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Public Health Open Access Research Article 14 min read

Circadian Biting patterns of Major Lymphatic Filariasis Vectors from Northwest Nigeria

Lawal N, Idoko AS, Abdullahi H and Imam AA*
* Corresponding author
ISSN: 2578-5001  10.23880/phoa-16000158  Received: July 14, 2020  Published: July 27, 2020
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Keywords
Anopheles gambiae Lymphatic Filariasis Culex quinquefasciatus
Abstract

The circadian biting pattern of mosquito vectors is an important determinant of malaria and lymphatic filariasis (LF) transmission. Good knowledge of the vector host-seeking behavior, its outdoor/indoor biting preference, and nocturnal biting time is essential component for vector control strategies in Northwest Nigeria. The aim of this study was to assess the circadian biting time and identify the mosquito composition morphologically and molecularly. Mosquitoes were collected outdoor by human landing catches (HLC) using mouth aspirator from Batagarawa town and Gajerar Giwa village. Mosquitoes were morphologically and molecularly identified as Anopheles gambiae (M and S forms), Anopheles funestus s.s and Culex quinquefasciatus. The peak biting time for Culex quinquefasciatus were relatively similar across the study communities and the nocturnal biting rates showed an increased night time peak between 11pm to 12am. However, the peak biting time of Anopheles gambiae s.l also varied between study communities and the nocturnal biting rates showed an increased night time peak between 9pm to 10pm and then 11pm to 12am but, the peak biting time for Anopheles funestus s.l which was found only in Gajerar Giwa village and the nocturnal biting rates showed an increased night time peak between 12am to 1am. This finding serves as a basis for creating an improved vector control strategies that target specific species, and if combine with other interventions could result in the elimination of malaria and LF transmission from Northwest Nigeria.

Introduction

Knowledge of the vector’s biting behavior is an essential component in determining the vectorial capacity. The latter, in its classical form, comprises the parasite’s extrinsic incubation period, the ratio of mosquitoes to humans, mosquito survival through one day, and human biting rates (HBRs) [1]. Sound knowledge of the vector’s biting behavior is an important prerequisite to better understand and reduce malaria and lymphatic filariasis (LF) transmission. While endophagic species take their blood meal predominantly inside human dwellings, exophagic species feed mostly outdoors. Similarly, with regard to resting behavior, endophilic species commonly prevail indoors while exophilic species rest mostly outdoors. Anopheline species, such as Anopheles gambiae Patton

(Diptera: Culicidae) and Anopheles funestus Giles (Diptera: Culicidae), are known to prefer humans for their blood meal (anthropophilic), while others, such as Anopheles quadriannulatus Theobald (Diptera: Culicidae) and Anopheles melas Theobald (Diptera: Culicidae), feed predominantly but not exclusively on animals (zoophilic) [2]. Current malaria and LF vector control in sub-Saharan African (SSA) relies heavily on indoor insecticidal interventions using indoor residual spraying (IRS) and long lasting insecticidal nets (LLINs) [3, 4, 5]. The scale up of IRS and LLINs during the last decade has substantially reduced malaria and LF incidence in many parts of SSA [6, 7]. These interventions reduce the density, feeding frequency and longevity of malaria and LF vectors by killing the vectors with insecticides or blocking their contact with humans [8, 9] and primarily target malaria and LF vectors that feed indoors and at night on sleeping humans [4]. The difference in the biting pattern contributes significantly in the transmission pattern of diseases among various hosts during different seasons. The rapid change in the ecosystem due to several factors such as the increase in global warming [10], unplanned urbanization, deforestation, changing human behavior [11], availability of hosts are found affecting the behavior of mosquitoes and thereby enhancing vector borne pathogens transmission [12]. This study was designed to monitor circadian biting patterns of Culicine and Anopheline mosquitoes as a function of seasonal variation in Northwest Nigeria.

Methods

Study Site and Human Landing Catches (HLC) Method

Mosquitoes were collected using the Human Landing Catches (HLC) method [13]. Briefly, two collectors were made to sit in a dark room, with their legs exposed. Torch lights, switched on intermittently, were used to locate host-seeking mosquitoes. Landing mosquitoes that are ready to take a blood meal were then aspirated using a mouth aspirator. Trapped mosquitoes were then released into paper cups covered with mosquito netting and labeled to indicate the hour of collection (Figure 1):

  • Batagarawa town, Batagarawa Local Government in the Sahel Savannah of Katsina State (12°54′17″N, 7°37′11″E), is a semi-urban area characterized by fewer of Anopheles gambiae s.l breeding sites.
  • Sahel Savannah of Gajerar Giwa village (12095′21′N, 7075′19′E) in Rimi Local Government of Katsina State, where rice and vegetables irrigation are practiced using water from Ajiwa dam, characterized by large number of Anopheles funestus s.l breeding sites.
Figure 1: Adopted from google map.
Click to enlarge
Figure 1: Adopted from google map.

