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Blackwell Publishing Ltd

RESEARCH PAPER

Seasonal distribution and species composition of daytime biting mosquitoes

Waseem AKRAM

1

, Faisal HAFEEZ

1

, Unsar Naeem ULLAH

1

, Yeon Kook KIM

2

, Aftab HUSSAIN

3

and Jong-Jin LEE

2

1 Department of Agri-Entomology, University of Agriculture, Faisalabad, Pakistan2 Chonbuk National University, Chonju, South Korea3 Council of Governments (COG), Environmental Health Bureau, Arlington, Virginia, USA

Correspondence

Waseem Akram, Department of Agri-Entomology, University of Agriculture, Faisalabad, Pakistan. Email: areeba14@yahoo.com

Received 8 September 2008; accepted 28 September 2008.

doi: 10.1111/j.1748-5967.2009.00204.x

Abstract

Adults and immatures of

Aedes

mosquito populations were collected at temperaturesbetween 40 and 44

°

C (summer), while larvae were collected at 0

°

C (winter). Majormosquito activities were observed from February to mid-December at variouscollection sites that yielded high populations of

Aedes

spp. from May to September,and high populations of

Culex

spp. and

Anopheles

spp. from March to September.In June to July, mosquito activity was suspended because the relative humidity washigh (70%); a result of the monsoon rains. In August, with temperature rangingfrom 38 to 42

°

C, the populations of

Culex

,

Anopheles

and

Aedes

began to increase(36.8, 32.1 and 26.3%, respectively). Population estimates (through standardprototype Centers for Disease Control (CDC) and Biogents (BG)-sentinel) andspecies composition of

Aedes

in forest habitats indicated a rapid increase inthe populations of

Ae. albopictus

(52.3%),

Ae. aegypti

(19.1%) and

Ae. vittatus

(28.5%) following the rainy season in July. Areas positive for

Ae. albopictus

hadidentical population levels and distribution ranges of

Ae. vittatus

, however,there were no

Ae. aegypti

in

Ae. albopictus

areas from August to September. Thepopulation level, seasonal distribution, habitat and areas of adult activity markedby global positioning system (GPS) coordinates are being used for reference andfor species composition data of

Anopheles

spp. (2),

Culex

spp. (10) and

Aedes

spp.(5) in addition to associated temperature, relative humidity and physico-chemicalfactors of larval habitat. Global meteorological changes have caused an expansionof the active period, leading to the mosquito’s possibility of being a vector of diseaseincreasing, resulting in the spread of dengue fever.

Key words:

Aedes

, seasonal distribution, species composition.

Introduction

Mosquitoes are an annoyance as well as potentialtransmitters of zoonotic and human diseases, and as suchthey affect outdoor and recreational activities. Species varyin their diurnal and nocturnal activities with regards totemperatures at which they are active as well as theirenvironment (urban and forest habitats) and seasonal

population densities. In Pakistan, some species are activethroughout the year, even at temperatures <0

°

C. Seasonalchanges are cyclic and largely predictable, and represent thestrongest external influence in relative population densities(Fretwell 1972; Wingfield & Kenagy 1991; Blank 1992).Despite the pervasive nature of seasonality, exploring itsconsequences for population dynamics is a challenge forecologists, partly because seasonal mechanisms can be

W. Akram

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difficult to pinpoint empirically and can generate complexpopulation fluctuation (Nunn & Altizer 2006). The effect ofvarious forces on the environment has been discussed byworkers such as Reisen

et al.

(1981), Savage

et al.

(1989),Almiron and Brewer (1996) and Rajnikant

et al.

(1998), whodemonstrated interspecific associations of mosquitoes withphysicochemical and biological composition of mosquitobreeding sites. Aditya

et al.

(2006) conducted studies oflarval habitats and temporal variations in mosquito diversityfor a qualitative and quantitative assessment of mosquitodistribution.

Abiotic factors play a vital role in mosquito developmentand thus affect population density (Bradshaw 2000).Precipitation and environmental conditions followingperiods of rainfall (wind speed and temperature) affectbiting activity. Rainfall and accumulated water, watervelocity, and upper and daily temperature limits (Hanson1995) define the accumulation of thermal units necessaryfor larval development and emergence of mosquitopopulations.

Over-populated urban areas and the lack of recycling unitslike artificial containers (cans, vases, water tanks) increasesthe potential for increased container breeding of mosquitoesand transmission of disease in densely populated humandwellings. Environmental factors have thus resulted in theestablishment of new mosquito breeding areas and overallthe geographical distribution of mosquitoes has been extendedto new localities. In light of growing environmental concernsand increasing temperatures, we focused our studies on annualdistributional patterns of selected mosquito species fromvarious larval habitats.

