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It is essential to hunt for new technologies that promote sustainable practices for agroecosystems; thus, the bioprospecting of beneficial microorganisms complementing with mutation induction techniques to improve their genomic, metabolic, and functional traits is a promising strategy for the development of sustainable microbial inoculants. Bacillus cabrialesii subsp. cabrialesii strain TE3T, a previously recognized plant growth-promoting and biological control agent, was subjected to UV mutation induction to improve these agro-biotechnological traits. Dilutions were made which were spread on Petri dishes and placed under a 20 W UV lamp at 10-min intervals for 60 min. After the UV-induced mutation of this strain, 27 bacterial colonies showed morphological differences compared to the wild-type strain; however, only a strain named TE3T-UV25 showed an improvement in 53.6% of the biocontrol against Bipolaris sorokiniana vs. the wild-type strain, by competition of nutrient and space (only detected in the mutant strain), as well as diffusible metabolites. Furthermore, the ability to promote wheat growth was evaluated by carrying out experiments under specific greenhouse conditions, considering un-inoculated, strain TE3T, and strain TE3T-UV25 treatments. Thus, after 120 days, biometric traits in seedlings were quantified and statistical analyses were performed, which showed that strain TE3T-UV25 maintained its ability to promote wheat growth in comparison with the wild-type strain. On the other hand, using bioinformatics tools such as ANI, GGDC, and TYGS, the Overall Genome Relatedness Index (OGRI) and phylogenomic relationship of mutant strain TE3T-UV25 were performed, confirming that it changed its taxonomic affiliation from B. cabrialesii subsp. cabrialesii to Bacillus subtilis. In addition, genome analysis showed that the mutant, wild-type, and B. subtilis strains shared 3654 orthologous genes; however, a higher number of shared genes (3954) was found between the TE3T-UV25 mutant strain and B. subtilis 168, while the mutant strain shared 3703 genes with the wild-type strain. Genome mining was carried out using the AntiSMASH v7.0 web server and showed that mutant and wild-type strains shared six biosynthetic gene clusters associated with biocontrol but additionally, pulcherriminic acid cluster only was detected in the genome of the mutant strain and Rhizocticin A was exclusively detected in the genome of the wild-type strain. Finally, using the PlaBase tool, differences in the number of genes (17) associated with beneficial functions in agroecosystems were detected in the genome of the mutant vs. wild-type strain, such as biofertilization, bioremediation, colonizing plant system, competitive exclusion, phytohormone, plant immune response stimulation, putative functions, stress control, and biocontrol. Thus, the UV-induced mutation was a successful strategy to improve the bioactivity of B. cabrialesii subsp. cabrialesii TE3T related to the agro-biotecnology applications. The obtained mutant strain, B. subtilis TE3T-UV25, is a promising strain to be further studied as an active ingredient for the bioformulation of bacterial inoculants to migrate sustainable agriculture.

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Abstract Somatic characters are shared by many Chelodesmidae groups, and generic placement and species identifications traditionally have been based on gonopodal morphology. Female genitalic characters have been largely neglected and are rarely photographed or illustrated. This is rather unfortunate as the morphology of female genitalia presents important characters and may be decisive for developing a more robust family classification. We describe the heretofore unknown female of Sandalodesmus araujoi (Schubart, 1946), previously known only from the male holotype collected in São Paulo, Brazil in December 1943; discuss the utility of female genitalic characters for species delineation in Sandalodesmus; and report the first case of a mass occurrence in the Chelodesmidae. While an attempt at a formal diagnosis of Sandalodesmus females based on genitalic characters is premature, the vulvar morphology of the three taxa examined in this study suggests that female genitalia are species-specific. Some characters (i.e., asymmetric valves, presence of digitiform projections and reduction of setae on the internal basal portion of the valves) are constant between the species, suggesting utility for generic-level delineation. Mass occurrences of millipedes are typically unpredictable and likely related to variations in environmental conditions and/or anthropogenic modifications of natural habitats. Although the mass occurrence of S. araujoi reported herein was only observed once, the event coincides with the mating period of millipedes during the rainy season in Brazil. On the other hand, the region where the species was found has been the target of intense urban development, including replacement of natural habitats with residential areas, which may have influenced its population dynamics.

