Background
In the last three years, many countries have reported significant increases in malaria cases, according to the World Health Organization (WHO) latest malaria report [
1]. In 2018, the number of malaria cases worldwide was 228 million cases [
2], which is a reduction from 237 millions in 2010, but since then progress towards global elimination of malaria has been stalling [
3,
4], with 93% of cases occurring in the WHO African region [
2]. The reasons for the slowdown differed across specific regions and countries, but contributing factors included insufficient funding, a lack of interventions to prevent spread of the disease, risks posed by conflict in malaria endemic regions, irregular climate patterns [
4], and the rapid emergence of both parasite and mosquito vector resistance to drugs and insecticides [
1]. Emergence and spread of resistance to pyrethroids, organophosphates and carbamates is a particular threat, as most malaria control programmes rely heavily on these broad-spectrum insecticides to reduce vector populations [
5,
6].
As a consequence of these growing problems, the WHO called for research and development of alternative approaches in controlling vector-borne diseases, thus decreasing the usage of insecticides [
7]. Integrated Vector Management (IVM) efforts are now oriented towards controlling
Anopheles either at the larval stage and/or at the adult stage by means of microbial control, namely fungi and bacteria [
8,
9]. Many of these approaches are now focusing on the use of genetically engineered microorganisms to either block the development of the malaria parasite within the
Anopheles vector [
10‐
14], or target the vector itself [
10,
15]. Despite intensive efforts to develop entomopathogenic bacteria as biocontrol agents against malaria vectors, the strains under investigation have not met expectations due to some functional and practical limitations [
8,
9]. For example, bacteria such as
Bacillus thuringiensis israelensis (Bti) and
Bacillus sphaericus (Bs) show no residual persistence post-application [
8]. Among promising entomopathogenic bacteria
Wolbachia, only a few strains are known to be associated with
Anopheles gambiae [
16].
Interestingly, Ramirez and collaborators in 2014, showed that
Chromobacterium sp. Panama (
C. sp_P) isolated from the midgut of field-collected
Aedes aegypti, has unique properties: it can kill larvae and adults of multiple mosquito species, and it exerts in vitro anti-
Plasmodium and anti-dengue virus activity suggesting that it could be a highly potent candidate for developing tools against current and future mosquito-borne diseases [
10].
Chromobacterium violaceum is a Gram-negative facultative anaerobic and non sporing-coccobacillus bacterium. This bacterium is rarely human pathogenic
. It is part of the normal flora of water and soil of tropical and sub-tropical regions of the world. It produces a natural antibiotic called violacein [
17]. Some strains of
Chromobacterium have already been developed for agricultural pest control [
18]. Short et al
. have shown that
Chromobacterium sp. Panama
C.sp_P exposure have important effects on mosquito fitness and mosquito physiology including some transgenerational impacts [
19]. In the same study, they have also shown that mosquito exposure to cell-free
C.sp_P-conditioned media could elicit detoxification, xenobiotic response, and stress response genes within female mosquito midgut [
19]. This phenomenon is also shown when mosquitoes are exposed to common chemical insecticides used for vector control and this highlights the potential of using
Chromobacterium for vector borne diseases control, including malaria.
In this study, the impact of infection of malaria vector (An. coluzzii) with an indigenous burkinabè strain of C. violaceum isolated from both wild caught adults and larvae of An. gambiae was examined. Using a logistically simple method of infection, cotton balls soaked with sugar meal containing bacteria, assessments of the pathogenicity of this local strain of C. violaceum against adult mosquitoes and its impact of mosquito blood feeding and fecundity were carried out.
Discussion
Chromobacterium violaceum has shown an oral toxicity in a population of malaria vector
An. coluzzii that is highly resistant to pyrethroids. With a medium concentration, this local strain of
Chromobacterium surpassed the mortality of 80% thresholds of WHO over a week.
