Environ Biol Fish (2011) 91:1–5 DOI 10.1007/s10641-011-9779-1 On our origins David L G Noakes Received: 13 January 2010 / Accepted: 22 February 2011 / Published online: 10 March 2011 # Springer Science+Business Media B.V 2011 We marked 2009 devoted to origins It was our way of acknowledging the coincidence of 2009 as the bicentennial of the birth of Charles Darwin, the sesquicentennial of his publication of The Origin of Species (Darwin 1859) - and the 170th anniversary of his marriage A remarkable number and variety of books and articles devoted to Darwin were published to recognize the year (e.g., Quammen 2008; Dawkins 2009; Ruse 2009; van Wyhe 2009) Springer Academic Publishers recognized it as The Year of Darwin (Fig 1) It is fitting to add our acknowledgement, given the importance of evolution to Environmental Biology of Fishes I invited Ian Potter to provide his article on the origin of Environmental Biology of Fishes (Potter 2010) Ian is in the unique position to provide that important information, as the only other remaining active member from the original Editorial Board of this journal (I was responsible for Notes and Reviews) Furthermore, he and Bill Beamish were the origin of the name of our journal! In this Editorial I will comment more specifically on the origin of Environmental Biology of Fishes and the development to the current state of our journal D L G Noakes (*) Fisheries & Wildlife Department, Oregon State University, Room 120 Nash Hall, Corvallis, OR 97331-3803, USA e-mail: David.Noakes@oregonstate.edu Origins, evolution and development are important to our journal, as they are to all of modern biology Perhaps more than some other journals, Environmental Biology of Fishes emphasizes an evolutionary perspective Our evolutionary emphasis shows not only in the articles we publish but also in the interests and activities of our Editorial Board Of course we rely heavily on our external reviewers for their expertise and advice, and our panel of external reviewers is critical for reviews of our manuscripts I will consider the Editorial Board and our published articles as the basis for my comments on the origin and development of Environmental Biology of Fishes I have taken the information from the public forum, in the tables of contents, the lists of Advisory Editors, the Acknowledgements for Reviewing published in every volume, and of course most importantly the articles we publish In addition, I have drawn upon the complete editorial files we maintain to provide critical details on the current status of the journal As Ian Potter described in his article (Potter 2010), our journal originated about 35 years ago, during a time of rapid growth for science and academia, at least in North America and Western Europe Many changes have taken place over the ensuing quarter century, including recent economic upheavals, dramatic changes in technology and marked shifts in our perspectives on the natural world Certainly the economic, social and other aspects of the historical context are important, but they are not the subjects of Fig The year of Darwin poster, Springer Academic Publishers my consideration in this editorial I am concerned with our science in our research articles and the affiliations of the authors and reviewers of those articles as indicators of the nature of our journal We have stated from Volume 1, Issue that Environmental Biology of Fishes is an international journal that publishes original studies on ecology, life history, epigenetics, behavior, physiology, morphology, systematics and evolution I will review how we have accomplished that over our first quarter century For my comparison I chose two issues of Environmental Biology of Fishes: Volume 1, Number (30 August 1976), the first issue, and Volume 85, Number (May 2009) from a recent complete volume of the journal Volume 1, Number had 14 articles (111 pages) and was the only issue in that year (Volume 1, Number was published 15 March 1977) Volume 85, Number had 15 articles (88 pages) so it appears that there might not be much difference in the overall “package” of the individual issues Of course we now publish 12 issues each year so there is an enormous quantitative difference in the annual output of the Environ Biol Fish (2011) 91:1–5 journal That comparison alone documents the very considerable growth of an order of magnitude in our journal in both the total content and the production rate of our journal So our growth has been very considerable, but what about the content? What have we demonstrated in our science, and how has that developed over time? I will focus my comparison on these two individual issues of the journal to judge the content and coverage, recognizing that the first issue represents the production for an entire year and the recent issue is the production for a single month A number of important differences emerge from this comparison, and they tell us a great deal about the development of our journal I begin with a consideration of the Editorial Board The original Editorial Board consisted of 12 individuals, including the Editor-in-Chief and 11 Advisory Editors (The list of Advisory Editors on the front cover of the first issue does not agree with the list on page 2, but the number of Advisory Editors was stated as 11 in the Preface) Those individuals were affiliated with academic or other scientific institutions in six countries: Australia, Canada, the USA, England, Israel and Germany All but one of the original Editorial Board were males, the single exception (and an exceptional individual she remains) was Eugenie Clark The Editorial Board in 2009 consisted of 21 members, including the Editor-in-Chief and 20 Advisory Editors, 17 male and four female The 2009 Advisory Editors were affiliated with academic or other scientific institutions in eight countries: the USA, Canada, France, Argentina, Mexico, Japan, Australia and China The number of members, the composition, and the international representation of the Editorial Board have all changed significantly since the founding of the journal in what must be interpreted as progressive ways Volume 1, Number consisted of a Preface, an Invited Editorial, two Main research articles, three Notes, three Essays, three Book Reviews and Translation Proposals by authors