Influence of GST- and P450-based metabolic resistance to pyrethroids on blood feeding in the major African malaria vector Anopheles funestus

Insecticide resistance genes are often associated with pleiotropic effects on various mosquito life-history traits. However, very little information is available on the impact of insecticide resistance on blood feeding process in mosquitoes. Here, using two recently detected DNA-based metabolic markers in the major malaria vector, An. funestus, we investigated how metabolic resistance genes could affect the blood meal intake. After allowing both the field F1 and lab F8 Anopheles funestus strains to feed on the human arm for 30 minutes, we assessed the association between key parameters of blood meal process including, probing time, feeding duration, blood feeding success, blood meal size, and markers of glutathione S-transferase (L119F-GSTe2) and cytochrome P450 (CYP6P9a_R)—mediated metabolic resistance. None of the parameters of blood meal process was associated with L119F-GSTe2 genotypes. By contrast, for CYP6P9a_R, homozygous resistant mosquitoes were significantly more able to blood-feed than homozygous susceptible (OR = 3.3; CI 95%: 1.4–7.7; P = 0.01) mosquitoes. Moreover, the volume of blood meal ingested by CYP6P9a-SS mosquitoes was lower than that of CYP6P9a-RS (P<0.004) and of CYP6P9a-RR (P<0.006). This suggests that CYP6P9a gene is inked with the feeding success and blood meal size of An. funestus. However, no correlation was found in the expression of CYP6P9a and that of genes encoding for salivary proteins involved in blood meal process. This study suggests that P450-based metabolic resistance may influence the blood feeding process of Anopheles funestus mosquito and consequently its ability to transmit malaria parasites.

The authors have declared that no competing interests exist. NO    Insecticide resistance genes are often associated with pleiotropic effects on various mosquito 28 life-history traits. However, very little information is available on the impact of insecticide 29 resistance on blood feeding process in mosquitoes. Here, using two recently detected DNA-30 based metabolic markers in the major malaria vector, An. funestus, we investigated how 31 metabolic resistance genes could affect blood meal intake.

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After allowing both the field F1 and lab F8 Anopheles funestus strains to feed on the human 33 arm for 30 minutes, we assessed the association between key parameters of blood meal process 34 including, probing time, feeding duration, blood feeding success, blood meal size, and markers 35 of glutathione S-transferase (L119F-GSTe2) and cytochrome P450 (CYP6P9a_R) -mediated 36 metabolic resistance. None of the parameters of blood meal process was associated with L119F-37 GSTe2 genotypes. In contrast, for CYP6P9a_R, homozygote resistant mosquitoes were 38 significantly more able to blood-feed than homozygote susceptible (OR = 3.3; CI 95%: 1.4-7.7; 39 P =0.01) mosquitoes. Moreover, the volume of blood meal ingested by CYP6P9a-SS 40 mosquitoes was lower than that of CYP6P9a-RS (P<0.004) and of CYP6P9a-RR (P<0.006).

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This suggests that CYP6P9a gene affects the feeding success and blood meal size of An. 42 funestus. However, no correlation was found in the expression of CYP6P9a and that of genes 43 encoding for salivary proteins involved in blood meal process.

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This study suggests that P450-based metabolic resistance may increase the blood feeding ability 45 of malaria vectors and potentially impacting their vectorial capacity.

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Malaria remains a major public health scourge in sub-Sahara Africa despite significant progress 52 made since the 2000s in reducing its burden [1]. This disease is caused by a Plasmodium 53 parasite transmitted by Anopheles mosquito species while taking a blood meal on humans.      In contrast, little is known on the impact of metabolic resistance as DNA-based markers were 88 not previously available for this mechanism; thereby limiting the ability to investigate its 89 physiological impact on the blood feeding process in mosquitoes. However, taking advantage (the CYP6P9a-R) in An. funestus. This marker showed that mosquitoes carrying this P450-97 resistant allele survived and succeeded in blood feeding more often than did susceptible 98 mosquitoes when exposed to insecticide-treated nets [24]. The design of assays for both GST-99 and P450-based resistance now offers a great opportunity to explore how the blood feeding 100 process is influenced by metabolic resistance mechanism in malaria vectors and further assess 101 how resistance may impact the vectorial capacity of mosquitoes to transmit malaria in the 102 natural environment. 103 Here, we investigated the effect of metabolic resistance to pyrethroids on the blood feeding 104 process in An. funestus, using the two DNA-based metabolic resistance markers: L119F-GSTe2 105 and CYP6P9a-R [22,24]. Specifically, we assessed the association between the genotypes of 106 these metabolic resistance markers and key parameters of blood feeding including mosquito 107 probing time, feeding duration and the blood meal size. in Yaoundé where they were kept for 4-5 days until they became fully gravid and were then 118 induced to lay eggs using the forced eggs-laying method [26]. The eggs were placed in paper 119 cups containing water to hatch, after which the larvae were transferred in to trays and reared to 120 adults. To assess the effect of CYP6P9a marker, F8 progenies were generated from reciprocal  The duration of probing and blood feeding was assessed using a batch of 120 F1 female field-139 collected mosquitoes. For this purpose, mosquitoes were individually transferred in polystyrene 140 plastic cups covered with net. They were allowed to rest for 15 min before observations began.

