Ecology

Results of chemical analysisThe results of the study showed that the concentrations of Cd and Pb among all analyzed fruit samples (n = 242) were below the associated LOQs in only 87 and 96 samples, respectively. Similarly, in vegetable samples (n = 128) we found that Cd and Pb concentrations were below the LOQ in 31 and 69 samples, respectively. The levels of the Cd and Pb in the analyzed food samples were compared and contrasted with the maximum levels in foodstuffs regulated by legal acts: Commission Regulation (EU) No 488/2014 of 12 May 2014 amending Regulation (EC) No 1881/2006 as regards maximum levels of cadmium in foodstuffs and Commission Regulation (EU) 2015/1005 of 25 June 2015 amending Regulation (EC) No 1881/2006 as regards maximum levels of lead in certain foodstuffs3,4. It was found that in 12 food samples, the Cd content exceeded the maximum acceptable level. Among the fruit samples, this result was observed in: frozen raspberries (n = 1; 122% of maximum level) and frozen strawberries (n = 1; 114% of maximum level). In the case of vegetables, this result was observed in: fresh beetroots (n = 2; 203% and 670% of maximum level), frozen carrot (n = 1; 113% of maximum level), fresh celery (n = 4; 130%, 150%, 345%, 356% of maximum level) and processed tomatoes (n = 3; 102%, 112%, 134% of maximum level). The maximum permissible Pb level was exceeded in 3 analyzed food samples: fresh beetroot (n = 1; 135% of maximum level), frozen carrot (n = 1; 117% of maximum level) and 1 sample of frozen tomatoes in which the Pb concentration was up to 1074% of the acceptable limit (Table 5).Table 5 The number and type of food samples in which the maximum level of Cd or Pb has been exceeded.Full size tableTables 6 and 7 present the mean and SD, as well as the minimum and maximum values for the Cd and Pb contents in each of the analyzed fruits (Table 6) and vegetables (Table 7). Heavy metals concentrations were reported in mg/kg f.m. (fresh mass) in the fresh, frozen and processed products, while the content of Cd and Pb in dried products were presented in mg/kg d.w. (dry weight). Lack of a value in the tables means that the Cd or Pb value was below the LOQ for that particular sample.Table 6 The mean value, standard deviation, minimum and maximum values ​​of Cd and Pb concentrations in particular types of fruit samples.Full size tableTable 7 The mean value, standard deviation, minimum and maximum values of Cd and Pb concentrations in particular types of vegetable samples.Full size tableThe analysis of Cd and Pb contents in all food products is necessary due to the possibility of assessing the health risks associated with consumption of contaminated ready-to-eat different types of food. A review of the scientific literature showed that the issue of food contamination with heavy metals is discussed by several researchers. However, they mostly include only fresh fruits and vegetables. Additionally, there is a little data concerning the level of heavy metals contamination of vegetables and fruits cultivated in other European countries in the available literature. Consequently, the results presented in this paper may form the basis for further research on the scale of food contamination with heavy metals such as Pb and Cd.Among fruits such as apples, pears, raspberries and strawberries, the highest average values of both Cd and Pb were observed in dried products (Cd: 0.023, 0.015, 0.116, 0.131 mg/kg d.w., respectively; Pb: 0.127, 0.036, 0.111, 0.161 mg/kg d.w., respectively). In cranberry samples, the highest levels of Cd were determined in fresh fruits (0.008 mg/kg f.m.), while Pb—in processed products (0.01 mg/kg f.m.). In the case of grape samples, the same average Cd concentration was recorded in both dried and fresh products (0.001 mg/kg), while the highest Pb content was observed in processed products (0.07 mg/kg f.m.). In most fruit samples the lowest average Cd concentrations were determined in processed products (grapes, pears, raspberries and strawberries—0.0004, 0.0008, 0.009, 0.003 mg/kg f.m., respectively), while Pb—in fresh fruits (cranberries, grapes, pears—0.004, 0.005, 0.008 mg/kg f.m.) or processed (raspberries and strawberries—0.011 and 0.006 mg/kg f.m.). In apple samples, the same average Pb value was recorded in both fresh fruit and processed products (0.009 mg/kg f.m.).The content of Cd and Pb in fruits, in the results available in the literature, is very diverse. The demonstrated average Cd content in apples (0.001 mg/kg f.m.) is lower compared to studies from other regions of the world, including Great Britain (0.002 mg/kg f.m.)23. The amounts of Cd in raspberries and strawberries tested in Poland were higher compared to those investigated by Norton et al. (2015) (0.002 mg/kg f.m. vs 0.011 mg/kg f.m. and 0.002 mg/kg f.m. vs 0.018 mg/kg f.m.)23. Additionally, in samples collected in Turkey and Serbia, the Cd content in the analyzed products was below the LOQ24,25.Our results of Pb values in fruit samples are similar to those reported by some researchers and the range of values presented for this element in other analyses were very wide. However, as in the case of Cd content in apples purchased in Poland, Pb concentrations in these fruits (0.009 mg/kg f.m.) were also lower than other studies—minimum of 200%23. The average Pb content in grapes (0.009 mg/kg f.m.) was comparable to that obtained by Bağdatlıoğlu et al. (2010) (0.006 mg/kg f.m.)24. The results of author’s research regarding the content of Pb in raspberries (0.012 mg/kg f.m.) exceeded 2.5 times those published by Norton et al. (2015)23. Pb concentrations in strawberries (0.009 mg/kg f.m.) compared to other studies are in their lower range (0.010 mg/kg–0.027 mg/kg f.m.)23,24.The highest average concentrations of Cd were determined in fresh vegetables (beetroot and celery—0.235 and 0.152 mg/kg f.m., respectively) and dried—carrots and tomatoes (0.2 and 0.103 mg/kg d.w.), while Pb—in frozen vegetables (beetroots and tomatoes—0.173 and 0.294 mg/kg f.m.), as well as dried (carrots and celery—0.206 and 0.259 mg/kg d.w.). For most samples, the lowest average Cd and Pb levels were observed in processed products (beetroots, carrots, celery). Exceptions were samples of tomatoes—the lowest average Cd and Pb concentration values were observed in fresh foodstuffs (0.003 and 0.016 mg/kg f.m., respectively).Analyses conducted by other scientists indicate lower average Cd content in fresh beetroots (0.018–0.09 mg/kg f.m.)23,26 and higher by almost 600% in the case of Pb (0.58 mg/kg f.m.)26 compared to our research (Cd—0.235 mg/kg f.m.; Pb—0.095 mg/kg f.m.). Only the British study has shown lower Pb content (0.033 mg/kg f.m.)23. Our results—concentration of Cd (0.041 mg/kg f.m.) and Pb (0.027 mg/kg f.m.) in fresh carrot samples were similar to those obtained by other authors from the same territory in Poland, but also those from Great Britain, China or Brazil—Cd values ranged from 0.014 mg/kg f.m. to 0.03 mg/kg f.m., while Pb from 0.023 mg/kg f.m. to 0.971 mg/kg f.m.23,26,27,28. In the scientific literature we found only individual articles regarding celery heavy metal contamination. Guerra et al. (2012) showed 3 times lower Cd content in this vegetable—0.05 mg/kg f.m.26. The concentration of Pb in Brazilian research indicates higher content (0.47 mg/kg f.m.) than those obtained in this study (0.031 mg/kg f.m.)26. Tomatoes are the most frequently analyzed products, probably due to the easiness and simplicity of processing. Our analysis showed relatively low concentration of Cd and Pb in fresh tomatoes (Cd—0.003 mg/kg f.m.; Pb—0.016 mg/kg f.m.). In the most available scientific data Cd levels were in the range of 0.028 mg/kg f.m. to 0.033 mg/kg f.m., and Pb from 0.078 mg/kg f.m. to 0.18 mg/kg f.m.26,28. Only Norton et al. (2015) and Bagdatlioglu et al. (2010) noted lower or equal Cd and Pb values in the corresponding product23,24.Massadeh et al. (2018) in Jordan determined Pb and Cd of various canned fruits and canned vegetables including canned juice (pineapple), canned tomato sauce, canned whole carrots and canned green beans. They showed metal concentration levels in the samples were in the range of 0.50–0.60 mg/kg f.m. for Cd and 2.6–3.0 mg/kg f.m. for Pb29. These results significantly exceed the values shown in present study, as well as the results presented by Domagała-Świątkiewicz and Gąstoł (2012) in the analysis of vegetable juices (beetroot, carrot, celery)30.The high contamination found in vegetables might be closely related to the pollutants in irrigation water, farm soil, fertilizers and also industrial and low pollution household emissions. Differences in levels of contamination between fruits and vegetables may result from the specificity of the geographical area from which they are collected, their diverse capacity to accumulate heavy metals, as well as the way they are processed. It should be pointed out that in polluted environments (soil, water, and air), the presence of toxic metals in elevated concentrations is not uncommon. Due to the structure of consumption of various groups of food products both in Poland and other countries, a significant risk of exposure to heavy metals is associated with the consumption of fruits and vegetables, which are one of the main elements of the diet. Unfortunately, complete elimination of elements such as Cd or Pb from these products is impossible, and the technological processes used in food production can only remove a small part of the impurities from selected products or even contribute to their increased contamination. Thus, there is a need for regular monitoring of heavy metals on every kind of foodstuff, not only in fresh products, in order to estimate the health risk from heavy metals in the human food chain.Statistical analysisANOVAFor the purpose of ANOVA carried out to detect significant differences in the heavy metal concentrations of the four types of food (fresh, dried, frozen, and processed), samples with concentration value below the LOQ were removed from the analysis. In the case of Cd concentration, the value of F statistic was 11.15 for fruits and 4.049 for vegetables, leading to significant results with p-values below 0.001 and 0.01 respectively. For the of Pb concentration, the ANOVA results were even more extreme with F values of 56.59 for fruits and 7.13 for vegetables with associated p-values being below 0.001 in both cases. These results show that there is strong evidence to believe that mean Cd and Pb contents in the four types of fruits and vegetables are not equal (Table 8).Table 8 Analysis of variance (ANOVA) for variates in four groups.Full size tableOutlier analysisThe boxplots depicted in Fig. 1 were used to illustrate the outlier analysis for Cd and Pb. Each plot shows the median of the observations along with the lower quartile (Q1) and the upper quartile (Q3). The highest and the lowest observations are shown by the whiskers. From Fig. 1a, there appears to be two outliers in the dried fruits with values 0.277 and 0.210. From Fig. 1b, there seems to be six outliers in the fresh vegetables with values of 0.203, 0.670, 0.260, 0.690, 0.300 and 0.712. In Fig. 1c, we see two outliers in the processed fruits with values of 0.127 and 0.047. Finally, Fig. 1d shows that there is one one outlier in the frozen vegetable category with the value of 0.537.Figure 1Outlier analysis in case: Cd concentration in: (a) fruits, (b) vegetables, and Pb concentration in: (c) fruits, (d) vegetables.Full size imageOutliers associated with high Cd and Pb values in fruit and vegetable samples may be the result of sample contamination during technological processes or vegetables/fruits cultivation in a polluted agricultural area.Post-hoc multiple comparisonSince the ANOA results indicated significant differences among the mean concentrations of Cd and Pb both in fruits and vegetables, to further detect the specific different means, the Tukey HSD test22 was applied. The results are presented in Fig. 2. For the Cd concentration, comparison of all pairs of means indicated that the content of Cd in dried fruits is significantly different from mean concentrations of other types of food namely fresh, frozen, and processed fruits, see Fig. 2a. In the case of vegetables, the mean Cd contents of fresh and processed vegetables are different, see Fig. 2b, although mean Cd content of frozen and fresh vegetables are also significantly different if a significance level of 10% is used. Upon analyzing the mean concentrations of Pb in fruits, we found that the mean content of dried fruits was significantly different from the other three types, namely fresh, frozen and processed, see Fig. 2c. For the Pb concentrations in vegetables, a highly significant difference was detected between the means of processed and dried vegetables. In addition, mean Pb concentrations of fresh versus dried and processed versus frozen vegetables were significantly different, see Fig. 2d.Figure 2Post-hoc Multiple Comparison Tukey-Test of Cd and Pb in all samples of fruits and vegetables; differences in Cd mean concentration of: (a) fruits, (b) vegetables; differences in Pb mean concentration of: (c) fruits, (d) vegetables.Full size image More