Morphological Identification

The working bench was cleaned with 70% ethanol. The mosquitoes brought from the study sites were separated using morphological features [14, 15, 16, 17] to separate the mosquitoes into anophelines (Anopheles mosquitoes) and culicines (Aedes, Culex, and Mansonia mosquitoes). Each mosquito was picked with forceps into a petri dish and examined under a dissection (stereo) microscope (Olympus SZ60). All Anopheles mosquitoes were identified using the pale and dark band patterns on the costal margins of their wings. All culicines are also grouped based on the absence of the pale and dark band patterns on the costal margins of their wings and into their respective genera using morphological identification keys. In all genera of mosquitoes, the males were distinguished from the females by the presence of their plumose (bushy) antennae. The different species that had been morphologically identified were recorded on appropriate entomological data recording forms/sheets [18].

DNA Extraction from Mosquitoes Collected from Study Sites

Genomic DNA was extracted by Livak [19] protocol with little modification from the carcasses of mosquitoes after homogenizing with sterile plastic pestles in 100μl bender buffer. The homogenate was incubated at 65°C for 30min, followed by the addition of 14μl 8M K-acetate (to final concentration of 1M). The mixture was vortexed and centrifuged at 14,000rpm for 20min at 4°C using Eppendorf® Centrifuge 5415C. The supernatant was transferred into new 1.5ml Eppendorf tube. Two hundred microlitres of 100% ethanol were added to the supernatant. The mixture was vortexed and centrifuged at 14,000rpm for 15min at 4oC and this was incubated at -40°C for an hour, spun at 10,000rpm for 10min and supernatant poured off. The pellet was then rinsed with 100μl of 70% ethanol, spun at 10,000rpm for 5min. The supernatant was discarded and dried-up the pellet by leaving the tubes open on bench for an hour. The pellet was suspended in 100μl dH2O then incubated at 65°C for 10min.

Molecular Identification of Anopheles Mosquito Species

Extracted DNA from morphologically identified mosquitoes was used as DNA templates for PCR. Each PCR reaction mixture of 25μl contained 1×PCR buffer (Sigma, USA), 200μM each of the four deoxyribonucleotide triphosphates (dNTPs), 10μM each of the forward and reverse oligonucleotide primers, and 0.125units of Taq Polymerase enzyme (Sigma, USA). One microlitre of the genomic DNA was used as template for the amplification reaction.

After DNA amplification, the PCR products were electrophoresed in 2% agarose gel containing 0.5μg/ml ethedium bromide in 1×TAE buffer using a mini gel system (BIORAD USA). Eight microlitres of each sample was mixed with 1μl of 5×Orange G loading dye and loaded into a well of the gel. The voltage of the power unit was set at 100V and run for an hour. A photograph of the gel was obtained using a UV trans-illuminator (UPC, USA) and a Polaroid camera of film type 667 (Polaroid, USA). The sizes of the PCR products were estimated by comparison with the mobility of a 100 base pair molecular weight size marker (Sigma).

Molecular Identification of Culex quinquefasciatus

Extracted DNA from morphologically identified Culex mosquitoes was used as DNA templates for PCR in which 5µl of extracted DNA was added to 1.5ml eppendorf tube then followed by acepip (F) 0.4µl, ace gun (F) 0.8µl and B12465 (B) 0.8µl and 2µl of 1x Tag A, 0.1µl of MgCl, 11.92µl of dH2O and 0.1µl of Kappa Tag were added to the reaction mixture to form a total reaction volume of 20µl. The cycling conditions used were 95°C for 5min followed by 40 cycles of denaturation at 94°C for 30sec, annealing at 54°C for 30sec and extention at 72°C for 10min then a final extention at 72°C for 10min. Electrophoresis using 1.5% agarose gel stained in ethidium bromide was run for 30min after loading 3μl PCR product.

Results

Mosquito Sampling and Identification

From the study communities, 50 Anopheles gambiae s. l complex were randomly selected for molecular specie identification. It was observed that 25(50%), 3(6%) and 16(32%) in Figure 2 were Anopheles coluzzii (formerly M form), Anopheles arabiensis and Anopheles gambiae s.s (formerly S form) respectively. However Anopheles Funestus s.l. collected, 50 were randomly selected for molecular identification.