Chromobacterium violaceum exerted an important entomopathogenic activity even in the presence of other microorganisms from mosquito microbiota, because non-aseptic mosquitoes were used during bioassays. Indeed, The strain of Chromobacterium used in this study could be a natural and specific
Anopheles-pathogens because it was originally isolated from wild larvae and adults of
An. gambiae s.l. in western Burkina Faso. In contrast, previous mosquito-
Chromobacterium strains were isolated from
Aedes aegypti midgut [
10]. The current strain of
Chromobacterium virulence results corroborate those found by Ramirez et al
. that also showed a low survival rate of
An. gambiae and
Ae. aegypti after a blood meal containing
Chromobacterium sp at 10
8 bacterial cells/ml. However the entomopathogenic effect of
Chromobacterium species upon
An. gambiae s.l. is still to be elucidated. A number of potential virulence factors may contribute to mosquitocidal effect, including production of the pigment violacein, siderophores, hydrogen cyanide, and secreted chitinases [
26]. In addition, some strains
Chromobacterium are capable of forming biofilms in vitro, though whether biofilm formation occurs within the mosquito midgut remains untested [
10]. Bacterial biofilms are structured clusters of bacterial cells coated with a polymeric matrix and attached to a surface. The biofilm protects bacteria and allows them to survive in harsh environmental conditions and to resist the immune response of the host. The ability to form a biofilm is now recognized as a characteristic of many entomopathogenic microorganisms [
27].
The results from the present study indicated that the rapid death of mosquitoes from
C. violaceum, however is not the only part of the story. Once infected with
C. violaceum, mosquitoes are less inclined to blood feed which mainly appear a common effect of fungal infection in mosquitoes and non for bacterial infections [
23,
28]. The reduction of mosquito willingness to blood feed appears stronger as the bacterial infection progresses and can contribute to significant reductions in host feeding as early as day 6, essentially accelerating transmission blocking. What is interesting here, is that when the effects of blood feeding are added in risk of malaria transmission is essentially reduced to zero within 4 day of bacterial exposure and never recovers.
Chromobacterium violaceum infection in mosquitoes could share the same physiological impact in term of blood feeding reduction as with fungi. Fungal infection actually increases mosquito metabolic rate and reduces flight propensity and flight stamina. Poor flight performance has been strongly associated with reductions in the mobile energy reserves of the host [
29,
30]. Further specific studies are needed to access the impact of
C. violaceum infection on mosquito flight ability associated with blood feeding propensity to withdraw more conclusions.
One of the important results this study is the disruption of mosquito reproduction after infection with
C. violaceum. Indeed, bioassays showed that
C. violaceum inhibited egg development in the ovaries of
An. coluzzii mosquitoes and Short et al
. [
19] have shown that the exposure to
Chromobacterium (
C. sp_P) impacts female mosquito transgenerational fitness with no change in fecundity. From the current study, egg exposures to
C. violaceum appear to negatively impact ovarian follicles development. Female mosquitoes require blood meals for their egg maturations. Newly emerged female mosquitoes come out from the larval stage with theirs follicles at first stage (Stage I) need blood meals to complete the development of their ovaries to the follicle stage V. In the current experiments, 24 h of exposure to
C. violaceum reduced and subsequently stopped the maturation of the follicle because they have not reached the stage V for the most of them.
Chromobacterium violaceum could secrete a hormone or substance that directly affects the development of the ovaries and eggs. This substance could indirectly inhibit the action of the juvenile hormones (JHs) in mosquitoes. The juvenile hormones are mainly responsible for the development of ovaries and eggs in mosquitoes [
31]. The biochemical and physiological mechanisms governing the reduction of mosquito fecundity following
C. violaceum infection remain to be elucidated. On other hand, a few
C.violaceum infected females of
An. coluzzii that were able to lay a couple of eggs were not viable or had a low hatching rate.
C. violaceum infection seems to share these vertical transmission (mother to offspring) characteristics with some bacteria as
Serratia sp,
Pantoa agglomerans,
Asaia sp [
14,
31‐
35]. In addition, experiments are needed to be performed to check if
C. violaceum plays a sort of cytoplasmic incompatibility properties as some strains of
Wolbachia [
36] within their mosquito host
. In the simplest case, crossing-bioassays between
C. violaceum infected and uninfected
An. coluzzii in order to see if early embryonic arrest occurs when uninfected females mate with infected males.
Acknowledgements
We are very grateful to Gnada Kobo Daniel, Eli Kabré, Athur Djibougou, Ouattara Abel Kader, Saré Issiaka for their technical contributions to the field and lab works.
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