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Aims Phosphate chemical fertilizers are costly and raise concerns about environmental pollution through industrial production. The use of phosphate rock (PRs) emerges as a more sustainable alternative for agriculture. The aim of this work was to isolate phosphate rock-solubilizing bacteria (PRSB) from maize mycorrhizosphere, having growth promoting traits and that will be arbuscular mycorrhizae fungi (AMF) compatible. Methods Bacteria were isolated from the mycorrhizosphere of maize and tested for rock phosphate solubilization, production of organic acids, phosphatases and phytase activities, and growth promotion traits. The capacity of some strains to enhance the dry weight of maize plants was determined in a growth chamber experiment. The compatibility of the selected strains with Rhizophagus irregularis under in vitro conditions was also tested. Results Out of 118 isolates from maize, eight belonging to Asaia lannaensis , Rahnella sp., Pantoea sp., and Pseudomonas sp . were found to be the best PRSB. On solid media, all strains mobilized P from tricalcium phosphate, hydroxyapatite, and PRs. A. lannaensis was the only PRSB showing visible solubilization of AlPO 4 and FePO 4 . All the PRSBs solubilized PR by producing D-gluconic acid and 2-ketogluconic acid and by lowering the pH. Most strains presented IAA and siderophore production and different biofilm formation and motility capacities. All strains improved the dry weight of maize seedlings compared with non-inoculated plants. The results proved that PRSBs were able to grow on R. irregularis hyphae as the sole in vitro C source. Conclusions Bacteria isolated from the mycorrhizosphere of maize shows effective solubilization of phosphorus from PR with different reactivity levels. The traits of these bacteria as growth promoters and their biocompatibility with AMF show their potential as inoculants. Improvement of the agronomic effectiveness of PRs is relevant for developing countries that use PRs directly as P-fertilizers (less expensive than soluble P-fertilizers) for sustainable agriculture.

Background The male mosquito microbiome may be important for identifying ideal candidates for disease control. Among other criteria, mosquito-associated symbionts that have high localization in both male and female mosquitoes and are transmissible through both vertical and sexual routes are desirable. However, mosquito microbiome studies have mainly been female-focused. In this study, the microbiota of male and female Anopheles gambiae sensu lato (s.l.) were compared to identify shared or unique bacteria. Methods Late larval instars of Anopheles mosquitoes were collected from the field and raised to adults. Equal numbers of males and females of 1-day-old non-sugar-fed, 4–5-day-old sugar-fed and post-blood-fed females were randomly selected for whole-body analyses of bacteria 16S rRNA. Results Results revealed that male and female mosquitoes generally share similar microbiota except when females were blood-fed. Compared to newly emerged unfed mosquitoes, feeding on sugar and/or blood increased variability in microbial composition (⍺-diversity), with a higher disparity among females (39% P  = 0.01) than in males (29% P  = 0.03). Elizabethkingia meningoseptica and Asaia siamensis were common discriminants between feeding statuses in both males and females. While E. meningoseptica was particularly associated with sugar-fed mosquitoes of both sexes and sustained after blood feeding in females, A. siamensis was also increased in sugar-fed mosquitoes but decreased significantly in blood-fed females (LDA score > 4.0, P  < 0.05). Among males, A. siamensis did not differ significantly after sugar meals. Conclusions Results indicate the opportunities for stable infection in mosquitoes should these species be used in bacteria-mediated disease control. Further studies are recommended to investigate possible host-specific tissue tropism of bacteria species which will inform selection of the most appropriate microbes for effective transmission-blocking strategies.

Mosquitoes transmit many pathogens responsible for human diseases, such as malaria which is caused by parasites in the genus Plasmodium . Current strategies to control vector-transmitted diseases are increasingly undermined by mosquito and pathogen resistance, so additional methods of control are required. Paratransgenesis is a method whereby symbiotic bacteria are genetically modified to affect the mosquito’s phenotype by engineering them to deliver effector molecules into the midgut to kill parasites. One paratransgenesis candidate is Asaia bogorensis , a Gram-negative bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. Previously, engineered Asaia strains using native signals to drive the release of the antimicrobial peptide, scorpine, fused to alkaline phosphatase were successful in significantly suppressing the number of oocysts formed after a blood meal containing P . berghei . However, these strains saw high fitness costs associated with the production of the recombinant protein. Here, we report evaluation of five different partner proteins fused to scorpine that were evaluated for effects on the growth and fitness of the transgenic bacteria. Three of the new partner proteins resulted in significant levels of protein released from the Asaia bacterium while also significantly reducing the prevalence of mosquitoes infected with P . berghei . Two partners performed as well as the previously tested Asaia strain that used alkaline phosphatase in the fitness analyses, but neither exceeded it. It may be that there is a maximum level of fitness and parasite inhibition that can be achieved with scorpine being driven constitutively, and that use of a Plasmodium specific effector molecule in place of scorpine would help to mitigate the stress on the symbionts.

