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Year : 2019  |  Volume : 56  |  Issue : 3  |  Page : 207-211

Effects of insecticide resistance on the reproductive potential of two sub-strains of the malaria vector Anopheles coluzzii

1 Unité d'Entomologie Médicale, Institut Pasteur de Dakar, Dakar, Sénégal
2 Laboratoire d'Ecologie Vectorielle et Parasitaire, Departement de Biologie Animale, Université Cheikh Anta Diop de Dakar, Dakar, Sénégal

Date of Submission24-Apr-2018
Date of Acceptance29-Sep-2018
Date of Web Publication09-Jul-2020

Correspondence Address:
Dr Ibrahima Dia
Unité d'Entomologie Médicale, Institut Pasteur de Dakar, 36 Avenue Pasteur, BP 220, Dakar
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.289401

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Background & objectives: The emergence and spread of insecticide resistance in African malaria vectors raise concerns over the control of malaria disease. Therefore, the implementation of better control strategies need a thorough understanding of the effects and mechanisms of resistance on vector adaptation capacities. We studied the effects of insecticide resistance on the reproductive potential of two laboratory sub-strains of the malaria vector Anopheles coluzzii characterised by phenotypic resistance/susceptibility to DDT.
Methods: The two sub-strains were selected from a laboratory strain of An. coluzzii using WHO test tubes. For each sub-strain, the number of produced and hatched eggs, developmental time, mosquito stages mortality, sex ratio and insemination rates after dissection of spermathecae were compared as measures of reproductive potential.
Results: Overall, the susceptible sub-strain produced higher but not significant mean numbers of eggs. However, the mean numbers of hatched eggs, larvae, pupae and adults were significantly lower than those of the resistant substrain. The mean time from egg–hatching to adult–emergence, egg–flooding to hatching, I instar to pupae and pupae to adult were similar between the two sub-strains. The mortality rates at the pupal stage were significantly different between the two sub-strains. Of the dissected spermathecae, 85.1% of the females from the resistant sub-strain were fertilized compared to 66.1% of the females from the susceptible sub-strain (p <0.0001). The resistant sub-strain produced more females in comparison to the susceptible sub-strain (respective mean sex ratio 1.37 vs 1.03, p = 0.01).
Interpretation & conclusion: The results show differential life history traits between the two sub-strains of the malaria vector An. coluzzii, particularly fertility, insemination rate and sex ratio. They may have varied implications for insecticide resistance spread, monitoring and management; and hence underscore the need of further investigations before any generalization.

Keywords: Anopheles coluzzii; insecticide resistance; reproductive potential; Senegal

How to cite this article:
Sy FA, Faye O, Diallo M, Dia I. Effects of insecticide resistance on the reproductive potential of two sub-strains of the malaria vector Anopheles coluzzii. J Vector Borne Dis 2019;56:207-11

How to cite this URL:
Sy FA, Faye O, Diallo M, Dia I. Effects of insecticide resistance on the reproductive potential of two sub-strains of the malaria vector Anopheles coluzzii. J Vector Borne Dis [serial online] 2019 [cited 2023 Mar 31];56:207-11. Available from: http://www.jvbd.org//text.asp?2019/56/3/207/289401

  Introduction Top

Malaria vector control in Africa is mainly based on the use of residual insecticides in human environment[1]. Long-lasting insecticide impregnated nets (LLINs) and indoor residual spraying (IRS) are currently the most used tools. Their concomitant use with high coverage has proven their effectiveness in the reduction of malaria associated morbidity and mortality[2]. Thus, the recent recorded successes contributed to the consideration of malaria elimination as a feasible objective[3]. However, this ambition is accompanied by an increase of resistance in malaria vectors to all the classes of insecticide available, which can substantially dilute the efforts of malaria vector control for the elimination of the disease. The mechanisms involved include target site insensitivity, enhanced enzymatic detoxification and behavioural adaptation to avoid the contact with the insecticides[4],[5]. Their possible consequences include variable fitness costs[6],[7],[8]. For these reasons, detailed information on the adaptive changes in vectors are required to set-up alternative control strategies and to design a thorough insecticide resistance management plan. The insecticide resistance management is primarily based on the understanding of the fitness costs associated with resistant gene that modulate the behavioural adaptation resulting from the selection of genetically inherited traits or phenotypic plasticity in response to implemented control measures.

