Vector competence of Simulium oyapockense s.l. and S. incrustatum for Onchocerca volvulus:...

Acta Tropica 94 (2005) 139–158

Diurnal biting periodicity of parousSimulium(Diptera:Simuliidae) vectors in the onchocerciasis Amazonian focus

M.-E. Grilleta,b, N.J. Villamizarb, J. Cortezb, H.L. Frontadob,1,M. Escalonab, S. Vivas-Martınezb,c, M.-G. Basanezb,d,∗

a Laboratorio de Biolog´ıa de Vectores, Instituto de Zoolog´ıa Tropical, Facultad de Ciencias, Universidad Central de Venezuela,Apartado Postal 47072, Caracas 1041-A, Venezuela

b Centro Amazónico para Investigación y Control de Enfermedades Tropicales “Simón Bolıvar” (CAICET),Apartado Postal 59, Puerto Ayacucho 7101, Amazonas, Venezuela

c Departamento de Medicina Preventiva y Social, Escuela Luis Razetti, Facultad de Medicina, Universidad Central de Venezuela,Caracas 1041-A, Venezuela

d Department of Infectious Disease Epidemiology, Faculty of Medicine (St. Mary’s Campus), Imperial College,Norfolk Place, London W2 1PG, UK

Received 3 September 2004; received in revised form 1 January 2005; accepted 2 February 2005

Abstract

We describe the hourly patterns of parous biting activity of the three main simuliid vectors of human onchocerciasis in theAo nomamiv uming anu typ ge and1 aily cycles,p romotedb The resultsa d control.©

K iasis;S

M

0

mazonian focus straddling between Venezuela and Brazil, namely,Simulium guianenses.l. Wise;S. incrustatumLutz, andS.yapockenses.l. Floch and Abonnenc. Time series of the hourly numbers of host-seeking parous flies caught in five Yaillages during dry, rainy, and their transition periods from 1995 to 2001 were investigated using harmonic analysis (assnderlying circadian rhythm) and periodic correlation (based on Spearman’sr). ParousSguianenses.l. showed a bimodal activiattern, with a minor peak in mid-morning and a major peak at 16:00 h.S. incrustatumexhibited mainly unimodal activity durinither early morning or midday according to locality.S. oyapockenses.l. bit humans throughout the day mainly between 10:006:00 h but also showed bimodal periodicity in some localities. Superimposed on the endogenous, species-specific darous activity showed variation according to locality, season, air temperature and relative humidity, with biting being py warmer and drier hours during wet seasons/periods and reduced during hotter times in dry seasons or transitions.re discussed in terms of their implications for blackfly biology and ecology as well as onchocerciasis epidemiology an2005 Elsevier B.V. All rights reserved.

eywords: Simulium guianenses.l.;S. incrustatum; S. oyapockenses.l.; Time-series; Harmonic analysis; Host-seeking activity; Onchocercouthern Venezuela

∗ Corresponding author. Tel.: +44 207 5943295; fax: +44 207 4023927.E-mail address:[email protected] (M.-G. Basanez).

1 Present address: Instituto de Altos Estudios de Salud Publica “Dr. Arnoldo Gabaldon”, IAES-MSDS, Apartado Postal 2113,aracay 2101-A, Aragua, Venezuela.

001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.actatropica.2005.02.002

140 M.-E. Grillet et al. / Acta Tropica 94 (2005) 139–158

1. Introduction

Human onchocerciasis transmission and severity isthe result of continuous exposure of individual hosts tothe bites ofSimuliumLatreille vectors ofOnchocercavolvulusLeuckart. Consequently, quantification of thedegree of host exposure, measured by vector densityand the biting rate of flies on humans is important toparameterize models for transmission dynamics andcontrol (Renz and Wenk, 1987; Dye, 1994; Basanez etal., 2002). The most commonly used indicator of thedegree of human exposure in onchocerciasis studies isthe annual biting rate (Duke, 1968). This parameter,which describes the yearly number of flies attemptingto obtain a blood-meal on a human, represents moretruly a ‘landing’ than a ‘biting’ rate thus overestimatingexposure. Being a summary measure, the annual bitingrate gives no real indication of the temporal and spatialvariation in both blackfly abundance and human expo-sure (Renz, 1987). Investigation of the monthly, daily,and hourly variation in blackfly biting activity, and inparticular of the parous component of the fly popula-tion, would permit identification of months of the yearand hours of the day during which transmission of on-chocerciasis is potentially highest (Renz, 1987). Ide-ally, these studies should be accompanied by estimatesof the amount of time that populations at risk spendon activities that expose them to vector bites duringthese months and hours, in order to obtain more accu-rate estimates of contact rates for transmission models(

hilics ns-m ela,nL .l.S tude,l bit-i on-t llyt cus( ;G -i thehns 40p high

vector competence (Takaoka et al., 1984; Basanez etal., 1995; Grillet et al., 2000, 2001). S. incrustatumplays a secondary vectorial role in hyperendemic set-tings, predominating mainly during the rains, rainy-dry and early dry seasons when its parous (and prob-ably survival) rates are highest (median = 180 parousbites/person-day; range = 60–390) (Grillet et al., 2001).S. oyapockenses.l. is the prevalent species at low-land hypo- and mesoendemic forested areas, whereit bites in very large numbers (median = 1920 parousbites/person-day; range = 1500–3300) during the dryand rainy seasons, compensating for its low vectorcompetence (Shelley et al., 1987; Basanez et al., 1988,1995; Vivas-Martınez et al., 1998; Grillet et al., 2000,2001).

