Philippa Kaur

All the results were reported relative to the control, unless specifically stated to the contrary or for clarity.Growth of cucumber plants in response to different biofertilizersSoilThere was no significant difference in cucumber growth before microbial fertilizer was applied. However, some microbial fertilizers significantly increased cucumber height and stem diameter when they were applied within 4 weeks from when the seedlings were planted (Fig. 1a,b,e,f). In the second week, SHZ and SMF increased plant height by 11.2 and 9.5%, respectively. In the third week, S267, SBS, SBH, SM and SHZ increased plant height by 12.0, 13.8, 15.0, 20.5 and 26.9%, respectively (Fig. 1a). In the fourth and fifth weeks, some treatments significantly increased cucumber height. In the second and third weeks, S267 significantly increased stem diameter by 21.2 and 16.8% (Fig. 1b).Figure 1Effect of different biofertilizer treatments on the growth of cucumber seedlings produced in soil or substrate in a greenhouse. S267 = Trichoderma Strain 267 added to soil; SBH = Bacillus subtilis and T. harzianum biofertilizers added to soil; SBS = B. subtilis biofertilizer added to the soil; SM = Compound biofertilizer added to soil; SHZ = T. harzianum biofertilizer added to soil; SCK = Untreated soil. US267 = T.267 biofertilizer added to substrate; USBH = B. subtilis and T. harzianum biofertilizers added to substrate; USBS = B. subtilis biofertilizer added to substrate; USM = Compound biofertilizer added to substrate; USHZ = T. harzianum biofertilizer added to substrate; USCK = Untreated substrate.Full size imageOver the subsequent 5 weeks, some microbial fertilizer treatments decreased cucumber height and stem diameter (Fig. 1g,h).SubstrateThere were no significant differences in cucumber growth before microbial fertilizer microbial fertilizer was applied (Fig. 1c,d,g,h). However, within 4 weeks of applying the microbial fertilizer, each biofertilizer treatment applied significantly increased cucumber height (Fig. 1c). US267 and USHZ significantly increased cucumber height by 39.8–75.4% and 56.1–86.1%, respectively. US267, USM and USHZ significantly increased the stem diameter by 76.8–108.9%, 71.1–97.6% and 80.4–122.4%, respectively (Fig. 1d).Over the subsequent 5 weeks, US267, USM and USHZ treatments continued to significantly increase cucumber height and stem diameter (Fig. 1g,h).Changes in the taxonomic composition of soil-borne fungal pathogensSoilBiofertilizers application significantly reduced the taxonomic composition of soil-borne fungal pathogens at different times during the cucumber growth period (Tables 1 and 2). Fusarium spp. were significantly reduced (T, 63.8% reduction, P  More

Schimper, A. F. W. Plant Geography upon a Physiological Basis (Clarendon Press, 1903).Alexander, J. M., Diez, J. M. & Levine, J. M. Novel competitors shape species’ responses to climate change. Nature 525, 515–518 (2015).Article 
ADS 
CAS 
PubMed 

Google Scholar 
HilleRisLambers, J., Harsch, M. A., Ettinger, A. K., Ford, K. R. & Theobald, E. J. How will biotic interactions influence climate change-induced range shifts? Ann. N. Y. Acad. Sci. 1297, 112–125 (2013).PubMed 

Google Scholar 
Chesson, P. Mechanisms of maintenance of species diversity. Annu. Rev. Ecol. Syst. 31, 343–366 (2000).Article 

Google Scholar 
Loarie, S. R., Weiss, S. B., Hamilton, H., Branciforte, R. & Kraft, N. J. B. The geography of climate change: implications for conservation biogeography. Divers. Distrib. 16, 476–487 (2010).Article 

Google Scholar 
Callaway, R. M. et al. Positive interactions among alpine plants increase with stress. Nature 417, 844–848 (2002).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Dybzinski, R. & Tilman, D. Resource use patterns predict long‐term outcomes of plant competition for nutrients and light. Am. Nat. 170, 305–318 (2007).Article 
PubMed 

Google Scholar 
Hautier, Y., Niklaus, P. A. & Hector, A. Competition for light causes plant biodiversity loss after eutrophication. Science 324, 636–638 (2009).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Levine, J. M. & HilleRisLambers, J. The importance of niches for the maintenance of species diversity. Nature 461, 254–257 (2009).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Kraft, N. J. B., Godoy, O. & Levine, J. M. Plant functional traits and the multidimensional nature of species coexistence. Proc. Natl Acad. Sci. USA 112, 797–802 (2015).Article 
ADS 
CAS 
PubMed 
PubMed Central 

Google Scholar 
Knapp, A. K. et al. Rainfall variability, carbon cycling, and plant species diversity in a mesic grassland. Science 298, 2202–2205 (2002).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Sandel, B. et al. Contrasting trait responses in plant communities to experimental and geographic variation in precipitation. New Phytol. 188, 565–575 (2010).Article 
PubMed 

Google Scholar 
Esch, E. H., Ashbacher, A. C., Kopp, C. W. & Cleland, E. E. Competition reverses the response of shrub seedling mortality and growth along a soil moisture gradient. J. Ecol. 106, 2096–2108 (2018).Article 

Google Scholar 
Alon, M. & Sternberg, M. Effects of extreme drought on primary production, species composition and species diversity of a Mediterranean annual plant community. J. Veg. Sci. 30, 1045–1061 (2019).Article 

Google Scholar 
Chesson, P. Updates on mechanisms of maintenance of species diversity. J. Ecol. 106, 1773–1794 (2018).Article 

Google Scholar 
Barabás, G., D’Andrea, R. & Stump, S. M. Chesson’s coexistence theory. Ecol. Monogr. 88, 277–303 (2018).Article 

Google Scholar 
Ellner, S. P., Snyder, R. E., Adler, P. B. & Hooker, G. An expanded modern coexistence theory for empirical applications. Ecol. Lett. 22, 3–18 (2019).Article 
ADS 
PubMed 

Google Scholar 
Adler, P., Hillerislambers, J. & Levine, J. A niche for neutrality. Ecol. Lett. 10, 95–104 (2007).Article 
PubMed 

Google Scholar 
Germain, R. M., Mayfield, M. M. & Gilbert, B. The ‘filtering’ metaphor revisited: competition and environment jointly structure invasibility and coexistence. Biol. Lett. 14, 20180460 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
Pau, S. et al. Predicting phenology by integrating ecology, evolution and climate science. Glob. Change Biol. 17, 3633–3643 (2011).Article 
ADS 

Google Scholar 
Fargione, J. & Tilman, D. Niche differences in phenology and rooting depth promote coexistence with a dominant C4 bunchgrass. Oecologia 143, 598–606 (2005).Article 
ADS 
PubMed 

Google Scholar 
Godoy, O., Kraft, N. J. B. & Levine, J. M. Phylogenetic relatedness and the determinants of competitive outcomes. Ecol. Lett. 17, 836–844 (2014).Article 
PubMed 

Google Scholar 
Díaz, S. et al. The global spectrum of plant form and function. Nature 529, 167–171 (2016).Article 
ADS 
PubMed 

Google Scholar 
Kunstler, G. et al. Plant functional traits have globally consistent effects on competition. Nature 529, 204–207 (2016).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Diffenbaugh, N. S., Swain, D. L. & Touma, D. Anthropogenic warming has increased drought risk in California. Proc. Natl Acad. Sci. USA 112, 3931–3936 (2015).Article 
ADS 
CAS 
PubMed 
PubMed Central 

Google Scholar 
Swain, D. L., Langenbrunner, B., Neelin, J. D. & Hall, A. Increasing precipitation volatility in twenty-first-century California. Nat. Clim. Change 8, 427–433 (2018).Article 
ADS 

Google Scholar 
Chesson, P. Geometry, heterogeneity and competition in variable environments. Phil. Trans. R. Soc. Lond. B 330, 165–173 (1990).Article 
ADS 

Google Scholar 
Aronson, J., Kigel, J., Shmida, A. & Klein, J. Adaptive phenology of desert and Mediterranean populations of annual plants grown with and without water stress. Oecologia 89, 17–26 (1992).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Santa Barbara County Public Works water resources hydrology: historical rainfall data: daily and monthly rainfall. County of Santa Barbara http://www.countyofsb.org/pwd/water/downloads/hydro/421dailys.pdf (2019).Kandlikar, G. S., Kleinhesselink, A. R. & Kraft, N. J. B. Functional traits predict species responses to environmental variation in a California grassland annual plant community. J. Ecol. 110, 833–844 (2022).Article 
CAS 

Google Scholar 
Cleland, E. E. et al. Sensitivity of grassland plant community composition to spatial vs. temporal variation in precipitation. Ecology 94, 1687–1696 (2013).Article 
PubMed 

Google Scholar 
Usinowicz, J. et al. Temporal coexistence mechanisms contribute to the latitudinal gradient in forest diversity. Nature 550, 105–108 (2017).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Kandlikar, G. S., Johnson, C. A., Yan, X., Kraft, N. J. B. & Levine, J. M. Winning and losing with microbes: how microbially mediated fitness differences influence plant diversity. Ecol. Lett. 22, 1178–1191 (2019).PubMed 

Google Scholar 
Kleinhesselink, A. R., Kraft, N. J. B., Pacala, S. W. & Levine, J. M. Detecting and interpreting higher order interactions in ecological communities. Ecol. Lett. 25, 1604–1617 (2022).Article 
PubMed 

Google Scholar 
Saavedra, S. et al. A structural approach for understanding multispecies coexistence. Ecol. Monogr. 87, 470–486 (2017).Article 

Google Scholar 
Levine, J. I., Levine, J. M., Gibbs, T. & Pacala, S. W. Competition for water and species coexistence in phenologically structured annual plant communities. Ecol. Lett. 25, 1110–1125 (2022).Article 
PubMed 

Google Scholar 
Farrior, C. E. et al. Resource limitation in a competitive context determines complex plant responses to experimental resource additions. Ecology 94, 2505–2517 (2013).Article 
PubMed 

Google Scholar 
Harrison, S., Grace, J. B., Davies, K. F., Safford, H. D. & Viers, J. H. Invasion in a diversity hotspot: exotic cover and native richness in the Californian serpentine flora. Ecology 87, 695–703 (2006).Article 
PubMed 

Google Scholar 
Pérez-Harguindeguy, N. et al. New handbook for standardised measurement of plant functional traits worldwide. Aust. J. Bot. 61, 167–234 (2013).Article 

Google Scholar 
Godoy, O. & Levine, J. M. Phenology effects on invasion success: insights from coupling field experiments to coexistence theory. Ecology 95, 726–736 (2014).Article 
PubMed 