from Canada, Poland and the USA The two Main articles dealt with a study estimating the fish production of a freshwater lake in Canada, and the physiology of skipjack tuna (Katsuwonus pelamis) The three shorter notes dealt with teleost embryology, fish production, and technical aspects of rearing fish larvae Volume 85, Number included an Editorial, and 14 articles that dealt with threatened fishes of the Environ Biol Fish (2011) 91:1–5 world, telemetry and behavior, field studies of spawning behavior, seasonal reproductive physiology, retinal structure and function, behavior, salinity tolerance, exotic and invasive species, impacts of habitat change on fish condition, and conservation of deep-sea fishes Authors of the articles were from Canada, the USA, China, Uganda, Spain, Croatia, Costa Rica, Japan, Venezuela, New Zealand, Bolivia, and Germany The articles published in Volume 1, Number were reviewed by 11 reviewers, 10 from Canada and from Australia For my comparison to the recent status of our journal I have chosen the period from May to 31 August 2009 as representative and comparable to Volume 1, Number (although again this comparison is year for the first issue and only months for the recent issue) During that period in 2009 we received 126 manuscripts from authors with affiliations in 13 countries (we had multiple authors from China for several manuscripts in the special issue on Chinese fishes), and we solicited reviews from external reviewers in 31 countries (Table 1) I repeat my earlier gratitude to this multitude of external reviewers who contribute so much to the journal As we saw with the published articles and the Editorial Board, the coverage of our journal has clearly continued to grow not only in volume but also in scientific breadth and international coverage As I have noted in previous Editorials, a number of independent metrics, including impact factor, also demonstrate this significant growth in the coverage and content of our journal Environmental Biology of Fishes continues its growth and development as a truly international journal The special issue dedicated to Chinese Fishes (Volume 86, Number 1) is a recent example of our increasing coverage and impact I have given details of the continued increases in the number and breadth of manuscripts handled, and the impact factor for the journal in previous Editorials (Noakes 2003, 2008) Development of the journal will continue, with special issues forthcoming on otoliths, threatened fishes and fish conservation, elasmobranch feeding behavior, and salmon biology, in addition to the latest research in contributed manuscripts I will continue this series of Editorials to document those developments I conclude this Editorial with some more personal comments on growth, development and the year 2009 I presented the invited keynote address at the International Charr Symposium in Sterling, Scotland Table Summary of authors and reviewers of manuscripts received during the period of May until 31 August 2009, according to their current affiliation addresses (in addition to the Advisory Editors who also review manuscripts, and whose affiliations are provided in the text) Author affiliation Reviewer affiliation Argentina Brazil Australia Canada Austria China (multiple) Brazil Croatia Canada Germany China (multiple) Hong Kong Czech Republic Japan Finland Mexico France South Africa Germany Spain Greece Thailand Iceland USA India Venezuela Italy Japan Mexico Mongolia Nepal Portugal Scotland South Africa Spain Sweden Taiwan Turkey United Kingdom Uruguay USA Venezuela in June 2009, where the focus was on Darwin (“Charles and Charr: 200 Years On”) During that address I emphasized the continuity and the connections we all have through our science to Darwin, his ideas and his influence It is a relatively simple exercise to trace our individual scientific genealogies through our undergraduate and graduate advisors, as well as our intellectual contacts For some of us our scientific heritage will lead, more or less directly, to Darwin I hasten to add the obvious point, that Environ Biol Fish (2011) 91:1–5 Fig The Fitzwilliam Museum in Cambridge, England with the special Darwin exhibition for 2009 (photograph David L G Noakes) Darwin did not personally supervise any undergraduate or graduate students so our connections cannot literally lead directly to him Nonetheless, ideas are as important to our scientific heritage as are the people, perhaps more so in many cases Those ideas will leave trails as clear as the signatures of our supervisors on academic transcripts Fortunately, we have historians and philosophers who seek out and document these connections and academic tracks and trails in science for us (Ruse 2009) After my presentation at Stirling University, I revisited Edinburgh University—the site of my first academic position and coincidentally the location of Darwin’s first attempt at a university education Darwin withdrew from the unsatisfactory experience of his intended medical education at Edinburgh, but his biological education clearly benefited from his contacts there with Professor Robert Grant, his studies of lumpfish, Cyclopterus lumpus, and other marine creatures, and perhaps most significantly from his training in the preparation of study specimens by John Edmonston, a former slave who had experience in tropical expeditions (Berra 2009) From Edinburgh I traveled on to Cambridge, where Darwin had completed his formal university education, and where I toured a number of the special Darwin exhibitions staged for the year (Fig 2) Finally, a visit to Oxford where I had spent a sabbatical year myself, and where the famous confrontation took place between Samuel Wilberforce and Thomas H Huxley, the great defender of Darwin after publication of The Origin of Species A personal scientific pilgrimage, for sure, although I had visited all those places, and many of the other Darwin sites, Shrewsbury, Down House, Westminster Abbey and the Natural History Museum many times before That is a pilgrimage every biologist should take Fig Annie Proctor Richardson, at 92 years of age, seated in a chair now in my possession (photograph courtesy of Jean Noakes Park) Environ Biol Fish (2011) 91:1–5 My final point is poignant and most personal (Fig 3) The lady in the photo