Up and Away Article Worksheet 1. TITLE: Up and away: ontogenic transference as a pathway for aerial dispersal of microplastics microplastics as a pathway fortitle aerial dispersal a.Do you find this adequately andofconcisely describes what the authors were evaluating in their report? Is it engaging? If not, what would you use as a title? 2. INTRODUCTION a. In your own words, provide background and important concepts/processes that specifically apply to this experimental study: b. What is the purpose of this study? c. Clearly state the hypothesis (or hypotheses, if more than one) that is/are being evaluated in this experiment: d. Based on background information and your hypothesis, what are the expected results or predictions of outcomes you can make for this experiment? e. Define and explain how 5 keywords were used within the context of the Al-Jaibachi, et al. experiment. 3.MATERIALS AND METHODS a.In the lab report write up process, this section should be detailed enough for any person to be able to repeat your experiment. In your own words, describe the key equipment, tools and procedures used in the study. A significant amount of supporting information can be found in the supplementary pdf within the pre-lab reading assignment. b. Describe how data were analyzed and summarized (which statistical methods and computer software were used, if applicable) 4. Results : provides a space to present and summarize key fidings of an experimental study in a purely objective manner that often includes tables, figures and/or plotsof figure 1 vs figure 2 in this experiment? a. What was the purpose b. What type of data is summarized in Figure 1? What type of statistical analysis summary is illustrated in figure 1? c. Describe the important experimental outcomes/trends that can be inferred from the data summarized in Fig 1? The table below summarizes the numerical values represented in Fig 1 of the article. Treatment (n=5 per experimental group) Single exposure Mixed exposure Life Stage Larvae Pupae Adult 2um: mean (+ SE) 3047.2 + (278.4) 1045.6 + (366.5) 40.2 + (10.5) 15um: mean (+ SE) 279.0 + (96.8) 107.4 + (6.0) 0.0+ (0.0) 2um: mean (+ SE) 3952.2 + (743.7) 1653.4 + 301.1) 16.0 + (2.6) 15um: mean (+ SE) 244.4 + (32.9) 65.2 + (30.0) 0.4 + (0.4) 5. Discussion : a self contained story that ties together your introduction and section a. Using the summary presented in figure 1 and the photos presented in figure 2, interpret the r state the main findings of this study: b. Was/were the hypothesis(es) and prediction(s) supported by the results of the study? Provide experimental evidence (if hypothesis is supported) or alternative explanations (if hypothesis no regarding the experimental result findings. What factor(s) may have directly affected the results experimental outcome for this study? c. What is the relevance of the presence of microplastic particles in the Malpighian tubles? Whi size was tranferred in greater number? What was the effect of single vs. mixed particle size trea d. Based on the findings of the Up, Up and Away publication, what would you propose as a good experiment to perform? together your introduction and results os presented in figure 2, interpret the results and ted by the results of the study? Provide specific ernative explanations (if hypothesis not supported) ) may have directly affected the results or articles in the Malpighian tubles? Which particle ect of single vs. mixed particle size treatment? on, what would you propose as a good followup Community ecology rsbl.royalsocietypublishing.org Up and away: ontogenic transference as a pathway for aerial dispersal of microplastics Research Rana Al-Jaibachi1, Ross N. Cuthbert1,2 and Amanda Callaghan1 1 Cite this article: Al-Jaibachi R, Cuthbert RN, Callaghan A. 2018 Up and away: ontogenic transference as a pathway for aerial dispersal of microplastics. Biol. Lett. 14: 20180479. http://dx.doi.org/10.1098/rsbl.2018.0479 Received: 3 July 2018 Accepted: 23 August 2018 Subject Areas: ecology, environmental science Keywords: food chain, ontogeny, life stage, Malpighian tubules, microplastics, Culex pipiens Author for correspondence: Amanda Callaghan e-mail: a.callaghan@reading.ac.uk Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9. figshare.c.4215785. Ecology and Evolutionary Biology, School of Biological Sciences, University of Reading, Harborne Building, Reading RG6 6AS, UK 2 Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, Belfast BT9 7BL, UK RNC, 0000-0003-2770-254X; AC, 0000-0002-2731-3352 Microplastics (MPs) are ubiquitous pollutants found in marine, freshwater and terrestrial ecosystems. With so many MPs in aquatic systems, it is inevitable that they will be ingested by aquatic organisms and be transferred up through the food chain. However, to date, no study has considered whether MPs can be transmitted by means of ontogenic transference, i.e. between life stages that use different habitats. Here, we determine whether fluorescent polystyrene beads could transfer between Culex mosquito life stages and, particularly, could move into the flying adult stage. We show for the first time that MPs can be transferred ontogenically from a feeding (larva) into a non-feeding (pupa) life stage and subsequently into the adult terrestrial life stage. However, transference is dependent on particle size, with smaller 2 mm MPs transferring readily into pupae and adult stages, while 15 mm MPs transferred at a significantly reduced rate. MPs appear to accumulate in the Malpighian tubule renal excretion system. The transfer of MPs to the adults represents a potential aerial pathway to contamination of new environments. Thus, any organism that feeds on terrestrial life phases of freshwater insects could be impacted by MPs found in aquatic ecosystems. 1. Introduction Microplastics (MPs) are ubiquitous pollutants found in marine, freshwater and terrestrial ecosystems [1–3]. There is little doubt that plastic and MP pollution is a major environmental concern globally. Despite this, there is relatively little research into the impact of MPs on freshwater ecosystems, with most research concentrating on marine systems and organisms [2]. MPs have been defined as plastic particles smaller than 5 mm in size [4,5]. However, this simple description covers a wide range of types, including, among others, polypropylene, polyethylene and polystyrene MPs entering the environment in different shapes and sizes, including fibres, pellets and cosmetic beads [6,7]. MPs are categorized based on their origin as primary or secondary types, depending on whether they were released into the environment as MPs ( primary) or have degraded to that size in the environment (secondary) [8,9]. MPs pass through terrestrial environments in household wastewater [2,10]. Rivers can subsequently deliver MPs into the sea and lakes, where they can be found in high concentrations [11 –13]. MPs are ingested by aquatic organisms and can be transferred through the food chain in both freshwater and marine environments [14 – 18]. However, to date, no study has considered whether MPs can be transmitted by means of ontogenic transference, i.e. between life stages that use different habitats. Freshwater environments are inhabited by insects that spend their juvenile stages in water but their adult stages in the terrestrial environment. Such & 2018 The Author(s) Published by the Royal Society. All rights reserved. (a) single (b) mixed 2 4000 larvae 3000 2000 1000 rsbl.royalsocietypublishing.org no. MPs (mean ± s.e.) 5000 0 1600 1200 pupae no. MPs (mean ± s.e.) (d) 2000 800 400 (e) 60 no. MPs (mean ± s.e.) 0 48 (f) adults 36 24 12 0 2 15 particle size (mm) 2 15 particle size (mm) Figure 1. Uptake counts of MPs across larval (a,b), pupal (c,d) and adult (e,f ) Culex mosquito stages following single (a,c,e) and mixed (b,d,f ) exposures to 2 and 15 mm beads. Means are +s.e. (n ¼ 5 per experimental group). insects include mayflies, dragonflies, midges and mosquitoes, most of which are eaten by terrestrial vertebrates. This raises the potential for MPs to enter terrestrial ecosystems from freshwater habitats aerially via transference to adult invertebrate life stages. Here, we thus determine whether 2 and 15 mm fluorescent polystyrene beads could transfer between insect life stages and, particularly, could move into the flying adult stage. Fluorescent beads were selected to enable MPs to be easily detected in the non-feeding stages and also to allow an investigation of location within the body during metamorphosis. The Culex pipiens mosquito complex was selected as a model for this study given their worldwide distribution and broad habitat preference [19]. Mosquitoes develop through four feeding larval instars and a non-feeding pupal stage, and finally emerge into a flying adult. 