Our results have also suggested that seasonal fluctu-ations of parous populations are correlated mainly withriver level, with the dry season and the transition pe-riods between seasons potentially contributing most toonchocerciasis transmission (Grillet et al., 2001). Lessresearch has been conducted, however, on the patternsof hourly variation in biting activity for these simuliidspecies. Knowledge of these patterns would be impor-tant not only to help quantifying exposure but also toaid the design of sampling protocols that would maxi-mize the efficiency of rapid entomological assessment(REntA) methods for the monitoring and evaluation ofivermectin-based control programmes (Basanez et al.,1998; Vieira et al., 2005).

iv-s

ofge-h asid-

ks

sea-andthe,tyhicoftion

d

Bockarie and Davies, 1990; Renz et al., 1987).We have already shown that three anthropop

imuliid species are involved in onchocerciasis traission in the Amazonian focus of southern VenezuamelySimulium guianenseWise s.l.,S. incrustatumutz, andS. oyapockenseFloch and Abonnenc such studies have also revealed that spatial (alti

ocality) and temporal (seasonal) variation in theng activity of these species, together with their crasting vectorial efficiencies contribute differentiao exposure and transmission in this endemic foBasanez et al., 1988; Vivas-Martınez et al., 1998rillet et al., 2001). S. guianenses.l. prevails dur

ng the transition between dry and wet seasons inighland hyperendemic areas. In these areas,S. guia-enses.l. acts as the main vector ofO. volvulusde-pite its relatively low parous biting rate (median =arous bites/person-day; range = 4–350), due to its

Blackflies exhibit a 24-h pattern of rest and actity, presumably driven by natural circadian rhythmtimed or adapted to the predictable daily cyclelight and darkness, but readily modifiable by (exonous) weather and environmental conditions, sucwind speed, rainfall pattern, temperature and humity (Wenk, 1981; Crosskey, 1990). Adult females bitefrom dawn to dusk, but most activity occurs in peaaround particular times of the day.

Daily biting peaks are dependent on species,son, climatic conditions, parity status (e.g., parsnullipars seeking bloodmeals at different times ofday), location and host availability (Duke, 1968; Wenk1981; Crosskey, 1990). Since host-seeking periodiciis highly regulated by the duration of the gonotrop(blood feeding and oviposition) cycle, biting activityparous females may provide insights into the duraof such cycle (Cheke, 1995) and the timing of oviposi-tion (Duke, 1968). Short gonotrophic cycles and bloo

M.-E. Grillet et al. / Acta Tropica 94 (2005) 139–158 141

feeding soon after oviposition may translate into higherbiting frequency and increased vectorial potential ofthe simuliid species (Collins et al., 1981). The inter-val between two consecutive bloodmeals forms part ofthe calculation of the vector biting rate on humans, aparameter which appears twice (squared) in the expres-sions for vectorial capacity and basic reproductive ratioof the parasite (Dietz, 1982; Basanez and Boussinesq,1999). Consequently, epidemiological models are sen-sitive to variations in this parameter (Kennedy andBasanez, 2002; Kennedy et al., 2003).

The question of interest in this study was, there-fore, whether or not there is a discernable periodicityin the diurnal host-seeking activity of the three mainO.volvulusvectors in the Amazonian focus, an area whereresearch has so far been sparse (but seeLacey andCharlwood, 1980; Narbaiza, 1986; Basanez, 1992;Shelley et al., 1997). While Narbaiza (1986)andBasanez (1992)worked mainly in the Sierra Parimahighlands (≥250 m altitude), we focus here on lo-calities situated below 250 m. Our work forms partof a larger investigation into entomological risk fac-tors of human onchocerciasis, that together with par-asitological (Botto et al., 1997, 1999; Vivas-Martınezet al., 2000a) and epidemiological (Vivas-Martınez etal., 1998, 2000b; Carabin et al., 2003) research aimsat providing locale-specific data for parameterizationof mathematical models for the population dynamicsand control of the infection (Basanez and Ricardez-Esquinca, 2001).

2

2

fly-ce uti rnV ri,H longt sys-t 40 m( l andr pec-tT ature

of 26–27◦C, lying in a very humid tropical forest(Huber et al., 1984). Annual rainfall (3750–5000 mm)is seasonally unimodal, with May to August being therainiest months and October to March the driest. Thetransition between the dry and the wet season takesplace around April, and that between the rainy and dryseason around September (seeFig. 1 of Grillet et al.,2001).

2.2. Entomological methods

In each community, and during one to five consecu-tive days per visit, host-seeking flies that landed on twohuman attractants from the village were caught withmanual aspirators by a team of two collectors (work-ing during alternate hours) during the first 30 min ofeach hour, from 07:00 to 18:30 h. This amounted to12 half-hour intervals or sampling units (hereafter re-ferred to as the collection hour) per sampling day. Fe-males were collected before procuring a blood-meal,thus what we refer to as biting rates are more trulylanding rates. However, we will assume, for the pur-poses of this paper, that the number of flies that wouldhave obtained a blood-meal (had they been allowed todo so) is proportional to the number of females alight-ing. Whenever possible, the collectors were the samethroughout the study (NJV and JC) in order to min-imize variations resulting from individual differencesin catching ability. In the field, all hourly-caught flieswere anaesthetized with chloroform vapour, identifiedt s ac-co thek .( 01,w othd achc achc airt edi re-s e-t

2

kingp iod-

. Materials and methods

.1. Study area

Descriptions of the study area and selection ofollection sites have been presented elsewhere (Grillett al., 2001). Entomological sampling was carried o

n five riverine Yanomami communities in southeenezuela: Maweti-theri, Mahekoto-theri, Awei-theasupiwei-theri, and Pashopeka-theri, situated a

he Ocamo–Putaco and Orinoco–Orinoquito riverems. The study villages were located between 1Maweti) and 240 m (Pashopeka) above sea leveanged in microfilarial prevalence between, resively, 24 and 80% (Vivas-Martınez et al., 1998).he area is characterized by an average temper

o species, counted, and dissected for parity statuording to criteria suggested byWenk (1981). Tax-nomic identification to morphospecies followedeys byRamırez-Perez et al. (1982)andShelley et al1997). Collections took place between 1995 and 20ith communities visited more than once to cover bry and rainy seasons (and their transitions) for eommunity. During each collecting day and site at eommunity, initial and final half-hourly readings ofemperature (◦C) and relative humidity (RH, expressn %) were conducted from 07:00 to 18:30 h with,pectively, a minimum–maximum mercury thermomer and a wet–dry bulb thermohygrometer.