Google Scholar  More

Yang, X., Quam, M. B., Zhang, T. & Sang, S. Global burden for dengue and the evolving pattern in the past 30 years. J. Travel Med. 28, taab146 (2021).Simmons, C. P., Farrar, J. J., van Vinh Chau, N. & Wills, B. Dengue. N. Engl. J. Med. 366, 1423–1432 (2012).Article 
CAS 
PubMed 

Google Scholar 
Brady, O. J. et al. Refining the global spatial limits of dengue virus transmission by evidence-based consensus. PLoS Negl. Trop. Dis. 6, e1760 (2012).Article 
PubMed 
PubMed Central 

Google Scholar 
Morin, C. W., Comrie, A. C. & Ernst, K. Climate and dengue transmission: Evidence and implications. Environ. Health Perspect. 121, 1264–1272 (2013).Article 
PubMed 
PubMed Central 

Google Scholar 
Brady, O. J. et al. Global temperature constraints on Aedes aegypti and Ae. albopictus persistence and competence for dengue virus transmission. Parasites Vectors 7, 338 (2014).Betanzos-Reyes, Á. F. et al. Association of dengue fever with Aedes spp. abundance and climatological effects. Salud Pública de México 60, 12 (2017).WHO. Dengue and severe dengue. (2022).Gubler, D. J. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev. 11, 480–496 (1998).Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 
Delatte, H. et al. Aedes albopictus, vecteur des virus du chikungunya et de la dengue à La Réunion : biologie et contrôle. Parasite 15, 3–13 (2008).Article 
CAS 
PubMed 

Google Scholar 
Kles, V., Michault, A., Rodhain, F., Mevel, F. & Chastel, C. A serological survey regarding Flaviviridae infections on the island of Reunion (1971–1989). Bull. Soc. Pathol. Exot. 1990(87), 71–76 (1994).
Google Scholar 
Pierre, V. et al. Epidémie de dengue 1 à la Réunion en 2004. Journal de Veille Sanitaire (2005).Vincent, M. et al. From the threat to the large outbreak: dengue on Reunion Island, 2015 to 2018. Eurosurveillance 24, (2019).Cellule Santé Publique France en Région, ARS. Situation de la dengue à La Réunion au 15 décembre 2020. https://www.lareunion.ars.sante.fr/avec-le-retour-de-lete-agissons-des-maintenant-contre-la-dengue (2020).Agence Régionale de Santé. Communiqué de presse: dengue à La Réunion. Situation au 28 juillet 2021. https://www.lareunion.ars.sante.fr/system/files/2021-07/2021-07-28-Dengue-Situation à La Réunion_0.pdf (2021).Hafsia, S. et al. Overview of dengue outbreaks in the southwestern Indian Ocean and analysis of factors involved in the shift toward endemicity in Reunion Island: A systematic review. PLoS Negl. Trop. Dis. 16, e0010547 (2022).Article 
PubMed 
PubMed Central 

Google Scholar 
Reiter, P., Fontenille, D. & Paupy, C. Aedes albopictus as an epidemic vector of chikungunya virus: Another emerging problem?. Lancet. Infect. Dis 6, 463–464 (2006).Article 
PubMed 

Google Scholar 
Njenga, M. K. et al. Tracking epidemic Chikungunya virus into the Indian Ocean from East Africa. J. Gen. Virol. 89, 2754–2760 (2008).Article 
CAS 

Google Scholar 
Soumahoro, M.-K. et al. The Chikungunya epidemic on La Réunion Island in 2005–2006: a cost-of-illness study. PLoS Negl. Trop. Dis. 5, e1197 (2011).Article 
PubMed 
PubMed Central 

Google Scholar 
Larrieu, S., Balleydier, E., Renault, P., Baville, M. & Filleul, L. [Epidemiological surveillance du chikungunya on Reunion Island from 2005 to 2011]. Médecine tropicale : Revue du Corps de Santé colonial 72 Spec No, 38–42 (2012).Soghigian, J. et al. Genetic evidence for the origin of Aedes aegypti, the yellow fever mosquito, in the southwestern Indian Ocean. Mol. Ecol. 29, 3593–3606 (2020).Article 
CAS 
PubMed 
PubMed Central 

Google Scholar 
Kotsakiozi, P. et al. Population structure of a vector of human diseases: Aedes aegypti in its ancestral range Africa. Ecol. Evol. 8, 7835–7848 (2018).Article 
PubMed 

Google Scholar 
MacGregor, M. E. Aedes (Stegomyia) mascarensis, MacGregor: A new Mosquito from Mauritius. Bull. Entomol. Res. 14, 409–412 (1924).Article 

Google Scholar 
Salvan, M. & Mouchet, J. Aedes albopictus et Aedes aegypti à l’Ile de La Réunion. Ann. Soc. Belg. Med. Trop. 74, 323–326 (1994).CAS 
PubMed 

Google Scholar 
Bagny, L., Delatte, H., Quilici, S. & Fontenille, D. Progressive Decrease in Aedes aegypti distribution in Reunion Island since the 1900s. J. Med. Entomol. 46, 1541–1545 (2009).Article 
PubMed 

Google Scholar 
Le Vassal, J. J. paludisme à l’Ile de La Réunion. Per Gli Stud Della Maria 8, 18–27 (1907).
Google Scholar 
Delatte, H. et al. Geographic distribution and developmental sites of Aedes albopictus (Diptera: Culicidae) during a chikungunya epidemic event. Vector-Borne Zoon. Dis. 8, 25–34 (2008).Article 
CAS 

Google Scholar 
Hamon, J. Etudes biologique et systématique des Culicinae de l’Ile de La Réunion. Mem. Inst. Scient. Madagascar 4, 521–541 (1953).
Google Scholar 
Bouyer, J. & Lefrançois, T. Boosting the sterile insect technique to control mosquitoes. Trends Parasitol. 30, 271–273 (2014).Article 
PubMed 

Google Scholar 
Haramboure, M. et al. Modelling the control of Aedes albopictus mosquitoes based on sterile males release techniques in a tropical environment. Ecol. Model. 424, 109002 (2020).Article 

Google Scholar 
Bouyer, J., Yamada, H., Pereira, R., Bourtzis, K. & Vreysen, M. J. B. Phased conditional approach for mosquito management using sterile insect technique. Trends Parasitol. 36, 325–336 (2020).Article 
PubMed 

Google Scholar 
Bouyer, J. & Vreysen, M. J. B. Yes, irradiated sterile male mosquitoes can be sexually competitive!. Trends Parasitol. 36, 877–880 (2020).Article 
CAS 
PubMed 

Google Scholar 
Organization, W. H. Guidelines for laboratory and field testing of mosquito larvicides. WHO/CDS/WHOPES/GCDPP/2005.13 (2005).World Health Organization and Special Programme for Research and Training in Tropical Diseases and World Health Organization. Department of Control of Neglected Tropical Diseases and World Health Organization. Epidemic and Pandemic Alert. Dengue: Guidelines for diagnosis, treatment, prevention and control. (World Health Organization, 2009).Yap, H. H. Preliminary report on the color preference for oviposition by Aedes albopictus (Skuse) in the field. Southeast Asian J. Trop. Med. Public Health 6, 1–2 (1975).
Google Scholar 
Yap, H. H., Lee, C. Y., Chong, N. L., Foo, A. E. S. & Lim, M. P. Oviposition site preference of Aedes albopictus in the laboratory. J. Am. Mosquito Control Assoc. Mosquito News 11, 128–132 (1995).CAS 
PubMed 

Google Scholar 
Marin, G., Mahiba, B., Arivoli, S. & Tennyson, S. Does colour of ovitrap influence the ovipositional preference of Aedes aegypti Linnaeus 1762 (Diptera: Culicidae). Int. J. Mosq. Res 7, 11–15 (2020).CAS 

Google Scholar 
Claudel, I. et al. To bait or not to bait? Optimizing the use of adult mosquito traps for monitoring arbovirus vector populations in La Réunion Island. (2022). https://doi.org/10.21203/rs.3.rs-1798972/v1.Cleveland, W. S. Visualizing data. (Hobart press, 1993).Lamigueiro, Ó. P. Displaying time series, spatial, and space-time data with R. (Chapman; Hall/CRC, 2018).Yoshioka, M. et al. Diet and density dependent competition affect larval performance and oviposition site selection in the mosquito species Aedes albopictus (Diptera: Culicidae). Parasites Vectors 5, (2012).Wong, J., Stoddard, S. T., Astete, H., Morrison, A. C. & Scott, T. W. Oviposition site selection by the dengue vector Aedes aegypti and its implications for dengue control. PLoS Negl. Trop. Dis. 5, e1015 (2011).Article 
PubMed 
PubMed Central 

Google Scholar 
Hawley, W. A. The biology of aedes albopictus. J. Am. Mosquito Control Assoc. Suppl 1, 1–39 (1988).Delatte, H., Gimonneau, G., Triboire, A. & Fontenille, D. Influence of temperature on immature development, survival, longevity, fecundity, and gonotrophic cycles of Aedes albopictus, vector of Chikungunya and dengue in the Indian Ocean. J. Med. Entomol. 46, 33–41 (2009).Article 
CAS 
PubMed 

Google Scholar 
Daugherty, M. P., Alto, B. W. & Juliano, S. A. Invertebrate carcasses as a resource for competing Aedes albopictus and Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 37, 364–372 (2000).Article 
CAS 
PubMed 

Google Scholar 
Papaj, D. R. & Rausher, M. D. Individual variation in host location by phytophagous insects. Herbivorous Insects: Host seeking behavior and mechanisms 77–127 (1983).Valladares, G. & Lawton, J. H. Host-plant selection in the holly leaf-miner: Does mother know best?. J. Anim. Ecol. 60, 227 (1991).Article 

Google Scholar 
Ellis, A. M. Incorporating density dependence into the oviposition preference-offspring performance hypothesis. J. Anim. Ecol. 77, 247–256 (2008).Article 
PubMed 

Google Scholar 
Juliano, S. A., OMeara, G. F., Morrill, J. R. & Cutwa, M. M. Desiccation and thermal tolerance of eggs and the coexistence of competing mosquitoes. Oecologia 130, 458–469 (2002).Costanzo, K. S., Kesavaraju, B. & Juliano, S. A. Condition-specific competion in container mosquitoes: The role of non-competing life-history stages. Ecology 86, 3289–3295 (2005).Article 
PubMed 