2. Material and methods For additional details of all methods and analyses, see the electronic supplementary material. Two types of MPs were used: a 2 mm fluorescent yellowgreen carboxylate-modified polystyrene (density 1.050 g cm23, excitation 470 nm; emission 505 nm, Sigma-Aldrich, UK) and a 15.45 + 1.1 mm fluorescent dragon green polystyrene (density 1.06 g cm23 (5  106 particles ml21), excitation 480 nm; emission 520 nm, Bangs Laboratories, Inc., USA). Four treatments were used: a control with no MPs, a treatment of 8  105 2 mm particles ml21, a treatment of 8  102 15 mm particles ml21 and a 1 : 1 mixture of both treatments. Each replicate (five per treatment) contained 10 third instar C. pipiens larvae in a 50 ml glass beaker filled with 50 ml of tap water. The control and all treatments contained 100 mg of pelleted guinea pig food. Treatments were assigned randomly to a position on the laboratory bench to reduce experimental error. One random individual was removed from each beaker when every mosquito had moulted into the fourth instar, and again when they pupated or emerged as adults. All samples were then placed in separate 1.5 ml Eppendorf tubes and stored at 2208C prior to examination. MPs were extracted from mosquitoes by homogenization and filtration. The filter membrane was examined using an epi-fluorescent microscope (Zeiss Axioskop) under a 20 lens to count the number of fluorescent MPs. Adults were further dissected under a binocular stereo microscope (0.7 – 4.5) to extract Biol. Lett. 14: 20180479 (c) 3 (a) rsbl.royalsocietypublishing.org the gut and quantify the numbers of MPs under the epi-fluorescent microscope [20]. All data were analysed using the statistical software R v. 3.4.2 [21]. MP counts were analysed using generalized linear models assuming a quasi-Poisson distribution. Uptake of MPs was examined with respect to ‘particle size’, ‘treatment’ and ‘life stage’. We performed model simplification via stepwise removal of non-significant effects. Tukey’s tests were used post hoc for multiple comparisons. No MPs were found in control groups of any mosquito life stage. Densities of MPs were significantly different between life stages (F2,56 ¼ 160.42, p , 0.001), with MP numbers significantly falling as mosquitoes moved between successive ontogenic levels (all p , 0.001) (figure 1; electronic supplementary material, table S1 and S2). MP transference to adults was confirmed by fluorescent microscopy where the beads were detected in the adult abdomen, specifically inside the Malpighian tubules (figure 2). Significantly more 2 mm particles were found in mosquito life stages than 15 mm particles overall (F1,58 ¼ 303.98, p , 0.001). MPs uptake was also significantly greater overall in mixed exposure treatments (F1,55 ¼ 6.00, p ¼ 0.02). Although 2 mm particles were transferred to adults in all instances, we found no transference of 15 mm particles following single treatment exposures. However, in the mixed MPs treatment, transference to adults of both 2 and 15 mm particles was evidenced (figure 1). Biol. Lett. 14: 20180479 3. Results 100 mm (b) Malpighian tubules 4. Discussion Here, we show for the first time that MPs can be transferred ontogenically from a feeding (larval) into a non-feeding ( pupal) life stage and subsequently into the flying (adult) life stage. Transference through to adults was found in both MP sizes, although the larger 15 mm MPs were not ingested as readily as the 2 mm MPs. Dissection of mosquito adults showed that 2 mm MPs accumulated in the renal excretion system of Malpighian tubules which, unlike the gut, pass from larvae to adult stages without visible reorganization [22]. This has been demonstrated previously to provide a physical transport system between stages during