peerj_json_files/PeerJ_Json_135.json

{
    "v1_Abstract": "Substantial quantities of small plastic particles, termed \u201cmicroplastic,\u201d have been found in many areas of the world ocean, and have accumulated in particularly high densities on the surface of the subtropical gyres. While plastic debris has been documented on the surface of the North Pacific Subtropical Gyre (NPSG) since the early 1970s, the ecological implications remain poorly understood. Organisms associated with floating objects, termed the \u201crafting assemblage,\u201d are an important component of the NPSG ecosystem. These objects are often dominated by abundant and fast-growing gooseneck barnacles (Lepas spp.), which predate on plankton and larval fishes at the sea surface. To assess the potential effects of microplastic on the rafting community, we examined the gastrointestinal tracts of 385 barnacles collected from the NPSG for evidence of plastic ingestion. We found that 33.5% of the barnacles had plastic particles present in their gastrointestinal tract, ranging from one plastic particle to a maximum of 30 particles. Particle ingestion was positively correlated to capitulum length, and no blockage of the stomach or intestines was observed. The majority of ingested plastic was polyethylene, with polypropylene and polystyrene also present. Our results suggest that barnacle ingestion of microplastic is relatively common, with unknown trophic impacts on the rafting community and the NPSG ecosystem.",
    "v1_col_introduction": "introduction  : Oceanic litter, termed \u201cmarine debris\u201d or \u201cplastic pollution,\u201d is a matter of increasing\nscientific and public concern (STAP-GEF 2011, U.S. Environmental Protection Agency 2011,\nConvention on Biological Diversity and STAP-GEF 2012). The durability and longevity that\nmake plastic a useful substance also leads to its persistence in the marine environment, with\nconsequences that include entanglement, damage to habitats, species transport, and ingestion\n(National Research Council 2009). One study estimated that more than 267 species have been\ndocumented to ingest plastic (Allsopp et al. 2006), including mammals (Eriksson and Burton\n2003, Jacobsen et al. 2010), seabirds (Moser and Lee 1992, Ryan 2008, van Franeker et al. 2011),\nturtles (Schuyler et al. 2013), and a wide variety of fishes (Possatto et al. 2011, Lusher et al.\n2013, Anastasopoulou et al. 2013). Negative effects of plastic ingestion may include intestinal\nblockage, diminished feeding stimulus, lowered steroid hormone levels, delayed ovulation and\nreproductive failure (Azzarello and Van Vleet 1987, Derraik 2002). Because oceanic plastic\ndebris can contain high levels of hydrophobic toxins (Endo et al. 2005, Frias et al. 2010, Rios et\nal. 2010, Rochman et al. 2013), ingestion of plastic debris may also increase toxic exposure\n(Teuten et al. 2009, Gassel et al. 2013).\nMost plastic ingestion has been documented in vertebrates (Convention on Biological\nDiversity and STAP-GEF 2012), but the extent of plastic ingestion in marine invertebrates\nremains poorly known. Laboratory experiments suggest that many invertebrate species ingest plastic (reviewed in Wright et al. 2013). Suspended plastic particles (2-60 \u03bcm in diameter) were\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n22\n23\n24\nPeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nsuccessfully fed to calanoid copepods, cladocerans, and salps in the context of studying particle\nsize selectivity (Burns 1968, Wilson 1973, Frost 1977, Kremer and Madin 1992). In laboratory\nstudies focused specifically on the incidence of plastic ingestion, plastic particles were readily\nconsumed by an assortment of zooplankton (Cole et al. 2013) and benthic invertebrates\n(Thompson et al. 2004, Browne et al. 2008, Graham and Thompson 2009, Wegner et al. 2012,\nvon Moos et al. 2012, Besseling et al. 2013). However, the evidence from natural ecosystems is\nfar sparser. To date, we are aware of only three studies that have found in situ plastic ingestion in\ninvertebrates: sandhopper amphipods (Talitrus saltator; Ugolini et al. 2013), Norway lobster\n(Nephrops norvegicus; Murray and Cowie 2011), and flying squid (Ommastrephes bartrami; Day\n1988 cited in Laist 1997).\nThough plastic pollution has been documented in the North Atlantic and North Pacific\nsubtropical gyres since the early 1970s (Carpenter and Smith 1972, Wong et al. 1974, Day and\nShaw 1987, Moore et al. 2001, Law et al. 2010, Goldstein et al. 2012), the ecological\nimplications have been relatively little studied. In this open ocean ecosystem, the majority of\nmarine debris are small particles (termed \u201cmicroplastic,\u201d less than 5 mm in diameter) that float at\nthe sea surface (Hidalgo-Ruz et al. 2012), though wind mixing moves some particles deeper\n(Kukulka et al. 2012). Floating plastics in these areas are primarily comprised of polyethylene,\nwith polypropylene and polystyrene also present (Rios et al. 2007, Goldstein 2012). Ingestion has\nbeen found in surface-feeding seabirds (Fry et al. 1987, Avery-Gomm et al. 2012) and epipelagic\nand mesopelagic fishes (Boerger et al. 2010, Davison and Asch 2011, Jantz et al. 2013, Choy and Drazen 2013), but the biota most likely to be impacted by microplastic pollution is the neuston, a specialized community associated with the air-sea interface which includes both zooplankton and substrate-associated rafting organisms (Cheng 1975).\nRafting organisms in the open ocean are increasingly associated with floating plastic\ndebris, which has supplemented natural substrates such as pumice and macroalgae (Thiel and\n25\n26\n27\n28\n29\n30\n31\n32\n33\n34\n35\n36\n37\n38\n39\n40\n41\n42\n43\n44\n45\n46\n47\n48\n49\nPeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nGutow 2005a). Two species of lepadomorph barnacles (Lepas anatifera and Lepas pacifica) are widespread throughout the North Pacific Subtropical Gyre (NPSG) and frequently dominate the rafting assemblage (Tsikhon-Lukanina et al. 2001). (A third species, Lepas (Dosima) fascularis, forms its own float at the end of the juvenile stage and drifts independently, and is therefore not a major component of the rafting assemblage; Newman and Abbott 1980.) These barnacles are omnivorous, feeding opportunistically on the neustonic zooplankton, and are said to \u201chold a singular position in having more sources of food to draw upon than any other organisms in the neuston (Bieri 1966).\u201d The barnacles are themselves preyed upon by omnivorous epipelagic crabs and the rafting nudibranch Fiona pinnata (Bieri 1966, Davenport 1992).\nIn this study, we hypothesized that Lepas\u2019 indiscriminate feeding strategy and position at\nthe sea surface could cause this species to ingest microplastic, with unknown implications for NPSG ecology. To this end, we examined the gastrointestinal tracts of 385 Lepas from the NPSG for evidence of microplastic ingestion.",