1.Stearns, S.C. The Evolution of Life Histories. https://doi.org/10.1046/j.1420-9101.1993.6020304.x (Oxford University Press/Wiley, 1992).2.Dingle, H. The evolution of life histories. in Population Biology (Wöhrmann K., Jain S.K. eds.). https://doi.org/10.1007/978-3-642-74474-7_9 (Springer, 1990).3.Yang, L., Walck, J. L. & El-Kassaby, Yousry, A. Roles of the environment in plant life-history tradeoffs. in Advances in Seed Biology (Jimenez-Lopez, J.C. ed.) 674. https://doi.org/10.5772/intechopen.70312.4.Hamrick, J. L. & Godt, M. J. W. Effects of life history traits on genetic diversity in plant species. Philos. Trans. R. Soc. Lond.. Ser. B Biol. Sci. 351, 1291–1298 (1996).5.Hobbs, R. J., Higgs, E. & Harris, J. A. Novel ecosystems: Implications for conservation and restoration. Trends Ecol. Evol. 24, 599–605 (2006).Article 

Google Scholar 
6.Prozherina, N., Freiwald, V., Rousi, M. & Oksanen, E. Interactive effect of springtime frost and elevated ozone on early growth, foliar injuries and leaf structure of birch (Betula pendula). New Phytol. 159, 623–636 (2003).CAS 
Article 

Google Scholar 
7.Zvereva, Elena, L., Roitto, M. & Kozlov, M. V. Growth and reproduction of vascular plants in polluted environments: A synthesis of existing knowledge. Environ. Rev. 18, 355–367 (2010).8.Niinemets, Ü. Responses of forest trees to single and multiple environmental stresses from seedlings to mature plants: Past stress history, stress interactions, tolerance and acclimation. For. Ecol. Manag. 260, 1623–1639 (2010).Article 

Google Scholar 
9.Possen, B. J. H. M. et al. Adaptability of birch (Betula pendula Roth) and aspen (Populus tremula L.) genotypes to different soil moisture conditions. For. Ecol. Manag. 262, 1387–1399 (2011).10.Řehounková, K. & Prach, K. Life-history traits and habitat preferences of colonizing plant species in long-term spontaneous succession in abandoned gravel–sand pits. Basic Appl. Ecol. 11, 45–53 (2010).Article 

Google Scholar 
11.Franiel, I. The Biology and Ecology of Betula pendula Roth on Post-Industrial Waste Dumping Grounds: The Variability Range of Life History Traits (Silesia University, 2012).
Google Scholar 
12.Kompała-Ba̧ba, A. & Ba̧ba, W. Participation of grasses (Poaceae) in the communities, which developed on iron smelter affected lands in the Silesian Uplands. Fragm. Florist. Geobot. Pol. 20, 267–284 (2013).13.Kompała-Bąba, A. & Bąba, W. The spontaneous succession in a sand-pit—The role of life history traits and species habitat preferences. Polish J. Ecol. 61, 13–22 (2013).
Google Scholar 
14.Darbah, J. N. T. et al. Impacts of elevated atmospheric CO2 and O3 on paper birch (Betula papyrifera): Reproductive fitness. Sci. World J. 7, 240–246 (2007).Article 

Google Scholar 
15.Alvarez-Cansino, L., Zunzunegui, M., Díaz Barradas, M. C. & Esquivias, M. P. Gender-specific costs of reproduction on vegetative growth and physiological performance in the dioecious shrub Corema album. Ann. Bot. 106, 989–998 (2010).16.Zvereva, E. L., Roitto, M. & Kozlov, M. V. Growth and reproduction of vascular plants in polluted environments: A synthesis of existing knowledge. Environ. Rev. 18, 355–367 (2010).CAS 
Article 

Google Scholar 
17.Franiel, I. & Babczyńska, A. The growth and reproductive effort of Betula pendula Roth in a heavy-metals polluted area. Polish J. Environ. Stud. 20, 1097–1101 (2011).CAS 

Google Scholar 
18.Lancaster, L. T., Morrison, G. & Fitt, R. N. Life history trade-offs, the intensity of competition, and coexistence in novel and evolving communities under climate change. Philos. Trans. R. Soc. B Biol. Sci. 372, (2017).19.Obeso, J. R. Costs of reproduction in ilex aquifolium: Effects at tree, branch and leaf levels. J. Ecol. 85, 159–166 (1997).Article 

Google Scholar 
20.Obeso, J. R. The costs of reproduction in plants. New Phytol. 155, 321–348 (2002).Article 

Google Scholar 
21.Cipollini, M. L. & Stiles, E. W. Costs of reproduction in Nyssa sylvatica: Sexual dimorphism in reproductive frequency and nutrient flux. Oecologia 86, 585–593 (1991).ADS 
Article 

Google Scholar 
22.Seidling, W., Starfinger, U. & Stöcklin, J. Plant population ecology. Prog. Bot. 55, 345–370 (1994).Article 

Google Scholar 
23.Possen, B. J. H. M. et al. Variation in 13 leaf morphological and physiological traits within a silver birch ( Betula pendula ) stand and their relation to growth. Can. J. For. Res. 44, 657–665 (2014).Article 

Google Scholar 
24.Körner, C. Limitation and stress—Always or never?. J. Veg. Sci. 14, 141–143 (2003).
Google Scholar 
25.Giuliani, C., Lazzaro, L., Mariotti Lippi, M., Calamassi, R. & Foggi, B. Temperature-related effects on the germination capacity of black locust (Robinia pseudoacacia L., Fabaceae) seeds. Folia Geobot. 50, 275–282 (2015).26.Wolkovich, E. M. et al. Warming experiments underpredict plant phenological responses to climate change. Nature https://doi.org/10.1038/nature11014 (2012).Article 
PubMed 

Google Scholar 
27.Koski, V. & Rousi, M. A review of the promises and constraints of breeding silver birch (Betula pendula Roth) in Finland. For. Int. J. For. Res. 78, 187–198 (2005).
Google Scholar 
28.Marguí, E., Queralt, I., Carvalho, M. L. & Hidalgo, M. Assessment of metal availability to vegetation (Betula pendula) in Pb–Zn ore concentrate residues with different features. Environ. Pollut. 145, 179–184 (2007).Article 

Google Scholar 
29.Hynynen, J. et al. Silviculture of birch (Betula pendula Roth and Betula pubescens Ehrh.) in northern Europe. Forestry 83, 103–119 (2010).30.Frouz, J. et al. Development of canopy cover and woody vegetation biomass on reclaimed and unreclaimed post-mining sites. Ecol. Eng. 84, 233–239 (2015).Article 

Google Scholar 
31.Řehounková, K., Lencová, K. & Prach, K. Spontaneous establishment of woodland during succession in a variety of central European disturbed sites. Ecol. Eng. 111, 94–99 (2018).Article 

Google Scholar 
32.Franiel, I. & Więski, K. Leaf features of silver birch (Betula pendula Roth). Variability within and between two populations (uncontaminated vs Pb-contaminated and Zn-contaminated site). Trees 19, 81–88 (2005).33.Kozlov, M. V. & Zvereva, E. L. Industrial barrens: Extreme habitats created by non-ferrous metallurgy. Rev. Environ. Sci. Bio/Technol. 6, 231–259 (2007).CAS 
Article 

Google Scholar 
34.Zvereva, E. L. & Kozlov, M. V. Growth and reproduction of dwarf shrubs, Vaccinium myrtillus and V. vitis-idaea, in a severely polluted area. Basic Appl. Ecol. https://doi.org/10.1016/j.baae.2004.11.003 (2005).35.Kozlov, M. V. Pollution resistance of mountain birch, Betula pubescens subsp. czerepanovii, near the copper–nickel smelter: Natural selection or phenotypic acclimation? Chemosphere 59, 189–197 (2005).36.Kondracki, J. Geografia fizyczna Polski. (Physical Geography of Poland). (PWN (in Polish), 2001).37.Pełka-Gościniak, J. Environmental aspects of relief transformation (Silesian Upland, Southern Poland). Environ. Socio-Econ. Stud. 2, 13–20.38.Tomusiak, R. et al. Age tables for silver birch (Betula pendula Roth) trees for early succession stands on abandoned agricultural lands. Sylwan 158, 579–589 (2014).
Google Scholar 
39.Szymkiewicz, B. Tablice Zasobności i Przyrostu Drzewostanów Ważniejszych Gatunków Drzew Leśnych. (Państwowe Wydawnictwo Rolnicze i Leśne, 2001).40.Dubois, H., Verkasalo, E. & Claessens, H. Potential of Birch (Betula pendula Roth and B. pubescens Ehrh.) for forestry and forest-based industry sector within the changing climatic and socio-economic context of Western Europe. Forests 11, 336 (2020).41.Ostrowska, A., Gawliński, S. & Szczubiałka, Z. Metody Analizy i Oceny Właściwości Gleb i Roślin. Katalog (Methods for Analysis and Assessment of Soil and Plant Properties) Catalog. (Instytut Ochrony Środowiska, 1991).42.Baskin, C. C. & Baskin, J. M. Seeds : Ecology, Biogeography, and Evolution of Dormancy and Germination. (Elsevier, 2014).43.Soltani, E., Ghaderi-Far, F., Baskin, C. C. & Baskin, J. M. Problems with using mean germination time to calculate rate of seed germination. Aust. J. Bot. 63, 631–635 (2015).Article 

Google Scholar 
44.Flores, P., Poggi, D., García, S. M., Catraro, M. & Gariglio, N. Effects of pre-stratification storage conditions on black walnut seed post-stratification germination capacity. Int. J. Fruit Sci. 17, 29–40 (2017).Article 

Google Scholar 
45.Ranal, M. A., de Santana, D. G., Ferreira, W. R. & Mendes-Rodrigues, C. Calculating germination measurements and organizing spreadsheets. Rev. Bras. Botân. 32, 849–855 (2009).Article 

Google Scholar 
46.Matthews, S. & Khajeh Hosseini, M. Mean germination time as an indicator of emergence performance in soil of seed lots of maize (Zea mays). Seed Sci. Technol. 34, 339–347 (2006).47.Ter Braak, C. J. F. & Šmilauer, P. Canoco Reference Manual and User’s Guide: Software for Ordination, Version 5.0. (Microcomputer Power, 2012).48.Inc., D. Dell Statistica (Data Analysis Software System, Version 13). (2016).49.Team, R. D. C. R: A Language and Environment for Statistical Computing. (R Foundation for Statistical Computing, 2008).50.Nicotra, A. B. et al. Plant phenotypic plasticity in a changing climate. Trends Plant Sci. 15, 684–692 (2010).CAS 
Article 

Google Scholar 
51.Gray, S. B. & Brady, S. M. Plant developmental responses to climate change. Dev. Biol. 419, 64–77 (2016).CAS 
Article 

Google Scholar 
52.Dahl, Å. E. & Fredrikson, M. The timetable for development of maternal tissues sets the stage for male genomic selection in Betula pendula (Betilaceae). Am. J. Bot. 83, 895–902 (1996).Article 

Google Scholar 
53.Kozlov, M. V. & Zvereva, E. L. Reproduction of mountain birch along a strong pollution gradient near Monchegorsk, Northwestern Russia. Environ. Pollut. 132, 443–451 (2004).CAS 
Article 

Google Scholar 
54.Sultan, S. E. Phenotypic plasticity in plants: A case study in ecological development. Evol. Dev. 5, 25–33 (2003).Article 

Google Scholar 
55.Zvereva, E. L. & Kozlov, M. V. Effects of pollution-induced habitat disturbance on the response of willows to simulated herbivory. J. Ecol. 89, 21–30 (2001).Article 

Google Scholar 
56.Eränen, J. K. Local adaptation of mountain birch to heavy metals in subarctic industrial barrens. For. Snow Landsc. Res. 80, 161–167 (2006).
Google Scholar 
57.Neuvonen, S., Nyyssonen, T., Ranta, H. & Kiilunen, S. Simulated acid rain and the reproduction of mountain birch [Betula pubescens ssp. tortuosa (Ledeb.) Nyman]: A cautionary tale. New Phytol. 118, 111–117 (1991).58.Cuinica, L. G., Abreu, I., Gomes, C. R. & Esteves da Silva, J. C. G. Exposure of Betula pendula Roth pollen to atmospheric pollutants CO, O3 and SO2. Grana 52, 299–304 (2013).59.Pasonen, H.-L., Pulkkinen, P. & Kärkkäinen, K. Genotype-environment interactions in pollen competitive ability in an anemophilous tree, Betula pendula Roth. Theor. Appl. Genet. 105, 465–473 (2002).Article 

Google Scholar 
60.Sarvas, R. On the flowering of birch and the quality of seed crop. Commun. Inst. For. Fenn. 1–38 (1952).61.Midmore, E., McCartan, S., Jinks, R. & Cahalan, C. Using thermal time models to predict germination of five provenances of silver birch (Betula pendula Roth) in southern England. Silva Fenn. 49, 1–12 (2015).62.Bojarczuk, K. et al. Effect of polluted soil and fertilisation on growth and physiology of Silver Birch (Betula pendula Roth.) seedlings. Polish J. Environ. Stud. 11, 483–492 (2002).63.Wierzbicka, M. & Rostański, A. Microevolutionary changes in ecotypes of calamine waste heap vegetation near Olkusz, Poland: A review. Acta Bot. Cracoviensia 44, 7–10 (2002).
Google Scholar 
64.Mahdi, T. & Whittaker, J. B. Do birch trees (Betula pendula) grow better if foraged by wood ants?. J. Anim. Ecol. 62, 101–116 (1993).Article 

