A Time Point Proteomic Analysis Reveals Protein Dynamics of Plasmodium Oocysts.

The oocyst is a sporogonic stage of Plasmodium development that takes place in the mosquito midgut in about 2 weeks. The cyst is protected by a capsule of unknown composition, and little is known about oocyst biology. We carried out a proteomic analysis of oocyst samples isolated at early, mid, and late time points of development. Four biological replicates for each time point were analyzed, and almost 600 oocyst-specific candidates were identified. The analysis revealed that, in young oocysts, there is a strong activity of protein and DNA synthesis, whereas in mature oocysts, proteins involved in oocyst and sporozoite development, gliding motility, and invasion are mostly abundant. Among the proteins identified at early stages, 17 candidates are specific to young oocysts. Thirty-four candidates are common to oocyst and the merosome stages (sporozoite proteins excluded), sharing common features as replication and egress. Western blot and immunofluorescence analyses of selected candidates confirm the expression profile obtained by proteomic analysis.

Combination of computational techniques and RNAi reveal targets in Anopheles gambiae for malaria vector control.

Increasing reports of insecticide resistance continue to hamper the gains of vector control strategies in curbing malaria transmission. This makes identifying new insecticide targets or alternative vector control strategies necessary. CLassifier of Essentiality AcRoss EukaRyote (CLEARER), a leave-one-organism-out cross-validation machine learning classifier for essential genes, was used to predict essential genes in Anopheles gambiae and selected predicted genes experimentally validated. The CLEARER algorithm was trained on six model organisms: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Mus musculus, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and employed to identify essential genes in An. gambiae. Of the 10,426 genes in An. gambiae, 1,946 genes (18.7%) were predicted to be Cellular Essential Genes (CEGs), 1716 (16.5%) to be Organism Essential Genes (OEGs), and 852 genes (8.2%) to be essential as both OEGs and CEGs. RNA interference (RNAi) was used to validate the top three highly expressed non-ribosomal predictions as probable vector control targets, by determining the effect of these genes on the survival of An. gambiae G3 mosquitoes. In addition, the effect of knockdown of arginase (AGAP008783) on Plasmodium berghei infection in mosquitoes was evaluated, an enzyme we computationally inferred earlier to be essential based on chokepoint analysis. Arginase and the top three genes, AGAP007406 (Elongation factor 1-alpha, Elf1), AGAP002076 (Heat shock 70kDa protein 1/8, HSP), AGAP009441 (Elongation factor 2, Elf2), had knockdown efficiencies of 91%, 75%, 63%, and 61%, respectively. While knockdown of HSP or Elf2 significantly reduced longevity of the mosquitoes (p<0.0001) compared to control groups, Elf1 or arginase knockdown had no effect on survival. However, arginase knockdown significantly reduced P. berghei oocytes counts in the midgut of mosquitoes when compared to LacZ-injected controls. The study reveals HSP and Elf2 as important contributors to mosquito survival and arginase as important for parasite development, hence placing them as possible targets for vector control.

Wolbachia and Asaia Distribution among Different Mosquito Vectors Is Affected by Tissue Localization and Host Species.

Microbial communities play an important role in the fitness of mosquito hosts. However, the factors shaping microbial communities in wild populations, with regard to interactions among microbial species, are still largely unknown. Previous research has demonstrated that two of the most studied mosquito symbionts, the bacteria Wolbachia and Asaia, seem to compete or not compete, depending on the genetic background of the reference mosquito host. The large diversity of Wolbachia-Asaia strain combinations that infect natural populations of mosquitoes may offer a relevant opportunity to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction. We surveyed Wolbachia and Asaia in 44 mosquito populations belonging to 11 different species of the genera Anopheles, Aedes, and Culex using qualitative PCR. Through quantitative PCR, the amounts of both bacteria were assessed in different mosquito organs, and through metagenomics, we determined the microbiota compositions in some selected mosquito populations. We show that variation in microbial community structure is likely associated with the species/strain of mosquito, its geographical position, and tissue localization. Together, our results shed light on the interactions among different bacterial species in the microbial communities of mosquito vectors, and this can aid the development and/or improvement of methods for symbiotic control of insect vectors.

Dietary fiber supplementation increases Drosophila melanogaster lifespan and gut microbiota diversity.

