Efficacy of the Microbial Larvicide VectoMax®G against Anopheles gambiae s.l. and Culex spp. Larvae under Laboratory and Open Field Trial Experiments in the City of Yaoundé, Cameroon

Background: With the rapid expansion of insecticide resistance limiting the effectiveness of insecticide-based vector control interventions, integrated control strategies associating larviciding could be appropriate to improve current control efforts. The present experimental study assesses laboratory and field efficacy of the larvicide VectoMaxG on Anopheline and Culicine larval stages in Yaoundé. Methods: The effect of the larvicide VectoMaxG, a combination of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs), on larval development was assessed during both laboratory and open field trial experiments. Laboratory experiments permitted the evaluation of five different concentrations with four replicates/experiments. Laboratory experiments were conducted with Anopheles coluzzii “Ngousso” and Culex quinquefasciatus laboratory strains. Open field trials were conducted using sixteen plastic containers with a diameter of 0.31 m buried in an array of four rows with 4 containers each. Distance between rows and between containers in a row was 1 meter. This experiment permitted to test the How to cite this paper: Edmond, K., Aurelie, F.D.G., Nadège, S.-C., Roland, B., Landre, D.-D., Abdou, T., Serges, D., Flobert, N., Parfait, A.-A., Sinclair, W.C. and Christophe, A.-N. (2022) Efficacy of the Microbial Larvicide VectoMax®G against Anopheles gambiae s.l. and Culex spp. Larvae under Laboratory and Open Field Trial Experiments in the City of Yaoundé, Cameroon. Advances in Entomology, 10, 34-51. https://doi.org/10.4236/ae.2022.101003 Received: October 15, 2021 Accepted: November 27, 2021 Published: November 30, 2021


Background
Vector control in Africa heavily relies on insecticide-based interventions such as Long-lasting insecticide nets (LLINs) and indoor residual spraying (IRS) [1]. However, the effectiveness of these measures is affected by a certain number of limits including the rapid expansion of insecticide and behavioural resistance in vector populations [2] [3] [4]. To minimize the dependence on chemical insecticides, there is an urgent need to explore alternative measures for mosquito control. One such alternative control approach is to include larviciding as an additional intervention in urban settings [5] [6]. In Africa, the use of larval source management (LSM) as an additional tool for integrated vector management (IVM) has become increasingly requested in different epidemiological contexts [7]. Larviciding has been at the forefront of control strategies that successfully eliminated malaria in many places [8].
Larviciding is a vector control intervention that consists of regular application Bti has a broad spectrum that could be targeted and has a rapid control effect and low potential for resistance development in the field [11] [12]. Moreover, to become resistant to Bti, an individual must develop resistance mechanisms to each of the four toxins, namely Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Acontained in each Bti spore [13]. Nowadays, there have been no reports of resistance to Bti in mosquito populations despite it having been applied for decades in several countries [13] [14]. However, some biotic and abiotic factors such as mosquito species, the rate of ingestion, the density and age of the larvae, the temperature and the organic matter content have been reported to affect the efficacy of biolarvicide formulations in the field [12] [15]. Unlike Bti, which requires clean water to be effective, Bs can provide good control of larvae in polluted water which is the preferential breeding habitats of Culex [12] [16] [17]. Bacillus sphaericus has an extended residual activity. However, there have been several reports suggesting development of resistance to Bs in many places [18] [19] [20], indicating that resistance management strategies are needed in operational programmes that use Bs. A mixture of Bti and Bs may represent a potentially effective approach and may prevent emergence of resistance to biolarvicides [21]. In this study, the effect of the larvicide VectoMax ® G combining Bacillus thuringiensis israelensis strain AM65-52 and Bacillus sphaericus strain 2362 formulation (VectoMax ® G) on Culex and Anopheline larvae was tested to determine the effectiveness of different doses and residual effect of the larvicide in both laboratory and open field trial experiments.