Google Scholar 
Sanchez, M. & Probst, J.-M. Distribution and conservation status of the Manapany day gecko, Phelsuma inexpectata MERTENS, 1966, an endemic threatened reptile from Réunion Island (Squamata: Gekkonidae). Cahiers scientifiques de l’océan Indien occidental 2, (2011).Braks, M. A. H., Honório, N. A., Lounibos, L. P., De-Oliveira, R. L. & Juliano, S. A. Interspecific competition between two invasive species of container mosquitoes, Aedes aegypti and Aedes albopictus (Diptera: Culicidae), in Brazil. Ann. Entomol. Soc. Am. 97, 130–139 (2004).Article 

Google Scholar 
Moore, C. G. & Fisher, B. R. Competition in mosquitoes.1 Density and species ratio effects on growth, mortality, fecundity, and production of growth retardant2. Ann. Entomol. Soc. Am. 62, 1325–1331 (1969).Madeira, N. G., Macharelli, C. A. & Carvalho, L. R. Variation of the Oviposition Preferences of Aedes aegypti in Function of Substratum and Humidity. Mem. Inst. Oswaldo Cruz 97, 415–420 (2002).Article 
CAS 
PubMed 

Google Scholar 
Bellini, R. et al. Use of the sterile insect technique against Aedes albopictus in Italy: first results of a pilot trial. in Area-wide control of insect pests 505–515 (Springer, 2007).Boussès, P., Dehecq, J. S., Brengues, C. & Fontenille, D. Inventaire actualisé des moustiques (Diptera : Culicidae) de l’île de La Réunion, océan Indien. Bulletin de la Société de pathologie exotique 106, 113–125 (2013).Article 
PubMed 

Google Scholar 
Zeileis, A., Kleiber, C. & Jackman, S. Regression models for count data in R. J. Stat. Softw. 27, (2008).Sileshi, G. Selecting the right statistical model for analysis of insect count data by using information theoretic measures. Bull. Entomol. Res. 96, 479–488 (2006).CAS 
PubMed 

Google Scholar 
Guthery, F. S., Burnham, K. P. & Anderson, D. R. Model selection and multimodel inference: A practical information-theoretic approach. J. Wildl. Manag. 67, 655 (2003).Article 

Google Scholar 
Hurvich, C. M. & Tsai, C.-L. Model selection for extended quasi-likelihood models in small samples. Biometrics 51, 1077–1084 (1995).Article 
CAS 
PubMed 
MATH 

Google Scholar 
Burnham, K. P. & Anderson, D. R. Model selection and multimodel inference: a practical information-theoretic approach. 496 (Springer-Verlag, 2002).Manly, B. F. J. Randomization, bootstrap and Monte Carlo methods in biology. 399 (CRC Press / Chapman & Hall, 2006). https://doi.org/10.1201/9781315273075.Holm, S. A simple sequentially rejective multiple test procedure. Scand. J. Stat. 65–70 (1979).R Core Team. R: A Language and Environment for Statistical Computing. (R Foundation for Statistical Computing, 2022).Lesnoff, M. & Lancelot, R. aods3: analysis of overdispersed data using S3 methods. (2018).Barton, K. MuMIn: Multi-Model Inference. (2022).Xie, Y., Dervieux, C. & Riederer, E. R Markdown Cookbook. (Chapman; Hall/CRC, 2020). https://doi.org/10.1201/9781003097471. More

Cavalleri, A., Masumoto, M., Minaei, K., Mound, L. & Ulitzka, M. R. ThripsWiki – providing information on the World’s thrips. https://thrips.info/wiki/Main_Page (2022).Kirk, W. D. J., de Kogel, W. J., Koschier, E. H. & Teulon, D. A. J. Semiochemicals for thrips and their use in pest management. Annu. Rev. Entomol. 66, 101–119. https://doi.org/10.1146/annurev-ento-022020-081531 (2021).Article 
CAS 
PubMed 

Google Scholar 
Mota-Sanchez, D. & Wise, C. J. The Arthropod Pesticide Resistance Database. Available at http://www.pesticideresistance.org (2022).von Kèler, S. Entomologisches Wörterbuch. mit besonderer Berücksichtigung der morphologischen Terminologie. 3rd edn. (Akademie, Berlin, 1963).
Google Scholar 
Pascini, T. V. & Martins, G. F. The insect spermatheca: an overview. Zoology Jena (Germany) 121, 56–71. https://doi.org/10.1016/j.zool.2016.12.001 (2017).Article 

Google Scholar 
Bode, W. Der Ovipositor und die weiblichen Geschlechtswege der Thripiden (Thysanoptera, Terebrantia). Z. Morph. Tiere (Zeitschrift für Morphologie der Tiere) 81, 1–53; https://doi.org/10.1007/BF00290072 (1975).Moritz, G. Zur Morphologie und Anatomie des Fransenflüglers Aeolothrips intermedius Bagnall, 1934 (Aeolothripidae, Thysanoptera, Insecta) III. Mitteilung: Das Abdomen. Investigation on the Morphology and Anatomy in Aeolothrips intermedius Bagnall, 1934 (Aeolothripidae, Thysanoptera, Insecta) 3. The Abdomen. Zoologische Jahrbücher/ Abteilung für Anatomie und Ontogenie der Tiere 108, 293–340 (1982).Moritz, G. Die Ontogenese der Thysanoptera (lnsecta) unter besonderer Berücksichtigung des Fransenflüglers Hercinothrips femoralis (O. M. REUTER, 1891) (Thysanoptera, Thripidae, Panchaetothripinae) VI. Imago – Abdomen. The Ontogenesis of Thysanoptera (Insecta) with Special Reference to the Panchaetothripine Hercinothrips femoralis (O. M. REUTER, 1891) (Thysanoptera, Thripidae, Panchaetothripinae) VI. Imago – Abdomen. Zoologische Jahrbücher/ Abteilung für Anatomie und Ontogenie der Tiere 119, 157–217 (1989).Heming, B. S. Postembryonic development of the female reproductive system in Frankliniella fusca (Thripidae) and Haplothrips verbasci (Phlaeothripidae) (Thysanoptera). Misc. Publ. Entomol. Soc. Am. 7, 197–234 (1970).
Google Scholar 
Heming, B. S. History of the germ line in male and female thrips. In Thrips Biology and Management. International conference on Thysanoptera: Towards Understanding Thrips Management, Vermont, edited by B. L. Parker, M. Skinner & T. Lewis (Springer, Berlin, 1995), pp. 505–535.Dallai, R., Del Bene, G. & Lupetti, P. Fine structure of spermatheca and accessory gland of Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Int. J. Insect Morphol. Embryol. 25, 317–330; https://doi.org/10.1016/0020-7322(95)00018-6 (1996).Jordan, K. Anatomie und Biologie der Physapoda. Zeitschrift für wissenschaftliche Zoologie 47, 541–620 (1888).
Google Scholar 
Bournier, A. L`appareil gènital femelle de Caudothrips buffai Karny et sa pompe spermatige (Thysan.). Bulletin de la Sociètè Entomologique de France 67, 203–207 (1962).Dhileepan, K. & Ananthakrishnan, T. Impact of sex-limited and alary polymorphism on spermathecal diversity and reproductive behaviour in some mycophagous Tubulifera. Proc. Indian Natl. Acad. Sci. 4, 329–336 (1987).
Google Scholar 
Bhatti, J. S. The spermatheca as a useful character for species differentiation in Coleothrips Haliday (Insecta: Terebrantia: Aeolothripidae). J. Pure Appl. Zool. 1, 111–116 (1988).
Google Scholar 
Klocke, F. Beiträge zur Anatomie und Histologie der Thysanopteren. Zeitschrift für wissenschaftliche Zoologie 128, 1 (1926).
Google Scholar 
Priesner, H. Die Thysanopteren Europas (F. Wagner Verlag, Wien, 1926–1928).Bode, W. Spermienstruktur und Spermatohistogenese bei Thrips validus Uzel (Insecta, Thysanoptera). Zoologische Jahrbücher/ Abteilung für Anatomie und Ontogenie der Tiere 109, 301–318 (1983).
Google Scholar 
Moritz, G. Thripse. Fransenflügler, Thysanoptera. 1st ed. (Westarp Wissenschaften, Hohenwarsleben, 2006).Bournier, A. Contribution à l’étude de la parthénogénèse des thysanoptères et de sa cytologie. Archives de Zoologie expérimentale et générale 93, 219–318 (1956).
Google Scholar 
Heming, B. S. Postembryonic development of the male reproductive system in Frankliniella fusca (Thripidae) and Haplothrips verbasci (Phlaeothripidae) (Thysanoptera). Misc. Publ. Entomol. Soc. Am. 7, 235–272 (1970).
Google Scholar 
Krueger, S., Jilge, M., Mound, L. & Moritz, G. B. Reproductive behavior of Echinothrips americanus (Thysanoptera: Thripidae). J. Insect Sci. 17; https://doi.org/10.1093/jisesa/iex043 (2017).Krueger, S. & Moritz, G. Sperm ultrastructure in arrhenotokous and thelytokous Thysanoptera. Arthropod Struct. Dev. 64, 101084; https://doi.org/10.1016/j.asd.2021.101084 (2021).Lewis, T. Thrips. Their biology, ecology and economic importance (Academic Press, London, 1973).Jacobs, W. & Seidel, F. Systematische Zoologie, Insekten (Fischer, 1975).
Google Scholar 
Gotoh, A., Ito, F. & Billen, J. Vestigial spermatheca morphology in honeybee workers, Apis cerana and Apis mellifera, from Japan. Apidologie 44, 133–143; https://doi.org/10.1007/s13592-012-0165-6 (2013).Gotoh, A., Billen, J., Hashim, R. & Ito, F. Degeneration patterns of the worker spermatheca during morphogenesis in ants (Hymenoptera: Formicidae). Evol. Dev. 18, 96–104; https://doi.org/10.1111/ede.12182 (2016).Schoeters, E. & Billen, J. The importance of the spermathecal duct in bumblebees. J. Insect Physiol. 46, 1303–1312; https://doi.org/10.1016/S0022-1910(00)00052-4 (2000).Gobin, B., Ito, F., Peeters, C. & Billen, J. Queen-worker differences in spermatheca reservoir of phylogenetically basal ants. Z. Zellforsch (Zeitschrift für Zellforschung und Mikroskopische Anatomie) 326, 169–178; https://doi.org/10.1007/s00441-006-0232-2 (2006).Gobin, B., Ito, F., Billen, J. & Peeters, C. Degeneration of sperm reservoir and the loss of mating ability in worker ants. Die Naturwissenschaften 95, 1041–1048; https://doi.org/10.1007/s00114-008-0420-x (2008).Gotoh, A., Billen, J., Hashim, R., Ito, F. Comparison of spermatheca morphology between reproductive and non-reproductive females in social wasps. Arthropod. Struct. Dev. 37, 199–209; https://doi.org/10.1016/j.asd.2007.11.001 (2008).Gotoh, A., Billen, J., Tsuji, K., Sasaki, T. & Ito, F. Histological study of the spermatheca in three thelytokous parthenogenetic ant species, Pristomyrmex punctatus, Pyramica membranifera and Monomorium triviale (Hymenoptera: Formicidae). Acta Zool. 93, 200–207; https://doi.org/10.1111/j.1463-6395.2010.00498.x (2012).Buffa, P. Studi intorno al ciclo partenogenetico dell´ Heliothrips haemorrhoidales (Boúche). REDIA 7, 71–109 (1911).
Google Scholar 
Bene, G. D., Cavallo, V., Lupetti, P. & Dallai, R. Ultrastructure of the accessory gland in the parthenogenetic thrips Heliothrips haemorrhoidalis (Bouché) (Thysanoptera. Thripidae). Int. J. Insect Morphol. Embryol. 27, 255–261; https://doi.org/10.1016/S0020-7322(98)00018-X (1998).Nakao, S. & Yabu, S. Ethological and chemical discrimination between thelytokous and arrhenotokous Thrips nigropilosus Uzel, with discussion of taxonomy. Jpn. J. Appl. Entomol. Zool. 42, 77–83; https://doi.org/10.1303/jjaez.42.77 (1998).Article 
CAS 

