2. MATERIALS AND METHODS
2.1 Study area
The study was undertaken in fourteen (14) localities in two bioclimatic
zones in Burkina Faso (Fig. 1). The bioclimatic zones include: i)
Sudan-sahelian (dry savannah) which extends throughout much of the
central part of Burkina Faso and ii) Sudan-savannah (humid savannah)
located in the south and west part of the country. Study area is
characterized by a rainy period from May to September with average
annual rainfall about 1200mm (Namountougou et al. 2019) .
2.2 Cotton pests control
strategies
Overall, in the western and central Burkina Faso, cotton remains the
main industrial crop and around 90% of total pesticides are applied for
its protection. (Ouédraogo et al. 2011). Three strategies to control
cotton pests are recommended in Burkina Faso and deployed in the
different study sites: i) Conventional cotton cultivation (Banfora,
Diébougou, Houndé, Gaoua, Kombissiri, Koubri, Orodara, Soumousso and
Vallée du Kou) with high rates of insecticide treatment (N=9 sites).
Insecticide applications were done throughout the cotton season (May to
September) according to three treatment windows and depending on the
abundance and pest species concerned
(Table S1). These treatments are based on the temporal rotation of
different insecticides including PY, OP/ Carbamates (CX) and
neonicotinoids; ii) Organic cotton cultivation (Dano and Tiéfora)
without chemical use (N=2 sites) still known as “coton bio”. It has
been introduced in Burkina Faso since 2004 but its large-scale
production has been supported since 2006 by some development partners
who promote this technonology; organic cotton areas extend on limited
areas (Table S2); iii) transgenic cotton (Bt ) cultivation (Fada
N’Gourma, Koupéla and Manga) with low insecticide use (N= 3 sites). In
2008, as regards emergency and spread of insecticide resistance in
cotton pest populations, transgenic cotton was adopted and implemented
into Burkina Faso to control pest caterpillars such as Helicoverpa
armigera (Raj et al. 2002, Chaturvedi 2007), a pest species that causes
severe damages. In this technology, no PY and OP are used.
2.3 Mosquito sampling and
rearing
Mosquito larvae were sampled during the rainy season of the years 2008,
2009, 2013 and 2014 from their natural breeding sites in the 14
localities throughout the three cotton cultivation areas of Burkina
Faso. In each locality and each date of sampling, all instars of larvae
were collected in at least 10 different breeding sites. Specimens
collections were pooled per locality. Larvae were brought back to the
insectarium of IRSS/Centre Muraz in Bobo-Dioulasso, reared under
standard controlled conditions (temperature 25±2°C, relative Humidity:
80± 10% RH and12:12h Light-Dark) and fed with Tetramin
baby® fish food every day until adultood. Upon
emergence, we identified mosquitoes morphologically according to
standard identification keys described by Gillies MT et Coetzee M
(1987). Only, An. gambiae complex females were selected and
maintained alive on 10% sugar solution until they were used for
bioassays.
2.4 Insecticide susceptibility test
Bioassays were performed on 3–4 days old unfed female of An.
gambiae complex mosquitoes using WHO test kits (WHO 1998, 2013).
Insecticide-treated filter papers at the diagnostic dose as recommended
by WHO (WHO 1998, 2016) were tested including: two pyrethroids (PY),
Permethrin 0.75% and Deltamethrin 0.05%, one organochlorine, DDT 4%,
one carbamate, Bendiocarb 0.1%, and one organophosphate,
chlorpyriphos-methyl 0.4%. DDT and Permethrin were tested only in 2008
and 2009 while the three others insecticide were tested in 2008, 2009,
2013 and 2014. It is important to note that in Burkina Faso, permethrin
was the reference insecticide until 2014 for long-lasting insecticide
nets (LLINs) and after that date it was replaced by deltamethrin which
is now the insecticide of preference for LLINs. DDT was chosen to verify
the presence of cross-resistance to pyrethroids. The others insecticides
belonging to the OP and CX families are used as reference insecticides
for IRS. Four replicates of batches of 20–25 test mosquitoes were
exposed for 1 hour to each insecticide-treated paper. The laboratory
reference strain susceptible to all insecticides, An. gambiaeKisumu, has been tested on all insecticides impregnated papers as a
control. Control mosquitoes (N=20–25 females per test population and
the Kisumu strain) were tested for the same time to untreated filter
papers. After exposure time, mosquitoes were transferred into
insecticide-free observation tubes of the tests kit and maintained on
10% sucrose solution at 25 to 28°C. Final mortality in test and control
mosquitoes was examined 24 h after exposure. For further molecular
analysis, the samples of mosquitoes tested to different insecticides
were kept individually at -20 °C according to their phenotypic state
(dead or alive).
2.5 Molecular analysis
Thirty females referring to An. gambiae complex per site were
randomly chosen within the group of bioassays specimens for molecular
tests. They were identified to species member of the An. gambiaecomplex according to PCR described by Santolamazza et al. ( 2008), and
this sampling was considered as representative of the mosquito
populations being tested in each locality (Chouaïbou et al. 2008). Their
genotype at the kdr locus was determined using the diagnostic
tests described by Martinez-Torres et al. (Martinez-Torres et al. 1998).
2.6 Statistical analyses
The threshold of susceptibility was fixed at 98% mortality rate for the
six active molecules according to the WHO’s protocol. When the mortality
rates were between 90 and 98%, the population was considered as
“suspected resistant”. Mortality rates below 90% indicate the
presence of insecticide resistance (WHO 2016). Genotypic frequencies at
the kdr L1014F loci in An. gambiae populations were
compared to Hardy-Weinberg expectations using the Fischer exact test
procedure implemented in GenePop (ver.4.1.4) software (Raymond and
Rousset 1995).
The impact of cotton pest management practices on kdr L1014Ffrequencies within An. gambiae complex populations was determined
using generalized mixed effect models (GLMM). Models were fitted using
the glmmTMB function run in the R software (The R Development Core Team
2008). Post-hoc Tukey method was used to do multiple comparison among
modalities of the fixed terms using the ‘emmeans’ function of the
‘emmeans’ package (Russell et al. 2019). Also, the impact of cotton pest
control practices on kdr L1014F frequencies in An. gambiaes.l. populations was assessed using a Kruskall-Wallis test and a Dunn’s
post-hoc test for multiple comparisons (Dinno 2017). A difference is
considered as significant when the p-value is less than 0.05.