Philippa Kaur

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Bacterial strains, media and batch culturesWe used the wildtype strain Vibrio natriegens ATCC 14048 and Alteromonas macleodii sp. 4B03 (non-clumping variant) isolated from marine particles [8]. Strains were cultured in Marine Broth (MB, Difco 2216) and grown overnight at 25 °C. In total, 1 ml of cell culture was centrifuged (13,000 rpm for 2 min) in a 1.5 ml microfuge tube. After discarding the supernatant, the cells were washed with 1 ml of MBL minimal medium medium without carbon source. Cells were centrifuged again and the cell pellet was resuspended in 1 ml of MBL (marine minimal medium) [30, 34] adjusted to an 0.002 OD600. Cells from these cultures were used for experiments in MBL minimal medium containing 0.1% (weight/volume) Pentaacetyl-Chitopentaose (Megazyme, Ireland). The carbon source was added to the MBL minimum medium and filter sterilized using 0.22 μm Surfactant-Free Cellulose Acetate filters (Corning, USA). A total of 500 µl of the prepared cultures (250 µl + 250 µl for co-cultures) were added to 9.5 ml of MBL + 0.1 % chitopentaose (v/w) in serum flasks. This resulted in a starting OD of 0.0001. The flasks included a stirrer and were sealed with a rubber seal. Serum flasks were stored on a bench top magnetic stirrer (500 rpm) and connected to the microfluidics setup via Hamilton NDL NO HUB needles (ga21/135 mm/pst 2).MicrofluidicsMicrofluidics experiments were performed as described previously [35,36,37,38]. Cell growth was imaged within mother machine channels of 25 × 1.4 × 1.26 μm (length × width × height). Within these channels, cells could experience the batch culture medium that diffused through the main flow channels. The microfluidic device consisted of a PDMS flow cell (50 µm/23 µm). The PDMS flow cell was fabricated by mixing the SYLGARD 184 Silicone Elastomer Kit chemicals 10:1 (w/v), pouring the mix on a master waver and hardening it at 80 °C for 1 h. The solid PDMS flow cell was cut out of the master waver and holes were pierced at both ends of each flow channel prior to binding it to a cover glass (Ø 50 mm) by applying the “high” setting for 30 s on the PDC-32G Plasma Cleaner by Harrick Plasma. The flow cell was connected via 40 mm Adtech PTFE tubing (0.3 mm ID × 0.76 mm OD) to a Ismatech 10 K Pump with 40 mm of Ismatech tubing (ID 0.25 mm, OD 0.90 mm) which again was connected via 80 mm Adtech PTFE tubing (0.3 mm ID × 0.76 mm OD) via a 5 mm short Cole-Parmer Tygon microbore tubing (EW-06418-03) (ID 0.762 mm OD 2.286 mm) connector tubing to a Hamilton NDL NO HUB needle (ga21/135 mm/pst 2) that was inserted into the feeding culture. During the whole experiment the pump flow was set to 1.67 µl/min (0.1 ml/h).Time-lapse microscopyMicroscopy imaging was done using fully automated Olympus IX81 or IX83 inverted microscope systems (Olympus, Japan), equipped with a ×100 NA1.3 oil immersion, phase contrast objective, an ORCA-flash 4.0 v2 sCMOS camera (Hamamatsu, Japan), an automated stage controller (Marzhauser Wetzlar, Germany), shutter, and laser-based autofocus system (Olympus ZDC 1 and 2). Detailed information about the microscopy setup has been described by D’Souza et al. [39]. Channels on the same PDMS Chip were imaged in parallel, and phase-contrast images of each position were taken every 5 min. The microscopy units and PDMS chip were maintained at room temperature. All experiments were run at a flow rate of 0.1 ml h−1, which ensures nutrients enter the chamber through diffusion. Four biological replicates were performed. These replicates consist of four independent microfluidics channels (two for each of the strains). These channels were connected to one of two independent batch cultures.The microscopy dataset consists of 200 mother machine channels; 49 channels for the degrader on co-culture, 51 for the degrader on mono-culture, 40 for the cross-feeder on mono-culture and 60 for the cross-feeder on co-culture.Image analysisImage processing was performed using a modified version of the Vanellus image analysis software (Daan Kiviet, https://github.com/daankiviet/vanellus), together with Ilastik [40] and custom written Matlab scripts.Movies were registered to compensate for stage movement and cropped to the region of growth channels. Subsequently, segmentation was done on the phase contrast images using Ilastik’s supervised pixel classification workflow and cell tracking was done using the Vanellus build-in tracking algorithm.After visual curation of segmentation and tracking for each mother machine and at every frame growth parameters were calculated using custom written matlab scripts [36]. Lengths of individual cells were estimated by finding the cell center line by fitting a third-degree polynomial to the cell mask; then the cell length was calculated as the length of the center line between the automatically detected cell pole positions (see Kiviet et al. [33] for details).We quantified cell growth by calculating single-cell elongation rates r from measured cell length trajectories: L(t) = L(0)∙e^(r ∙ t). Cell lengths and growth rates varied drastically over the time course of the experiment; we thus developed a robust procedure that can reliably estimate elongation rates both for large fast-growing cells as well as for small non-growing cells. We first log-transformed cell lengths, which were subsequently smoothed over a moving time window with a length of 5 h (60 time points). We used a second order local regression using weighted linear least squares (rloess method of Matlab smooth function) in order to minimize noise while maintaining sensitivity to changes in elongation rates. Subsequently the instantaneous elongation rate was estimated as the slope of a linear regression over a moving time window of 30 min (7 time points). Time points for which the fit quality was bad (χ2  > 10−4) were removed from the analysis [32]. All parameters were optimized manually by visually inspecting the fitting procedure of many cell length trajectories randomly selected from across all replicates.As cells are continuously lost from the mother machine channels it is non-trivial to calculate the total amount of biomass produced in the chip. We thus need to estimate this quantity from the observed single-cell elongation rates. Specifically, we estimated the total amount of biomass produced per individual mother machine until a given time point as:$$B_T = e^{Delta tmathop {sum}limits_{i = 1}^T { < r_i > } }$$Where is the average growth rate of all cells in a given replicate at time point i, and where Δt is the time interval between two timepoints. By using the average growth rate, we ignore the variation in growth rates between cells. However, it is difficult to calculate population growth when growth rates vary both with time and between cells and the current method still allows us to capture the overall effect of interactions on cell growth.Datasets and statistical analysisAll microfluidics experiments were replicated four times. No cells were excluded from the analysis after visual curation. For V. natriegens 2227 cells were analyzed on mono-culture, and 1707 cells were analyzed on co-culture. For A. macleodii 2657 cells were analyzed on mono-culture, and 3901 cells were analyzed on co-culture. Each mother machine channel was treated as an independent sample. All statistical analysis was performed in Rstudio v1.2.5033. Percent increases were calculated using the relative differences of estimated between the corresponding values. For mixed effect models analysis the LmerTest package (Version 3.1-3) [41] with the following equation were used: y ~ Batch + (1 | Replicate) The Tukey Post hoc test was performed using the Multcomp package (Version 1.4-15) [42].Chitinase assayDegrader and Cross-feeder cells were cultured in Marine Broth (MB, Difco 2216) and grown overnight at 25 °C. In total, 1 ml of cell culture was centrifuged (13,000 rpm for 2 min) in a 1.5 ml microfuge tube. After discarding the supernatant the cells were washed with 1 ml of MBL minimal medium medium without carbon source. Cells were centrifuged again and the cell pellet was resuspended in 1 ml of MBL adjusted to an 0.002 OD600. A total of 10 µl of cell culture was added to 190 µl of MBL containing 0.1% Chitopentaose (w/v). Cultures were grown to exponential phase in a plate reader (Eon, BioTek) at 25 °C. Cell free supernatants were generated by sterile filtering cultures using a multi-well filter plate (AcroPrep) into a fresh 96 well plate. Chitinase activity of cell free supernatants was measured using a