Dietary fiber has been shown to have multiple health benefits, including a positive effect on longevity and the gut microbiota. In the present study, Drosophila melanogaster has been chosen as an in vivo model organism to study the health effects of dietary fiber supplementation (DFS). DFS extended the mean half-life of male and female flies, but the absolute lifespan only increased in females. To reveal the underlying mechanisms, we examined the effect of DFS on gut microbiota diversity and abundance, local gut immunity, and the brain proteome. A significant difference in the gut microbial community was observed between groups with and without fiber supplementation, which reduced the gut pathogenic bacterial load. We also observed an upregulated expression of dual oxidase and a modulated expression of Attacin and Diptericin genes in the gut of older flies, possibly delaying the gut dysbiosis connected to the age-related gut immune dysfunction. Brain proteome analysis showed that DFS led to the modulation of metabolic processes connected to mitochondrial biogenesis, the RhoV-GTPase cycle, organelle biogenesis and maintenance, membrane trafficking and vesicle-mediated transport, possibly orchestrated through a gut-brain axis interaction. Taken together, our study shows that DFS can prolong the half-life and lifespan of flies, possibly by promoting a healthier gut environment and delaying the physiological dysbiosis that characterizes the ageing process. However, the RhoV-GTPase cycle at the brain level may deserve more attention in future studies.

Tripartite interactions comprising yeast-endobacteria systems in the gut of vector mosquitoes.

It is shown that bacteria use yeast as a niche for survival in stressful conditions, therefore yeasts may act as temporary or permanent bacterial reservoirs. Endobacteria colonise the fungal vacuole of various osmotolerant yeasts which survive and multiply in sugar-rich sources such as plant nectars. Nectar-associated yeasts are present even in the digestive system of insects and often establish mutualistic symbioses with both hosts. Research on insect microbial symbioses is increasing but bacterial-fungal interactions are yet unexplored. Here, we have focused on the endobacteria of Wickerhamomyces anomalus (formerly Pichia anomala and Candida pelliculosa), an osmotolerant yeast associated with sugar sources and the insect gut. Symbiotic strains of W. anomalus influence larval development and contribute digestive processes in adults, in addition to exerting wide antimicrobial properties for host defence in diverse insects including mosquitoes. Antiplasmodial effects of W. anomalus have been shown in the gut of the female malaria vector mosquito Anopheles stephensi. This discovery highlights the potential of utilizing yeast as a promising tool for symbiotic control of mosquito-borne diseases. In the present study, we have carried out a large Next Generation Sequencing (NGS) metagenomics analysis including W. anomalus strains associated with vector mosquitoes Anopheles, Aedes and Culex, which has highlighted wide and heterogeneous EB communities in yeast. Furthermore, we have disclosed a Matryoshka-like association in the gut of A stephensi that comprises different EB in the strain of W. anomalus WaF17.12. Our investigations started with the localization of fast-moving bacteria-like bodies within the yeast vacuole of WaF17.12. Additional microscopy analyses have validated the presence of alive intravacuolar bacteria and 16S rDNA libraries from WaF17.12 have identified a few bacterial targets. Some of these EB have been isolated and tested for lytic properties and capability to re-infect the yeast cell. Moreover, a selective competence to enter yeast cell has been shown comparing different bacteria. We suggested possible tripartite interactions among EB, W. anomalus and the host, opening new knowledge on the vector biology.

Anopheline mosquito saliva contains bacteria that are transferred to a mammalian host through blood feeding.

Introduction: Malaria transmission occurs when Plasmodium sporozoites are transferred from the salivary glands of anopheline mosquitoes to a human host through the injection of saliva. The need for better understanding, as well as novel modes of inhibiting, this key event in transmission has driven intense study of the protein and miRNA content of saliva. Until now the possibility that mosquito saliva may also contain bacteria has remained an open question despite the well documented presence of a rich microbiome in salivary glands. Methods: Using both 16S rRNA sequencing and MALDI-TOF approaches, we characterized the composition of the saliva microbiome of An. gambiae and An. stephensi mosquitoes which respectively represent two of the most important vectors for the major malaria-causing parasites P. falciparum and P. vivax. Results: To eliminate the possible detection of non-mosquito-derived bacteria, we used a transgenic, fluorescent strain of one of the identified bacteria, Serratiamarcescens, to infect mosquitoes and detect its presence in mosquito salivary glands as well as its transfer to, and colonization of, mammalian host tissues following a mosquito bite. We also showed that Plasmodium infection modified the mosquito microbiota, increasing the presence of Serratia while diminishing the presence of Elizabethkingia and that both P. berghei and Serratia were transferred to, and colonized mammalian tissues. Discussion: These data thus document the presence of bacteria in mosquito saliva, their transfer to, and growth in a mammalian host as well as possible interactions with Plasmodium transmission. Together they raise the possible role of mosquitoes as vectors of bacterial infection and the utility of commensal mosquito bacteria for the development of transmission-blocking strategies within a mammalian host.