.3. Variables of interest

We focused mainly on the number of host-seearous flies per hour to describe diurnal biting per


icity. Patterns of variation were analyzed both betweeni (i = 1–12) hours of a day and betweenj (j = 1–n) con-secutive days (n= 3–5). However, and in order to ob-tain an overall picture per species, locality, and sea-son, the average number of parous flies per collec-tion hour (Pi) was estimated using the geometric meanof Williams, which normalized the distribution of flycounts (Williams, 1937):

Pi =j=n∏

j=1

(Pi,j + 1)

1/n

− 1 (1)

The (asymmetrical) confidence intervals around thePi values were estimated according toKirkwood andSterne (2003).

2.4. Data analysis

Analyses were conducted according to species, lo-cality and season. The sequence of observations, i.e.,log(Pi,j + 1), was ordered along the (independent) timeaxis (hoursi (from 07:00 to 18:00) within each of theconsecutivej (from 1 ton) collection days and consid-ered as a time series dataset (36–60 log-transformedobservations per sampling visit). Assuming a cyclicaltrend in the data series (this is, a circadian cycle withperiod,T= 24 h), we aimed at identifying statisticallysignificant maxima or biting peaks in the population ofparous flies. To this end, we carried out a harmonic orp e,1 00o useste eri-o ana-t lyt

x

In Eq. (2), c is a constant that fits the position ofthe cosine along the abscissa, so that it correspondsto the time of minimum and maximum values in thedataset. Whenc= 0, for instance, the function peaks at12:00 h. In order to detect the extent to which the orig-inal data series of log(Pi,j + 1) observations exhibitedconcordant periodic variation with the transformedx′variable, correlation analyses using Spearman’sr wereconducted between these both variables by shiftingx′with respect to the number of parous flies, with time-lags ofc units (c= 1–6). The positive sign in Eq.(2)shifts the series forwards (towards 18:00 h) and the neg-ative sign shifts the series backwards (towards 06:00 h).The results thus obtained were plotted in a periodogram(Legendre and Legendre, 1998). Positively significantcorrelation(s) identifies the maximum (or maxima) bit-ing peak(s) during the day. Hence, the purpose of theanalysis is to identify which daylight hours best ex-plain the variance observed in the response variabley= log(Pi,j + 1). However, and for graphical purposesonly we show the (untransformed) hourly number ofparous flies per person (Pi,j ) versus collection hour andsuperimpose the best (most significant) periodic func-tion(s) in the figures described in Section3 of this re-port.

Separately, cross-correlation analyses were alsoconducted between the log-transformed numbers ofparous flies and hourly air temperature and relativehumidity values, shifting the series of the two lattervariables with respect toy with time-lags ofk-hours( n-d or-r sis.S ria-t d bya A).

ther ryv er-a a

F flies pe ur)f peka-t sis focuso seaso Awei-theri);b pril 199 The righth ry and (l t axis) en lines)i broken

eriodic correlation analysis (Legendre and Legendr998), which is well suited to the study of small (<1bservations) ecological data series. This method

he first term of a Fourier series (Eq.(2)) to analyzecological cyclic phenomena in which sinusoidal pdicity (e.g., circadian) may be expected. The expl

ory variablex (=hoursi of the day) is consequentransformed into a cyclic variablex′,

′ = cos

(2π(x ± c)

T

)(2)

ig. 1. Lower panels: Williams’ mean-hourly number of parousorSimuliumguianenses.l. in the Yanomami localities of (a) Pashof southern Venezuela. White diamonds and thin solid line: drylack circles and thick solid line: dry–rainy transition period (Aand axis of (a) (on a scale of 0–20) refers to both the Februa

ines on left axis) and relative humidity (%; thinner lines on righn (a) and for April 1999 (black solid lines) and May 2001 (grey

k= 0–2) and using Spearman’sr (Legendre and Legere, 1998). Since lagging the series did not improve celations, onlyk= 0 was used in subsequent analypatial (locality) and seasonal (sampling month) va

ions of the meteorological variables were explorenalysis of variance (one factor, fixed-effects ANOV

Finally, we explored the relationship betweenesponse variabley= log(Pi,j + 1) and the explanatoariables: hour (as, a cyclic variable), hourly tempture (in◦C), and relative humidity (in %) by using

r person (Pi) ± 95% confidence intervals vs. time of day (collection hoheri (Ps) and (b) Awei-theri (Aw) in the Amazonian onchocercian (February 2001 in Pashopeka-theri and November 1998 in9); grey triangles and broken line: rainy season (May 2001).May 2001 data. Upper panels: average-hourly temperatures◦C; thicker

for February 2001 (black solid lines) and May 2001 (grey broklines) in (b).


multiple regression model:

y = β0 + β1x′ + β2temp+ β3RH (3)

where the hourly temperature (temp) and relative hu-midity (RH) are the averages of the half-hourly read-ings corresponding to each collection hour. All statis-tical analyses were conducted using STATISTICATM

(StatSoft, 1994) and they were considered significantatP< 0.05.

2.5. Ethical considerations and conflicts of interest

The leaders of the communities were informed ofthe objectives of the study prior to the commencementof collections and participants freely consented to actas blackfly attractants. The presence of entomologicalteams in the communities was followed shortly by med-ical teams who conducted parasitological evaluationsand distributed mass ivermectin treatment. All fly at-tractants were therefore treated. There are no conflictsof interest to report.

