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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 4  |  Page : 308-314

Entomological status of Anopheles sergentii and the first molecular investigation of its insecticide-resistant genes, kdr and ace-1 in Morocco


1 Laboratory of Vector-Borne-Diseases, Institut Pasteur du Maroc, Casablanca; Health and Environment Laboratory, Hassan II University of Casablanca, Aïn Chock Faculty of Sciences, Morocco
2 Medical Entomology laboratory, Institut National d'Hygiène, Rabat, Morocco
3 Health and Environment Laboratory, Hassan II University of Casablanca, Aïn Chock Faculty of Sciences, Morocco
4 Harris County Public Health, Mosquito & Vector Control Division, Houston, Texas, USA
5 Laboratory of Vector-Borne-Diseases, Institut Pasteur du Maroc, Casablanca, Morocco

Date of Submission08-Sep-2018
Date of Acceptance06-Sep-2019
Date of Web Publication30-Nov-2020

Correspondence Address:
Dr. M Sarih
Institut Pasteur du Maroc, 1, Place Louis Pasteur, Casablanca–20360
Morocco
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.302033

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  Abstract 

Background & objectives: Prior to their elimination in 1974 and 2004, respectively, Plasmodium falciparum and P. vivax were the main native malaria parasites involved in disease transmission in Morocco. Imported cases of human malaria are still reported from the country. Anopheles labranchiae in northern Morocco and An. sergentii in the southern regions are the main malaria vectors. The bionomics and insecticide susceptibility of An. sergentii are poorly understood and need to be further studied to enhance the epidemiological surveillance of this important malaria vector.
Methods: The adults and larvae of Anopheles sergentii were collected during the mosquito breeding season in 2015 and 2016 and environmental characteristics of their breeding sites were recorded. Blood meals were analyzed using PCR-RFLP. Alongside, the WHO routine susceptibility tests with DDT (4%) and malathion (5%) were conducted and An. sergentii specimens were screened for knockdown resistance (kdr) and acetyl cholinesterase encoding (ace-1) gene mutations.
Results: Anopheles sergentii was observed during the summer and autumn seasons, feeding mainly on sheep, cows and also on humans. The WHO bioassays revealed complete susceptibility to DDT and malathion. Analysis of the sequences of the voltage-gated sodium channel gene revealed the absence of the kdr “Leu-Phe” mutation and PCR-RFLP revealed the absence of the G119S mutation in the ace-1.
Interpretation & conclusion: With the increasing number of imported cases of human malaria in Morocco, the indiscriminate feeding behavior of this species may pose an infectious medical threat. Fortunately, the absence of insecticide resistance can ensure, for now, the efficiency of insecticides, as a part of the vector control program in controlling An. sergentii in Morocco.

Keywords: Blood meal; breeding site; insecticide susceptibility; insecticide-resistant gene; vector


How to cite this article:
Filali O B, Faraj C, Kabine M, Debboun M, Sarih M. Entomological status of Anopheles sergentii and the first molecular investigation of its insecticide-resistant genes, kdr and ace-1 in Morocco. J Vector Borne Dis 2019;56:308-14

How to cite this URL:
Filali O B, Faraj C, Kabine M, Debboun M, Sarih M. Entomological status of Anopheles sergentii and the first molecular investigation of its insecticide-resistant genes, kdr and ace-1 in Morocco. J Vector Borne Dis [serial online] 2019 [cited 2021 Oct 18];56:308-14. Available from: https://www.jvbd.org/text.asp?2019/56/4/308/302033


  Introduction Top


For centuries, human malaria was endemic in Morocco. Plasmodium falciparum was the main human malaria parasite followed by P. vivax, which became the predominant species[1] after 1950. A national malaria control program[2] was launched in 1965. The use of insecticides was the main strategy for controlling malaria vectors in Morocco until 2001, using DDT indoor residual spraying for the adult mosquitoes control and organophosphates, mainly temephos (Abate 500 EC) for larval control. Treatment of the parasite reservoir and chemoprophylaxis were also used to prevent the spread of malaria. This program resulted in the elimination of P. falciparum transmission in 1974, and no locally acquired cases of P. vivax have been reported[2] since 2004. Since the elimination of autochthonous (local) transmission of malaria, a vector control program was established to prevent its possible return. It is mainly based on larval control involving integrated mosquito management using environmental methods; larvivorous fish (Gambusia holbrooki) as biological control and insecticides as the last option[2]. It has to be noted that pyrethroids are largely used in agriculture and public health as aerosols or sprays to get rid of mosquitoes’ nuisance inside people homes.

