Ecology

Inhibition of cell signaling by commensal streptococci
To study how S. mutans ComRS signaling could be impacted by the presence of a competing species, we empirically optimized a dual-species model system (Fig. 1a) in which a strain of S. mutans carrying the promoter regions of comS or comX (PcomS, PcomX) fused to a codon-optimized green fluorescence protein (gfp) reporter gene could be cocultured with wild-type strains of Streptococcus gordonii DL1, Streptococcus sanguinis SK150, or S. sp. A12. All experiments were performed in chemically defined medium (CDM) [38, 39] because activation of the ComRS circuit occurs spontaneously in CDM as cell density increases, with no need for addition of synthetic XIP or overexpression of the gene for the XIP precursor (comS) (Supplementary Fig. 1). CDM is also heavily buffered with phosphate, which is advantageous because ComRS signaling is optimal at neutral pH values [40, 41]. The buffer also prevents the generation of strongly acidic conditions by S. mutans, which is detrimental to the comparatively acid-sensitive commensal Streptococcus spp.
Fig. 1: Loss of S. mutans peptide signaling in presence of competitor.

a An oral Streptococcus spp. competitor strain (blue) was cocultured in chemically defined medium (CDM) with an S. mutans PcomX::gfp reporter strain (green). As cell density of the reporter strain increases during growth, the XIP peptide that originates from the comS gene will be produced and accumulates extracellularly. XIP is then reimported into the cell through the Opp oligopeptide permease, binds to ComR and activates the comX promoter. Additionally, intracellular signaling occurs with ComS binding directly to ComR. The reporter strain harbors a plasmid, pDL278, carrying a copy of gfp that is driven by the comX promoter (PcomX) to monitor ComRS signaling activation. b Cocultures of the S. mutans PcomX::gfp reporter strain grown with either S. mutans UA159 (control, green circles), S. gordonii DL1 (blue squares), S. sanguinis SK150 (orange triangles), or S. sp. A12 (red diamonds). Colored, non-connected symbols represent relative fluorescent units (RFUs) plotted on the left y-axis, while black, connected lines with symbols represent growth of the cocultures over the course of the experiment measured by optical density at 600 nm plotted on the right y-axis. Data are averages from three biological replicates of the experiment. c Percentage of each species remaining within the coculture after 18 h of monitoring, determined by colony forming unit (CFU) plating. The PcomX::gfp reporter strain is represented in the orange bars, while the competitor, listed on the left y-axis, is represented in blue. Average of collected CFUs is shown to the right. Data represent averages from three biological replicates of the experiment that was conducted in panel (b). d Cocultures of the S. mutans PcomX::gfp reporter strain in which 5 µM sXIP was added prior to the start of the experiment. e Cocultures of the S. mutans PcomX::gfp reporter strain that contains a plasmid that overexpresses the XIP peptide precursor, ComS. Control represents the PcomX::gfp reporter that contained an empty vector only.