Mosquito Populations
Laboratory assays were carried out with 3 rd instar larvae of An. coluzzii "Ngousso" and Cx. quinquefasciatus laboratory colonies. These mosquito strains were originally colonized from Anopheles s.l. and Culex spp. larvae collected respectively in 2006 and 2017 in the city of Yaoundé and maintained at the OCEAC insectary. The open field trials were conducted with larvae from wild An. gambiae s.l. and Culex spp. females that naturally oviposited or were added in the experimental containers.

Laboratory Assays
The granular formulations of VectoMax ® G were tested in the laboratory to determine the minimum effective dosages, from the recommended dose by the manufacturer. A total of 50 to 200 third instar larvae were exposed in different plastic containers containing different concentration of the larvicide. Test concentrations were obtained after sequential dilution of recommended dose by the manufacturer (500 mg/m 2 ). The formulation of VectoMax ® G was weighed and sprinkled on the water surface of the containers.
The bioassays were run with five different concentrations of VectoMax ® G (ranging from 500 to 25 mg/m 2 ). Each experiment contained a control (distilled water only). The experiments were run in four replicates at the same time and the entire experiments were carried out on three different occasions. All trials were conducted at ambient temperatures ranging from 25˚C to 27˚C and larvae were not fed during the experiments.
To determine laboratory efficacy of biolarvicide, mean larval mortality of mosquitoes in each concentration was calculated over a 12 hour period. Third instars (laboratory strain) of An. coluzzii and Cx. quinquefasciatus were used.
Concerning the assessment of the residual activity of VectoMax ® G at different doses, only An. coluzzii "Ngousso" larvae were introduced into the test containers at day 0 post-treatment. Different mosquito larval batches were tested at day 1, day 3, day 7, day 14, day 28, and day 35. Larval mortality was recorded 24 hours after experiment with each dose and dead larvae were removed. Moribund larvae were considered as dead and included in the analyses. When mortality exceeded 10% in the controls, the experiment was discarded and repeated.

Open Field Trials
Open field trials with VectoMax ® G were performed between July and September 2018, corresponding to the dry season and the beginning of the rainy season. Artificial ponds were created following the experimental design described in Fillinger et al. [23]. Sixteen plastic containers with a diameter of 0.31 m each and about 30 cm long were buried into an array of four rows with 4 containers each.
Distance between rows and between containers in a row was 1 meter. Soil from known Anopheles breeding sites was added to each container, providing a stan- mg/m 2 . Prior to experiments, 80 Anopheles (60 first and second instar and 20 third and fourth) and 20 Culex spp. (10 first and second instar and 10 third and fourth) field-collected larvae were placed in each container. Anopheles s.l. and Culex spp. larvae were collected from surrounding natural habitats for which recent studies indicated that 91.1% of anopheline larvae were An. coluzzii and 8.9% An. gambiae s.s. [24] and 79.4% Cx. quinquefasciatus [25].
The respective concentrations of biolarvicide (VectoMax ® G) were applied evenly on the water surface of each container, by hand. All containers were examined daily and larval count was performed in all 16 containers, pupae were removed. Immature mosquitoes were classified in three categories: early instars (first and second), late instars (third and fourth) and pupae. All larvae were classified to genus and development stage and then returned to their respective containers.

Data Analyses
From the bioassay results, lethal concentration (LC50 and LC95) values were determined using log-probit regression analysis in WINDEL software version 32. LC50 represents the concentration of the larvicide killing 50% of larvae and LC95% is the concentration of the larvicide killing 95% of larvae. The percentage reduction in larval mosquito densities was calculated using the formula of Mulla [26] which takes into account natural changes (for instance through predation) occuring at the same level and rate in both treated and un-

Laboratory Experiments
Mortality Rates of Mosquito Larvae Exposed to Different Concentrations of VectoMax ® G Larval mortality was determined after 12 hours exposition to VectoMax ® G as a ratio of death and exposed larvae. The mortality results showed that third instar larvae of An. coluzzii and Cx. quinquefasciatus were susceptibles to all serial dilutions of the VectoMax ® G with 100% mortality recorded within 4 -12 hours and 6 -9 hours after exposition respectively (Table 1 and Table 2). It was observed that the time taken to achieve 100% mortality varied according to the larvicide concentrations. With concentration of 500 mg/m 2 (representing the minimal  Mortality rates for each species at different doses are shown in Table 3. No dead was observed in control containers.

