Ectomycorrhizal fungi are shared between seedlings and adults in a monodominant Gilbertiodendron dewevrei rain forest in Cameroon

Ectomycorrhizal networks may facilitate the establishment and survival of seedlings regenerating under the canopies of tropical forests and are often invoked as a potential contributor to monodominance. We identified ectomycorrhizal fungi in a monodominant Gilbertiodendron dewevrei (Fabaceae) rain forest in Cameroon, using sporocarps and ectomycorrhizae of three age categories (seedlings, intermediate trees, and large trees) and tentatively revealed nutrient transfer through ectomycorrhizal networks by measuring spontaneous isotopic (¹³C and ¹⁵N) abundances in seedlings. Sporocarp surveys revealed fewer ectomycorrhizal fungal taxa (59 species from 1030 sporocarps) than molecular barcoding of ectomycorrhizal roots (75 operational taxonomic units from 828 ectomycorrhizae). Our observations suggest that ectomycorrhizal fungal diversity is similar to that in other mixed tropical forests and provide the first report of the Tuber‐Helvella lineage in a tropical forest. Despite some differences, all age categories of G. dewevrei had overlapping ectomycorrhizal fungal communities, with families belonging to Thelephoraceae, Russulaceae, Sebacinaceae, Boletaceae, and Clavulinaceae. Of the 49 operational taxonomic units shared by the three age categories (65.3% of the ectomycorrhizal fungal community), 19 were the most abundant on root tips of all categories (38.7% of the shared taxa), supporting the likelihood of ectomycorrhizal networks. However, we obtained no evidence for nutrient transfer from trees to seedlings. We discuss the composition of the ectomycorrhizal fungal community among the G. dewevrei age categories and the possible role of common ectomycorrhizal networks in this rain forest.     

Response of field-grownCasuarina equisetifolia to inoculation withFrankia strain ORS 021001 entrapped in alginate beads

A large scale field experiment (ca 1 ha) was carried out in Senegal, to evaluate the response ofCasuarina equisetifolia to inoculation withFrankia strain ORS 021001 entrapped in alginate beads. Biomasses (expressed as dry weight or total nitrogen) of assimilatory branchlets, wood and roots, and nodules were measured in uninoculated and inoculated trees, randomly sampled 1,2 and 3 years after transplantation in the field. When biomasses were expressed as dry weight, increases due to inoculation were similar at the three sampling dates, 45, 36 and 40%, respectively. When biomasses were expressed as total nitrogen, the response to inoculation with time was much higher in the 2nd year than in the 1st and 3rd year. N₂ fixation, estimated using the difference method reached 2.48, 12.25 and 13.44 g N₂ fixed annually per tree. Correspondingly, nodule dry weights, expressed in g per tree, were 2.5, 12.18 and 22.75 at the end of the 1 st, 2nd and 3rd year, respectively. In spite of the positive response of field-grownCasuarina equisetifolia to inoculation, the decrease of N₂ fixation observed in the third year was probably due to unfavorable climatic conditions coupled with insect attacks at the beginning of the third year.     

Propagation of Casuarina equisetifolia through axillary buds of immature female inflorescences cultured in vitro

The study of the actinorhizal symbiosis in Casuarina equisetifolia requires an homogenous plant material. Consequently, we devised a method of micropropagation based on the use of immature female inflorescences (IFI) as explants. IFI excised from an adult tree formed multiple buds after 4-week incubation on Murashige and Skoog medium with 0.05 mol 1^–1 NAA and 11.1 mol 1^–1 BAP. The axillary buds evolved into 5–6 cm long shoots 5 weeks after the transfer of IFI on a similar medium except for the addition of activated charcoal. Rooting of the shoots was obtained on a third medium, without BAP or charcoal, but with 1 mol 1^–1 NAA. The plantlets were transferred into soil. Their growth was satisfactory and no plagiotropic tendency was observed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - IFI immature female inflorescences - NAA -naphtaleneacetic acid     

Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry: An Emerging Tool for the Rapid Identification of Mosquito Vectors

Background

The identification of mosquito vectors is typically based on morphological characteristics using morphological keys of determination, which requires entomological expertise and training. The use of protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is increasingly being used for the routine identification of bacteria, has recently emerged for arthropod identification.