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When the PcomX::gfp reporter strain was cocultured with wild-type S. mutans UA159 (control), robust ComRS signaling was observed as cell density increased (Fig. 1b). However, when cocultured with a competitor Streptococcus spp., no signal from the S. mutans reporter could be detected above background levels; i.e., the nonspecific fluorescence generated by an S. mutans strain that did not contain a copy of the gfp gene. The lack of fluorescence in the cocultures with commensals was not due to growth inhibition of S. mutans as the reporter strain constituted 10 ± 3%, 37 ± 5%, or 54 ± 3% of the total colony forming units (CFUs) recovered after 18 h of coculturing with S. gordonii DL1, S. sanguinis SK150, or S. sp. A12, respectively (Fig. 1c). The quantity of S. mutans cells in the commensal cocultures compared favorably with the recovery of the reporter strain (54 ± 5%) in coculture with wild-type S. mutans UA159. Of note, the fact that equal proportions of reporter and wild-type S. mutans were recovered from cocultures demonstrated that the presence of the GFP gene fusion did not compromise the fitness of the reporter strain, further verified by growth rate comparisons between wild-type and reporter strains (Supplementary Fig. 1).
Two strategies were implemented to try to recover active ComRS signaling by the reporter strain during cocultivation with commensal streptococci. First, synthetic XIP was added to the cocultures to a final concentration of 5 µM just prior to the beginning of the fluorescence monitoring phase of the experiments, and cocultures were observed as above. No detectable fluorescence signal was recorded above background in the cocultures, with or without exogenously added XIP (Fig. 1d). Second, a plasmid encoding a copy of the XIP precursor comS under the control of a highly expressed constitutive promoter (P23) [42] was introduced into the S. mutans reporter strain; we previously reported that overexpression of comS could strongly activate PcomX [28]. However, no increase in GFP expression was observed in cocultures of the comS overexpressing strain with the commensals, whereas signaling was greatly enhanced when cocultured with strain UA159 as a control (Fig. 1e).
To ensure these observations were not limited to only planktonic growth conditions, we examined S. mutans ComRS signaling in cocultured biofilm populations. While almost all cells harboring the PcomX::gfp reporter were GFP-positive in the control biofilms (coculture of the reporter with wild-type S. mutans), confocal imaging of biofilms containing competitor streptococci uniformly showed that almost no S. mutans cells were expressing detectable GFP (Fig. 2a). However, in some frames (0.22 × 0.22 mm frames, ~30,000 S. mutans cells per frame), a small number of cells (1–3 cells per frame) were GFP-positive. When 3D renderings of these areas within the biofilm were constructed, GFP-positive cells were found close to the substratum (Fig. 2b and Movie S1, same area of biofilm as top panel of Fig. 2b). Also, PcomX-active cells were not necessarily confined to distinct S. mutans microcolonies, and in some cases could be seen adjacent to the competitor streptococci, which carried a constitutively expressed red fluorescent protein (DsRed2) for their identification. To quantify the different types of cells in the biofilm populations, we physically dispersed the biofilms by sonication and analyzed the populations by flow cytometry (Supplementary Fig. 2). About 1 in 10,000 S. mutans cells counted displayed activation of PcomX within the biofilms, which was similar to the proportions of GFP-expressing cells in planktonic growth conditions (Supplemental Table 1).
Fig. 2: S. mutans peptide signaling in coculture biofilms.

a 3D volume projections of imaged biofilms in the XY-orientation (from the top looking down). Each biofilm contains either S. mutans UA159 with a constitutive gfp reporter plasmid (top row), or the PcomX::gfp reporter plasmid (bottom row) that was cocultured with either S. mutans (control; left), S. gordonii DL1 (middle), or S. sp. A12 (right) who all constitutively produce DsRed2. To the right of each expanded color image is the black and white image capture of each individual channel: blue (top), green (middle), and red (bottom). b Zoomed image frames of PcomX-active cells within cocultured biofilms with S. gordonii DL1. The images captured are a single z plane near or at the biofilm substratum. Two different areas of the biofilm (top and bottom rows) were imaged. Each panel represents one color channel of blue (SYTO 42 stained; total cells), green (PcomX::gfp positive cells), or red (S. gordonii P23::DsRed2) followed by the merged image on the far right. The top panel of (b) is the same area of biofilm shown in Movie S1.

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Commensal signaling inhibition is dependent on cell contact
Changes in phenotypes that are observed when two different species of bacteria are cocultured can usually be induced by secreted molecules from one of the bacterial strains [1]. We suspected that molecule(s) secreted by the competitor strains are required for shutting down cell–cell signaling in S. mutans. To explore this hypothesis, we cultured the competitors individually overnight and collected the supernatant fluids after centrifugation. The supernates were then filter sterilized, pH adjusted from ~6.3 to 7.0 with NaOH, and carbohydrate was added back to achieve a final concentration of added glucose to 20 mM. We then inoculated our reporter strain into the commensal supernates and monitored fluorescence activity (Fig. 3a). Surprisingly, ComRS signaling was readily observed in all supernates. In fact, reporter activity tended to be higher in the supernates of competitors compared to controls.
Fig. 3: Cell contact dependence in signaling inhibition.