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.

This study was conducted to investigate the antibacterial and antiadhesive activities of ethanol extracts from five edible plant parts: cinnamon bark ( Cinnamomum zeylanicum ), licorice root ( Glycyrrhiza radix ), nettle leaves ( Urtica dioica ), green tea leaves ( Camellia sinensis ), and elderberry flowers ( Sambucus nigra ). The chemical constituents of the extracts were identified using high-performance liquid chromatography and liquid chromatography plus mass spectrometry. Six strains of Asaia lannensis and Asaia bogorensis bacteria isolated from spoiled commercial fruit-flavored noncarbonated mineral water were used. Bacterial adhesion to polystyrene as an attachment substrate in culture media supplemented with 10% plant extract was evaluated using luminometric measurement of the ATP extracted from adhered cells. The viability of the adhered and planktonic cells was assessed using the plate count method, and the relative adhesion coefficient was calculated. All tested crude extracts contained flavonols (kaempferol, quercetin, and their derivatives), flavanols (catechin and derivatives), flavanones (glabrol, licorice glycoside A, and liquiritin), and phenolic acids (gallic, quinic, chlorogenic, neochlorogenic, caffeic, coumaric, and ferulic). The culture medium with 10% elderberry extract provided the least favorable environment for all tested bacterial strains. Extracts from green tea, cinnamon, and licorice also had significant inhibitory effects on the adhesion of the tested bacterial strains. This research suggests that the addition of selected edible plant extracts could improve the microbial stability of noncarbonated soft drinks.

Deadly pathogens and parasites are transmitted by vectors and the mosquito is considered the most threatening vector in public health, transmitting these pathogens to humans and animals. We are currently witnessing the emergence/resurgence in new regions/populations of the most important mosquito-borne diseases, such as arboviruses and malaria. This resurgence may be the consequence of numerous complex parameters, but the major cause remains the mismanagement of insecticide use and the emergence of resistance. Biological control programmes have rendered promising results but several highly effective techniques, such as genetic manipulation, remain insufficiently considered as a control mechanism. Currently, new strategies based on attractive toxic sugar baits and new agents, such as Wolbachia and Asaia, are being intensively studied for potential use as alternatives to chemicals. Research into new insecticides, Insect Growth Regulators, and repellent compounds is pressing, and the improvement of biological strategies may provide key solutions to prevent outbreaks, decrease the danger to at-risk populations, and mitigate resistance.

Microbiome composition has been associated with insecticide resistance in malaria vectors. However, the contribution of major symbionts to the increasingly reported resistance escalation remains unclear. This study explores the possible association of a specific endosymbiont, Asaia spp., with elevated levels of pyrethroid resistance driven by cytochrome P450s enzymes and voltage-gated sodium channel mutations in Anopheles funestus and Anopheles gambiae. Molecular assays were used to detect the symbiont and resistance markers (CYP6P9a/b, 6.5 kb, L1014F, and N1575Y). Overall, genotyping of key mutations revealed an association with the resistance phenotype. The prevalence of Asaia spp. in the FUMOZ_X_FANG strain was associated with the resistance phenotype at a 5X dose of deltamethrin (OR = 25.7; p = 0.002). Mosquitoes with the resistant allele for the markers tested were significantly more infected with Asaia compared to those possessing the susceptible allele. Furthermore, the abundance correlated with the resistance phenotype at 1X concentration of deltamethrin (p = 0.02, Mann-Whitney test). However, for the MANGOUM_X_KISUMU strain, findings rather revealed an association between Asaia load and the susceptible phenotype (p = 0.04, Mann-Whitney test), demonstrating a negative link between the symbiont and permethrin resistance. These bacteria should be further investigated to establish its interactions with other resistance mechanisms and cross-resistance with other insecticide classes.