Materials and methods

Seasonal distribution and species composition studies wereconducted from May 2006 to October 2007 at selected sitesacross different environmental and population zones ofPunjab province. Longitude, latitude and elevation ofsampled sites were determined using a global positioningsystem (GPS) (Magellan Explorist 600; China).

Mosquito sampling

Adult mosquitoes were collected either by L-shaped aspirator(Fulton, USA) from indoors or by long-handle aspirator (Mag-Lite, USA) from concealed places such as within bamboo,wooden blocks and automobile tires. Hand nets (6

×

5 ft) werealso used for collection during the morning, midday, afternoonand evening. Collection of immature mosquitoes was conductedconcurrently by the dipping and netting methods accordingto WHO (1975) guidelines. Identification was based on adultas well as immature characters using available taxonomic keys.Natural and man-made (container) collections were made at

each site. In addition, artificial containers (e.g. automobile tiresand painted cans) were placed and sampled at specific sites.

Species associations were identified for both adult andimmature collections. Habitat characteristics (e.g. water depth,natural/artificial, permanent/temporary, shady/lighted, vegetationcondition and turbidity) for each of the collection sites wererecorded. This study focused on the seasonal distribution ofvarious mosquito species (Nicolescu 1995) for which thesampling of adults with different trapping/sampling methodsand larvae was carried out from 2006 to 2007.

Adults

Centers for Disease Control (CDC) miniature, Biogents (BG)-sentinal and magnetic light traps operated with octanolwere used to sample adult mosquitoes during 2006 to 2007(Hatfield

et al

. 1985). Adult collections were conducted at15-day intervals from May 2006 to October 2007. The trapswere hung approximately 1.7–2.0 m above ground levelfrom a tree branch in a protected location. After each 24 hof collection, mosquitoes were released in the cages andprovided with a blood meal in the cage (laboratory rat) and a10% sugar solution. Both male and female mosquitoes werecaptured in the traps, however, the number and proportion ofeach sex varied by type of trap and species. Generally speaking,fewer males than females were trapped. The egg potentialand the duration of the life stages of the mosquitoes wererecorded, and dead individuals were collected and identified.

Larvae

Mosquito larvae were collected by a standard mosquito larvaldipper (Clarke, USA) on days when adult trap collection wasset out. All artificial habitats, such as ponds, plastic bags, rainpools, puddles, tins and discarded tires, were sampled forlarvae. The number of dips was based on the area of thesample site and the amount of water present. In cases wherefew mosquito larvae were collected, additional samples weremade to ensure uniform coverage of the entire locality. Thelarvae and pupae were removed from each scoop with an eyedropper (Kent

et al

. 1987) and were placed into 250 mLplastic bottles with water from the sampling site. The larvaewere classified on the basis of instar and were placed in Petridishes. Fourth instars were preserved in vials containing 35%isopropyl alcohol and a drop of glycerin (Akram & Lee2004). Adults were obtained from pupae, and were later usedfor identification purposes. Date-wise data on the number oflarvae and pupae were collected. A brief description of thehabitat and the mapped locality (GPS) were recorded for alllarval and adult collection sites. The habitat descriptionincluded detailed tabulation of parameters comprising thearea of the habitat, plant community and type of bottom(aquatic fauna and flora, including leaves, grasses, mud etc).

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The quality of water was assessed by measuring conductivity,pH, temperature, dissolved oxygen and total dissolved salt (TDS).Because the maximum larval life cycle is approximately9 days during the summer months, all collection sites wererevisited during this period to observe change in larval abundanceand species composition over time.

Mosquito trap sites, 2006

Traps were made at six sampling sites from May 2006 to October2007. Collection sites were at Multan (1), Chechawatni (2),Dera Ghazi Khan (DGK) (3), Sialkot (4), Changa Manga (5)a forest area near Faisalabad (6), Gujranwala (7), Sarghoda(8), Chakwal (9), Rawalpindi (10), Taxilla (11), Wah (12),Chattar (13) and Murree (14). These sites were located infour ecological zones of Punjab (Anonymous 2004). Themajor type of habitat in these sites was plain to desert, sub-mountainous to mountainous, and dry to relatively wet areascharacterized by natural water reservoirs, and man-madepolluting sources in the form of plastic bags, cans, vases,pots, fast food wrappers etc. Populations collected near dams,rivers and seasonally flooded ditches were also observedhaving temporary pools and ponds. Habitat types were inurban, suburban or rural areas. The traps were installed over3 days during the peak monsoon season in forest areas thatwere visited by picnickers, and had water canals, abundantand diverse thick vegetation and fauna.

Ecological Zone 1

These sites were characterized as being in the center ofcities with large human populations. The areas includeddisposal tanks (1), covered and uncovered sewerage lines(2), open garbage containers (3), hospital garbage outlets(4), hospital disposals bags, syringes and medicines (5),hedges (6), domestic vegetable gardens (7), bins (8) andgarages (9).

