The study was conducted at the Laboratoire d’Ecologie Vectorielle et Parasitaire (LEVP) of Cheikh Anta Diop University (UCAD), Dakar. The laboratory operates an insectary maintaining several Anopheles strains, including An.coluzzii strain from Cameroon, An.arabiensis strain from Dakar, maintained for several generations, and the An.gambiaes.s. (Kisumu) strain from Kenya. The facility also includes a controlled experimental station for larvicide evaluation.

Novaluron, 1-[chloro-4-(1, 1, 2-trifluoro-methoxyethoxy) phenyl]-3-(2,6-difluorobenzoyl) urea] is an IGR developed by Makhteshim-Agan Industries Ltd and supplied by Syngenta [24]. It acts as a chitin synthesis inhibitor (CSI) belonging to the benzoylurea chemical class.

2.3.1. Biological Material

Stage III and IV larvae, as well as pupae from two Anopheles species were used in laboratory bioassays: An.coluzzii, a laboratory strain maintained for several generations and known to be susceptible to DDT and pyrethroids, An.arabiensis, a laboratory-maintained strain. To determine the activity range of the product, larvae and pupae were initially exposed to concentrations ranging from 0.01 µg/l and 1000 µg/l. Next, sixteen (16) concentrations were selected: five (5) doses for stage III larvae (0.01 µg/l; 0.05 µg/l; 0.1 µg/l; 0.5 µg/l and 1 µg/l); 5 doses for stage IV larvae (1 µg/l; 2 µg/l; 3 µg/l; 4 µg/l and 5 µg/l) and 6 doses for pupae (100 µg/l; 200 µg/l; 300 µg/l; 600 µg/l; 700 µg/l and 750 µg/l ug/l).

2.3.2. Preparation of Test Solutions

A stock solution was prepared following WHO recommendations [23]. A 1% stock volume was obtained by adding 10 ml of the product stock solution to 990 ml of distilled water, yielding a concentration of 1000 mg/L. This solution was then diluted ten times by mixing 2 ml of the stock solution with 18 ml of distilled water. Serial dilutions were subsequently prepared, and aliquots (100 - 1000 µl depending on the target concentration) were added to enamel-coated trays containing 250 ml of tap water and 25 larvae, or plastic cups containing 100 ml and 25 pupae. Disposable pipettes were used for transfer, with separate pipettes for treating and control batches to avoid cross-contamination. Each concentration was tested with four replicates, and an equivalent number of controls. The entire experiment was repeated three times on separate days. Larvae were fed every two days with Tetramin® fish food and development status (larvae, pupae and adults) were recorded daily until all individuals had either emerged or died. Live pupae from each tray were transferred to plastic cups containing 25 ml of water from the original tray for emergence monitoring.

2.3.3. Environmental Conditions

During the tests, temperature ranged from 24 to 27˚C, with a relative humidity of 80% ± 10% and a 12:12 light-dark photoperiod.

2.3.4. Effect of Novaluron on Pupation Rate

The effect of novaluron on larval mortality was assessed to determine pupation inhibition rates (PI%). Moribund larvae, dead larvae and pupae that had not fully completely separation from the larval exuviae were classified as affected by novaluron. Data from all replicates were pooled to calculate the mean number of larvae impacted per concentration.

The semi-natural tests were conducted at the experimental station located on the premises of the former Geological and Mining Research Office (GMRO), which currently hosts the Laboratoire d’Ecologie Vectorielle et Parasitaire (LEVP).

The station consists of a 20 m² enclosed structure, delimited by a 1 m high wall topped with a plastic mosquito net extending to a transparent plastic roof. This setup allows natural sunlight to penetrate while preventing the containers from being filled by rainwater and avoiding contamination from external mosquitoes or other insects. Inside the enclosure, ten white-bottomed plastic batches (48 cm × 37 cm × 23 cm) were arranged on the ground in two rows of five, spaced at least 1 m apart. Each bin was covered with a removable mosquito net equipped with a sleeve to facilitate the collection of pupae and emerging adults during follow-up.

Following the two laboratory experiments, DD was established (double of EI₉₉), and two monitoring cycles were initiated at the experimental station using third-instar larvae of the two Anopheles species tested. For each species, three concentrations, expressed as multiples of the DD, were evaluated. Monitoring of An.coluzzii was carried out for 33 days at concentrations of 0.6 µg/l, 3 µg/l and 6 µg/l and that of An.arabiensis was carried out for 40 days at concentrations 1.5 µg/l, 7 µg/l and 15 µg/l.