Figure 2: Gel electro pho re gram for the identification of members of the An. gambiae s.l complex. Lane M=100bp molecular weight marker, Lane 1,3,8,12,13,15,16,17,21,23,24,25,27,28,30 at 470bps=Anopheles coluzzii (formerly M form), Lane 2,4,5,6,7,9,10,11,14,23,26,28 at 240bps=Anopheles gambiae s.s (formerly S form), Lane 18-20 at 220bps=Anopheles arabiensis.
Click to enlarge
Figure 2: Gel electro pho re gram for the identification of members of the An. gambiae s.l complex. Lane M=100bp molecular weight marker, Lane 1,3,8,12,13,15,16,17,21,23,24,25,27,28,30 at 470bps=Anopheles coluzzii (formerly M form), Lane 2,4,5,6,7,9,10,11,14,23,26,28 at 240bps=Anopheles gambiae s.s (formerly S form), Lane 18-20 at 220bps=Anopheles arabiensis.

Figure 2: Gel electro pho re gram for the identification of members of the An. gambiae s.l complex. Lane M=100bp molecular weight marker, Lane 1,3,8,12,13,15,16,17,21,23,24,25,27,28,30 at 470bps=Anopheles coluzzii (formerly M form), Lane 2,4,5,6,7,9,10,11,14,23,26,28 at 240bps=Anopheles gambiae s.s (formerly S form), Lane 18-20 at 220bps=Anopheles arabiensis.

Out of these 43(86%) in Figure 3 were predominantly Anopheles Funestus s.s from Gajerar Giwa village. Out of the large number of Culex spp that were morphologically identified, 50 Culex mosquitoes were randomly selected for molecular specie identification. The results showed 47(92.5%) in Figure 4 were predominantly Culex quenquifasciatus from the study communities.

Figure 3: Gel electrophoregram for the identification of members of the Anopheles Funestus s.l complex. Lane M=100bp molecular weight marker, Lane 1-48 at 460bps=Anopheles Funestus s.s.
Click to enlarge
Figure 3: Gel electrophoregram for the identification of members of the Anopheles Funestus s.l complex. Lane M=100bp molecular weight marker, Lane 1-48 at 460bps=Anopheles Funestus s.s.
Figure 4: Gel electrophoregram for the identification of members Culex spp complex. Lane M=100bp molecular weight marker, Lane 1-31 at 274bps=Culex quenquifasciatus.
Click to enlarge
Figure 4: Gel electrophoregram for the identification of members Culex spp complex. Lane M=100bp molecular weight marker, Lane 1-31 at 274bps=Culex quenquifasciatus.

Circadian Biting Time

The peak biting times for Culex spp were relatively similar across the study communities Figure 5, with showing a unimodal distribution. The nocturnal biting rates showed an increased night time peak between 11pm to 12am.

Figure 5: Circadian biting pattern of Culex quinquefasciatus at the study communities, from 6pm to 6am.
Click to enlarge
Figure 5: Circadian biting pattern of Culex quinquefasciatus at the study communities, from 6pm to 6am.

However, the peak biting time of Anopheles gambiae s.l also varied between study communities Figure 6, with showing bimodal distributions. The nocturnal biting rates showed an increased night time peak between 9pm to 10pm and then 11pm to 12am but, the peak biting time for Anopheles funestus s.l which was found only in Gajerar Giwa village Figure 7, with showing single distribution. The nocturnal biting rates showed an increased night time peak between 12am to 1am.

Figure 6: Circadian biting of Anopheles gambiae s.l at the study communities, from 6pm to 6am.
Click to enlarge
Figure 6: Circadian biting of Anopheles gambiae s.l at the study communities, from 6pm to 6am.
Figure 7: Circadian biting of Anopheles funestus s.l at the study communities, from 6pm to 6am.
Click to enlarge
Figure 7: Circadian biting of Anopheles funestus s.l at the study communities, from 6pm to 6am.

An important observation is that in most sampling communities, 70% or more of mosquitoes were collected between 5am to 6am in the morning. The outdoor biting pattern of the mosquitoes appeared to exceed the indoor biting pattern in both study communities.

Discussion

This present study indicated that Anopheles funestus s.s recorded 56.5% was the most abundant vector species caught in only Gajerar Giwa village. But Culex quinquefasciatus recorded 21.3% was caught from all the study communities followed by Anopheles gambiae s.l complex (13.4%) in which (50%) Anopheles coluzzii (formerly M form), (6%) Anopheles arabiensis and (32%) Anopheles gambiae s.s (formerly S form). These observations are corroborated by reports from Dar es Salaam by Mwakitalu E, et al. [20]. The higher abundance of Anopheles funestus s.s complex in the present study might be because mosquitoes were collected during the rainy season in which the Ano. Funestus s.s population was very high. The observed Anopheles Funestus s.s abundance has important implications in the transmission of both malaria and lymphatic filariasis. It is therefore likely that the incline in Anopheles funestus s.s abundance observed in this present study will have comparatively more impact on the transmission of lymphatic filariasis than that of malaria.