Google Scholar 
Arakaki, N., Miyoshi, T. & Noda, H. Wolbachia-mediated parthenogenesis in the predatory thrips Franklinothrips vespiformis (Thysanoptera: Insecta). Proc. R. Soc. B Biol. Sci. 268, 1011–1016; https://doi.org/10.1098/rspb.2001.1628 (2001).Kumm, S. & Moritz, G. First detection of Wolbachia in arrhenotokous populations of thrips species (Thysanoptera: Thripidae and Phlaeothripidae) and its role in reproduction. Environ. Entomol. 37, 1422–1428. https://doi.org/10.1603/0046-225X-37.6.1422 (2008).Article 
PubMed 

Google Scholar 
Moritz, G. The biology of thrips is not the biology of their adults: a developmental view. In Thrips and Tospovirus: Proceedings of the 7th International Symposium on Thysanoptera, edited by L. A. Mound & R. Marullo (Australian National Insect Collection CSIRO, Canberra, 2002), pp. 259–267.Moritz, G., Schäfer, E., Kumm, S., Steller, A. & Tschuch, G. D. Alien-Thrips: Suocerathrips linguis – Biologie und Verhalten. Mitteilungen der Deutschen Gesellschaft für allgemeine und angewandte Entomologie 14, 177–181 (2004).
Google Scholar 
Gehlsen, U. Ernährungssystem, Verhalten und Wehrsekret des subsozialen Phlaeothripinen Suocerathrips linguis MOUND & MARULLO, 1994 (Insecta, Thysanoptera, Tubulifera). PhD-thesis (Martin-Luther University Halle-Wittenberg, Germany, 2009).Kumm, S. Reproduction, progenesis and embryogenesis of thrips (Thysanoptera, Insecta). Dissertation. Martin-Luther-Universität Halle-Wittenberg, 2002.Krueger, S., Mound, L. A. & Moritz, G. B. Offspring sex ratio and development are determined by copulation activity in Echinothrips americanus MORGAN 1913 (Thysanoptera: Thripidae). J. Appl. Entomol. 140, 462–473; https://doi.org/10.1111/jen.12280 (2016).Oetting, R. D., Beshear, R. J., Liu, T.-X., Braman S. K., & Baker, J. R. Biology and identification of thrips on greenhouse ornamentals. Univ. Ga. Res. Bull. 414, 20 (1993).Mound, L. A., Nielsen, M.-C. & Hastings, A. Thysanoptera Aotearoa. Thrips of New Zealand. Available at https://keys.lucidcentral.org/keys/v3/nz_thrips/index.html.Schindelin, J. et al. Fiji an open-source platform for biological-image analysis. Nat. Methods 9, 676–682. https://doi.org/10.1038/nmeth.2019 (2012).Article 
CAS 
PubMed 

Google Scholar 
Uzel, H. Monographie der Ordnung Thysanoptera (Nabu Oress, Charleston SC, United States, 1895).Marzo L. De. Dettagli anatomici dei genitali interni in Melanthrips fuscus (Sulzer) e altri tisanotteri. Entomologica 36, 109–119. https://doi.org/10.15162/0425-1016/747 (2002).Melis, A. Nuove osservazioni anatomo-istologiche sui diversi stati postembrionali del Liothrips oleae Costa. REDIA 21, 263–334 (1934).
Google Scholar 
van der Kooi, C. J. & Schwander, T. On the fate of sexual traits under asexuality. Biol. Rev. 89, 805–819. https://doi.org/10.1111/brv.12078 (2014).Article 
PubMed 

Google Scholar 
Sloan, N. S. & Simmons, L. W. The evolution of female genitalia. J. Evol. Biol. 32, 882–899. https://doi.org/10.1111/jeb.13503 (2019).Article 
PubMed 

Google Scholar 
Buffa, P. Tisanotteri esotici esistenti nel Museo Civico di Storia Naturale di Genova. REDIA 5, 157–172 (1909).
Google Scholar 
Osborn, H. Note on a New Species of Phloeothrips, with description. Proc. Iowa Acad. Sci. 3, 228 (1895).
Google Scholar 
Mound, L. A. & Marullo, R. New thrips on mother-in-law`s tongue. Entomol. Mon. Mag. 130, 95–98 (1994).
Google Scholar 
Zur Strassen, R. Die terebranten Thysanopteren Europas und des Mittelmeer-Gebietes (Goecke und Evers, Keltern, 2003).Davies, R. G. The postembryonic development of the female reproductive system in Limothrips cerealium Haliday (Thysanoptera: Thripidae). Proc. Zool. Soc. Lond. 136, 411–437. https://doi.org/10.1111/j.1469-7998.1961.tb05883.x (1961).Article 

Google Scholar 
Gerber, G. H. Evolution of the methods of spermatophore formation in pterygotan insects. Can. Entomol. 102, 358–362 (1970).Article 

Google Scholar 
Dallai, R., Afzelius, B. A., Lanzavecchia, S. & Bellon, P. L. Bizarre flagellum of thrips spermatozoa (Thysanoptera, Insecta). J. Morphol. 209, 343–347. https://doi.org/10.1002/jmor.1052090309 (1991).Article 
CAS 
PubMed 

Google Scholar 
Paccagnini, E., Mencarelli, C., Mercati, D., Afzelius, B. A. & Dallai, R. Ultrastructural analysis of the aberrant axoneme morphogenesis in thrips (Thysanoptera, Insecta). Cell Mot. Cytoskel. 64, 645–661. https://doi.org/10.1002/cm.20212 (2007).Article 

Google Scholar 
Paccagnini, E., Lupetti, P., Afzelius, B. A. & Dallai, R. New findings on sperm ultrastructure in thrips (Thysanoptera, Insecta). Arthropod. Struct. Dev. 38, 70–83. https://doi.org/10.1016/j.asd.2008.07.004 (2009).Article 
PubMed 

Google Scholar 
Paccagnini, E., Mercati, D., Giusti, F., Conti, B. & Dallai, R. The spermatogenesis and the sperm structure of Terebrantia (Thysanoptera, Insecta). Tissue & cell 42, 247–258. https://doi.org/10.1016/j.tice.2010.04.008 (2010).Article 

Google Scholar 
Pitnick, S., Wolfner, M. F. & Dorus, S. Post-ejaculatory modifications to sperm (PEMS). Biol. Rev. Camb. Philos. Soc. 95, 365–392. https://doi.org/10.1111/brv.12569 (2020).Article 
PubMed 

Google Scholar 
Karr, T. L., Swanson, W. J. & Snook, R. R. The evolutionary significance of variation in sperm–egg interactions. In Sperm biology. An evolutionary perspective, edited by T. R. Birkhead. 1st ed. (Academic Press/Elsevier, Amsterdam, 2009), pp. 305–365.Friedländer, M., Jeshtadi, A. & Reynolds, S. E. The structural mechanism of trypsin-induced intrinsic motility in Manduca sexta spermatozoa in vitro. J. Insect Physiol. 47, 245–255. https://doi.org/10.1016/s0022-1910(00)00109-8 (2001).Article 
PubMed 

Google Scholar 
Hughes, M. & Davey, K. G. The activity of spermatozoa of Periplaneta. J. Insect Physiol. 15, 1607–1616. https://doi.org/10.1016/0022-1910(69)90181-4 (1969).Article 

Google Scholar 
Longo, G. et al. Ultrastructural changes in sperm of Eyprepocnemis plorans (Charpentier) (Orthoptera: Acrididae) during storage of gametes in female genital tract. Inverteb. Reprod. Dev. 24, 1–6. https://doi.org/10.1080/07924259.1993.9672325 (1993).Article 

Google Scholar 
Makielski, S. K. The structure and maturation of the spermatozoa of Sciara coprophila. J. Morphol. 118, 11–41. https://doi.org/10.1002/jmor.1051180103 (1966).Article 
CAS 
PubMed 

Google Scholar 
Giuffrida, A. & Rosati, F. Changes in sperm tail of Eyprepocnemis plorans (Insects, Orthoptera) as a result of in vitro incubation in spermathecal extract. Inverteb. Reprod. Dev. 24, 47–52. https://doi.org/10.1080/07924259.1993.9672330 (1993).Article 

Google Scholar 
Giuffrida, A., Focarelli, R., Lampariello, R., Thole, H. & Rosati, F. Purification and properties of a 35 kDa glycoprotein from spermathecal extract of Eyprepocnemis plorans (Insecta, Orthoptera) with axonemal cytoskeleton disassembly activity. Insect Biochem. Mol. Biol. 26, 347–354. https://doi.org/10.1016/0965-1748(95)00095-X (1996).Article 
CAS 

Google Scholar 
Arakaki, N., Noda, H. & Yamagishi, K. Wolbachia-induced parthenogenesis in the egg parasitoid Telenomus nawai. Entomol. Exp. Appl. 96, 177–184. https://doi.org/10.1046/j.1570-7458.2000.00693.x (2000).Article 

Google Scholar 
Pannebakker, B. A. et al. Sexual functionality of Leptopilina clavipes (Hymenoptera: Figitidae) after reversing Wolbachia-induced parthenogenesis. J. Evol. Biol. 18, 1019–1028. https://doi.org/10.1111/j.1420-9101.2005.00898.x (2005).Article 
CAS 
PubMed 