metamorphosis for Pseudomonas bacteria and seems to be important for ontogenic transmission from larvae to adults [23]. Few 15 mm MPs were transferred into adults, suggesting that MP size is an important factor in ontogenic transfer which could be related to the transfer and accumulation of MPs in the Malpighian tubes. Although the translocation mechanism of MPs to the Malpighian tubules is unclear in mosquitoes, analysis of fish, fiddler crab and marine mussels has demonstrated that MPs can be translocated from gastrointestinal tracts into other tissues in a wide range of phyla [24 –26]. Malpighian tubules have an entry point to the gut between the mid- and hindgut of mosquitoes, but the flow of fluid is from the Malpighian tubules to the hindgut [27]. Diptera are known to produce structures called concretions in the Malpighian tubules which have been shown to sequester heavy metals [28]. However, it is unlikely that this pathway would operate with a solid MP. 100 mm Figure 2. Epi-fluorescent microscope images showing fluorescent MP particles within (a) the abdomen of an adult mosquito before dissection and (b) the abdominal Malpighian tubules following dissection. Our results have important implications because any aquatic life stage that is able to consume MPs and transfer them to their terrestrial life stage is a potential vector of MPs onto novel aerial and terrestrial habitats. Ingestion of MP-contaminated organisms by terrestrial organisms is not new [29]. Indeed, the widespread distribution of MPs in marine environments has meant that animals such as fish and shellfish sold for human consumption are contaminated with a range of plastics with a consequent transference of MPs between trophic levels [24]. Unlike MP fibres, which are common in the air and atmosphere, there has been no evidence for MP beads being transported into the air [30]. We have demonstrated here that species with aquatic and terrestrial life stages can harbour MPs through their life history. Adults are predated on emergence by many animals including dipteran flies Empididae and Dolichopodididae, while resting predominantly by spiders and in flight they are the prey of dragonflies, damselflies, birds (such as swallows and swifts) and bats [31]. Where many insects are emerging Data accessibility. Data files are available in the electronic supplementary to conception and design, or acquisition of data, or analysis and interpretation of data; were involved in drafting the article or revising it critically for important intellectual content; approved the final version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Competing interests. We declare we have no competing interests. Funding. A.C. is funded by the University of Reading. R.A.-J. is self-funded and R.N.C. is funded through the Department for the Economy, Northern Ireland. Acknowledgements. We thank Natali Ortiz-Perea for assisting with mosquito colony rearing. References 1. Sighicelli M, Pietrelli L, Lecce F, Iannilli V, Falconieri M, Coscia L, Di Vito S, Nuglio S, Zampetti G. 2018 Microplastic pollution in the surface waters of Italian subalpine lakes. Environ. Pollut. 236, 645 –651. (doi:10.1016/j.envpol.2018.02.008) 2. Wagner M, Lambert S. 2018 Freshwater microplastics. Cham, Switzerland: Springer International Publishing. 3. Mason SA, Welch V, Neratko J. 2018 Synthetic polymer contamination in bottled water. New York, NY: Fredonia State University. 4. Eriksen M, Lebreton LCM, Carson HS, Thiel M, Moore CJ, Borerro JC, Galgani F, Ryan PG, Reisser J. 2014 Plastic pollution in the world’s oceans: more than 5 trillion plastic pieces weighing over 250 000 tons afloat at sea. PLoS ONE 9, e111913. (doi:10.1371/ journal.pone.0111913) 5. Imhof HK, Ivleva NP, Schmid J, Niessner R, Laforsch C. 2013 Contamination of beach sediments of a subalpine lake with microplastic particles. Curr. Biol. 23, 1–15. (doi:10.1016/j.cub.2013.09.001) 6. Andrady AL, Neal MA. 2009 Applications and societal benefits of plastics. Phil. Trans. R. Soc. B 364, 1977 –1984. (doi:10.1098/rstb.2008.0304) 7. 