is Annie Proctor Richardson, my paternal great-grandmother She was born in 1865, the year Abraham Lincoln died Abraham Lincoln was born 12 April 1809, the same day and the same year as Charles Darwin, as every biologist should know Annie was 17 years old when Charles Darwin died (1882) I was 17 years old the year Annie died (1959) Of course I had known her well—she was the matriarch of the family, a tiny little lady dressed in black most of the time How sad, I now realize, that I never talked to her about anything of substance Of course there is absolutely no reason to believe that she had any experience or any opinion related to Charles Darwin They certainly had nothing in common in terms of social or economic status Charles and Emma Darwin had as many as 10 servants and staff in Down House Annie Richardson worked “in service” as a young girl in England, that is she was a servant in the house of one of the wealthy families where she lived near Kendal, England She lived the difficult life of a working class family with little formal education and few opportunities for any social or economic advancement No doubt that grinding poverty and those dreary prospects influenced her decision to immigrate to Canada with her family early in the 20th century Her family included the daughter (also named Annie) who would become my grandmother and who lived to visit me as a graduate student at the University of California at Berkeley and later read my postcards from Edinburgh and Oxford This pilgrimage was thus also intensely personal Acknowledgements Suzanne Mekking, Lynn Bouvier and Martine van Bezooijen provided advice, encouragement, data summaries and constructive comments for this manuscript Bill Beamish, Hiroya Kawanabe and Michael Ruse provided the critical comments and insightful suggestions I needed from trusted colleagues Jeff Noakes commented on the historical aspects of the manuscript and Pat Noakes corrected my recollections Jean Noakes Park provided the photo of Annie Proctor Richardson and reviewed the manuscript for consistency I thank Springer Academic Publishers for their continued support for Environmental Biology of Fishes, and particularly for their recognition of The Year of Darwin The Oregon Department of Fish and Wildlife and the Fisheries and Wildlife Department of Oregon State University provide my current academic home and continuing support References Berra TM (2009) Charles Darwin The concise story of an extraordinary man The Johns Hopkins University Press, New York, p 114 Darwin CR (1859) On the origin of species by natural selection John Murray, London, p 502 Dawkins R (2009) The greatest show on earth The evidence for evolution Free Press, New York, 470 pp Noakes DLG (2003) Changes and continuity Environ Biol Fish 66:1–2 Noakes DLG (2008) Growth and development Environ Biol Fish 85:1–2 Potter IC (2010) On the origin: environmental biology of fishes Environ Biol Fish 87:275–276 Quammen D (2008) Charles Darwin on the origin of species The illustrated edition Sterling, New York, p 544 Ruse M (2009) Philosophy after Darwin: classic and contemporary readings Princeton University Press, Princeton, p 592 van Wyhe J (2009) Darwin in Cambridge Christ’s College, Cambridge, p 75 Environ Biol Fish (2011) 91:7–13 DOI 10.1007/s10641-010-9753-3 Sexual dimorphism of drumming muscles in European hake (Merluccius merluccius) Anne-Laure Groison & Olav S Kjesbu & Marc Suquet Received: December 2009 / Accepted: 11 November 2010 / Published online: December 2010 # The Author(s) 2010 This article is published with open access at Springerlink.com Abstract Dissections of mature and non-mature European hake males and females (N=142) collected in waters off the western coast of Norway and in the Bay of Biscay (France) in 2004–2006 demonstrate for the first time that this gadoid species contains drumming muscles There were differences in drumming muscles weight with body length, sex and maturity stage This study shows that the difference between females and males is primarily manifested during the spawning season, seen both in the French and Norwegian samples For the mature females the drumming muscles dry weight increases only slightly, if at all, with increase in total length For mature males there is a corresponding rapid increase There does not seem to be any consistent difference between the average dry weight of the drumming muscles in immature male and immature and mature female hake of the same length, tested on the Norwegian samples A.-L Groison (*) Department of Biology, University of Bergen, P.O Box 7803, Thormøhlensgate 55, 5020 Bergen, Norway e-mail: Anne-Laure.Groison@imr.no A.-L Groison : O S Kjesbu Institute of Marine Research, Nordnesgaten 50, P.O Box 1870, Nordnes 5817 Bergen, Norway M Suquet IFREMER, PFOM / ARN, 29840 Argenton, France Our results suggest that male hake, like the males of other gadoids studied, may produce sounds in the context of spawning Keywords Drumming muscles Merluccius merluccius Sexual dimorphism Sound production Spawning Introduction More than 800 fishes from 109 families are known to produce sounds, though this is likely to be an underestimate (Rountree et al 2003) It is evident that most of these sounds are deliberate rather than incidental These sounds have a role in communication, i.e., are used as exchange of information between individual fish as part of their social behaviour (Hawkins and Myrberg 1983) Thus, fish produce sounds in a variety of contexts Sounds are produced by some species when disturbed or when approached by a predator Likewise, sounds are also produced by fish which are competing with one another for food or space (Ladich and Myrberg 2006) In many sound-producing fish males produce sounds during courtship of the female to advertise their nest sites, to attract the female, and promote courtship and spawning (Myrberg and Lugli 2006) The gas-filled swimbladder is a characteristic feature of the viscera of teleost fish It contributes to the ability of a fish to control its buoyancy, and thus to stay at the current water depth without having to waste energy in depthcompensating swimming activity Another function of the gas bladder is the use as a resonating chamber to produce or receive sound and in some species is equipped with drumming muscles (DM) for