a Growth and fluorescence of S. mutans PcomX::gfp reporter strain in spent supernatant fluids of either S. mutans UA159 (control, green circles), S. gordonii DL1 (blue squares), S. sanguinis SK150 (orange triangles), or S. sp. A12 (red diamonds). Depiction on top shows methods used to treat supernatant fluids following harvesting and prior to reporter strain inoculation. Overnight cultures of selected strains where centrifuged, spent supernates removed, filter sterilized, the pH was adjusted to 7.0 and 20 mM additional glucose was added. The PcomX::gfp reporter strain was then inoculated and monitored for 18 h in a Synergy 2 multimode plate reader. b Growth of cocultures in a transwell apparatus. The PcomX::gfp reporter strain was first inoculated in 0.1 mL of CDM medium in a 96-well microtiter plate. The transwell plate was then overlaid on top of the 96-well plate, and 0.1 mL of CDM inoculated with either S. mutans UA159 (control, green circles), S. gordonii DL1 (blue squares), S. sanguinis SK150 (orange triangles), or S. sp. A12 (red diamonds) was added to the top chamber, as shown. Cultures of the reporter strain and competitor were separated by a 0.4 µM pore size polycarbonate filter membrane. Fluorescence (RFUs) of the reporter strain was monitored for 18 h.

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In another experiment to confirm these results, we grew competitor and our reporter strains together in a transwell apparatus, so that both bacterial strains shared the same growth medium, but were physically separated by 0.4 µm pore size polycarbonate membrane that would allow passage of small molecule(s) between the chambers (Fig. 3b). Even in the transwell system, cell signaling was robust in cocultures containing competitor species. This result is consistent with data showing that the proximity of live commensal cells with S. mutans prior to signal activation is required for the signaling inhibition.
Impairment of S. mutans cell signaling by oral commensals is conserved across species
We next screened a collection of low-passage oral streptococci that had been previously genome sequenced [43] to determine whether the ability to inhibit S. mutans ComRS signaling was conserved across commensal species and to assess whether the presence or absence of certain genes might contribute to inhibition of peptide signaling. Ten different low-passage clinical isolates of S. gordonii, ten isolates of S. sanguinis, and five isolates of S. sp. A12-related organisms [19] were cocultured with our S. mutans ComRS signaling reporter. The S. sp. A12-related organisms included strains classified as A12-like (A13 and BCC21), as Streptococcus australis (G1 and G2), or as Streptococcus parasanguinis (A1). Interestingly, significant production of GFP by S. mutans was evident when cultured with one isolate of S. sanguinis (BCC64) and with three isolates that were classified as A12-related (BCC21, G1 and G2) (Fig. 4a). However, these results were most likely due to the inability of these isolates to grow well within the CDM medium during the course of the experiment (Supplementary Fig. 3). In fact, after 18 h of monitoring, these isolates comprised  0.1 after 12 h as monitored using a Bioscreen system, see Supplementary Fig. 3) inhibited PcomX activation. Thus, if a commensal strain could grow in CDM, even somewhat poorly, it could completely inhibit ComRS signaling.
Fig. 4: Conservation of ComRS signaling antagonism across oral isolates.

a Relative fluorescent units (RFUs) of the S. mutans PcomX::gfp reporter strain cocultured with clinical oral isolates of either S. gordonii, S. sanguinis or S. sp. A12-like strains. Relative fluorescent units were recorded after coculture inoculation at 1:1 ratio and 12 h of incubation at 37 °C. Results from four biological replicates of the experiment are shown. b RFUs after 12 h of incubation of the PcomX::gfp reporter harbored in various S. mutans clinical isolates. The PcomX::gfp reporter strain was cocultured with either S. mutans UA159 (control; black dots and bars) or an oral competitor streptococci (S. sp. A12, red dots and bars).