commercially available fluorometric chitinase assay kit (CS1030, Sigma-Aldrich) following the protocol. In short, 10 µl of sterile supernatant was added to 90 µl of the assay mix. The solution was incubated in the dark at 25 °C for 40 min before measuring fluorescence (Excitation 360 nm, Emission 450 nm) in a plate reader (Synergy MX, Biotek). Logarithmic chitinase activity per OD600 was analyzed for eight replicates.Chitinase activity in units per ml was calculated using a standard concentration. Using the following Formula: ({{{{{rm{Units}}}}}}/{{{{{rm{ml}}}}}} = frac{{left( {{{{{{rm{FLU}}}}}} – {{{{{rm{FLUblank}}}}}}} right) times 1.9 times 0.3 times {{{{{rm{DF}}}}}}}}{{{{{{{rm{FLUstandard}}}}}} times {{{{{rm{time}}}}}} times {{{{{rm{Venz}}}}}}}})Here, FLU indicates measured fluorescence, DF indicates the dilution factor, and V indicates the volume of the sample in ml [43].Acetate assayCell cultures were prepared and grown in serum flasks as described above. At different time intervals 1 ml of culture was removed and OD600 was measured. Cultures were filter sterilized using 0.22 μm Surfactant-Free Cellulose Acetate filters (Corning, USA) into a 1.5 ml microfuge tube. Cell free supernatants were stored at −4 °C until they were used for acetate measurements. Acetate concentrations were measured using a colorimetric assay kit (MAK086, Sigma-Aldrich) following the protocol. In short, 50 µl of cell free supernatant was added to 50 µl of assay mix. The solution was incubated in the dark at 25 °C for 30 min. Acetate concentrations were measured in a plate reader (Eon, Biotek) at 450 nm [44].Growth on spend mediaDegrader and Cross-feeder cells were cultured in Marine Broth (MB, Difco 2216) and grown overnight at 25 °C. In total, 1 ml of cell culture was centrifuged (13,000 rpm for 2 min) in a 1.5 ml microfuge tube. After discarding the supernatant, the cells were washed with 1 ml of MBL minimal medium medium without carbon source. Cells were centrifuged again and the cell pellet was resuspended in 1 ml of MBL adjusted to an 0.002 OD600. A total of 10 µl of cell culture was added to the 190 µl cell free supernatant described above. Cultures were grown in a plate reader (Eon, BioTek) at 25 °C. Cell free supernatants after this growth assay were generated by sterile filtering cultures using a multi-well filter plate (AcroPrep) into a fresh 96 well plate. These supernatants were used as described above to measure acetate levels after growth on spend media. More

Study species and spider collectionAustralomisidia ergandros is a subsocial spider inhabiting South-Eastern Australia. They live in communal kin-groups in nests usually built with leaves from Eucalyptus trees bound by silk threads. Group size usually ranges from 5 to 45 spiderlings. Groups are comprised of the offspring of a single female who provides maternal care until her death28. Offspring continue to live in groups for 5 to 7 months after the mother’s death29,30. One of the females inherit the natal nest while the remaining females disperse to found new nests. It is not entirely clear if A. ergandros inbreed with natal kin or if spiders show a mandatory pre-mating dispersal.We collected 29 A. ergandros nests from a population along Yass River Road in New South Wales, Australia (34° 55′ 20.50′′ S, 149° 6′ 15.53′′ E) in February 2016. At this time of year, the spiderlings are very young and the presence of immigrants, who might influence the extent of social foraging, is improbable9,31. For our experiments, we transferred the original nests to the laboratory at Macquarie University in Sydney.Group composition effectsOur experiments spanned a duration of 56 days. To investigate group composition (cooperators vs. defectors) effects, we first assessed the hunting types of individuals within ‘initial’ groups (phase 1) and subsequently composed and tested ‘sorted’ groups of cooperators or defectors only (phase 2). The formation of initial groups was dictated by special requirements. Basically, we randomly selected up to 30 individuals per original nest and split these individuals into two to three initial groups of ten (Nnests = 10, N groups = 25). Each selected individual received a unique color mark (©Plaka-Farbe) and was weighed to the nearest 0.01 mg on an electronic balance (Mettler Toledo New Classic MS). Each group was then transferred to a petri dish (100 mm in diameter) which served as the test arena for the hunting type assessment. An acclimatization period of four days ensured that the spiders weaved silk threads which amplify vibrations by prey32.Phase 1We assessed hunting types with a modified version of the ‘communal feeding experiment’ originally used by Dumke et al.16 to establish hunting specialization in A. ergandros. For each initial group, we completed 7 feeding trials over 24 days (1 trial every 4 days), during which we offered living Musca domestica flies and observed the foraging behaviour of all group members (Fig. 1). Each fly was weighed before being placed into the petri dish and either removed after two hours if not captured, or after two hours post capture. For each trial, we documented the attack latency, the attacker IDs and the IDs of the feeding individuals in 10-min intervals over two hours. From these data, we determined the feeding frequency of each individual (i.e. the number of trials it was feeding) and calculated the proportions to which it cooperated vs. defected. We thus obtained comparable quantifications of hunting types16. All individuals except those that died during the assessment (56 of 250 spiders) were weighed two days after the last trial to assess weight gain1 (= log (end weight1/start weight133).Phase 2Following phase 1, we regrouped individuals into ‘sorted’ groups of cooperators or defectors only, and this time gave three days acclimatization time since the re-grouping took one day. We formed experimental cooperator groups by selecting nine to ten individuals with the highest cooperating tendencies from the original colony. Next, we formed experimental defector groups analogously from that same pool (Fig. 1). Thus, we achieved paired relatedness between cooperator groups and defector groups, to control for nest origin and nest experience (matched pairs design). We further ensured comparability of cooperator groups and defector groups in the individuals’ physical state (details in Supplementary Methods). Owing to mortality in three nests and restricted possibilities to realize balanced conditions between groups in two nests, we could establish five cooperator-defector group pairs with nine individuals per group.To explore group composition effects on social foraging behaviour and individual fitness payoffs, we tested each sorted group over another seven feeding trials over 24 days. The trials were conducted in exactly the same manner as for the feeding type assessment (phase 1). From the recorded data (attack latency, IDs of attackers, IDs of feeding individuals), we calculated a set of variables that quantified social foraging behaviour (data points per trial and group). To examine individual fitness payoffs, we checked the petri dishes for dead individuals and noted their IDs prior to every trial. As an additional fitness payoff measure for those individuals still alive at the end of phase 2, we determined individual weight gain2 (= log (end weight2/start weight2)).The role of sex in cooperator vs. defector typesTo examine the role of sex in cooperation-defection scenarios, we collected another eight nests from Yass River Road in June 2016. Around this time, A. ergandros individuals reached the subadult stage, at which sex can be visually determined17. Three nests contained subadult males and females in sufficient numbers, so that we formed three groups, each with ten males and ten females from the same nest (in total: N males = 30, N females = 30). All group members were weighed and color marked before they were tested in another, extended feeding type assessment over ten trials.Based on the IDs of attackers and individuals that hunted in these trials, we generated social network graphs and visualized the foraging interactions within groups31,34,35. Individuals were represented by ‘nodes’; a directed line (‘edge’) was drawn from one node to another if the specific individual had cooperated by sharing prey with the other. The lines received weights reflecting the frequency of the respective interaction. We quantified individual prey sharing tendencies using the node-level metric out-strength: the weight sum of all outgoing edges from a particular node35. This