3. Results

A total of 91,121 flies were caught during 118 sam-pling days between 1995 and 2001. Temperature andrelative humidity were very similar from one day to thenext for each sampling visit, but there was much varia-t enm lativeh itswF r-a ity,r5 -p ax-i esw anelso

3.1. Simulium guianense s.l.

This species prevailed in the villages of Pashopeka-and Awei-theri particularly during the dry–rainy tran-sition (April), being more abundant in the former(Fig. 1a) than in the latter (Fig. 1b) community. Al-though also collected in Hasupiwei- and Mahekoto-theri, its very low numbers in these villages precludedany analysis for these localities.S. guianensewas vir-tually absent in Maweti-theri. Overall, the host-seekingbehaviour of parous females showed a bimodal activ-ity at variable times of the day depending mainly onseason and to a lesser extent on locality (Fig. 1).

In the transition between the dry and rainy seasons,when the mean fly density reached its highest levels (upto 38 parous flies/person-hour at Pashopeka in April1999;Fig. 1a), parousS. guianenses.l. bit during ev-ery daylight hour, with two distinct maxima of activity(a lower peak in the morning, and a higher peak be-tween 15:00 and 17:00 h). According to the harmonicanalysis, the afternoon biting peak showed a statisti-cally significant correlation with 16:00 h at Pashopeka(rS = 0.30,n= 54,P< 0.01;Fig. 2b).

In the rainy season (May 2001 inFig. 1a forPashopeka- andFig. 1b for Awei-theri), there wasa considerable reduction of morning parous activ-ity, whereas the afternoon peak remained the highestand, again, showed a statistically significant correlationwith 16:00 h at Awei-theri (rS = 0.61,n= 36,P< 0.01;Fig. 2c). By the middle of the dry season (February2 iths peka(c ingl thed

wasl thef r al-t ifi-c re-s

F . flies c s) fore e of ha variationi ary 200 dotted line).( c funct ay 2001);h d line).

ion within each day. (The results of ANOVA betweean temperature and hour, and between mean reumidity and hour for all localities and sampling visere, respectively,F= 42.85, d.f. = 11,P< 0.001 and= 21.35, d.f. = 11,P< 0.001.) Within the day, tempeture was correlated inversely with relative humidanging from 23◦C and 96% at 07:00 h to 37◦C and1% at 12:00 h (rS =−0.84,P< 0.05,n= 276; all samles, localities, months, and hours). In general, m

mum temperature (and minimum humidity) valuere reached between 11:00 and 15:00 h (upper pf Figs. 1, 3, and 5).

ig. 2. Time series of the hourly number of parousS. guianenses.lach of the collection days in a sampling period, and amplitud

n parous fly density at (a) Pashopeka-theri, dry season (Februb) Pashopeka-theri, dry–rainy transition (April 1999); harmoniarmonic function peak = 15:00 (dashed line) and 16:00 (dotte

001) parous activity became mainly matinal, wignificant peaks at 07:00 and 09:00 h at PashorS = 0.50 for both peaks;n= 36;P< 0.05;Fig. 2a). Byontrast, at Awei-theri activity always peaked durate afternoon (17:00 h) throughout the whole ofry season (November 1998;Fig. 1b).

Relative humidity was higher and temperatureower at Awei-theri than at (Pashopeka) despiteact that Pashopeka (240 m) is located at a higheitude than Awei-theri (162 m). (There were signant differences between the five localities withpect to temperature:F= 9.26, d.f. = 4,P< 0.001, and

oming to feed on human attractant (solid grey lines on left axirmonic function(s) (broken lines on right axis) that best explain1); harmonic function peaks = 07:00 (dashed line) and 09:00 (

ion peak = 16:00 (dashed line). (c) Awei-theri, rainy season (M


Table 1Results of the multiple regression model (Eq.(3)) between the log-transformed number of parous flies per hour (y= log(Pi,j + 1)) and theexplanatory variables of hour (as a cyclic variable,x′), hourly temperature (temp), and relative humidity (RH)

Species Locality (date) Best predictor variable Coefficient (βi ± S.E.) t d.f. R2 P value

S. guianense Ps (February 2001) x′ (07:00 h) β1 = 0.52± 0.06 3.6 35 0.27 <0.001S. guianense Aw (May 2001) x′ (16:00 h) β1 = 0.58± 0.07 4.1 35 0.33 <0.001S. incrustatum Ps (January 1997) x′ (13:00 h) β1 = 0.32± 0.28 2.5 53 0.11 <0.05S. incrustatum Ps (June 1995) x′ (07:00 h) βι = 0.40± 0.07 3.3 58 0.16 <0.05S. incrustatum Ps (September 1997) x′ (07:00 h) β1 = 0.57± 0.07 5.3 59 0.40 <0.001S. incrustatum Aw (April 1999) RH β3 =−0.74± 1.73 −5.0 59 0.31 <0.05S. incrustatum Aw (May 2001) x′ (12:00 h) β1 = 0.65± 0.14 4.9 35 0.42 <0.001

Only the results for the significant coefficients are shown (Ps: Pashopeka-theri; Aw: Awei-their).

relative humidity:F= 2.93, d.f. = 4,P< 0.05, respec-tively.) There was more between-season variation inthese variables at Pashopeka than at Awei-theri (up-per panels ofFig. 1a and b, respectively). Duringthe rainy season of May 2001, the biting activity ofS. guianensewas positively associated with increas-ing temperature at both Pashopeka (rS = 0.41,n= 36,P< 0.05) and Awei-theri (rS = 0.42,n= 36,P< 0.05),with warmer hours (25–29◦C) promoting more bit-ing activity than cooler ones (<25◦C), and negativelyrelated with increasing humidity (rS =−0.35,n= 36,P< 0.05 andrS =−0.24,n= 36,P< 0.05, respectively),with a very wet time, i.e., >90% RH reducing fly ac-tivity (upper panels ofFig. 1a and b). The oppositewas observed at Pashopeka during February 2001 (dryseason), when correlations of parous flies were neg-ative with temperature (rS =−0.41, n= 36, P< 0.05)and positive with RH (rS = 0.45,n= 36,P< 0.05). Thehigh temperatures (>30◦C) and low humidity values(<60%) reached between midday and early afternoon,presumably, concentrated parous biting activity duringmorning hours (February 2001;Figs. 1a and 2a). InAweitheri, and during the dry–rainy transition of April1999, biting activity tracked temperature (rS = 0.33,n= 60,P< 0.01) and was inversely related with humid-ity (rS =−0.39,n= 60,P< 0.01) (Fig. 1b).