Google Scholar 
65.Barantal, S. et al. Contrasting effects of tree species and genetic diversity on the leaf—Miner communities associated with silver birch. Oecologia 189, 687–697 (2019).ADS 
Article 

Google Scholar 
66.Santamour, Frank, S. & Greene, A. European hornet damage to ash and birch trees. J. Arboric. 12, 273–279 (1986).67.Klingeman, B., Oliver, J. & F., H. Who’s doin’ all that chewin’? The European hornet. Tenn. Green Times 2, 34–36 (2001).68.Ylioja, T., Roininen, H., Heinonen, J. & Rousi, M. Susceptibility of Betula pendula clones to Phytobia betulae, a dipteran miner of birch stems. Can. J. For. Res. 30, 1824–1829 (2000).Article 

Google Scholar 
69.Franiel, I. Development of Betula pendula seedlings growing on the Silesia Steelworks dumping grounds in Katowice. Acta Agrophys. 51, 51–55 (2001).
Google Scholar  More

1.Díaz et al. Summary for Policymakers of the Global Assessment.pdf.2.Kehoe, L. et al. Biodiversity at risk under future cropland expansion and intensification. Nat. Ecol. Evolut. 1, 1129–1135 (2017).Article 

Google Scholar 
3.Hendershot, J. N. et al. Intensive farming drives long-term shifts in avian community composition. Nature 579, 393–396 (2020).ADS 
CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
4.Bommarco, R., Kleijn, D. & Potts, S. G. Ecological intensification: Harnessing ecosystem services for food security. Trends Ecol. Evol. 28, 230–238 (2013).PubMed 
Article 
PubMed Central 

Google Scholar 
5.Michael, D. R., Wood, J. T., O’Loughlin, T. & Lindenmayer, D. B. Influence of land sharing and land sparing strategies on patterns of vegetation and terrestrial vertebrate richness and occurrence in Australian endangered eucalypt woodlands. Agr. Ecosyst. Environ. 227, 24–32 (2016).Article 

Google Scholar 
6.Tittonell, P. Ecological intensification of agriculture—Sustainable by nature. Curr. Opin. Environ. Sustain. 8, 53–61 (2014).Article 

Google Scholar 
7.Willer, E. H., Schlatter, B., Trávní, J., Kemper, L. & Lernoud, J. The World of Organic Agriculture Statistics and Emerging Trends 2020. 337.8.Reganold, J. P. & Wachter, J. M. Organic agriculture in the twenty-first century. Nat. Plants 2 (2016).9.Connor, D. J. Organic agriculture cannot feed the world. Field Crop Res. 106, 187–190 (2008).Article 

Google Scholar 
10.Seufert, V. & Ramankutty, N. Many shades of gray—The context-dependent performance of organic agriculture. Sci. Adv. 3, e1602638 (2017).11.Smith, O. M. et al. Landscape context affects the sustainability of organic farming systems. Proc. Natl. Acad. Sci. 117, 2870–2878 (2020).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
12.Bengtsson, J., Ahnström, J. & Weibull, A.-C. The effects of organic agriculture on biodiversity and abundance: A meta-analysis: Organic agriculture, biodiversity and abundance. J. Appl. Ecol. 42, 261–269 (2005).Article 

Google Scholar 
13.Tuck, S. L. et al. Land-use intensity and the effects of organic farming on biodiversity: A hierarchical meta-analysis. J. Appl. Ecol. 51, 746–755 (2014).PubMed 
PubMed Central 
Article 

Google Scholar 
14.Lichtenberg, E. M. et al. A global synthesis of the effects of diversified farming systems on arthropod diversity within fields and across agricultural landscapes. Glob. Change Biol. 23, 4946–4957 (2017).ADS 
Article 

Google Scholar 
15.Lori, M., Symnaczik, S., Mäder, P., De Deyn, G. & Gattinger, A. Organic farming enhances soil microbial abundance and activity—A meta-analysis and meta-regression. PLOS ONE 12, e0180442 (2017).16.Kleijn, D., Rundlöf, M., Scheper, J., Smith, H. G. & Tscharntke, T. Does conservation on farmland contribute to halting the biodiversity decline?. Trends Ecol. Evol. 26, 474–481 (2011).PubMed 
Article 
PubMed Central 

Google Scholar 
17.Birkhofer, K., Ekroos, J., Corlett, E. B. & Smith, H. G. Winners and losers of organic cereal farming in animal communities across Central and Northern Europe. Biol. Cons. 175, 25–33 (2014).Article 

Google Scholar 
18.Mackie, K. A., Müller, T., Zikeli, S. & Kandeler, E. Long-term copper application in an organic vineyard modifies spatial distribution of soil micro-organisms. Soil Biol. Biochem. 65, 245–253 (2013).CAS 
Article 

Google Scholar 
19.Buchholz, J. et al. Soil biota in vineyards are more influenced by plants and soil quality than by tillage intensity or the surrounding landscape. Sci. Rep. 7 (2017).20.Hole, D. G. et al. Does organic farming benefit biodiversity?. Biol. Cons. 122, 113–130 (2005).Article 

Google Scholar 
21.Power, A. G. Ecosystem services and agriculture: Tradeoffs and synergies. Philos. Trans. R. Soc. B Biol. Sci. 365, 2959–2971 (2010).Article 

Google Scholar 
22.Peigné, J. et al. Earthworm populations under different tillage systems in organic farming. Soil Tillage Res. 104, 207–214 (2009).Article 

Google Scholar 
23.Biondi, A., Desneux, N., Siscaro, G. & Zappalà, L. Using organic-certified rather than synthetic pesticides may not be safer for biological control agents: Selectivity and side effects of 14 pesticides on the predator Orius laevigatus. Chemosphere 87, 803–812 (2012).ADS 
CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
24.Mehrabi, Z., Seufert, V., Ramankutty, N. The conventional versus alternative agricultural divide: A response to Garibaldi et al. Trends Ecol. Evolut. 32, 720–721 (2017).25.Tscharntke, T., Klein, A. M., Kruess, A., Steffan-Dewenter, I. & Thies, C. Landscape perspectives on agricultural intensification and biodiversity – ecosystem service management. Ecol. Lett. 8, 857–874 (2005).Article 

Google Scholar 
26.Gámez-Virués, S. et al. Landscape simplification filters species traits and drives biotic homogenization. Nat. Commun. 6 (2015).27.Holzschuh, A., Steffan-Dewenter, I. & Tscharntke, T. Agricultural landscapes with organic crops support higher pollinator diversity. Oikos 117, 354–361 (2008).Article 

Google Scholar 
28.Muneret, L., Auriol, A., Thiéry, D. & Rusch, A. Organic farming at local and landscape scales fosters biological pest control in vineyards. Ecol. Appl. 29, e01818 (2019).29.Gabriel, D. et al. Scale matters: The impact of organic farming on biodiversity at different spatial scales: Scale matters in organic farming. Ecol. Lett. 13, 858–869 (2010).PubMed 
Article 
PubMed Central 

Google Scholar 
30.Agreste. Pratiques Phytosanitaires en Viticulture. Campagne 2016. (2020)31.Agreste. La Viticulture Bio en Nouvelle-Aquitaine: Un Dynamisme à Tous les Stades de la Filière. (2020).32.Gruber, S. & Claupein, W. Effect of tillage intensity on weed infestation in organic farming. Soil Tillage Res. 105, 104–111 (2009).Article 

Google Scholar 
33.Pfingstmann, A. et al. Contrasting effects of tillage and landscape structure on spiders and springtails in vineyards. Sustainability 11, 2095 (2019).Article 

Google Scholar 
34.Dittmer, S. & Schrader, S. Longterm effects of soil compaction and tillage on Collembola and straw decomposition in arable soil. Pedobiologia 44, 527–538 (2000).Article 

Google Scholar 
35.Kolb, S., Uzman, D., Leyer, I., Reineke, A. & Entling, M. H. Differential effects of semi-natural habitats and organic management on spiders in viticultural landscapes. Agric. Ecosyst. Environ. 287, 106695 (2020).36.Birkhofer, K. et al. Relationships between multiple biodiversity components and ecosystem services along a landscape complexity gradient. Biol. Cons. 218, 247–253 (2018).Article 

Google Scholar 
37.Kratschmer, S. et al. Tillage intensity or landscape features: What matters most for wild bee diversity in vineyards?. Agric. Ecosyst. Environ. 266, 142–152 (2018).Article 

Google Scholar 
38.Ullmann, K. S., Meisner, M. H. & Williams, N. M. Impact of tillage on the crop pollinating, ground-nesting bee, Peponapis pruinosa in California. Agric. Ecosyst. Environ. 232, 240–246 (2016).Article 

Google Scholar 
39.Jiang, X., Wright, A. L., Wang, X. & Liang, F. Tillage-induced changes in fungal and bacterial biomass associated with soil aggregates: A long-term field study in a subtropical rice soil in China. Appl. Soil. Ecol. 48, 168–173 (2011).Article 

Google Scholar 
40.Zuber, S. M. & Villamil, M. B. Meta-analysis approach to assess effect of tillage on microbial biomass and enzyme activities. Soil Biol. Biochem. 97, 176–187 (2016).CAS 
Article 

Google Scholar 
41.Luff, M. L. The biology of the ground beetle Harpalus rufipes in a strawberry field in Northumberland. Ann. Appl. Biol. 94, 153–164 (1980).Article 

Google Scholar 
42.Shearin, A. F., Reberg-Horton, S. C. & Gallandt, E. R. Direct effects of tillage on the activity density of ground beetle (Coleoptera: Carabidae) weed seed predators. Environ. Entomol. 36, 1140–1146 (2007).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
43.Rundlöf, M. et al. Seed coating with a neonicotinoid insecticide negatively affects wild bees. Nature 521, 77–80 (2015).ADS 
PubMed 
Article 
CAS 
PubMed Central 

Google Scholar 
44.Martin, E. A. et al. The interplay of landscape composition and configuration: new pathways to manage functional biodiversity and agroecosystem services across Europe. Ecol. Lett. 22, 1083–1094 (2019).PubMed 
Article 
PubMed Central 

Google Scholar 
45.Goded, S., Ekroos, J., Azcárate, J. G., Guitián, J. A. & Smith, H. G. Effects of organic farming on plant and butterfly functional diversity in mosaic landscapes. Agric. Ecosyst. Environ. 284, 106600 (2019).46.Rusch, A., Valantin-Morison, M., Sarthou, J.-P. & Roger-Estrade, J. Multi-scale effects of landscape complexity and crop management on pollen beetle parasitism rate. Landsc. Ecol. 26, 473–486 (2011).Article 

Google Scholar 
47.Tamburini, G., De Simone, S., Sigura, M., Boscutti, F. & Marini, L. Conservation tillage mitigates the negative effect of landscape simplification on biological control. J. Appl. Ecol. 53, 233–241 (2016).Article 

Google Scholar 
48.Le Féon, V. et al. Intensification of agriculture, landscape composition and wild bee communities: A large scale study in four European countries. Agric. Ecosyst. Environ. 137, 143–150 (2010).Article 

Google Scholar 
49.Sousa, J. P. et al. Changes in Collembola richness and diversity along a gradient of land-use intensity: A pan European study. Pedobiologia 50, 147–156 (2006).Article 

Google Scholar 
50.Vanbergen, A. J. et al. Scale-specific correlations between habitat heterogeneity and soil fauna diversity along a landscape structure gradient. Oecologia 153, 713–725 (2007).ADS 
PubMed 
Article 
PubMed Central 

Google Scholar 
51.Lehmitz, R., Russell, D., Hohberg, K., Christian, A. & Xylander, W. E. R. Active dispersal of oribatid mites into young soils. Appl. Soil. Ecol. 55, 10–19 (2012).Article 

Google Scholar 
52.Concepción, E. D., Díaz, M. & Baquero, R. A. Effects of landscape complexity on the ecological effectiveness of agri-environment schemes. Landsc. Ecol. 23, 135–148 (2008).Article 

Google Scholar 
53.Tscharntke, T. et al. Landscape moderation of biodiversity patterns and processes – Eight hypotheses. Biol. Rev. 87, 661–685 (2012).PubMed 
Article 
PubMed Central 