Google Scholar 
Stouthamer, R., Russell, J. E., Vavre, F. & Nunney, L. Intragenomic conflict in populations infected by Parthenogenesis Inducing Wolbachia ends with irreversible loss of sexual reproduction. BMC Evol. Biol. 10, 229. https://doi.org/10.1186/1471-2148-10-229 (2010).Article 
PubMed 
PubMed Central 

Google Scholar 
Sänger, K. & Helfert, B. Comparative studies on number and position of the micropyles and the shape of the eggs of Saga pedo, S. natoliae and S. ephippigera (Orthoptera: Tettigoniidae). Entomologia 19, 49–56. https://doi.org/10.1127/ENTOM.GEN/19/1994/049 (1994).Article 

Google Scholar 
Gottlieb, Y. & Zchori-Fein, E. Irreversible thelytokous reproduction in Muscidifurax uniraptor. Entomol. Exp. Appl. 100, 271–278. https://doi.org/10.1046/j.1570-7458.2001.00874.x (2001).Article 

Google Scholar 
Stouthamer, R., Breeuwer, J. A. J. & Hurst, G. D. D. WOLBACHIA PIPIENTIS. Microbial manipulator of arthropod reproduction. Annu. Rev. Microbiol. 53, 71–102. https://doi.org/10.1146/annurev.micro.53.1.71 (1999).Article 
CAS 
PubMed 

Google Scholar 
Schwander, T., Crespi, B. J., Gries, R. & Gries, G. Neutral and selection-driven decay of sexual traits in asexual stick insects. Proc. Biol. Sci. 280, 20130823. https://doi.org/10.1098/rspb.2013.0823 (2013).Article 
PubMed 
PubMed Central 

Google Scholar 
Werren, J. H., Baldo, L. & Clark, M. E. Wolbachia: master manipulators of invertebrate biology. Nat. Rev. Micro. 6, 741–751. https://doi.org/10.1038/nrmicro1969 (2008).Article 
CAS 

Google Scholar  More

The link between SDGs and NRRPThe Italian National Recovery and Resilience Plan (NRRP) is part of the Next Generation EU (NGEU) program, the 750-billion-euro package, consisting of about half of grants, agreed by the European Union in response to the pandemic crisis. The main component of the NGEU program is the Recovery and Resilience Facility (RRF), which has a duration of six years, from 2021 to 2026, and a total size of €672.5 billion (€312.5 billion grants, the remaining €360 billion loans at subsidized rates).The Plan is developed around three strategic axes shared at European level: digitalization and innovation, ecological planning and social inclusion.The missions of the NRRP are as follows:

Mission 1: Digitalization, innovation, competitiveness, culture and tourism

Mission 2: Green revolution and ecological transition

Mission 3: Infrastructure for sustainable mobility

Mission 4: Education and research

Mission 5: Cohesion and inclusion

Mission 6: Health.

With the aim of encouraging the debate on the use of sustainability indicators for monitoring the progress of the PNRR, a mapping of the correspondences between the 17 Sustainable Development Goals and the 6 Missions provided for by the NRRP is proposed (Fig. 1). In this way it is possible to identify the SDGs indicators that can be useful tools for achieving the missions of the NRRP.Figure 1Relationships between SDGs indicators and NRRP missions.Full size imageOf particular interest for the purposes of our work is Mission 2 (Green Revolution and Ecological Transition) of NRRP. It provides for investments and reforms for the circular economy and to improve waste management, strengthen separate collection infrastructure and modernize or develop new waste treatment plants. Substantial tax incentives are provided to increase the energy efficiency of buildings, to achieve progressive decarbonization, to increase the use of renewable energy sources. In addition, the Mission devotes resources to enhancing the capacity of electricity grids, their reliability, security, and flexibility (Smart Grid) and water infrastructure. The Mission also includes the issues of territorial security, with prevention and restoration interventions in the face of significant hydrogeological risks, the protection of green areas and biodiversity, and those related to the elimination of water and soil pollution, and the availability of water resources.The main components of this mission are:

M2C1: Circular economy and sustainable agriculture

M2C2: Renewable energy, hydrogen, grid, and sustainable mobility

M2C3: Energy efficiency and upgrading of buildings

M2C4: Protection of land and water resources.

The analysis of Mission 2 (Green Revolution and Ecological Transition) finds ample space in the SDGs creating important interconnections between the different indicators present in the individual Goals and the objectives of the Mission itself.The SDGs indicators to support the NRRPThe SDGs indicators selected for the analysis of Mission 2 (Green Revolution and Ecological Transition) of the NRRP, are descripted in Table 1. We considered 13 indicators, selected from Goals 2, 6, 7, 11, 12 and 15 which may be of significant interest for the achievement of Mission 2. These indicators will then be attributed to the individual components of the mission.Table 1 Goal, indicators, measures e source of SDGs data.Full size tableThe indicators were chosen based on their relevance to the objectives of the mission and on the availability of data on a regional basis. For each main component we can use the following indicators:

M2C1: Circular economy and sustainable agriculture:

– Share of utilized agricultural area invested by organic crops

– Growth rate of organic crops

– Delivery of municipal waste to landfill.

– Separate waste collection

M2C2: Renewable energy, hydrogen, grid and sustainable mobility:

M2C3: Energy efficiency and upgrading of buildings

M2C4: Protection of land and water resources

– Irregularities in water distribution

– Sealing and soil consumption per capita

– Soil sealing from artificial cover

– Fragmentation of the natural and agricultural territory

– Incidence of urban green areas on the urbanized surface of cities.

The SDGs indicators at the level of territorial distribution in ItalyWe carry out a first analysis by territorial distribution for the different sets of main components of Mission 2.From a first analysis of the M2C1 indicators (Circular Economy and Sustainable Agriculture) it emerges that the share of agricultural area destined for organic crops is greater, especially in the Center and in the South of Italy. In 2019, the extent of organic farming in Italy reached 15.8% of the utilized agricultural area, almost double the EU average. However, the annual growth rate of the areas converted to organic farming or in the process of conversion (+ 1.8%) is the lowest since 2012 and is negative in the South, where for the second consecutive year there is a decrease (− 2.1% in the 2-year period 2017–2019). The dynamics of organic farming is an index of the spread of sustainable agricultural practices, which must be accompanied by measures that also consider the pressure on the environment generated by agriculture (Table 2).Table 2 M2C1 indicators—Circular economy and sustainable agriculture by territorial distribution (year 2019).Full size tableAlso, in the Central and Southern Italy area there is the greatest delivery of waste to landfills. Waste cycle management is crucial for living conditions and global health. The share of municipal waste landfilled is steadily decreasing at national level. In 2019, in fact, the part sent to landfill is equal to 20.9% of the total, down compared to the previous year (21.5%). The separate collection of municipal waste represents a further important step in view of the objective of reducing the amount of waste returned to the environment and, more specifically, of the delivery of waste to landfills. The 18.5 million tons of differentiated RU in 2019 represent 61.3% of national production, a share almost doubled compared to ten years ago and up from last year by 3.1 percentage points. Despite the evident progress, Italy is still marked by a considerable delay compared to the regulatory objectives, having not yet reached, in 2019, the target of 65% of separate collection planned for 2012. Critical issues are also observed in relation to the substantial territorial gaps, which disadvantage the Center and the South compared to the North, despite the distances have been reduced in recent years.
Regarding the M2C2 Mission (Renewable Energy, Hydrogen, Network and Sustainable Mobility), national and international energy policies have been committed for years to the enhancement of renewable energy sources, with the aim of decarbonizing the economy and guaranteeing the commitments made in the field of climate change. In 2019, one year after the expiry of the objectives of the European Union’s Climate-Energy Package, fourteen Member States, including Italy, exceeded the target assigned at national level. In Italy, the overall share of energy from renewable sources in gross final consumption (CFL) of energy, equal to 18.2% (Table 3), a percentage slightly lower than the average of the EU27 (19.7%), is placed for the sixth consecutive year above the 17% target set for our country. However, for Italy to achieve the ambitious programs defined by the 2020 National Integrated Energy and Climate Plan, which set a 30% target for renewables by 2030, a further boost to production from renewable sources is necessary. The resources introduced by the National Recovery and Resilience Plan (NRRP) to achieve the “green revolution and ecological transaction” include significant investments in the energy field, focusing, among other components, on a further strengthening of the Sources from Renewable energy (FER).Table 3 M2C2 indicators—Renewable energy, hydrogen, network and sustainable mobility by territorial distribution (year 2019).Full size tableThe M2C3 Mission (Energy Efficiency and Upgrading of Buildings) devotes resources to enhancing the capacity of electricity grids, their reliability, safety, and flexibility (Smart Grid). Consistent with the objectives of reducing energy consumption pursued by European policies, the Italian figure for 2019 confirms the process of reducing Italian energy intensity, which marks a further contraction of 1.3%, reaching an overall negative balance compared to the last decade of 11.8%, with an average annual rate of change of − 1.2% (Table 4). The reduction in energy intensity is largely attributable to the effect of the measures in favor of energy efficiency, which, between 2011 and 2019, resulted in energy savings of 12 Mtoe/year, equal to 77% of the 2020 target set by the National Action Plan for Energy Efficiency 2017. A further acceleration of energy efficiency is expected, in the coming years, because of the investment plan envisaged by the NRRP, also linked to the redevelopment of the public and private building stock. At the sectoral level, the reduction in energy intensity is driven by improvements in industry, which, despite the slight increase in the last year, in 2019, with 92 toes per million euros, shows a decrease compared to 2009 of 17%, with an average annual rate of change of − 1.8%.Table 4 M2C3 indicators—Energy efficiency and requalification of buildings by territorial distribution (year 2019).Full size tableThe M2C4 Mission (Protection of the territory and water resources) also includes the issues of territorial safety, with prevention and recovery interventions, the protection of green areas and those related to the elimination of water and soil pollution.Italy is among the European countries of the Mediterranean area that use groundwater, springs and wells the most; these represent the most important resource of fresh water for drinking water use on the Italian territory (84.8% of the total withdrawn). The efficiency of municipal drinking water distribution networks has been steadily deteriorating since 2008 for more than half of the regions. The share of families who complain of irregularities in the water supply service in their home is stable (equal to 8.6% in 2019) with more accentuated values in the Center and South of Italy (Table 5).Table 5 M2C4 indicators—Protection of land and water resources by territorial distribution (year 2019).Full size tableLand degradation, understood as loss of ecological functionality, is monitored through the dynamics of land consumption, which Italy has committed to zero by 2030 with the National Strategy for Sustainable Development (2017). The “consumed” soil is that occupied by urbanization and made impermeable by artificial roofing (soil sealing). Excessive fragmentation of open spaces, however, is also a factor of degradation, since the barriers made up of buildings and infrastructures interrupt the continuity of ecosystems, making even unoccupied but not large enough spaces ecologically inert and unproductive. Moreover, in a fragile territory such as Italy, land consumption is also a significant factor of hydrogeological risk and deterioration of the landscape. The index of sealing and land consumption per capita in 2019 increases for the fifth consecutive year, resulting in 357 m2 per inhabitant. The soil sealed by artificial covers is equal to 7.1% of the national territory (8.5% in the North, 6.7% in the Center, 5.9% in the South).According to Ispra estimates, 44.3% of Italy’s natural and agricultural land has a high or very high degree of fragmentation. A joint representation of the variations in fragmentation and soil sealing over the last two years summarizes recent trends in land consumption and their impact on the environment and landscape.A further objective for 2030 is to provide universal access to safe, inclusive, and accessible public green spaces, for women and children, the elderly, and people with disabilities. In 2019 the incidence of urban green areas on the urbanized surface of cities is equal to 8.5% in Italy with slightly higher values in the North and less elevated in the South. More