Ecological Zone 2

These sites included rice growing areas and the humanpopulation. Mosquitoes were trapped in temporary andpermanent water catchment areas within a kilometer of ricefields that represented the boundaries of farmhouses and cropareas. Immature mosquitoes were collected from puddles;adults were trapped from rice fields during the day andevening and from human dwellings at night. The mosquitopopulation was recorded from livestock farms. Nets (6

×

5 ft)were generally placed in the farmhouses for the collection ofmosquitoes from farm animals.

Ecological Zone 3

This site generally included mosquitoes collected frompools, small dark caves and thick vegetation along with waterreservoirs, standing or flowing water, near old temples.

Adults were identified under a M3300-D Swift (China)microscope. The egg potential and the duration of the adults wererecorded. Data collected was subjected to statistical analysis.

Results and discussion

Data collected from May 2006 to June 2007 on seasonaldistribution and species composition of mosquitoes revealedthat for 14 of the total 17 months of collection (Fig. 1), thepopulation of mosquitoes was high during different times ofthe year in Chakwal (9), Changa Manga (5), Chattar (13),Chechawatni (2), DGK (3), Faisalabad (6), Gujranwala (7),Murree (14), Multan (1), Rawalpindi (10), Sarghoda (8),Sialkot (4), Taxilla (11) and Wah (12). Our findings aresimilar to those of Rifaat

et al.

(1970) and Alten

et al.

(2000),who reported that the daytime biting mosquito speciescaptured throughout the study period were most abundant inlate summer (Fig. 1). Overall, the population of

Aedes

washigh from May to September, with peaks between mid-Juneto end-September, followed by a decline to December thatpersisted to March of the next year. A downward populationtrend was recorded between 45 to 50

°

C with relativehumidity of 45%, however, in the shady areas even duringJune to July high populations of

Aedes

were recorded. Formost of the mosquito species, more individuals werecollected during the high season months (March to October)than in the low season months (January to Mid February).Average population counts for

Anopheles

,

Culex

and

Aedes

were in the ratio to 50 : 90 : 18 (Fig. 1a). The population thatwas increasing revealed

Culex

(36.8%),

Anopheles

(32.1%)and

Aedes

(26.3%) in greater numbers in August withtemperature more or less in the range of 40 ± 2

°

C.

Habitat characteristics of breeding areas

Different types of trap have been used to collect mosquitoes,with varying degrees of captured individuals. The typesof attractant used in these traps include carbon dioxide andoctenol (1-octen-3-ol), a component of ox odor (Clements1999) also known to occur in human sweat. Takken and Kline(1989) were the first to investigate the attractiveness of theattractants to mosquitoes and found octenol and carbondioxide to be synergistic in attracting different mosquitoes.

Aedes

populations showed peaks in limited areas (notwidely distributed in all collection sites) such as tires (9) anddisposal areas of hospitals (5) (where most of a hospital’sused items are dumped) – these are effective breeding sitesof daytime biting mosquitoes. Bushes around the homes (6).This study has been the first in which five species of

Aedes

were collected from Punjab.

Aedes aegypti

was unique inthe sense that it had a limited distribution and was confinedto Lahore and Sheikhupura (Fig. 2).

Culex

(Figs 3,4) and itsspecies were found to be active even during the daytime in

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farmhouses where livestock and poultry were kept (Davidson& Peairs 1966). It was the only population other than

Aedes

that was collected. Felipe

et al.

(1990) reported that

Culex pipiens

and

Anopheles

is present in rural environments. Females

concentrate inside cattle shelters in high densities, reachingseveral thousand individuals per shelter in some areas andsome periods of time. In our study, maximum populationsof

Culex

were recorded in human dwellings and animal

Figure 2 Aedes and the most commonly recorded species in 2006.At Habitat 2, Aedes albopictus was found in tires. At Habitat 6, Ae. albopictuswas found in hedges and small disposables (syringes, cut drip bags etc.).

Figure 1 (a) Temperature (�) versus totaltraps (�). Population trapped during May toApril. (b) Population status and related urbanhabitats.

Figure 3 Culex species and associated habitats.

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barns, followed by

Aedes

, which was high in barns only(Fig. 5).

Adults trapped through the BG-Sentinel (BGS) designedin our laboratory were primarily used to sample

Aedesalbopictus

and

Ae. aegypti

. Human sweat in used vests andsocks along with octenol-dipped cotton buds were used aslures. The BGS traps were found to be more effective thanCDC traps in attracting

Aedes

(Williams

et al.

2006; Maciel-de-Freitas

et al.