    "v2_Abstract": "Substantial quantities of small plastic particles, termed \u201cmicroplastic,\u201d have been found in many areas of the world ocean, and have accumulated in particularly high densities on the surface of the subtropical gyres. While plastic debris has been documented on the surface of the North Pacific Subtropical Gyre (NPSG) since the early 1970s, the ecological implications remain poorly understood. Organisms associated with floating objects, termed the \u201crafting assemblage,\u201d are an important component of the NPSG ecosystem. These objects are often dominated by abundant and fast-growing gooseneck barnacles (Lepas spp.), which predate on plankton and larval fishes at the sea surface. To assess the potential effects of microplastic on the rafting community, we examined the gastrointestinal tracts of 385 barnacles collected from the NPSG for evidence of plastic ingestion. We found that 33.5% of the barnacles had plastic particles present in their gastrointestinal tract, ranging from one plastic particle to a maximum of 30 particles. Particle ingestion was positively correlated to capitulum length, and no blockage of the stomach or intestines was observed. The majority of ingested plastic was polyethylene, with polypropylene and polystyrene also present. Our results suggest that barnacle ingestion of microplastic is relatively common, with unknown trophic impacts on the rafting community and the NPSG ecosystem.",
    "v2_col_introduction": "introduction  : Oceanic litter, termed \u201cmarine debris\u201d or \u201cplastic pollution,\u201d is a matter of increasing\nscientific and public concern (STAP-GEF 2011, U.S. Environmental Protection Agency 2011,\nConvention on Biological Diversity and STAP-GEF 2012). The durability and longevity that\nmake plastic a useful substance also leads to its persistence in the marine environment, with\nconsequences that include entanglement, damage to habitats, species transport, and ingestion\n(National Research Council 2009). One study estimated that more than 267 species have been\ndocumented to ingest plastic (Allsopp et al. 2006), including mammals (Eriksson and Burton\n2003, Jacobsen et al. 2010), seabirds (Moser and Lee 1992, Ryan 2008, van Franeker et al. 2011),\nturtles (Bjorndal et al. 1994, Bugoni et al. 2001), and a wide variety of fishes (Possatto et al.\n2011, Lusher et al. 2013, Anastasopoulou et al. 2013). Negative effects of plastic ingestion may\ninclude intestinal blockage, diminished feeding stimulus, lowered steroid hormone levels,\ndelayed ovulation and reproductive failure (Azzarello and Van Vleet 1987, Derraik 2002).\nBecause oceanic plastic debris can contain high levels of hydrophobic toxins (Endo et al. 2005,\nFrias et al. 2010, Rios et al. 2010, Rochman et al. 2013), ingestion of plastic debris may also\nincrease toxic exposure (Teuten et al. 2009, Gassel et al. 2013).\nMost plastic ingestion has been documented in vertebrates (Convention on Biological\nDiversity and STAP-GEF 2012), but the extent of plastic ingestion in marine invertebrates\nremains poorly known. Laboratory experiments suggest that many invertebrate species ingest\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n22\n23\nPeerJ reviewing PDF | (v2013:07:631:0:1:NEW 30 Jul 2013)\nR ev ie w in g M an\nus cr ip t\nplastic (reviewed in Wright et al. 2013). Suspended plastic particles (2-60 \u03bcm in diameter) were\nsuccessfully fed to calanoid copepods, cladocerans, and salps in the context of studying particle\nsize selectivity (Burns 1968, Wilson 1973, Frost 1977, Kremer and Madin 1992). In laboratory\nstudies focused specifically on the incidence of plastic ingestion, plastic particles were readily\nconsumed by an assortment of zooplankton (Cole et al. 2013) and benthic invertebrates\n(Thompson et al. 2004, Browne et al. 2008, Graham and Thompson 2009, Wegner et al. 2012,\nvon Moos et al. 2012, Besseling et al. 2013). However, the evidence from natural ecosystems is\nfar sparser. To date, we are aware of only three studies that have found in situ plastic ingestion in\ninvertebrates: sandhopper amphipods (Talitrus saltator; Ugolini et al. 2013), Norway lobster\n(Nephrops norvegicus; Murray and Cowie 2011), and flying squid (Ommastrephes bartrami; Day\n1988 cited in Laist 1997).\nThough plastic pollution has been documented in the North Atlantic and North Pacific\nsubtropical gyres since the early 1970s (Carpenter and Smith 1972, Wong et al. 1974, Day and\nShaw 1987, Moore et al. 2001, Law et al. 2010, Goldstein et al. 2012a), the ecological\nimplications have been relatively little studied. In this open ocean ecosystem, the majority of\nmarine debris are small particles (termed \u201cmicroplastic,\u201d less than 5 mm in diameter) that float at\nthe sea surface (Goldstein et al. 2012b), though wind mixing moves some particles deeper\n(Kukulka et al. 2012). Floating plastics in these areas are primarily comprised of polyethylene,\nwith polypropylene and polystyrene also present (Rios et al. 2007, Goldstein 2012). Ingestion has\nbeen found in surface-feeding seabirds (Fry et al. 1987, Avery-Gomm et al. 2012) and epipelagic\nand mesopelagic fishes (Boerger et al. 2010, Davison and Asch 2011, Jantz et al. 2013, Choy and\nDrazen 2013), but the biota most likely to be impacted by microplastic pollution is the neuston, a\nspecialized community associated with the air-sea interface which includes both zooplankton and substrate-associated rafting organisms (Cheng 1975).\n24\n25\n26\n27\n28\n29\n30\n31\n32\n33\n34\n35\n36\n37\n38\n39\n40\n41\n42\n43\n44\n45\n46\n47\nPeerJ reviewing PDF | (v2013:07:631:0:1:NEW 30 Jul 2013)\nR ev ie w in g M an\nus cr ip t\nRafting organisms in the open ocean are increasingly associated with floating plastic\ndebris, which has supplemented natural substrates such as pumice and macroalgae (Thiel and\nGutow 2005a). Two species of lepadomorph barnacles (Lepas anatifera and Lepas pacifica) are\nwidespread throughout the North Pacific Subtropical Gyre (NPSG) and frequently dominate the rafting assemblage (Tsikhon-Lukanina et al. 2001). (A third species, Lepas (Dosima) fascularis, forms its own float at the end of the juvenile stage and drifts independently, and is therefore not a major component of the rafting assemblage; Newman and Abbott 1980.) These barnacles are omnivorous, feeding opportunistically on the neustonic zooplankton, and are said to \u201chold a singular position in having more sources of food to draw upon than any other organisms in the neuston (Bieri 1966).\u201d The barnacles are themselves preyed upon by omnivorous epipelagic crabs (Planes spp.) and the rafting nudibranch Fiona pinnata (Bieri 1966, Davenport 1992).\nIn this study, we hypothesized that Lepas\u2019 indiscriminate feeding strategy and position at\nthe sea surface could cause this species to ingest microplastic, with unknown implications for NPSG ecology. To this end, we examined the gastrointestinal tracts of 385 Lepas from the NPSG for evidence of microplastic ingestion.",
    "v1_text": "results : Of the 385 barnacles examined, 129 individuals (33.5%) had ingested plastic (Fig 2, Table 1). These included 243 Lepas anatifera and 85 Lepas pacifica (57 barnacles could not be 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t identified to species), of which 90 L. anatifera, 34 L. pacifica, and 5 Lepas spp. contained plastic. Forty-one of the barnacles that ingested plastic had one plastic particle in their stomach or intestines, 26 individuals had two particles, and 57 individuals contained three or more particles, to a maximum of 30 particles (Fig 3A). Overall, the number of ingested particles was positively correlated to capitulum length (Kendall\u2019s tau=0.099, p=0.015). However, when we considered only barnacles that had ingested plastic, the correlation between plastic ingestion and capitulum length was not significant (Kendall\u2019s tau=-0.080, p=0.229). Individuals with a capitulum length between 2 and 3 cm consumed the greatest number plastic particles (Fig 3B). With the exception of one individual, all the barnacles that consumed plastic had a capitulum length of 1.7 cm or greater. In total, 518 plastic particles were recovered from barnacle digestive tracts. Of these, 