The strain of An. coluzzii mosquitoes were collected at Soumousso (4˚02'45''W; 11˚00'46''N) using a mouth aspirators (see Figure 1 ). Soumousso is a village situated approximately 55 km southeast of Bobo-Dioulasso. Soumousso has a Sudanian climate and is characterized by Guinean savannah vegetation, with an

average rainy season occurring from May to October and annual rainfall ranging between 1000 and 1200 mm. The village is bordered by a semi-permanent stream [13] [14] .

Breeding habitats for mosquitoes include swamps, rainwater puddles, and semi-permanent quarries, which are conducive to the development of Anopheles mosquitoes. The following five species of malaria vectors are present in the area: An. coluzzii, An. gambiae s.s., An. funestus, An. nili, and An. Arabiensis [15] [16] .

Prior to laboratory analyses, we confirmed the presence of Microsporidia MB in mosquitoes using Polymerase Chain Reaction (PCR). This allowed us to distinguish between MB⁺ (infected) and MB⁻ (uninfected) mosquitoes.

After insemination, female mosquitoes were individually oviposited in plastic cups containing approximately 35 mL of tap water. Each female that had laid eggs was stored at −20˚C in 1.5 mL Eppendorf tube.

DNA extraction was performed according to the protocol established by Myriam and Cécile (2003) [17] .

For the PCR, specific primers (MB_F: 5' ATA GTA TAC TCG CAA GAG TG 3' and MB_R: 5' CTG TTA TAG CCT CTT TC 3') [6] , were used to amplify the 18S rRNA gene region. The reaction mixture for each sample consisted of 15 μL, including 7.5 μL of ultrapure water, 3 μL of master mix, 0.75 μL of each primer, and 3 μL of purified DNA. The PCR cycling conditions included an initial denaturation at 95˚C for 15 minutes, followed by 35 cycles of denaturation at 95˚C for 1 minute, annealing at 60˚C for 1 minute and 30 seconds, extension at 72˚C for 1 minute, and a final extension at 72˚C for 5 minutes.

The resulting PCR products were analyzed using 2% agarose gel electrophoresis with ethidium bromide staining, migrated for 1 hour, and visualized under UV light at 254 nm. The expected band size was 250 bp (see Appendix 1). After visualization, eggs from MB⁺ parents were separated from MB⁻ eggs, and the eggs were hatched separately to obtain stage 2 (L2) larvae.

In the insectarium, adult mosquitoes were housed in cages measuring 30 cm × 30 cm × 30 cm, with a density of 200 mosquitoes per cage. They were maintained at a temperature of 27˚C and a relative humidity of 75%, with a photoperiod synchronized with sunset. Mosquitoes were provided with a 5% glucose solution for sustenance, and females were fed rabbit blood for their blood meals. After feeding, females continued to be kept under standard insectarium conditions.

Eggs were collected by placing water-soaked blotting paper in Petri dishes, where females deposited their eggs. The eggs were dried for 24 hours, washed with 0.2% bleach, and filtered before hatching in a plastic tray containing 1 liter of dechlorinated tap water. The water was allowed to stand for at least 24 hours to let chlorine dissipate. After hatching, larvae were maintained under similar conditions as the eggs, with a density of approximately 200 larvae per tray and fed daily with cat food at a rate of 0.06 g/L. Every two days, the water in the larval tanks was replaced to minimize bacterial growth until pupation. Larvae and egg trays were organized on shelves. Pupae were collected with a Pasteur pipette and placed in beakers containing about 70 mL of tap water before being transferred to rearing cages for emergence, thus completing the cycle.

To assess the effects of larval diet on the life-history traits of Microsporidia MB-infected An. coluzzii, we used Tetramin brand fish food at three different concentrations: 0.18 g/L, 0.03 g/L (the average quantity used in our laboratory), and 0.0075 g/L (representing low and high quantities based on Sadia et al. [18] . The food quantities were measured using an Explorer Pro (OHAUS®) precision balance.

Each food quantity was tested on two groups of larvae: 150 MB⁺ larvae divided into three replicates of 50 larvae each, and 150 MB⁻ larvae serving as a control group, also divided similarly. Each replicate was fed daily with the assigned food quantity until reaching the pupal stage. After pupation, live pupae were transferred to rearing cages for emergence. The following parameters were measured: larval development time, pupation rate, emergence rate, and sex ratio.

To evaluate the effects of temperature and relative humidity on the life-history traits of Microsporidia MB-infected An. coluzzii, we established three temperature levels (21˚C, 27˚C, and 39˚C) and three relative humidity conditions (50% RH, 75% RH, and 80% RH). These parameters were selected to represent both extreme and average environmental conditions recorded in the Hauts-Bassins region [19] .

Three temperature-humidity combinations were tested: 21˚C - 80% RH, 39˚C - 50% RH, and 27˚C - 75% RH. Each combination was applied to groups of 150 MB⁺ larvae and 150 MB⁻ larvae, each divided into three replicates of 50 larvae. Each replicate was placed in a Sanyo Versatile climatic chamber configured to maintain the designated conditions until pupation. Upon completion of pupation, pupae were transferred to rearing cages for emergence. The same four parameters as previously mentioned were measured.