2006). Environmental factors such as temperatureand relative humidity were found to be promising predictorsof mosquito distribution (Pemola & Jauhari 2005). One ofthe most important factors favoring the distribution of

Aedes

was vegetation, which favors larval propagation and adultswarming in shady areas (Fig. 1). Habitat and climate bothhave a correlation with the mosquito species that arepresent in an area. Therefore, it was felt that a comprehensiveunderstanding of qualitative and quantitative parameters(physicochemical and biological) of breeding habitatsare essential, as these generally represent a specific mosquitospecies with its seasonal progression, as has been observedwhen we surveyed rural, urban and suburban areas of Punjab.The association of species was ultimately related to thesimilarity of their breeding habitats. The present study is thefirst attempt to understand mosquito associations in differentbreeding habitats in various localities of Punjab, where

daytime biting mosquitoes have been breeding extensively,and thus severe problems with dengue fever have arisen.

Positive breeding habitats and their quantitative characters(dissolved oxygen (DO), electric conductivity (EC), pH) andqualitative characters (natural/artificial, permanent/temporary,shady/lighted, stagnant water, flora and fauna, turbidity)play an important role in defining the presence or absenceof different mosquito species. The breeding habitats ofmosquitoes (Hall 1987) used in the present studies wereold tires, cans, water channel beds, rice fields, tanks, forestpools, ditches, streams, tree holes, artificial fiber objectsand containers. Breeding habitats such as water channelsand artificial rock pools were the richest habitats, with amaximum number of mosquito larvae of all stages in limitedquantities of water. We recorded most of the genera withtheir species from natural habitats in quiet/stagnant condi-tions at shallow depths – less than a tablespoon of water.

Aedes

, in particular total number of species, preferredpartially shady and temporary water habitats (Akram & Lee2004). In the artificial rock pools, very limited vegetationwith most of the water had very high mosquito populationsand the change in the altitude defined species diversity;that is, at low altitude

Ae. albopictus

was abundant while athigher attitude only

Ae. vittatus

was common. Mosquitospecies from seepage pools, rice fields, tanks, streams androck holes, partially shady habitats with slow-flowing tur-bid–foul and clear water were the main characters of water inthe old civilization (Fig. 4). Populations recorded from aban-doned areas included

Aedes

,

Culex

,

Armigers

and

Anophe-les

.

Culex

generally ranked as the leading genus, followedby

Aedes

in pools and temples.

Armigers

had compara-tively large populations in natural water reservoirs and vege-tation.

Anopheles

was present only in vegetation.

Aedes

species mostly preferred the habitats with dense vegeta-tion, while

Culex

had no preference for any particular habitat(Fig. 5). Immatures were also found in tree holes in which thewater depth varied from 0.1–0.5 m. Seepage pools, forestpools, ditches, rock holes, tree holes and containers were thecommon breeding habitats. The larvae of

Ae. albopictus

werefound to breed (Linley

et al.

1991) in all habitats exceptditches, with a preference for shady to partially shadyareas. Our study results are in agreement with the findingsof Savage

et al.

(1990), Rajmankova et al. (1991, 1992),Rodriguez et al. (1993), Almiron and Brewer (1996) andRajnikant et al. (1996), as almost all of these researchersused the same set of habitat conditions, physico-chemicalcharacteristics of the water, and the flora and fauna livinginside it. All of these researchers were of the view that thedistribution of the diversified flora and fauna in the water is thebest indicator of the total mosquito population in any particulartype of habitat, irrespective of the species. In the presentstudy, mosquito immatures found in turbid water were almostalways Culicines, which is similar to the findings of Sattler

Figure 4 Different genera associated with particular habitats.

Figure 5 Mosquitoes and associated environments.

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et al. (2005). Overall, Ae. albopictus and Cx. pipens were themost abundant species, active for more than 10 months in ayear, while for rest of the species collected, activity was foronly 3 to 7 months.

The forest area Changa Manga is species rich area: it hadalmost all of the mosquito species that were collected fromother parts of the Punjab province. Population estimates(through standard prototype CDC and BG-sentinel traps)and species composition of Aedes in forest indicated a rapidincrease in the population of Ae. albopictus (52.3%), Ae. aegypti(19.1%) and Ae. vittatus (28.5%) following rain in July.

Considering the results of the present study in comparisonto earlier findings, it has been found that positive associationsbetween mosquito species might result from a commonpreference for a particular habitat, while negative associationsmight cause variation in preference for a particular habitat.Maximum immature association was recorded in habitatssuch as forest areas, artificial rock pools at various heightsand seepage pools, probably based on high survival rate,ovipositional preference and favorable physico-chemicalcharacteristics of these habitats, ranging from shady to partlyshady. The co-existence of more than one species in a habitatat a given time indicates that mosquito species of the samenature and preference interact with each other. For a betterunderstanding of the factors (physical and chemical charactersof water) we are continuously working to explore all of thefactors that are responsible in the distribution of the speciesand their richness.

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