Resumo Os caracteres somáticos são compartilhados por muitos grupos de Chelodesmidae, e o posicionamento genérico e as identificações de espécies tradicionalmente têm sido baseadas na morfologia do gonópodo. Carateres genitais das fêmeas foram amplamente negligenciados e raramente são fotografados ou ilustrados. Isso é lamentável, pois a morfologia da genitália feminina apresenta características importantes e pode ser decisiva para o desenvolvimento de uma classificação mais robusta. Neste trabalho, descrevemos a até então desconhecida fêmea de Sandalodesmus araujoi (Schubart, 1946), anteriormente conhecida apenas pelo holótipo macho coletado em São Paulo, Brasil, em dezembro de 1943; discutimos a utilidade de caracteres genitais femininos para delineamento de espécies em Sandalodesmus; e relatamos o primeiro caso de ocorrência em massa para Chelodesmidae. Embora uma tentativa de diagnose formal para fêmeas de Sandalodesmus com base em caracteres genitais seja prematura, a morfologia vulvar dos três táxons examinados neste estudo, sugere que a genitália feminina é espécie-específica. Alguns caracteres (e.g. válvulas assimétricas, presença de projeções digitiformes e redução de cerdas na margem interna das válvas) são constantes entre as espécies do gênero, sugerindo utilidade para delineamento em nível genérico. Ocorrências em massa de milípedes são tipicamente imprevisíveis e provavelmente relacionadas a variações nas condições ambientais e/ou modificações antropogênicas de habitats naturais. Embora a ocorrência em massa de S. araujoi aqui relatada tenha sido observada apenas uma vez, o evento coincide com o período de reprodução dos milípedes durante a estação chuvosa no Brasil. Por outro lado, a região onde a espécie foi encontrada tem sido alvo de intenso desenvolvimento urbano, incluindo substituição de habitats naturais por áreas residenciais, o que pode ter influenciado sua dinâmica populacional.

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Membrane vesicles (MVs) are envelope-derived extracellular sacs that perform a broad diversity of physiological functions in bacteria. While considerably studied in pathogenic microorganisms, the roles, relevance, and biotechnological potential of MVs from environmental bacteria are less well established. Acidithiobacillaceae family bacteria are active players in the sulfur and iron biogeochemical cycles in extremely acidic environments and drivers of the leaching of mineral ores contributing to acid rock/mine drainage (ARD/AMD) and industrial bioleaching. One key aspect of such a role is the ability of these bacteria to tightly interact with the mineral surfaces and extract electrons and nutrients to support their chemolithotrophic metabolism. Despite recent advances in the characterization of acidithiobacilli biofilms and extracellular matrix (ECM) components, our understanding of its architectural and mechanistic aspects remains scant. Using different microscopy techniques and nano-tracking analysis we show that vesiculation is a common phenomenon in distant members of the Acidithiobacillaceae family, and further explore the role of MVs in multicellular colonization behaviors using 'Fervidacidithiobacillus caldus' as a bacterial model. Production of MVs in 'F. caldus' occurred in both planktonic cultures and biofilms formed on sulfur surfaces, where MVs appeared individually or in chains resembling tube-shaped membranous structures (TSMSs) important for microbial communication. Liquid chromatography-mass spectrometry data and bioinformatic analysis of the MV-associated proteome revealed that 'F. caldus' MVs were enriched in proteins involved in cell-cell and cell-surface processes and largely typified the MVs as outer MVs (OMVs). Finally, microbiological assays showed that amendment of 'F. caldus' MVs to cells and/or biofilms affects collective colonizing behaviors relevant to the ecophysiology and applications of these acidophiles, providing grounds for their exploitation in biomining.