In 2010, Morocco was declared malaria-free by the WHO[3]. However, recently in 2015, the Ministry of Health reported more than 500 cases of imported malaria (mainly P. falciparum) in Morocco[4]. The increase in foreign student numbers from endemic countries, tourism, and deployment of army and police forces in foreign endemic areas have contributed to the importation of malaria into Morocco.

Epidemiological data indicate that, An. labranchiae (Felleroni) in the north and An. sergentii (Theobald) in the south are the two major human malaria mosquito vectors in Morocco[5],[6]. However, recently, An. sergentii has spread from south to north Morocco[6],[7], and thus it warranted its study, particularly with the consistent increase in gametocyte positive samples[4],[8],[9],[10],[11],[12] observed over the period of 2010–2014. Known as the “oasis vector” or the “desert malaria vector,” An. sergentii can adjust to the extreme climate of the North African Sahara[13],[14]. In Egypt, An. sergentii is also considered to be the main vector of P. falciparum malaria because it has been found naturally infected in the Siwa oasis and the Faiyum governorate [15]. Recently, naturally infected An. sergentii have also been reported in the Republic of Yemen feeding on humans and animals, both indoors and outdoors[16],[17],[18].

Unfortunately, there is a lack of data regarding the susceptibility of An. sergentii to insecticides. Thus, this study examined for the first time, the insecticide susceptibility and host preferences of An. sergentii in southern Morocco.


  Material & Methods Top


Study sites

The study was conducted in locality of Ait Daoud, Agafay (31°29258.93N; 8°10217.13W) in Marrakesh region located 327 km southwest of the Moroccan capital Rabat. The predominant climate is hot semi-arid. Mosquitoes were collected from April to October in 2015 and 2016. The presence of larvae was investigated on several points in the urban ditches in the outskirts of the city of Marrakesh, troughs near crop fields, and different randomly selected areas along the edges of the stream of N’fis River [Figure 1]. Adult mosquitoes were collected in animal shelters and human dwellings randomly chosen in the Ait Daoud locality. The houses visited are mainly of traditional type, built with adobe. Inside the houses there are stables for cattle and sheep. Irrigation canals run through this locality. The vegetation is dominated mainly by cactus and wheat fields. Agriculture remains the main source of income for the inhabitants.
Figure 1: Anopheles sergentii larval site located on Oued N'fis near the selected buildings used for the collection of adults mosquitoes in Marrakesh city, Morocco.

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Mosquito sampling

Four random buildings were selected for the collection of adult mosquitoes. The collections of resting fauna was conducted monthly from April to October in two human dwellings and one animal shelter that was divided into two sections (one for cows and the other for sheep) in each building for three consecutive nights. Mosquitoes were collected using pyrethroid spray catches (PSCs). The methods involved covering the floor with a white sheet, spraying inside the buildings, and collecting the mosquitoes that were knocked down after 10 mins. Four miniature CDC light traps were placed monthly over night in each building, for three consecutive nights (one in animal shelters, two in human habitations and one outdoor) from 2000–0600 h.

Larvae of An. sergentii were collected and their density was estimated at each larval habitat using dippers and counted the number of larvae per 10 dips in each investigated area. Larvae were transported to the insectary at the Pasteur Institute of Casablanca, maintained under standard conditions (25–28°C, ~70–80% relative humidity and with a 16h : 8h photoperiod), fed on cat kibbles and reared to the adult stage. Emerged adults were used in the insecticide susceptibility bioassays and stored at –20 °C to screen for kdr and acetyl cholinesterase encoding gene (ace-1) mutations.

Mosquito identification

Mosquitoes were identified using a software for the identification of the Culicidae in the Mediterranean area[19] enabling An. sergentii to be distinguished from other Anopheles. The identification of An. sergentii was molecularly confirmed by sequencing ribosomal DNA second internal transcribed spacer (ITS2) markers[20].

