Mosquito Egg Raft Distribution Is Affected by Semiochemicals: Indication of Interspecific Competition.

Numerous species of animals alter their behavior in response to increasing competition. To do so, they must possess the ability to detect the presence and density of interspecific competitors. We studied the role of semiochemicals released by increasing densities of larval Culiseta longiareolata Macquart on female oviposition habitat selection in two field experiments. Similarly to C. longiareolata larvae, subordinate Culex laticinctus Edwards are periphyton grazers who dwell in rain-filled pools in the Mediterranean region. We show that C. laticinctus females oviposited significantly less in mesocosm pools that were treated with crowding signals originating from C. longiareolata larvae. In the second experiment, we placed a similar number of larvae directly inside the 50 L mesocosms. These low-density mesocosms did not affect C. laticinctus oviposition but were attractive to conspecific oviposition. These results increase our understanding of the female ability to detect species-specific signals, indicating increased larval competition.

Fish microbiota repel ovipositing mosquitoes.

The mere presence of predators causes prey organisms to display predation-avoidance strategies. Predator presence is often communicated through predator-released chemical signals. Ovipositing female mosquitoes of several species are repelled by unknown signals released from larvivorous fish. It was previously suggested that in many cases, a predator's microbiota plays an important role in the release of these signals; however, this mechanism is still poorly understood. In this study, we looked into the effects of the microbiota originating from the larvivorous Gambusia affinis (Baird and Girard) on the oviposition behaviour of gravid female mosquitoes. We used fish with altered microbiota and bacterial isolates in a set of outdoor mesocosm experiments to address this aim. We show that interference with the fish microbiota significantly reduces fish's repellent effect. We further show that the bacterium Pantoea pleuroti, isolated from the skin of the fish, repels oviposition of Culex laticinctus Edwards and Culiseta longiareolata Macquart mosquitoes similarly to the way in which live fish repel them. Our results highlight the importance of bacteria in the interspecies interactions of their hosts. Furthermore, this finding may lead to the development of an ecologically friendly mosquito repellent, that may reduce the use of larvivorous fish for mosquito control.

Quantification of bacterial adhesion to tissue in high-throughput kinetics.

Bacterial adhesion to tissue is the starting point for many pathogenic processes and beneficial interactions. The dynamics and speed of adhesion (minutes) make high-resolution temporal kinetic data important, but this capability is absent from the current toolset. We present a high-throughput method with a second-to-minute kinetic resolution, testing the adhesion of Pseudomonas aeruginosa PAO1 wild-type, flagella-, pili-, and quorum-sensing mutants to human embryonic kidney (HEK293) cells. Adhesion rates were in good correlation with HEK293 confluence, and the ways in which various bacterial mutations modified adhesion patterns are in agreement with the published literature. This simple assay can facilitate drug screening and treatment development as well as provide a better understanding of the interactions of pathogenic and probiotic bacteria with tissues, allowing the design of interventions and prevention treatments.

Effect of Drinking Water Salt Content on the Interaction between Surfactants and Bacteria.

Sodium dodecyl sulfate (SDS) is a common surfactant used in various hygienic products. Its interactions with bacteria were studied previously, but the three-way interaction between surfactants, bacteria, and dissolved salts in the context of bacterial adhesion has not been studied. Here, we examined the combined effects of SDS (at concentrations typical of everyday hygienic activities) and salts, sodium chloride, and calcium chloride (at concentrations typically found in tap water) on the adhesion behavior of the common opportunistic pathogen Pseudomonas aeruginosa. We found that bacterial adhesion in the absence of SDS was dependent on the cation concentration rather than the total ionic strength and that combined treatment with several millimolar NaCl and SDS can increase bacterial adhesion. The addition of low concentrations of SDS (2 mM) to tens to hundreds millimolar concentrations of NaCl, typical of systems that suffer seawater incursion, reduced bacterial adhesion dramatically. Combined treatment with Ca+2 (in concentrations typical of those found in hard water) and SDS produced a small increase in total adhesion but a dramatic increase in the strength of adhesion. We conclude that the type and concentration of salts in water can have a considerable effect on the efficacy of soap in reducing bacterial adhesion and should be taken under consideration in critical applications. IMPORTANCE Surface-adhering bacteria are a reoccurring problem in many settings, including households, municipal water systems, food production facilities, and hospitals. Surfactants, and specifically sodium dodecyl sulfate (also known as SDS/SLS), are commonly used to remove bacterial contamination, but data regarding the interaction of SDS with bacteria and especially the effects of water-dissolved salts on this interaction are lacking. Here, we show that calcium and sodium ions can dramatically affect the efficacy of SDS on bacterial adhesion behavior and conclude that salt concentrations and ion species in the water supply should be considered in SDS applications.