sound production Sounds are produced by contracting DM associated with the swimbladder and thereby vibrating the swimbladder wall (Jones and Marshall 1953; Brawn 1961) It is known that one particular family, the Gadidae, includes a number of vocal species (Hawkins and Rasmussen 1978), including haddock (Melanogrammus aeglefinus) (Hawkins and Chapman 1966), lythe (Pollachius virens) (Hawkins and Rasmussen 1978), tadpole fish (Raniceps raninus) (Hawkins and Rasmussen 1978) and Atlantic cod (Gadus morhua) (Brawn 1961) In their work on cod Nordeide et al (2008) found that the DM mass was similar in both sexes a couple of months prior to spawning but became sexually dimorphic at the onset of spawning and continued being sexually dimorphic (bigger in males) for several months after the termination of spawning To date, no studies have investigated whether European hake (Merluccius merluccius) possess drumming muscles This is somewhat surprising in view of the importance of this species, and the fact that the presence of drumming muscles have been reported in other gadoid species European hake is a semi-demersal, multiple batch spawner found in waters from Mauritania to Norway It is believed that hake spawning and reproduction occur at depth ranging 100–200 m (Alvarez et al 2001; Olivar et al 2003) The peak spawning time of hake is in March in waters south of the Bay of Biscay (France), and occurs progressively later at higher latitudes (Casey and Pereiro 1995) In the present study we sampled wild hake males and females from French (Bay of Biscay) and Norwegian waters at different times of the year to search for the presence of drumming muscles and, if so, to quantify variation among individuals in drumming muscles size Specifically, our objectives were to (a) record differences in drumming muscle appearance and mass in relation to sex, spawning status, and body size; and (b) compare hake drumming muscles with what has been observed in other gadoids Environ Biol Fish (2011) 91:7–13 Material and methods Fish collection A total of 142 wild European hake were sampled offshore Western Norway (Nw) (61°34′N, 5° 56′W) and in the Bay of Biscay, France (Fr) (47°44′N, 4°2′W) (Table 1) Each fishing trip typically lasted 1–3 days Non-mature fish were captured by trawl in Nw waters while mature fish were captured at both locations by gillnets set overnight at depths of between 30–180 m over sandy sea bottom Recently dead fish (few hours) were retrieved from the gillnets Fish dissection The fish were transported to laboratory to be dissected within 12–32 h of sampling, and all showed muscles attached to the swimbladder on both sides For each individual examined we recorded total body length (N= 140; TL to the nearest 0.1 cm was measured for all fish except for one individual with damaged tail), total (i.e., ungutted) body mass (N=138; TW) and gonad mass (N=61) (to the nearest 0.1 g consulting only gonads which were not smashed or deteriorated by stripping) Sex and maturity stage (immature, ripening, ripe/ spawning, and spent) were recorded Only two groups of individuals were considered: “spawning” (sp.) for ripening or ripe/spawning individuals (N=69) and “non-spawning” (n sp.) for immature or spent individuals (N=73) The pair of DM was easily separated from the surrounding tissue using forceps After excision, DM were dried at 65°C for days to obtain dry weight to the nearest 0.001 mg (N=141) The following fish characteristics were calculated: h i condition factor (K ¼ Total weight=ðTLÞ3  100, N = 138); gonadosomatic index (GSI ¼ ½Gonad weight=TWŠ  100 in %, N=61), and hepatosomatic index (HSI ¼ ½Liver weight=TWŠ  100 in%, N=71) Statistical analysis Data were presented as means ± SD Measured and calculated characteristics of the dissected fish were combined (Table 2) Statistical analyses were performed using the software SigmaStat 3.1 Statistical significant difference between two groups were tested at the probability level 0.05 using Student t-test (when data were distributed normally and variances were not Environ Biol Fish (2011) 91:7–13 Table Summary of spawning and nonspawning European hake captured in the Bay of Biscay (France, Fr) and in waters western Norway (Nw) at different dates in 2004–2005–2006 Spawning fish, N= Non spawning fish, N= Date Origin Females Males Females Males 20 March 2006 Fr 10 0 04 April 2006 Fr 14 0 17 August 2005 Nw 22 August 2006 Nw 1 0 23 August 2004 Nw 0 23 August 2005 Nw 1 01 September 2005 Nw 12 September 2006 Nw 0 27 September 2004 Nw 0 10 13 12 October 2004 Nw 0 14 13 13 October 2006 Nw 0 2 30 November 2004 Nw 0 15Nw+15Fr 15Nw+24Fr 36Nw 37Nw Total significantly different) or Mann-Whitney Rank Sum test (if one of these two previous conditions, or both, were invalidated) As the fish dissection resulted in an uneven number of left (N=141) and right DM (N= 120) a pilot analysis was run to test for any differences in dry weight between them, which turned out not to be the case (Mann-Whitney rank sum test, P>0.05) Therefore, in the following analysis dry weight of the left DM were used and named DM ANCOVA with total length as covariate was used to test for differences in drumming muscle mass in relation to sex and spawning status (i.e “spawning” (sp.) for ripening or ripe/spawning individuals and “non-spawning” (n sp.) for immature or spent individuals) Relationships between DM dry weight and characteristics of sp and n sp individuals (TL, TW, K, GSI and HSI) were investigated with Pearson correlations Correlations were investigated separately for Norwegian and French fish and for males and females Table Mean ± SD values of fish characteristics measured on spawning and non-spawning hake for male and female (Fem.) captured in the Bay of Biscay (France, Fr) and western Norway waters (Nw): total length (TL), total weight (TW), dry weight of the left drumming muscles (DM), gonadosomatic index (GSI), condition factor (K) and hepatosomatic index (HSI) Maturation state Origin Non-spawning Nw Spawning Nw Fr Sex N TL (cm) Results Fish and observations of drumming muscles Nw-spawning individuals for both sexes showed significant higher GSIs compared to Fr-spawning individuals (Table 2) The present study showed the presence of DM in hake: a pair of muscular structures is located at the anterior end of the