In those localities or sampling visits in which thebiting behaviour of parousS. guianensewas simulta-neously and significantly explained by the three ex-

planatory variables of hour, temperature, and humid-ity, most of the variation was explained by hour ratherthan by changes in the remaining two covariates asshown by the multiple regression results summarized inTable 1.

3.2. Simulium incrustatum

This species was collected solely at Pashopeka- andAwei-theri, being less abundant in the former (max-imum mean-hourly density of 28 parous flies/person)than in the latter (45 parous flies/person). At Pashopekavector density was higher during the rainy–dry tran-sition (September 1997) and dry (November 1998)seasons than during the rains (June 1995) (Fig. 3a).At Awei-theri, by contrast, parous biting activity wasless intense during the rainy–dry transition (Septem-ber 1998) than during the dry (November 1998) season(Fig. 3b). However, it must be stressed that these com-parisons are made between snapshots taken during dif-ferent years, and that perhaps more importantly are theconsistent patterns of activity that emerge across theyears. In this respect, correlation and harmonic anal-yses indicated the existence of a primarily unimodalpattern of diurnal activity for parousS. incrustatum,with the timing of maximum biting showing variabil-ity according to locality and to a lesser extent accordingto season. At Pashopeka, most biting activity was con-centrated in early morning (07:00–09:00 h) significant

F s flies ionh ashop in solidl line: ra s and thicks tembe fers to theJ cker lin xis)f olid lin 1998 (blacks

ig. 3. Lower panels: Williams’ mean-hourly number of parouour) forSimulium incrustatumin the Yanomami localities of (a) P

ine: dry season (November 1998); grey triangles and brokenolid line: rainy–dry transition (September 1997 in (a) and Sepune 1995 data. Upper panels: average-hourly temperatures (◦C; thior June 1995 (grey broken lines) and September 1997 (black solid lines) in (b).

per person (Pi) ± 95% confidence intervals vs. time of day (collecteka-theri (Ps) and (b) Awei-theri (Aw). White diamonds and th

iny season (June 1995 in (a) and July 1997 in (b); black circler 1998 in (b)). The right hand axis of (a) (on a scale of 0–20) rees on left axis) and relative humidity (%; thinner lines on right aes) in (a) and for July 1997 (grey broken lines) and September


peaks (rS = 0.60,n= 60, P< 0.01 for both 07:00 and08:00 peaks, September 1997, andrS = 0.40,n= 48,P< 0.01 for peak at 07:00, September 1998;Fig. 4a;rS = 0.56, n= 57, P< 0.01 for both 08:00 and 09:00peaks, November 1998;Fig. 4b). During these months(rainy–dry and dry seasons), parous fly activity wasnegatively associated with air temperature (rS =−0.35,n= 60, P< 0.01) and positively associated with rela-tive humidity (rS = 0.27,n= 60,P< 0.05) (upper panel

in Fig. 3a). This was particularly true in September1997, when high temperatures (>30◦C) and low hu-midity (<60%) values were reached during midday andearly afternoon. However, in January 1997 and May2001, when early morning temperatures were as lowas 23–24◦C, and relative humidity as high as 94%, thebiting peak shifted towards midday (data not shown).In fact, biting activity was occasionally significantlyexplained by changes in air temperature and relative

Fd(llA

ig. 4. Time series of the hourly number of host-seeking parousS. incrusays in a sampling period, and amplitude of harmonic function(s) (broa) Pashopeka-theri, rainy–dry transition; September 1997: dark greyine) and 08:00 (dotted line); (b) Pashopeka-theri, dry season (Novembine); (c) Awei-theri, rainy season (July 1997); harmonic function peakwei-theri, rainy–dry transition (September 1998); harmonic function

tatumflies (solid grey lines on left axis) for each of the collectionken lines on right axis) that best explain variation in parous fly density atand September 1998: light grey; harmonic function peaks = 07:00 (dasheder 1998); harmonic function peaks = 08:00 (dashed line) and 09:00 (dotted

s = 12:00 (dashed line), 13:00 (dotted line) and 14:00 (dot-dash line); (d):peaks = 09:00 (dashed line) and 10:00 (dotted line).


Fig. 4. (Continued).

humidity. For instance, early morning or late afternoonlower temperatures and wetter periods were associatedwith reduced parous activity during those periods inthe dry–rainy (April) and rainy (July) seasons at Awei-theri (July 1997;Fig. 3b), or during the dry season atPashopeka (November 1998;Fig. 3a).

In Awei-theri, and during the rains biting densityof parous females peaked between 12:00 and 15:00 h(Fig. 3b), and this was corroborated by correlation andharmonic analyses (rS = 0.36,rS = 0.33 andrS = 0.28,n= 59, P< 0.05 for, respectively, maxima at 12:00,13:00, and 14:00 h, July 1997;Fig. 4c), although there

was a small lull in biting around noon. During Septem-ber 1998 in particular, when midday temperatures andrelative humidity were, respectively, as high as 34◦Cand as low as 70%, peak biting activity shifted towardsmid-morning (08:00–11:00 h;Fig. 3b), and this wasalso highlighted by statistically significant peaks tak-ing place at 09:00 (rS = 0.56,n= 48,P< 0.01) and 10:00(rS = 0.54,n= 48,P< 0.01) hours in the harmonic anal-ysis (Fig. 4d). In general, however, the biting behaviourof parousS. incrustatumwas best explained by hourthan by daily changes in temperature and/or relativehumidity (Table 1).


3.3. Simulium oyapockense s.l.

This species occurred mainly at Maweti-theri (withan average of up to 420 parous flies/person-hourin May 1995), followed by Mahekoto-theri (260parous flies/person-hour in April 1999) and Hasupiwei-theri (140 parous flies/person-hour in April 1999)(Fig. 5a–c, respectively). In Pashopeka- and Awei-therithis species was less abundant but high parous fly densi-ties were seen during the dry season (February) of 2001(350 and 180 parous flies/person-hour in, respectively,Pashopeka- and Awei-theri).