Butchart, S. H. M. et al. Shortfalls and solutions for meeting national and global conservation area targets. Conserv. Lett. 8, 329–337 (2015).Article 

Google Scholar 
McDonald-Madden, E. et al. ‘True’ conservation progress. Science 323, 43–44 (2009).Article 
CAS 
PubMed 

Google Scholar 
Corson, C. et al. Everyone’s Solution? Defining and redefining protected areas at the Convention on Biological Diversity. Conservation and Society 190–202. https://www.jstor.org/stable/26393154?seq=1#metadata_info_tab_contents (Accessed 1st March 2022) (2014).Caro, T. & Berger, J. Can behavioural ecologists help establish protected areas?. Philos. Trans. R. Soc. B 374, 20180062 (2019).Article 

Google Scholar 
Barnes, M. D. et al. Wildlife population trends in protected areas predicted by national socio-economic metrics and body size. Nat. Commun. 7, 12747 (2016).Article 
ADS 
CAS 
PubMed 
PubMed Central 

Google Scholar 
Watson, J. E. M. et al. Bolder science needed now for protected areas. Conserv. Biol. 30, 243–248 (2016).Article 
PubMed 

Google Scholar 
Joppa, L. N. & Pfaff, A. High and far: Biases in the location of protected areas. PLoS One 4, e8273 (2009).Article 
ADS 
PubMed 
PubMed Central 

Google Scholar 
Runge, C. A., Martin, T. G., Possingham, H. P., Willis, S. G. & Fuller, R. A. Conserving mobile species. Front. Ecol. Environ. 12, 395–402 (2014).Article 

Google Scholar 
Thirgood, S. et al. Can parks protect migratory ungulates? The case of the Serengeti wildebeest. Anim. Conserv. 7, 113–120 (2004).Article 

Google Scholar 
Craigie, I. D. et al. Large mammal population declines in Africa’s protected areas. Biol. Conserv. 143, 2221–2228 (2010).Article 

Google Scholar 
Geldmann, J. et al. Effectiveness of terrestrial protected areas in reducing habitat loss and population declines. Biol. Conserv. 161, 230–238 (2013).Article 

Google Scholar 
Hansen, A. J. & DeFries, R. Ecological mechanisms linking protected areas to surrounding lands. Ecol. Appl. 17, 974–988 (2007).Article 
PubMed 

Google Scholar 
Beresford, A. E. et al. Poor overlap between the distribution of Protected Areas and globally threatened birds in Africa. Anim. Conserv. 14, 99–107 (2011).Article 

Google Scholar 
Haynes, G. Elephants (and extinct relatives) as earth-movers and ecosystem engineers. Geomorphology 157–158, 99–107 (2012).Article 
ADS 

Google Scholar 
Terborgh, J., Davenport, L. C., Ong, L. & Campos-Arceiz, A. Foraging impacts of Asian megafauna on tropical rain forest structure and biodiversity. Biotropica 50, 84–89 (2018).Article 

Google Scholar 
Galanti, V., Preatoni, D., Martinoli, A., Wauters, L. A. & Tosi, G. Space and habitat use of the African elephant in the Tarangire-Manyara ecosystem, Tanzania: Implications for conservation. Mamm. Biol. 71, 99–114 (2006).Article 

Google Scholar 
Williams, C. et al. Elephas maximus. The IUCN Red List of Threatened Species. e.T7140A45818198. https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.T7140A45818198.en. Accessed on 15 February 2022. (2020)Stokke, S. & Du Toit, J. T. Sexual segregation in habitat use by elephants in Chobe National Park, Botswana. Afr. J. Ecol. 40, 360–371 (2002).Article 

Google Scholar 
Chowdhury, S. et al. Protected areas in South Asia: Status and prospects. Sci. Total Environ. 811, 152316 (2022).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Goswami, V. R. et al. Community-managed forests and wildlife-friendly agriculture play a subsidiary but not substitutive role to protected areas for the endangered Asian elephant. Biol. Conserv. 177, 74–81 (2014).Article 

Google Scholar 
Fernando, C., Weston, M. A., Corea, R., Pahirana, K. & Rendall, A. R. Asian elephant movements between natural and human-dominated landscapes mirror patterns of crop damage in Sri Lanka. Oryx https://doi.org/10.1017/S0030605321000971 (2022).Article 

Google Scholar 
Santini, L., Saura, S. & Rondinini, C. Connectivity of the global network of protected areas. Divers. Distrib. 22, 199–211 (2016).Article 

Google Scholar 
Brennan, A. et al. Functional connectivity of the world’s protected areas. Science 376, 1101–1104 (2022).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Kumar, M. A. & Singh, M. Behavior of Asian elephant (Elephas maximus) in a land-use mosaic: Implications for human-elephant coexistence in the Anamalai Hills, India. Wildl. Biol. Pract. 6, 69–80 (2010).
Google Scholar 
Rathnayake, C. W. M., Jones, S., Soto-Berelov, M. & Wallace, L. Human–elephant conflict and land cover change in Sri Lanka. Appl. Geogr. 143, 102685 (2022).Article 

Google Scholar 
Chan, A. N. et al. Landscape characteristics influence ranging behavior of Asian elephants at the human-wildlands interface in Myanmar. Mov. Ecol. 10, 1–15 (2022).Article 

Google Scholar 
Magioli, M. et al. Land-use changes lead to functional loss of terrestrial mammals in a Neotropical rainforest. Perspect. Ecol. 19, 161–170 (2021).
Google Scholar 
Fernando, P. et al. The future of Asian elephant conservation: Setting sights beyond protected area boundaries. in Conservation Biology in Asia 252–260 (2006).Kumar, M. A., Vijayakrishnan, S. & Singh, M. Whose habitat is it anyway? Role of natural and anthropogenic habitats in conservation of charismatic species. Trop. Conserv. Sci. 11, 194008291878845 (2018).Article 

Google Scholar 
Sirua, H. Nature above people: Rolston and “fortress” conservation in the South. Ethics Environ. 11, 71–96 (2006).Article 

Google Scholar 
Keerthipriya, P. et al. Musth and its effects on male–male and male–female associations in Asian elephants. J. Mammal. 101, 259–270 (2020).Article 

Google Scholar 
Eisenberg, J. F., Mckay, G. M. & Jainudeen, M. R. Reproductive behavior of the Asiatic elephant (Elephas maximus maximus). Behaviour 38, 193–225 (1971).Article 
CAS 
PubMed 

Google Scholar 
Fernando, P. et al. Ranging behavior of the Asian elephant in Sri Lanka. Mamm. Biol. Zeitschrift für Säugetierkd. 73, 2–13 (2008).Article 

Google Scholar 
Hollister-Smith, J. A., Alberts, S. C. & Rasmussen, L. E. L. Do male African elephants, Loxodonta africana, signal musth via urine dribbling?. Anim. Behav. 76, 1829–1841 (2008).Article 

Google Scholar 
LaDue, C. A., Vandercone, R. P. G., Kiso, W. K. & Freeman, E. W. Behavioral characterization of musth in Asian elephants (Elephas maximus): Defining progressive stages of male sexual behavior in in-situ and ex-situ populations. Appl. Anim. Behav. Sci. 251, 105639 (2022).Article 

Google Scholar 
LaDue, C. A., Goodwin, T. E. & Schulte, B. A. Concentration-dependent chemosensory responses towards pheromones are influenced by receiver attributes in Asian elephants. Ethology 124, 387–399 (2018).Article 

Google Scholar 
Goldenberg, S. Z., de Silva, S., Rasmussen, H. B., Douglas-Hamilton, I. & Wittemyer, G. Controlling for behavioural state reveals social dynamics among male African elephants, Loxodonta africana. Anim. Behav. 95, 111e119 (2014).Article 

Google Scholar 
Chave, E. et al. Variation in metabolic factors and gonadal, pituitary, thyroid, and adrenal hormones in association with musth in African and Asian elephant bulls. Gen. Comp. Endocrinol. 276, 1–13 (2019).Article 
CAS 
PubMed 

Google Scholar 
Glaeser, S. S. et al. Characterization of longitudinal testosterone, ocrtisol, and musth in male Asian Elephants (Elephas maximus), effects of aging, and adrenal responses to social changes and health events. Animals 12, 1332 (2022).Article 
PubMed 
PubMed Central 

Google Scholar 
de Silva, S., Ranjeewa, A. D. G. & Kryazhimskiy, S. The dynamics of social networks among female Asian elephants. BMC Ecol. 11, 17 (2011).Article 
PubMed 
PubMed Central 

Google Scholar 
Nandini, S., Keerthipriya, P. & Vidya, T. N. C. Group size differences may mask underlying similarities in social structure: A comparison of female elephant societies. Behav. Ecol. 29, 145–159 (2018).Article 

Google Scholar 
de Silva, S. & Wittemyer, G. A comparison of social organization in Asian elephants and African Savannah elephants. Int. J. Primatol. 33, 1125–1141 (2012).Article 

Google Scholar 
Nandini, S., Keerthipriya, P. & Vidya, T. N. C. Seasonal variation in female Asian elephant social structure in Nagarahole-Bandipur, southern India. Anim. Behav. 134, 135–145 (2017).Article 

Google Scholar 
de Silva, S., Schmid, V. & Wittemyer, G. Fission-fusion processes weaken dominance networks among female Asian elephants in a productive habitat. Behav. Ecol. 28, 243–252 (2017).Article 