swimbladder, close to its ventral wall (Nw female: Fig 1a; Nw male: Fig 1b) Note that the DM are rounded at the posterior end but slightly pointed anteriorly and considerably larger in TW (g) DM (mg) GSI (%) K HSI (%) Fem 37 28.7±15.2 327.2±815.1 3.12±5.41 1.56±2.89 0.57±0.06 2.60±1.58 Male 37 27.1±11.6 143.7±166.1 10.15±23.74 0.17±0.05 0.56±0.05 1.58±0.03 Fem 15 75.5±7.5 3085.3±1273.1 23.27±8.96 9.38±4.25 0.69±0.08 4.98±5.20 Male 15 69.1±8.7 2477.1±852.2 225.7±123.6 4.12±2.78 0.69±0.06 2.71±1.06 Fem 15 64.7±13.0 2120.1±1423.1 18.38±9.41 5.10±2.03 0.71±0.06 3.33±0.83 Male 24 45.7±13.4 784.3±690.3 98.75±78.29 1.44±0.88 0.63±0.07 2.20±1.07 10 Fig Dissection of spawning hake caught on 18 August 2005 in waters off Western Norway Total length (TL), total weight (TW) and the dry weight of the left drumming muscles (DM) are indicated (a) Dissection of a spawning female TL=83 cm, TW=4 270 g and dry weight of left drumming muscles=22 mg (b) Dissection of a spawning male TL=80 cm, TW=3 440 g and dry weight of left drumming muscles=266 mg Environ Biol Fish (2011) 91:7–13 Environ Biol Fish (2011) 91:103–115 Sharp GD, Pirages S (1978) The distribution of red and white swimming muscles, their biochemistry, and the biochemical phylogeny of selected scombrid 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Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice Nucleic Acids Res 22:4673–4680 Tseng MC, Jean CT, Tsai WL, Chen NC (2009) Distinguishing between two sympatric Acanthopagrus species from Dapeng Bay, Taiwan, using morphometric and genetic characters J Fish Biol 74:357–376 Ward RD, Elliott NG, Grewe PM (1995) Allozyme and mitochondrial DNA separation of Pacific northern bluefin tuna, Thunnus thynnus orientalis (Temminck and Schlegel), from Southern bluefin tuna, Thunnus maccoyii (Castelnau) Mar Freshw Res 46:921–930 Ward RD, Elliott NG, Innes BH, Smolenski AJ, Grewe PM (1997) Global population structure of yellowfin tuna, Thunnus albacares, inferred from allozyme and mitochondrial DNA variation Fish Bull 95:566–575 Wu GCC, Chiang HC, Chen KS, Hsu CC, Yang HY (2009) Population structure of albacore (Thunnus alalunga) in the northwestern Pacific Ocean inferred from mitochondrial DNA Fish Res 95:125–131 Zhao L, Zhang J, Liu Z, Funk SM, Wei F, Xu M, Li M (2008) Complex population genetic and demographic history of the salangid, Neosalanx taihuensis, based on cytochrome b sequences BMC Evol Biol 8:201 doi:10.1186/1471-21488-201 Environ Biol Fish (2011) 91:119–120 DOI 10.1007/s10641-011-9778-2 Threatened fishes of the world: Puntius tumba Herre, 1924 (Cyprinidae) Gladys Boransing Ismail & Pedro T Escudero Received: 14 February 2009 / Accepted: 22 February 2011 / Published online: 12 March 2011 # Springer Science+Business Media B.V 2011 Keywords Threatened fish Endemic species Puntius tumba Common name: Tumba Local name: Tumba (Maranao) Conservation status: Vulnerable A2cde— IUCN Red List (1996) Identification: D 10; A 9; P 17; V 10 Maximum size 115 mm SL 30 g Body elongate; silver coloration with yellow tinge along the lateral line and gill cover, dorsal and pectoral fins pale orange, ventral and anal fins orange, caudal fin yellow orange Four barbels, two on maxillary and two on mandible Specimens may have one to three black lateral dots, the one closest to the caudal peduncle most visible Scales cycloid Illustration from Escudero et al (1980) Distribution: Endemic to Lake Lanao and Lanao Plateau, Philippines (Herre 1953) 7° 50′ 0″, 124° 20′ 0″ Abundance: Consistently less than 0.2% of the fish in the market in Lake Lanao and its outflow (Escudero et al 1980, Escudero and Demoral 1983, Escudero 1994, G B Ismail (*) Department of Fisheries and Wildlife, Oregon State University, Nash Hall, Corvallis, OR 97331–4501, USA e-mail: gladys_ismail@yahoo.com P T Escudero College of Fisheries, Mindanao State University, Marawi City 9700 Lanao del Sur, Philippines Ismail unpublished) Habitat and ecology: Very low in abundance based on the market surveys (Escudero et al 1980, Escudero and Demoral 1983, Escudero 1994; Ismail unpubl data) In 2008 only caught from Agus River, the only outlet of Lake Lanao, and none from the lake itself Reproduction: No published information Threats: Introduction of nonnative species like Hypseleotris agilis, and Tilapia mossambica (Kornfield and Carpenter 1984), overexploitation due to its high price in the market and the hydroelectric dams along the Agus River Dams may have altered the habitat and may have caused the extinction of the endemic Mandibularca resinus that previously thrived in the river Conservation actions: Successfully cultured at the College of Fisheries, Mindanao State University, Marawi Conservation recommendation: Research on life history, biology and evolution One of the only two remaining endemic species left in Lake Lanao Create and implement law to protect Lake Lanao and its endemic species through the Bureau of Fisheries and Aquatic Resources 120 References Escudero PT (1994) Lake Lanao fisheries: problems and recommendations Philipp Biota 27(1):8–18 Escudero PT, Demoral MA (1983) Preliminary studies on the biology and fishery of Hypseleotris agilis Herre (Eleotridae) J Fish Aquac 4(1–2):3–89 Escudero PT, Gripaldo OM, Sahay NM (1980) Biological studies of the Glossogobius giurus (Hamilton & Buchanan) and the Environ Biol Fish (2011) 91:119–120 Puntius sirang (Herre) in Lake Lanao J Fish Aquac 1(1):1– 154 Herre AW (1953) Checklist of Philippine fishes United States Government Printing Office, Washington D.C., pp 125– 126 Kornfield I, Carpenter KE (1984) Cyprinids of Lake Lanao, Philippines: taxonomic validity, evolutionary rates and speciation scenarios In: Echelle AE, Kornfield I (eds) Evolution of fish species flocks University of Maine Press, Orono, pp 69–84 Environ Biol Fish (2011) 91:121–126 DOI 10.1007/s10641-010-9756-0 Hearing sensitivity in two black bass species using the auditory brainstem response approach Daniel E Holt & Carol E Johnston Received: 12 March 2010 / Accepted: 23 November 2010 / Published online: 16 December 2010 # Springer Science+Business Media B.V 2010 Abstract Recently, several bioacoustic studies have focused on the red eye bass (Micropterus coosae) One of these studies documented sound production, while the other played back sounds produced