metric comprehensively reflects an individual’s prey sharing tendency, as it incorporates the frequency and the spread of prey sharing behaviour. To visualize social networks and calculate the individuals’ out-strengths, we used the software UCINET 636.Statistical analysesAll model analyses were performed in R version 3.2.2, whereas all social network analyses were conducted in UCINET 636.Group composition effectsWe modelled the effect of group composition on social foraging behaviour separately for each response variable with binomial or gamma GEEs (generalized estimation equations). GEEs are adequate to analyse data from repeated measurements over time within same groups because they allow adjustment for the dependence of these measurements37. Defining the dependence structure of our data, we set sorted-group ID as a grouping variable and specified the temporal correlation AR-1. Group composition constituted the explanatory variable of interest, fly weight and group size were included as additional variables to control for prey mass and mortality. An exception was the model for the scrounging degree, in which group size was controlled by the variable itself. We assessed the significance of group composition effects by dropping each explanatory variable in turn and then comparing the full model to its nested models based on Wald test statistics. The least significant variable was removed, and model comparisons were repeated until all remaining variables were significant.Mortality was compared between cooperator groups and defector groups using a Chi-squared test. The difference between group compositions in individual weight gain2 was analysed in a GLS (generalized least squares) model that incorporated an exchangeable correlation structure with sorted-group ID as the grouping variable.Sex differencesWe conducted a node-based Monte Carlo randomization test to determine whether the observed difference in mean out-strength between sexes deviated significantly from the difference expected if producing associations occurred randomly and hence independent of sex. The observed data were shuffled in 10,000 node-label randomizations that preserved group membership. The sum of the differences between mean male out-strength (σm) and mean female out-strength (σf) within groups was used as the test statistic A (A = sumnolimits_{(i = 1)}^{3} {left( {sigma_{{(m_{i} )}} – sigma_{{left( {f_{i} } right)}} } right)}^{ – }), where i denotes group identity. To produce a probability value, we compared the observed test statistic to the distribution of random test statistics drawn from the 10,000 Monte Carlo simulations34. More

Funicular bodies: structure, function, and developmentThe ultrastructural and functional complexity of funicular bodies in bryozoans25,46,47 (our data), as well as the reduction of the genome in their symbiotic bacteria (e.g., Bugula neritina48), point at a long-term co-evolution between these organisms, also suggesting that infected bryozoan colonies spend a significant part of their energy budget supporting numerous bacteria inside FBs. All known prokaryote symbionts are apparently non-pathogenic for bryozoans49,62. Instead, the overall evidence indicates a mutualistic relationship. Symbiotic bacteria are known to produce toxic substances (bryostatins in Bugula63; bryoanthrathiophene in Watersipora64) that protect bryozoan larvae from predators42,44,45. Another potential role of bacterial secondary metabolites is the chemical defense of early developmental stages during larval settlement and metamorphosis40,65. Such chemical defence may also prevent epibiotic overgrowth of the bryozoan colonies by other bacteria and algae (reviewed in66,67). Similar functions can be assumed for symbiotic prokaryotes of Dendrobeania fruticosa, although experimental evidence is still required.FBs with bacterial symbionts in colonies of D. fruticosa show signs of high specialization. The walls of the funicular body completely isolate its internal cavity from the surrounding zooidal cavity: the cells of the outer layer overlap each other, whereas cells of the inner layer have tight Z-shaped contacts. Such isolation probably creates a specific environment inside the symbiont-containing space that helps maintain a growing population of bacterial symbionts. A massive protein-synthesizing apparatus was observed in the cells of the inner layer. In addition, the ‘pocket’-like structure of the internal cavity of FB and the abundance of cytoplasmic processes, some of which protruded deep into this cavity, contribute to increasing both the general inner surface area of FB and the contact area between its inner cells and bacteria. From their side, bacteria provide this contact by numerous pili. Numerous pits and microvesicles associated with the apical membrane of the inner cells imply an active exchange of substances between the host and its symbionts (e.g., nutrition provided to growing and multiplying bacteria and absorption of the potential wastes they produce).The ultrastructure and functional morphology of FBs in Aquiloniella scabra, the only species in which the ultrastructure of these organs was described, are very similar to those in D. fruticosa. Its FBs also consist of two cell types, but the inner cell layer is formed by one or a few cells, whereas external cells form a multilayered envelope25. In both species, the main function of FBs was considered to be the chambers/organs for symbiont incubation and nourishment.FBs with symbiotic bacteria have been found in several bryozoan species from four different families20,25,47,49,51 (our data). Remarkably, all these families (Bugulidae, Beaniidae, Candidae, Epistomiidae) belong to the clade Buguloidea. Encapsulated aggregations of bacteria within the zooidal cavity were also described in two species of Watersipora from the phylogenetically distant family Watersiporidae (Smittinoidea)62,68. In that case, prokaryotes (“mollicutes”) were also enveloped by flattened bryozoan cells, although the current scarcity of data makes it difficult to compare these cell aggregations with funicular bodies.Among Buguloidea, FBs show the same basic structure, although species described by Lutaud49, Dyrynda and King51, and Mathew with co-authors46 based on light microscopy require re-examination by TEM. Moreover, FBs are in all instances associated with the funicular system of the colony. This similarity could indicate the single origin of the bacterial symbiosis and FBs within Buguloidea. Still, it remains unknown (although rather probable) whether that system transports nutrients from feeding polypides to FBs because no communication between the lacunae of the funicular cords and the FB internal cavity was detected. Another, more likely option, however, is the independent acquisition of bacteria (see also below) and a similar ‘reaction’ of host tissues to invaders, resulting in the formation of bacterial organs (FB) with a similar bauplan. The presence of incapsulated bacteria in non-related Watersipora supports the second interpretation. Answering this question will require both ultrastructural studies and molecular identification of bacteria.As for the initial source of somatic cells and development of FBs, two variants have been suggested. Describing the development of FBs in Bugulina turbinata, Lutaud49 stated that the epithelial cells of the cystid wall were transformed into the inner cell layer of the FB, whereas peritoneal cells formed its external lining. By contrast, while studying Aquiloniella scabra, Karagodina with co-authors25 suggested that bacteria are engulfed by one of the funicular cells, which becomes a ‘bacteriocyte’ that is later enveloped by neighboring funicular cells. In the latter case, FBs are considered to be modified expanded parts of the funicular system. This is consistent with experiments by Sharp and co-authors40, who detected groups of labeled bacteria in the funicular cords of Bugula neritina, and also multiple bacteria developing inside enlarged funicular cords (in fact, very large FBs) in the related Paralicornia sinuosa20.Our TEM study of FBs in D. fruticosa showed that they are not swollen parts of the funicular cords, as was stated by Vishnyakov with co-authors for B. neritina20. It is more likely that the funicular cords and processes of their cells contact the external cell layer of FBs. According to the third scenario that we present here, the inner cell layer of FBs in D. fruticosa, as well as in other studied bugulids, most likely originates from the coelomocytes which accumulate bacteria