Google Scholar 
54.Desneux, N., Decourtye, A. & Delpuech, J.-M. The sublethal effects of pesticides on beneficial arthropods. Annu. Rev. Entomol. 52, 81–106 (2007).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
55.Naveed, M. et al. Simultaneous loss of soil biodiversity and functions along a copper contamination gradient: When soil goes to sleep. Soil Sci. Soc. Am. J. 78, 1239–1250 (2014).ADS 
Article 
CAS 

Google Scholar 
56.Eijsackers, H., Beneke, P., Maboeta, M., Louw, J. P. E. & Reinecke, A. J. The implications of copper fungicide usage in vineyards for earthworm activity and resulting sustainable soil quality. Ecotoxicol. Environ. Saf. 62, 99–111 (2005).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
57.Le Provost, G. et al. Land-use history impacts functional diversity across multiple trophic groups. Proc. Natl. Acad. Sci. 117, 1573–1579 (2020).PubMed 
Article 
CAS 
PubMed Central 

Google Scholar 
58.Muneret, L. et al. Organic farming expansion drives natural enemy abundance but not diversity in vineyard-dominated landscapes. Ecol. Evol. https://doi.org/10.1002/ece3.5810 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
59.Lechenet, M., Dessaint, F., Py, G., Makowski, D. & Munier-Jolain, N. Reducing pesticide use while preserving crop productivity and profitability on arable farms. Nat. Plants 3 (2017).60.Le Féon, V. et al. Solitary bee abundance and species richness in dynamic agricultural landscapes. Agric. Ecosyst. Environ. 166, 94–101 (2013).Article 

Google Scholar 
61.McCravy, K. & Ruholl, J. Bee (Hymenoptera: Apoidea) diversity and sampling methodology in a midwestern USA deciduous forest. Insects 8, 81 (2017).PubMed Central 
Article 

Google Scholar 
62.Bano, R. & Roy, S. Extraction of Soil Microarthropods: A Low Cost Berlese-Tullgren Funnels Extractor. 4.63.Grueber, C. E., Nakagawa, S., Laws, R. J. & Jamieson, I. G. Multimodel inference in ecology and evolution: Challenges and solutions: Multimodel inference. J. Evol. Biol. 24, 699–711 (2011).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
64.Burnham, K. P. & Anderson, D. R. Multimodel inference: Understanding AIC and BIC in model selection. Sociol. Methods Res. 33, 261–304 (2004).MathSciNet 
Article 

Google Scholar 
65.Bartoń, K. MuMIn: Multi-Model Inference. R Package Version 1.43.17. https://CRAN.R-project.org/package=MuMIn (2020).66.R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/ (2020).67.Hartig, F. DHARMa: Residual Diagnostics for Hierarchical (Multi-Level/Mixed) Regression Models (2020). R Package Version 0.3.3.0. https://CRAN.R-project.org/package=DHARMa68.Bates, D., Maechler, M., Bolker, B., Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67(1), 1–48. https://doi.org/10.18637/jss.v067.i01 (2015). More

1.Mutreja, A. et al. Evidence for several waves of global transmission in the seventh cholera pandemic. Nature 477, 462–465 (2011).CAS 
Article 

Google Scholar 
2.Ali, M., Nelson, A. R., Lopez, A. L. & Sack, D. A. Updated global burden of cholera in endemic countries. PLoS Negl. Trop. Dis. 9, e0003832 (2015).Article 

Google Scholar 
3.Bompangue, D. N. et al. Lakes as source of cholera outbreaks, Democratic Republic of Congo. Emerg. Infect. Dis. 14, 798–800 (2008).Article 

Google Scholar 
4.Weill, F. X. et al. Genomic history of the seventh pandemic of cholera in Africa. Science 358, 785–789 (2017).CAS 
Article 

Google Scholar 
5.Hounmanou, Y. M. G. et al. Genomic insights into Vibrio cholerae O1 responsible for cholera epidemics in Tanzania between 1993 and 2017. PLoS Neglect Trop. D. 13, e0007934 (2019).Article 

Google Scholar 
6.Colwell, R. R. Global climate and infectious disease: the cholera paradigm. Science 274, 2025–2031 (1996).CAS 
Article 

Google Scholar 
7.Singleton, F., Attwell, R., Jangi, M. & Colwell, R. Influence of salinity and organic nutrient concentration on survival and growth of Vibrio choleraein aquatic microcosms. Appl. Environ. Microbiol. 43, 1080–1085 (1982).CAS 
Article 

Google Scholar 
8.Kirschner, A. K. T. et al. Rapid growth of planktonic Vibrio cholerae non-O1/non-O139 strains in a large alkaline lake in Austria: Dependence on temperature and dissolved organic carbon quality. Appl. Environ. Microb. 74, 2004–2015 (2008).CAS 
Article 

Google Scholar 
9.Reid, P. C. et al. The continuous plankton recorder: concepts and history, from Plankton Indicator to undulating recorders. Prog. Oceanogr. 57, 117–173 (2003).Article 

Google Scholar 
10.Vezzulli, L. et al. Climate influence on Vibrio and associated human diseases during the past half-century in the coastal North Atlantic. Proc. Natl. Acad. Sci. USA. 113, E5062–E5071 (2016).CAS 
Article 

Google Scholar 
11.Huq, A. et al. Detection, isolation, and identification of Vibrio cholerae from the environment. Curr. Protoc. Microbiol. https://doi.org/10.1002/9780471729259.mc06a05s26 (2012).12.Thompson, F. L. et al. Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis. Appl. Environ. Microb. 71, 5107–5115 (2005).CAS 
Article 

Google Scholar 
13.Vezzulli, L. et al. GbpA as a novel qPCR target for the species-specific detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in environmental, stool, and historical continuous plankton recorder samples. s. PLoS ONE 10, e0123983 (2015).Article 

Google Scholar 
14.Alam, M. et al. Toxigenic Vibrio cholerae in the aquatic environment of Mathbaria, Bangladesh. Appl. Environ. Microb. 72, 2849–2855 (2006).CAS 
Article 

Google Scholar 
15.Senoh, M. et al. Isolation of viable but nonculturable Vibrio cholerae O1 from environmental water samples in Kolkata, India, in a culturable state. Microbiol. Open 3, 239–246 (2014).CAS 
Article 

Google Scholar 
16.Vezzulli, L. et al. Whole-genome enrichment provides deep insights into vibrio cholerae metagenome from an African river. Microb. Ecol. 73, 734–738 (2017).CAS 
Article 

Google Scholar 
17.Kaboré, S. et al. Occurrence of Vibrio cholerae in water reservoirs of Burkina Faso. Res Microbiol 169, 1–10 (2018).Article 

Google Scholar 
18.Bwire, G. et al. Environmental surveillance of Vibrio cholerae O1/O139 in the five African Great Lakes and other major surface water sources in Uganda. Front. Microbiol. 9, 1560 (2018).Article 

Google Scholar 
19.Vezzulli, L., Baker-Austin, C., Kirschner, A., Pruzzo, C. & Martinez-Urtaza, J. Global emergence of environmental non-O1/O139 Vibrio cholerae infections linked with climate change: a neglected research field? Environ. Microbiol. 22, 4342–4355 (2020).CAS 
Article 

Google Scholar  More

Mesocosm setupThe mesocosm experiment AQUACOSM VIMS-Ehux was carried out between 24th May (day 0) and 16th June (day 23) 2018 in Raunefjorden at the Marine Biological Station Espegrend, Norway (60°16′11 N; 5°13′07E) as previously described [7]. Four light-transparent enclosure bags were filled with surrounding fjord water (day −1; pumped from 5 m depth), and continuously mixed by aeration (from day 0 onwards). Each bag was supplemented with nutrients at a nitrogen to phosphorous ratio of 16:1 (1.6 µM NaNO3 and 0.1 µM KH2PO4 final concentration) on days 0–5 and 14–17, whereas on days 6, 7, and 13 only nitrogen was added. Nutrient concentrations were measured daily [18].Enumeration of phytoplankton cells by flow cytometryFor E. huxleyi enumeration by flow cytometry, water samples were collected in 50 mL tubes from ~1 m depth. Water samples were pre-filtered using 40 µm cell strainers and immediately analyzed with an Eclipse iCyt flow cytometer (Sony Biotechology, Champaign, IL, USA) as previously described [19]. A total volume of 300 µl with a flow rate of 150 µl min−1 was analyzed. A threshold was applied on the forward scatter to reduce background noise. Four groups of phytoplankton populations were identified in distinct gates by plotting the autofluorescence of chlorophyll (em: 663–737 nm) versus phycoerythrin (em: 570–620 nm) and side scatter: calcified E. huxleyi (high chlorophyll and high side scatter), Synechococcus (high phycoerythrin), nanophytoplankton including calcified and non-calcified E. huxleyi (high chlorophyll and phycoerythrin), and picophytoplankton (low chlorophyll and low phycoerythrin) [20]. See Fig. S1 for further details of gating strategy.Enumeration of EhV-like particles and bacteria by flow cytometryFor EhV and bacteria counts, 200 µl of sample were fixed a final concentration of 0.5% glutaraldehyde for one hour at 4 °C and flash frozen in liquid nitrogen. For analysis, they were thawed and stained with SYBR gold (Invitrogen, Carlsbad, CA, USA) that was diluted 1:10,000 in 0.2 μm filtered TE buffer (10:1 mM Tris:EDTA, pH 8), incubated for 20 min at 80 °C and cooled to room temperature [21]. Bacteria and EhV-like particles were counted and analyzed using an Eclipse iCyt flow cytometer (ex: 488 nm, em: 500–550 nm), and identified by comparing to reference samples containing fixed EhV201 and bacteria from lab cultures. EhV gating was very stringent in order to minimize the misidentification of other large viruses such as Micromonas pusilla virus (MpV) in the samples (see Fig. S2 for further details of gating strategy for EhV counts).Enumeration of extracellular EhV by qPCRWater samples (1–2 l) were sequentially filtered by vacuum through polycarbonate filters with a pore size of 20 µm (47 mm; Sterlitech, Kent, WA, US), then 2 µm (Isopore, 47 mm; Merck Millipore, Cork, Ireland), and finally 0.22 µm (Isopore, 47 mm; Merck Millipore). Filters were immediately flash-frozen in liquid nitrogen and stored at −80 °C until further processing. DNA was extracted from the 0.22 µm filters using the DNeasy PowerWater kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Each sample was diluted 100 times, and 1 µl was then used for qPCR analysis. EhV abundance was determined by qPCR for the major capsid protein (mcp) gene [22] using the following primers: 5ʹ-acgcaccctcaatgtatggaagg-3ʹ (mcp1F[23],) and 5ʹ-rtscrgccaactcagcagtcgt-3ʹ (mcp94Rv; Mayers, K. et al., unpublished). All reactions were carried out in technical triplicates. For all reactions, Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) was used as described by the manufacturer. Reactions were performed on a QuantStudio 5 Real-Time PCR System equipped with the QuantStudio Design and Analysis Software version 1.5.1 (Applied Biosystems, Foster City, CA, USA) as follows: 50 °C for 2 min, 95 °C for 5 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. Results were calibrated against serial dilutions of EhV201 DNA at known concentrations, enabling exact enumeration of viruses. Samples showing multiple peaks in melting curve analysis or peaks that were not corresponding to the standard curves were omitted.Vesicle concentration and separationLab samplesE. huxleyi CCMP2090 was grown in 20 l filtered sea water (FSW) supplemented with K/2 nutrient mix at 18 °C, 16:8 h light:dark cycle, 100 μmol photons m−2 s−1. Uninfected cultures were grown to ~ 106 cells ml−1. Infected cultures were inoculated with EhV201 at a multiplicity of infection (MOI) of ~1:1 plaque forming unit (pfu) per cell and incubated under normal growth conditions for 120 h, at which time the culture had cleared. The entire 20 l volume was then filtered through a GF/C filter (Whatman, Maidstone, United Kingdom) followed by an 0.45 µm PVDF filter (Durapore, Merck Millipore) to eliminate cells and cellular debris.Mesocosm samplesOn days 2, 4, 5, 8, 12, 15, 18, and 23 we collected 25 l from bags 1–4 and combined them to produce one sample of 100 l for each sampling time. The samples were pre-filtered using a 200 µm nylon mesh, and then filtered through a GF/C filter (Whatman) followed by an 0.45 µm PVDF filter (Durapore, Merck Millipore) to eliminate cells and cellular debris.Particle concentrationParticles in the flow-through from the filtration stage were concentrated on a 100 kDa tangential flow filter (Spectrumlabs, Repligen, Waltham, Massachusetts, USA) to a final volume of ~500 ml. At this stage, mesocosm samples were stored in the dark at +4 oC and shipped back to the home lab. All samples were further concentrated to a final volume of 1–2 ml using 100 kDa Amicon-ultra filters (Merck Millipore).Vesicle separationVesicles were separated from other particles (including viruses) using an 18–35% OptiPrep gradient (MilliporeSigma, St. Louis, Missouri, USA). Gradients were centrifuged in an ultracentrifuge for 12 h at 200,000 × g. Fractions (0.5 ml) were collected from the top of the gradient and the fraction material was cleaned by washing three times and resuspended in 0.02 µm-filtered FSW using 100 kDa Amicon-ultra filters (Merck Millipore). Vesicles were detected in fractions with densities of 1.05–1.07 g ml–1 (fractions 3–5 from the top).Vesicle concentration in samples from lab cultures was measured by NTA using the NanoSight NS300 instrument (Malvern Instruments, Malvern, UK) equipped with a 488 nm laser module and NTA V3.2 software. Samples were diluted so that each field of view contained 20–100 particles. Three 60 s videos were recorded for each biological replicate, representing different fields of view. All the videos for a given experiment were processed using identical settings (screen gain of one and detection threshold of five).RNA extraction and sequencingIn order to eliminate RNA molecules that are not packed into vesicles, we subjected vesicle samples to RNase treatment prior to RNA extraction. Samples were incubated for 60 min at 37 oC with 10 pg µl−1 of RNase A (Bio Basic, Toronto, Canada). RNase activity was inactivated by adding 100 unites of Protector RNase Inhibitor (Roche, Basel, Switzerland). Total RNA (including RNA from ~18 nucleotides or more) was extracted using the miRNeasy kit according to the manufacturer’s instructions (Qiagen). Libraries were prepared using the TruSeq Small RNA Library kit (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. Each sample was indexed twice with the same index, one with polynucleotide kinase I treatment (according to manufacturer’s instructions, NEB, Ipswich, Massachusetts, USA) and one without. After 15 cycles of PCR amplification, libraries were cleaned with the QIAquick PCR Purification Kit according to the manufacturer’s instructions (Qiagen). Libraries were sequenced on the NextSeq platform (Illumina).sRNA bioinformatics analysisLow-quality read ends were trimmed and adaptors were removed using the cutadapt program [24], version 1.18. Reads shorter than 17 bp after the trimming were removed from further analyses. The remaining reads were mapped to an E. huxleyi integrated reference transcriptome shortly described in [6] using the RSEM software [25], version 1.3.1, with the default option of bowtie, version 1.1.2 [26]. Genes that had at least 5 reads in any of the samples were selected. For the heatmap (Fig. 1d), read counts were scaled to one million reads mapped to the E. huxleyi transcriptome and log2 transformed.Effect of vesicles on natural populations—experimental design and analysisOn days 14 and 20 of the mesocosm experiment (blue and red arrows in Fig. 1a, respectively), we combined equal volumes of water samples from bags 1–4 and filtered them through a 10 µm nylon mesh to eliminate zooplankton predators. We then supplemented the natural populations with f/50 nutrient mix and divided them into flasks, each containing 10 ml. In total, 30 flasks were treated with vesicles from uninfected lab cultures of E. huxleyi CCMP2090, at a ratio of ~500 vesicles cell−1 (calcified E. huxleyi determined by flow cytometry), and then all flasks were incubated in a growth chamber (15 °C, 16:8 h light:dark cycle, 100 μmol photons m−2 s−1). Once a day, samples were taken for flow cytometric quantification of live cells (see “Enumeration of phytoplankton cells by flow cytometry” above), or fixed for virus and bacteria counts (see “Enumeration of EhV-like particles and bacteria by flow cytometry” above). For statistical analysis, we used two-tailed t test with equal variance.Decay rate of EhV virions- experimental design and analysisTo determine the decay rate of infectivity of natural EhV virions, water was sampled from bag 4 on day 18, at a time point when viral infection was detected (green cross in Fig. 1a). This sample was filtered through a 0.45 PVDF filter (Durapore, Merck Millipore) to eliminate algal and most bacteria cells. EhV-like particles were counted by flow cytometry as described above and divided into nine tubes, each containing 1 ml. Triplicate samples were either treated with vesicles from EhV201-infected (VirusVesicles) or uninfected (controlVesicles) lab cultures (see above) at a ratio of ten vesicles per EhV-like particle, or not treated at all. All tubes were incubated in an on-land mesocosm facility that mimics the light and temperature conditions found at ~ 1 m depth within the fjord water. We used the most probable number (MPN) method [27] to determine the half-life of EhV within these samples. Briefly, a series of five-fold dilutions was prepared for each sample. Each dilution (10 μl) was then added, in eight technical replicates, to 100 μl of exponentially growing E. huxleyi CCMP374 cultures in multi-well plates and incubated under normal growth conditions for five days. This was repeated for four consecutive days for all samples. Clearance (infection) of the cells in the multi-wells was measured using an EnSpireTM 2300 Multilabel Reader (PerkinElmer, Turku, Finland) set to in vivo fluorescence (ex:460 nm, em:680 nm). MPN was calculated using the MPN calculation program, version 5 [28]. For the samples treated with controlvesicles, we could only obtain a positive MPN value for one time point, as the decay was faster than expected. Therefore, the minimum detectable infectivity values were used in order to calculate the maximum possible half-life. For statistical analysis, each treatment was compared to the untreated control, using ANOVA with Dunnett’s post-hoc test. More