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Somatic characters are shared by many Chelodesmidae groups, and generic placement and species identifications traditionally have been based on gonopodal morphology. Female genitalic characters have been largely neglected and are rarely photographed or illustrated. This is rather unfortunate as the morphology of female genitalia presents important characters and may be decisive for developing a more robust family classification. We describe the heretofore unknown female of Sandalodesmus araujoi (Schubart, 1946), previously known only from the male holotype collected in São Paulo, Brazil in December 1943; discuss the utility of female genitalic characters for species delineation in Sandalodesmus; and report the first case of a mass occurrence in the Chelodesmidae. While an attempt at a formal diagnosis of Sandalodesmus females based on genitalic characters is premature, the vulvar morphology of the three taxa examined in this study suggests that female genitalia are species-specific. Some characters (i.e., asymmetric valves, presence of digitiform projections and reduction of setae on the internal basal portion of the valves) are constant between the species, suggesting utility for generic-level delineation. Mass occurrences of millipedes are typically unpredictable and likely related to variations in environmental conditions and/or anthropogenic modifications of natural habitats. Although the mass occurrence of S. araujoi reported herein was only observed once, the event coincides with the mating period of millipedes during the rainy season in Brazil. On the other hand, the region where the species was found has been the target of intense urban development, including replacement of natural habitats with residential areas, which may have influenced its population dynamics.

Os caracteres somáticos são compartilhados por muitos grupos de Chelodesmidae, e o posicionamento genérico e as identificações de espécies tradicionalmente têm sido baseadas na morfologia do gonópodo. Carateres genitais das fêmeas foram amplamente negligenciados e raramente são fotografados ou ilustrados. Isso é lamentável, pois a morfologia da genitália feminina apresenta características importantes e pode ser decisiva para o desenvolvimento de uma classificação mais robusta. Neste trabalho, descrevemos a até então desconhecida fêmea de Sandalodesmus araujoi (Schubart, 1946)

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Quorum sensing (QS) is a bacterial cell-cell communication system with genetically regulated mechanisms dependent on cell density. Canonical QS systems in gram-negative bacteria possess an autoinducer synthase (LuxI family) and a transcriptional regulator (LuxR family) that respond to an autoinducer molecule. In Gram-positive bacteria, the LuxR transcriptional regulators "solo" (not associated with a LuxI homolog) may play key roles in intracellular communication. Arthrobacter sp. UMCV2 is an actinobacterium that promotes plant growth by emitting the volatile organic compound N, N-dimethylhexadecylamine (DMHDA). This compound induces iron deficiency, defense responses in plants, and swarming motility in Arthrobacter sp. UMCV2. In this study, the draft genome of this bacterium was assembled and compared with the genomes of type strains of the Arthrobacter genus, finding that it does not belong to any previously described species. Genome explorations also revealed the presence of 16 luxR-related genes, but no luxI homologs were discovered. Eleven of these sequences possess the LuxR characteristic DNA-binding domain with a helix-turn-helix motif and were designated as auto-inducer-related regulators (AirR). Four sequences possessed LuxR analogous domains and were designated as auto-inducer analogous regulators (AiaR). When swarming motility was induced with DMHDA, eight airR genes and two aiaR genes were upregulated. These results indicate that the expression of multiple luxR-related genes is induced in actinobacteria, such as Arthrobacter sp. UMCV2, by the action of the bacterial biocompound DMHDA when QS behavior is produced.

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Pseudomonas aeruginosa is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. Rhamnolipids play a pivotal role in biofilm formation and motility phenotypes in P. aeruginosa, possibly acting as wetting agents and mediating chemotactic stimuli. However, no biochemical mechanism or gene regulatory network has been investigated in regard to rhamnolipids' modulation of those group behaviors. Using DNA microarrays, we investigated the transcriptomic profiles in the stationary phase of growth of wild-type P. aeruginosa PAO1 and a rhlA-mutant strain, unable to produce rhamnolipids. A total of 134 genes were differentially expressed, comprising different functional categories, indicating a significant physiological difference between the rhamnolipid-producing and -non-producing strains. Interestingly, several flagellar genes are repressed in the mutant strain, which directly relates to the inability of the rhlA-minus strain to develop a swarming-motility phenotype. Supplementation with exogenous rhamnolipids has partially restored flagellar gene expression in the mutant strain. Our results show significant evidence that rhamnolipids, the major biosynthetic products of rhlABC pathway, seem to modulate gene expression in P. aeruginosa.