Insecticide susceptibility bioassays

Susceptibility tests were performed in accordance with the WHO guidelines[21]. Four sets of 20–25 non blood-fed and 3–5 day-old female mosquitoes were exposed to insecticide-treated filter papers in accordance with the WHO discriminating concentrations and exposure times[22]. The insecticides used were DDT (4%) and Malathion (5%). The number of mosquitoes knocked down with DDT exposure was recorded at intervals of 10 min. After exposure to both insecticides, mosquitoes were transferred into the holding tube and provided a 10% sugar solution and kept for 24 hr, after which mortality was recorded. Two sets of 20 unfed 3–5 day-old females were exposed to insecticide-free papers as controls.

The knockdown time values for 50% (the median knockdown time ‘KdT50’) and 90% (KdT90) of exposed mosquitoes to DDT were estimated using a log time probit model (WinDL software; CIRAD–CA/MABIS; Montpellier, France).

DNA extraction

DNA was isolated from individual adult mosquitoes after homogenization of the sample using DNAzol, according to the manufacturer’s instructions (Invitrogen, Thermo Fisher Scientific, MA, USA).

Blood meal analysis

The blood-fed An. sergentii were analysed to determine the host source of the blood meals.

The PCR amplification was performed as described earlier[23]. Briefly, each 25 μl PCR reaction consisted of 0.1 mM each dNTP, 5 μl of 5× reaction buffer, 1.25 mM MgCl2, 0.4 μM of each primer dinF and foruR and 1U/μl of taq polymerase enzyme. Reaction conditions were 95°C for 1 min followed by 36 cycles of 95°C for 30 s, 58°C for 50 s, and 72°C for 40s, with a final extension at 72°C for 5 min.

The amplification resulted in a specific sharp band of 358 bp of the CytB gene of vertebrate mtDNA. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) digestion of PCR products was performed in a 25μl solution containing 15μl PCR product, 2.5μl enzyme buffer, and 5U of the appropriate restriction enzymes (XhoI and TaqI).

Using the TaqI enzyme, three profiles were detected under the UV light:

  • Two bands of 220 bp and 135 bp which corresponded to the cows’ profile
  • One original band of 358 bp which corresponded to the sheep’s profile
  • Three bands of 358, 220 and 135 bp which corresponded to a mixture of the cow and sheep’s blood


For the XhoI enzyme, it resulted in the detection of the following profiles:

  • Two bands of 250 bp and 100 bp which corresponded to the human blood profile
  • One original band of 358 bp which indicated that no human blood was detected in the sample analyzed.


Detection of kdr mutation

Fragment of the voltage-gated sodium channel (VGSC) gene including the IIS6 coding region on the exon 20, with the whole sequence of intron from 60 specimens of An. sergentii adults were amplified by PCR according to the protocol of Dinparast et al[24] using primers, dinF and foruR:

Forward primer (dinF): 5′-TGG ATT GAA TCA ATG TGG GAT TG-3′

Reverse primer (foruR): 5′-AAG GAT GAA GAA CCG AAA TTG GAC-3′.

Each 25 μl PCR reaction consisted of 0.1 mM each dNTP, 5 μl of 5× reaction buffer, 2 mM MgCl2, 1 μM each primer dinF and foruR and 1U/μl of taq polymerase enzyme. Reaction conditions were 95°C for 5 min followed by 30 cycles of 1 min for each step of 95, 58, and 72°C, with a final extension at 72°C for 15 min. The expected sizes of PCR products of around ~211bp were confirmed by electrophoresis on a 1.5% agarose gel.

Sequencing of the VGSC

The final PCR products of 211bp were purified using the ExS-Pure™ Enzymatic PCR Cleanup Kit (NimaGen BV, The Netherlands), and sequenced using the same amplification primers and BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The sequencer used was an ABI PRISM® 3130 DNA automated sequencer (Applied Biosystems Inc., Foster City, CA).

Detection of ace-1 mutation

The ace-1 was amplified according to the protocol of Weill M et al[25] using the degenerated primers Moustdir1 (5′-CCGGGNGCSACYATGTGGAA-3′) and Moustrev 1 (5′-ACGATMACGTTCTCYTCCGA-3′). Each 25 μl PCR reaction consisted of 0.1 mM each dNTP, 5 μl of 5× reaction buffer, 1.25 mM MgCl2, 0.4 μM each primer Moustdir1 and Moustrev1 and 0.06U/μl of taq polymerase enzyme. Reaction conditions were: 35 cycles (94 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min). The PCR products of 194 bp were then digested using AluI enzyme at 37 °C for 3h.