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During the blood intake, each mosquito was filmed with a Digital HD Video Camera (Canon  To determine the blood meal size for both strains, batches of 25 mosquitoes grouped according 150 to their weight were allowed to bite on a human arm. In this case, neither the probing time nor 151 the feeding duration was recorded. After the trial, the whole abdomen of successfully fed   The L119F-GSTe2 mutation was genotyped using gDNA extracted from carcasses of field-185 collected strains following an allele specific PCR diagnostic assay previously described [23]. 186 The primers sequences are given in table S1. PCR was performed in Gene Touch thermalcycler  Midori Green Advance DNA Stain (Nippon genetics Europe GmbH) and visualised using a gel 210 imaging system to confirm the product size (450bp). Then, the PCR product was incubated at 211 65°C for 2 hours. This was done in 0.2ml PCR strip tubes using 5µl of PCR product, 1µl of 212 cutSmart buffer, 0.2µl of 2 units of Taq1 enzyme (New England Biolabs) and 3.8µl of dH20.

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Size separation was done on a 2.0% agarose gel stained with Midori Green Advance DNA Stain 214 at 100V for 30 minutes. The gel was visualised using the gel imaging system.  Table 1). This absence of correlation between the L119F-GSTe2 genotypes and the weight of 271 mosquitoes was confirmed at the allele level (OR=1; 95%: CI: 0.5-2.0; p =0.5) showing that 272 the L119F mutation may not impact the weight of this An. funestus field population (Table 1). 273 In contrast, a significant association was observed between CYP6P9a genotypes and the weight 274 of mosquito (χ 2 = 29.54, p<0. 0001). Indeed, proportions of RR and RS genotypes were higher 275 than that of SS in the lowest weight class, whereas, for larger weight, mosquitoes with SS 276 genotype were more abundant (67.2%). This association is further supported by odds ratio

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Out of the 120 mosquitoes that were individually filmed to assess the influence of insecticide  Regarding the blood feeding duration, it was observed that the median and mean time for a 331 mosquito to have a full blood meal was 249.5 seconds and 303 ± 181 seconds respectively, with a minimum = 68 seconds and a maximum =772 seconds. The feeding duration was longer 333 (median=269s) in L/L119 mosquitoes compared to L119F-RS (229.5s) and 119F/F-RR (214s) 334 but the difference was not statistically significant (p=0.19, Kruskal-Wallis test). 374 Impact of metabolic resistance on blood feeding success 375 The present study revealed that CYP6P9a but not the L119F-GSTe2 mutation could impact the  of influence of the CYP6P9a gene on the expression level of salivary gland genes involved in 468 the blood feeding process observed in the present study appears to indicate that the association 469 found between this gene and the size of blood meal taken by An. funestus mosquito might not 470 be related to the expression of these salivary genes encoding proteins which mediate the blood 471 meal process.

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This study revealed that GSTe2-mediated resistance does not affect the blood meal intake of 473 An. funestus mosquitoes, whereas CYP6P9a-based resistance to pyrethroids is associated with 474 a feeding success and a higher blood meal size. However, this influence on Anopheles funestus 475 blood meal intake is not associated with differential expression of major salivary gland proteins 476 involved in blood-feeding. Given the rapid growth of insecticide resistance, it would be 477 interesting to study how this association could impact the fecundity and the vectorial capacity