Native entomopathogenic Metarhizium spp. from Burkina Faso and their virulence against the malaria vector Anopheles coluzzii and non-target insects

Genetically enhanced Metarhizium pingshaense are being developed for malaria vector control in Burkina Faso. However, not much is known about the local prevalence and pathogenicity of this fungus, so we prospected mosquitoes and plant roots (a common habitat for Metarhizium spp.) for entomopathogenic fungi. Our investigations showed that Metarhizium spp. represented between 29–74% of fungi isolated from plant root rhizospheres in diverse collection sites. At low spore dosages (1 × 106 conidia/ml), two mosquito-derived M. pingshaense isolates (Met_S26 and Met_S10) showed greater virulence against Anopheles coluzzii (LT80 of ~7 days) than isolates tested in previous studies (LT80 of ~10 days). In addition, the local isolates did not cause disease in non-target insects (honeybees and cockroaches). Our work provides promising findings for isolating local Metarhizium strains for application in mosquito biological control and for future transgenic biocontrol strategies in Burkina Faso.


Background
Unlike mosquitocidal bacteria and viruses, ascomycete fungi can infect and kill insects without being ingested. As with chemical insecticides, tarsal contact alone is sufficient to kill mosquitoes [1]. Despite intensive efforts to develop entomopathogenic fungi as biocontrol agents against malaria vectors, the strains under investigation have not met expectations due to their poorer efficacy relative to cheaper chemical insecticides [2]. The United States Department of Agriculture (USDA) ARSEF collection (the world's largest collection of entomopathogenic fungi) has more than 12,000 isolates of insect pathogenic fungi. Of these, only 156 are from sub-Saharan Africa (South Africa and Benin are the source of 40 and 36 isolates, respectively), with none from Burkina Faso. The mosquitocidal activity of Metarhizium has been enhanced by engineering them to express insect-selective neurotoxins [3][4][5], and a transgenic strain of Metarhizium pingshaense is being evaluated in semi-field trials in Burkina Faso [5]. We speculate that future development of transgenic fungi worldwide will preferentially use local isolates as these may be better adapted to kill local mosquitoes and survive harsh local conditions (i.e. rainy season heat, sunlight and humidity) than exotic strains. However, the distribution and properties of indigenous Burkinabe Metarhizium spp. have not been characterized. The first objective of this study was to prospect for the presence and distribution of local Metarhizium strains. As well as prospecting mosquitoes, we also sampled rhizosphere soils (i.e. the soil in the vicinity of plant roots that is influenced by root secretions), as some Metarhizium spp. are abundant in the rhizosphere and may function as symbionts promoting plant growth. The plant-beneficial effects of Metarhizium species correlate with their association with roots and are mediated via plant hormones [6]. The second objective was to evaluate the pathogenicity of local Metarhizium isolates against wild-caught, insecticideresistant Anopheles coluzzii. Finally, we also assessed the pathogenicity of the local isolates against American cockroaches and honeybees as representative non-target or beneficial species.

Fungal collection, isolation and morphological identification
Collections were carried out on a monthly basis during the 2015 rainy season (from July to September) from plant roots and wild-caught mosquitoes. Our three collection sites were the Kou Valley (11°23'N, 4°24'W), a rice crop area; Bana (11°9'41"N, 4°10'30"W), a savanna and forested area; and Soumousso (11°04'N, 4°03'W), a savanna and corn crop area (Fig. 1). One hundred and fifty-five plants were sampled from these three different agro-ecological sites. We followed the protocol described in [7] to collect rhizosphere soil and isolate fungi. The fungal selective medium contained 42 g potato-dextrose agar, 0.5 g chloramphenicol and 0.6 g cetyl trimethylammonium bromide per liter.
Overall, 300 mosquitoes were collected from 3 types of resting sites (inhabited houses, abandoned houses and outdoor piles of wood). Mosquitoes were brought to the IRSS/Centre Muraz insectary, where they were fed on 6% sterile glucose ad libitum. Approximately 22% of collected mosquitoes (67 mosquitoes) died within 2 weeks and were plated on selective medium for fungal isolations.  isolates from rhizospheres or mosquitoes were identified using macro-morphological characters, such as conidiogenesis, estimation of radial growth, spore color and mycelia texture of the isolates on PDA media according to Humber [8]. In addition, we used microscopic morphology to identify Metarhizium spp. spores as described by Fernandes et al. [9]. Met_S10 and Met_S26 were confirmed as Metarhizium pingshaense through amplification and Sanger sequencing of the intron-rich region of translation elongation factor 1-α [10].

Fungal virulence on mosquitoes, honeybees and cockroaches
Initial screens on mosquitoes revealed two promising isolates (Met_S10 and Met_S26) isolated from mosquito cadavers from Soumousso and Bana, respectively, that readily grew on PDA and were highly virulent (Additional file 1: Table S1): these strains were therefore chosen for further characterization.

Bioassay on mosquitoes
For bioassays, we used An. coluzzii adult mosquitoes reared from larval collections at the Kou Valley, Burkina Faso. Mosquitoes from this area are known to be highly resistant to multiple insecticides [5,11]. We carried out bioassays with local M. pingshaense isolates Met_S10 and Met_S26. A M. pingshaense strain that has been used as the foundation for development of transgenic mosquito control technologies was used as a positive control; this strain was engineered to constitutively express red fluorescent protein (RFP) [5]. Expression of RFP provides a fluorescent tag for following infection processes without altering virulence. We used an atomizer protocol for infections, as described previously [12]. Three serial concentrations were used: 1 × 10 8 ; 1 × 10 7 ; and 1 × 10 6 conidia/ml. We confirmed that this inoculation technique was able to deliver a repeatable inoculating dose (mean ± SE): 276 ± 16 spores per mosquito with 1 × 10 8 ; 211 ± 13 spores per mosquito with 1 × 10 7 spores/ml; and 44 ± 3 spores per mosquito with 1 × 10 6 . Mortality was counted twice daily over two weeks.

Results and discussion
We bioassayed honeybees and cockroaches with the local strains and Met_RFP. However, even at the highest spore dosage (1 × 10 8 conidia/ml), these fungi did not significantly increase mortality compared to controls containing no conidia (Table 2). Fewer than 5% of honeybees and cockroaches died during the bioassays, and no mycosis was observed on any cadavers. This is in agreement with previous studies that report Met_RFP is a specialist to Culicidae [5]. The host ranges of different Metarhizium strains are chiefly controlled by recognition events on the cuticle [19], and the cuticles of  honeybees, cockroaches and mosquitoes would likely have many topographical and chemical differences.
Despite being more virulent than other WT Metarhizium strains, the Burkinabe Anopheles-derived isolates are still significantly less effective than transgenic strains expressing arthropod toxins [5]. However, our results suggest that these native Burkinabe Metarhizium strains would make attractive candidates for transgenic virulence enhancement and subsequent use as transgenic biocontrol agents.

Conclusion
Native fungal isolates may offer a superior alternative to introducing a foreign biocontrol strain, as they may be better adapted to both kill local mosquitoes and survive local conditions. There are also regulatory and ecological advantages to using strains already present in the country or in the ecosystem. This study provides a promising precedent for isolating local Metarhizium strains for application in mosquito biological control, and it lays a foundation for future transgenic biocontrol projects in Burkina Faso.

Additional files
Additional file 1: Table S1. Preliminary infections data on mosquitoes.