maculatus s.l. also showed a broad feeding time but an earlier mean feeding time of 23:40 and extending until dawn (Table 3).

3.2. Vector sur6i6al Parous rates were determined on mosquitoes caught in Jan–Feb 1993. All mosquitoes were dissected, but since the bulk of mosquitoes came from carabao catches, the parous rates reflect the carabao baited catch. An. fla6irostris had a high parous rate of 0.76 implying an average survival of 4.2 feeds (Saul et al., 1990). An. maculatus s.l. gave a similar parous rate for all collections of 0.72.

3.3. Host seeking preferences Over 7 times more An. fla6irostris were caught in carabao-baited traps than in parallel human landing catches (Table 4). An. maculatus s.l. proved to be even more zoophilic, with nearly 50 times more mosquitoes being caught in a carabao baited trap than on a person. Too few An. litoralis and An. mangyanus were caught to give an accurate measure of host preference, but they appear to be similar to An. fla6irostris. No substantial variation in the host-seeking preference of An. fla6irostris was seen with season (Fig. 3).

3.4. Indoor/outdoor feeding preferences Overall, An. fla6irostris showed little preference for indoor or outdoor feeding, with 375 caught indoors and 478 outdoors. There was a significant difference in the relative numbers caught indoors and outdoors, with outdoor feeding predominating in the evening (18:00 – 20:00) and indoor biting before dawn (04:00–06:00) (P B 0.001, Table 3, by goodness of fit test). Peak feeding occurred approximately 1 h later indoors (01:35 for indoor; 00:37, outdoor). There was no significant association between the relative number caught indoor and outdoor and the place and period of collection. In contrast, An. maculatus s.l. appeared to have a much clearer preference for outdoor feeding. The number of mosquitoes collected outdoors was nearly 3 times higher than those caught indoors.

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4. Discussion These studies confirm the relative importance of An. fla6irostris as a principal vector in Morong, but at the same time show the difficulties in obtaining quantitative data with which to assess vectorial capacity in these low endemic areas. Even at peak season the average number caught was 3 mosquitoes per person-night for An. fla6irostris and much less for An. litoralis and An. mangyanus. Direct confirmation of the vector status by measuring sporozoite rates would be desirable. However, because of both the low endemicity of malaria, and the low abundance of mosquitoes, not surprisingly, attempts to determine sporozoite rates by dissections and by an immunoradiometric assay to detect the circumsprozoite protein failed (data not shown). An. mangyanus has a limited flight range and might have been abundant elsewhere. However, exhaustive coverage of fresh water larval breeding sites in the municipality yielded only a single larva. In addition, adult collection sites covered sitios where most of the transmission occurred. Thus, it is unlikely that An. mangyanus plays a significant role in malaria transmission in this area. An. litoralis is a brackish water breeder. As the collecting stations used to monitor vectors were not close to this species’ potential breeding sites, it is more likely that its contribution may have been underestimated, particularly in foci of malaria cases found in the north of the study area, beside the sea (Belizario et al., 1997). Even in areas with relatively high malaria prevalence, its abundance was very low. Since it is a strong flyer, nowhere in Morong would be outside the potential flight range from its preferred brackish breeding sites (Baisas, 1975). It is unlikely to be a significant vector in these areas. An. maculatus s.l. occurred more frequently. The abundance of adults and larvae closely paralleled An. fla6irostris, although other reports would suggest that An. maculatus is normally found in elevated areas. Its survival was also similar to An. fla6irostris. Thus the relative, potential contribution of these two mosquitoes depends on their anthropophilic behaviour. Observations on host seeking preference showed that 50 times more mosquitoes were caught in a carabao baited trap than on human bait. However, it was difficult to estimate how this translates into the human feeding rate. In these sitios there were fewer carabaos than people and there were other animals, especially goats, dogs and chickens. Further studies using traps baited with these animals may provide further information on the species feeding habit. The best estimates would come from direct measurements from blood meal analysis or from measurement of mosquito infection rates (Saul et al., 1990). In this area, both are impractical. Blood meal analysis requires mosquitoes to be sampled at random. Repeated attempts to find resting, fed mosquitoes failed and it is nearly impossible to obtain sufficient wild caught infected mosquitoes for an analysis of infection rates. Analysis of An. maculatus is further complicated by its uncertain taxonomic status. It is not clear if the two sibling species described by Rattanarithikul and Harbach (1990) occur in this area. If the population is markedly heterogeneous with respect to host feeding preferences with a small proportion feeding almost