De novo genome assembly of the invasive mosquito species Aedes japonicus and Aedes koreicus.

BACKGROUND: Recently, two invasive Aedes mosquito species, Ae. japonicus and Ae. koreicus, are circulating in several European countries posing potential health risks to humans and animals. Vector control is the main option to prevent mosquito-borne diseases, and an accurate genome sequence of these mosquitoes is essential to better understand their biology and to develop effective control strategies. METHODS: A de novo genome assembly of Ae. japonicus (Ajap1) and Ae. koreicus (Akor1) has been produced based on a hybrid approach that combines Oxford Nanopore long-read and Illumina short-read data. Their quality was ascertained using various metrics. Masking of repetitive elements, gene prediction and functional annotation was performed. RESULTS: Sequence analysis revealed a very high presence of repetitive DNA and, among others, thermal adaptation genes and insecticide-resistance genes. Through the RNA-seq analysis of larvae and adults of Ae. koreicus and Ae. japonicus exposed to different temperatures, we also identified genes showing a differential temperature-dependent activation. CONCLUSIONS: The assembly of Akor1 and Ajap1 genomes constitutes the first updated collective knowledge of the genomes of both mosquito species, providing the possibility of understanding key mechanisms of their biology such as the ability to adapt to harsh climates and to develop insecticide-resistance mechanisms.

Delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts.

BACKGROUND: In recent years, acetic acid bacteria have been shown to be frequently associated with insects, but knowledge on their biological role in the arthropod host is limited. The discovery that acetic acid bacteria of the genus Asaia are a main component of the microbiota of Anopheles stephensi makes this mosquito a useful model for studies on this novel group of symbionts. Here we present experimental results that provide a first evidence for a beneficial role of Asaia in An. stephensi. RESULTS: Larvae of An. stephensi at different stages were treated with rifampicin, an antibiotic effective on wild-type Asaia spp., and the effects on the larval development were evaluated. Larvae treated with the antibiotic showed a delay in the development and an asynchrony in the appearance of later instars. In larvae treated with rifampicin, but supplemented with a rifampicin-resistant mutant strain of Asaia, larval development was comparable to that of control larvae not exposed to the antibiotic. Analysis of the bacterial diversity of the three mosquito populations confirmed that the level of Asaia was strongly decreased in the antibiotic-treated larvae, since the symbiont was not detectable by PCR-DGGE (denaturing gradient gel electrophoresis), while Asaia was consistently found in insects supplemented with rifampicin plus the antibiotic-resistant mutant in the diet, and in those not exposed to the antibiotic. CONCLUSIONS: The results here reported indicate that Asaia symbionts play a beneficial role in the normal development of An. stephensi larvae.

Bacterial Symbionts in Ceratitis capitata.

Ceratitis capitata (Diptera: Tephritidae) is responsible for extensive damage in agriculture with important economic losses. Several strategies have been proposed to control this insect pest including insecticides and the Sterile Insect Technique. Traditional control methods should be implemented by innovative tools, among which those based on insect symbionts seem very promising. Our study aimed to investigate, through the 16S Miseq analysis, the microbial communities associated with selected organs in three different medfly populations to identify possible candidates to develop symbiont-based control approaches. Our results confirm that Klebsiella and Providencia are the dominant bacteria in guts, while a more diversified microbial community has been detected in reproductive organs. Concertedly, we revealed for the first time the presence of Chroococcidiopsis and Propionibacterium as stable components of the medfly's microbiota. Additionally, in the reproductive organs, we detected Asaia, a bacterium already proposed as a tool in the Symbiotic Control of Vector-Borne Diseases. A strain of Asaia, genetically modified to produce a green fluorescent protein, was used to ascertain the ability of Asaia to colonize specific organs of C. capitata. Our study lays the foundation for the development of control methods for C. capitata based on the use of symbiont bacteria.

Increased biting rate and decreased Wolbachia density in irradiated Aedes mosquitoes.