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We also tested several genomically and phenotypically diverse isolates of S. mutans [44, 45], both in coculture with our PcomX::gfp reporter in the UA159 background (Supplementary Fig. 4) and against competitor Streptococcus spp., after transformation of the S. mutans strains with the PcomX reporter plasmid (Fig. 4b). Various levels of spontaneous activation of the PcomX::gfp reporter were observed among the different S. mutans strains in monocultures in CDM, consistent with recent reports showing strain-dependent differences in S. mutans peptide signaling [46]. One isolate, Smu107 (R221), had undetectable levels of GFP in monoculture in CDM alone. All others showed activity above baseline. However, when cocultured with S. sp. A12, ComRS signaling was inhibited to an extent similar to that observed with strain wild-type UA159. Therefore, the ability to obstruct ComRS signaling is conserved among isolates of S. gordonii, S. sanguinis, and A12-related streptococci, and inhibition by commensals is similarly conserved in genomically diverse isolates of S. mutans.
Relatively small proportions of live commensal streptococci can inhibit signaling
To verify that the ability of the competitor species to grow (viability) was required for inhibition of peptide signaling, we used two different treatments of the competitor species S. sp. A12 after it was grown to mid-exponential phase: 80 °C for 0.5 h in a heating block (Fig. 5a) or treatment with 4% paraformaldehyde for 1 h at ambient temperature (Fig. 5b). After treatment, the inactivated commensal cells were washed and resuspended in fresh CDM and then mixed with the S. mutans reporter strain to begin the experiment. With heat-treated cells, some ComRS signal activity was evident, but not near the levels seen with S. mutans-only controls. However, when the paraformaldehyde-treated cells were used, the competitor did not inhibit signaling and fluorescence, with levels being similar to the S. mutans-only control. Importantly, we determined that there was a greater number of live cells, by plating and counting CFUs, for the competitor after heat treatment, compared to paraformaldehyde fixing (Supplementary Fig. 5), which likely explains the difference in effects on PcomX activation. These results support that metabolically active and growing competitors are required for S. mutans ComRS signaling obstruction.
Fig. 5: Importance of oral competitor cell density in signaling inhibition.

Cocultures of the S. mutans PcomX::gfp reporter strain with untreated or treated cells by either a 0.5 h heat inactivation at 80 °C or b 1 h suspension in 4% paraformaldehyde. Data represent averages from three biological replicates. c Dilution of an oral competitor streptococci (S. sp. A12) in coculture with the S. mutans PcomX::gfp reporter strain. Legend (top left) refers to the amount of S. sp. A12 within the coculture at the time of initial inoculation. Bottom: addition of either control (UA159; blue squares) or an oral competitor streptococci (S. gordonii DL1; orange triangles) at 4.5 h to a growing culture of the S. mutans PcomX::gfp strain when competence activation was d fully detected, e beginning to be detected, or f not yet detected. See Supplementary Fig. 7 for comparisons at 4.5 and 12 h, specifically.

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Based on the intermediate inhibitory effects seen with reduced proportions of a live competitor on our reporter strain, i.e. with heat-treated cells, we tested whether some minimal proportion of live competitor was required to exert effects on ComRS signaling. We utilized S. sp. A12 and varied the percentage of S. mutans and S. sp. A12 in the cocultures, after determining that the proportions of cells recovered after 18 h were similar to the proportions in the initial inocula (Supplementary Fig. 6). Complete inhibition of S. mutans ComRS signaling occurred when S. sp. A12 constituted ≥6.3% of the initial inoculum (Fig. 5c). At 3.1 or 1.6% of S. sp. A12, reporter activity was detectable, but at lower levels than when no S. sp. A12 was present. No difference in S. mutans reporter activity was observed when  More

Hundreds of people in the nineteenth-century United States reported seeing the Gloucester Sea Serpent (above), which was probably a marine creature bedecked with fishing debris. Credit: Museum of Fine Arts, Boston

Fisheries
30 September 2020

Centuries-old ‘unidentified marine objects’ hint that sea creatures have been getting entangled in fishing lines since before the invention of plastic.

‘Sea serpents’ spotted around Great Britain and Ireland in the nineteenth century were probably whales and other marine animals ensnared in fishing gear — long before the advent of the plastic equipment usually blamed for such entanglements.
The snaring of sea creatures in fishing equipment is often considered a modern phenomenon, because the hemp and cotton ropes used in the past degraded more quickly than their plastic counterparts. But Robert France at Dalhousie University in Truro, Canada, identified 51 probable entanglements near Great Britain and Ireland dating as far back as 1809.
France analysed 214 accounts of ‘unidentified marine objects’ from the early nineteenth century to 2000, looking for observations of a monster that had impressive length, a series of humps protruding above the sea surface and a fast, undulating movement through the water. France says that such accounts describe not sea serpents but whales, basking sharks (Cetorhinus maximus) or other marine animals trailing fishing gear such as buoys or other floats.
Such first-hand accounts could help researchers to construct a better picture of historical populations of marine species and the pressures they faced, France says. More