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The Burkholderia genus has high ecological and nutritional versatility, having species capable of causing diseases in animals, humans, and plants. During chronic infections in humans, biofilm formation is a characteristic often associated with strains from different species of this genus. However, there is still no information on the formation of biofilms by plant pathogenic strains of B. cenocepacia (Bce) lineages IIIA and IIIB and B. gladioli pv. alliicola (Bga), which are associated with onion bacterial scale rot in the semi-arid region of northeast Brazil. In this study, we performed an in vitro characterization of biofilm formation ability in different culture media by the phytopathogenic strains of Bce and Bga and investigated its relationship with swarming motility. Our results indicated the existence of an intraspecific variation in biofilm formation capacity in vitro by these bacteria and the existence of a negative correlation between swarming motility and biofilm formation for strains of Bce lineage IIIB. In addition, histopathological analyses performed using optical microscopy and scanning electron microscopy revealed the formation of biofilm in vivo by Bce strains in onion tissues.

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Bacterial chemotaxis is the directed movement of motile bacteria in gradients of chemoeffectors. This behavior is mediated by dedicated signal transduction pathways that couple environment sensing with changes in the direction of rotation of flagellar motors to ultimately affect the motility pattern. Azospirillum brasilense uses two distinct chemotaxis pathways, named Che1 and Che4, and four different response regulators (CheY1, CheY4, CheY6, and CheY7) to control the swimming pattern during chemotaxis. Each of the CheY homologs was shown to differentially affect the rotational bias of the polar flagellum and chemotaxis. The role, if any, of these CheY homologs in swarming, which depends on a distinct lateral flagella system or in attachment is not known. Here, we characterize CheY homologs' roles in swimming, swarming, and attachment to abiotic and biotic (wheat roots) surfaces and biofilm formation. We show that while strains lacking CheY1 and CheY6 are still able to navigate air gradients, strains lacking CheY4 and CheY7 are chemotaxis null. Expansion of swarming colonies in the presence of gradients requires chemotaxis. The induction of swarming depends on CheY4 and CheY7, but the cells' organization as dense clusters in productive swarms appear to depend on functional CheYs but not chemotaxis per se. Similarly, functional CheY homologs but not chemotaxis, contribute to attachment to both abiotic and root surfaces as well as to biofilm formation, although these effects are likely dependent on additional cell surface properties such as adhesiveness. Collectively, our data highlight distinct roles for multiple CheY homologs and for chemotaxis on swarming and attachment to surfaces.

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Vibrio parahaemolyticus cells transit from free-swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic diguanylate monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase (PDE) is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called trigger phosphodiesterases, and hence we named the product of VP1881 TpdA, for trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while, its overexpression from an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible promoter showed a positive effect on both swimming and swarming motility and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes, as well as that of the biofilm-related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of these social behaviors. IMPORTANCE c-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, and hence not many examples of these type of signaling proteins have been reported in the literature. Here, we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.

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Flagellar-mediated motility is a crucial virulence factor in many bacterial species. A dual flagellar system has been described in aeromonads; however, there is no flagella-related study in the emergent human pathogen Aeromonas dhakensis. Using 46 clinical A. dhakensis, phenotypic motility, genotypic characteristics (flagellar genes and sequence types), biochemical properties and their relationship were investigated in this study. All 46 strains showed swimming motility at 30 °C in 0.3% Bacto agar and carried the most prevalent 6 polar flagellar genes cheA, flgE, flgG, flgH, flgL, and flgN. On the contrary, only 18 strains (39%) demonstrated swarming motility on 0.5% Eiken agar at 30 °C and they harbored 11 lateral flagellar genes lafB, lafK, lafS, lafT, lafU, flgCL, flgGL, flgNL, fliEL, fliFL, and fliGL. No association was found between biochemical properties and motility phenotypes. Interestingly, a significant association between swarming and strains isolated from pus was observed (p = 0.0171). Three strains 187, 277, and 289 isolated from pus belonged to novel sequence types (ST522 and ST524) exhibited fast swimming and swarming profiles, and they harbored > 90% of the flagellar genes tested. Our findings provide a fundamental understanding of flagellar-mediated motility in A. dhakensis.