99% were degraded fragments and 1% were monofilament line. None of the pre-production pellets known as \u201cnurdles\u201d were found. The median diameter of ingested particles was 1.41 mm, and the median surface area 1.00 mm2, smaller than the median diameter of 1.78 mm and median surface area of 1.27 mm2 for all particles collected in nets during 2009 (Fig 4, KolmogorovSmirnov test p<0.001). The smallest particle ingested by barnacles had a maximum diameter of 0.609 mm and the largest (a long thin fragment) a maximum diameter of 6.770 mm. No blockage of the stomach or intestine was observed, and particles did not accumulate in any area of the digestive tract. All particles were of a plausible size to pass through the anus. Of the randomly selected subset of 219 ingested plastic particles that were analyzed for plastic type, 58.4% were polyethylene, 5.0 % were polypropylene, and 1.4% were polystyrene. As noted in the Methods section, we were unable to identify 35% of the subset due to darker pigmentation in these particles, which caused melting under the Raman spectrophotometer. Of the 29 barnacles that had ingested more than one piece of plastic, 66% contained more than one type of plastic. The plastic types of 12 floating debris items to which barnacles were attached 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t were more diverse than those of ingested particles. Four substrates were polystyrene, 3 were polyethylene, 2 were polypropylene, 2 were polyethylene terephthalate, and one was tire rubber. acknowledgements : We thank the captain, crews, and students of the SEAPLEX cruise on the R/V New Horizon and Sea Education Association cruises S-242 and S-243 on the SSV Robert C. Seamans. Assistance from L. Sala, M.D. Ohman, and E. Zettler made this project possible. methods : Floating debris items with attached gooseneck barnacles (Fig 1A) were opportunistically collected during the 2009 Scripps Environmental Accumulation of Plastic Expedition (SEAPLEX) and two 2012 Sea Education Association (SEA) research cruises onboard the SSV Robert C. Seamans: S242, an undergraduate voyage from Honolulu, HI to San Francisco, CA (mid-June to mid-July 2012), and S243, the Plastics at SEA: North Pacific Expedition from San Diego, CA to Honolulu, HI (early October to mid-November 2012; Fig 1A). Collection occurred by several means, including 1) from the vessel using a long-handled dip net (335 \u03bcm mesh, 0.5m diameter mouth); 2) incidentally during neuston net (335 \u03bcm mesh, 0.5 x 1.0m mouth) tows at the air-sea interface; and 3) from small boat surveys within 0.5 km of each vessel when sea conditions were calm. No specific permissions were required for these samples, since they were 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t taken in international waters and did not involve protected species. Seven debris items were sampled on SEAPLEX and 29 by SEA (5 during S242 and 24 on S243). Stations within 8.5 km of each other were combined for a total of 19 sampling locations within in the northeastern Pacific Ocean (Fig 2, Table 1). During SEAPLEX, the entire piece of debris with attached barnacles was preserved in 5% Formalin buffered with sodium borate. When the item was too large to be preserved (e.g., a fishing buoy), barnacles were removed and preserved separately. On SEA cruises, as many barnacles as possible to a maximum of 50 were removed from the debris object and preserved in 10% ethanol. Where feasible, a fragment of the item itself was also sampled. In the laboratory, capitulum length was measured using a ruler and species identification (L. anatifera or L. pacifica) determined for all intact individuals (Fig 1B). Barnacles less than 0.8 cm were present, but not sampled in this study. Barnacles greater than approximately 0.8 cm in length were dissected and the contents of their stomach and intestinal tract examined under a dissecting microscope (6-25x magnification as needed). Barnacles were cut open with a scalpel, and the intestinal tract removed and placed in a separate section of the petri dish. The intestinal tract was opened lengthwise, and the contents examined systematically both visually and with forceps. To avoid cross-contamination between samples, each barnacle was dissected in unique, clean petri dish and the scalpel was thoroughly rinsed with deionized water between each samples. Only microplastic fragments and monofilament that were clearly present inside the intestine were considered. Fine microfibers were discounted, as they could not be distinguished from airborne contamination. Because the vast majority of microplastic found were relatively large degraded fragments (>0.5 mm in diameter), visual examination was sufficient to confirm that the microplastic was present in the intestine, and not a result of contamination (Fig 1C). Plastic particles found in the stomach or intestine were quantified, photographed digitally against a ruler for size assessment, rinsed with fresh water and stored in a glass vial for later 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t analyses. The maximum diameter (feret diameter) and two-dimensional area of each particle were digitally measured with the software package NIH ImageJ (Rasband 2012). On the SEAPLEX cruise in 2009, we also measured the diameter and area of all plastic particles captured in surfacetowed plankton nets (number of particles=30,518) using NIH ImageJ-based tools in the Zooprocess software calibrated against manual measurements (Gilfillan et al. 2009, Gorsky et al. 2010). We identified the type of plastic recovered from a randomly selected subset of barnacles (Barnacles N=42; particles N=219). A Raman spectrometer (PeakSeeker Pro-785 with AmScope operated at 10 \u2013 50 mW and 5 \u2013 20 second integration time; Raman Systems MII, Inc/Agiltron, Inc., Woburn, MA) and associated RSIQ software were used to identify plastic type. The Raman spectrum for each plastic piece was compared to a reference library of known plastic types for identification. Particles of clear, white, gray and pale-colored (light blues and greens, oranges and yellows) plastics yielded high quality Raman spectra and were readily identifiable. Those that were darker (medium to dark blues, reds and greens as well as black; 35% of particles subjected to Raman spectroscopy) were heated by the laser beam and melted even at the lowest possible power and integration time settings, resulting in no usable spectra. We also identified a subset of the debris items to which the barnacles were attached. Fragments of 18 objects were collected for analysis, but 6 could not be identified due to darker pigmentation due to melting under the laser. Statistics and figures were generated with the R statistical environment, version R-2.15.1 (R Development Core Team 2012) and QuantumGIS, version 1.8.0-Lisboa (QGIS Development Team 2013). discussion : Our results show that 33.5% of lepadid barnacles collected from the NPSG ingested microplastic, and that the sizes and types of ingested particles were approximately representative of microplastic found on the NPSG surface. Plastic ingestion in these barnacles may therefore be explained by non-selective suspension feeding while exposed to high concentrations of microplastic. The percentage of barnacles observed with ingested plastic in this study is higher than the 9.2% found in NPSG micronektonic fishes (Davison and Asch 2011) and the 19-24.5% found in larger mesopelagic fishes (Jantz et al. 2013, Choy and Drazen 2013). It is likely that barnacles encounter microplastic more frequently than vertically migrating mesopelagic