1.Sokol, J. Troubled treasure. Fossils in Burmese amber offer an exquisite view of dinosaur times—and an ethical minefield. Science (23 May 2019).2.Lawton, G. Blood amber: the exquisite trove of fossils fueling war in Myanmar. New Scientist (1 May 2019).3.Lawton, G. Military now controls Myanmar’s scientifically important amber mines. New Scientist (30 August 2019).4.Rayfield, E. J., Theodor, J. M. & Polly, P. D. Fossils from conflict zones and reproducibility of fossil-based scientific data. Society of Vertebrate Paleontology (SVP) (21 April 2020).5.Barrett, P. M. & Johanson, Z. J. Syst. Palaeontol. 18, 1059 (2020).Article 

Google Scholar 
6.Engel, M. S. Nature 584, 525 (2020).CAS 
Article 

Google Scholar 
7.Barrett, P. M. & Johanson, Z. Nature 674, 586 (2020).
Google Scholar 
8.Poinar, G. & Ellenberger, S. Geoconserv. Res 3, 12–16 (2020).
Google Scholar 
9.Takai, M. et al. J. Hum. Evol. 84, 1–15 (2015).Article 

Google Scholar 
10.Barber, A. J., Khin Zaw & Crow, M. J. (eds) Myanmar: Geology, Resources and Tectonics Geological Society, London, Memoirs Vol. 48 (Geological Society of London, 2017).11.Jaeger, J.-J. et al. Proc. R. Soc. B 287, 20202129 (2020).Article 

Google Scholar 
12.Khin Zaw, Win Swe, Barber, A. J., Crow, M. J. & Yin Yin Nwe in Myanmar: Geology, Resources and Tectonics Geological Society, London, Memoirs Vol. 48 (eds Barber, A. J., Khin Zaw & Crow, M. J.) 1–17 (Geological Society of London, 2017).13.Phyo, M. M. et al. Minerals 10, 195 (2020).CAS 
Article 

Google Scholar 
14.GIAC (Geodynamics of India-Asia Collision) Final Report, A Joint Project of Scientific Co-operation between Total Myanmar Exploration and Production (TMEP), Unocal, Universities of Myanmar and Thailand, Myanmar Oil and Gas Enterprise (MOGE) and Ecole Normale Superieure (ENS) (TMEP, Unocal, Univ. Myanmar, Univ. Thailand, MOGE, ENS, 1999). More

College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao, ChinaChao Shi, Hao-hong Cai, Ri-xin Jiang & Shuo WangKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, ChinaChao Shi & Hua PengDepartment of Ecology & Evolutionary Biology, University of Kansas, Lawrence, KS, USAMichael S. EngelShanghai World Expo Museum, Shanghai, ChinaJi YuanKey Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaMing BaiDepartment of Entomology, College of Plant Protection, China Agricultural University, Beijing, ChinaDing YangCollege of Life and Environmental Sciences, Minzu University of China, Beijing, ChinaChun-lin LongCollege of Life Science, Shandong Normal University, Jinan, ChinaZun-tian ZhaoSouth China Botanical Garden, Chinese Academy of Sciences, Guangzhou, ChinaDian-xiang ZhangState Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, ChinaXian-chun ZhangState Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology and Center for Excellence in Life and Palaeoenvironment, Chinese Academy of Sciences, Nanjing, ChinaYong-dong WangSchool of Environment, Earth, and Ecosystem Sciences, The Open University, Milton Keynes, UKRobert A. SpicerCAS Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, ChinaRobert A. SpicerS.W., M.S.E., D.-X.Z., X.-C.Z., H.P., Y.-D.W. and R.A.S. conceived the idea and drafted the initial manuscript, with contributions from all other authors. All authors jointly revised the paper. More

1.Koch, E. & McFall-Ngai, M. Model systems for the study of how symbiotic associations between animals and extracellular bacterial partners are established and maintained. Drug Discov. Today Dis. Models 28, 3–12 (2018).PubMed 
Article 

Google Scholar 
2.Lee, K. H. & Ruby, E. G. Effect of the squid host on the abundance and distribution of symbiotic Vibrio fischeri in nature. Appl. Environ. Microbiol. 60, 1565–1571 (1994).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
3.Kremer, N. et al. Initial symbiont contact orchestrates host-organ-wide transcriptional changes that prime tissue colonization. Cell Host Microbe 14, 183–194 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
4.Bongrand, C. & Ruby, E. G. Achieving a multi-strain symbiosis: strain behavior and infection dynamics. ISME J. 13, 698–706 (2019).PubMed 
Article 

Google Scholar 
5.McFall-Ngai, M. J. The importance of microbes in animal development: lessons from the squid-vibrio symbiosis. Annu. Rev. Microbiol. 68, 177–194 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
6.Jones, B. W. & Nishiguchi, M. K. Counterillumination in the Hawaiian bobtail squid, Euprymna scolopes Berry (Mollusca: Cephalopoda). Mar. Biol. 144, 1151–1155 (2004).Article 

Google Scholar 
7.Graf, J. & Ruby, E. G. Host-derived amino acids support the proliferation of symbiotic bacteria. Proc. Natl Acad. Sci. USA 95, 1818–1822 (1998).CAS 
PubMed 
Article 

Google Scholar 
8.McFall-Ngai, M. J. & Ruby, E. G. Developmental biology in marine invertebrate symbioses. Curr. Opin. Microbiol. 3, 603–607 (2000).CAS 
PubMed 
Article 

Google Scholar 
9.Moriano-Gutierrez, S. et al. The noncoding small RNA SsrA is released by Vibrio fischeri and modulates critical host responses. PLoS Biol. 18, e3000934 (2020).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
10.Schwartzman, J. A. & Ruby, E. G. Stress as a normal cue in the symbiotic environment. Trends Microbiol. 24, 414–424 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
11.Nyholm, S. V. & McFall-Ngai, M. J. A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner. Nat. Rev. Microbiol. https://doi.org/10.1038/s41579-021-00567-y (2021).12.Schwartzman, J. A. et al. The chemistry of negotiation: rhythmic, glycan-driven acidification in a symbiotic conversation. Proc. Natl Acad. Sci. USA 112, 566–571 (2015). In this study, the host’s delivery of chitin-derived N-acetylglucosamine is shown to develop 4 weeks after hatching, and this chitin is apparently delivered by haemocytes that lyse in the crypts only at night. A nocturnal acidification of the crypts results, and a model for how this outcome enhances bioluminescence is described.CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
13.Heath-Heckman, E. A. et al. Bacterial bioluminescence regulates expression of a host cryptochrome gene in the squid-vibrio symbiosis. mBio https://doi.org/10.1128/mBio.00167-13 (2013).Article 
PubMed 
PubMed Central 

Google Scholar 
14.Koropatnick, T. A. et al. Microbial factor-mediated development in a host-bacterial mutualism. Science 306, 1186–1187 (2004).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
15.Bouskra, D. et al. Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 456, 507–510 (2008).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
16.Thaiss, C. A. et al. Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis. Cell 159, 514–529 (2014).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
17.Ruby, E. G. Symbiotic conversations are revealed under genetic interrogation. Nat. Rev. Microbiol. 6, 752–762 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
18.Bongrand, C. & Ruby, E. G. The impact of Vibrio fischeri strain variation on host colonization. Curr. Opin. Microbiol. 50, 15–19 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
19.Colton, D. M. & Stabb, E. V. Rethinking the roles of CRP, cAMP, and sugar-mediated global regulation in the Vibrionaceae. Curr. Genet. 62, 39–45 (2016).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
20.Mandel, M. J. & Dunn, A. K. Impact and Influence of the natural Vibrio-squid symbiosis in understanding bacterial-animal interactions. Front. Microbiol. 7, 1982 (2016).PubMed 
PubMed Central 
Article 