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Eriksen M, Mason S, Wilson S, Box C, Zellers A, Edwards W, Farley H, Amato S. 2013 Microplastic 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. pollution in the surface waters of the Laurentian Great Lakes. Mar. Pollut. Bull. 77, 177–182. (doi:10.1016/j.marpolbul.2013.10.007) Fischer EK, Paglialonga L, Czech E, Tamminga M. 2016 Microplastic pollution in lakes and lake shoreline sediments—a case study on Lake Bolsena and Lake Chiusi (central Italy). Environ. Pollut. 213, 648 –657. (doi:10.1016/j.envpol.2016.03.012) Su L, Xue Y, Li L, Yang D, Kolandhasamy P, Li D, Shi H. 2016 Microplastics in Taihu Lake, China. Environ. Pollut. 216, 711–719. (doi:10.1016/j.envpol.2016. 06.036) Aljaibachi R, Callaghan A. 2018 Impact of polystyrene microplastics on Daphnia magna mortality and reproduction in relation to food availability. PeerJ 6, e4601. (doi:10.7717/peerj.4601) Cole M, Lindeque P, Fileman E, Halsband C, Goodhead R, Moger J, Galloway TS. 2013 Microplastic ingestion by zooplankton. Environ. Sci. 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Exp. 5, e228. (doi:10.3791/228) R Development Core Team. 2017 R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. 22. Clements AN. 1992 The biology of mosquitoes. Volume 1: development, nutrition and reproduction. London: Chapman and Hall. 23. Chavshin AR, Oshaghi MA, Vatandoost H, Yakhchali B, Zarenejad F, Terenius O. 2015 Malpighian tubules are important determinants of Pseudomonas transstadial transmission and longtime persistence in Anopheles stephensi. Parasit. Vectors 8, 1 –7. (doi:10.1186/s13071-015-0635-6) 24. Von Moos N, Burkhardt-Holm P, Köhler A. 2012 Uptake and effects of microplastics on cells and tissue of the blue mussel Mytilus edulis L. after an experimental exposure. Environ. Sci. Technol. 46, 11 327–11 335. 25. Brennecke D, Ferreira EC, Costa TMM, Appel D, da Gama BAP, Lenz M. 2015 Ingested microplastics (.100mm) are translocated to organs of the tropical fiddler crab Uca rapax. Mar. Pollut. 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Dris R, Gasperi J, Saad M, Mirande C, Tassin B. 2016 Synthetic fibers in atmospheric fallout: A source of microplastics in the environment? Mar. Poll. Bull. 104, 290 – 293. 31. Medlock JM, Snow KR. 2008. Natural predators and parasites of British mosquitoes: a review. Eur. Mosquito Bull. 25, 1 –11. 4 Biol. Lett. 14: 20180479 material. Authors’ contribution. All authors provided substantial contributions rsbl.royalsocietypublishing.org from a highly contaminated site, the possibility of contamination of these predators could be high. While mosquitoes were used here as a model organism, any freshwater insect that can ingest MPs will likely equally transmit plastics into a terrestrial adult stage. This has implications for organisms that feed on adult stages with aerial and terrestrial animals accordingly open to MP exposure and transference appearing to occur at a higher rate for smaller MPs. Up and away: ontogenic transference as a pathway for aerial dispersal of microplastics Rana Al-Jaibachi1, Ross N. Cuthbert1,2, Amanda Callaghan1 Supplementary material Materials and methods Preparation of microplastics Two types of MPs were used in the experiments that differed in size and fluorescent property. The first was a 2µm (density 1.050g/cm3) fluorescent yellow-green carboxylate-modified polystyrene (excitation 470nm; emission 505nm), supplied by Sigma-Aldrich, UK. The 2 µm MPs were stored as a stock suspension (2.5 mg mL-1) in distilled water and mixed using a vortex prior to dilutions. The second was a 15.45 + 1.1 µm fluorescent dragon green polystyrene (excitation 480nm; emission 520nm) supplied by Bangs Laboratories Inc., USA (lot no. 12980) as 1% solid polystyrene microspheres (density 1.06 g/cm3 (5x106 particles/ml)). Unlike the first MP type, the 15 µm MPs were prepared prior to experiments. One millilitre was decanted from the stock solution into a 1.5ml Eppendorf tube and centrifuged at 9000 rpm for 10 min. The supernatant was discarded and 1 ml of distilled water was added. The solution was then resuspended by using the vortex and centrifuged again at the same speed and duration. This process was repeated two more times. Mosquito colonies Larvae of the mosquito complex Culex pipiens were