by prey items in order to determine their attractiveness to M coosae Surprisingly, the hearing ability of fishes in the genus Micropterus has received very little attention The need for audiograms describing hearing in Micropterus is apparent This study utilized the auditory brainstem response (ABR) approach to determine hearing sensitivity in terms of both sound pressure level (SPL) and particle acceleration in two black bass species, the red eye bass (M coosae) and the Alabama bass (M henshalli) Audiograms produced in this study expressed in both SPL and particle acceleration showed a positive relationship between hearing threshold and frequency Micropterus coosae was most sensitive to frequencies that overlap with the peak frequencies of their vocalizations, and the vocalizations of a prey species, Cyprinella trichroistia Bass hearing sensitivities at lower frequencies, measured in terms of particle acceleration, were similar to several sciaenid species D E Holt (*) : C E Johnston Department of Fisheries and Allied Aquaculture, Auburn University, 203 Swingle Hall, Auburn, AL 36849, USA e-mail: holtdan@auburn.edu Keywords Acceleration Bass Centrarchidae Pressure Sound Introduction Recently, studies investigating sound production and sound interception (Myrberg 1981) have been conducted on the red eye bass, Micropterus coosae The first of these studies attempted to determine whether M coosae intercepted the spawning vocalizations of a common prey item, the tricolor shiner (Cyprinella trichroistia; Holt and Johnston 2009) The results of this study showed that M coosae was not attracted to the sounds of C trichroistia One limitation of this study was that the hearing sensitivity of M coosae was unknown, and was assumed to be similar to those of other closely related species The second study documented and described sound production for the first time in M coosae (Johnston et al 2008) Sounds were produced by male M coosea during aggressive interactions, suggesting a role of acoustics for intraspecific communication The discovery of sound production in M coosae opens the door for many more studies on the functional significance of sounds in this genus The need for audiograms describing the hearing capabilities in M coosae is obvious Without knowledge of the hearing range and sensitivity of any organism, one can only speculate as to whether sounds produced by the organism can be detected 122 by conspecifics, or what environmental sounds (biotic or abiotic) may be of importance to the organism The auditory brainstem response (ABR) was first applied to fishes by Corwin et al (1982), and has become a common electrophysiological method for determining hearing range and thresholds in fishes Bass lack a peripheral adaptation (such as the Weberian apparatus) to transmit vibrations from the swim bladder to the inner ear, and are therefore thought to be less sensitive to the pressure component of sound than fishes with such a specialization This is not to say that they are completely insensitive to pressure Previous categorization of fishes into two broad groups (specialists and generalists) based on hearing and physiology has recently come under scrutiny by Popper and Fay (2010), who propose a continuum of pressure detection in fishes Based on the fact that they have a swim bladder, but not have any type of connection between it and the ear, M coosae and M henshalli would fall somewhere between the middle and motion only end of the continuum For this reason, it is important to attempt to characterize hearing in terms of both particle motion and pressure This study utilizes ABR to determine the hearing abilities of two black bass species, M coosae and M henshalli Because these species can likely detect both pressure and particle motion, audiograms expressed in both pressure and particle acceleration are presented Constructing audiograms for M coosae allows us to compare the hearing ability of M coosae to the spectral components of their signals, and provides an important step for determining what other environmental sounds may be important to M coosae Hearing was also tested in M henshalli as a comparison within the genus using the same ABR methods Data from M salmoides (Yan, unpublished data, pers comm.) is also included, and was gathered using a different ABR method Methods Micropterus henshalli were collected from Uphapee Creek, Alabama (32.444610˚N, −85.648055˚W) on November 3, 2008 Micropterus coosae were collected from Saugahatchee Creek, Alabama (32.619306˚N, −85.633655˚W) on 27 February 2009 All subjects included in this study (5 M coosae and M henshalli) were tested between March and 12 March 2009 Environ Biol Fish (2011) 91:121–126 Hearing thresholds were determined using the ABR technique The test tank was a 79 cm section of PVC pipe capped on both ends with a 16.5×52 cm access hole cut in the top Water was filled to a depth of 23 cm, and an underwater speaker (University Sound UW-30, Oklahoma City, OK) was suspended 10 cm from the waters surface and 39 cm from the left end of the tank according to the perspective of Fig (both measurements taken from the center of the speakers’ forward facing surface) Molding clay was wrapped around test subjects (up to the pectoral fins) for restraint, and a thin strip of clay was placed over the head, just anterior of the eyes to reduce head movement The molding clay was placed on an iron ring stand, and suspended in front of the speaker so that the fish’s head was 7.5 cm from the center of the speakers’ forward facing surface, and 11 cm under the surface of the water Methods for the presentation of stimuli and determination of thresholds followed very closely those of Wright et al (2005), and so they will be mentioned only briefly here A recording electrode (Rochester Electro-Medical, Inc., Tampa, FL) was placed directly above the brainstem, a reference electrode was placed anterior to the eyes near the nostrils, and a ground electrode was placed in the clay surrounding the fish Tone generation and presentation were performed using software and hardware from Tucker-Davis Technologies (TDT, Gainesville, FL) Tone bursts were 10 ms in duration with ms rise-fall times and gated using a Hanning window Frequencies tested included 100, 200, 300, 400, 500, 600, 700, 800, 1,000, and 