Data analysis

The knockdown time for 50% (KdT50) and 90% (KdT90) of exposed mosquitoes to DDT were estimated using a log time probit model (WinDL software). The sequences were examined and analyzed using Chromas v.2.6.2 software (Technelysium Pty Ltd, South Brisbane QLD 4101, Australia) and were identified and compared to similar sequences using basic local alignment search tool (BLAST) available at http://blast.ncbi.nlm.nih.gov.

Ethical statement: Not applicable


  Results Top


Mosquito identification and breeding site characteristics

The investigated sites were negative for An. sergentii from April to June of 2015 and 2016. A total of 386 adults were captured mostly by the PSCs. Among the Anopheles mosquitoes collected using the PSCs, 207 adults were morphologically and molecularly identified as An. sergentii and 179 adults were morphologically identified as An. dthali (n = 101) and An. cinereus (n = 78). The number of mosquitoes captured by CDC traps was insignificant with a total of 24 Anopheles that were morphologically identified as An. dthali (n = 6) and An. sergentii (n = 18).

Different sites were investigated for the presence of An. sergentii larvae; urban ditch and troughs were found negative after two years of surveillance, and six different areas along the edges of a stream of Oued N’fis were found positive as breeding sites [Figure 1]. A total of 490 larvae of Anopheles were collected from the edges of oued N’fis in Marrakesh from July to October, 2015 and 2016; 272 were identified as An. sergentii and 218 as An. dthali (n = 14) and An. cinereus (n = 204).

[Table 1] shows the environmental parameters of the breeding sites recorded during the 2-yr breeding season of An. sergentii.
Table 1: Environmental parameters of An. sergentii larval habitats in Marrakesh, Morocco

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Blood meal analysis

DNA isolated from blood-fed mosquitoes was used as a template in the PCR reactions and most host DNAs were successfully amplified. Blood sources were detected in 100% of 164 engorged female mosquitoes collected in animal shelters and inside human habitats using PSCs, with 6.097% (10/164) from humans, 25% (41/164) from cows, and 26.829% (44/164) from sheep, whereas, multiple blood meals/mixed from sheep and cows were detected in 42.073 % (69/164).

Insecticide susceptibility bioassay

The WHO susceptibility test conducted on An. sergentii revealed complete susceptibility to DDT and Malathion, with 100% knockdown in the first hour of exposure and 100% mortality rate for both insecticides. All mosquitoes used for the control test showed 0% mortality after 24 h. [Table 2] shows the KdT50 and KdT90 values.
Table 2: Mortality percentage of An. Sergentii after 1 h exposure to 4% DDT, 5% Malathion

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Detection of kdr mutation

Genomic DNA from 60 specimens of An. sergentii was extracted, and the region flanking the VGSC, including the IIS6 coding region and full sequence of intron I, was amplified. The PCR products were sequenced from both ends, resulting in a 211 bp fragment. The sequence has been submitted to GenBank under the accession number MF919398.

The analysis of DNA sequences indicated that the 1014F polymorphism of VGSC was not found in any of the An. sergentii species examined compared with An. gambiae (Y13592.1) [Figure 2] and [Figure 3]. The BLAST analysis of the sequences showed a 92% similarity between An. gambiae and An. sergentii. The comparison of the coding region also showed a number of differences in nucleotide sequence between the two species. Three were located in the 1st, 2nd, and 3rd positions and resulted in a change in the amino acid sequence (glutamic acid in the An. gambiae sequence and isoleucine in the An. sergentii sequence) [Figure 3].
Figure 2: Electropherogram of the DNA sequencing of the voltage gated sodium channel (VGSC) fragment from An. sergentii. The box indicates the wild-type allele, 1014L (TTA).

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Figure 3: Alignment (clustal) of the amplified coding region of the voltage gated sodium channel (VGSC) nucleic sequences from the Moroccan strain of An. sergentii (GenBank accession No. MF919398) compared with An. gambiae (GenBank accession No. Y13592.1) sequence

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Ace-1 mutation

The 60 specimens of An. sergentii mosquitoes analyzed for kdr mutations were also genotyped for ace-1 mutation at codon position 119 (G119S) using PCR-RFLP. The ace-1 mutation was not detected in any of the tested An. sergentii specimens.


