Insecticide Resistance Exerts Signi cant Fitness Costs in Immature Stages of Anopheles Gambiae in Western Kenya

Joyce K Osoro Kenya Medical Research Institute Maxwell Gesuge Machani KEMRI: Kenya Medical Research Institute Eric Ochomo KEMRI: Kenya Medical Research Institute Christine Wanjala Masinde Muliro University of Science and Technology Elizabeth Omukunda Masinde Muliro University of Science and Technology Stephen Munga KEMRI: Kenya Medical Research Institute Andrew K. Githeko KEMRI: Kenya Medical Research Institute Guiyun Yan UC Irvine: University of California Irvine Yaw A. Afrane (  yaw_afrane@yahoo.com ) University of Ghana Medical School https://orcid.org/0000-0001-6576-523X

Conclusion: The study showed that pyrethroid resistance in An. gambiae had a tness cost on their preimaginal development time and survival. Insecticide resistance delayed the development and reduced the survivorship of An. gambiae larvae. The study ndings are important in understanding the tness cost of insecticide resistance vectors that could contribute to shaping resistant management strategies.

Background
The development and spread of insecticide resistance threaten the control of vectors of infectious diseases in sub-Saharan Africa (1). The continued use of insecticides for public health interventions and agricultural purposes seems to have generated high selective pressure on mosquito populations leading to the development of insecticide resistance in mosquito vectors (2)(3)(4). Resistance to insecticides in malaria vectors has mainly been linked to the overexpression of detoxifying enzymes or enzyme structural changes that increase metabolic activity and target-site modi cation (5,6). This ability to resist insecticides through different mechanisms may present a tness cost to resistant genotypes with negative effects in their development, reproductive aspects and vector competence which could affect the vectorial capacity of the malaria vectors (7).
Environmental selection pressure may select for certain phenotypes that will adapt to the new environment. It is hypothesized that phenotypic changes in an organism may have deleterious effects when the organism returns to its old environment (8). For instance, resistant mosquito genotypes are believed to have an adaptive advantage in the insecticide environment resulting in increased resistance levels and this tends to decrease in the absence of insecticides suggesting the existence of a tness cost (9). The development and maintenance of resistant mechanisms in mosquitoes are thought to divert energy and resources associated with the primary physiological process, such as fecundity and longevity of individuals leading to a biological cost (8,10). Overexpression of metabolic enzymes and genes in resistant mosquitoes are thought to reallocate primary energetic resources from other life-history traits e.g. egg production and larval development to maintain secondary metabolic pathways involved in defence resulting in a tness cost (11). Changes in the insecticide target site may result in a tness cost if the molecular alteration or the expressed genes are essential for the viability of the insect impairing the resistant individuals' development and reproductive tness (9) Fitness costs associated with resistance have been reported to affect primary biological characteristics in resistant Culex and Aedes mosquitoes (9,(12)(13)(14)(15)(16)(17). Studies by Alout et al (18) have also documented the tness cost of insecticide resistance alleles on the vector competence of resistant phenotypes (19). Currently, little is known about the effects of major insecticide resistance mechanisms on the life-history parameters of An. gambiae the major malaria vector in Africa. This presents a gap that needs to be lled for better designing of malaria control strategies as insecticide resistance is increasingly reported, threatening the already achieved progress in malaria reduction in Africa. This study assessed the tness cost effects of insecticide resistance on the development and survival of immature An. gambiae from western Kenya.

Mosquito population used in the study
The colonies of An. gambiae s.s used in this study consisted of a pyrethroid-resistant selected colony (hereafter R colony) originating from Bungoma in western Kenya and an unselected colony (hereafter S colony) from the same site (Machani et al, 2020, in press). The R colony was selected using 0.05% deltamethrin at every generation. The S colony was not selected at any generation but a subset of this colony was checked for resistance after every other generation. The sixth generation of the R colony and the thirteenth generation of the S colony were used for this study. The 6th generation of the R colony used had an average mortality rate of 20% whilst the 13th generation of the S colony had an average mortality rate of 97.3% when exposed to 0.05% deltamethrin (supplementary Fig.1). The S colony had almost lost resistance after 9 generations without insecticide selection pressure (Mortality: 92%). The two knockdown resistance mutations (kdr) L1014S and L1014F were at high frequency (0.77 and 0.23 respectively) in the R colony compared to the S colony (L1014S-0.98; L1014F-0) (Machani et al 2020, in press).
Monooxygenase played a major role in resistance in the selected colony used, with an 11% increase in the enzyme activity compared to the unselected colony that exhibited a 42% reduction in the enzyme activity in relation to the parent population. The An. gambiae Kisumu reference laboratory strain which has been colonized since 1954 and is free of any detectable insecticide resistance mechanism was used as a control susceptible strain in all bioassays.
The mosquito colonies were reared in the insectary at KEMRI/CGHR under standard conditions (25 ± 2 °C; 80% ± 4% relative humidity with a 12 h: 12 h light/dark cycle). Larvae were fed on tetramin baby sh food and brewer's yeast daily and adults maintained in a 10% sugar solution.