Disinfection of SARS-CoV-2 by UV-LED 267 nm: comparing different variants.

UV irradiation is an efficient tool for the disinfection of viruses in general and coronavirus specifically. This study explores the disinfection kinetics of SARS-CoV-2 variants wild type (similar to the Wuhan strain) and three variants (Alpha, Delta, and Omicron) by 267 nm UV-LED. All variants showed more than 5 logs average reduction in copy number at 5 mJ/cm2 but inconsistency was evident, especially for the Alpha variant. Increasing the dose to 7 mJ/cm2 did not increase average inactivation but did result in a dramatic decrease in the inactivation inconsistency making this dose the recommended minimum. Sequence analysis suggests that the difference between the variants is likely due to small differences in the frequency of specific UV extra-sensitive nucleotide sequence motifs although this hypothesis requires further experimental testing. In summary, the use of UV-LED with their simple electricity need (can be operated from a battery or photovoltaic panel) and geometrical flexibility could offer many advantages in the prevention of SARS-CoV-2 spread, but minimal UV dose should be carefully considered.

Bacterial detoxification of plant defence secondary metabolites mediates the interaction between a shrub and frugivorous birds.

Many plants produce fleshy fruits, attracting fruit-eating animals that disperse the seeds in their droppings. Such seed dispersal results in a conflict between the plant and the animal, as digestion of seeds can be highly beneficial to the animal but reduces plant fitness. The plant Ochradenus baccatus uses the myrosinase-glucosinolates system to protect its seeds. We show that hydrolysis of the O. baccatus fruit glucosinolates by the myrosinase enzyme inhibited digestive enzymes and hampered digestion in naïve individuals of the bird Pycnonotus xanthopygos. However, digestion in birds regularly feeding on O. baccatus fruits was unaffected. We find that Pantoea bacteria, dominating the gut of these experienced birds as well as the fruits, thrive on glucosinolates hydrolysis products in culture. Augmentation of Pantoea protects both naïve birds and plant seedlings from the effects of glucosinolates hydrolysis products. Our findings demonstrate a tripartite interaction, where the plant-bird mutually beneficial interactions are mediated by a communal bacterial tenant.

Evaluation of the biocontrol activity of Frateuria defendens-derived metabolites against mollicutes.

Frateuria defendens is a candidate biocontrol agent that has been shown to reduce phytoplasma-related disease symptoms in grapevines and periwinkle plants. While a crude filtrate prepared from F. defendens can inhibit mollicute growth, the specific growth parameters for this bacterium, necessary to enhance this protective inhibitory response, remain unknown. Moreover, the separation of filtrate preparations from bacterial cells via centrifugation and filtration is laborious and time-consuming. As such, the present study was conducted to define the optimal growth conditions associated with maximal inhibitory activity of F. defendens and to establish a better approach to separating these bacterial cells from their secreted metabolites. To conduct these analyses, F. defendens was cultured in a range of media types, while associated inhibitory effects were tested in vitro using Spiroplasma melliferum as a model mollicute bacterium, and in planta using phytoplasma-infected periwinkle plantlets. These analyses revealed F. defendens growth patterns change based upon media composition, with filtrates prepared from a specific rich medium (S-medium) exhibiting beneficial activities, including the inhibition of S. melliferum and enhanced plant growth. When F. defendens cells were grown within semi-permeable, membrane-coated Small Bioreactor Platform (SBP) capsules, they could be more readily separated from the secreted metabolite fraction, obviating the need for filtration and/or centrifugation. This study is the first to have reported the use of SBP capsules to separate bacterial cells from their secreted metabolites under sterile conditions while retaining the ability of these metabolites to inhibit S. melliferum growth and to benefit the host plant. The results highlight promising new approaches to the effective biocontrol of phytoplasma-driven diseases in grapevines and other economically important plant species.