fishes due to the barnacles\u2019 consistent location at the air-sea interface. Since barnacles probably clear their guts in a matter of hours (Ritz 2008), it is likely that a higher percentage of the barnacle population interacts with microplastic than is presented here. Unfortunately, due to logistical considerations on both cruises, barnacle samples were not usually concurrent with neuston tows. Since neustonic microplastic is highly spatially heterogeneous (Ryan et al. 2009), we are thus unable directly compare neustonic microplastic concentrations with incidence of barnacle ingestion. However, the sampling area is known to contain high concentrations of neustonic microplastic (Moore et al. 2001, Goldstein et al. 2012). The objects to which the barnacles are attached may also shed microplastic particles, increasing the likelihood of those particles being ingested by the local rafting community. However, the microplastics ingested by individual barnacles in this study were of multiple plastic types and colors, suggesting they are taking in particles from the surrounding water rather than 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t solely from the substrate to which they are attached. Lepadid barnacles are known to be very nonselective feeders. For example, L.anatifera opportunistically ingests a wide variety of zooplankton and even fills its gut with sand when stranded on the beach (Howard and Scott 1959). L. anatifera can also readily consume large prey items up to 5 mm in diameter, larger than the majority of microplastic debris (Patel 1959). Less is known about the feeding habits of L. pacifica, but it is presumed to have a similar feeding ecology as L. anatifera and other lepadid barnacles (Crisp and Southward 1961, Cheng and Lewin 1976). To avoid difficulties in identifying plastic with darker pigmentation, future studies might consider supplementing Raman spectroscopy with density analysis (Moret-Ferguson et al. 2010), or utilizing Fourier transform infrared spectroscopy when available (Rios et al. 2007, Goldstein 2012). We found only one barnacle with a capitulum length of less than 1.7 cm that had ingested plastic. This observation implies that barnacles may need to reach a certain size before plastic ingestion is possible, perhaps due to the size of the cirri or oral opening. However, our study used visual methods to identify microplastic in barnacle gut contents, and spectroscopic methods or chemical digestion of the tissue are needed to positively identify plastic particles smaller than approximately 300 \u03bcm (Claessens et al. 2011, Hidalgo-Ruz et al. 2012). It is therefore possible that plastic ingestion in the smaller barnacles was not detected in this study. Assessing the ecological significance of plastic ingestion in pelagic invertebrates and fishes remains a challenge. Even in relatively well-studied species, it has been difficult to link plastic ingestion to mortality. For example, two studies of Laysan and black-footed albatross chicks did not find a linkage between cause of death and plastic ingestion (Sileo et al. 1990, Sievert and Sileo 1993), though a third study linked plastic ingestion with lower body weight in adult birds (Spear et al. 1995). Most studies on invertebrates have been relatively short-term investigations that have not found acute negative effects (Thompson et al. 2004, Browne et al. 2008, Graham and Thompson 2009), with the exception of an inflammatory immune response in 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t mussels (von Moos et al. 2012). In zooplankton, the presence of non-edible particles can reduce the rate of feeding on edible particles (Huntley et al. 1983, Ayukai 1987, Cole et al. 2013), and physical interference with sensory apparatus may occur in very high-plastic environments (Cole et al. 2013). The lepadid barnacles in this study did not show evidence of acute harm (e.g., intestinal blockage or ulceration), though negative long-term effects cannot be ruled out. Plastic ingestion may also lead to increased body loads of persistent organic pollutants in both vertebrates and invertebrates (Teuten et al. 2009, Yamashita et al. 2011, Gassel et al. 2013, Besseling et al. 2013), but it is not known whether this occurs in barnacles, or has population- level ramifications in any taxa (Gouin et al. 2011). For example, a modeling study based on lugworms (Arenicola marina) did not find a significant toxicological risk from plastic-adsorbed pollutants (Koelmans et al. 2013). Because L.anatifera appear to survive well in the laboratory (Patel 1959), more detailed studies may be possible. If barnacles are an important prey item, it is possible that their ingestion of plastic particles could transfer plastic or pollutants through the food web. Plastic particles found in fur seals (Eriksson and Burton 2003), piscivorous fishes (Davison and Asch 2011), and crabs (Farrell and Nelson 2013) have been linked to consumption of contaminated prey. The only documented predator of rafting Lepas spp. is the nudibranch Fiona pinnata (Bieri 1966), though it is probable that omnivorous rafting crabs also consume barnacles to some extent (Davenport 1992, Frick et al. 2011). Relatively low rates of predation on these barnacles may explain Lepas\u2019 place as one of the most abundant members of the North Pacific subtropical rafting community (Newman and Abbott 1980, Thiel and Gutow 2005b). For example, one study found that L. pacifica was excluded from nearshore kelp forests by the fish Oxyjulis californica, but was able to inhabit floating kelp paddies in high densities when O. californica was absent (Bernstein and Jung 1979). Studies of the diets of fishes associated with Fish Aggregating Devices (FADs) have found that fishes associated with floating objects rarely feed directly on the fouling community (Ibrahim et 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t al. 1996, Nelson 2003, Vassilopoulou et al. 2004). The likelihood of predators ingesting plastic by feeding on barnacles may therefore be relatively low. While plastic ingestion in taxa such as sea turtles (Schuyler et al. 2013) and cetaceans (Jacobsen et al. 2010) is clearly detrimental, the implications of widespread plastic ingestion in Lepas remain uncertain. Since little is known about the trophic structure and connectivity of both the rafting and drifting components of the neuston, additional studies are necessary to determine the impacts of microplastic ingestion on the rafting community and the larger pelagic ecosystem. a) a dense aggregation of lepas spp. barnacles growing on a buoy and attached line, : collected in October 2012. B) Basic anatomy of Lepas denoting the capitulum, which includes the body and its enclosing plates, and the peduncle, the muscular stalk that attaches the barnacle to the substrate. C) Microplastic ingested by an individual barnacle. PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t Figure 2 Ingestion of microplastic by barnacles across the study area. Circles indicate sampling stations and dark fill indicates the proportion of barnacles that had ingested microplastic at each site. Station coordinates, sample sizes, and ingestion proportions are given in Table 1. PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t Figure 3 Number of microplastic particles ingested by barnacles. (N=385). B) Frequency distribution of ingestion by capitulum length (N=369; sample size is smaller than above since capitulum length was not measured for 16 barnacles). Black bars are the number of individual barnacles that ingested plastic and grey bars are the