2.4.1. Collection of Wild Larvae

Anophelescoluzzii larvae used for the tests under semi-natural conditions came from the insectarium. The wild larvae used at the experimental station are of the An.arabiensis species, collected from the Dakar suburbs. In fact, entomolgical studies to characterize larval habitats, as well as the molecular identifications carried out annually by the LEVP, always revealed the exclusive presence of An.arabiensis in Dakar [25][26]. Larval surveys were conducted at larval breeding sites to collect aquatic stages of wild An.arabiensis used during the trials. Larvae were sampled using the dipping method [27], which consists in approaching the water body with caution, visually inspecting it for the presence of Anopheles larvae, and dipping a ladle or tray into calm, sunlit, shallow areas where larvae are most likely to concentrate. The characteristics and geographical coordinates of each surveyed breeding site were recorded. The Anopheles larvae collected were sorted by developmental stage and transferred into containers filled with water from their original breeding site for transportation to the experimental station.

2.4.2. Experimental Setup

Under semi-natural conditions, a total of eight batches were used, i.e. four batches per species (one for each concentration and one for control). Each treated batch contained 3 liters of dechlorinated tap water, 50 larvae and the volume of Novaluron solution required to achieve the desired concentration. The two control batches contained only 50 larvae and 3 liters of dechlorinated tap water. New lots of 50 larvae were introduced into the containers whenever the previous lots were depleted due to adult emergence or mortality. No additional water was added to the containers during the monitoring period, although a slight drop in the water level was noted.

In both laboratory and semi-natural conditions, test validity was determined based on larval or pupal mortality and pupation rates observed in the control groups. The test was considered invalid if the pupation rate in the control was greater than 10% within 24 hours following treatment, or control mortality was ≥20%. The test was considered valid if control mortality was < 5% and the pupation was < 10%. When control mortality ranged between 5% and 20%, emergence in the treated groups was corrected using Abbott’s formula (1925).

As with all insect growth regulators, the efficacy and persistence of Novaluron were assessed by calculating the emergence inhibition rates, cumulative mortality and pupation inhibition rate using the following formulas:

Emergence Inhibition Rate (EIR)

T is the percentage survival in the treated group and C is the percentage survival in the control group

Cumulative Mortality (CM)

CM (%) = LM + PM + AM

LM is the Larval mortality; PM the pupal mortality and AM the Adult mortality

Pupation Inhibition (PI)

T is the percentage of pupation in the treated batch and C is the percentage of pupation in the control group.

The concentrations causing 50% and 99% inhibition (IC₅₀ and IC₉₉) were determined using dose-response curve analysis in XLStat version 2022.

A chi-square test was used to compare emergence inhibition, larval, pupal and adult mortality rates, as well as the percentage of adult emergence between treatment groups. Statistical significance was set at p ≤ 0.05.

Larvae tests: Dose-response assays showed that Novaluron affected adult emergence in An.coluzzii and An.arabiensis at very low concentrations. For L3, inhibition of emergence was observed at doses ranging from 0.01 to 0.5 µg/l, whereas for L4, effective concentrations ranged from 1 to 6 µg/l (P = 0.337 and P = 0.957), respectively.

The minimum doses that completely inhibited adult emergence were estimated at 0.03 - 0.07 µg/l for L3 and 3.65 - 5.05 µg/l for L4.

Pupation inhibition rates were generally high for all concentrations tested on both species at the L3 and L4, ranging from 58.33 to 100%, except for the 0.01 µg/l concentration in An.arabiensis (25%, P < 0.05). However, none of the concentrations tested on L4 achieved complete inhibition of pupation (Table 1).

Emergence inhibition rates (EIR) were also high for both species, ranging from 54.54 to 100%.

The data from the three independent dose-response series showed consistent trends for both species. They were therefore pooled by concentration to estimate overall emergence inhibition (EI); EI₅₀, and EI₉₉ and DD (Tables1-2). For L3, EI₅₀and EI₉₉ values were low: 0.145 and 0.29 µg/l for An.coluzzii; 0.37 and 0.69 µg/l for An. arabiensis. For L4, higher concentrations were required: 1.95 and 3.61 µg/l An.coluzzii strain; 2.76 and 5.02 µg/l for An.arabiensis (Table 2).

Table 1. Pupation and adult (inhibition rate) of pupation in Anophelescoluzzii (insectary strain) and Anophelesarabiensis (Dakar population) exposed at the L3 and L4 stages to different concentrations of Novaluron.

Table 2. EI₅₀, EI₉₉ values, 95% confidence intervals (CI) and diagnostic doses (DD) for L3, L4 and pupal stages of Anophelescoluzzii and Anophelesarabiensis exposed to Novaluron.