Models are developed for a specific purpose and by the need to address certain questions about real systems. Models therefore focus on aspects of the real system that are considered important in answering these questions. Consequently, different models exist for the same system. Without knowing its purpose, it is impossible to assess whether a model’s outputs can be used to support decisions affecting the real world.
Model purposes fall into three main categories: demonstration, understanding, and prediction. Given these different purposes, models also reflect different scopes. Models for demonstration are designed to explore ideas, demonstrate the consequences of certain assumptions, and thereby help communicate key concepts and mechanisms. For example, at the onset of the Covid-19 pandemic simple mathematical models were used to demonstrate how lowering the basic reproduction value, R0, would lead to “flattening the curve” of infections over time. This is an important logical prediction that helped to make key decisions, but it does not, and cannot, say anything about how effective interventions like social distancing are in reducing R0.
Models for understanding are aimed at exploring how different components of a system interact to shape observed behavior of real systems. For example, a model can mechanistically represent movement and contact rates of individuals. The model can be run to let R0 emerge and then explore how R0 changes with interventions such as social distancing. Such models are not necessarily numerically precise, but they provide mechanistic understanding that helps to evaluate the consequences of alternative management measures.
Finally, models for prediction focus on numerical precision. They tend to be more detailed and complex and rely heavily on data for calibration. Their ability to make future projections therefore depends on the quality of data used for model calibration. Such models still do not predict the future with precision, as this is impossible11, but they provide important estimates of alternative future scenarios12.
Decision makers can benefit from all three types of models if they use them according to their given purpose. Modelers should therefore state a model’s purpose clearly and upfront. By asking this first screening question, one of the most common misuses of models can be prevented: using them for purposes for which they were not designed13. More

Overview of molecular determination of Anopheles species
Out of the 3556 Anopheles mosquitoes, 87.1% (3099/3556) were determined by species-specific PCRs or multiplex-PCRs and sequencing as major species An. gambiae sensu stricto (hereafter referred to as An. gambiae) (1440), An. arabiensis (718), and An. funestus sensu stricto (hereafter referred to as An. funestus) (941) in the five study sites (Fig. 1, Table 1, Supplementary Fig. S1). A subset of 21 randomly selected individuals from each major species identified by PCRs were confirmed by ITS2 sequencing based on similarity ( > 98%) to the sequences of anopheline voucher species retrieved from NCBI GenBank database (Supplementary Fig. S2).
Figure 1

Maps of sampling sites and Anopheles species distribution in western Kenya. (a) distribution of Anopheles major species; (b) distribution of Anopheles rare species. Pie-chart showed the abundance of Anopheles specimens for each site. The maps were generated using ArcGIS Pro 2.6 software. Map source: ESRI, CGIAR, and USGS (available at: www.esri.com).

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Table 1 Species composition of Anopheles mosquitoes determined by molecular approaches in western Kenya.
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The remaining 457 collected anophelines (12.9%) were classified into 18 rare species groups based on ITS2 sequence homology. Except for two species groups (An. sp.18 and An. sp.19), the ITS2 sequences of all the species were identified as different species based on their similarity ( > 98%) to the sequences of Anopheles voucher species retrieved from NCBI GenBank database (Supplementary Fig. S2). The ITS2 sequences of two species could not match with similarity  > 98% threshold to reference anopheline sequences or known vector species in GenBank databases, suggesting the existence of novel cryptic species.
Pairwise comparison of ITS2 sequence similarities of the 21 Anopheles species indicated that except for one pair with 98.5% identity between An. gambiae and An. arabiensis, all pairs showed a similarity of 90% or less with confirmed species classifications (Supplementary Table S1). Phylogenetic tree analysis indicated that the 21 species belong to two different Subgenus (Subgenus Cellia Theobald and Subgenus Anopheles Meigen) in five species series groups, including Myzomyia, Neocellia, Pyretophorus, Cellia, and Myzorhynchus series (Fig. 2, Supplementary Table S2). The two new species An. sp.18 and An. sp.19 as well as An. sp.17 (a recently reported species13) belong to two different series groups, and An. sp.18 belongs to a different Subgenus (Subgenus Anopheles Meigen). The ITS2 sequence of An. sp.9 is homogenous with that of An. theileri (GenBank acc. JN994151) and An. sp. 9 BSL-2014 (GenBank acc. KJ522821)14. The ITS2 sequences obtained in the study are available in GenBank with accession numbers: MT408564-MT408584.
Figure 2