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The development of smart nanoparticles (NPs) became a trend to enhance the delivery of drugs. In the present work, Tobramycin (TB), an aminoglycoside antibiotic that displays several undesirable side effects, has been encapsulated into cationic Eudragit®E100 (E100) NPs for the treatment of infections caused by Pseudomonas aeruginosa. Combination with neutral Eudragit®NE30D (NE30D) NPs containing resveratrol (RSV), a strong natural antioxidant, increased the antimicrobial activity of TB (75% higher than free TB). NPs were stabilized with 1.0% (w/v) poloxamer 188 (P188) or poloxamer 407 (P407) as surfactants. E100 NPs showed 83.3 ± 8.5%, and 70.1 ± 2.7 encapsulation efficiency (EE) of TB with P188 and P407 coatings, respectively. The presence of NPs was confirmed by DLS and TEM studies. TB was controlled released from NPs for 6 h. Hemotoxicity tests of NPs in the range of MIC values on human blood gave negative results. Analysis of Surface Plasmon Resonance verified that NE30D/P407/RSV does not interact with plasma proteins BSA, IgG or fibrinogen, besides E100/P188/TB interact with BSA, findings that are compatible with a negligible in vivo clearance of the nanovehicles. The obtained results show a potential binary fluid composed of two NPs to highly improve the effectiveness of conventional antibiotics.

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Aim: To determine phenotypically the anti quorum-sensing (QS) activity of 30 volatile organic products (VOPs) through the inhibition of swarming motility and pyoverdine production in Pseudomonas aeruginosa. Materials & methods: Twenty-four essential oils and six small volatile organic compounds randomly selected were screened for their anti-QS activity by violacein inhibition on Chromobacterium violaceum. The VOPs with positive results were subsequently evaluated for swarming motility and pyoverdine production on P. aeruginosa determining the colony diameter and fluorescence under UV light, respectively. Results: Fifty percent of VOPs tested showed strong violacein inhibition, 40% presented anti-swarming activity and 33% inhibited pyoverdine production. Conclusion: Our data demonstrate that VOPs have a great potential to inhibit virulence factors mediated by QS in P. aeruginosa.

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Silver nanoparticles (AgNPs), as well as silver ions, are described as toxic to a broad spectrum of microorganisms, especially bacteria. In contrast to this, a current trend is to develop and carry out the in vitro cultivation of microorganisms, facilitating the study of interactions between populations of cells and species. Thus, the goal of this study was to evaluate the behavior, growth, and swarming formation of bacteria under conditions of co-culture in solid medium modified with AgNPs. The aqueous extract from the leaves of Handroanthus serratifolius was used to chemically reduce silver nitrate (AgNO3) solution, forming AgNPs. This synthesis route was performed in an aqueous medium at 50 °C for 3 h. The hydrodynamic diameter (HD) and polydispersity index (PdI) were obtained by dynamic light scattering (DLS), and Zeta potential (ZP) of the AgNPs were measured by electrophoretic mobility. Atomic force microscopy (AFM) was used to evaluate the shape of the AgNPs. Luria Bertani (LB) medium was used for the liquid culture steps and for the solid medium, bacterial agar was added. Solutions containing AgNPs or AgNO3 were added at final concentrations of 256, 128, or 64 µM. Subsequently, microorganism Escherichia coli ATCC® 8739 and Staphylococcus aureus ATCC® 25923 were plated with AgNPs, AgNO3, and control media. Analyses of the AgNPs showed an average HD of 76.02 ± 3.08 nm, PdI of 0.461 ± 0.012, and ZP of -21.5 ± 2.2 mV; in addition, AgNPs were nearly spherical. The solid culture medium elaborated and modified with AgNPs at the concentrations of 256 and 128 µM inhibited the growth of the tested microorganisms and decreased the swarming formation. However, those media modified at a concentration of 64 µM did not induce any alteration in the growth and proliferation of the microorganisms. Furthermore, it was observed that plates containing modified culture media with 128 µM, increased proximity between both co-cultured bacteria occurred. Thus, the application of AgNPs in solid culture media becomes a promising and potentially reproducible strategy for evaluating the behavior, swarming formation, and toxicity of AgNPs, making the understanding of possible bactericidal or bacteriostatic effects, and also colonizing strategies.