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exclusively on humans and the remainder on animals, then the vectorial capacity for the human feeders could be as high as 0.8 at the peak of the transmission season. If the population was homogeneous, then given a higher zoophilic nature than An. fla6irostris, the contribution of An. maculatus to transmission would be negligible. Similar problems occur for estimating the vectorial capacity of An fla6irostris. Because of its high parous rate, this species has the potential for a substantial vectorial capacity in spite of its relatively low abundance. If all feeds occurred on humans, mean peak vectorial capacity would be 2.4 (assuming 4 feeding cycles in the extrinsic incubation period). However, this is an upper limit and the true value is likely to be much lower. In the comparison of host seeking preference, approximately 8 times more mosquitoes were caught from the carabao baited trap than from humans. This is in agreement with the report by Russell et al. (1963) who described its feeding behaviour as zoophilic although Schultz (1993) reported that An. fla6irostris at a study site on the southern island of Palawan had more anthropophagic tendencies. We have no information on the relative feeding preferences on other domestic animals in Morong. If the measured host seeking preference is indicative of the human blood index, then the mean vectorial capacity would be 0.3 over all sitios sampled. Human landing catches at Anahao and Matiko peaked during the low transmission season. On the basis of the prevalence of high seropositivity, neither of these sitios would be regarded as having relatively high malaria prevalence (Belizario et al., 1997). A focal epidemic of malaria occurred in Anahao in 1991 (Cheng et al., 1993). The impression is that malaria in these sitios is episodic rather than continuous and presumably relates to periodic mosquito abundance. The finding of peak mosquito numbers in the wet season suggests that breeding of An. fla6irostris has occurred in sites other than the usual stream pool environment and larvae were found in rice fields and irrigation canals at Anahao in the wet season. Although these may not make a substantial contribution to overall transmission, they may be important in the maintenance of malaria during the low transmission season. Further work is required to clarify this issue. More An. fla6irostris were caught on humans outdoors in agreement with the observations of Catangui et al. (1985). However, the difference between outdoor and indoor catches was not great, suggesting that this vector has little preference and that the place of feeding will reflect the distribution of people during the time the mosquito is seeking a meal. Catangui et al. (1985) also noted that transmission normally takes place both inside and outside the houses. The slightly later mean feeding time indoors suggests that the open house construction in the area may increase searching time but still allows the vectors to enter easily and leave soon after feeding (Hamon et al., 1970; Schultz, 1993). The feeding times observed in our study are in broad agreement with the peak feeding times of 23:30 to 02:30 reported by Chow (1970), although our data indicate a broader range extending from 20:00 to 04:00, especially in the peak transmission season. Espino et al. (1997) observed human behaviour through the night. Although there was some activity through the night, most people were asleep by 20:00.

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The results of this study have a number of implications for better malaria control specifically in Morong, but generally in similar relatively low endemic regions. Approximately half the malaria cases detected by active case detection or passive case detection occur in a small number of sitios (Belizario et al., 1997). These sitios are located in close proximity to sites where An. fla6irostris larvae were found (e.g. compare Fig. 1 with Fig. 1 of Belizario et al., 1997). Furthermore, in these sitios,the close association between seasonal abundance of An. fla6irostris and malaria, strongly suggests that the transmission is occurring within these sitios. This localised transmission, the late night biting profile of An. fla6irostris, and its propensity to feed on carabao should make this transmission particularly amenable to control with treated bed nets. Similarly, its localised breeding sites in pools in stream beds and the close association between vector abundance and malaria also suggests that environmental management could lead to substantial reductions in both mosquito numbers and malaria cases. However, detailed investigation of the local beliefs about malaria (Espino et al., 1997) shows that a high proportion of people do not believe that mosquitoes play a key role in transmission. Until education programs can reverse this view, better malaria control through vector control is likely to be difficult to implement.

Acknowledgements We wish to thank the staff of the Malaria Control Service in Bataan and the Rural Health Unit in Morong; the residents of Morong for their kindness and cooperation; the technical assistance of Dr Ponciano Epino and Leonard Bueno in the collection of mosquitoes and the technical support of the staff of the Department of Parasitology and Medical Entomology, Research Institute for Tropical Medicine. Special thanks are due to Annelyn Aguirre for statistical assistance, Dr Mary Ann Lansang, Des Foley and Dr Joan Bryan for useful comments on the manuscript. This research was supported by a National Insititutes of Health Tropical Medicine Research Center Grant No. SRC (55) 5P50 AI 030601-02.

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