obtained from colonies reared at the University of Reading, UK. Colonies were maintained at a temperature of 25°C, a relative humidity (RH) of 70+5% and a photoperiod of 16:8 light:dark. Adult mosquitoes were maintained in 32.5cm3 net cages. Each cage contained shallow containers of cotton wool soaked with a 10% sucrose solution that was topped up three times per week. A 250ml black oviposition cup (30cm diameter) one third filled with cool tap water was also placed in each cage. Each colony cage was fed overnight twice a week with horse blood using a Hemotek blood feeder covered with stretched parafilm. Eggs were collected three times per week and placed in a clean bowl one third filled with cool tap water, covered by an elasticated net cover. They were fed three times per week with a quarter-teaspoon of ground guinea pig food. The water was replaced once a week by straining the larvae through a flour sieve. After 10-14 days, larvae pupated and then emerged as adults. Adults were collected using an electric pooter to place them in a new cage, with each cage containing a maximum of 500 mosquitoes. Treatments for investigating uptake, transfer of MP on mosquito life stages Four treatments were used; a control with no microplastics, a treatment of 8x105 2µm particles/ml, a treatment of 8x102 15µm particles/ml, and a 1:1 mixture of both treatments. Each replicate (five per treatment) contained ten 3rd instar C. pipiens larvae in a 50ml glass beaker filled with 50ml of tap water. The control and all treatments contained 100mg of pelleted guinea pig food. Treatments were assigned randomly to a position on the laboratory bench to reduce experimental error. Measurement of the uptake, transfer and effect of MPs on mosquitoes One individual from each beaker was removed from each treatment when the mosquitoes moulted into the 4th instar, and again when they pupated. Mosquitoes were washed twice with distilled water before being placed in a 1.5ml Eppendorf tube. Adults that survived eclosion were collected immediately after emergence and placed in a 1.5ml Eppendorf tube. All samples were stored in a -20 ºC freezer. MPs were prepared for counting by homogenising the frozen individuals in situ using a glass pestle (Fisher Sciences Loughborough, UK) for one minute in 500µl of distilled water. Individuals treated with 2 µm MPs and mixed sizes were filtered through a nucleopore tracketched membrane (Whatman, UK) of < 0.1 µm. Those exposed to 15 µm MPs were filtered through a nucleopore track-etched membrane (Whatman, UK) of < 10 µm using a glass vacuum filter holder connected to a manual air pump. The membrane was examined under an epifluorescent microscope (Zeiss Axioskop) under a 20x lens to count the number of fluorescent MPs. Surviving adult mosquitoes from each treatment were examined under the epi-fluorescent microscope to check that no MPs were attached to the body. Adults were dissected following (Coleman et al., 2007) protocol under a binocular stereo microscope (0.7-4.5X) to extract the gut and examine the presence of MPs under the epi-fluorescent microscope. Table S1. Mean number of microplastics taken up by Culex pipiens life stages under single and mixed exposures to 2µm and 15µm particles. Means are ±SE (n=5 per experimental group). Treatment Single exposure Life stage 2µm (±SE) Mixed exposure 15µm (±SE) 2µm (±SE) 15µm (±SE) Larvae 3047.2 (±278.4) 279.0 (±96.8) 3952.2 (±743.7) 224.4 (±32.9) Pupae 1045.6(±366.5) 107.4 (±6.0) 1653.4 (±301.1) 65.2 (±30.0) Adult 40.2 (±10.5) 0.0 (±0.0) 16 (±2.6) 0.4 (±0.4) Table S2 Raw data: number of microplastics taken up by Culex pipiens life stages under single and mixed exposures to 2µm and 15µm particles. Means are ±SE (n=5 per experimental group). Treatments Mosquito life stages Larvae Larvae Larvae Larvae Larvae Pupae Pupae Pupae Pupae Pupae Adults Adults Adults Adults Adults Single exposure 2µm 15µm 2988 190 2500 264 4000 91 3248 650 2500 200 2232 123 1094 100 1314 118 488 90 100 106 23 0 78 0 29 0 48 0 23 0 Mixed exposure 2µm 15µm 1043 160 4500 182 5048 280 4970 325 4200 175 2169 61 2568 180 1308 21 1072 14 1150 50 13 0 11 2 12 0 24 0 20 0 
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