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Magnetic nanoparticles (MNPs) are a specific type of nanomaterial whose applications are widespread into several fields including biomedicine as a smart drug targeter and environmental engineering due to their interactions with contaminants. Lately, the use of MNPs has also been demonstrated in structuring three-dimensional (3D) cultures of mammalian cells. However, MNPs application to other cell types is still limited. In this sense, some planktonic microorganisms when adhered to surfaces perform the swarming phenomenon to guarantee the expansion of the colony and to guarantee more niches. Therefore, the aim of this study was to produce MNPs coated with four carbohydrates (galactose - gal, glucose - glu, sucrose - suc, and maltose - mal) aiming microorganism culture applications and also for possible 3D arrays. The results showed that carbohydrate-coated MNPs showed hydrodynamic diameters ranging from 100 to 200 nm and that their coatings influenced the chemical behavior in different ways. Indeed, when subjected to biological tests to determine their potential level of cytotoxicity, it was found that in concentrations of 1 mM, 800, 600, and 400 µM (iron equivalent), there was not any alteration on growth of model microorganisms when visually evaluated. Besides, magnetization of bacteria was promoted in different ways as well as the modulation of swarming formation in Escherichia coli when exposed to MNP-Glu. In sum, MNPs coated with carbohydrates and even uncoated were atoxic to bacteria and one of them was able to modulate E. coli swarming formation showing the potential for applications in 3D cultures of bacteria.

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The facultative plant endophyte Azospirillum brasilense Sp245 synthesizes two high-molecular-weight lipopolysaccharides, LPSI and LPSII, which comprise identical d-rhamnan O-polysaccharides and, presumably different core oligosaccharides. Previously, using random insertion mutagenesis, we constructed the LpsII- mutant KM139 of strain Sp245 that possessed an Omegon-Km insertion in plasmid AZOBR_p6. Here, we found that in KM139, Omegon-Km disrupted the coding sequence AZOBR_p60126 for a putative glycosyltransferase related to mannosyltransferases and rhamnosyltransferases. To verify its function, we cloned the AZOBR_p60126 gene of strain Sp245 in the expression vector plasmid pRK415 and transferred the construct pRK415-p60126 into KM139. In the complemented mutant KM139 (pRK415-p60126), the wild-type LPSI+ LPSII+ profile was recovered. We also compared the swimming and swarming motilities of strains Sp245, Sp245 (pRK415), KM139, KM139 (pRK415), and KM139 (pRK415-p60126). All these strains had the same flagellar-dependent swimming speeds, but on soft media, the LpsI+ LpsII- strains KM139 and KM139 (pRK415) swarmed significantly faster than the other LpsI+ LpsII+ strains. Such interstrain differences in swarming motility were more pronounced on 0.4% than on 0.5% soft agar plates. These data show that the AZOBR_p60126-encoded putative glycosyltransferase significantly affects the lipopolysaccharide profile and, as a consequence, the social motility of azospirilla.

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Pectobacterium is a diverse genus of phytopathogenic species from soil and water that cause infection either to restricted or multiple plant hosts. Phylogenetic analysis and metabolic fingerprinting of large numbers of genomes have expanded classification of Pectobacterium members. Pectobacterium brasiliense sp. nov has been elevated to the species level having detached from P. carotovorum. Here we present two P. brasiliense strains BF20 and BF45 isolated in Mexico from Opuntia and tobacco, respectively, which cluster into two different groups in whole genome comparisons with other Pectobacterium. We found that BF20 and BF45 strains are phenotypically different as BF45 showed more severe and rapid symptoms in comparison to BF20 in the host models celery and broccoli. Both strains produced similar levels of the main autoinducers, but BF45 shows an additional low abundant autoinducer compared to strain BF20. The two strains had different levels of c-di-GMP, which regulates the transition from motile to sessile lifestyle. In contrast to BF45, BF20 had the highest levels of c-di-GMP, was more motile (swarming), non-flocculant and less proficient in biofilm formation and exopolysaccharide production. Genomic comparisons revealed that differences in c-di-GMP accumulation and perhaps the associated phenotypes might be due to unique c-di-GMP metabolic genes in these two strains. Our results improve our understanding of the associations between phenotype and genotype and how this has shaped the physiology of Pectobacterium strains.