Google Scholar 
21.Aschtgen, M. S. et al. Insights into flagellar function and mechanism from the squid-vibrio symbiosis. NPJ Biofilms Microbiomes 5, 32 (2019).PubMed 
PubMed Central 
Article 

Google Scholar 
22.Stabb, E. V. & Visick, K. L. in The Prokaryotes (eds Rosenberg, E., DeLong, E. F., Lory, S., Stackebrandt, E. & Thompson, F.) 497–532 (Springer, 2013).23.Nawroth, J. C. et al. Motile cilia create fluid-mechanical microhabitats for the active recruitment of the host microbiome. Proc. Natl Acad. Sci. USA 114, 9510–9516 (2017). This work provides the first glimpse into the cilium-driven fluid mechanics that position V. fischeri cells to reach and settle in ‘quiet zones’ on the light organ surface, permitting a selective ‘recruitment’ of this microorganism from the planktonic environment.CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
24.Altura, M. A. et al. The first engagement of partners in the Euprymna scolopes-Vibrio fischeri symbiosis is a two-step process initiated by a few environmental symbiont cells. Environ. Microbiol. 15, 2937–2950 (2013). Aggregations of only a few V. fischeri cells are observed to initiate normal host responses, and reveal that aggregation is a two-part process that begins with bacterial attachment to the cilia.PubMed 
PubMed Central 

Google Scholar 
25.Nyholm, S. V., Stabb, E. V., Ruby, E. G. & McFall-Ngai, M. J. Establishment of an animal-bacterial association: recruiting symbiotic vibrios from the environment. Proc. Natl Acad. Sci. USA 97, 10231–10235 (2000).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
26.Yip, E. S., Geszvain, K., DeLoney-Marino, C. R. & Visick, K. L. The symbiosis regulator RscS controls the syp gene locus, biofilm formation and symbiotic aggregation by Vibrio fischeri. Mol. Microbiol. 62, 1586–1600 (2006).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
27.Koehler, S. et al. The model squid-vibrio symbiosis provides a window into the impact of strain- and species-level differences during the initial stages of symbiont engagement. Environ. Microbiol. https://doi.org/10.1111/1462-2920.14392 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
28.Morris, A. R. & Visick, K. L. Control of biofilm formation and colonization in Vibrio fischeri: a role for partner switching? Environ. Microbiol. 12, 2051–2059 (2010).CAS 
PubMed 
PubMed Central 

Google Scholar 
29.Norsworthy, A. N. & Visick, K. L. Gimme shelter: how Vibrio fischeri successfully navigates an animal’s multiple environments. Front. Microbiol. 4, 356 (2013).PubMed 
PubMed Central 
Article 

Google Scholar 
30.Shibata, S., Yip, E. S., Quirke, K. P., Ondrey, J. M. & Visick, K. L. Roles of the structural symbiosis polysaccharide (syp) genes in host colonization, biofilm formation, and polysaccharide biosynthesis in Vibrio fischeri. J. Bacteriol. 194, 6736–6747 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
31.Yip, E. S., Grublesky, B. T., Hussa, E. A. & Visick, K. L. A novel, conserved cluster of genes promotes symbiotic colonization and sigma-dependent biofilm formation by Vibrio fischeri. Mol. Microbiol. 57, 1485–1498 (2005).CAS 
PubMed 
Article 

Google Scholar 
32.Bassis, C. M. & Visick, K. L. The cyclic-di-GMP phosphodiesterase BinA negatively regulates cellulose-containing biofilms in Vibrio fischeri. J. Bacteriol. 192, 1269–1278 (2010).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
33.Chavez-Dozal, A., Hogan, D., Gorman, C., Quintanal-Villalonga, A. & Nishiguchi, M. K. Multiple Vibrio fischeri genes are involved in biofilm formation and host colonization. FEMS Microbiol. Ecol. 81, 562–573 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
34.Tischler, A. H., Lie, L., Thompson, C. M. & Visick, K. L. Discovery of calcium as a biofilm-promoting signal for Vibrio fischeri reveals new phenotypes and underlying regulatory complexity. J. Bacteriol. 200, e00016–e00018 (2018). This article expands our understanding of the regulatory controls and signals leading to biofilm formation by identifying calcium as a signal that induces a coordinate upregulation of Syp- and cellulose-dependent biofilm formation and revealing the sensor kinase HahK as a new biofilm regulator.CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
35.Ziemba, C., Shabtai, Y., Piatkovsky, M. & Herzberg, M. Cellulose effects on morphology and elasticity of Vibrio fischeri biofilms. NPJ Biofilms Microbiomes 2, 1 (2016).PubMed 
PubMed Central 
Article 

Google Scholar 
36.Ray, V. A., Driks, A. & Visick, K. L. Identification of a novel matrix protein that promotes biofilm maturation in Vibrio fischeri. J. Bacteriol. 197, 518–528 (2015).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
37.Shibata, S. & Visick, K. L. Sensor kinase RscS induces the production of antigenically distinct outer membrane vesicles That depend on the symbiosis polysaccharide locus in Vibrio fischeri. J. Bacteriol. 194, 185–194 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
38.Hussa, E. A., Darnell, C. L. & Visick, K. L. RscS functions upstream of SypG to control the syp locus and biofilm formation in Vibrio fischeri. J. Bacteriol. 190, 4576–4583 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
39.Mandel, M. J., Wollenberg, M. S., Stabb, E. V., Visick, K. L. & Ruby, E. G. A single regulatory gene is sufficient to alter bacterial host range. Nature 458, 215–218 (2009).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
40.Ray, V. A., Eddy, J. L., Hussa, E. A., Misale, M. & Visick, K. L. The syp enhancer sequence plays a key role in transcriptional activation by the sigma54-dependent response regulator SypG and in biofilm formation and host colonization by Vibrio fischeri. J. Bacteriol. 195, 5402–5412 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
41.Visick, K. L. & Skoufos, L. M. Two-component sensor required for normal symbiotic colonization of Euprymna scolopes by Vibrio fischeri. J. Bacteriol. 183, 835–842 (2001).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
42.Norsworthy, A. N. & Visick, K. L. Signaling between two interacting sensor kinases promotes biofilms and colonization by a bacterial symbiont. Mol. Microbiol. 96, 233–248 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
43.Thompson, C. M., Marsden, A. E., Tischler, A. H., Koo, J. & Visick, K. L. Vibrio fischeri biofilm formation prevented by a trio of regulators. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.01257-18 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
44.Brooks, J. F. II & Mandel, M. J. The histidine kinase BinK Is a negative regulator of biofilm formation and squid colonization. J. Bacteriol. 198, 2596–2607 (2016).PubMed 
PubMed Central 
Article 

Google Scholar 
45.Pankey, M. S. et al. Host-selected mutations converging on a global regulator drive an adaptive leap by bacteria to symbiosis. eLife https://doi.org/10.7554/eLife.24414 (2017). Evolutionary pathways that can lead to symbiotic colonization are revealed in this elegant study that follows the serial passage of a non-colonizing strain through many E. scolopes juveniles, resulting in altered, symbiosis-competent strains.Article 

Google Scholar 
46.Morris, A. R., Darnell, C. L. & Visick, K. L. Inactivation of a novel response regulator is necessary for biofilm formation and host colonization by Vibrio fischeri. Mol. Microbiol. 82, 114–130 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
47.Morris, A. R. & Visick, K. L. The response regulator SypE controls biofilm formation and colonization through phosphorylation of the syp-encoded regulator SypA in Vibrio fischeri. Mol. Microbiol. 87, 509–525 (2013).CAS 
PubMed 
Article 

Google Scholar 
48.Brooks, J. F. II et al. Global discovery of colonization determinants in the squid symbiont Vibrio fischeri. Proc. Natl Acad. Sci. USA 111, 17284–17289 (2014). This large-scale investigation of colonization factors provides important information on genetic requirements for symbiosis and provides a wealth of data for hypothesis generation that will foster many subsequent studies.CAS 
PubMed 
Article 

Google Scholar 
49.Thompson, C. M. & Visick, K. L. Assessing the function of STAS domain protein SypA in Vibrio fischeri using a comparative analysis. Front. Microbiol. 6, 760 (2015).PubMed 
PubMed Central 
Article 

Google Scholar 
50.Rotman, E. R. et al. Natural strain variation reveals diverse biofilm regulation in squid-colonizing Vibrio fischeri. J. Bacteriol. https://doi.org/10.1128/JB.00033-19 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
51.Bongrand, C. et al. A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior. ISME J. 10, 2907–2917 (2016). This study of the genomes and behaviours of a collection of a number of squid symbionts propelled the field from the near-exclusive study of a single isolate, ES114, into new and exciting directions with the genomic sequencing of dominant strains that contain numerous additional genetic sequences and factors.CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
52.Newell, P. D., Boyd, C. D., Sondermann, H. & O’Toole, G. A. A c-di-GMP effector system controls cell adhesion by inside-out signaling and surface protein cleavage. PLoS Biol. 9, e1000587 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
53.Christensen, D. G., Marsden, A. E., Hodge-Hanson, K., Essock-Burns, T. & Visick, K. L. LapG mediates biofilm dispersal in Vibrio fischeri by controlling maintenance of the VCBS-containing adhesin LapV. Mol. Microbiol. 114, 742–761 (2020). This article addresses a major long-standing question concerning the initiation of the light organ association; specifically, how do aggregated V. fischeri cells release themselves and migrate into host tissue? One factor may be an adhesin-cleaving protease, which is kept in check by a c-di-GMP-responsive protein, and can promote symbiont dispersal from biofilms.CAS 
PubMed 
Article 

Google Scholar 
54.Fidopiastis, P. M. et al. Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization. J. Bacteriol. 194, 3995–4002 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
55.Davidson, S. K., Koropatnick, T. A., Kossmehl, R., Sycuro, L. & McFall-Ngai, M. J. No means ‘yes’ in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association. Cellul. Microbiol. 6, 1139–1151 (2004).CAS 
Article 

Google Scholar 
56.Wang, Y. et al. Vibrio fischeri flavohaemoglobin protects against nitric oxide during initiation of the squid-Vibrio symbiosis. Mol. Microbiol. 78, 903–915 (2010).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
57.Stabb, E. V. Should they stay or should they go? Nitric oxide and the clash of regulators governing Vibrio fischeri biofilm formation. Mol. Microbiol. 111, 1–5 (2019).CAS 
PubMed 

Google Scholar 
58.Thompson, C. M., Tischler, A. H., Tarnowski, D. A., Mandel, M. J. & Visick, K. L. Nitric oxide inhibits biofilm formation by Vibrio fischeri via the nitric oxide sensor HnoX. Mol. Microbiol. 111, 187–203 (2019). This publication provides insight into the complex role in symbiosis of the squid-produced defence molecule NO by uncovering its ability to inhibit biofilm formation via the NO sensor HnoX, a finding that suggests that NO may influence the location or timing of biofilm formation and/or promote dispersal during symbiotic initiation.CAS 
PubMed 
Article 

Google Scholar 
59.Singh, P., Brooks, J. F. II., Ray, V. A., Mandel, M. J. & Visick, K. L. CysK plays a role in Biofilm formation and colonization by Vibrio fischeri. Appl. Environ. Microbiol. 81, 5223–5234 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
60.Raina, J. B., Fernandez, V., Lambert, B., Stocker, R. & Seymour, J. R. The role of microbial motility and chemotaxis in symbiosis. Nat. Rev. Microbiol. 17, 284–294 (2019).CAS 
PubMed 
Article 

Google Scholar 
61.Brennan, C. A., DeLoney-Marino, C. R. & Mandel, M. J. Chemoreceptor VfcA mediates amino acid chemotaxis in Vibrio fischeri. Appl. Environ. Microbiol. 79, 1889–1896 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
62.Graf, J., Dunlap, P. V. & Ruby, E. G. Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri. J. Bacteriol. 176, 6986–6991 (1994).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
63.Millikan, D. S. & Ruby, E. G. FlrA, a sigma54-dependent transcriptional activator in Vibrio fischeri, is required for motility and symbiotic light-organ colonization. J. Bacteriol. 185, 3547–3557 (2003).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
64.Millikan, D. S. & Ruby, E. G. Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization. J. Bacteriol. 186, 4315–4325 (2004).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
65.Wolfe, A. J., Millikan, D. S., Campbell, J. M. & Visick, K. L. Vibrio fischeri sigma54 controls motility, biofilm formation, luminescence, and colonization. Appl. Environ. Microbiol. 70, 2520–2524 (2004).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
66.O’Shea, T. M. et al. Magnesium promotes flagellation of Vibrio fischeri. J. Bacteriol. 187, 2058–2065 (2005).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
67.Ruby, E. G. & Asato, L. M. Growth and flagellation of Vibrio fischeri during initiation of the sepiolid squid light organ symbiosis. Arch. Microbiol. 159, 160–167 (1993).CAS 
PubMed 
Article 