To determine the prevalence of Microsporidia MB in adult mosquitoes from the previous phases, we confirmed infection status using conventional PCR. For each factor studied, DNA was extracted following 2% CTAB method. The amplification products were analyzed via 2% agarose gel electrophoresis, to determine the prevalence of Microsporidia MB in adult mosquitoes.

Data were analyzed using R software (version 4.1.2). We used the Kruskal-Wallis test to compare the larval development times of MB⁺ mosquitoes versus their MB⁻ congeners for each factor studied. Graphics were generated using ggplot 2. The Chi-2 test was used to compare pupation rates, emergence rates, sex ratios and prevalence of Microsporidia MB in adults at emergence, for each factor studied. All tests were performed at a significance level of 5% (p < 0.05).

The various parameters measured were calculated as follows:

DLD = date of stage 2 larvae − date of pupation

$\text{PR}\left(\text{\%}\right)=\frac{\text{Total number othe pupae}}{\text{Initial number of larvae}}\times 100$;

$\text{TE}\left(\text{\%}\right)=\frac{\text{Total number of adult mosquitoes emerged}}{\text{Initial number of pupae}}\times 100$;

$\text{Sex-ratio}\left(\text{\%}\right)=\frac{\text{Total number of male mosquitoes}}{\text{Total number of adult mosquitoes}}\times 100$;

The prevalence of Microsporidia MB in mosquitoes was determined using the following formula:

$\text{Prevalence}\left(\%\right)=\frac{\text{Number of postive mosquitoe specimens to}MB}{\text{Total number of mosquitoes testde by PCR}}\times 100$.

The detailed protocol was submitted to the Comité d’Ethique Institutionnel (CEI) of the Institut de Recherche en Sciences de la Santé (IRSS/DRO) in Bobo-Dioulasso, Burkina Faso, for authorization to conduct the study.

The impact of diet on larval development time was assessed by counting the number of pupae daily during pupation. For the low food quantity, the average development time was 9.11 days for MB-positive (MB⁺) larvae and 8.79 days for MB-negative (MB⁻) larvae. For the medium quantity, development times were 4.56 days for MB⁺ larvae and 4.74 days for MB⁻ larvae. For the high quantity, MB⁺ larvae averaged 2.98 days, while MB⁻ larvae averaged 3.65 days ( Figure 2 ). Statistical analysis indicated significant differences in development times across food quantities (Kruskal-Wallis test, χ² = 21.897; df = 2; p < 0.0001). However, no significant difference was found between MB⁺ and MB⁻ larvae (Kruskal-Wallis test, χ² = 17.807; df = 1; p = 0.9850).

A total of 841 pupae were collected, with 420 from MB⁻ larvae and 421 from MB⁺ larvae across the three food quantities tested. At the low quantity, 128 MB⁺ pupae (85.33%) and 125 MB⁻ pupae (83.33%) were recorded. For the medium quantity, 148 MB⁺ pupae (98.67%) and 147 MB⁻ pupae (98%) were collected. At the high quantity, 145 MB⁺ pupae (96.67%) and 148 MB⁻ pupae (98.67%) were obtained ( Figure 3 ). Statistical tests revealed no significant difference in the numbers of MB⁺ and MB⁻ pupae across food quantities (Kruskal-Wallis test, χ² = 21.897; df = 1; p > 0.05).

In total, 714 adult mosquitoes were collected, with 336 from MB⁺ larvae and 378 from MB⁻ larvae. At the low quantity, 71 MB⁺ adults (55.47%) and 100 MB⁻ adults (80%) were recorded. For the medium quantity, 133 MB⁺ adults (89.86%) and 135 MB⁻ adults (91.84%) emerged. At the high quantity, 132 MB⁺ adults (91.03%) and 143 MB⁻ adults (96.62%) were collected ( Figure 4 ). Statistical analyses showed a significantly lower number of MB⁺ adults compared to MB⁻ adults at the low quantity (chi-squared test, χ² = 16.271; df = 1; p < 0.0001). For the medium and

high quantities, emergence rates were similar between the two groups (chi-squared test, p > 0.05).

Overall, males outnumbered females ( Figure 5 ). At the low food quantity, MB⁺ mosquitoes had a higher percentage of females (54.84%), while MB⁻ mosquitoes showed a male predominance (58.67%). At the medium quantity, MB⁺ mosquitoes had a male ratio of 58.21%, while MB⁻ mosquitoes displayed an equal sex ratio (50% for each sex). At the high quantity, males were predominant in both groups. Nevertheless, statistical analysis indicated no significant difference in sex ratios between MB⁺ and MB⁻ mosquitoes (chi-squared test, χ² = 4.3406; df = 5; p = 0.5015).