ParousS. oyapockenses.l. displayed quite differ-ent biting patterns from those ofS. guianenseandS.incrustatum, with highly variable biting activity ac-cording to locality and sampling periods (Fig. 5). AtMaweti-theri (Fig. 5a), parous flies showed a relativelyconstant activity throughout the day, perhaps more pro-nounced between 10:00 and 16:00 h, but without anyprominent peak being picked up by the harmonic anal-ysis. In January 1997, a more clearly bimodal patternwas observed, with an early morning peak (starting at07:00 h), and a second (minor) peak between 16:00and 17:00 h (data not shown). Similarly, a relatively

F flies pe ur)f aweti-t n line)a transi koto-theri( broken lack thinl rcles a theri (Hs)irS(N(ll

ig. 5. Lower panels: Williams’ mean-hourly number of parousor Simulium oyapockenses.l. in the Yanomami localities of (a) Mnd June (white squares and thin solid line), and the rainy–dryMh) in the dry season of February 1997 (grey diamonds andine), and the rainy–dry transition of September 1997 (black ci
n the dry season of February 1997 (white diamonds and black thin liainy season of August 1997 (grey triangles and broken line); (d) Paeptember 1998 (black circles and thick line) and the dry seasons of
grey diamonds and broken line). In (d) and (e) the data for Septembovember 1998 are also plotted on the right axis. Upper panels: avera

%; thinner lines on right axis) for May (broken lines) and Septemberines) 1997 in (b); August 1997 (broken lines) and April 1999 (solid linines) in (d) and (e).

r person (Pi) ± 95% confidence intervals vs. time of day (collection hoheri (Mw) in the rainy seasons of May (grey triangles and broketion of September (black circles and thick line) 1995; (b) Maheline); the dry–rainy transition of April 1999 (white circles and bnd thick line on the right hand scale of 0–200); (c) Hasupiwei-
ne); dry–rainy transition of April 1999 (black circles and thick line), andshopeka-theri (Ps) and (e) Awei-theri (Aw) in the rainy–dry transition ofNovember 1998 (white diamonds and thin black line) and February 2001er 1998 are plotted on the right hand scale of 0–100. In (d) the data for
ge-hourly temperatures (◦C; thicker lines on left axis) and relative humidity(solid lines) 1995 in (a); February (broken lines) and September (solid

es) in (c); and September 1998 (solid lines) and February 2001 (broken


constant pattern of activity was observed throughoutthe day during the dry season and rainy–dry transi-tion (respectively February and September 1997) atMahekoto-theri (Fig. 5b). The pattern observed dur-ing the dry–rainy transition of April 1999 tends toecho that of the rainy–dry transition (Fig. 5b). AtHasupiwei-theri, parous fly activity showed a morebimodal pattern, with biting peaks during the morn-ing (08:00–11:00) and afternoon (15:00–17:00) hours(Fig. 5c). However, none of the Spearman correlations

in the harmonic analysis were statistically significantfor Hasupiwei.

In Pashopeka, where the parous fly density ofS.oyapockensewas low by comparison to that of thelower altitude localities of Maweti-, Mahekoto-, andHasupiwei-theri (<200 m), parous biting activity in-tensified in the afternoon, peaking between 15:00 and18:00 h (Fig. 5d) with a significant maximum de-tected at 17:00 h (rS = 0.43,n= 46,P< 0.01, Septem-ber 1998;Fig. 6a). By contrast, and during February

Fcdr

ig. 6. Time series of the hourly number of parousS. oyapockenses.l. fliollection days in a sampling period, and amplitude of harmonic functensity variation at (a): Pashopeka-theri, rainy–dry transition (Septemainy–dry transition (September 1998: light grey) and dry season (No

es caught on human host (solid lines on left axis) for each of theion(s) (broken lines on right axis) that best explain variation in parous flyber 1998); harmonic function peak = 17:00 (dashed line); (b): Awei-theri,

vember 1998: dark grey); harmonic function peak = 17:00 (dashed line).


2001, when the density of parousS. oyapockenseatthis locality was unusually high, parous biting activ-ity was concentrated in the morning (from 08:00 to12:00) followed by a steady decline during the after-noon (Fig. 5d). Finally, the dominant trend in the host-seeking parous population at Awei-theri was primar-ily unimodal, with parous females predominantly bit-ing between 15:00 and 18:00 h during the more typicaldensities of September and November 1998 (Fig. 5e)with a significant peak again at 17:00 h (rS = 0.41,n= 48,P< 0.01, September 1998, andrS = 0.50,n= 57,P< 0.01, November 1998;Fig. 6b), or shifting towardsthe morning (between 08:00 and 11:00 h) during the un-usually high fly densities recorded in February 2001,with a minor (non-significant) peak at 17:00 h (Fig. 5e).

Mostly, host-seeking behaviour ofS. oyapockensein relation to the recorded meteorological variablestended to be positively correlated with air temper-ature and inversely correlated with relative humid-ity in Maweti (respectively,rS = 0.35 andrS =−0.24,n= 53, P< 0.05 andP= 0.07 in May 1995;rS = 0.42and rS =−0.47, n= 22, P< 0.05 in September 1995;Fig. 5a); Mahekoto (rS = 0.34 andrS =−0.30,n= 60,P< 0.01 in September 1997;rS =−0.36 (RH),n= 60,P< 0.01 in February 1997;Fig. 5b); Hasupiwei-theri(rS =−0.39 (RH), n= 54, P< 0.01 in August 1997;Fig. 5c); Pashopeka (rS = 0.33 (temperature),n= 46,P< 0.05 in September 1998;Fig. 5d), and Awei-theri(rS = 0.42 andrS =−0.56 n = 36,P< 0.01 in February2001;Fig. 5e). In particular during the rains, warmer( oler( thel es andr

4

uth-e dic-i n iss r de-g thercs bit-i tly,m -

modal activity, with biting peaks during the morningor, under some circumstances, at midday.S. oyapock-enses.l., on the contrary, bit throughout the day, mainlybetween 10:00 and 16:00 h (although bimodal period-icity was sometimes observed). These results are inbroad agreement with those of previous studies (Laceyand Charlwood, 1980; Narbaiza, 1986; Shelley et al.,1997).