Google Scholar 
68.Beeby, M. et al. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold. Proc. Natl Acad. Sci. USA 113, E1917–E1926 (2016).CAS 
PubMed 
Article 

Google Scholar 
69.Deloney-Marino, C. R. & Visick, K. L. Role for cheR of Vibrio fischeri in the Vibrio-squid symbiosis. Can. J. Microbiol. 58, 29–38 (2012).CAS 
PubMed 
Article 

Google Scholar 
70.Ruby, E. G. et al. Complete genome sequence of Vibrio fischeri: a symbiotic bacterium with pathogenic congeners. Proc. Natl Acad. Sci. USA 102, 3004–3009 (2005).CAS 
PubMed 
Article 

Google Scholar 
71.Nikolakakis, K., Monfils, K., Moriano-Gutierrez, S., Brennan, C. A. & Ruby, E. G. Characterization of the Vibrio fischeri fatty acid chemoreceptors, VfcB and VfcB2. Appl. Environ. Microbiol. 82, 696–704 (2015).PubMed 
Article 
CAS 

Google Scholar 
72.Mandel, M. J. et al. Squid-derived chitin oligosaccharides are a chemotactic signal during colonization by Vibrio fischeri. Appl. Environ. Microbiol. 78, 4620–4626 (2012). While it was long-expected that V. fischeri might sense and be attracted to squid-produced molecules to facilitate directed migration into the light organ crypts, this work is the first to identify squid-produced molecules, chitin oligosaccharides, that function as a chemotactic signal promoting colonization.CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
73.Bennett, B. D., Essock-Burns, T. & Ruby, E. G. HbtR, a heterofunctional homolog of the virulence regulator TcpP, facilitates the transition between symbiotic and planktonic lifestyles in Vibrio fischeri. mBio https://doi.org/10.1128/mBio.01624-20 (2020). Comparisons of V. fischeri with the related pathogen Vibrio cholerae reveal that a regulator conserved among Vibrio spp. plays very different roles in the interactions of these two microorganisms with their respective hosts.Article 
PubMed 
PubMed Central 

Google Scholar 
74.Brennan, C. A. et al. A model symbiosis reveals a role for sheathed-flagellum rotation in the release of immunogenic lipopolysaccharide. eLife 3, e01579 (2014). A surprising role for flagellar rotation in the release of lipopolysaccharide molecules that promote squid development is revealed in this work, providing a novel function for the flagellar sheath.PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
75.Stabb, E. V. & Millikan, D. S. in Defensive Mutualism in Microbial Symbiosis Vol. 27 (eds White, J. F. & Torres, M. S.) 85–98 (CRC Press, 2009).76.Bose, J. L., Rosenberg, C. S. & Stabb, E. V. Effects of luxCDABEG induction in Vibrio fischeri: enhancement of symbiotic colonization and conditional attenuation of growth in culture. Arch. Microbiol. 190, 169–183 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
77.Visick, K. L., Foster, J., Doino, J., McFall-Ngai, M. & Ruby, E. G. Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. J. Bacteriol. 182, 4578–4586 (2000).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
78.Verma, S. C. & Miyashiro, T. Niche-specific impact of a symbiotic function on the persistence of microbial symbionts within a natural host. Appl. Environ. Microbiol. 82, 5990–5996 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
79.Dunn, A. K., Millikan, D. S., Adin, D. M., Bose, J. L. & Stabb, E. V. New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Appl. Environ. Microbiol. 72, 802–810 (2006).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
80.Essock-Burns, T., Bongrand, C., Goldman, W. E., Ruby, E. G. & McFall-Ngai, M. J. Interactions of symbiotic partners drive the development of a complex biogeography in the squid-vibrio symbiosis. mBio 11, e00853-20 (2020).PubMed 
PubMed Central 
Article 

Google Scholar 
81.Sycuro, L. K., Ruby, E. G. & McFall-Ngai, M. Confocal microscopy of the light organ crypts in juvenile Euprymna scolopes reveals their morphological complexity and dynamic function in symbiosis. J. Morphol. 267, 555–568 (2006).PubMed 
Article 
PubMed Central 

Google Scholar 
82.Koch, E. J., Miyashiro, T., McFall-Ngai, M. J. & Ruby, E. G. Features governing symbiont persistence in the squid-vibrio association. Mol. Ecol. 23, 1624–1634 (2014).PubMed 
Article 
PubMed Central 

Google Scholar 
83.Wollenberg, M. S., Preheim, S. P., Polz, M. F. & Ruby, E. G. Polyphyly of non-bioluminescent Vibrio fischeri sharing a lux-locus deletion. Environ. Microbiol. 14, 655–668 (2012).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
84.Chun, C. K. et al. Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association. Proc. Natl Acad. Sci. USA 105, 11323–11328 (2008).CAS 
PubMed 
Article 

Google Scholar 
85.McFall-Ngai, M., Heath-Heckman, E. A., Gillette, A. A., Peyer, S. M. & Harvie, E. A. The secret languages of coevolved symbioses: insights from the Euprymna scolopes-Vibrio fischeri symbiosis. Semin. Immunol. 24, 3–8 (2012).PubMed 
Article 

Google Scholar 
86.Moriano-Gutierrez, S. et al. Critical symbiont signals drive both local and systemic changes in diel and developmental host gene expression. Proc. Natl Acad. Sci. USA 116, 7990–7999 (2019).CAS 
PubMed 
Article 

Google Scholar 
87.Verma, S. C. & Miyashiro, T. Quorum sensing in the squid-Vibrio symbiosis. Int. J. Mol. Sci. 14, 16386–16401 (2013).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
88.Stabb, E. V., Schaefer, A., Bose, J. L. & Ruby, E. G. in Chemical Communication Among Bacteria (eds Winans, S. C. & Bassler, B. L.) 233–250 (ASM Press, 2008).89.Lupp, C. & Ruby, E. G. Vibrio fischeri uses two quorum-sensing systems for the regulation of early and late colonization factors. J. Bacteriol. 187, 3620–3629 (2005).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
90.Kimbrough, J. H. & Stabb, E. V. Comparative analysis reveals regulatory motifs at the ainS/ainR pheromone-signaling locus of Vibrio fischeri. Sci. Rep. 7, 11734 (2017).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
91.Kimbrough, J. H. & Stabb, E. V. Substrate specificity and function of the pheromone receptor AinR in Vibrio fischeri ES114. J. Bacteriol. 195, 5223–5232 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
92.Studer, S. V., Mandel, M. J. & Ruby, E. G. AinS quorum sensing regulates the Vibrio fischeri acetate switch. J. Bacteriol. 190, 5915–5923 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
93.Cao, X. et al. The novel sigma factor-like regulator RpoQ controls luminescence, chitinase activity, and motility in Vibrio fischeri. mBio https://doi.org/10.1128/mBio.00285-11 (2012).Article 
PubMed 
PubMed Central 

Google Scholar 
94.Studer, S. V. et al. Non-native acylated homoserine lactones reveal that LuxIR quorum sensing promotes symbiont stability. Environ. Microbiol. 16, 2623–2634 (2014).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
95.Boettcher, K. J. & Ruby, E. G. Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes. J. Bacteriol. 172, 3701–3706 (1990).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
96.Septer, A. N. & Stabb, E. V. Coordination of the arc regulatory system and pheromone-mediated positive feedback in controlling the Vibrio fischeri lux operon. PLoS ONE 7, e49590 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
97.Stabb, E. V. Could positive feedback enable bacterial pheromone signaling to coordinate behaviors in response to heterogeneous environmental cues? mBio https://doi.org/10.1128/mBio.00098-18 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
98.Bose, J. L. et al. Bioluminescence in Vibrio fischeri is controlled by the redox-responsive regulator ArcA. Mol. Microbiol. 65, 538–553 (2007).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
99.Lyell, N. L. et al. Cyclic AMP receptor protein regulates pheromone-mediated bioluminescence at multiple levels in Vibrio fischeri ES114. J. Bacteriol. 195, 5051–5063 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
100.Lyell, N. L., Dunn, A. K., Bose, J. L. & Stabb, E. V. Bright mutants of Vibrio fischeri ES114 reveal conditions and regulators that control bioluminescence and expression of the lux operon. J. Bacteriol. 192, 5103–5114 (2010).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
101.Septer, A. N., Lyell, N. L. & Stabb, E. V. The iron-dependent regulator fur controls pheromone signaling systems and luminescence in the squid symbiont Vibrio fischeri ES114. Appl. Environ. Microbiol. 79, 1826–1834 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
102.Stoudenmire, J. L. et al. An iterative, synthetic approach to engineer a high-performance PhoB-specific reporter. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.00603-18 (2018). This study not only provides a road map for synthetic promoter engineering in V. fischeri but also uncovers evidence for possible microenvironments present within different crypts of the E. scolopes light organ.Article 
PubMed 
PubMed Central 

Google Scholar 
103.Bose, J. L. et al. Contribution of rapid evolution of the luxR-luxI intergenic region to the diverse bioluminescence outputs of Vibrio fischeri strains isolated from different environments. Appl. Environ. Microbiol. 77, 2445–2457 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
104.Dunn, A. K. Vibrio fischeri metabolism: symbiosis and beyond. Adv. Microb. Physiol. 61, 37–68 (2012).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
105.Schwartzman, J. A. & Ruby, E. G. A conserved chemical dialog of mutualism: lessons from squid and vibrio. Microbes Infect. 18, 1–10 (2016).PubMed 
Article 
PubMed Central 

Google Scholar 
106.Pan, S. et al. Model-enabled gene search (MEGS) allows fast and direct discovery of enzymatic and transport gene functions in the marine bacterium Vibrio fischeri. J. Biol. Chem. 292, 10250–10261 (2017).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
107.Thompson, L. R. et al. Transcriptional characterization of Vibrio fischeri during colonization of juvenile Euprymna scolopes. Environ. Microbiol. 19, 1845–1856 (2017).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
108.Wier, A. M. et al. Transcriptional patterns in both host and bacterium underlie a daily rhythm of anatomical and metabolic change in a beneficial symbiosis. Proc. Natl Acad. Sci. USA 107, 2259–2264 (2010). In the first dual transcriptional study of an animal host and its symbionts, gene expression in both partners is shown to be regulated over a day–night cycle, revealing a daily remodelling of the crypt epithelial cells and a night-time provision of chitin to the symbionts.CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
109.Sun, Y., Verma, S. C., Bogale, H. & Miyashiro, T. NagC represses N-acetyl-glucosamine utilization genes in Vibrio fischeri within the light organ of Euprymna scolopes. Front. Microbiol. 6, 741 (2015).PubMed 
PubMed Central 

Google Scholar 
110.Wasilko, N. P. et al. Sulfur availability for Vibrio fischeri growth during symbiosis establishment depends on biogeography within the squid light organ. Mol. Microbiol. 111, 621–636 (2019). This study sheds light on both the nutritional adaptability of V. fischeri and the complex biogeography of the light organ by demonstrating that this symbiont uses different sulfur sources within different regions of the light organ.CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
111.Septer, A. N. et al. Bright luminescence of Vibrio fischeri aconitase mutants reveals a connection between citrate and the Gac/Csr regulatory system. Mol. Microbiol. 95, 283–296 (2015).CAS 
PubMed 
Article 

Google Scholar 
112.Lyell, N. L. & Stabb, E. V. Symbiotic characterization of Vibrio fischeri ES114 mutants that display enhanced luminescence in culture. Appl. Environ. Microbiol. 79, 2480–2483 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
113.Lyell, N. L. et al. An expanded transposon mutant library reveals that Vibrio fischeri delta-aminolevulinate auxotrophs can colonize Euprymna scolopes. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.02470-16 (2017).Article 
PubMed 
PubMed Central 

Google Scholar 
114.Colton, D. M., Stoudenmire, J. L. & Stabb, E. V. Growth on glucose decreases cAMP-CRP activity while paradoxically increasing intracellular cAMP in the light-organ symbiont Vibrio fischeri. Mol. Microbiol. 97, 1114–1127 (2015).CAS 
PubMed 
Article 

Google Scholar 
115.Miyashiro, T. et al. The N-acetyl-D-glucosamine repressor NagC of Vibrio fischeri facilitates colonization of Euprymna scolopes. Mol. Microbiol. 82, 894–903 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
116.Adin, D. M., Visick, K. L. & Stabb, E. V. Identification of a cellobiose utilization gene cluster with cryptic beta-galactosidase activity in Vibrio fischeri. Appl. Environ. Microbiol. 74, 4059–4069 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
117.Pan, M., Schwartzman, J. A., Dunn, A. K., Lu, Z. & Ruby, E. G. A single host-derived glycan impacts key regulatory nodes of symbiont metabolism in a coevolved mutualism. mBio 6, e00811 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
118.Boettcher, K. J., McFall-Ngai, M. J. & Ruby, E. G. Bioluminescence in the symbiotic squid Euprymna scolopes is controlled by a daily biological rhythm. J. Comp. Physiol. 179, 65–73 (1996).Article 