In Africa, DDT resistance of Anopheles gambiae s.s. populations was reported early in the 1960s in Burkina Faso[9] and then confirmed in the 1990s in Senegal in An. arabiensis populations[9],[10]. In a study conducted in 20 sympatric sites of An. arabiensis, An. coluzzii and An. gambiae, in Senegal insecticide bioassays showed a general trend with resistance to DDT and pyrethroids and susceptibility to organophosphates and carbamates[11]. However, despite the description of the observed resistance, few studies have been carried out on the mechanisms involved as well as the adaptive related effects. As this situation is currently observed almost everywhere in Africa[12], the present work investigated the effects of insecticide resistance on the reproductive potential of the malaria vector An. coluzzii.

  Material & Methods Top

Anopheles coluzzii colony and rearing

In this study, we used the Yaounde strain of An. coluzzii colonized in 2008 from specimens collected in the Essos outskirt in Yaounde, Cameroon. It was maintained in standard conditions at a temperature of 27±2 °C, 75±5% relative humidity and a 12:12 h photoperiod. Larvae were maintained in 30×21×6.5 cm trays at a density of approximately 300 I instar larvae (L1) per tray with 1500 ml of deionised water. They were fed with a diet of finely grounded Novobel Flakes® (Animalis®, Paris, France). Pupae were collected daily and placed in small plastic cups that were introduced in adult cages for emergence. Emerging adults were kept in 30×30×30 cm Lumite Screen Collapsible Cages (BioQuip products, Rancho Dominguez, California, USA). They were fed with 10% (w/v) sucrose solution and females were blood-fed biweekly on Guinea pig and allowed to oviposit in Petri dish lined with a wet filter paper.

Selection of the sub-strains

The laboratory An. coluzzii colony was used to select two sub-strains (susceptible and resistant) based on their status to DDT [Figure 1]. For the susceptible sub-strain, 5 to 7 days old females from the parent colony were provided with two blood meals and fully-fed females separated in individual cardboard cup with a Petri dish lined with a wet filter paper to enable laying eggs. The adults were thereafter exposed to 4% DDT for one h and kept under observation for 24 h[13]. Egg batches from dead females were pooled and the resulting adults represented the susceptible sub-strain. For the selection of the resistant sub-strain, 5 to 7 days old females from the parent colony were exposed to 4% DDT for 1 h and after 24 h observation, alive specimens were regrouped and represented the resistant sub-strain [Figure 1]. All the sub-strains were maintained in the same conditions like for the An. coluzzii originated colony.
Figure 1: Flow chart showing the procedure of selecting of two substrains of Anopheles coluzzii.

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Experimental procedure

In total 30 females aged between 5–7 days from each sub-strain were selected and provided with two blood meals. They were then placed individually in labelled cardboard cup with a damp Petri dish lined with a filter paper to enable them to lay eggs. For each female replicate, the filter paper was removed and replaced daily until no laid eggs were observed and all laid eggs counted and placed in rearing pans (one pan for the progeny of each female replicate). The larvae were fed daily and larval pans were checked daily and the pupae counted and transferred into plastic cups that were then introduced in cages for adult emergence (one cage for each female progeny). For each progeny, the number and sex of emerging adults were recorded and 5–7 days after emergence, emerging females were submitted to spermathecae dissection to identify fertilized and unfertilized status. The terminalia and segment IX were removed and the golf-ball-like spermathecae were isolated, transferred to a slide and viewed under 100 × magnification on a compound microscope. For inseminated females, the thread-like spermatozoa were observed to exhibit a rotational movement while no such movements were observed in non-inseminated females.

Data analysis

The number of eggs and the rate of hatched eggs were recorded as measures of fertility and fecundity. In addition, for each female, the times from the first laid egg to the first hatched larvae, the time from larvae to pupae as well as from pupae to adult were recorded in numbers of days. For each sub-strain, the mortality rates were estimated at each stage (larvae, pupae and adults). The length in days from egg hatching to adult emergence as well as the ratio of female to male emergences were recorded for each of the 60 families. The insemination rate was calculated as the proportion of inseminated females to the total females dissected.