Four phosphate rock-solubilizing bacteria (PRSB) (Asaia lannensis Vb1, Pseudomonas sp. Vr14, Rahnella sp. Sr24, and Pantoea sp. Vr25) isolated from the mycorrhizosphere of maize were evaluated as inoculants, individually or in different combinations, together with Rhizophagus irregularis DAOM 197198, under greenhouse conditions. To create an effective bacterial mix for P solubilization and growth promotion, parameters such as the ability of the strains (individually or in mixture) to mobilize phosphate rock (PR), their biocompatibility, and their capacity for biofilm formation over PR particles and root colonization were tested. Accompanying PR solubilization, the bacteria produced organic acids and reduced the pH of the medium, produced exopolysaccharides, and had variable capacity to colonize the roots of maize plantlets. In low-phosphorus soil amendment with PR, all strains, regardless of their capacity to solubilize PR, increased dry weight, nutrient (N, P, and K) uptake, and the percentage of indigenous arbuscular mycorrhizae (iAM) root colonization in maize plants, compared to the non-inoculated control. However, mixed inoculation of the strains showed significantly better results. Addition of Rhizophagus irregularis resulted in further growth stimulation. Overall results showed that two combinations—Rahnella sp. Sr24 + Pantoea sp. Vr25 + R. irregularis, and Pantoea sp. Vr25 + Pseudomonas sp. Vr14 + R. irregularis—were better inoculants. We concluded that an effective PRSB combination of biocompatible strains with capacity for PR solubilization, successful biofilm formation, effective root colonization, different growth promotion traits, and the addition of AM has potential as inoculants for more sustainable agriculture in low-phosphorus soils amended with low-reactivity PR.

, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the  genera, but has recently been found in  s.l. populations in West Africa.  As there are numerous  species that have the capacity to transmit malaria, we analysed a range of species across five malaria endemic countries to determine  prevalence rates, characterise novel  strains and determine any correlation between the presence of  ,   and the competing bacterium  .  adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017.  Molecular analysis was undertaken using quantitative PCR, Sanger sequencing,  multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial   gene.  : Novel  strains were discovered in five species:  ,  s.s.,  ,  and   species A, increasing the number of  species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with  supergroup B strains.  We also provide evidence for resident strain variants within  . species A.   is the dominant member of the microbiome in  and   species A but present at lower densities in  .  Interestingly, no evidence of  co-infections was seen and   infection densities were shown to be variable and location dependent.  The important discovery of novel  strains in  provides greater insight into the prevalence of resident  strains in diverse malaria vectors.  Novel  strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other  mosquito species, which could be used for population replacement or suppression control strategies.

Mosquitoes are efficient vectors of multiple human and animal pathogens. The biology of mosquitoes is strongly affected by their associated microbiota. The mosquito microbiota has a profound impact on multiple biological processes ranging from reproduction to disease transmission. Interestingly, the adult mosquito microbiota is largely derived from the larval microbiota, which in turn is dependent on the microbiota of their water habitat. The larval microbiota not only plays a crucial role in larval development but also has a significant impact on the adult stage of the mosquito. By precisely engineering the larval microbiota, it is feasible to alter larval development and other life history traits of the mosquitoes. Bacteriophages, given their host specificity, can serve as a tool for modulating the microbiota. For this proof-of-principle study, we selected representative strains of five common Anopheles mosquito-associated bacterial genera, namely, Enterobacter , Serratia , Pseudomonas , Elizabethkingia , and Asaia . Our results with monoaxenic cultures showed that Anopheles larvae with Enterobacter and Pseudomonas displayed normal larval development with no significant mortality. However, monoaxenic Anopheles larvae with Elizabethkingia showed delayed larval development and higher mortality. Serratia and Asaia gnotobiotic larvae failed to develop past the first instar. We isolated and characterized three novel bacteriophages (EP1, SP1, and EKP1) targeting Enterobacter , Serratia, and Elizabethkingia, respectively, and utilized a previously characterized bacteriophage (GH1) targeting Pseudomonas to modulate larval water microbiota. Gnotobiotic Anopheles larvae with all five bacterial genera showed reduced survival and larval development with the addition of bacteriophages EP1 and GH1, targeting Enterobacter and Pseudomonas , respectively. The effect was synergistic when both EP1 and GH1 were added together. Our results demonstrate a novel application of bacteriophages for mosquito control. IMPORTANCE Mosquitoes are efficient vectors of multiple human and animal pathogens. The biology of mosquitoes is strongly affected by their associated microbiota. Because of the important role of the larval microbiota in mosquito biology, the microbiota can potentially serve as a target for altering mosquito life-history traits. Our study provides proof of principle that bacteriophages can be used as tools to modulate the mosquito larval habitat microbiota and can, in turn, affect larval development and survival. These results highlight the utility of bacteriophages in mosquito microbiota research and also provide a new potential mosquito control tool.