Google Scholar 
119.Kremer, N. et al. The dual nature of haemocyanin in the establishment and persistence of the squid-vibrio symbiosis. Proc. Biol. Sci. 281, 20140504 (2014).PubMed 
PubMed Central 

Google Scholar 
120.Stabb, E. V. Shedding light on the bioluminescence “paradox”. ASM News 71, 223–229 (2005).
Google Scholar 
121.Septer, A. N., Bose, J. L., Dunn, A. K. & Stabb, E. V. FNR-mediated regulation of bioluminescence and anaerobic respiration in the light-organ symbiont Vibrio fischeri. FEMS Microbiol. Lett. 306, 72–81 (2010).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
122.Dunn, A. K. Alternative oxidase activity reduces stress in Vibrio fischeri cells exposed to nitric oxide. J. Bacteriol. https://doi.org/10.1128/JB.00797-17 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
123.Dunn, A. K. & Stabb, E. V. Genetic analysis of trimethylamine N-oxide reductases in the light organ symbiont Vibrio fischeri ES114. J. Bacteriol. 190, 5814–5823 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
124.Septer, A. N., Wang, Y., Ruby, E. G., Stabb, E. V. & Dunn, A. K. The haem-uptake gene cluster in Vibrio fischeri is regulated by Fur and contributes to symbiotic colonization. Environ. Microbiol. 13, 2855–2864 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
125.Graf, J. & Ruby, E. G. Novel effects of a transposon insertion in the Vibrio fischeri glnD gene: defects in iron uptake and symbiotic persistence in addition to nitrogen utilization. Mol. Microbiol. 37, 168–179 (2000).CAS 
PubMed 
Article 

Google Scholar 
126.Eickhoff, M. J. & Bassler, B. L. Vibrio fischeri siderophore production drives competitive exclusion during dual-species growth. Mol. Microbiol. 114, 244–261 (2020).CAS 
PubMed 
Article 

Google Scholar 
127.Ferretti, P. et al. Mother-to-infant microbial transmission from different body sites shapes the developing infant gut microbiome. Cell Host Microbe 24, 133–145 e135 (2018).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
128.Zheng, H. et al. Division of labor in honey bee gut microbiota for plant polysaccharide digestion. Proc. Natl Acad. Sci. USA 116, 25909–25916 (2019).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
129.Aschtgen, M. S. et al. Rotation of Vibrio fischeri flagella produces outer membrane vesicles that induce host development. J. Bacteriol. 198, 2156–2165 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
130.Aschtgen, M. S., Wetzel, K., Goldman, W., McFall-Ngai, M. & Ruby, E. Vibrio fischeri-derived outer membrane vesicles trigger host development. Cell. Microbiol. 18, 488–499 (2016).CAS 
PubMed 
Article 

Google Scholar 
131.Lynch, J. B. et al. Ambient pH alters the protein content of outer membrane vesicles, driving host development in a beneficial symbiosis. J. Bacteriol. https://doi.org/10.1128/JB.00319-19 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
132.Franzenburg, S. et al. Distinct antimicrobial peptide expression determines host species-specific bacterial associations. Proc. Natl Acad. Sci. USA 110, E3730–E3738 (2013).CAS 
PubMed 
Article 

Google Scholar 
133.Chen, F. et al. Bactericidal permeability-increasing proteins shape host-microbe interactions. mBio 8, e00040-17 (2017).PubMed 
PubMed Central 
Article 

Google Scholar 
134.Heath-Heckman, E. A. et al. Shaping the microenvironment: evidence for the influence of a host galaxin on symbiont acquisition and maintenance in the squid-Vibrio symbiosis. Environ. Microbiol. 16, 3669–3682 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
135.Wang, Y. et al. H-NOX-mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri. Proc. Natl Acad. Sci. USA 107, 8375–8380 (2010).CAS 
PubMed 
Article 

Google Scholar 
136.Schwartzman, J. A. et al. Acidic pH promotes lipopolysaccharide modification and alters colonization in a bacteria-animal mutualism. Mol. Microbiol. 112, 1326–1338 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
137.Kwong, W. K. & Moran, N. A. Gut microbial communities of social bees. Nat. Rev. Microbiol. 14, 374–384 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
138.Murfin, K. E. et al. Xenorhabdus bovienii strain diversity impacts coevolution and symbiotic maintenance with Steinernema spp. nematode hosts. mBio 6, e00076 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
139.Wollenberg, M. S. & Ruby, E. G. Population structure of Vibrio fischeri within the light organs of Euprymna scolopes squid from two Oahu (Hawaii) populations. Appl. Environ. Microbiol. 75, 193–202 (2009). This is the first comparative genome-level study of light organ symbionts both between and within adult squid, suggesting that on average each crypt of an organ is colonized by one or two V. fischeri cells, potentially creating crypt-separated, clonal lineages within a polyclonal organ.CAS 
PubMed 
Article 

Google Scholar 
140.Tomich, M., Planet, P. J. & Figurski, D. H. The tad locus: postcards from the widespread colonization island. Nat. Rev. Microbiol. 5, 363–375 (2007).CAS 
PubMed 
Article 

Google Scholar 
141.Gyllborg, M. C., Sahl, J. W., Cronin, D. C. III., Rasko, D. A. & Mandel, M. J. Draft genome sequence of Vibrio fischeri SR5, a strain isolated from the light organ of the Mediterranean squid Sepiola robusta. J. Bacteriol. 194, 1639 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
142.Bongrand, C. et al. Using colonization assays and comparative genomics to discover symbiosis behaviors and factors in Vibrio fischeri. mBio https://doi.org/10.1128/mBio.03407-19 (2020).Article 
PubMed 
PubMed Central 

Google Scholar 
143.Coryell, R. L. et al. Phylogeographic patterns in the Philippine archipelago influence symbiont diversity in the bobtail squid-Vibrio mutualism. Ecol. Evol. 8, 7421–7435 (2018).PubMed 
PubMed Central 
Article 

Google Scholar 
144.Soto, W., Rivera, F. M. & Nishiguchi, M. K. Ecological diversification of Vibrio fischeri serially passaged for 500 generations in novel squid host Euprymna tasmanica. Microb. Ecol. 67, 700–721 (2014).PubMed 
PubMed Central 
Article 

Google Scholar 
145.Soto, W., Travisano, M., Tolleson, A. R. & Nishiguchi, M. K. Symbiont evolution during the free-living phase can improve host colonization. Microbiology 165, 174–187 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
146.Fidopiastis, P. M., von Boletzky, S. & Ruby, E. G. A new niche for Vibrio logei, the predominant light organ symbiont of squids in the genus Sepiola. J. Bacteriol. 180, 59–64 (1998).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
147.Dillon, M. M., Sung, W., Lynch, M. & Cooper, V. S. Periodic variation of mutation rates in bacterial genomes associated with replication timing. mBio https://doi.org/10.1128/mBio.01371-18 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
148.Dillon, M. M., Sung, W., Sebra, R., Lynch, M. & Cooper, V. S. Genome-wide biases in the rate and molecular spectrum of spontaneous mutations in Vibrio cholerae and Vibrio fischeri. Mol. Biol. Evol. 34, 93–109 (2017).CAS 
PubMed 
Article 

Google Scholar 
149.Wollenberg, M. S. & Ruby, E. G. Phylogeny and fitness of Vibrio fischeri from the light organs of Euprymna scolopes in two Oahu, Hawaii populations. ISME J. 6, 352–362 (2012).CAS 
PubMed 
Article 

Google Scholar 
150.Koch, E. J. et al. The cytokine MIF controls daily rhythms of symbiont nutrition in an animal-bacterial association. Proc. Natl Acad. Sci. USA 117, 27578–27586 (2020).CAS 
PubMed 
Article 

Google Scholar 
151.Sun, Y. et al. Intraspecific competition impacts Vibrio fischeri strain diversity during initial colonization of the squid light organ. Appl. Environ. Microbiol. 82, 3082–3091 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
152.Speare, L. et al. Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host. Proc. Natl Acad. Sci. USA 115, E8528–E8537 (2018). The finding that V. fischeri engages in biological ‘warfare’ to become the sole colonizer of a given crypt has provided new insight into the dynamics and processes controlling light organ population structure and strain competition in nature.CAS 
PubMed 
Article 

Google Scholar 
153.Speare, L., Smith, S., Salvato, F., Kleiner, M. & Septer, A. N. Environmental viscosity modulates interbacterial killing during habitat transition. mBio https://doi.org/10.1128/mBio.03060-19 (2020).Article 
PubMed 
PubMed Central 

Google Scholar 
154.Guckes, K. R. et al. Incompatibility of Vibrio fischeri strains during symbiosis establishment depends on two functionally redundant hcp genes. J. Bacteriol. https://doi.org/10.1128/JB.00221-19 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
155.Guckes, K. R., Cecere, A. G., Williams, A. L., McNeil, A. E. & Miyashiro, T. The bacterial enhancer binding protein VasH promotes expression of a Type VI secretion system in Vibrio fischeri during symbiosis. J. Bacteriol. https://doi.org/10.1128/JB.00777-19 (2020).Article 
PubMed 
PubMed Central 

Google Scholar 
156.Bultman, K. M., Cecere, A. G., Miyashiro, T., Septer, A. N. & Mandel, M. J. Draft genome sequences of type VI secretion system-encoding Vibrio fischeri strains FQ-A001 and ES401. Microbiol. Resour. Announc. https://doi.org/10.1128/MRA.00385-19 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
157.Doino, J. A. & McFall-Ngai, M. J. A transient exposure to symbiosis-competent bacteria induces light organ morphogenesis in the host squid. Biol. Bull. 189, 347–355 (1995).CAS 
PubMed 
Article 

Google Scholar 
158.Dunn, A. K., Martin, M. O. & Stabb, E. V. Characterization of pES213, a small mobilizable plasmid from Vibrio fischeri. Plasmid 54, 114–134 (2005).CAS 
PubMed 
Article 

Google Scholar 
159.Lyell, N. L., Dunn, A. K., Bose, J. L., Vescovi, S. L. & Stabb, E. V. Effective mutagenesis of Vibrio fischeri by using hyperactive mini-Tn5 derivatives. Appl. Environ. Microbiol. 74, 7059–7063 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
160.Stoudenmire, J. L., Black, M., Fidopiastis, P. M. & Stabb, E. V. Mutagenesis of Vibrio fischeri and other marine bacteria using hyperactive mini-Tn5 derivatives. Methods Mol. Biol. 2016, 87–104 (2019).CAS 
PubMed 
Article 

Google Scholar 
161.Pollack-Berti, A., Wollenberg, M. S. & Ruby, E. G. Natural transformation of Vibrio fischeri requires tfoX and tfoY. Environ. Microbiol. 12, 2302–2311 (2010).PubMed 
PubMed Central 

Google Scholar 
162.Visick, K. L., Hodge-Hanson, K. M., Tischler, A. H., Bennett, A. K. & Mastrodomenico, V. Tools for rapid genetic engineering of Vibrio fischeri. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.00850-18 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
163.Burgos, H. L. et al. Multiplexed competition in a synthetic squid light organ microbiome using barcode-tagged gene deletions. mSystems 5, e00846-20 (2020).PubMed 
PubMed Central 
Article 

Google Scholar 
164.Brooks, J. F. II, Gyllborg, M. C., Kocher, A. A., Markey, L. E. & Mandel, M. J. TfoX-based genetic mapping identifies Vibrio fischeri strain-level differences and reveals a common lineage of laboratory strains. J. Bacteriol. 197, 1065–1074 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
165.Califano, G. et al. Draft genome sequence of Aliivibrio fischeri strain 5LC, a bacterium retrieved from gilthead seabream (Sparus aurata) larvae reared in aquaculture. Genome Announc. 3, e00593-15 (2015).PubMed 
PubMed Central 
Article 

Google Scholar 
166.Hehemann, J.-H. et al. Adaptive radiation by waves of gene transfer leads to fine-scale resource partitioning in marine microbes. Nat. Commun. 7, 12860 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
167.Nikolakakis, K., Lehnert, E., McFall-Ngai, M. J. & Ruby, E. G. Use of hybridization chain reaction-fluorescent in situ hybridization to track gene expression by both partners during initiation of symbiosis. Appl. Environ. Microbiol. 81, 4728–4735 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar  More