number of individual barnacles that did not ingest plastic. Bins of capitulum length are greater than the first value, and less than or equal to the second value (e.g., >0.5 cm and <=1.0 cm). Percentages of ingestion by size class are as follows: 6.7%, 0, 23.2%, 43.9%, 45.2%, 35.3%, 25.0%, 40.0%, 0. PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t Figure 4 Size of microplastic particles ingested by barnacles. Size\u2013frequency distributions for A) maximum diameter and B) two-dimensional surface area of particles ingested by barnacles (black; N=507) compared to of all microplastic particles collected in 2009 (grey; N=30,518). Note: 518 microplastic particles were recovered from barnacles, but 11 were lost before they could be photographed for this analysis. PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Station locations and proportion of microplastic ingestion. PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t Station ID Date of collection Latitude (\u00b0N) Longitude (\u00b0W) Total no. barnacles Proportion with plastic Proportion without plastic S242-021-DN 1-Jul-12 36.135 154.957 5 0.80 0.20 S242-023-DN 2-Jul-12 37.672 152.163 20 0.00 1.00 S243-083-DN 31-Oct-12 27.000 146.782 10 0.00 1.00 S243-069-DN 27-Oct-12 30.057 145.057 15 0.47 0.53 S243-055-057058-DN 24-Oct-12 30.140 141.220 80 0.68 0.33 S243-051-052DN 23-Oct-12 30.230 140.690 34 0.18 0.82 U39.F32 15-Aug-09 34.076 140.474 53 0.40 0.60 S243-046-DN 22-Oct-12 31.330 140.338 52 0.42 0.58 S3.F6 10-Aug-09 32.911 140.320 2 0.00 1.00 S242-031-NT 6-Jul-12 39.178 140.160 12 0.00 1.00 S4.F30-F26 14-Aug-09 34.090 139.870 9 0.33 0.67 S242-032-DN 6-Jul-12 39.270 139.570 10 0.10 0.90 S2.F22U40.F11 9-Aug-09 32.050 137.928 15 0.07 0.93 F13 9-Aug-09 32.075 137.223 1 1.00 0.00 S243-032-DN 16-Oct-12 33.563 135.432 17 0.59 0.41 S242-035-DN 8-Jul-12 39.717 135.325 10 0.00 1.00 S243-025-027DN 14-Oct-12 33.700 133.460 13 0.00 1.00 S243-023-DN 13-Oct-12 33.051 132.445 14 0.00 1.00 S243-011-DN 9-Oct-12 33.493 127.715 13 0.00 1.00 PeerJ reviewing PDF | (v2013:07:631:1:0:NEW 25 Sep 2013) R ev ie w in g M an us cr ip t barnacles and ingestion microplastic. :",
    "v2_text": "results : Of the 385 barnacles examined, 129 individuals (33.5%) had ingested plastic (Fig 1). These included 243 Lepas anatifera and 85 Lepas pacifica (57 barnacles could not be identified to species), of which 90 L. anatifera, 34 L. pacifica, and 5 Lepas spp. contained plastic. Forty-one of the barnacles that ingested plastic had one plastic particle in their stomach or intestines, 26 individuals had two particles, and 57 individuals contained three or more particles, to a maximum of 30 particles (Fig 2a). Overall, the number of ingested particles was positively correlated to capitulum length (Fig 2b, Kendall\u2019s tau=0.099, p=0.015). However, when we considered only barnacles that had ingested plastic, the correlation between plastic ingestion and capitulum length was not 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 PeerJ reviewing PDF | (v2013:07:631:0:1:NEW 30 Jul 2013) R ev ie w in g M an us cr ip t significant (Kendall\u2019s tau=-0.080, p=0.229). Individuals with a capitulum length between 2 and 3 cm consumed the greatest number plastic particles. With the exception of one individual, all the barnacles that consumed plastic had a capitulum length of 1.7 cm or greater. In total, 518 plastic particles were recovered from barnacle digestive tracts. Of these, 99% were degraded fragments and 1% were monofilament line. None of the pre-production pellets known as \u201cnurdles\u201d were found. The median diameter of ingested particles was 1.41 mm, and the median surface area 1.00 mm2, smaller than the median diameter of 1.78 mm and median surface area of 1.27 mm2 for all particles collected in nets during 2009 (Fig 3, Kolmogorov-Smirnov test p<0.001). The smallest particle ingested by barnacles had a maximum diameter of 0.609 mm and the largest (a long thin fragment) a maximum diameter of 6.770 mm. No blockage of the stomach or intestine was observed, and particles did not accumulate in any area of the digestive tract. All particles were of a plausible size to pass through the anus. Of the randomly selected subset of 219 ingested plastic particles that were analyzed for plastic type, 58.4% were polyethylene, 5.0 % were polypropylene, and 1.4% were polystyrene. As noted in the Methods section, we were unable to identify 35% of the subset due to darker pigmentation in these particles, which caused melting under the Raman spectrophotometer. Of the 29 barnacles that had ingested more than one piece of plastic, 66% contained more than one type of plastic. The plastic types of 12 floating debris items to which barnacles were attached were more diverse than those of ingested particles. Four substrates were polystyrene, 3 were polyethylene, 2 were polypropylene, 2 were polyethylene terephthalate, and one was tire rubber. acknowledgements : We thank the captain, crews, and students of the SEAPLEX cruise on the R/V New Horizon and Sea Education Association cruises S-242 and S-243 on the SSV Robert C. Seamans. Assistance from L. Sala, M.D. Ohman, and E. Zettler made this project possible. discussion : Our results show that 33.5% of lepadid barnacles collected from the NPSG ingested microplastic, and that the sizes and types of ingested particles were approximately representative of microplastic found on the NPSG surface. Plastic ingestion in these barnacles may therefore be 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 PeerJ reviewing PDF | (v2013:07:631:0:1:NEW 30 Jul 2013) R ev ie w in g M an us cr ip t explained by non-selective suspension feeding while exposed to high concentrations of microplastic. The percentage of barnacles observed with ingested plastic in this study is higher than the 9.2% found in NPSG micronektonic fishes (Davison and Asch 2011) and the 19-24.5% found in larger mesopelagic fishes (Jantz et al. 2013, Choy and Drazen 2013). It is likely that barnacles encounter microplastic more frequently than vertically migrating mesopelagic fishes due to the barnacles\u2019 consistent location at the air-sea interface. The objects to which the barnacles are attached may also shed microplastic particles, increasing the likelihood of those particles being ingested by the local rafting community. However, the microplastics ingested by individual barnacles in this study were of multiple plastic types and colors, suggesting they are taking in particles from the surrounding water rather than solely from the substrate to which they are attached. Lepadid barnacles are known to be very nonselective feeders. For example, L.anatifera opportunistically ingests a wide variety of zooplankton and even fills its gut with sand when stranded on the beach (Howard and Scott 1959). L. anatifera can also readily consume large prey items up to 5 mm in diameter, larger than the majority of microplastic debris (Patel 1959). Less is known about the feeding habits of L. pacifica, but it is presumed to have a similar feeding ecology as L. anatifera and other lepadid barnacles (Crisp and Southward 1961, Cheng and Lewin 1976). We found only one barnacle with a capitulum length of less than 1.7 cm that had ingested plastic. This observation implies that barnacles may need to reach a certain size before plastic ingestion is possible, perhaps due to the size of the cirri or oral opening. However, our study used visual methods to identify microplastic in barnacle gut contents, and spectroscopic methods or chemical digestion of the tissue are needed to positively identify plastic particles smaller than approximately 300 \u03bcm (Claessens et al. 2011, Hidalgo-Ruz et al. 2012). It is therefore possible that plastic ingestion in the smaller barnacles was not detected in this study. 