2,000 Hz for M coosae, and 100, 200, 400, 600, 800, 1,000, and 2,000 Hz for M henshalli Stimulus intensities were calibrated by measuring the voltage with a Brüel and Kjaer type 8103 hydrophone (sensitivity 27.1 μV/Pa), Brüel and Kjaer type 2635 charge amplifier, and a GW GOS6xxG dual trace Oscilloscope The hydrophone was placed at the location of the fish’s ears prior to the fish being placed in the test tank Voltage of each tone was converted into dB re μPa, and corrections were made so that the level played during the trials was accurate During trials, intensities at each frequency were increased in dB increments until evoked potentials had been obtained 15 dB above threshold (Fig 2a, b) In order to cancel stimulus artifacts, traces from tones played at opposite phases (90˚ and 270˚) were averaged Thresholds for each frequency Environ Biol Fish (2011) 91:121–126 123 Fig ABR tank setup Underwater speaker was suspended 7.5 cm from fish’s head Cutout of tank side in this figure is for illustrative purposes only Figure produced using Google Sketchup 7.0 were defined as the dB level at which the evoked potential spike was no longer visible above background noise in the stereotypical ABR response This method of threshold determination has been used in numerous ABR studies (Higgs et al 2001), and has been shown to be comparable to more quantitative methods (Mann et al 2001) Calibration in the absence of the test subject may have slightly affected the sound field, but it has been performed by Casper and Mann (2006) To verify that stimulus tones were not being altered by the holding apparatus (ring stand and clay), after the experiments Fig Evoked potential traces at threshold, dB below threshold, and 15 dB above threshold to a 100 Hz tone burst and b 400 Hz from M coosae Figure modified using Microsoft Paint we calibrated the tones with the hydrophone directly next to a fish’s head in the holding apparatus We then removed the holding apparatus containing the fish without altering the hydrophone position and replayed the calibrated tones At all frequencies, dB levels between the two conditions differed by less than dB Background noise levels in the test tank were determined at the fish’s location using the Brüel and Kjaer hydrophone with the holding apparatus in place and the speaker powered, but not playing A power spectrum was generated from a s selection of the background noise and was converted to spectrum level by subtracting 10log (analysis bandwidth; Fig 3a) Appropriate calibration coefficients were applied to the spectrum levels using the gain from all instrumentation to achieve dB re μPa The magnitude of particle acceleration associated with the tones played during ABR trials was calculated for each dB level used Acceleration was calculated from the pressure gradient measured between two hydrophones Two Hi-Tech HTI-96MIN hydrophones (sensitivity −164.4 re V/μPa) were fixed cm apart For each frequency used in the hearing tests, pure tones were played between 105 and 150 dB (at dB increments) and recorded simultaneously by both hydrophones The midpoint between the acoustical centers of each hydrophone was placed at the location of the fish’s head (7.5 cm from the speaker) The waveforms were then subtracted, and the RMS voltage was calculated from the resulting waveform RMS voltage was then converted into Pascals (Pa), and then into a pressure gradient (Pa/m) by dividing by the distance between hydrophones Acceleration (m·s−2) was calculated by dividing the pressure gradient by the density of fresh 124 Environ Biol Fish (2011) 91:121–126 underwater speaker, particle accelerations of tones below 95 dB were unable to be calculated Particle accelerations of tones between 80 and 100 dB were therefore extrapolated using data from 105 to 150 dB Results Fig Audiograms of M coosae (solid line with solid diamonds), M henshalli (broken line with hollow circles), and M salmoides (solid line with hollow triangles; data from Yan, unpublished data, personal communication) for a mean SPL in dB re: μPa, and b mean acceleration (M salmoides absent) Average hearing threshold is given at each frequency for five individuals of each species Exception is for M coosae at 2,000 Hz, at which a response was obtained from only one individual a Baseline noise is provided as a power spectrum (solid line with no markers) Baseline noise does not apply to M salmoides Graphs generated using Microsoft Excel and modified using Microsoft Paint water (997.8 kg·m−3) The equation for the above calculation is shown in Eq 1: sffiffiffiffiffiffiffiffiffiffiffiffi ! ! n X x2i  S Ä d Ä r ð1Þ Evoked potential traces at 100 and 200 Hz appeared different than traces at higher frequencies Latencies and duration were typically longer for evoked potentials at 100 and 200 Hz than at higher frequencies Evoked potentials at 100 and 200 Hz also showed multiple spikes within a larger dip and rise, while higher frequencies showed a single spike (Fig 2a, b) Both the pressure and particle acceleration audiograms of M coosae show that sensitivity decreases in a fairly linear fashion as frequency increases (Fig 3a, b) A similar pattern is seen in M henshalli In both M coosae and M henshalli, greatest hearing sensitivity occurs at 100 Hz with a threshold of 86 dB (4.0e−4 m s2) for M coosae, and a threshold of 90 dB (6.6e−4 m s2) for M henshalli Thresholds were clearly detectable for all five individuals of M coosae at all frequencies except 2,000 Hz, at which a threshold was obtained for only one individual Thresholds could not be obtained for frequencies above 600 Hz in M henshalli A notable increase in sensitivity was seen in M coosae for both SPL and particle acceleration below 400 Hz Background noise was generally around 55–60 dB (>20 dB lower than the lowest hearing threshold) A number of spikes in background noise were found at 60, 120, 180, 300, 420, 540, and 660 Hz Discussion i¼1 where xi is a sample point resulting from the subtraction of the two original time waveforms, S is the sensitivity of the hydrophone, d is the distance between hydrophones, and ρ is the density of fresh water This was done in three orthogonal axes, and the magnitude of particle acceleration was calculated using Eq 2: pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð x2 þ y2 