  Discussion Top


In Morocco, two mosquito species are considered as the principal vectors of malaria, An. labranchiae in the north of the country and An. sergentii in the south. This study represents the first work investigating the bionomics and resistance status of the southern Moroccan An. sergentii specifically in the region of Marrakesh where an alarming increase of the imported P. falciparum malaria cases is detected each year[4],[8],[9],[10],[11],[12].

This work was divided into two phases; in the first one, we investigated the presence of the species, characterized its larval breeding sites, determined its seasonal activity and identified blood meal sources of fed females, and in the second, we investigated its susceptibility to the insecticides.

During the two-year surveillance study, regular searches revealed the presence of An. sergentii during the summer and autumn seasons in Morocco. The summer-autumnal activity of this species has already been reported in Morocco in previous studies[1],[5],[7]. The breeding sites were identified in water courses within the rural zone of the city of Marrakesh, with clear and sunlit water. The presence of An. sergentii larvae were often associated with An. dthali and An. cinereus larvae. The adults were generally found resting inside animal shelters and much less in human rooms. The number of adults collected outside the buildings, using CDC traps was significantly low.

Identification of An. sergentii blood meals showed a tendency to feed on animals. However, since 6% of its blood meals analyzed corresponded to human blood, implicatings it as having an important role in a possible re-introduction of malaria in southern Morocco. This finding is in agreement with earlier results recorded from the northern Moroccan population[7] and from the Egyptian and Yemeni strains[16],[17].

Because An. labranchiae and An. sergentii are the only reported malaria vectors in Morocco, the second part of this study elaborated on the work previously done on An. labranchiae, located in north Morocco, through the evaluation of the insecticide resistance of An. sergentii. The adults of An. sergentii showed no resistance to any of the two tested insecticides. Even after years of treatment with DDT, An. sergentii didn’t appear to develop resistance to this insecticide as in contrast to An. labranchiae which has shown low to moderate resistance to DDT even after years of not using it[26],[27],[28].

One of this study’s objectives was to investigate and detect the kdr mutation as it confers resistance to pyrethroids and DDT and the ace-1 mutation that confers resistance to organophosphates and carbamates. Due to the low density of An. sergentii, only two insecticides were used for testing, based on the results of previous studies[26],[27],[28] conducted on An. labranchaie that showed resistance to DDT and susceptibility to pyrethroids.

Sequencing of VGSC, provided for the first time greater insights into the nucleotide composition of the wild populations of An. sergentii. It also revealed the presence of several variants in VGSC compared with An. gambiae in the partial sequence of the exon 20 that also contained the IIS6 coding region. However, three nucleotide substitutions resulted in the modification of the amino acid composition from glutamic acid to isoleucine. No earlier studies have described this substitution as a mutation that confers resistance to DDT and pyrethroid insecticides. These results are important for the generation of a regional genomic data to understand the mechanisms of insecticide resistance.

The results of PCR-RFLP also confirmed the absence of the G119S mutation in all of the analyzed mosquitoes. Susceptibility to organophosphorus insecticides (malathion, fenthion and fenitrothion) and to pyrethroids (permethrin, lambdacyalothrine and deltamethrine) and carbamates (propoxur) used in Morocco has already been reported in other mosquitoes including An. labranchiae[26],[27],[28]. However, DDT is no longer in use, which minimizes the development and spread of resistance, and therefore, pyrethroids and carbamates remain as efficient alternative insecticides to use considering that malathion is not prioritized in the vector control program because of its toxicity.


  Conclusion Top


From the results of this study, we concluded that An. sergentii was found resting indoors mainly in the summer and autumn. It demonstrated a marked zoophilic feeding but also showed it can feed on humans. Anopheles sergentii could potentially be responsible for the re-emergence of malaria in Morocco, considering the high vulnerability of the country due to the intensive circulation of gametocyte carriers coming from endemic countries. However, the absence of insecticide resistance mutations, mainly kdr and ace-1, indicates that pyrethroids and organophosphates can be efficient in the control of An. sergentii using an integrated vector control program, resulting in minimizing the risk of malaria resurgence.

Conflict of interest

There are no potential conflict of interests to declare.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

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