via phagocytosis. Such solitary cells were described inside the zooidal cavity in B. neritina20. Instead of being digested, engulfed bacteria could trigger the coelomocyte divisions resulting in the formation of the inner cell layer. In contrast, in A. scabra, the coelomocyte can remain single or undergo only a few divisions. We propose that the external cell layer of FB in D. fruticosa originates from the funicular cells because these cell types are ultrastructurally similar. Finally, it is also possible that the exact process and sources of FB formation differ in different species.Multiple and lobed FBs found in two zooids of D. fruticosa could indicate a potential mode of their multiplication. The case of P. sinuosa requires additional study, but currently we believe that its bacteria-bearing ‘funicular cords’ are very large, elongated FBs, as well (see20).Symbiont circulation in the bryozoan life cycleThe taxonomic diversity of bryozoan hosts and their symbiotic bacteria—supported by a variety of sites in the bryozoan zooids and larvae where symbionts have been found—unambiguously point to multiple independent origins of symbiotic associations between bacteria and cheilostome Bryozoa20,25.Rod-shaped bacteria are the most common symbionts in the superfamily Buguloidea. Although superficially similar, these bacteria strongly differ in their maximum size, suggesting the presence of different procaryote species. Thus, bacteria detected in the larvae of Bugulina simplex and in FBs of Aquiloniella scabra can reach 10 μm in length25,69, while healthy symbionts (see below) inside coelomocytes and presumably peritoneal cells of Bugula neritina were only 2.5 μm long20. The maximum length of bacteria in FBs of D. fruticosa never exceeded 5 μm. Coccoid bacteria in the tentacles of B. neritina were 0.5–0.7 μm in diameter20. Else, oval or irregularly-shaped mycoplasma-like α-Proteobacteria were detected in the genus Watersipora62,68,70. By contrast, the symbionts identified in B. neritina and B. simplex belong to γ-Proteobacteria69,71,72.Apart from FBs, prokaryote symbionts were described extracellularly in colonies of different cheilostome species: in vestibular glands of autozooids, inside polymorphic zooids (avicularia)38,39,50, in tentacles and funicular cords20. They have also been found intracellularly: inside coelomocytes, epithelial and peritoneal cells of the body wall, and pharyngeal cells20,50. In addition, Woollacott and Zimmer73 described bacteria in the ‘channels’ of the funicular cords associated with brood chambers. However, the TEM image they published shows bacteria inside large vacuoles of the funicular cells—seemingly not in the lacunae between these cells, recalling the aforementioned idea of a ‘bacteriocyte’. Finally, bacteria have also been found in the pallial sinus of bryozoan larvae (62,68,74; reviewed in72). All these diverse data have led to two opposite views on the acquisition and circulation of symbionts in the bryozoan life cycle.Discovery of bacteria in both colonies and larvae of B. neritina was regarded as possible evidence of their transmission from larvae to adults74. This assumption was experimentally proven using both labeled bacteria and their metabolites (bryostatins) in the larvae and preancestrulae developing from them40. Moreover, the presence of bacteria within brood chambers (ovicells) in this species was considered as proof for the next step—the transition of symbionts from the colony to the incubated larvae, i.e., the vertical transfer of symbionts. Symbiotic bacteria populating larvae (and making them unpalatable for predators) are incorporated into the preancestrular tissues during larval metamorphosis, and then found inside zooidal buds in early colonies (a symbiont association with the host cells was not specified) and funicular cords of rhizoids in adult colonies40.The next step of the bacterial development could be the formation of FBs as a locus of symbiont reproduction. Mature FBs, full of bacteria, were considered to be the starting point for the transfer of prokaryotes (by an unknown mechanism) from the zooid to the brood cavity (via funicular cords associated with both FB and ovicells), and then to the incubated larvae47. Light microscopic data demonstrated: (1) the association of FBs with tube-like funicular cords47, and (2) the presence of groups of ‘bacterial bodies’ (small aggregations of bacteria) inside the ooecial vesicle (membranous-epithelial ‘plug’ that closes the entrance to the ovicell; mentioned in B. neritina47, and shown in images of the related Bugulina flabellata75,76,77). In addition, ultrastructural data proved the presence of bacteria inside funicular cords, more precisely—inside their funicular cells (see above), “extending to the ooecial vesicle” (73, p. 362). Elsewhere, Sharp and co-authors (40, p. 697) used fluorescence microscopy to demonstrate the presence of bacteria “within the ovicells”, and suggested that they are transported there across the colony via funicular cords that also house bacteria. Combined, all these data imply that bacteria move from FBs to the ooecial vesicle, accumulate there, and then somehow enter the brood cavity, which contains a larva, either through or in-between the epithelial cells and the cuticle of the ooecial vesicle. Findings of bacteria inside larvae and adult colonies of two species from the non-related genus Watersipora62,68 further strengthened the hypothesis of vertical transfer, which has subsequently been widely accepted by many authors20,30,47,78. Despite extensive TEM studies, no bacteria have been found inside the funicular cords in B. neritina (Vishnyakov & Ostrovsky, unpublished data), which contradicts the data of Sharp and coauthors40 obtained by fluorescent microscopy. Accordingly, it was suggested that coelomocytes carry symbionts to the ooecial vesicle instead20.Nonetheless, the hypothesis of vertical transfer faces serious objections based on life history, molecular and morphological data. In Dendrobeania fruticosa in the White Sea, for example, larval production occurs predominantly in autumn (mainly in the distal parts of branches, Fig. 1A), and no bacteria are present in colonies during this period. In addition, molecular population studies revealed that B. neritina is a complex of sibling species, both symbiotic and aposymbiotic, some of which live in sympatry, with the horizontal transfer between colonies being the most parsimonious explanation for the distribution of bacteria between siblings79. A study of the genome of symbiotic bacteria showed that they may be able to live outside the host48, which is consistent with the hypothesis of horizontal transfer (which is not the same as environmental transmission, see below).TEM data showed no communication between the FB cavity and lacunae of the funicular cords in the studied species, in particular in B. neritina (Vishnyakov & Ostrovsky, unpublished data), Aquiloniella scabra25 and D. fruticosa (this study). Coelomocytes (and presumed peritoneal cells) with bacteria embedded in their cytoplasm were indeed recorded inside the ooecial vesicle in B. neritina20. Nevertheless, extensive TEM studies of B. neritina ovicells at various stages of placental development (Vishnyakov and Ostrovsky, unpublished data) have not revealed bacteria between placental cells adjoining a developing embryo. The fact that these cells are provided with both tight and adherens junctions (e.g., B. neritina and Bicellariella ciliata73,80), and additionally are covered by a cuticle (albeit thin) raises the question of whether coelomocytes with bacteria and/or bacteria alone can move through the very thick hypertrophied placental epithelium. Interestingly, Miller with co-authors48 detected a gene encoding chitinase in the genome of the symbiont of B. neritina that could potentially be used for cuticle piercing.Another opportunity for the vertical transfer of symbionts is their transport via the supraneural coelomopore during oviposition (see20,81). In this case, free bacteria in the cavity of the maternal zooid could stick to the ovulated oocyte before its transfer into the brood cavity via the coelomopore. However, free bacteria were never recorded in the zooidal cavity. For the Watersipora species, an assumed variant of symbiont transmission is through a strand of mucus extending from the maternal zooid to the released larva and tethering it for a few minutes68.Environmental transmission, when bacteria are acquired from the surrounding seawater, is an alternative option for symbiont acquisition. It may