Life table experiments
Three parameters were evaluated to examine the tness cost: mean larval development time (L1-Pupal), pupal emergence) and daily survival. The parameters were measured under semi-eld conditions and focused on the difference between mosquitoes raised in the presence (selected colony) and the absence of insecticide selection pressure (unselected colony). The Kisumu susceptible laboratory strain was used as a control.

Experimental design
A total of 27 semi-natural habitats (9 replicates per colony) were created using plastic washbasins (35 cm in diameter and 15 cm deep) at KEMRI/CGHR compound in Kisumu according to the method described by Afrane et al (20). Two kilograms of soil from breeding sites and ve litres of rainwater were added to each washbasin. Two holes (3 cm in diameter) were created near the top edge of each washbasin to maintain a constant water level when it rained. The holes were covered with a screen (mesh size _ 200 micrometres) to prevent larvae from being washed away (21,22). Thirty (30) 2-hour old larvae from the three colonies were placed separately in different basins. Each washbasin was covered with a nylon netting to prevent predators and wild mosquitoes from ovipositing eggs in the washbasin. The surviving larvae in each washbasin were checked and counted daily and their numbers were recorded. The stage of development of individual larvae was also recorded to measure the development time per each larval instar. Pupae were picked, recorded and transferred to pupal cups, which were then placed in cages for adult emergence. Pupae were monitored daily and the number and sex of emerging adults recorded. All larvae from the three colonies were reared through adults in semi-eld conditions. The mean length of time from the rst instar to adult emergence for each sex, as well as the ratio of male to female emergences was recorded for each colony. The experiment was repeated four times.

Data analysis
Mean larval development time was de ned as the average time of the rst instar larvae to develop into adults. Mean pupation time was calculated as the average time taken for the rst instar larva to pupate. The male and female development time was recorded differently because they take different times to emerge. The pupation rate was calculated as the percentage of the rst instar larvae that emerged to pupae. The emergence rate was calculated as the percentage of the pupae that emerged to adults. Analysis of variance (ANOVA) was conducted to determine the effects of insecticide resistance on the pupation time, larval development time, pupation rate and emergence rate of the R colony, S colony and the Kisumu reference An. gambiaes.s. Tukey HSD post hoc tests were used to determine the statistical signi cance of the difference in larval development time, pupation rate and emergence rate among the selected, unselected and the Kisumu reference colonies. Kaplan-mier survival test was used in the testing for differences in larval survivorship among the selected, unselected and the Kisumu reference mosquitoes. The level of signi cance was set at 0.05 for all tests.

Effect of insecticide resistance on larval development
The mean development time from rst instar (L1) to second instar (L2) in the R colony was 4.2 ± 0.2, while the S colony was 3.4 ± 0.1 and 3.4 ± 0.1 for the Kisumu strain (F 2,63 =44.43, P < 0.0001; Table 1). The average length of larval development time (L1-L2) for the resistant colony was 0.8 days longer compared to the unselected colony. The time for R colony to develop from rst instar (L1) to third instar (L3) was 6.9 ± 0.2 days while the S colony was 4.9 ± 0.2 and 4.8 ± 0.2 days for the Kisumu colony. The development time (L1-L3) for R colony was 2 days longer compared to the other colonies (F 2,63 =44.61, P < 0.0001). The mean pre-imaginal development time from rst instar (L 1 ) to fourth instar (L 4 ) of the R colony was 8.8 ± 0.2, while the S colony was 6.6 ± 0.2 and 6.3 ± 0.2 for Kisumu laboratory susceptible mosquitoes. The R colony took a signi cantly longer period (2 days) to develop from L 1 -L 4 with respect to the S colony and Kisumu strain (F 2,63 =47.06, P < 0.0001). Pupation and emergence times between the selected and unselected colonies The R colony reached pupal stage 10-11 days after hatching as L1, whilst the S colony took 6-7 days. Development time from L1 to pupal stage was signi cantly longer in R colony than in the S colony (mean: 10.28 ± 0.3 vs 7.5 ± 0.2; F 2,63 =39.45, P < 0.0001, Table 2). The Kisumu strain took 7.9 ± 0.2 days to pupate.  Table 3). The sex ratio of females to males was signi cantly different for R colony 1: 1.21 (t = 2.5248, df = 42, P < 0.0154) and S colony 1: 1.07 (t = 2.2525, df = 42, P < 0.029). Although the proportions of males to females was high in the Kisumu strain (51.7 vs 48.3%), this was not statistically signi cant (t = 0.854, df = 42, P > 0.05).