Google Scholar 
de Silva, S., Ranjeewa, A. D. G. & Weerakoon, D. Demography of Asian elephants (Elephas maximus) at Uda Walawe National Park, Sri Lanka based on identified individuals. Biol. Conserv. 144, 1742–1752 (2011).Article 

Google Scholar 
Ginsberg, J. R. & Young, T. P. Measuring association between individuals or groups in behavioural studies. Anim. Behav. 44, 377–379 (1992).Article 

Google Scholar 
Csardi, G. & Nepusz, T. The igraph software package for complex network research. InterJournal, Complex Syst. 5, 1–9 (2014).
Google Scholar 
Liechti, J. I. & Bonhoeffer, S. A time resolved clustering method revealing longterm structures and their short-term internal dynamics. arXiv:1912.04261 (2020).Farine, D. R. & Whitehead, H. Constructing, conducting and interpreting animal social network analysis. J. Anim. Ecol. 84, 1144–1163 (2015).Article 
PubMed 
PubMed Central 

Google Scholar 
Wikramanayake, E. D. et al. An ecology-based method for defining priorities for large mammal conservation: The tiger as case study. Conserv. Biol. 12, 865–878 (2008).Article 

Google Scholar 
Chundawat, R. S., Sharma, K., Gogate, N., Malik, P. K. & Vanak, A. T. Size matters: Scale mismatch between space use patterns of tigers and protected area size in a tropical dry forest. Biol. Conserv. 197, 146–153 (2016).Article 

Google Scholar 
Tucker, M. A. et al. Moving in the Anthropocene: Global reductions in terrestrial mammalian movements. Science 359, 466–469 (2018).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Karanth, K. K. & DeFries, R. Nature-based tourism in Indian protected areas: New challenges for park management. Conserv. Lett. 4, 137–149 (2011).Article 

Google Scholar 
Brown, J. L. et al. Comparative endocrinology of testicular, adrenal and thyroid function in captive Asian and African elephant bulls. Gen. Comp. Endocrinol. 151, 153–162 (2007).Article 
CAS 
PubMed 

Google Scholar 
Slotow, R. et al. Older bull elephants control young males. Nature 408, 425–426 (2000).Article 
ADS 
CAS 
PubMed 

Google Scholar 
Poole, J. H., Lee, P. C., Njiraini, N. & & Moss, C. J. Longevity, competition, and musth: A long-term perspective on male reproductive strategies. in The Amboseli Elephants: A Long‐Term Perspective on a Long‐Lived Mammal 272–286 (2011).Poole, J. H. Announcing intent: The aggressive state of musth in African elephants. Anim. Behav. 37, 153–155 (1989).Article 

Google Scholar 
Poole, J. H. Mate guarding, reproductive success and female choice in African elephants. Anim. Behav. 37, 842–849 (1989).Article 

Google Scholar 
Poole, J. H. Rutting behavior in elephants: The phenomenon of musth in African elephants. Anim. Behav. 102, 283–316 (1987).
Google Scholar 
Foley, A. M. et al. Reproductive effort and success of males in scramble-competition polygyny: Evidence for trade-offs between foraging and mate search. J. Anim. Ecol. 87, 1600–1614 (2018).Article 
PubMed 

Google Scholar 
Fernando, P., Leimgruber, P., Prasad, T. & Pastorini, J. Problem-elephant translocation: Translocating the problem and the elephant?. PLoS One 7, e50917 (2012).Article 
ADS 
CAS 
PubMed 
PubMed Central 

Google Scholar 
Archie, E. A., Morrison, T. A., Foley, C. A. H., Moss, C. J. & Alberts, S. C. Dominance rank relationships among wild female African elephants, Loxodonta africana. Anim. Behav. 71, 117–127 (2006).Article 

Google Scholar 
Wittemyer, G. & Getz, W. M. Hierarchical dominance structure and social organization in African elephants, Loxodonta africana. Anim. Behav. 73, 671–681 (2007).Article 

Google Scholar 
Wittemyer, G., Getz, W. M., Vollrath, F. & Douglas-Hamilton, I. Social dominance, seasonal movements, and spatial segregation in African elephants: A contribution to conservation behavior. Behav. Ecol. Sociobiol. 61, 1919–1931 (2007).Article 

Google Scholar 
Gunaryadi, D., Sugiyo, & Hedges, S. Community-based human-elephant conflict mitigation: The value of an evidence-based approach in promoting the uptake of effective methods. PLoS One 12, e0173742 (2017).Article 
PubMed 
PubMed Central 

Google Scholar 
Wilson, G. et al. Between a rock and a hard place: Rugged terrain features and human disturbance affect behaviour and habitat use of Sumatran elephants in Aceh, Sumatra, Indonesia. Biodivers. Conserv. 30, 597–618 (2021).Article 

Google Scholar 
de Silva, S. et al. Demographic variables for wild Asian elephants using longitudinal observations. PLoS One 8, e82788 (2013).Article 
ADS 
PubMed 
PubMed Central 

Google Scholar 
LaDue, C. A., Eranda, I., Jayasinghe, C. & Vandercone, R. P. G. Mortality patterns of Asian elephants in a region of human–elephant conflict. J. Wildl. Manag. 85, 794–802 (2021).Article 

Google Scholar 
Ram, A. K. et al. Tracking forest loss and fragmentation between 1930 and 2020 in Asian elephant (Elephas maximus) range in Nepal. Sci. Rep. 11, 1–13 (2021).Article 
ADS 

Google Scholar 
Neupane, D., Kwon, Y., Risch, T. S. & Johnson, R. L. Changes in habitat suitability over a two decade period before and after Asian elephant recolonization. Glob. Ecol. Conserv. 22, e01023 (2020).Article 

Google Scholar 
de Silva, S. & Leimgruber, P. Demographic tipping points as early indicators of vulnerability for slow-breeding megafaunal populations. Front. Ecol. Evol. 7, 171 (2019).Article 

Google Scholar 
Rodrigo, M. Farmers move to occupy a critical elephant corridor in Sri Lanka. Mongabay (2021).de la Torre, J. A. et al. There will be conflict—Agricultural landscapes are prime, rather than marginal, habitats for Asian elephants. Anim. Conserv. 24, 720–732 (2021).Article 

Google Scholar 
Rood, E., Ganie, A. A. & Nijman, V. Using presence-only modelling to predict Asian elephant habitat use in a tropical forest landscape: Implications for conservation. Divers. Distrib. 16, 975–984 (2010).Article 

Google Scholar 
Evans, L. J., Goossens, B., Davies, A. B., Reynolds, G. & Asner, G. P. Natural and anthropogenic drivers of Bornean elephant movement strategies. Glob. Ecol. Conserv. 22, e00906 (2020).Article 

Google Scholar 
Morales-Hidalgo, D., Oswalt, S. N. & Somanathan, E. Status and trends in global primary forest, protected areas, and areas designated for conservation of biodiversity from the Global Forest Resources Assessment 2015. For. Ecol. Manag. 352, 68–77 (2015).Article 

Google Scholar 
Ellis, E. C. et al. People have shaped most of terrestrial nature for at least 12,000 years. Proc. Natl. Acad. Sci. U.S.A. 118, 1–8 (2021).Article 

Google Scholar 
Goswami, V. R., Vasudev, D. & Oli, M. K. The importance of conflict-induced mortality for conservation planning in areas of human–elephant co-occurrence. Biol. Conserv. 176, 191–198 (2014).Article 

Google Scholar 
de Silva, S. & Leimgruber, P. Demographic tipping points as early indicators of vulnerability for slow-breeding megafaunal populations. Front. Ecol. Evol. 7, 1–13 (2019).Article 
CAS 

Google Scholar 
Hettiarachchi, K. ‘Gathering’ shuns ‘brimming’ Minneriya. The Sunday Times (2021).Srinivasaiah, N., Kumar, V., Vaidyanathan, S., Sukumar, R. & Sinha, A. All-male groups in Asian elephants: A novel, adaptive social strategy in increasingly anthropogenic landscapes of southern India. Sci. Rep. 9, 1–11 (2019).Article 
ADS 
CAS 

Google Scholar 
de Silva, E. M. K. et al. Feasibility of using convolutional neural networks for individual-identification of wild Asian elephants. Mamm. Biol. https://doi.org/10.1007/S42991-021-00206-2 (2022).Article 

Google Scholar 
de Silva, S. The Elephant Attribute Recording System (EARS): A tool for individual-based research on Asian elephants. Gajah 40, 46 (2014).
Google Scholar 
Jainudeen, M. R., Katongole, C. B. & Short, R. V. Plasma testosterone levels in relation to musth and sexual activity in the male Asiatic elephant, Elephas maximus. J. Reprod. Fertil. 29, 99–103 (1972).Article 
CAS 
PubMed 

Google Scholar 
Farine, D. R. Animal social network inference and permutations for ecologists in R using asnipe. Methods Ecol. Evol. 4, 1187–1194 (2013).Article 

Google Scholar 
Whitehead, H. Analyzing Animal Societies (University of Chicago Press, 2008).Book 

Google Scholar 
Bates, D., Mächler, 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).Article 

Google Scholar 
Dekker, D., Krackhardt, D. & Snijders, T. A. B. Sensitivity of MRQAP tests to collinearity and autocorrelation conditions. Psychometrika 72, 563–581 (2007).Article 
MathSciNet 
PubMed 
PubMed Central 
MATH 

Google Scholar  More

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02 November 2022

Predicting the risk of extinction from climate change requires an understanding of the interactions between species. An analysis of how changes in rainfall affect competition between plant species offers a way of tackling this challenge. More