Electrochemical Detection of Waterborne Bacteria Using Bi-Functional Magnetic Nanoparticle Conjugates.

Detection of microbial contamination in water is imperative to ensure water quality. We have developed an electrochemical method for the detection of E. coli using bi-functional magnetic nanoparticle (MNP) conjugates. The bi-functional MNP conjugates were prepared by terminal-specific conjugation of anti-E. coli IgG antibody and the electroactive marker ferrocene. The bi-functional MNP conjugate possesses both E. coli-specific binding and electroactive properties, which were studied in detail. The conjugation efficiency of ferrocene and IgG antibodies with amine-functionalized MNPs was investigated. Square-wave voltammetry enabled the detection of E. coli concentrations ranging from 101-107 cells/mL in a dose-dependent manner, as ferrocene-specific current signals were inversely dependent on E. coli concentrations, completely suppressed at concentrations higher than 107 cells/mL. The developed electrochemical method is highly sensitive (10 cells/mL) and, coupled to magnetic separation, provides specific signals within 1h. Overall, the bi-functional conjugates serve as ideal candidates for electrochemical detection of waterborne bacteria. This approach can be applied for the detection of other bacteria and viruses.

Effective Removal of Acid Dye in Synthetic and Silk Dyeing Effluent: Isotherm and Kinetic Studies.

Here, we propose a low-cost, sustainable, and viable adsorbent (pine tree-derived biochar) to remove acid dyes such as acid violet 17 (AV), which is used in the silk dyeing industry. As a case study, the AV removal process was demonstrated using synthetic effluent and further as a proof of concept using real dye effluent produced from the Sirumugai textile unit in India. The pine tree-derived biochar was selected for removal of aqueous AV dye in batch and fixed-bed column studies. The adsorbent material was characterized for crystallinity (XRD), surface area (BET), surface morphology and elemental compositions (SEM-EDX), thermal stability (TGA), weight loss (DGA), and functional groups (FTIR). Batch sorption studies were performed to evaluate (i) adsorption at various pH values (at pH 2 to 7), (ii) isotherms (at 10, 25, and 35 °C) to assess the temperature effect on the sorption efficiency, and (iii) kinetics to reveal the effect of time, adsorbent dose, and initial concentration on the reaction rate. After systematic evaluation, 2 g/L biochar, 25 mg/L AV, pH 3, 40 °C, and 40 and 360 min in a completely mixed batch study resulted in 50 and 90% dye removal, respectively. The isoelectric point at pH 3.7 ± 0.2 results in maximum dye removal, therefore suggesting that monitoring the ratio of different effluent (acid/wash/dye) can improve the colorant removal efficiency. The Langmuir isotherm best fits with the sorption of AV to biochar, provided a maximal dye uptake of 29 mg/g at 40 °C, showing that adsorption was endothermic. Fixed-bed studies were conducted at room temperature with an initial dye concentration of 25 and 50 mg/L. The glass columns were packed with biochar (bed depth 20 cm, pore volume = 14 mL) at an initial pH of 5.0 and a 10 mL/min flow rate for 120 min. Finally, the regeneration of the adsorbent was achieved using desorption studies conducted under the proposed experimental conditions resulted in 90-93% removal of AV even after five cycles of regeneration.

Pseudomonas aeruginosa Mobbing-Like Behavior against Acanthamoeba castellanii Bacterivore and Its Rapid Control by Quorum Sensing and Environmental Cues.