potentially occur either via infection of brooded larvae inside the ovicell by bacteria entering the brood cavity from the external environment or via infection of larvae during the free-swimming period by bacteria from the water column. Published images by Sharp and co-authors40 showed the presence of both symbiotic bacteria and bryostatins both inside the ooecial vesicle and in the peripheral part of the brood cavity, close to the entrance of the ovicell. Although these authors stated that such close “locations of both the bacterial symbionts and the bryostatins demonstrate that the B. neritina–‘E. sertula’ association has a delivery system for both the symbionts and the bryostatins to embryos within the ovicell” (40, p. 699), we argue, based on the above-mentioned data, that this statement remains a probable yet unproven speculation. Our numerous unpublished TEM images indicate the presence of a large number of bacteria filling the brood space between the embryos and the ovicell wall in the brood chambers with and without developing larvae. These bacteria, attracted by some chemical signal(s), can enter the ovicells from outside and infect larvae. Until the transfer of bacteria through the wall of the ooecial vesicle or during oviposition is documented, environmental transmission remains the more probable method. Notably, recent studies on sponge microbiotas showed that the environmental transmission is widespread in this group of suspension feeders82.The hypothesis of external acquisition of bacteria by the bryozoan hosts leaves different infection pathways open. Some of these could potentially develop into vertical transfer. Beyond the infection of larvae, prokaryotes could enter feeding autozooids via the mouth (and further through the intestinal epithelium into the zooidal cavity), through the coelomopore—a presumed entrance for alien sperm76,81,83, or by direct infection of the tentacles (probably by penetration through the outer epithelium).We should stress that FBs were absent in zooidal buds and the youngest zooids with functional polypides in the growing branch tip of one colony of D. fruticosa collected in June. This suggests that bacteria are not transmitted from the older colony parts (and, thus, are not inherited from the founding larvae), but obtained from the external medium since older (and more proximal) zooids had FBs. This idea is supported by the lack of signs of transfer of bacteria between zooids via communication pores and their pore-cell complexes in Dendrobeania fruticosa and Aquiloniella scabra in our TEMs. In contrast, fluorescence microscopy showed symbionts in the non-feeding zooidal bud of the newly-formed small colony of B. neritina40. Bacteria were also present in the preancestrula formed during larval metamorphosis. It remains unknown whether they can move from the preancestrula to the bud along with coelomocytes, with growing funicular cords or both before the formation of transverse walls that isolate newly budded zooids. So, interzooidal transfer to budding sites is possible, and the youngest zooids with functional polypides in the growing tips of D. fruticosa could already receive bacteria too, but FBs were not yet developed. Thus the question of interzooidal/intracolonial transport of bacteria remains open.Two infection pathways—via larvae and by direct penetration through the tissues of the functional polypide, potentially exist in the same species. This is the case in B. neritina, which has morphologically different symbionts in FBs and in the tentacles20. Bacteria in the epithelial wall of the tentacle sheath and ooecial vesicle (see above) could potentially get there via both pathways, or enter the zooid via the coelomopore (third way), subsequently becoming entrapped by coelomocytes or cells of the cystid wall.Finally, the presence of bryozoan sibling species, some of which have symbionts while others do not, and the presence of symbiotic and aposymbiotic colonies within the same species79, suggests that bacteria can be lost and acquired anew at both short- and long-term time scales, as occurs in hermatypic corals and their symbiotic zooxanthellae84,85,86. In this light, it would be important to know whether FBs can develop anew in D. fruticosa after overwintering in the same zooids, or whether they appear only in newly budding zooids. What is the source of bacteria in overwintered colonies? Is it an external infection, or some ‘survivor’-cells (descendants of the bacterial pool from the larva that overwintered inside epithelial cells and/or coelomocytes), or both? This will require further study.Symbiont population dynamics in Dendrobeania fruticosa and its potential driversWhatever the route used by bacteria to enter zooids, they are apparently immediately ‘trapped’ by somatic cells. Free bacteria have never been observed inside the zooidal cavity, another argument against their passage through the wall of the ooecial vesicle to the ovicell.We have shown that regardless of the as-yet-unknown mode of FB development, these temporary organs and the symbionts inside them undergo seasonal changes. Early and mature FBs with non-modified morphology and ‘healthy’ bacteria were found in young zooids only in the colonies collected in June. In one of these colonies, older zooids contained FBs at the initial stage of degradation. In the same month, one colony possessed FBs either at the early-advanced degradation stage (in young zooids) still containing numerous bacteria or at the late-advanced and even terminal stages in old, presumably overwintered zooids. At the initial stage of modification, the slightly developed ‘interlayer’ space between the inner and outer FB cell layers contained fibrils (presumed virus-like particles). Interestingly, the cells of the inner lining apparently engulfed some of the bacterial cells by phagocytosis, supporting our interpretation of the origin of these cells from coelomocytes in this species.One to two months later (August–September), all examined FBs were either at the middle-, or the late-advanced stages of degradation. The number of bacteria in the FB internal cavity distinctly decreased, the inner layer of cells became thinner, and in some regions remained only as a double membrane. The protein-synthesis apparatus was seen only occasionally, and engulfed bacteria were no longer visible inside the inner cells. A wide ILS, formed between the cells of the outer and inner layers, contained abundant putative virus-like particles (Figs. 2, 10). All these FBs were recorded in feeding and non-feeding zooids in the non-growing distal parts of colony branches.Polypide recycling and a seasonal drop of planktonic food alone cannot explain these changes. Firstly, modified FBs were found in zooids with both degenerated and functioning polypides. Secondly, the initial stages of FBs degradation were detected in June when phytoplankton is abundant in the White Sea (e.g.87). We therefore propose the following scenario for the sequence of changes in FBs and their possible causes. In June, newly-formed zooids build funicular bodies containing bacteria that were acquired either from outside or via internal transfer from older colony parts. During that month, FBs begin to degrade. This process continues throughout the rest of the summer. The mid-advanced stage of FB degradation, with few modified bacteria surviving and distributed on the periphery of the FB cavity, was recorded in August. This stage is reminiscent of the final stage in Lutaud’s49 descriptions of the gradual destruction of FBs accompanied by the disappearance of bacterial symbionts in Bugulina turbinata. In late September, FBs change and bacteria disappear, probably through viral lysis (the induction of prophages) (Fig. 2). Young and non-modified FBs were never encountered in August and September, indicating that development of FBs occurs only in young zooids at the growing tips of colony branches in June.Vishnyakov et al.20 recently described the degradation of symbiotic prokaryotes in Bugula neritina and Paralicornia sinuosa accompanied by a change in bacterial morphology similar to bacteriophage-mediated lysis. Degradation process was accompanied by the appearance of polyhedral VLPs in B. neritina and by the formation of structures similar to the so-called metamorphosis-associated contractile complexes (MACs) in P. sinuosa. These complexes are phage-related structures whose activity eventually results in the cell lysis, see88. Although the fate of FBs was not analyzed in their paper, these two VLP variants were observed both inside the