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Abstract The genus Cyclocephala is common in Brazil (Coleoptera, Scarabaeidae, Dynastinae). The adults of some species are important pollinators, and the larvae develop in the soil, feed on organic matter, and contribute to nutrient cycle, but immatures of some species feed on plant roots, and some were registered causing damage in crops. The mating process of some phytophagous scarab beetles has a chemical recognition step, and the antenna is the main structure involved in the detection of odorants associated with insect communication. In the present study the mating behavior, life cycle, and antennal sensilla of C. putrida are described. The study was conducted at the Universidade Estadual de Mato Grosso do Sul, Cassilândia, Brazil. Adults were collected by a light trap installed from January 2016 to December 2017 and were taken to the laboratory for studies. Adults swarms are brief and were registered from January to February, and specimens were mostly collected at 20:00 to 22:00h. Chemical recognition may occur at least during one of the mating steps, during which the couple kept their antennae moving and the lamellae open, while females select males. In laboratory, the mating process lasted 7.5 minutes on average. The antennae of females have about 3399 sensilla and males about 4229 sensilla. Sensilla placodea types I, II, and III are the most abundant, and sensilla ampullacea, basiconica, and coeloconica are also present. The embryonic period lasted 16.0 days; first, second and third instars lasted 16.0, 48.3, and 165.3 days, respectively. The pupal period lasted 24.0 days. The period between egg deposition and adult emergency is about 271.5 days.

Resumo O gênero Cyclocephala é comum no Brasil (Coleoptera, Scarabaeidae, Dynastinae). Os adultos de algumas espécies são importantes polinizadores, e a larva desenvolve-se no solo, alimenta-se de matéria orgânica e contribui para a ciclagem de nutrientes, mas imaturos de algumas espécies alimentam-se de raízes de plantas, e alguns são registrados causando danos em plantas cultivadas. O processo de cópula de algumas espécies de Scarabaeidae fitófagos, apresentam reconhecimento químico, e nas antenas aparecem várias estruturas responsáveis pela detecção dos odores envolvidos na comunicação. No presente trabalho o comportamento de cópula, ciclo de vida e sensilas antenais de Cyclocephala putrida são descritos. O estudo foi conduzido na Universidade Estadual de Mato Grosso do Sul, Cassilândia, Brasil. Adultos foram coletados com armadilha luminosa instalada de Janeiro de 2016 a Dezembro de 2017, e levados para laboratório para estudos. Os adultos revoam por curto período de janeiro a fevereiro, e os espécimes foram coletados em maior quantidade das 20:00 às 22:00 h. O reconhecimento químico possivelmente ocorre durante as etapas que envolvem o comportamento de cópula, no qual o casal mantém as antenas em movimento e as lamelas abertas e as fêmeas selecionam os machos. Em laboratório a cópula dura 7,5 minutos em média. As antenas das fêmeas possuem 3399 sensilas e os machos 4229 sensilas, e as sensilas placódeas dos tipos I, II e III, foram as mais abundantes e sensilas ampuláceas, basicônicas e coelocônicas também estão presentes. O estágio de ovo durou 16,0 dias; o primeiro, segundo e terceiro instar duraram 16,0, 48,3 e 165,3 dias, respectivamente. O período pupal durou 24,0 dias. O período entre deposição dos ovos e emergência dos adultos é de 271,5 dias em média.