Prior to statistical comparison, the normality and ho- moscedasticity of the data were checked using Shapiro and Bartlett tests. The data were analysed accordingly using parametric or non-parametric procedures to test differences between the two sub-strains. Chi-squared tests were used to compare the proportions. For all tests, the alpha level was set at p <0.05. Statistical analysis was performed using R-software (v. 3.3.1).

  Results Top

Reproductive characteristics

Overall, the susceptible sub-strain produced higher but not significant mean numbers of eggs (Kruskal-Wallis : χ2 = 2.7, df = 1, p = 0.06) than the resistant sub-strain [Table 1]. The mean number of laid eggs per female for the susceptible sub-strain was 101.3 ± 9.4 while the resistant sub-strain laid a mean number of 82 ± 3.9 eggs per female. The mean numbers of larvae, pupae and adults produced by the resistant sub-strain were significantly higher than those of the susceptible sub-strain [Table 1]. The proportion of females produced by the resistant sub-strain was significantly higher than that of the susceptible sub-strain with 55.7% (996/1787) and 48.5% (668/1376) respectively (χ2 = 15.8, df = 1,p <0.0001).
Table 1: Reproductive characteristics for the resistant and susceptible sub-strains

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Developmental time and mortality rates

Adult emergence occurred at 9.4 ± 0.2 days and 9.1 ± 0.2 days after egg hatching, respectively for the resistant and susceptible sub-strains. Oviposition to hatching occurred 1.1 and 1.2 day for the resistant and susceptible sub-strains, respectively (Kruskal-Wallis : χ2 = 0.9, df = 1, p = 0.32) . The mean time from first instar to pupae was similar for both sub-strains (7 days for the resistant substrain and 6.7 days for the susceptible sub-strain, Kruskal-Wallis : χ2 = 14, df = 1, p = 0.2) with a range of 5–9 days for both the sub-strains. The majority of larvae pupated between 7 and 8 days following egg-hatching (73.3–96% for the resistant sub-strain and 80–96% for the susceptible sub-strain). The time spent between the pupal stage and adult-emergence varied between 1 and 2 days for both the sub-strains, the majority being observed after 1 day (70%, n = 21 for the resistant sub-strain and 90%, n = 20 for the susceptible sub-strain). The mean time from pupae to adult did not differ between two sub-strains (Kruskal- Wallis : χ2 = 3.7, df = 1, p = 0.06). The mortality rates from larval to adult stages were higher in adult stages for both the sub-strains [Figure 2]a. Significant difference was observed only at pupal stage [Figure 2]a.
Figure 2: Life history traits for the susceptible and resistant sub-strains: (a) Mortality rates at larval, pupal and adults stages. The bars represent 95% confidence interval; (b) Insemination rates; and (c) Sex ratios for the resistant and susceptible sub-strains. Error bars represent the standard error of the mean. The significance of differences is indicated (ns = not significant; * = p <0.05).

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Insemination rates and sex ratio

The dissection of spermathecae revealed that 85.1% (CI 95% = 82.1–87.9, n = 612) of the resistant sub-strain were fertilized compared to 66.1% (CI 95% = 62.8–69.2, n = 867) of the susceptible sub-strain [Figure 2]b. There was statistical significant difference between them (χ2 = 66.5, df = 1,p <0.0001). The mean sex ratios between the two sub-strains were significantly different (1.37 ± 0.10 for the resistant, 1.03 ± 0.09 for the susceptible sub-strain, p = 0.01) [Figure 2]c.