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 PeerJ reviewing PDF | (v2013:07:631:0:1:NEW 30 Jul 2013) R ev ie w in g M an us cr ip t Assessing the ecological significance of plastic ingestion in pelagic invertebrates and fishes remains a challenge. Even in relatively well-studied species, it has been difficult to link plastic ingestion to mortality. For example, two studies of Laysan and black-footed albatross chicks did not find a linkage between cause of death and plastic ingestion (Sileo et al. 1990, Sievert and Sileo 1993), though a third study linked plastic ingestion with lower body weight in adult birds (Spear et al. 1995). Most studies on invertebrates have been relatively short-term investigations that have not found acute negative effects (Thompson et al. 2004, Browne et al. 2008, Graham and Thompson 2009), with the exception of an inflammatory immune response in mussels (von Moos et al. 2012). In zooplankton, the presence of non-edible particles can reduce the rate of feeding on edible particles (Huntley et al. 1983, Ayukai 1987, Cole et al. 2013), and physical interference with sensory apparatus may occur in very high-plastic environments (Cole et al. 2013). The lepadid barnacles in this study did not show evidence of acute harm (e.g., intestinal blockage or ulceration), though negative long-term effects cannot be ruled out. Plastic ingestion may also lead to increased body loads of persistent organic pollutants in both vertebrates and invertebrates (Teuten et al. 2009, Yamashita et al. 2011, Gassel et al. 2013, Besseling et al. 2013), but it is not known whether this occurs in barnacles, or has population-level ramifications in any taxa (Gouin et al. 2011). For example, a modeling study based on lugworms (Arenicola marina) did not find a significant toxicological risk from plastic-adsorbed pollutants (Koelmans et al. 2013). Because L.anatifera appear to survive well in the laboratory (Patel 1959), more detailed studies may be possible. If barnacles are an important prey item, it is possible that their ingestion of plastic particles could transfer plastic or pollutants through the food web. Plastic particles found in fur seals (Eriksson and Burton 2003), piscivorous fishes (Davison and Asch 2011), and crabs (Farrell and Nelson 2013) have been linked to consumption of contaminated prey. The only documented 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 PeerJ reviewing PDF | (v2013:07:631:0:1:NEW 30 Jul 2013) R ev ie w in g M an us cr ip t predator of rafting Lepas spp. is the nudibranch Fiona pinnata, (Bieri 1966), though it is probable that omnivorous rafting crabs also consume barnacles to some extent (Davenport 1992, Frick et al. 2011). Relatively low rates of predation on these barnacles may explain Lepas\u2019 place as one of the most abundant members of the North Pacific subtropical rafting community (Newman and Abbott 1980, Thiel and Gutow 2005b). For example, one study found that L. pacifica was excluded from nearshore kelp forests by the fish Oxyjulis californica, but was able to inhabit floating kelp paddies in high densities when O. californica was absent (Bernstein and Jung 1979). The likelihood of predators ingesting plastic by feeding on barnacles may therefore be relatively low. While plastic ingestion in taxa such as sea turtles (Bugoni et al. 2001) and cetaceans (Jacobsen et al. 2010) is clearly detrimental, the implications of widespread plastic ingestion in Lepas remain uncertain. Since little is known about the trophic structure and connectivity of both the rafting and drifting components of the neuston, additional studies are necessary to determine the impacts of microplastic ingestion on the rafting community and the larger pelagic ecosystem. methods : Floating debris items with attached gooseneck barnacles were opportunistically collected during the 2009 Scripps Environmental Accumulation of Plastic Expedition (SEAPLEX) and two 2012 Sea Education Association (SEA) research cruises onboard the SSV Robert C. Seamans: S242, an undergraduate voyage from Honolulu, HI to San Francisco, CA (mid-June to mid-July 2012), and S243, the Plastics at SEA: North Pacific Expedition from San Diego, CA to Honolulu, HI (early October to mid-November 2012). Collection occurred by several means, including 1) from the vessel using a long-handled dip net (335 \u03bcm mesh, 0.5m diameter mouth); 2) incidentally during neuston net (335 \u03bcm mesh, 0.5 x 1.0m mouth) tows at the air-sea interface; 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 PeerJ reviewing PDF | (v2013:07:631:0:1:NEW 30 Jul 2013) R ev ie w in g M an us cr ip t and 3) from small boat surveys within 0.5 km of each vessel when sea conditions were calm. No specific permissions were required for these samples, since they were taken in international waters and did not involve protected species. Seven debris items were sampled on SEAPLEX and 29 by SEA (5 during S242 and 24 on S243). Stations within 8.5 km of each other were combined for a total of 19 sampling locations within in the northeastern Pacific Ocean (Fig 1, Table 1). During SEAPLEX, the entire piece of debris with attached barnacles was preserved in 5% Formalin buffered with sodium borate. When the item was too large to be preserved (e.g., a fishing buoy), barnacles were removed and preserved separately. On SEA cruises, as many barnacles as possible to a maximum of 50 were removed from the debris object and preserved in 10% ethanol. Where feasible, a fragment of the item itself was also sampled. In the laboratory, capitulum length was measured using a ruler and species identification (L. anitifera or L. pacifica) determined for all intact individuals. Barnacles greater than approximately 0.8 cm in length were dissected and the contents of their stomach and intestinal tract examined under a dissecting microscope. Barnacles less than 0.8 cm were present, but not sampled in this study. Plastic particles found in the stomach or intestine were quantified, photographed digitally against a ruler for size assessment, rinsed with fresh water and stored in a glass vial for later analyses. The maximum diameter (feret diameter) and two-dimensional area of each particle were digitally measured with the software package NIH ImageJ (Rasband 2012). On the SEAPLEX cruise in 2009, we also measured the diameter and area of all plastic particles captured in surface-towed plankton nets (N=30,518) using NIH ImageJ-based tools in the Zooprocess software, and calibrated against manual measurements (Gilfillan et al. 2009, Gorsky et al. 2010, Goldstein et al. 2012b). We identified the type of plastic recovered from a randomly selected subset of barnacles (Barnacles N=42; particles N=219). A Raman spectrometer (PeakSeeker Pro-785 with AmScope 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 PeerJ reviewing PDF | (v2013:07:631:0:1:NEW 30 Jul 2013) R ev ie w in g M an us cr ip t operated at 10 \u2013 50 mW and 5 \u2013 20 second integration time; Raman Systems MII, Inc/Agiltron, Inc., Woburn, MA) and associated RSIQ software were used to identify plastic type. The Raman spectrum for each plastic piece was compared to a reference library of known plastic types for identification. Particles of clear, white, gray and pale-colored (light blues and greens, oranges and yellows) plastics yielded high quality Raman spectra and were readily identifiable. Those that were darker (medium to dark blues, reds and greens as well as black; 35% of particles subjected to Raman spectroscopy) were heated by the laser beam and melted even at the lowest possible power and integration time settings, resulting in no usable spectra. We also identified a subset of the debris items to which the barnacles were attached. Fragments of 18 objects were collected for analysis, but 6 could not be identified due to darker pigmentation due to melting under the laser. Statistics and figures were generated with the R statistical environment, version R-2.15.1 (R Development Core Team 2012) and QuantumGIS, version 1.8.0-Lisboa (QGIS Development Team 2013).",