4.1. The role of endogenous, species-specificcycles

Species-specific hourly biting patterns were gen-erally consistent throughout the study period in spiteof seasonal and yearly variations, as hour of the daywas by and large the best predictor of parous fe-male biting activity at each of the study localities.This suggests that host-seeking periodicity in thesesimuliid species is largely determined by endogenousrhythms probably driven by blood feeding and oviposi-tion cycles.Dalmat (1955), working with Guatemalanblackflies (S. ochraceum, S. metallicum, S. callidum)andAlverson and Noblet (1976), investigating NorthAmerican simuliids (S. slossonaeamong others) foundthat time of the day was the variable best explaining ob-served patterns of host-seeking activity in such black-flies, and suggested the operation of circadian controlmechanisms. Similar arguments have been proposedby Lacey and Charlwood (1980)for Brazilian simuli-ids includingS. guianenses.l. However, it is clear thatb edo un-f ion,f par-t ns.

4

thel ngesi flu-e e andm , lo-c ationm thea hel onalv

and drier) hours promoted more biting than co<24◦C) and wetter periods (>90% RH). In none ofocalities was the biting behaviour ofS. oyapockens.l. simultaneously explained by hour, temperatureelative humidity.

. Discussion

In the Amazonian onchocerciasis focus of sorn Venezuela, it can be said that the biting perio

ty of the three vector species under investigatiotrongly species-specific but influenced, to a lesseree, by locality, season, and daily changes in weaonditions. The parous females ofS. guianenses.l.howed a predominantly bimodal activity pattern,ng mainly during mid-morning and more prominen

id-afternoon.S. incrustatumexhibited mainly uni

iting periodicity is also readily modifiable (promotr suppressed) by environmental (favourable or

avourable) factors that may influence host locateeding or egg-laying, helping to explain some deures from otherwise well-defined fly activity patter

.2. The role of overall fly abundance

For each species and locality, daily variation inevel of host-seeking activity may be caused by chan overall fly abundance. Adult fly abundance is innced, among other factors, by rates of emergencortality, which may vary seasonally. For instance

ally unsuitable breeding sites due to seasonal variight affect negatively the rate of recruitment todult population which, in turn, might influence t

evel of biting. We have already shown that seasariation of parous biting rates ofS. incrustatumand


S. oyapockenses.l. are strongly correlated with riverlevels and breeding site dynamics in the study locali-ties (Grillet et al., 2001). In this report,S. guianenses.l. andS. oyapockenses.l. showed a greater degree ofseasonal variation in biting pattern thanS. incrustatum,which could be explained in part by daily and seasonalvariations in the rates of recruitment to the biting popu-lation. It is expected that as fly density reaches its max-imum, observed periodicity will increasingly reflecttrue underlying host-seeking patterns as the rhythmsof individual flies will sink in phase (or in synchrony)with waves of communal emergence and ovipositionat population level (Corbet, 1966). InS. guianenses.l.,the greatest degree of variation in the timing of bitingpeaks was observed at Awei-theri, where the speciesshowed lower biting parous rates than at Pashopeka(Fig. 1b). The same applied toS. oyapockenses.l. atHasupiwei-theri (Fig. 5c). Our observations resemblethose ofRenz (1987)onS. damnosums.l. in northernCameroon.

4.3. The role of breeding and resting sites

Between-locality variation in the diurnal host-seeking patterns of parous flies could also be attributedto variations in the proximity of breeding (oviposit-ing) sites, resting (post-prandial) sites, blood host pop-ulations, and dispersal ability of parous flies. For in-stance,S. incrustatumshowed variation in the timingof its morning biting peaks between Pashopeka- andA is-tt orn-i itesa inP con-t ivef dingo formfla d-d sib-l eeno ,1 ari-a ,M op-p e ar-

eas) is that of differences in cytospecies compositionat these localities.

4.4. The role of microclimatic conditions

Changing meteorological conditions (at local level)are also an important source of biting variation. Diur-nal and locality-specific variations in tropical simuliidman-biting density have been associated mainly to vari-ations in air temperature and relative humidity (Dalmat,1955; Collins, 1979; Lacey and Charlwood, 1980). Inpractice, it is difficult to disentangle the possibly sepa-rate effect of each of these variables due to their closeand inverse relationship. In this study, we found that, forS. guianenseandS. incrustatum, parous biting activitywas, in general, promoted by warmer and drier hoursduring the rainy months, whereas the opposite relation-ship was observed during the drier and hotter months.Variation in the parous biting activity ofS. oyapockensetended to track that of temperature and be negativelycorrelated with that in relative humidity in all localitiesand seasons. Although we observed biting activity upto 34◦C and 51% RH, the temperatures that on averagepromoted most biting ranged from 24 to 29◦C, with ac-companying humidity ranging from 70 to 92%. Whenthese factors were outside of these optimal ranges theyhad just the opposite effect, i.e., when temperature andrelative humidity were much higher or lower than theirtypical values at each locality, a reduction or modifi-cation in the host-seeking activity was observed. Thisc ell-dta ityp t-iM oft dayi pro-n e lo-c isono nnal ttb , hu-m outt oreo rs of

wei-theri, with the former locality having a consently earlier peak of parousS. incrustatumactivity thanhe latter, where the peak occurred later in the mng perhaps indicating that suitably oviposition sre further away from the village in Awei-theri thanashopeka for this particular species. This would