Mobbing, group attack of prey on predator, is a behavior seen in many animal species in which prey animals use numbers and coordination to counter individually superior predators. We studied attack behavior of Pseudomonas aeruginosa toward the bacterivore Acanthamoeba castellanii. This behavior consists of directed motility toward and specific adhesion to the predator cells, enacted in seconds and responding to both prey and predator population densities. Attack coordination relies on remote sensing of the predator and the use of the Pseudomonas quinolone signal (PQS), a P. aeruginosa species-specific quorum sensing molecule. Mutants unable to produce the PQS show unspecific adhesion and reduced survival, and a corresponding increase in predator population occurs as a result of predation. The addition of an external PQS restored some predator-specific adherence within seconds, suggesting a novel response mechanism to this quorum sensing (QS) signal. Fast behavioral response of P. aeruginosa to PQS is also supported by the rate of signal accumulation in the culture, reaching relevant concentrations within minutes, enabling bacteria response to self population density in these short timescales. These results portray a well-regulated group attack of the bacteria against their predator, reacting within seconds to environmental cues and species-specific signaling, which is analogous in many ways to animal mobbing behavior. IMPORTANCE Pseudomonas aeruginosa was shown previously to attack amoebae and other predators by adhering to them and injecting them with virulent substances. In this work, we show that an active, coordinated group behavior is enacted by the bacteria to utilize these molecular components, responding to both predator and bacterial population density. In addition to their ecological significance, immediate behavioral changes observed in response to PQS suggest the existence of a fast QS signal cascade, which is different from canonical QS that relies on slow-to-respond gene regulation. Similar regulatory circuits may drive other bacterial adaptations and pathogenicity mechanisms and may have important clinical implications.

A sterol-defined system for quantitative studies of sterol metabolism in C. elegans.

This protocol describes the culturing of the nematode Caenorhabditis elegans (C. elegans) in a sterol-defined experimental system and the subsequent quantitative analysis of C. elegans sterols through gas chromatography-mass spectrometry. Although studied primarily in mammals, sterols are essential biomolecules for most eukaryotes. C. elegans cannot synthesize sterols and thus relies on the uptake of dietary sterols. Therefore, C. elegans is a powerful system to study metabolism in sterol-defined conditions that are described in our protocol. For complete details on the use and execution of this protocol, please refer to Shamsuzzama et al. (2020).

Investigation of glucosinolates in the desert plant Ochradenus baccatus (Brassicales: Resedaceae). Unveiling glucoochradenin, a new arabinosylated glucosinolate.

Here we describe the structure elucidation and quantification of six glucosinolates (GSLs) from the roots of the desert plant Ochradenus baccatus, Delile 1813 (family Resedaceae; order Brassicales). The structure elucidation was established on the corresponding enzymatically desulfated derivatives of the native GSLs of the plant. Among these GSLs we describe the previously undescribed 2″-O-(α-L-arabinopyranosyloxy)benzylglucosinolate (1a), for which we propose the name glucoochradenin. The other five glucosinolates (2a-6a) were (2S)-2-hydroxy-2-phenylethylglucosinolate (2a; glucobarbarin), 2″-O-(α-L-rhamnopyranosyloxy)benzylglucosinolate (3a), benzylglucosinolate (4a; glucotropaeolin), indol-3-ylmethylglucosinolate (5a; glucobrassicin) and phenethylglucosinolate (6a; gluconasturtiin), all elucidated as their desulfo-derivatives, 2b-6b respectively). Structures were elucidated by MS and 1D and 2D-NMR techniques, the identity of the arabinose verified by ion chromatography, and the absolute configuration of the sugar units determined by hydrolysis, coupling to cysteine methyl-ester and phenyl isothiocyanate followed by HPLC-MS analysis of the resulted diastereomers. Response factors were generated for desulfo-2″-O-(α-L-arabinopyranosyloxy)benzylglucosinolate and for desulfo-2″-O-(α-L-rhamnopyranosyloxy)benzylglucosinolate and all six GSLs were quantified, indicating that the root of O. baccatus is rich in GSLs (Avg. 61.3 ± 10.0 µmol/g DW and up to 337.2 µmol/g DW).