Methods

To investigate the usefulness of MALDI-TOF-MS as a mosquito identification tool, we tested protein extracts made from mosquito legs to create a database of reference spectra. The database included a total of 129 laboratory-reared and field-caught mosquito specimens consisting of 20 species, including 4 Aedes spp., 9 Anopheles spp., 4 Culex spp., Lutzia tigripes, Orthopodomyia reunionensis and Mansonia uniformis. For the validation study, blind tests were performed with 76 specimens consisting of 1 to 4 individuals per species. A cluster analysis was carried out using the MALDI-Biotyper and some spectra from all mosquito species tested.

Results

Biomarker mass sets containing 22 and 43 masses have been detected from 100 specimens of the Anopheles, Aedes and Culex species. By carrying out 3 blind tests, we achieved the identification of mosquito vectors at the species level, including the differentiation of An. gambiae complex, which is possible using MALDI-TOF-MS with 1.8 as the cut-off identification score. A cluster analysis performed with all available mosquito species showed that MALDI-Biotyper can distinguish between specimens at the subspecies level, as demonstrated for An gambiae M and S, but this method cannot yet be considered a reliable tool for the phylogenetic study of mosquito species.

Conclusions

We confirmed that even without any specific expertise, MALDI-TOF-MS profiling of mosquito leg protein extracts can be used for the rapid identification of mosquito vectors. Therefore, MALDI-TOF-MS is an alternative, efficient and inexpensive tool that can accurately identify mosquitoes collected in the field during entomological surveys.     

Resistance to DDT and pyrethroids and increased kdr mutation frequency in An. gambiae after the implementation of permethrin-treated nets in Senegal

IntroductionThe aim of this study was to evaluate the susceptibility to insecticides of An. gambiae mosquitoes sampled in Dielmo (Senegal), in 2010, 2 years after the implementation of Long Lasting Insecticide-treated Nets (LLINs) and to report the evolution of kdr mutation frequency from 2006 to 2010.MethodsWHO bioassay susceptibility tests to 6 insecticides were performed on adults F0, issuing from immature stages of An. gambiae s.l., sampled in August 2010. Species and molecular forms as well as the presence of L1014F and L1014S kdr mutations were assessed by PCR. Longitudinal study of kdr mutations was performed on adult mosquitoes sampled monthly by night landing catches from 2006 to 2010.FindingsNo specimen studied presented the L1014S mutation. During the longitudinal study, L1014F allelic frequency rose from 2.4% in year before the implementation of LLINs to 4.6% 0–12 months after and 18.7% 13–30 months after. In 2010, An. gambiae were resistant to DDT, Lambda-cyhalothrin, Deltamethrin and Permethrin (mortality rates ranging from 46 to 63%) but highly susceptible to Fenitrothion and Bendiocarb (100% mortality). There was significantly more RR genotype among An. gambiae surviving exposure to DDT or Pyrethroids. An. arabiensis represented 3.7% of the sampled mosquitoes (11/300) with no kdr resistance allele detected. An. gambiae molecular form M represented 29.7% of the mosquitoes with, among them, kdr genotypes SR (18%) and SS (82%). An. gambiae molecular form S represented 66% of the population with, among them, kdr genotype SS (33.3%), SR (55.6%) and RR (11.1%). Only 2 MS hybrid mosquitoes were sampled and presented SS kdr genotype.ConclusionBiological evidence of resistance to DDT and pyrethroids was detected among An. gambiae mosquitoes in Dielmo (Senegal) within 24 months of community use of LLINs. Molecular identification of L1014F mutation indicated that target site resistance increased after the implementation of LLINs.