    "url": "https://peerj.com/articles/185/reviews/",
    "review_1": "Tomas Perez-Acle \u00b7 Oct 2, 2013 \u00b7 Academic Editor\nACCEPT\nDear authors,\nThank you very much for your efforts to include the comments and solicitations of the reviewing panel. It is now our pleasure to publish your paper in accordance to the previous decision. Thanks a lot for considering PeerJ to be the showcase of this scientific contribution. Looking forward for your future contributions in this field.",
    "review_2": "Tomas Perez-Acle \u00b7 Aug 12, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nDear authors,\nDespite that I have made extensive efforts to find additional reviewers for your article entitled \u201cProtein signatures using electrostatic molecular surfaces in harmonic space\u201d, I haven't succeeded. In order to speed up the evaluation process of your article and considering my expertise in structure-function relationships in protein structure, I have acted as the second reviewer. As a whole, my overall decision is that your paper should be accepted for publication at PeerJ once all issues raised by the review process are properly addressed. Please find my review below (and you will find the Comments of Reviewer 1 below that).\n\nBasic Reporting:\nThe article entitled \u201cProtein signatures using electrostatic molecular surfaces in harmonic space\u201d from authors Carvalho, Vlachakis, Tsiliki, Vasileios, Megalooikonomou and Kossida, presents a novel approach to use the fourier spectrum computed from the electrostatic potential derived from protein surfaces as a comparative value among protein structures. According to the authors, this method reduces the 3D information available at the protein surface to 1D information where a specific distance metric in the harmonic space may be used to compare protein structures.\n\nExperimental design:\nBy relying on the Wiener-Khinchin theorem, the authors of this paper correlate different points of the electrostatic field computed from an APBS calculation by applying a Fourier transform of the power spectrum inferred over a regular grid over the protein surface. The authors evaluated their method by using a training set composed by 3 helicase proteins from HCV, including 2 crystal structures and 1 comparative model. They also included as decoys 1 HCV polymerase and 1 cAMP-dependant kinase.\n\nValidity of the findings:\nMinor concerns\n- Despite that the authors succeeded presenting a method to compare protein structures relying on 1D information, it is unclear how this method can be used to extract functional information from proteins. Moreover, one of the authors\u2019 claims is that this method allows \"a fast similarity search\". Though comparing 1D information is faster than 3D, the authors do not consider that their method requires a previous calculation of the electrostatic potential of every protein structure. Is the time needed to compute the electrostatic potential being considered as part of the method? On the other hand, in order to better evaluate the performance of their method, the authors should compare their results with well-established methods such as VASP and others.\n- Notoriously, the elegant approach by which the authors use the Fourier transform of the power spectrum computed on the electrostatic potential of protein surfaces, heavily contrast with the use of a simple \u201cdistance metric\u201d by which they compare the power spectrum of different proteins. The inclusion of a more robust method such as SVM or Kohonen maps to compare proteins, could improve their findings.\n- In order to simplify their calculations, the authors have assumed that electrostatic field is isotropic so that the power spectrum depends only on the distance between each pair of points. However, it is well known that in the surface of proteins is where the side chain of charged residues is actually located. At physiological pH, many residues can be charged forming dipoles that are sensitive to the charge flow. Therefore, It is unclear how this simplification can be used to account for changes in electrostatic potential due to flow of charges as in the case of MD simulations.\n\nMajor concerns:\n- Overall, the major concern behind this interesting work is the scarcity of the protein structure database that was used as test set. Despite that the inclusion of decoys is an elegant way to approximate to the real values of precision and recall, the authors should make a compelling effort to increase the size of both the training and the test set used to evaluate this study. They should also improve the statistics behind the evaluation of their method by taking a look at the actual predictive value (precision and recall).",
    "review_3": "Reviewer 1 \u00b7 Jul 22, 2013\nBasic reporting\nThe contribution report a new method of analysis of protein molecular surface based on Fourier analysis. The authors calculate a power spectrum of surfaces of the molecular electrostatic potential, to compare with other molecules in a structural database. This method allows a reduction of the three-dimensional information to one-dimensional information. They use a training set of the Hepatitis C viral proteins.\nThis approach shows a contribution to speed-up the comparison and classification of proteins. The method is mathematically correct and allows the calculation a power spectrum that can easily be comparing between different molecules.\nExperimental design\nThe training set is small to can show the real meaningful of the methods, in fact the author use only to structure, Helicase A and Helicase B, all the other structures were derivatives of one of them, so they present only small differences with the original, and in this case are not candidate to show the advantages of the methods. In fact, one of them present a power spectrum different to the non related protein (Polymerase) but the other present a power spectrum similar to the other non related protein ( kinase).\nValidity of the findings\nThe findings are based in a trainning set not robust, I will suggest use a bigger and robust training set, to show the real effect of the methods.\nAdditional comments\nThis a good contribution that need inclrese the training set and demeostrate tha capability of the powwr spectrum to differenciate non related protein.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Protein signatures using electrostatic molecular surfaces in harmonic space (v0.1)\". PeerJ https://doi.org/10.7287/peerj.185v0.1/reviews/1",
    "pdf_1": "https://peerj.com/articles/185v0.2/submission",
    "pdf_2": "https://peerj.com/articles/185v0.1/submission",