Pupae test: The pupal bioassay demonstrated the contact mode of action of Novaluron, with activity observed at concentrations ranging from 100 to 750 µg/l (Figure 1). The EI₅₀ and EI₉₉ values pupae were higher than those for larvae: 145 µg/l and 260 µg/l for An.coluzzii, 428.37 and 714.13 µg/l for An.arabiensis. Overall, An.coluzzii was significantly more susceptible to Novaluron than An.arabiensis (P = 0.03).

Figure 1.Cumulative mortality of Anophelescoluzzii (insectary strain) and Anophelesarabiensis (Dakar population) pupae and adults following exposure to Novaluron.

Anophelescoluzzii: The CM in the control batches was very low throughout the monitoring period. By day 11, all concentrations tested (0.6 µg/l, 3 µg/l and 6 µg/l) resulted in 100% CM and complete emergence inhibition (EIR = 100%). No pupation was observed at any of these doses.

By day 21, only the 6 µg/l concentration maintained complete CM. At 0.6 µg/l and 3 µg/l, it reached 86% and 94%, respectively. Larval mortality ranged from 68% at 0.6 µg/l to 82% at 6 µg/l, PM from 12% to 20%, and AM from 4% to 6%. Emergence inhibition rates ranged from 78.26% to 95. 65% at 0.6 µg/l (P between 0.057 and 0.924) (Figure 2 and Table 3).

At day 33, CM ranged between 68 and 86%. Larval and pupae mortality were similar respectively between the 0.6 µg/l and 3 µg/l doses (LM: 34% and 38%; PM: 24% and 28%, P = 0.91). Emergence Inhibition rate varied between 56.25% and 77.08%. No significant difference was detected between CM across the tested concentrations (P = 0.674) during the entire monitoring period. However, a significant difference was observed in the LM values for the 0.6 µg/l dose between day 11 and day 33 (100% vs. 34%, P < 0.001) (Figure 2 and Table 3).

Figure 2.Cumulative mortality (CM) of L3 larvae, pupae and adults of Anophelescoluzzii (insectary strain) following exposure to Novaluron in the experimental station trial. **: End of monitoring of the 50 larvae and reintroduction of 50 new larvae; *: End of monitoring of the 50 larvae.

Table 3. Mortality of Anophelescoluzzii (insectary strain) L3 larvae, pupae and adults after exposure to Novaluron over 33 days.

LM: Larval mortality; PM: pupal mortality; AM: Adult mortality; LA: Living adults; EIR: Emergence Inhibition Rate.

Anophelesarabiensis: As with An.coluzzii strain, mortality in the control batches was very low throughout the monitoring period. On Day 10, CM and an EIR of 100% were achieved with all tested concentrations (1.5 µg/l, 7.5 µg/l, and 15 µg/l). No pupation or emergence was observed (Figure 3 and Table4).

Figure 3. Cumulative mortality (CM) of L3 larvae, pupae and adults of Anophelesarabiensis (Dakar population) following exposure to Novaluron in the experimental station trial. **: End of monitoring of the 50 larvae and reintroduction of 50 new larvae; *: End of monitoring of the 50 larves.

On Day 19, only the 15 µg/l concentration provided complete CM; for the 1.5 µg/l and 7.5 µg/l, it was, respectively, 82% and 90%, with EIR ranging from 76.16% to 93.75%. Larval mortality ranged from 68% at 1.5 µg/l to 80% at 15 µg/l; PM, from 12 to 16%; and AM, from 6% to 20% were observed. No significant difference was observed between the CM and the EIR values for the three doses (P ranging from 0.058 to 1) (Figure 3 and Table4).

On Day 29, CM ranged from 76 to 92%, LM, 42% at 1.5 µg/l to 68% at 15 µg/l; PM was similar, from 20 to 28%. The EIR ranged from 63.82 to 87.23%. Larval mortality was very high compared to PM (P < 0.01) (Figure 3 and Table4).

On Day 40, CM ranged from 64 to 84%, and EIR ranged from 52.08 to 77.08%. Larval mortality were comparable, ranging from 40% at 1.5 µg/L to 50% at 7.5 µg/l; PM were also similar, ranging from 20 to 28%. The LM and EIR values were not significantly different (P = 0.052) (Table 4).

No significant difference was observed between the CM of the tested doses (P = 0.965) throughout the monitoring period (Figure 3).

Table 4.Mortality of L3 larvae, pupae and adults of An.arabiensis (Dakar population) after exposure to Novaluron over 40 days.

LM: Larval mortality; PM: Pupal mortality; AM: Adults mortality; LA: Living adults; EIR: Emergence Inhibition Rate.