BACKGROUND: Releasing considerable numbers of radiation-sterilized males is a promising strategy to suppress mosquito vectors. However, releases may also include small percentages of biting females, which translate to non-negligible numbers when releases are large. Currently, the effects of irradiation on host-seeking and host-biting behaviors have not been exhaustively investigated. Information is also lacking regarding the effects of sterilizing treatment on the endosymbiotic bacterium Wolbachia, which is known to affect the vector competence of infected mosquitos. METHODS: To ascertain the effects of irradiation on females, the pupae of two Aedes albopictus strains, differing in their natural or artificial Wolbachia infection type, and Aedes aegypti-which is not infected by Wolbachia-were treated with various doses of X-rays and monitored for key fitness parameters and biting behavior over a period of 2 weeks. The effect of radiation on Wolbachia was investigated by quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) analysis. RESULTS: Partial Aedes albopictus female sterility was achieved at 28 Gy, but the number of weekly bites more than doubled compared to that of the controls. Radiation doses of 35 and 45 Gy completely inhibited progeny production but did not significantly affect the survival or flight ability of Ae. albopictus females and caused a tripling of the number of bites per female per week (compared to untreated controls). These results were also confirmed in Ae. aegypti after treatment at 50 Gy. Wolbachia density decreased significantly in 45-Gy-irradiated females, with the greatest decreases in the early irradiation group (26 ± 2-h-old pupae). Wolbachia density also decreased as adults aged. This trend was confirmed in ovaries but not in extra-ovarian tissues. FISH analysis showed a strongly reduced Wolbachia-specific fluorescence in the ovaries of 13 ± 1-day-old females. CONCLUSIONS: These results suggest that, under sterile insect technique (SIT) programs, the vector capacity of a target population could increase with the frequency of the irradiated females co-released with the sterile males due to an increased biting rate. In the context of successful suppression, the related safety issues are expected to be generally negligible, but they should be conservatively evaluated when large-scale programs relying on imperfect sexing and high overflooding release ratios are run for long periods in areas endemic for arboviral diseases. Also, the effects of irradiation on the vector competence deserve further investigation.

Wolbachia in Aedes koreicus: Rare Detections and Possible Implications.

The emerging distribution of new alien mosquito species was recently described in Europe. In addition to the invasion of Aedes albopictus, several studies have focused on monitoring and controlling other invasive Aedes species, as Aedes koreicus and Aedes japonicus. Considering the increasing development of insecticide resistance in Aedes mosquitoes, new control strategies, including the use of bacterial host symbionts, are proposed. However, little is known about the bacterial communities associated with these species, thus the identification of possible candidates for Symbiotic Control is currently limited. The characterization of the natural microbiota of field-collected Ae. koreicus mosquitoes from North-East Italy through PCR screening, identified native infections of Wolbachia in this species that is also largely colonized by Asaia bacteria. Since Asaia and Wolbachia are proposed as novel tools for Symbiotic Control, our study supports their use for innovative control strategies against new invasive species. Although the presence of Asaia was previously characterized in Ae. koreicus, our study characterized this Wolbachia strain, also inferring its phylogenetic position. The co-presence of Wolbachia and Asaia may provide additional information about microbial competition in mosquito, and to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction in Ae. koreicus.

The yeast Wickerhamomyces anomalus (Pichia anomala) inhabits the midgut and reproductive system of the Asian malaria vector Anopheles stephensi.

While symbiosis between bacteria and insects has been thoroughly investigated in the last two decades, investments on the study of yeasts associated with insects have been limited. Insect-associated yeasts are placed on different branches of the phylogenetic tree of fungi, indicating that these associations evolved independently on several occasions. Isolation of yeasts is frequently reported from insect habitats, and in some cases yeasts have been detected in the insect gut and in other organs/tissues. Here we show that the yeast Wickerhamomyces anomalus, previously known as Pichia anomala, is stably associated with the mosquito Anopheles stephensi, a main vector of malaria in Asia. Wickerhamomyces anomalus colonized pre-adult stages (larvae L(1)-L(4) and pupae) and adults of different sex and age and could be isolated in pure culture. By a combination of transmission electron microscopy and fluorescent in situ hybridization techniques, W. anomalus was shown to localize in the midgut and in both the male and female reproductive systems, suggesting multiple transmission patterns.

Different mosquito species host Wickerhamomyces anomalus (Pichia anomala): perspectives on vector-borne diseases symbiotic control.