The prevalence of Microsporidia MB varied with food quantities. At the low quantity, 23 of 71 mosquitoes tested positive for Microsporidia MB, resulting in a prevalence of 32.39%. At the medium quantity, 66 out of 133 mosquitoes were positive (49.62%). For the high quantity, 56 of 125 mosquitoes tested positive, yielding a prevalence of 44.8% ( Figure 6 ). Statistical analysis indicated that larvae fed the low-quantity diet had a significantly lower prevalence of Microsporidia MB compared to those fed the medium (chi-squared test, χ² = 4.9088; df = 1; p = 0.02672) and high (chi-squared test, χ² = 4.7958; df = 1; p = 0.02853) quantities. No significant difference was observed between medium and high quantities (chi-squared test, χ² = 4.1053; df = 1; p = 1).

At 21˚C and 80% RH, the average larval development time was 5.94 days for MB⁺ larvae and 6.46 days for MB⁻. At 27˚C and 75% RH, it was 4.74 days for MB⁺ larvae and 4.56 days for MB⁻. At 39˚C and 50% RH, the average development time was 3.64 days for MB⁺ larvae and 3.79 days for MB⁻ larvae ( Figure 7 ). Statistical testing revealed that different temperatures and humidity levels led to varying development times (Kruskal-Wallis test, χ² = 11.7978; df = 2; p < 0.0001). No significant difference in development time was found between MB⁺ and MB⁻ larvae (chi-squared test, χ² = 21.1864; df = 1; p = 0.8530).

A total of 827 pupae were collected, with 422 from MB⁻ larvae and 405 from MB⁺ larvae across the three temperature-humidity combinations. At 21˚C and 80% RH, 143 MB⁺ and 142 MB⁻ pupae were recorded, resulting in pupation rates of 95.33% and 94.67%, respectively. At 27˚C and 75% RH, 148 MB⁺ pupae (98.67%) and 147 MB⁻ pupae (98%) were collected. At 39˚C and 50% RH, 114 MB⁺ pupae (76%) and 133 MB⁻ pupae (88.67%) were recorded ( Figure 8 ). Statistical analyses revealed a significantly lower number of MB⁺ pupae compared to MB⁻ pupae at 39˚C and 50% RH (chi-squared test, χ² = 7.4249; df = 1; p = 0.006433). No significant differences were detected between the two groups at other temperature-humidity combinations (chi-squared test, χ² = 23.8673; df = 1; p > 0.05).

A total of 579 adult mosquitoes were collected, comprising 313 from MB⁻ larvae and 266 from MB⁺ larvae. At 21˚C and 80% RH, 112 MB⁺ and 141 MB⁻ adults emerged, with emergence rates of 72.32% and 99.29%, respectively. At 27˚C and 75% RH, 133 MB⁺ adults (89.86%) and 135 MB⁻ adults (91.84%) were recorded. At 39˚C and 50% RH, only 19 MB⁺ adults (16.67%) and 40 MB⁻ adults (30.07%) emerged ( Figure 9 ). Statistical analysis showed that at 21˚C and 80% RH, the number of MB⁺ adults was significantly lower than that of MB⁻ adults (chi-squared test, χ² = 27.396; df = 1; p < 0.0001). This was also the case at 39˚C and 50% RH (chi-squared test, χ² = 5.3551; df = 1; p = 0.0207). At 27˚C and 75% RH, however, adult numbers were similar between the two groups (chi-squared test, χ² = 11.1485; df = 1; p = 0.7).

Overall, males predominated over females ( Figure 10 ). At 21˚C and 80% RH, MB⁺ mosquitoes exhibited a male ratio of 63.21%, while MB⁻ mosquitoes had a nearly equal sex ratio. At 27˚C and 75% RH, the male ratio for MB⁺ mosquitoes was 58.21%, with MB⁻ mosquitoes showing equal percentages for both sexes. At 39˚C and 50% RH, the trend reversed, with a higher female percentage (61.54%) observed in MB⁺ mosquitoes, while MB⁻ mosquitoes had a lower female percentage (38.89%). Statistical analysis indicated no significant difference in the sex ratio between MB⁺ Fand MB⁻ mosquitoes (chi-squared test, χ² = 6.6916; df = 5; p = 0.2446).

Prevalence of Microsporidia MB varied with temperature and humidity. At 39˚C and 50% RH, 3 of 19 tested mosquitoes were positive, yielding a prevalence of 15.79%. At 27˚C and 75% RH, 66 of 133 mosquitoes tested positive (49.62%). For larvae reared at 21˚C and 80% RH, 65 of 112 mosquitoes tested positive, resulting in a prevalence of 58.04% ( Figure 11 ). The prevalence at 39˚C and 50% RH was significantly different from that at 27˚C and 75% RH (chi-squared test, χ² = 6.3736; df = 1; p = 0.01158) and from that at 21˚C and 80% RH (chi-squared test, χ² = 9.983; df = 1; p = 0.00158). No significant difference was found between

mosquitoes reared at 27˚C and 75% RH compared to those at 21˚C and 80% RH (chi-squared test, χ² = 1.4075; df = 1; p = 0.2355).