rast with a situation in which female flies could arrrom many different, scattered, and suitable breer resting sites, producing a more steady and uniow of host-seeking flies such as in the case ofS. oy-pockenses.l. at Maweti- and Mahekoto-theri. In aition, variation in host-seeking patterns between

ing members of simuliid species complexes has bbserved within several Neotropical species (Shelley988). Hence, an alternative explanation for the vbility observed inS. oyapockenses.l. at Maweti-ahekoto- and Hasupiwei-theri (lowland areas) asosed to Awei-theri and Pashopeka (higher altitud

ould help explain observed variations around a wefined biting pattern (as withS. guianense), oscilla-

ions around a biting peak (S. incrustatum), or shiftsnd variability in the time of day when biting activeaked (S. oyapockense). A more steady level of bi

ng activity, as that observed inS. oyapockenses.l. ataweti could be the result of fairly constant values

emperature and relative humidity throughout then lowland forested areas in contrast with the moreounced variations registered at the higher altitudalities of Pashopeka- and Awei-theri. In a comparf vector bionomics in forested versus open sava

ocalities of West Africa,Crosskey (1990)pointed ouhat biting activity levels ofS. damnosums.l. tend toe more stable in the former (where temperatureidity and shade remain fairly constant through

he day) than in the latter (with little shade). In mpen settings the biting activity of savanna membe


the damnosumcomplex usually decreases at middayas temperature and humidity reach their highest andlowest levels, respectively, although unimodal activityis sometimes observed (Crosskey, 1955). In summary,subtle variations in microclimatic conditions among lo-calities, as well as the between-season variation withinlocalities could explain a great deal of the observedvariation in blackfly biting patterns.

4.5. Implications for the timing of blackflyoviposition

Our results suggest that gravidS. guianenses.l. fe-males may oviposit either early in the afternoon orperhaps around noon as indicated by the main bitingpeak of parous flies in mid-afternoon. Another ovipo-sition cycle would occur during late evening (sunset) orvery early in the morning (sunrise), with a subsequentpeak of parous host-seeking activity in mid-morning.Most (gonotrophically concordant) blackfly speciesseek their next blood meal within a few (2–4) hours af-ter laying eggs, with the preferred time for ovipositionbeing that around sunrise or sunset (Crosskey, 1990).Blood feeding soon after emergence and ovipositionhas been suggested to increase the vectorial potentialof otherwise relatively poor insect hosts forO. volvu-lus such asS. ochraceums.l. in Guatemala (Collins etal., 1981). Patterns of emergence and oviposition arepoorly known for the vectors in the Amazonian focusand deserve further investigation, particularly in thec sg dsu bit-i

4

id-a k ofot hiss iblet tiveb erem ciesh liidsi um-

ber of infective larvae per fly will be typically overdis-persed (with a few flies harbouring up to 13 infectivelarvae), inter-individual variation in attractiveness toflies may mean that some hosts might still receive a highnumber of L3 larvae. The contribution ofS. incrusta-tum to onchocerciasis transmission, in those localitieswhere this species prevails, will take place mainly inthe morning, whereas transmission byS. oyapockenses.l. in mesoendemic localities will occur throughoutthe day. Although this species is the least competentof the three, the hourly parous biting rate can be con-sistently higher than 100 per person in Maweti- andMahekoto-theri. We have already discussed the rela-tionship between vector competence, the vector bitingrate on humans, the intensity of onchocerciasis trans-mission, and the endemicity of the infection in hostpopulations in a comparative analysis between Meso-America and West Africa (Basanez et al., 2002) and inEcuador (Vieira et al., 2005).

4.7. Implications for ivermectin-based control

As well as describing biting patterns of parous fliesand indicating times of the day most likely to con-tribute to onchocerciasis transmission, the informationprovided in this paper, once analyzed together with in-fection and infectivity data, will be most useful for thedesign of sampling protocols aimed at detecting statis-tically meaningful changes in the intensity of transmis-sion during ivermectin-based control programmes. Re-c caseo trolt lua-tt er-il tede

A

in-i perO dyw amih rea r. J.

ase of species as epidemiologically important aS.uianenses.l. An early morning oviposition (aroununrise) is inferred forS. incrustatumfrom its mainlynimodal and early to mid-morning peak of parous

ng activity.

.6. Epidemiological implications

Our results suggest that mid-morning and mfternoon are potentially associated with highest risnchocerciasis transmission byS. guianenses.l. Given

he relatively low parous biting rate exhibited by tpecies (up to 48 flies per person-hour), it is posshat a considerable reduction of exposure to infecites could be achieved if human-vector contact winimized during these hours. However, this speas the highest vector competence of the three simu

nvestigated, and although the distribution of the n

ently, such information has been reported in thef the Ecuadorian program for onchocerciasis con

o provide clear guidelines for entomological evaion ofS.exiguums.l. andS.quadrivittatumthat will aidhe Onchocerciasis Elimination Program for the Amcas efforts in the region (Vieira et al., 2005). A simi-ar analysis for the Amazonian focus will be presenlsewhere.

cknowledgements

We are grateful to the Salesian Mission, the Mstry of Health authorities in Amazonas, and the Uprinoco Yanomami communities in which this stuas conducted. In particular, we thank the Yanomealth workers F. Guzman and L. Urdaneta. We also grateful for the logistic support provided by F


Bortoli and Drs. F. Armada, A. Arenas, C. Botto, M.Magris, S. Rojas, and L. Villegas. In the field, L. Bueno,P. Coronel, M. Frontado, and J. Zegarra provided in-valuable assistance. Pierre Legendre, at the Universityof Montreal, provided statistical advice. This study wassupported by the World Bank, the Pan American HealthOrganization (PAHO), the Wellcome Trust, the BritishCouncil Academic Link Program, and the Venezue-lan Council for Scientific and Technological Research(CONICIT). MGB thanks the Medical Research Coun-cil, UK for a Career Establishment Grant.

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Vector competence of Simulium oyapockense s.l. and S. incrustatum for Onchocerca volvulus:...

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