Open Field Trials
Anopheline and Culicine mosquito larvae were detected 6 to 7 days after the artificial habitat was set-up. Both early instars (L1 and L2 larvae) and late instar (L3 and L4 larvae) were recorded. The percentage reduction of Anopheles s.l.
larvae following VectoMax ® G application is shown in Table 4. Two rounds of treatments took place with each lasting more than 12 days. Different concentrations were tested during each round. The mean number of larvae including early, late instars and pupae in control and treated sites are shown in Figure 1. A 100% mortality rate was recorded within the first 24 hours after larviciding application. Over time in both treated and untreated containers, natural declines    T1  T2  T3  C  T1  T2  T3  C  T1 T2 T3 T1  T2  T3  T1  T2    Asterisks (*) indicate days with larvicide application. C = Control; T1, T2 and T3 day 0* ≠ T1, T2 and T3 day 13* (see Figure 1). and increases of larval densities were observed. In the first round of larvicide treatment, a reduction rate of 100% for late instar larvae was observed for up to six days after treatment. VectoMax ® G was very effective against the late instars reducing the population by 100% within 24 hours post treatment for all concentrations. Generally, the larvicide impact on the late instars remained high up to day 6 post treatment with VectoMax ® G (Figure 1). VectoMax ® G was effective against early instars of Anopheles s.l. with a reduction of 100% of the larval population within 24 hours. This effect lasted up to day 3 after application before recolonization of the sites occurs. Though the VectoMax ® G treatments resulted in 100% mortality of the early and late instars of Anopheles s.l. within 24 hours, an initial recolonization of treated sites by L1 larvae was generally observed three to four days after treatment. All concentrations tested, were equally effective up to 4 days post-treatment for early instar larvae and up to 6 days for late instars. During the first 6 days of round one and the first 8 days of round two, no pupae were found in the treated containers, meaning that the late instars were killed by the action of larvicide. The effects of VectoMax ® G on larval densities of Culex spp. and reduction rate compared to control containers is shown in Figure 2 and Table 5. Sublethal concentration of VectoMax ® G was not found to be effective against late instars of Culex larvae 24 hours post treatment. A 100% reduction was obtained after 48 hours. When supra doses were used 100% reduction and no recolonization of sites was observed up to seven days post-treatment. VectoMax ® G was effective against early instars of Culex spp. with a reduction of 81% -100% of the larval population within 24 hours depending on concentration/doses. This effect lasted up to day 5 after application. More interesting, pupation levels were very low in the treated ponds (Figure 2), which is considered the most important parameter for efficacy assessment of larval control measures [27].  C  T1  T2  T3  C  T1  T2  T3  C  T1  T2  T3  T1  T2  T3  T1  T2