Survivorship Among The Selected And Unselected
The R colony showed a longer survival time of 15 days, with a median survival length of 8 days compared to the S colony that survived for 12 days with a median survival length of 6 days (Fig. 1)

Discussion
Under an evolutionary perspective, it is hypothesised that genetic changes arising as a result of insecticides' selective pressure can present a tness cost to resistant insects bearing negative effects on their biological traits (23). The study assessed larval development time and survivorship of An. gambiae colonies, exhibiting different insecticide resistance status. The results of this study demonstrate the existence of tness cost n An. gambiae s.s immature stages associated with pyrethroid resistance. Overall larval development time and survival was compromised in the pyrethroid-selected colony compared to the unselected colony originating from the same background. The development time of the unselected colony was remarkably similar to that of the susceptible Kisumu reference strain.
The study observed prolonged development time from one larval instar to the other in the selected resistant colony when compared with the unselected colony whose development time was similar to the Kisumu colony. Majority of individuals from the unselected colony and the Kisumu strain reached the pupal stage in 6-7 days after the hatching of the rst instar whereas the selected colony took additional 3-4 days before pupation. These ndings present adaptive disadvantage on the resistant individuals as the amount of time spent in the natural breeding habitats in the eld may impact their survival rates due to exposure to natural predations. They are also likely to suffer temporary or permanent loss of habitats before emerging to adults, which may in turn have a direct consequence on the vectorial capacity (7). Similarly, studies on pyrethroid-resistant An. funestus, Culex quinquefasciatus, Aedes aegypti and Aedes albopictus have observed longer phase of larval development unlike their susceptible counterparts (12,(14)(15)(16)24).
Larval survivorship of the pyrethroid-resistant colony was low characterized by low pupation rates, high pupae mortality and decreased adult emergence compared to the unselected and Kisumu colonies. These could be possibly due to the accumulation of harmful effects of genes related to insecticide detoxifying enzymes or molecular alterations on the target (kdr mutations). The success in survivorship of the unselected or susceptible colony could be attributed to the loss of resistance in them that could enable them to focus most of their energy to growth enhancement metabolic processes. The low larval survivorship in the selected colony may present low vector population densities disabling effective malaria transmission by resistant mosquitoes. Similar studies have reported the negative effects associated with insecticide resistance on the biological characteristics of pyrethroid-resistant Aedes albopictus and Culex pipiens compared to their susceptible counterparts (17,25) It is important to highlight that monooxygenase enzyme was majorly implicated in the pyrethroid resistance of the selected colony even though kdr mutations were observed at high frequencies (Machani et al 2020, in press). It is likely that the overproduction of monooxygenase would have committed resources important for primary biological functions such as development to maintaining secondary functions i.e. insecticide detoxi cation (23). For instance, some studies have linked the staggered larval development time of resistant individuals with spending more time in the accumulation of nutrients to achieve the development threshold that triggers growth to the next stage as most of the resources are used to maintain resistance (26). The ndings of this study are similar to reports on An. funestus from West Africa harboring 119F-GSTe2 resistant alleles which exhibited delayed larval development compared to the population without the resistant alleles (27). The kdr mutation has been associated with a delay in the larval development of A. aegypti, (9,28). The observed negative effects associated with insecticide resistance may affect the spread of insecticide resistance genes in a population, as the resistant individuals are likely to take a longer time to develop and emerge as adults, unlike the susceptible ones. Based on this, resistance management tactics may rely on this reduced tness disadvantage to design integrated vector control management strategies with an aim of limiting the spread of insecticide resistance and maintaining the effectiveness of the existing vector control tools.