bacteria and in the FB internal space by Vishnyakov and co-authors20.In D. fruticosa, presumed VLPs in the form of spherical complexes (as clusters of straight filaments) are present inside bacteria and, together with their fragments, inside the ILS. The filaments were also observed in the free state, frequently curved (apparently flexible) in the internal cavity of FBs. ILS was mostly filled with ‘fibrils’ (potentially representing modified/corrupted filaments) and ‘globules’, although filaments were incidentally recorded inside ILS too. We suggest that, in D. fruticosa, filaments may represent bacteriophage virions. This interpretation is supported by their appearance being associated with the degradation of bacterial cells, as in the case of VLP in B. neritina and P. sinuosa. Nonetheless, the morphology of the spherical complexes built from the filaments in D. fruticosa is unique: they do not resemble any known group of bacterial viruses. We found these putative VLPs inside ILS between the outer and inner FB cell layers. It remains unclear whether they travel there from the FB internal cavity or self-assemble inside ILS from individual filaments that were incidentally met there too. We add that spherical complexes, complete or partial, were recorded inside ILS of the funicular bodies in non-overwintered (collected on 31 September 2019) and presumably overwintered (14 June 2021) zooids.A filamentous morphology is known from only one bacteriophage from the order Tubulavirales, which includes two families Inoviridae and Plectoviridae89. Although inoviruses or plectoviruses have never been reported to assemble in any regular macrocomplexes, the ability of the filamentous Pf phages (inoviruses) of Pseudomonas aeruginosa to form nearly regular liquid crystalline assemblages was recently demonstrated90 (see also review91). Interestingly the formation of such crystals required interactions with bacterial or eukaryotic polymeric molecules such as polysaccharides, DNA and probably mucin90,91.The development of filamentous phages in bacterial cells usually does not kill the cells because these viruses assemble, along with extrusion from the infected bacterium, without disrupting its cell wall (reviewed in92). However, cell death mediated by filamentous prophage induction has been reported in P. aeruginosa due to the emergence of so-called superinfective phage variants93,94,95. Accordingly, the degradation of bacteria in D. fruticosa associated with bacterial viruses is possible.Since the assembly of known filamentous phages is associated with their extrusion from the cell, virions should not accumulate inside bacteria. Although filamentous assembly intermediates may be present (see92), they are not expected to accumulate in such large quantities and/or form superstructures in the bacterial cytoplasm like the ones we found in D. fruticosa (Fig. 9). Our observations revealed nothing resembling the extrusion of a filamentous phage from the surface of a bacterial cell. Therefore, if the described filaments are indeed VLPs, they may represent a new type of bacterial virus.Even though many details remain unknown, we assume that the filaments, ‘globules’, ’fibrils’, and spherical structures in D. fruticosa are of viral origin. Their development following the total disappearance of bacteria in FBs indicates their bacteriophage nature. If so, our observations support the idea that viruses control the number of symbionts in their bryozoan host20, as has been reported in some insects85,86,96. More

Environmental parametersSampling locations, air temperature, water temperature, water depth, salinity, nutrient concentrations (NO3-N, NO2-N, NH4-N, SiO4, PO4-P), and incubation temperatures are shown in Table 1. The air and water temperatures of the studied locations were 11 and 16.6 °C, 3.1 and 3.8 °C, 3.1 and 3.7 °C, 24.5 and 25.9 °C, 24.5 and 18.8 °C, respectively in the Kuroshio Current, SPG surface layer, SPG chlorophyll maximum zone, STG surface layer, and STG chlorophyll maximum zone. At SPG, the values of different parameters were quite similar (p = 0.62, two-tail t-Test; at 5% level of significance) between surface (5 m) and chlorophyll maximum (37 m), indicating the vertical mixing in the upper water column. At STG, the values were relatively different (p = 0.39, two-tail t-Test; at 5% level of significance) between surface (5 m) and chlorophyll maximum (125 m), suggesting the vertical stratification of the water column. The in-situ (water) temperatures (6.4 °C, 0.2 °C, 0.3 °C and 4.2 °C) were lower than the incubation temperatures compared to those of Kuroshio Current, SPG surface layer, SPG chlorophyll maximum zone, and STG chlorophyll maximum zone, while 2.9 °C higher than the incubation temperature of the STG surface layer. Nutrient assays revealed a big difference in nutrient concentrations between SPG and STG; the waters from the station STG were nutrient-poor. The incubation temperatures of the onboard microcosms were 23 ± 1 °C, ~ 4 °C, and 23 ± 1 °C in the case of Kuroshio Current, SPG, and STG, respectively (Table 1).Table 1 Environmental properties of three water samples used in microcosm experiments. Microcosm experiments were conducted on board during the KH-14-2 cruise in May–June 2014.Full size tableBacterial cell densities and cell volumesAt initial incubation periods (12 h to 24 h), the cell densities between the glucose-amended and non-treated microcosms were similar (p = 0.74, two-tail t-Test; at 5% level of significance). Highly significant differences (p  More

Field approachOur searches uncovered 301 papers reporting field studies. After screening the titles abstracts, and full texts, we kept 130 articles for the analysis (Supplementary Fig. 1), from which we obtained 1342 observations regarding 57 Cx. mosquito species from 28 countries and 135 localities (Fig. 1A). Of these 1342 observations, 733 (54.61%) were classified as high quality, (i.e., the number of individuals tested was specified) (Supplementary Tables 1 and 2). The best represented countries were the USA (64.7%, number of observations = 869), Italy (9.3%, n = 125), and Iran (2.9%, n = 39). Based on mosquito field surveillance and individuals testing positive, we concluded that JES is distributed mainly in the Nearctic, Palearctic and Oriental regions (Fig. 1A).Figure 1(A) Weighted Minimum Infection Rates and (B) Weighted Transmission Efficiency of mosquito populations for JES. High-quality data. The size of circles represents the magnitude of the estimates. Map was generated using R software version 4.1.2 with the packages mapdata, maps and tydiverse (https://www.r-project.org) and edited with Inkscape (https://inkscape.org/es/).Full size imageWest Nile virusWNV was detected mainly in the USA (76.5%, number of observations = 826), Italy (4.9%, n = 53) and Iran (3.6%, n = 39) (Fig. 1A). We also recorded 23 species (57%, 41 species) interacting with this virus (Supplementary Tables 1 and 3). Cx. quinquefasciatus became naturally infected in North America [Infection Frequency (IF) = 2.33] (Table 1). We recorded WNV interacting with Cx. tritaeniorhynchus in Asia (IF = 1.02), with Cx. pipiens in Europe (IF = 1.74) and with Cx. antennatus, Cx. neavei, Cx. perexiguus, Cx. perfuscus, Cx. poicilipes, Cx. quinquefasciatus and Cx. tritaeniorhynchus in Africa (IF = 1) (Supplementary Table 3).Table 1 Description of the variables.Full size tableThe highest infection rates were found in North America in Cx. restuans [Standardized minimum infection rate (SMIR) = 56.01], and in Africa and Europe in Cx. pipiens (SMIR = 20.45 and 29.25, respectively). No positive SMIR values were reported in Asian mosquitoes, and Oceanic mosquitoes were not sampled for this virus (Fig. 2A and Supplementary Table 3). The highest infection risk or potential was recorded in species from the USA, such as Cx. restuans (Infection Risk (IR) = 69.50), Cx. pipiens (IR = 55) and Cx. tarsalis (IR = 52.16) (Fig. 3A and Supplementary Table 3). Finally, WNV lineage 1 was detected in Algeria, Turkey, Portugal, Mexico, Tunisia, Iran, Spain and Italy, lineage 2 in Italy, Bulgaria, Greece and the Czech Republic, and lineage 5 in India (Supplementary Table 2).Figure 2(A) Box plots for the Weighted Minimum Infection Rates and (B) Weighted Transmission Efficiency Rates for JES. Boxes indicate 2nd and 3rd quartiles, vertical lines upper and lower quartiles, and horizontal lines the median. Points indicate outliers. The Y axis was transformed to Sqrt (Square root).Full size imageFigure 3JES (A) Infection Risk and (B) Transmission Risk by mosquito