    "all_reviews": "Review 1: Tomas Perez-Acle \u00b7 Oct 2, 2013 \u00b7 Academic Editor\nACCEPT\nDear authors,\nThank you very much for your efforts to include the comments and solicitations of the reviewing panel. It is now our pleasure to publish your paper in accordance to the previous decision. Thanks a lot for considering PeerJ to be the showcase of this scientific contribution. Looking forward for your future contributions in this field.\nReview 2: Tomas Perez-Acle \u00b7 Aug 12, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nDear authors,\nDespite that I have made extensive efforts to find additional reviewers for your article entitled \u201cProtein signatures using electrostatic molecular surfaces in harmonic space\u201d, I haven't succeeded. In order to speed up the evaluation process of your article and considering my expertise in structure-function relationships in protein structure, I have acted as the second reviewer. As a whole, my overall decision is that your paper should be accepted for publication at PeerJ once all issues raised by the review process are properly addressed. Please find my review below (and you will find the Comments of Reviewer 1 below that).\n\nBasic Reporting:\nThe article entitled \u201cProtein signatures using electrostatic molecular surfaces in harmonic space\u201d from authors Carvalho, Vlachakis, Tsiliki, Vasileios, Megalooikonomou and Kossida, presents a novel approach to use the fourier spectrum computed from the electrostatic potential derived from protein surfaces as a comparative value among protein structures. According to the authors, this method reduces the 3D information available at the protein surface to 1D information where a specific distance metric in the harmonic space may be used to compare protein structures.\n\nExperimental design:\nBy relying on the Wiener-Khinchin theorem, the authors of this paper correlate different points of the electrostatic field computed from an APBS calculation by applying a Fourier transform of the power spectrum inferred over a regular grid over the protein surface. The authors evaluated their method by using a training set composed by 3 helicase proteins from HCV, including 2 crystal structures and 1 comparative model. They also included as decoys 1 HCV polymerase and 1 cAMP-dependant kinase.\n\nValidity of the findings:\nMinor concerns\n- Despite that the authors succeeded presenting a method to compare protein structures relying on 1D information, it is unclear how this method can be used to extract functional information from proteins. Moreover, one of the authors\u2019 claims is that this method allows \"a fast similarity search\". Though comparing 1D information is faster than 3D, the authors do not consider that their method requires a previous calculation of the electrostatic potential of every protein structure. Is the time needed to compute the electrostatic potential being considered as part of the method? On the other hand, in order to better evaluate the performance of their method, the authors should compare their results with well-established methods such as VASP and others.\n- Notoriously, the elegant approach by which the authors use the Fourier transform of the power spectrum computed on the electrostatic potential of protein surfaces, heavily contrast with the use of a simple \u201cdistance metric\u201d by which they compare the power spectrum of different proteins. The inclusion of a more robust method such as SVM or Kohonen maps to compare proteins, could improve their findings.\n- In order to simplify their calculations, the authors have assumed that electrostatic field is isotropic so that the power spectrum depends only on the distance between each pair of points. However, it is well known that in the surface of proteins is where the side chain of charged residues is actually located. At physiological pH, many residues can be charged forming dipoles that are sensitive to the charge flow. Therefore, It is unclear how this simplification can be used to account for changes in electrostatic potential due to flow of charges as in the case of MD simulations.\n\nMajor concerns:\n- Overall, the major concern behind this interesting work is the scarcity of the protein structure database that was used as test set. Despite that the inclusion of decoys is an elegant way to approximate to the real values of precision and recall, the authors should make a compelling effort to increase the size of both the training and the test set used to evaluate this study. They should also improve the statistics behind the evaluation of their method by taking a look at the actual predictive value (precision and recall).\nReview 3: Reviewer 1 \u00b7 Jul 22, 2013\nBasic reporting\nThe contribution report a new method of analysis of protein molecular surface based on Fourier analysis. The authors calculate a power spectrum of surfaces of the molecular electrostatic potential, to compare with other molecules in a structural database. This method allows a reduction of the three-dimensional information to one-dimensional information. They use a training set of the Hepatitis C viral proteins.\nThis approach shows a contribution to speed-up the comparison and classification of proteins. The method is mathematically correct and allows the calculation a power spectrum that can easily be comparing between different molecules.\nExperimental design\nThe training set is small to can show the real meaningful of the methods, in fact the author use only to structure, Helicase A and Helicase B, all the other structures were derivatives of one of them, so they present only small differences with the original, and in this case are not candidate to show the advantages of the methods. In fact, one of them present a power spectrum different to the non related protein (Polymerase) but the other present a power spectrum similar to the other non related protein ( kinase).\nValidity of the findings\nThe findings are based in a trainning set not robust, I will suggest use a bigger and robust training set, to show the real effect of the methods.\nAdditional comments\nThis a good contribution that need inclrese the training set and demeostrate tha capability of the powwr spectrum to differenciate non related protein.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Protein signatures using electrostatic molecular surfaces in harmonic space (v0.1)\". PeerJ https://doi.org/10.7287/peerj.185v0.1/reviews/1\nReview 4: nan\nReview 5: nan\nReview 6: \n",
    "LLM_extracted_review": "[\"Review 1: The paper is accepted for publication, and the authors are thanked for their efforts in addressing the comments from the reviewing panel.\\n\\nReview 2: \\n- Minor concerns include the unclear extraction of functional information from the method, the need to consider the time required for electrostatic potential calculations, and the suggestion to compare results with established methods like VASP. \\n- The use of a simple distance metric for comparing power spectra is criticized, with a recommendation to use more robust methods like SVM or Kohonen maps. \\n- The assumption of isotropy in the electrostatic field is questioned, particularly regarding its implications for charge flow in proteins. \\n- Major concerns highlight the small size of the protein structure database used for testing, suggesting the need for a larger training and test set and improved statistical evaluation of the method.\\n\\nReview 3: \\n- The method is mathematically correct and allows for the comparison of protein structures, but the training set is too small to demonstrate the method's effectiveness. \\n- The limited diversity in the training set, primarily consisting of derivatives of two helicase structures, raises concerns about the robustness of the findings. \\n- A larger and more diverse training set is recommended to better showcase the method's capabilities in differentiating unrelated proteins.\"]"
}