Genetic diversity and core subset selection in ex situ seed collections of the banana crop wild relative Musa balbisiana

Crop wild relatives (CWRs) play a key role in crop breeding by providing beneficial trait characteristics for improvement of related crops. CWRs are more efficiently used in breeding if the plant material is genetically characterized, but the diversity in CWR genetic resources has often poorly been assessed. Seven seed collections of Musa balbisiana, an important CWR of dessert and cooking bananas, originating from three natural populations, two feral populations and two ex situ field collections were retrieved and their genetic diversity was quantified using 18 microsatellite markers to select core subsets that conserve the maximum genetic diversity. The highest genetic diversity was observed in the seed collections from natural populations of Yunnan, a region that is part of M. balbisiana's centre of origin. The seeds from the ex situ field collections were less genetically diverse, but contained unique variation with regards to the diversity in all seed collections. Seeds from feral populations displayed low genetic diversity. Core subsets that maximized genetic distance incorporated almost no seeds from the ex situ field collections. In contrast, core subsets that maximized allelic richness contained seeds from the ex situ field collections. We recommend the conservation and additional collection of seeds from natural populations, preferentially originating from the species' region of origin, and from multiple individuals in one population. We also suggest that the number of seeds used for ex situ seed bank regeneration must be much higher for the seed collections from natural populations

Molecular detection of six high importance endosymbiotic bacteria in Belgian wild-caught mosquitoes

Introduction Several disease vectors presented a resistance to various pesticides currently used. One of an alternative solution was to use endosymbiotic bacteria because their probably interactive effects with their host. According to the introduction risks of mosquito born disease and their dispersion, we propose to investigate the prevalence of six endosymbiontic bacteria in wild-caught Culicidae in Belgium. Methods Eleven species of Belgian fields mosquitoes (Culex pipiens s.l., Cx. torrentium, Cx. hortensis, Anopheles claviger, An. maculipennis s.l., An. plumbeus, Culiseta annulata, Ochlerotatus geniculatus, Oc. dorsalis, Aedes albopictus and Coquillettidia richiardii) were used for the screening of six genera endosymbiotic bacteria (Wolbachia sp., Commamonas sp., Delftia sp., Pseudomonas sp., Acinetobacter sp. and Asaia sp.) according to their possible impact in mosquito biology. PCR was done for the screening and positives bands were sequenced and deposited in GenBank. Results Total of 144 larvae and 32 adults were used. Wolbachia, Pseudomonas, Acinetobacter and Asaia were found in mosquitoes with different proportions, according to stages (adults, larvae) with a predominance of Pseudomonas in all species, as far as Acinetobacter and Asaia also have a high prevalence. Commamonas and Delftia were absent from all species tested, either in larvae and in adults. Discussion Choice of endosymbiotic bacteria studied here was allowing of their importance in literature. For Pseudomonas, it showed that this bacteria could produced ovipositon attractants for mosquito. Acinetobacter was suggested efficient in transmission and maintenance within host populations. Asaia was capable of efficiently crossing body barriers and colonizing different organs. Wolbachia was currently the most studied bacteria which plays an important role in the genetic manipulation of the host. Present advances in understanding the mosquito–microbiota relationships may have a great impact in a better understanding of some traits of mosquito biology and in the development of innovative mosquito-borne disease-control strategies.Contribution à l’étude des endosymbiotes des Culicidae de Belgique : rôles dans la transmission virale et cible pour le contrôle des moustique

Using micro-injection technique to assess fungal toxicity in mosquito control

Topical application of insecticidal compounds allows directly exposing these substances on insect tissues and measuring their toxicity while ignoring many factors. However, this technique remains difficult to apply on mosquito larvae considering their aquatic lifestyle. Micro-injection could be used for the direct deposition of toxic compounds in the larvae. Capillaries exhibiting an injection tip with an external diameter of 0.5 mm have been designed from silica tubes. For each treatment, a capillary is mounted on a pump connected to a flow rate regulator. Culex quinquefasciatus larvae were injected with 10^7 spores/ml of entomopathogenic fungi (Aspergillus clavatus, Metarhizium anisopliae, Metarhizium sp.). Mortalities were recorded daily during 72h. The distribution of spores stained with methylene blue and injected into the body of larvae was also observed according to the system described. Results showed that spores were distributed over the whole body. The injection of Aspergillus clavatus, Metarhizium anisopliae and Metarhizium sp spores induced corrected mortalities of 62%, 53% and 57% after 72h, and differed statistically from control groups. Finally, post-mortem emergences of filaments from dead larvae were observed in the case of the three fungal strains confirming spore viability. Injection of inactivated spores (or inert bodies of similar size) could help to reject the hypothesis of a response due to the presence of foreign bodies

Biofilms from entomopathogenic fungi in mosquito control

Mosquitoes (Diptera: Culicidae) are zoonotic vectors of medical and veterinary importance. As part of an integrated vector control, metabolites secreted by entomopathogenic fungi could be developed as biopesticides. In this context, filamentous microorganisms growing on a support as biofilm in a liquid medium would offer several advantages in bioreactor regarding performances and metabolites recovery. The production of toxic metabolites by an entomopathogenic fungus Aspergillus flavus in such conditions was assessed. Three initial inoculum levels, i.e. 10^1, 10^3 and 10^6 spores/ml of PYG medium, have been tested in shake flask with or without support. Toxicity tests were performed on Culex quinquefasciatus larvae using dilutions of 1, 2, 4, 6, 8 and 10% of liquid cultures. The results indicated that A. flavus tends to form pellets in submerged culture; the size and the amount of pellets was affected by the initial inoculum level of spores. Under similar conditions, the filaments fixed on a support and didn’t appear in free form in the liquid. Toxicity tests revealed differences between both free and fixed forms. All combined conditions, LC50s ranging up to dilutions of 2.2 and 4.8% were observed within 48 hours. Secretomes could be compared between these culture conditions by proteomic and metabolomic approaches