Discussion
In this study, several doses of VectoMax ® G were used regarding the minimum reference dose recommended by the manufacturer, i.e., 500 mg/m 2 . Doses lower than the latter were used to estimate the effectiveness of the product in case of insufficient quantity or wrong weighing. Other doses greater than or equal to the standard dose were also used. The manufacturer recommends retreatment after 3 to 4 weeks under conventional weather conditions for normal and above normal doses and states that an appearance of stage 1, 2, or 3 larvae do not indicate a need for retreatment. Therefore, this study also examined whether the manu-  [30], and in Malaysia, the same number of days were reported with Bti [31].
Concerning the open field trial, we recorded a very low natural colonization of the containers by mosquitoes; this could be due to the presence of several potential and natural breeding sites/water collections not far away from the study site.
It is also important to point the fact that the size, depth, and populations have also been reported in other studies [27]. VectoMax ® G, was found to reduce both early and late instar stages. Surprisingly, late instars stages were found to be more affected by treatment and retreatment of sites.
The different applications of VectoMax ® G resulted in an effective reduction of the density of Anopheline and Culicine larvae and pupae (81% to 100%) with the normal and higher doses 24 hours after the treatment. Our results are similar to those of many studies such as the one conducted by Owolola et al. [32] in Lagos (Nigeria), where the small-scale field trial caused an effective inhibition of the emergence of An. gambiae s.s. and Cx. quinquefasciatus greater than 80%, as well as the study in Penang (Malaysia) by Ahmad et al. [33] reporting similar results on Cx. quinquefasciatus and Ae. aegypti. For sublethal doses, the efficacy of VectoMax ® G was good on Anopheles larvae and pupae (96% -100% reduction rate) but less important with Culex spp. (27% -83% larval reduction). Vec-toMax ® G was also found to be more effective on Anopheles larvae during the first day's post-treatment but less effective after one week. On the opposite, Culex spp. larvae were less affected during the first day post-treatment but displayed a high reduction rate several days after treatments. This could be explained by the difference in the feeding and resting behaviour between Anopheline and Culex mosquitoes [34] [35] [36]. Anopheles larvae feed mainly on the surface and generally only dive to escape from danger. They do not remain at depth as long as Culex spp. where the latter mainly feed. Because the larvicide crystals sediment after few days, they are during the first days more available to Anopheles which quickly consume the lethal quantity and then when they sediment they become available to Culex spp.
periments. The current literature reports different findings on the residual effect of microbial larvicides, Kinde-Gazard and Baglo [37] reported 9 days before larvae reappeared after larvicide treatment. Kroeger et al. [38] found in a study carried out in Ecuador and Peru that the effect of treatment could last 7 to 10 days.
In Eritrea, Shililu et al. [39] described an effect of up to three weeks of microbial larvicides. In Burkina Faso, Kenya, and Ghana, Dambach et al. [40], Fillinger et al. [23] and Nartey et al. [41] described an effect ranging between three and six days. Our findings are in line with these observations [42]   This is in line with the observation of Becker and Margalit [14] stating that the efficacy of the different formulations is influenced by the availability of Vec-toMax ® G crystals in the first 10 cm of the surface of a water column. In the present study, the high-water depth (30 cm) is a possible explanation for the low persistence of the residual effect of the product. It has also been reported that the presence of a high concentration of chlorine and iron seems to reduce the toxic activity of VectoMax ® G crystals [43]. Although being carefully washed, the containers may have retained chemical residues that could have reduced the persistence of the larvicide. It was also noted throughout the study that many competitors such as frog tadpoles, were regularly present in most containers. Although their numbers were not measured, they may be responsible to some extent for the reduced persistence of the product, as they consume the product and quickly make it unavailable to larvae. Organic pollution also acts on the effectiveness of the product by adsorption of the product crystals on organic particles, facilitating precipitation, which decreases their availability [44] [45] [46]. Despite these possible limiting factors, the present study highlighted the high efficacy of the biolarvicide VectoMax ® G against Anopheline and Culicine larvae.

Conclusion
Our results strongly suggest that the microbial larvicide VectoMax ® G has a high larvicidal effect on both Anopheles and Culex spp., the known vectors for Plasmodium and Lymphatic filariasis respectively. Given the high rate of malaria in Cameroon, successful and affordable vector control strategies, such as the use of microbial larvicides could be key for the successful elimination of malaria in urban settings.

Authors Contributions
KE and CAN conceptualized and designed the study; KE, DS, NE, BR, DDL, TA and S-CN performed the field experiments; KE performed laboratory experiments and statistical analysis. FG, NF, A-AP and WCS critically reviewed and amended the manuscript. KE and CAN interpreted, analysed data and wrote the manuscript with input from all authors. All the authors read and approved the final version.

Funding
This study received financial support from the Welcome Trust Senior Fellowship in Public Health and Tropical Medicine (202687/Z/16/Z) awarded to CAN.