Bacterial Adhesion Kinetics in a High Throughput Setting in Seconds-minutes Time Resolution.

Bacterial surface adhesion, the first step in many important processes including biofilm formation and tissue invasion, is a fast process that occurs on a time scale of seconds. Adhesion patterns tend to be stochastic and spatially heterogeneous, especially when bacteria are present in low population densities and at early stages of adhesion to the surface. Thus, in order to observe this process, a high degree of temporal resolution is needed across a large surface area in a way that allows several replicates to be monitored. Some of the current methods used to measure bacterial adhesion include microscopy, staining-based microtiter assays, spectroscopy, and PCR. Each of these methods has advantages in assaying aspects of bacterial surface adhesion, but none can capture all features of the process. In the protocol presented here, adapted from Shteindel et al., 2019 , fluorescently-labeled bacteria are monitored in a multi-titer setting using a standard plate fluorimeter and a dye that absorbs light in the fluorophore excitation and emission wavelengths. The advantage of using this dye is that it restricts the depth of the optic layer to the few microns adjacent to the bottom of the microtiter well, eliminating fluorescence originating from unattached bacteria. Another advantage of this method is that this setting does not require any preparatory steps, which enables reading of the sample to be repeated or continuous. The use of a standard multi-titer well allows easy manipulation and provides flexibility in experimental design.

Wavelength-dependent time-dose reciprocity and stress mechanism for UV-LED disinfection of Escherichia coli.

Ultraviolet (UV) disinfection efficiency by low-pressure (LP) mercury lamp depends on the UV fluence (dose): the product of incident irradiance (fluence rate) and exposure time, with correction factors. Time-dose reciprocity may not always apply, as higher UV-LP inactivation of E. coli was obtained at a higher irradiance over shorter exposure time, for the same UV fluence. Disinfection by UV LEDs is limited by low radiant flux compared to mercury LP lamps. Our goal was to determine the UV-LED time-dose reciprocity of E. coli for four different central LED wavelengths (265, 275, 285 and 295 nm) under different fluence rates. Inactivation kinetics determined at UV-LED265 was not affected by the fluence rate or exposure time for a given UV fluence. In contrast, UV-LED275, UV-LED285, and UV-LED295 led to higher inactivation at low fluence rate coupled to high exposure time, for the same UV fluence. The intracellular damage mechanisms for each LED central wavelength were determined by using the bioreporters RecA as an indicator of bacterial DNA damage and SoxS as an indicator of oxidative stress. For 265 nm, higher DNA damage was observed, whereas for 285 and 295 nm, higher oxidative stress (possibly due to reactive oxygen species [ROS] damage) was observed. ROS inactivation of E. coli was predicted to be more effective when keeping the ROS concentration low but allowing longer exposure, for a given UV fluence.

Assessment of saliva interference with UV-based disinfection technologies.