Laboratory and semi-field environment tests for the control efficacy of Metarhizium anisopliae formulated with neem oil (suneem) against Anopheles gambiae s.l. adult emergence

peer reviewedMetarhizium anisopliae was evaluated previously in Suneem formulation against malaria vector adults. However, their ability to control aquatic stages is not yet evaluated. In laboratory conditions: the lethal dose (LD90) of the formulation was determined on Anopheles gambiae larvae collected from breeding sites and evaluated into artificial vats at dry and rainy seasons. In laboratory conditions, the LD90 was 5.3 × 106 spores/ml in 48 h. In semi-field environment, the formulation had a great emergence inhibition of mosquito adult (P < 0.0001). The emergences rate at day 8 were 2.25 ± 0.03, 28.00 ± 1.07 and 97.25 ± 1.56 % in dry season for the oil formulation (OF), Suneem (S), and water control respectively. In rainy season, the emergences were 1.25 ± 0.15, 30.25 ± 1.23 and 98 ± 0.76 % respectively. No significant difference was observed between dry and rainy seasons (P=0.3). Therefore, M. anisopliae formulated with Suneem may provide a more sustainable management strategy for malaria vectors control at larval stages

Étude de l'origine phylogénétique de Aedes albopictus et de Aedes koreicus (Diptera, Culicidae), deux espèces de moustiques invasives en Belgique

The main ways of the introduction of invasive mosquito species in Europe were greatly facilitated by the importation of used tires and plants from the South of China (Dracaena sanderiana, called "Lucky bamboo"), which are a very good breeding sites of these mosquitoes (www.eidatlantique.eu). For Belgium, several species of exotic mosquitoes have already been listed such as Ochlerotatus japonicus japonicus, Aedes albopictus and A. koreicus. We have captured one male of A. albopictus in Belgium in July 2013 (Boukraa et al, 2013), and teams from other entomologist have been able to find several individuals in the months that have followed our discovery (personal communication). However, his presence was reported first in 2000 (Schaffner et al. 2004) but until 2012, no individuals have been found (Versteirt et al., 2013). We also could find larvae of A. koreicus in abundance. These discovery suggest that these two invasive species are being established in Belgium. Methods: Adult mosquitoes were collected by CO2-baited traps Mosquito Magnet Liberty Plus (MMLP) and immature stages by the dipping method from 8 sites of Belgium. Each individual was then determined morphologically. For A. albopictus and A. koreicus, reconfirmation by molecular method was performed with COI and ND5 mitochondrial primers and sequences were then aligned with those of mosquitoes available in databases by using BioEdit and Multialn softwares. The two gene sequences were concatenated to improve the reliability of the phylogenetic analysis and were carried out by using the Seaview software based on maximum likelihood (ML) methods. Trees were then constructed with the general time reversible (GTR) model, and branch supports were estimated by bootstrapping with 1000 replicates. Result: The aim of this work will focus on the study of the probable origin of these two species of Aedes, which are for the moment safe yet, but that might become dangerous in the event of massive outbreak. The knowledge of the probably origin of the two invasive mosquitoes allow take more protection against their way to enter in the country

Diversité bactérienne de moustiques provenant de différentes régions de Belgique et leur impact potentiel sur la transmission des maladies

Several vectors disease presented a resistance to various pesticides currently used. Endosymbiotic bacteria was an alternative solution found because of their probably interactive effects with their host. According to the introduction risks of these virus and disease dispersion, we propose to investigate the bacterial endosymbiont role in Culicidae in Belgium. Among the 30 species of mosquitoes identified in this country, about ten are considered as potential vectors of arboviruses. In this study, eleven species of Culicidae belonging to five genera (Culex pipiens s.l., Cx. torrentium, Cx. hortensis, Anopheles claviger, An. maculipennis s.l., An. plumbeus, Culiseta annulata, Ochlerotatus geniculatus, Oc. dorsalis, Aedes albopictus and Coquillettidia richiardii) mosquitoes fields from eight sites of Belgium were used for the screening of the presence of six genera endosymbiotic bacteria (Wolbachia sp, Commamonas sp, Delftia sp, Pseudomonas sp, Acinetobacter sp and Asaia sp) according their possible impact in mosquito biology. PCR was done for the screening of endosymbiotic bacteria mosquitoes studied. A total of 176 individuals (144 larvae and 32 adults) were used. Our results allowed us to confirm the absence of Wolbachia in An. clavigere, An. maculipennis s.l and Cx. torentium. Acinetobacter was found in every species. Current advances in understanding the mosquito–microbiota relationships may have a great impact in a better understanding of some traits of mosquito biology and in the development of innovative mosquito-borne disease-control strategies aimed to reduce mosquito vectorial capacity and/or inhibiting pathogen transmission.Contribution à l’étude des Culicidae exotiques, leur bactérie endosymbiotique Wolbachia face aux changements climatiques et les échanges commerciaux en Belgiqu