Birds and housing62 males and 8 females of house sparrows were caught with mist nets in September and October 2019 in several sites in Kraków, Poland. Before releasing them to the outdoor aviary located on the campus of the Jagiellonian University, Kraków, Poland, each bird was weighed and banded with a metal band. The aviary measured 3.5 m in width, 10.0 m in length, 2.5 m in height, and was outfitted with trees, bushes, perches, wooden shelters, a water source, and food dishes. Initially, birds were maintained with water and a mixture of seeds: wheat, barley, millet, and sunflower seeds, provided ad libitum. Additionally, they had access to sand with shells and sepia.Experimental designAfter a few weeks of acclimation to captivity, the aviary was divided into two separate parts (3.5 × 5 m): aviary no. 1 (A1) and aviary no. 2 (A2). At the same time male individuals were assigned to two crossed experimental treatments, ensuring that in each aviary birds originated from all sampled populations. The experiment comprised of two different treatments conducted simultaneously—one designed to simulate a deficiency in an environmental factor influencing colouration (the quality of available food), the other—to introduce physiological stress and facilitate trade-offs in the allocation of resources limited by the first treatment (an immune response induced by a bacteria-derived compound, S1).The dietary manipulation was achieved by feeding one group of birds with a low-quality protein food (diet reduced in exogenous amino acids, namely phenylalanine and tyrosine content, which are precursors essential for melanin synthesis; PT-reduced diet), and the other one with a wholesome diet (control diet). At the same time, two levels of immune challenge were achieved within each dietary group, by injecting half of the birds with either lipopolysaccharide (LPS) from the cell wall of Escherichia coli, or a 0.9% saline vehicle (as a control). Four females were placed in each group of males to alleviate interspecific conflicts occurring in all-male sparrow flocks, but they did not take part in the experiments. After three weeks of experiment, birds housed in A1 were moved to A2, whereas birds from A2 were moved to A1.Immune challengeBefore receiving injections, birds were first weighed and then transferred from the outdoor aviary to the laboratory. 31 house sparrows (from both dietary groups) were injected intraperitoneally with 0.026 mg LPS (serotype O55:B5, Sigma-Aldrich) diluted in 0.1 mL of 0.9% saline vehicle, so that each bird received a dose of ca. 1 mg/kg body mass, which had previously been shown to induce sickness behaviour in another passerine, the white-crowned sparrow, Zonotrichia leucophrys55. 31 control males were injected with the same volume (0.1 mL) of 0.9% saline vehicle. All individuals were injected twice throughout the experiment with an interval of three weeks between the injections. Birds were always injected at the same time in the morning and early afternoon (between 9:00 am and 12:30 pm).Diet manipulationDuring the six weeks of the experiment (S1), birds received synthetic diet ad libitum, which constituted of a mixture of protein (WPC80, free amino acids and whey protein isolate BiPRO GMP 9000 (Agropur Inc., Appleton, USA)), fats, carbohydrates, and fiber30. The ingredients were thoroughly mixed to produce small pellets (6 mm in diameter) that the sparrows consumed readily. The experimental diet had phenylalanine and tyrosine at 42% (N = 32) of their level in the control diet (N = 30)30. The food pellets were prepared by ZooLab (zoolab.pl/en/home, Sędziszów, Poland). Each bird was weighed before and after the experiment to monitor potential effects of diet on body mass of each animal. Following the experiment, during the next three consecutive days, the amount of food consumed by passerines within every 24 h (starting from 10 am each day to 10 am next day) was noted for both compartments of the aviary. Because of different numbers of individuals per aviary, an overall weight of food consumed in A1 and A2 was calculated per individual, respectively.Feathers samplingMoult of the black bib feathers was stimulated at the end of the moulting period occurring in natural conditions in early November. At day 1 of the dietary/immunological experiment (S1) a small area of the bib (around 25 mm2) was plucked from each male sparrow held in A1. At day 2 the same procedure was performed on individuals from A2. The time difference is orders of magnitude smaller than the timescale of feather growth and hence it would not affect the results in any way.Because the feather growth rate may differ during melanogenesis, with consequences for final colouration (if feathers grow at a faster rate, pigments may be deposited over a larger surface and therefore result in less intense colouration56, we measured the rate of feather development during the course of the experiment. After three weeks of the experiment, three feathers from the upper, central, and lower region of the previously plucked bib were plucked once again. The mass of the collected feathers was determined to the nearest 0.01 mg (XP26 Micro Balance, Mettler-Toledo, Greinfensee, Switzerland). The experiment was completed after six weeks after fully regrown and developed feathers from the bib and PC2 were sampled the second time (S1). Three feathers from the central part of previously plucked bib region were collected to perform transmission electron microscopy (TEM) imaging, whereas the feathers obtained from the rest of the regrown bib area were subjected to electron paramagnetic resonance (EPR) spectroscopy and feather microstructure analyses (greater spatial density of melanized barbs or barbules may affect colouration17.Feathers measurementsReflectance measurementsAn USB4000 spectrophotometer (range 300–700 nm) with the PX-2 Pulsed Xenon Lamp (Ocean Optics, Dunedin, FL, USA) and a bifurcated probe with 7 × 400 μm optical fibres, equipped with a permanently attached 3 mm long black collar, was used to quantify the brightness of the bib feathers collected at the end of the experiment. The measurements were taken with 90 ms integration time and the probe held at 90° to a feather’s surface. Calibration measurements of a Spectralon white standard (Ocean Optics. Largo, FL, USA) were taken every 15 min during measurements. The order in which the samples were measured was randomized in terms of belonging to the experimental group. From each sample (N = 62), seven feathers were chosen and stacked in one pile on a piece of black paper. Ten reflectance measurements were taken on each pile, avoiding distal, brighter parts of the feathers. The obtained spectra were averaged and smoothed in the package ‘pavo’57. Brightness was calculated as a sum of the reflectance values over all wavelengths of a spectrum, and its lower values were interpreted as those indicative of a more melanin-rich feathers (i.e., absorbing more light).Feather developmentEach feather (3 per individual; N = 62 individuals) was laid on a white card and covered by a microscope slide to flatten the naturally curved feathers. Digital photographs were taken using camera (Canon EOS 7D) and imported to ImageJ v1.52a Software (National Institutes of Health, USA). The lengths of fully developed and undeveloped (still in sheath) parts of each feather were measured. To estimate the degree of a feather’s development, the length of the developed part of the vane was divided by its total length (quill with rachis plus the developed vane, Fig. 4A).Figure 4House sparrow feathers sampled from bib after three weeks of the experiment. Feathers during development (A), a TEM cross-sections of feather sampled from bib after the experiment (B).Full size imageFeather densityBarb density measurements were performed on the sampled regrown black bib feathers (N = 2–3 for each individual; N = 62 individuals), but because of their sparser structure we calculated the number of non-down (i.e., rigid) barbs on both sides of the vane, and divided this number by two (to obtain an average single-sided number of barbs) and then by the length of the rachis.Melanosome density (TEM)Feathers sampled from the bib of male sparrows (N = 62) were fixed for transmission electron microscopy (TEM) analysis in a mixture of 0.25 M sodium hydroxide and 0.1% Tween for 20 to 30 min on a bench-top shaker. Next, the feathers were treated with formic acid and ethanol in the ratio of 2:3 for 2.5 h and dehydrated twice for 20 min in 100% ethanol. Samples were embedded in a mixture of the PolyBed 812 resin (20 ml), DDSA (9 ml), NMA (12 ml) and DMP-30 (0.82 ml). Resin infiltration was gradual from 15% resin content in ethanol through 50%, 70% to 100% without alcohol. Each step lasted for 24 h. Then, the feathers were placed in silicone embedding moulds (Agar Scientific) and transferred to an oven. The polymerization proceeded at the temperature of 60 °C for 16 h. The epoxy resin blocks were then trimmed to get rid of excess resin. The surface of each block was prepared by its trimming, starting from the end of the feather, to approximately 5 mm using a glass knife. Next, ultrathin sections (70 nm) were cut with a diamond knife (DIATOME A. G., Berno, Switzerland) on a microtome (UC7, Leica, Wetzlar, Germany) and collected on single slot grids coated with a formvar film. The sections were then contrasted in uranyl acetate and lead citrate for 3 min. They were viewed and photographed with a transmission electron microscope (TEM) JEOL 2100HT (Jeol Ltd, Tokyo, Japan) for the purpose of investigating the number and density of the embedded pigment granules. For each individual three photographs of the cross-sections from a similar feather region were selected. Melanosome density was measured as the number of melanin granules observed in the barb cross-section divided by its area. Images were analysed using Adobe Photoshop (cross-sections area) and ImageJ (number of melanosomes, Fig. 4B).Melanin content: electron paramagnetic resonance (EPR) spectroscopyQuality and quantity of melanin pigments58 in individual feather samples obtained from the bib of house sparrows (N = 57) were characterized using a Varian E3 spectrometer (Varian, Sunnyvale, LA, USA) equipped with a rectangular resonance (TE 102) cavity. Five milligrams of feathers per individual were placed inside the Wilmad finger quartz dewar WG-816-Q (Rototec-Spintec GmbH, Griesheim, Germany). Prior to inserting the vessel into the resonance cavity of the EPR spectrometer, feathers were pressed down the quartz finger to a height of approximately 0.5 cm to ensure comparable volumes of each sample. Measurements were performed at room temperature, at X-band (9.26–9.27 GHz frequency), using the following parameters: magnetic field range 3240–3340 Gs, microwave power 1 mW, modulation frequency 100 kHz, modulation amplitude and time constant—5 Gs and 0.3 s for quantitative analysis, 1 Gs and 0.1 s for qualitative analysis. An EPR signal was recorded as its first derivative, averaged from three consecutive scans, lasting 160 s each (giving a total of 480 s of scan time per EPR spectrum). Then, the following parameters were measured: peak-to-peak amplitude, area under the microwave absorption curve (the integral intensity of the recorded signal) and linewidth of the EPR absorption curve (ΔH;59).Statistical analysesStatistical analysis was performed in R (version 4.0.2,60) using a two-way ANOVA test, with bird’s diet (control vs. PT-reduced) and applied immune challenges (LPS vs. saline-injections) as the independent variables. The following parameters were used as the dependent variables: feathers reflectance (brightness), feather growth rate, feather density (number of barbs per mm), and melanisation level (expressed as the EPR spectrum amplitude measured in arbitrary units [a.u.]). The density of melanosomes was analysed by fitting a linear mixed-effects model. In this model, melanosome density was used as the dependent variable, with diet, immunological challenge, and slice ID as independent variables, and individual ID as a random-effect term. Additionally, to assess the reliability of measurements, the intraclass correlation coefficient (i.e., technical repeatability) was calculated. The models’ residuals were checked for normality and homoscedasticity. Mean food consumption per individual was analysed by the Friedman test. Body mass before and after the experiment was analysed by fitting a linear mixed-effect model. Body mass was used as the dependent variable, whereas diet, immunological challenge, and time as the independent variables, and individual ID as a random-effect term. The model included the following interaction terms: time × diet, time × injection, and diet × injection, and was reduced by removing the non-significant interactions. Results are reported with appropriate statistical tests and estimates (accompanied by standard errors) signifying relevant factor contrasts (relative to the reference group, which in all analyses was diet: control; injection: LPS, body mass: before experiment).
Ethical noteAll applicable national and institutional guidelines for the care and use of animals were followed. The research was performed under permit no. 25/2019 (with a supplementary permit no. 78/2020) from the 2nd Local Institutional Animal Care and Use Committee in Kraków. More