Worldwide shortages of personal protective equipment during COVID-19 pandemic has forced the implementation of methods for decontaminating face piece respirators such as N95 respirators. The use of UV irradiation to reduce bioburden of used respirators attracts attention, making proper testing protocols of uttermost importance. Currently artificial saliva is used but its comparison to human saliva from the UV disinfection perspective is lacking. Here we characterize UV spectra of human and artificial saliva, both fresh and after settling, to test for possible interference for UV-based disinfection. ASTM 2720 artificial saliva recipe (with either porcine or bovine mucin) showed many discrepancies from average (N = 18) human saliva, with different mucins demonstrating very different UV absorbance spectra, resulting in very different UV transmittance at different wavelength. Reducing porcine mucin concentration from 3 to 1.7 g/L brought UVA254 in the artificial saliva to that of average human saliva (although not for other wavelengths), allowing 254 nm disinfection experiments. Phosphate saline and modified artificial saliva were spiked with 8.6 log CFU/ml B. subtilis spores (ATCC 6633) and irradiated at dose of up to 100 mJ/cm2, resulting in 5.9 log inactivation for a saline suspension, and 2.8 and 1.1 log inactivation for ASTM-no mucin and ASTM-1.7 g/L porcine mucin 2 µL dried droplets, respectively. UVC irradiation of spores dried in human saliva resulted in 2.3 and 1.5 log inactivation, depending on the size of the droplets (2 vs 10 µL, respectively) dried on a glass surface. Our results suggest that in the presence of the current standard dried artificial saliva it is unlikely that UVC can achieve 6 log inactivation of B. subtilis spores using a realistic UV dose (e.g. less than 2 J/cm2) and the ATSM saliva recipe should be revised for UV decontamination studies.

Attitudes of food consumers at universities towards recycling human urine as crop fertiliser: A multinational survey dataset.

We present here a data set generated from a multinational survey on opinions of university community members on the prospect of consuming food grown with human urine as fertiliser and about their urine recycling perceptions in general. The data set comprises answers from 3,763 university community members (students, faculty/researchers, and staff) from 20 universities in 16 countries and includes demographic variables (age bracket, gender, type of settlement of origin, academic discipline, and role in the university). Questions were designed based on Ajzen's theory of planned behaviour to elicit information about three components of behavioural intention-attitudes, subjective norms, and perceived behavioural control. Survey questions covered perceived risks and benefits (attitudes), perceptions of colleagues (injunctive social norm) and willingness to consume food grown with cow urine/faeces (descriptive social norm), and willingness to pay a price premium for food grown with human urine as fertiliser (perceived behavioural control). We also included a question about acceptable urine recycling and disposal options and assessed general environmental outlook via the 15-item revised New Ecological Paradigm (NEP) scale. Data were collected through a standardised survey instrument translated into the relevant languages and then administered via an online form. Invitations to the survey were sent by email to university mailing lists or to a systematic sample of the university directory. Only a few studies on attitudes towards using human urine as fertiliser have been conducted previously. The data described here, which we analysed in "Willingness among food consumers at universities to recycle human urine as crop fertiliser: Evidence from a multinational survey" [1], may be used to further understand potential barriers to acceptance of new sanitation systems based on wastewater source separation and urine recycling and can help inform the design of future sociological studies.

Willingness among food consumers to recycle human urine as crop fertiliser: Evidence from a multinational survey.

Source-separating sanitation systems offer the possibility of recycling nutrients present in wastewater as crop fertilisers. Thereby, they can reduce agriculture's impacts on global sources, sinks, and cycles for nitrogen and phosphorous, as well as their associated environmental costs. However, it has been broadly assumed that people would be reluctant to perform the new sanitation behaviours that are necessary for implementing such systems in practice. Yet, few studies have tried to systematically gather evidence in support of this assumption. To address this gap, we surveyed 3763 people at 20 universities in 16 countries using a standardised questionnaire. We identified and systematically assessed cross-cultural and country-level explanatory factors that were strongly associated with people's willingness to consume food grown using human urine as fertiliser. Overall, 68% of the respondents favoured recycling human urine, 59% stated a willingness to eat urine-fertilised food, and only 11% believed that urine posed health risks that could not be mitigated by treatment. Most people did not expect to pay less for urine-fertilised food, but only 15% were willing to pay a price premium. Consumer perceptions were found to differ greatly by country and the strongest predictive factors for acceptance overall were cognitive factors (perceptions of risks and benefits) and social norms. Increasing awareness and building trust among consumers about the effectiveness of new sanitation systems via cognitive and normative messaging can help increase acceptance. Based on our findings, we believe that in many countries, acceptance by food consumers will not be the major social barrier to closing the loop on human urine. That a potential market exists for urine-fertilised food, however, needs to be communicated to other stakeholders in the sanitation service chain.