The genetic manipulation of the microbial community associated with hematophagus insects is particularly relevant for public health applications. Within mosquito populations, this relationship has been overlooked until recently. New advances in molecular biotechnology propose the genetic manipulation of mosquito symbionts to prevent the transmission of pathogens to humans by interfering with the obligatory life cycle stages within the insect through the use of effector molecules. This approach, defined as 'paratransgenesis', has opened the way for the investigation and characterization of microbes residing in the mosquito body, particularly those localised within the gut. Some interesting bacteria have been identified as candidates for genetic modification, however, endosymbiotic yeasts remain largely unexplored with little information on the symbiotic relationships to date. Here we review the recent report of symbiotic relationship between Wickerhamomyces anomalus (Pichia anomala) and several mosquito vector species as promising methods to implement control of mosquito-borne diseases.

Baru (Dipteryx alata Vogel), a woody species characteristic of Cerrado and its phytoremediation potential.

Baru (Dipteryx alata Vogel) is a native tree plant, widely distributed in Brazil, and has a growth and development in acidic soils like Cerrado, indicating a probable tolerance to adverse soil conditions, such as the high concentration of metals and the acidic pH. Due to the lack of information about the tolerance of this species to metals and the possibility of being used in the recovery of degraded areas and/or in phytoremediation, this work was developed with the objective of evaluating the in vitro germination and growth capacity of baru in medium supplemented with different concentrations of aluminum, iron, and manganese, as well as through chemical analysis, to determine the concentration of metals accumulated in cultivated plants in these conditions. The treatments consisted in different concentrations of metals: aluminum, Al3+ (0, 3.5, 7.0, 10.5, 21.0, or 42.0 mg L-1); iron, Fe3+ (0, 2.5, 4.9, 7.4, 14.7, or 29.4 mg L-1); and manganese, Mn2+ (0, 0.4, 0.8, 1.2, 2.4, or 4.8 mg L-1) added to the medium WPM. The tested values were based on using the lower concentration as the limit value, calculated based on risk to human health in accordance with CONAMA resolution 420/2009 for groundwater. At 60 days of cultivation, the percentage of germination, the average number of leaves, the length of the main root and the aerial part, the fresh and dry mass of the aerial part and the root system and the cations concentration Al3+, Fe3+ and Mn2+ in the plant biomass, were evaluated. The results showed that under the conditions in which the experiment was conducted, germination and in vitro growth of baru were not affected by the presence in high concentrations of any of the evaluated metals, with no differences in the percentage of germination and plant growth, as well as typical toxicity characteristics were not observed, such as changes in root morphology, chlorosis, or tissue oxidation. The absence of toxicity symptoms in baru plants, in the presence of Al3+, Fe3+, and Mn2+, indicate that the species is tolerant to these metals. The accumulation of Al3+ and Fe3+ in the plant biomass at the beginning of growth, simultaneously with the increase in the concentrations of these elements in the culture medium, indicates that this species can be used for phytoremediation, because it is a probable accumulator of these elements throughout its development, given the presence in significant concentrations of these elements also in the seeds.

Phylogenomics Reveals that Asaia Symbionts from Insects Underwent Convergent Genome Reduction, Preserving an Insecticide-Degrading Gene.

The mosquito microbiota is composed of several lineages of microorganisms whose ecological roles and evolutionary histories have yet to be investigated in depth. Among these microorganisms, Asaia bacteria play a prominent role, given their abundance in the gut, reproductive organs, and salivary glands of different mosquito species, while their presence has also been reported in several other insects. Notably, Asaia has great potential as a tool for the control of mosquito-borne diseases. Here, we present a wide phylogenomic analysis of Asaia strains isolated from different species of mosquito vectors and from different populations of the Mediterranean fruit fly (medfly), Ceratitis capitata, an insect pest of worldwide economic importance. We show that phylogenetically distant lineages of Asaia experienced independent genome reductions, despite following a common pattern, characterized by the early loss of genes involved in genome stability. This result highlights the role of specific metabolic pathways in the symbiotic relationship between Asaia and the insect host. Finally, we discovered that all but one of the Asaia strains included in the study possess the pyrethroid hydrolase gene. Phylogenetic analysis revealed that this gene is ancestral in Asaia, strongly suggesting that it played a role in the establishment of the symbiotic association between these bacteria and the mosquito hosts. We propose that this gene from the symbiont contributed to initial pyrethroid resistance in insects harboring Asaia, also considering the widespread production of pyrethrins by several plants.IMPORTANCE We have studied genome reduction within several strains of the insect symbiont Asaia isolated from different species/strains of mosquito and medfly. Phylogenetically distant strains of Asaia, despite following a common pattern involving the loss of genes related to genome stability, have undergone independent genome reductions, highlighting the peculiar role of specific metabolic pathways in the symbiotic relationship between Asaia and its host. We also show that the pyrethroid hydrolase gene is present in all the Asaia strains isolated except for the South American malaria vector Anopheles darlingi, for which resistance to pyrethroids has never been reported, suggesting a possible involvement of Asaia in determining resistance to insecticides.

Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species.

The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

Mosquito-bacteria symbiosis: the case of Anopheles gambiae and Asaia.

The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.

Plant functional traits are correlated with species persistence in the herb layer of old-growth beech forests.

This paper explores which traits are correlated with fine-scale (0.25 m2) species persistence patterns in the herb layer of old-growth forests. Four old-growth beech forests representing different climatic contexts (presence or absence of summer drought period) were selected along a north-south gradient in Italy. Eight surveys were conducted in each of the sites during the period spanning 1999-2011. We found that fine-scale species persistence was correlated with different sets of plant functional traits, depending on local ecological context. Seed mass was found to be as important for the fine-scale species persistence in the northern sites, while clonal and bud-bank traits were markedly correlated with the southern sites characterised by summer drought. Leaf traits appeared to correlate with species persistence in the drier and wetter sites. However, we found that different attributes, i.e. helomorphic vs scleromorphic leaves, were correlated to species persistence in the northernmost and southernmost sites, respectively. These differences appear to be dependent on local trait adaptation rather than plant phylogenetic history. Our findings suggest that the persistent species in the old-growth forests might adopt an acquisitive resource-use strategy (i.e. helomorphic leaves with high SLA) with higher seed mass in sites without summer drought, while under water-stressed conditions persistent species have a conservative resource-use strategy (i.e. scleromorphic leaves with low SLA) with an increased importance of clonal and resprouting ability.

Chimeric symbionts expressing a Wolbachia protein stimulate mosquito immunity and inhibit filarial parasite development.

Wolbachia can reduce the capability of mosquitoes to transmit infectious diseases to humans and is currently exploited in campaigns for the control of arboviruses, like dengue and Zika. Under the assumption that Wolbachia-mediated activation of insect immunity plays a role in the reduction of mosquito vectorial capacity, we focused our attention on the Wolbachia surface protein (WSP), a potential inductor of innate immunity. We hypothesized that the heterologous expression of this protein in gut- and tissue-associated symbionts may reduce parasite transmission. We thus engineered the mosquito bacterial symbiont Asaia to express WSP (AsaiaWSP). AsaiaWSP induced activation of the host immune response in Aedes aegypti and Anopheles stephensi mosquitoes, and inhibited the development of the heartworm parasite Dirofilaria immitis in Ae. aegypti. These results consolidate previous evidence on the immune-stimulating property of WSP and make AsaiaWSP worth of further investigations as a potential tool for the control of mosquito-borne diseases.

Inhibition of Asaia in Adult Mosquitoes Causes Male-Specific Mortality and Diverse Transcriptome Changes.

Mosquitoes can transmit many infectious diseases, such as malaria, dengue, Zika, yellow fever, and lymphatic filariasis. Current mosquito control strategies are failing to reduce the severity of outbreaks that still cause high human morbidity and mortality worldwide. Great expectations have been placed on genetic control methods. Among other methods, genetic modification of the bacteria colonizing different mosquito species and expressing anti-pathogen molecules may represent an innovative tool to combat mosquito-borne diseases. Nevertheless, this emerging approach, known as paratransgenesis, requires a detailed understanding of the mosquito microbiota and an accurate characterization of selected bacteria candidates. The acetic acid bacteria Asaia is a promising candidate for paratransgenic approaches. We have previously reported that Asaia symbionts play a beneficial role in the normal development of Anopheles mosquito larvae, but no study has yet investigated the role(s) of Asaia in adult mosquito biology. Here we report evidence on how treatment with a highly specific anti-Asaia monoclonal antibody impacts the survival and physiology of adult Anopheles stephensi mosquitoes. Our findings offer useful insight on the role of Asaia in several physiological systems of adult mosquitoes, where the influence differs between males and females.