species.Full size imageJapanese encephalitis virusJEV was detected mainly in Taiwan (21.6%, n = 24), Korea (18%, n = 20) and Australia (15.3%, n = 17) (Fig. 1A). We found 23 mosquito species interacting with JEV: Cx. vishnui was the one most frequently found to be positive (IF = 1.20), followed by Cx. tritaeniorhynchus (IF = 1.17), Cx. pipiens and Cx. annulus (IF = 0.98) in Asia, while the most susceptible species in Oceania were Cx. sitiens and Cx. gelidus (IF = 0.71) (Supplementary Table 3).The highest SMIR values were recorded in Asia in Cx. rubithoracis (62.38), Cx. annulus (47.68) and Cx. tritaeniorhynchus (28.16) (Fig. 2A and Supplementary Table 3), and Cx. fuscocephala had the highest estimated natural IR (Fig. 3A, Supplementary Table 3). Three genotypes were recorded: genotype I (strain VNKT/479/2007, VNKT/486/2007, and JEV Ishikawa12), genotype III (Tibet-Culex-JEV1-5), and genotype V (K12YJ1174). These were isolated in China, Vietnam, and Japan (Genotype I), Italy, China (Genotype III), and Korea (Genotype V) (Supplementary Table 2).Usutu virusField studies on USUV have been conducted in Europe, Africa, and Asia, most of them in Italy (66.6%, n = 72), Czechoslovakia (11.1%, n = 12) and Slovakia (7.4%, n = 8) (Fig. 1A). Six species were reported to be susceptible to natural infection. Cx. perexiguus had the highest IF and SMIR (1.30) (Supplementary Table 3). In Africa, Cx. antennatus (IF = 1), and in Asia Cx. pipiens (IF = 1) were the most likely to be positive, while Cx. pipiens had the highest IR (5.19) (Fig. 3A, Supplementary Table 3). The recorded strains were USU181_09/USU090-10/USU173_09 (Italy) and USU/Croatia/Zagreb-102/2018 (Italy).St. Louis encephalitis virusThe field studies on SLEV focused on North America (97.7%, n = 43) and Brazil (2.2%, n = 1). Three species were recorded interacting with this virus. Cx. erraticus had the highest IF (2.06), SMIR (2.06) and IR, followed by Cx. quinquefasciatus (North America) (IF = 0.73, SMIR = 1.97) (Fig. 2A, Supplementary Table 3).The highest estimated IR of JES was for Cx. pipiens (Europe), which can be naturally infected with WNV and USUV, followed by Cx. quinquefasciatus (North America), which can be infected with WNV and SLEV (Fig. 3A).Experimental approachExperimental studies were reported in 481 articles. After screening the titles, abstracts, and full texts, as well as opportunistic records, 95 articles remained for the analysis (Supplementary Fig. 2). From these we obtained 189 high quality observations of the TE of JES in 11 countries, 40 localities, and 21 species (Fig. 1B, Supplementary Table 1). The USA was the best represented country (54.4%, n = 103), followed by Germany (13.2%, n = 25) and Australia (12.6%, n = 24). There was, however, a notable lack of information on the vector competence of Cx. mosquitos for JES in many regions of the world, such as Central and South America, and Africa (Fig. 1B).The most common means of infection was oral (94.8%, 395 observations), while the rest were intrathoracic. Intrathoracic infection bypasses the midgut barrier so is not considered natural infection. We therefore carried out the subsequent analyses using only the data on oral infection (Supplementary Table 4).We used a generalised linear model (GLM) for the statistical analysis, which was conducted only on the WNV dataset (strain NY99), the only one with sufficient observations for the purpose (n = 63). We did not find a significant effect of viral titre, temperature, or days post infection on TE. However, more data with a wide range of values is necessary to confirm these observations. On the other hand, we found that the Extrinsic Incubation Period (as DPI) was shorter at higher temperatures (Fig. 4 and Supplementary Table 5).Figure 4Relationship between temperature and Days Post Infection for WNV strain NY99.Full size imageWest Nile virusMosquito populations from many locations on all continents have been studied for their vector competence for this virus, particularly in the USA (60.3% of observations, n = 96), Germany (15.7%, n = 25) and Australia (6.9%) (Fig. 1B). Our bibliographic research revealed 21 species of Cx. with the ability to transmit WNV under laboratory conditions (Supplementary Table 6). Cx. pipiens (North America) and Cx. tarsalis were the most frequently studied species and were the most efficient in transmitting the virus (Transmission Frequency (TF) = 2.33) (Table1). Cx. quinquefasciatus had the highest TF (1.70) in Africa, Cx. modestus in Europe (TF = 1.32), and Cx. annulirostris and Cx. quinquefasciatus in Oceania (TF = 1.48) (Supplementary Table 6).Concerning Standardized Transmission Rates (STE) estimates, Cx. quinquefasciatus had the highest values in the USA (STE = 1.63), Cx. pipiens in Europe (0.90), Cx. tritaeniorhynchus in Asia (1.8), Cx. neavei in Africa (0.17) and Cx. annulirostris in Oceania (2.45) (Fig. 2B, Supplementary Table 6). We found 20 different strains of WNV tested. The TE of the various WNV strains vary considerably, but lineage 1 was more efficient than lineage 2. There were also more studies on the lineage 1 strains (n = 11), which exhibited high variation (Fig. 5).Figure 5Box plots for WNV (A) lineages and (B) strains used to measure Weighted Transmission Rates.Full size imageJapanese encephalitis virusJEV has been studied mainly in mosquito populations from France (45%, n = 20) and Australia (34%, n = 15), but also the United Kingdom, India, Taiwan, New Zealand, and the USA (Fig. 1B). Six mosquito species are capable of transmitting JEV. Cx. pipiens (Europe) had the highest TF (1.85), while Cx. gelidus had high values of STE (1.73) (Fig. 3B and Supplementary Table 6).St. Louis encephalitis virusVector competence for SLEV has been studied in two countries: the USA (93.3%, n = 42) and Argentina (6.6%, n = 3), and 7 mosquito species have been investigated. Cx. nigripalpus was the most efficient in transmitting the virus (TF = 1.60), while Cx. pipiens had the highest STE (0.68) (Figs. 2B, 3B and Supplementary Table 6).Usutu virusStudies have also been conducted on the Usutu virus in mosquito populations in the USA (28.57%, n = 4), the United Kingdom (42.8%, n = 6) and Senegal (25%, n = 4), in particular on Cx. neavei, Cx. pipiens and Cx. quinquefasciatus (TF = 1). Cx. neavei also had the highest STE (0.79) (Fig. 3B).We found reports of JES transmission under laboratory conditions in 22 Cx. species, and natural infections in 32 species (55.1% of the total sample) in the field. Cx. pipiens complex (biotypes quinquefasciatus, pipiens, molestus and pallens) was the most common vector accounting for 36.9% (n = 660) of the experimental observations and 25.7% (n = 1342) of the field observations. With both approaches, WNV was the most common flavivirus, accounting for 80.4% of the field observations and 86.7% of the experimental data (Fig. 1A,B). Only WNV, therefore, had enough observations to make comparison between the experimental and field data possible. We were able to compare 16 mosquito species and found a high positive correlation between TF and IF (R = 0.57, p = 0.02) (Fig. 7).In summary, we found that the species with the highest infection-transmission risk (IRT) for WNV was Cx. restuans, for USUV it was Cx. pipiens (Europe), for SLEV Cx. quinquefasciatus (North America), and for JEV Cx. gelidus (Oceania) (Fig. 6 and Supplementary Tables 2 and 6).Figure 6JES infection-transmission risk by continent and flavivirus.Full size image More