  Discussion Top

In this work, we studied several life history traits of the progenies of two An. coluzzii sub-strains based on their phenotypic resistance/susceptibility to DDT. The results showed that the susceptible sub-strain produced higher, but not significant absolute numbers of eggs compared to the resistant sub-strain. However, the mean numbers of hatched eggs produced were higher in the resistant sub-strain. Thus, even if it is not possible to directly infer to fitness cost (among other limiting factors, the lack of information on the males that fertilized the parent females used as well as the genetic loci associated with the phenotypic differences in insecticide resistance), there is a clear association between the life history traits studied and insecticide resistance. Indeed, as observed in our study, substantial fitness costs linked to fecundity were observed[6] between the susceptible referenced strains Kisumu and the resistant strains AcerKis and KdrKis, (and were confirmed among the AcerKis strain, KisumuP and Acerdu-pliKis strain which harbour the Ace-1 duplicated allele confering less resistance[14]) than the Ace-1. Several factors could explain such results including the quantity of blood ingested during blood feeding as well as the number of blood meal taken to achieve eggs maturation. In our experiments, it is possible that during blood feeding, resistant females took a lower quantity of blood in comparison to susceptible females. In fact for their first gonotrophic cycle, Anopheles females commonly require two blood meals for egg maturation[15]. This, probably because the females use their first blood meal to compensate potential energitic deficit after emergence[16],[17]. This phenomenon could be more marked in the resistant sub-strain as during our experiments the two sub-strains were blood fed twice.

Other factors including differential behavioural characteristics can also affect the number of produced eggs in the resistant sub-strain. This is the case of the ability to feed on potential host. In their laboratory experimental conditions on blood meal acceptance and amount of ingested blood on the same host, Belinato et al[18] showed that temephos-resistant Ae. aegypti ingested 15% less blood than the Rockefeller susceptible strain. This decrease was followed by 21% reduction in the number of produced eggs. Similar observations were made between a field selected pyrethroid resistant Ae. aegypti and the Rockfeller reference strain with a significant reduction of the relative amount of ingested blood in the resistant strain[19].

Considering the highly significant mean number of hatched eggs observed for the resistant sub-strain, it appears that the lower number of produced eggs observed are compensated by an increase in the numbers of hatched larvae. Therefore, despite the two blood meals offered to the parent females used in this study, a plausible hypothesis is that an energetic rather than reproductive cost is associated with insecticide resistance in the resistant sub-strain females as observed elsewhere[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20]. This is probably a result of lower teneral reserves associated with insecticide resistance as previously observed in carbamate resistant Cx. pipiens[21]. Further studies taking into account blood meal completion or several blood feedings to estimate the energetic reserves required, could help to unravel this hypothesis.

The resistant sub-strain showed similar developmental times between the different mosquito stages compared to the susceptible sub-strain as already observed in An. funestus and Ae. aegypti between resistant females and their counterparts[20],[21],[22]. These results could suggest that insecticide resistance has no effect on the developmental time of the different stages. These results contrast with those of Li et al[23] on the study of the reproductive characteristics of deltametrin-resistant and susceptible Cx. pipi-enspallens. The egg and larval phases development were prolonged as well as the time from hatching to emergence in the deltametrin-resistant strain. Similarly, during a process of selection of a deltamethrin-resistant Ae. aegypti, deleterious effects on both development and reproduction were observed in the course of nine generations[19].

The mortality rates were higher in adult stages for both sub-strains and significantly different only at the pupal stage. These observations contrast with those of Assogba et al[14] wherin there was a significant lower mortality rate between the larval stages in the An. gambiae AcerKis, homozygous for the G119S mutation in the gene conferring resistance to organophosphates ace-1 and car- bamates compared to the Kisumu susceptible reference strain[14]. As in the present study, the two sub-strains were reared in the same conditions, these results suggest the involvement of some other mechanisms that need to be investigated at least for the pupal stage.

The insemination rates and sex ratio were significantly higher in the resistant sub-strain. This means a better male reproductive and females mating successes in the resistant sub-strain. However, these results contrast with other records that showed a lower reproductive activity of permethrin-resistant Ae. aegypti males associated to a lower insemination rate of permethrin-resistant females when compared to a susceptible field strain[24] and gammaHCH/dieldrin RR males and females resistant An. stephensi who mate assortatively[25]. As the insemination rate and sex ratio are literally related, this situation is decisive for subsequent generations and need more investigations. Such results are important and if confirmed could explain in part the rapid increase in insecticide resistance in many parts of Africa.

  Conclusion Top

This study highlights the differential life history traits between the two sub-strains of An. coluzzi, particularly fertility, insemination rate and sex ratio. These results may have many implications for insecticide resistance spread, monitoring and management and, therefore, highlight the need of further investigations before any generalization.

Conflict of interest: None

  References Top

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  [Figure 1], [Figure 2]

  [Table 1]

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