Molecular phylogenetic analysis of ITS2 sequences by Maximum Likelihood method. The phylogenetic tree was constructed using MEGA 7.0 software based on the Kimura 2-parameter model with 1000 bootstrap replicates. Pink filled diamonds showed the major species; red filled circles indicated the novel cryptic species tested positive for Plasmodium infections.

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Comparison of morphological and molecular identifications
Of the 3556 mosquitoes tested, 3226 (90.7%) samples with available morphological data were used to evaluate the accuracy of morphological identification as compared to molecular identification. Among the 3226 mosquito samples, 2192 (67.9%) individuals were morphologically identified as An. gambiae s.l., 938 (29.1%) as An. funestus, 94 (2.9%) as An. coustani, and the remaining 2 (0.1%) as An. pharoensis (Table 2). The An. gambiae s.l. complex and An. funestus group (An. funestus, An. cf.rivulorum, and An. leesoni) had a similar percentage of matches (gambiae complex: 85.8%, 1881/2192, funestus group: 85.2%, 799/938) between molecular assay and morphological identification, while only 53.2% of specimens morphologically identified as An. coustani were confirmed by the molecular assay (50/94). Based on molecular assays, An. gambiae s.l. complex had the lowest misidentification (4.3%), followed by An. funestus group (6.8%), while 18.0% (11/61) An. coustani specimens were morphologically misidentified as An. gambiae complex (9) or An. funestus (2). Based on morphological identification, less than 15% of specimens morphologically assigned to An. gambiae complex (14.2%) or An. funestus group (14.8%) were identified as other species, while there were 44 specimens morphologically assigned to An. coustani (46.8%) that were classified by molecular assay into 9 anopheline species, including An. rufipes (17), An. funestus (7), and An. gambiae complex (6). Overall, more than 60% (264/427) of the rare species were morphologically misidentified as An. gambiae s.l. Specifically, nearly 20% (81/427) and 7.2% (31/427) of rare species were misidentified as An. funestus and An. coustani, respectively, whereas only 11.7% (50/427) of the rare species were correctly identified as An. coustani. Altogether, 84.0% (2710/3226) identification alignment was observed between the morphological and molecular analysis (Table 2).
Table 2 Comparison of morphological and molecular identifications in Anopheles mosquitoes from western Kenya.
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Comparison of Anopheles species distributions and diversity
Overall, the three major species were found in all five study sites but at varying proportions. Anopheles funestus accounted for a large proportion (44.7–98.2%) of species observed throughout the five study sites (Fig. 1A, Table 1). Anopheles gambiae was the predominant species in two highland sites (56.7% in Emutete and 61.3% in Iguhu) and one lowland site (47.73% in Kombewa), whereas An. gambiae was nearly absent in Homa Bay (0.4%), a lowland site, and Kisii (0.6%), a highland site. An. arabiensis, was observed in high proportion in lowland areas (Homa Bay: 70.8% and Kombewa: 24.9%) than in highland areas, which ranged from 1.8% (Emutete) to 5.2% (Iguhu).
Seventeen of 18 rare species were identified in the highland areas, whereas only six rare species were detected in the lowland areas, suggesting that cryptic species might be more related to the sympatric An. gambiae than An. arabiensis. In lowland sites, the most abundant rare anopheline species was An. sp.15 (n = 17), followed by An. rufipes (n = 14) and An. cf.rivulorum (n = 14), whereas multiple rare species (such as An. christyi, An. sp.1, and An. sp.17) were identified in the highlands (Fig. 1B, Table 1).
A significantly higher species diversity was observed in the highland areas than in the lowland areas (Shannon index H, t-test, t =  − 6.59, df = 3419, p  More

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