In this study, three important lymphatic filariasis vectors in Northwest Nigeria were molecularly identified. Culex quinquefasciatus, Anopheles gambiae (M and S forms) and Anopheles funestus s.s were found to be the three human- biting mosquitos’ species occurring outdoors in the study area. The occurrence of the majority of biting is the early to middle phase of the night (during 09.00pm to 01.00am) and peak biting by Culex quinquefasciatus between 11.00pm and 12.00am is contrary with the already known time of peak biting by the majority of human-biting Culex quinquefasciatus which showed peak biting after midnight in West Africa [21] and East Africa where it is an established vector [22]. This study was done outdoors during the time the people usually stayed outdoors (up till 12.00am) and inside (12.00am- 06.00am) when they were normally indoors. It is known that abundance, attractiveness, and availability of the human host are influencing vector biting behavior [23]. The peak biting time of Anopheles gambiae varies between Batagarawa town and Gajerar Giwa village and the nocturnal biting rate peak between 9.00pm-10pm and 10.00pm-11.00pm respectively. This is consistent with the already established time of peak biting by the majority of human-biting microfilaria positive Anopheles gambiae, i.e. between 11.00pm and 05.00am hours, a period when most people are in bed and under nets if they have them [24, 25]. An. gambiae biting rate was moderate within the first 2hours after dusk and increased quickly towards a 6-hours period of high biting. This finding is new and contrasts with many other African research sites, where the respective main vectors showed higher variation in nocturnal biting activity, featuring pronounced biting peaks and periods of lower activity.

Kenea O, et al. [26] observed an early biting peak between 7.00pm and 8.00pm hours for the most important vector Anopheles ziemanni Grunberg (Diptera: Culicidae) in Ethiopia, while An. arabiensis showed two peaks, from 8.00pm to 10.00pm hours and around midnight. Similarly, a study from Ethiopia [27] found An. gambiae s.l. and mosquitoes from the Anopheles coustani complex to have two activity peaks, one between dusk and 10.00pm hours and a smaller in the early morning around 04.00am hours.

Anopheles funestus which was only found in Gajerar Giwa village and this could be attributed to the presence of more permanent water for breeding provided by a larger Ajiwa dam in this locality. Anopheles funestus is known to breed all year round and prefer permanent, stagnant water bodies such as shores of rivers and creeks, swamps or fish ponds for breeding, the peak biting rate showed an increase night time peak between 12.00am to 1.00am. Similar scenario was also reported in Zimbabwe by Sande S, et al. [28] that, the peak biting time of Anopheles funestus was between 10.00pm and 11.00pm hours, and around 03.00am hours.

Conclusion

From the study communities there were high vectors of lymphatic filariasis outdoor biting activity throughout the nights with marked single or multiple peaks. These biting patterns could be the consequence of mosquito tolerance to the recently achieved high LLIN and IRS coverage in the study communities. Higher activities of mosquito vectors of lymphatic filariasis were observed throughout the study nights, vectors contact at any time during the nights are high when people are not sleeping under a bed net. Undertaking the goal of further reducing malaria and lymphatic filariasis transmission might raise the need for additional vector control strategies that either decrease vector populations or reduce human-vector contact.

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@article{lawal2020,
  title   = {Circadian Biting patterns of Major Lymphatic Filariasis Vectors from Northwest Nigeria},
  author  = {Lawal N, Idoko AS, Abdullahi H and Imam AA},
  journal = {Public Health Open Access},
  year    = {2020},
  volume  = {4},
  number  = {2},
  doi     = {10.23880/phoa-16000158}
}
Lawal N, Idoko AS, Abdullahi H and Imam AA (2020). Circadian Biting patterns of Major Lymphatic Filariasis Vectors from Northwest Nigeria. Public Health Open Access, 4(2). https://doi.org/10.23880/phoa-16000158
TY  - JOUR
TI  - Circadian Biting patterns of Major Lymphatic Filariasis Vectors from Northwest Nigeria
AU  - Lawal N, Idoko AS, Abdullahi H and Imam AA
JO  - Public Health Open Access
PY  - 2020
VL  - 4
IS  - 2
DO  - 10.23880/phoa-16000158
ER  -