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Identification of windthrow eventsLandsat images from January 1st 2018 to December 31st 2019 were filtered on 20% or less of cloud coverage, and only the least cloudy image at each location was selected to make an image composite covering the entire Amazon region. In total, 395 least cloudy Landsat 8 images within the Amazon boundary during 2018–2019 were selected and displayed in false color (red: shortwave infrared band, green: near-infrared band, blue: red band) on Google Earth Engine for windthrow events identification (Supplementary Fig. 6). Hollow regions on Supplementary Fig. 6 (2.8% of the total area of the Amazon region) indicated that no clear images with 1 year before the identification were displayed in bright green colors (due to reflectance in near-infrared band from the pioneer species). “Old” windthrows account for ~80% of total identified windthrows, and they were verified using historical Landsat images that can go as far as 1984 (when Landsat 5 was launched). “Old” windthrows were validated once they were found with clear shape and more distinguish color on the historical Landsat images (Supplementary Fig. 7c). 10–15% of “old” windthrows without fan-shape were eliminated from this study because it was hard to identify if they were windthrows or other types of forest disturbance. The minimum size of windthrows identified in this study was 25,000 m2. This process generated the location and rough size of 1012 visible (both “old” and “new”) windthrow scars with fan-shaped patch, scattered small disturbance pixels tails, and an area of over 25,000 m2 (Supplementary Fig. 8). Based on a gap-size probability distribution function that simulates the entire disturbance gradient from all sizes of windthrows19, the proportion of total tree mortality represented by large windthrows ( >25,000 m2) identified in this study is 0.5–1.1%.Among 1012 visual identified windthrows, the occurrence year of 125 windthrows were identified using Landsat 5,7,8, MODIS, and TRMM dataset (Supplementary Table 2), and 38 windthrows from these 125 windthrows had clear remote sensing evidence to validate their occurring date (Supplementary Table 3). It is difficult to get the accurate year and date of occurrence of all identified windthrows. Previous studies showed that windthrows in the northwestern Amazon took ~20 years to recover to 90% of “pre-disturbance” biomass from all damage classes while forests in the central Amazon took ~40 years to recover40. The biomass recovery depends on the windthrow severity and time since disturbance33. Based on the recovery time (20–40 years) and the time of windthrow identification (2018–2019), we estimated that these 1012 windthrows most likely occurred within 30 years (between 1990 and 2019), and the estimated occurrence period was validated using the range of the occurrence year (1986–2019) of 125 windthrow cases.Windthrow density dataThe windthrow density shown in Fig. 1b was generated using 1012 windthrow points in QGIS45. We created a 2.5° by 2.5° grid map, and the windthrow density was calculated by counting the number of windthrows in each grid. These values were then converted to a density with units of counts of windthrows per 10,000 km2. We chose 2.5 degrees to aggregate the data to make sure that over 50% of grids have at least 1 windthrow event while still preserving the spatial distribution of mean afternoon CAPE over the Amazon. The contour lines displayed in Fig. 1c were generated using the “Contour” function on the windthrow density map in QGIS.Meteorological dataTo derive the correlation between windthrow density and meteorological variables, we used ERA 5 global reanalysis hourly CAPE on single levels from 1979 to present at 0.25° × 0.25° resolution provided by the European Center for Medium-Range Weather Forecasts. ERA 5 CAPE was computed by considering parcels of air departing at different pressure levels below the 35 kPa level, with maximum–unstable algorithm under a pseudo-adiabatic assumption46. Afternoon mean CAPE map was calculated as the average of hourly CAPE data from 17:00–23:00 UTC (13:00–19:00 local time in Amazon) over all the months between 1990 and 2019. We chose to average CAPE over 30 years because these windthrow events occurred in these 30 years and calculating the average can help capture the overall spatial pattern of CAPE and minimize the influence of interannual climate variability on windthrow events.To project future windthrow density in the Amazon for the end of the 21st century, we analyzed meteorological output from 10 ESMs that participated in CMIP6 (https://www.wcrp-climate.org/wgcm-cmip/wgcm-cmip6). The models used in this research were listed in Supplementary Table 1. We extracted daily surface temperature (tas), specific humidity (huss), surface pressure (ps), temperature (ta) from these models to calculate daily nondilute, near-surface-based, adiabatic CAPE. CMIP6 CAPE was calculated by considering the buoyancy of a near-surface parcel lifted adiabatically to a series of discrete pressure levels (100 kPa to 10 kPa in increments of 10 kPa). CMIP6 CAPE is calculated as follows:$${CAPE}=mathop{sum }limits_{i=1}^{10}{{{{{rm{d}}}}}}p{{{{{rm{H}}}}}}({b}_{i}){b}_{i}$$
(1)
Where ({{{{{rm{d}}}}}}p) = 10 kPa, H is the Heaviside unit step function, and ({b}_{i}=frac{1}{{rho }_{i}}-frac{1}{{rho }_{e,i}}), with ({rho }_{i}) being the parcel density at pressure level i and ({rho }_{e,i}) being the environmental density at pressure level i.The future projections in our analysis were based on SSP585, a high-emission scenario with high radiative forcing by the end of the century. We calculated mean daily CAPE over 1990–2015 as current CMIP6 CAPE and mean daily CAPE over 2070–2099 as future CMIP6 CAPE. Since different approaches were used to calculate ERA 5 CAPE and CMIP6 CAPE47, the absolute CAPE values of the two datasets are not comparable. Therefore, for each ESM model, we scaled future CMIP6 CAPE by multiplying, grid-wise, the delta CAPE generated from an individual model in CMIP6 with the ERA 5 current mean afternoon CAPE (Fig. 1c) as follows:$${delta},{CAPE}=(CAPE_{CMIP6_{,future}},-CAPE_{CMIP6_current})/CAPE_{CMIP6_current}$$
(2)
$$CAP{E}_{scaled_CMIP6_,future}=(1+delta,CAPE)times CAP{E}_{ERA5}{_}_{current}$$
(3)
The delta CAPE indicated the projected increase in CAPE from 1990–2015 to the end of the 21st century. In this way, a scaled CMIP6 future CAPE map was generated for each model, and an ensemble-mean scaled CMIP6 CAPE map over 10 ESM models can be found in Supplementary Fig. 5b. The scaled CMIP6 future CAPE values were within plausible range compared to the ERA 5 current mean afternoon CAPE values, and both current and future CAPE maps were used to produce the increase in area with high CAPE values ( >1023 J kg−1) in Table 1. However, it is worth noting that the scaling with relative changes in delta CAPE (%) is more sensitive to CMIP historical baseline conditions than absolute changes of CAPE (J kg−1), which will likely introduce a larger scaled spread (min/max CAPE changes).The increase in area with storm-favorable environments was calculated as follows:$$Increase=(are{a}_{future}-area_{current})/are{a}_{current}$$
(4)
Where areacurrent is the area of CAPE  > 1023 J kg−1 for current ERA 5 CAPE, and areafuture is the area of CAPE  > 1023 J kg−1 for the scaled CMIP6 future CAPE.A model of windthrow densityWe developed a model based on the relationship between satellite-derived windthrow density and mean afternoon CAPE from the ERA 5 reanalysis over 1990–2019. The non-parametric model provides a look-up table of windthrow density as a function of CAPE within the range of observations. Counts of observed windthrow events and Amazon’s area were separately binned by CAPE using the same bins, producing two histograms of CAPE. The ratio of the former to the latter gives the density of windthrow events (windthrow events per area) as a function of CAPE. To avoid noise at the tails of the histograms, the six CAPE bins were chosen such that each bin would have about the same number of windthrow events (either 168 or 169). The total number of windthrow events is given by the sum over bins of the product of windthrow density and area. The minimum and maximum of current ERA 5 mean afternoon CAPE was 42 and 1549. The minimum CAPE value of the first bin was extended to 0 and the maximum CAPE value of the last bin was extended to infinity under the assumption that the windthrow density is similar for neighboring values. Based on the windthrow density and CAPE relationship used in the model, it is the increase in the area with high CAPE that then leads to an increase in the number of windthrow events.It is worth noting that the future windthrow density produced by models may be underestimated because the windthrow observations within regions with high CAPE were incomplete due to high cloud coverage. Moreover, the non-parametric model makes the conservative assumption that the windthrow density does not increase at higher, as-yet unobserved values of mean afternoon CAPE.Future projections of windthrow densityWe combined the non-parametric relationship (Fig. 2a) with the future CAPE map generated from the ten CMIP6 ESMs (adjusted by ERA 5 mean CAPE values) to estimate the changes in windthrow density at the end of the 21st century. We estimated uncertainties for windthrow density projections by combining information about model-to-model differences. The analysis yielded a set of 10 estimates. The overall windthrow density increase and uncertainty were estimated using the mean increase and one standard deviation from the ensemble of the 10 models. More