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BACKGROUND: Anopheles albimanus is a malaria vector in Central America, northern South America and the Caribbean. Although a public health threat, An. albimanus precopulatory mating behaviors are unknown. Acoustics play important roles in mosquito communication, where flight tones allow males to detect and attract potential mates. The importance of sound in precopulatory interactions has been demonstrated in Toxorhynchites brevipalpis, Aedes aegypti, Culex quinquefasciatus and Anopheles gambiae; convergence in a shared harmonic of the wing beat frequency (WBF) during courtship is thought to increase the chance of copulation. To our knowledge, An. albimanus precopulatory acoustic behaviors have not been described to date. Here, we characterized An. albimanus (i) male and female flight tones; (ii) male-female precopulatory acoustic interactions under tethered and free flight conditions; and (iii) male-male acoustic interactions during free flight. RESULTS: We found significant increases in the WBFs of both sexes in free flight compared to when tethered. We observed harmonic convergence between 79% of tethered couples. In free flight, we identified a female-specific behavior that predicts mate rejection during male mating attempts: females increase their WBFs significantly faster during mate rejection compared to a successful copulation. This behavior consistently occurred during mate rejection regardless of prior mating attempts (from the same or differing male). During group flight, males of An. albimanus displayed two distinct flying behaviors: random flight and a swarm-like, patterned flight, each associated with distinct acoustic characteristics. In the transition from random to patterned flight, males converged their WBFs and significantly decreased flight area, male-male proximity and the periodicity of their trajectories. CONCLUSIONS: We show that tethering of An. albimanus results in major acoustic differences compared to free flight. We identify a female-specific behavior that predicts mate rejection during male mating attempts in this species and show that male groups in free flight display distinct flying patterns with unique audio and visual characteristics. This study shows that An. albimanus display acoustic features identified in other mosquito species, further suggesting that acoustic interactions provide worthwhile targets for mosquito intervention strategies. Our results provide compelling evidence for swarming in this species and suggests that acoustic signaling is important for this behavior.

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Many plant bacterial pathogens monitor their group behaviour and their population density via production of N-acyl homoserine lactone signals which regulate the expression of several genes via the LuxI/R homologs. This regulatory network, termed quorum sensing (QS), is present in the soybean bacterial pathogen Pseudomonas savastanoi pv glycinea (Psg). The sequenced genomes of two strains of Psg, race 4 and B076, contain an N-acyl homoserine lactone (AHL) based LuxI/R QS system named AhlI/R. While studying the QS system of Psg strains race 4 and B076 isolated in USA, LMG5066 in New Zealand and IBSBF355 in Brazil, we found that B076, LMG5066 and IBSBF355 possess a point mutation in the ahlR gene that causes a frameshift resulting in a truncated AhlR protein. Psg race 4 does not possess the mutation in ahlR and the QS system is functional. The same mutation in the ahlR gene was found to be also present in 9 of 19 Psg strains isolated from diseased soybean in Illinois. Phenotypic analysis of strains showed that swarming motility is repressed whereas phosphate solubilisation was activated by QS in Psg. Analysing the secretome, we also found that four proteins were under QS regulation.

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We investigated the genes kpsMTII, iucD, sfaDE, afaBC, papA and papC, (proposed to be involved in extra-intestinal pathogenic Escherichia coli-ExPEC), phylogroup classification and the in vitro swimming and swarming motility in 50 E. coli isolated from bovine mastitis with different clinical severity scores (mild, moderate and severe). The aforementioned genes were detected in 12 (n = 12/50; 24·0%) isolates. kpsMTII and iucD were the most frequent genes identified in six (n = 6/50; 12·0%) and four (n = 4/50; 8·0%) of the isolates, respectively. In only one (n = 1/50; 2·0%) isolate, more than one gene was simultaneously identified: iucD and kpsMTll were detected whereas sfaDE and afaBC were not detected. Mild, moderate and severe clinical signs were observed in 40·0% (n = 20/50), 28·0% (n = 14/50) and 32·0% (n = 16/50) of the cases. Commensal phylogroups B1 (n = 19/50; 38·0%) and A (n = 19/50; 38·0%) were prevalent; whereas pathogenic phylogroups B2 and D were observed in only 10·0% (n = 5/50). Swarming and swimming motilities were observed in 90·0% (n = 45/50) and 68·0% (n = 34/50) of the isolates, respectively; and there was a significant association (P = 0·0036) between swarming motility and severe clinical cases (score 3). To the best of our knowledge, this is the first study where clinical severity of bovine mastitis cases and the genes proposed to classify ExPEC were assessed in relation to swarming and swimming motility. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli is classified as extra-intestinal (ExPEC) when strains contain at least two of the genes kpsMTII, iucD, sfaDE, afaBC and papA and/or papC. We investigated in vitro motility and the presence of these genes in 50 E. coli isolated from bovine mastitis with different clinical scores (mild, moderate and severe). Clinical severity was not associated with the genes studied. Swarming motility was associated with severe cases (score 3) of clinical mastitis. Results of this study contribute to a better understanding of the factors that determine the severity of clinical mastitis.

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