þ z Þ ð2Þ where x, y, and z are the particle accelerations in each orthogonal axis Due to electrical noise from the Hearing thresholds determined by ABR should not be interpreted as absolute due to the inability of ABR to measure all levels of sound processing in the brain Hearing thresholds determined by ABR are typically higher than thresholds determined by more classical methods However, ABR does provide hearing sensitivity relative to other frequencies, and has been shown, in some cases, to provide thresholds similar to those obtained through more classical methods (Kenyon et al 1998; Kojima et al 2005) Audiograms in both SPL and particle acceleration show that hearing sensitivity Environ Biol Fish (2011) 91:121–126 in M henshalli and M coosae is greatest at low frequencies, particularly below 300 Hz These results corroborate the earlier studies on sound production in M coosae Johnston et al (2008) found the dominant frequency of sounds produced by M coosae during aggressive encounters to be 170 Hz, which, according to the results of this study, is within the species’ most sensitive range of hearing This finding supports the proposition by Johnston that the sounds produced by M coosae are used for intraspecific communication The results from this study also provide evidence against the possibility that in Holt and Johnston (2009), M coosae did not respond to C trichroistia’s signals because they were undetectable Cyprinella trichroistia signals are broadband, and most of the power occurs at low frequencies (near 100 Hz), which is well within the most sensitive portion of M coosae’s hearing Baseline noise levels were generally much lower than thresholds for M coosae and M henshalli Spikes in the baseline noise were electrical noise from the underwater speaker This was confirmed through the attenuation of the spikes as the hydrophone was moved farther away from the speaker The electrical noise was not audible, and should not have caused threshold elevations in M coosae or M henshalli The shape of M coosae and M henshalli audiograms are rather unusual in that they are more linearly shaped than most audiograms Audiograms generally take a U shape, with thresholds increasing at both high and low frequency limits of hearing However, this is not always the case, as can be seen with Astronotus ocellatus in Kenyon et al (1998) We believe the linear shape of the audiograms in the current study may be due in part to input from the lateral line system at 100 and 200 Hz Because the lateral line is maximally sensitive to particle accelerations between 100 and 200 Hz (Münz 1985; Alfons et al 1992), the hearing threshold at these frequencies may be decreased The lack of a specialized mechanism for transferring motions induced on the gas bladder by the pressure component of sound to the ears may explain why hearing sensitivity does not improve at higher frequencies The only other bass audiograms we know of are for M salmoides (Jones 2005; Yan unpublished data, pers comm.) Jones shows a distinct elevation in sensitivity at 300 Hz, which does not begin to decrease until below 100 Hz The results from Jones complement ours in that the greatest sensitivity occurs at frequen- 125 cies below 300 Hz However, because methodology associated with Jones’ audiogram is lacking, comparisons can only be made loosely The audiogram of M salmoides hearing based on Yan’s results shares some similarities with that of M coosae above 300 Hz However, a sharp increase in threshold is seen at 300 Hz, and frequencies below this level were not tested by Yan in order to minimize stimulation of the lateral line A difference in ABR setup may also contribute to the sharp threshold elevation in M salmoides Because Yan’s methodology involved playing stimulus tones from above the water surface, the ratio of particle acceleration to pressure was likely different from our study, and may explain our increased sensitivity at frequencies of 300 Hz and less Because so few studies have reported hearing sensitivity in both SPL and particle acceleration, comparison to other species in terms of particle acceleration is difficult However, it is worth mentioning that the sensitivity of M coosae to particle accelerations of 4.0e−4 m s2 at 100 Hz and 1.1e−3 m s2 at 200 Hz is comparable to sensitivities of several sciaenid species (Leiostomus xanthurus, Cynoscion regalis, Micropogonias undulatus, and Sciaenops ocellatus) tested by Horodysky et al (2008) Though there is some disagreement, all audiograms presented agree that sensitivity increases continuously from higher frequencies down to at least 400 Hz The results from this study, which does not attempt to exclude lateral line stimulation, show much greater sensitivity at frequencies below 300 Hz Because vocalizations of M coosae occur below 200 Hz, particle acceleration is likely detected by the lateral line during intraspecific communication Future studies investigating the importance of acoustic communication should attempt to incorporate particle acceleration into the characterization of involved sounds Acknowledgments We thank Dennis Higgs for technical assistance in setting up the ABR system and David Mann for assistance in calculating particle accelerations We would also like to thank Hong Young Yan for providing M salmoides audiogram data Thanks to Patricia Speares, Nicole Kierl, and Sean Holder for help collecting and maintaining specimens References Alfons B, Kroese A, Schellart NAM (1992) Velocity-and acceleration-sensitive units in the trunk lateral line of the trout J Neurophysiol 68:2212–2221 126 Casper BM, Mann DA (2006) Evoked potential audiograms of the nurse shark (Ginglymostoma cirratum) and the yellow stingray (Urobatis jamaicensis) Environ Biol Fish 76:101–108 Corwin JT, Bullock TH, Schweitzer J (1982) The auditory brainstem response in five vertebrate classes Electroencephalogr Clin Neurophysiol 54:629–641 Higgs DM, Souza MJ, Wilkins HR, Presson JC, Popper AN (2001) Age-and size-related changes in the inner ear and hearing ability of the adult zebrafish (Danio rerio) J Assoc Res Otolaryngol 03:174–184 Holt DH, Johnston CE (2009) Signaling without the risk of illegitimate receivers: predators respond to the acoustic signals of Cyprinella (Cyprinidae)? Environ Biol Fish 84:347–357 Horodysky AZ, Brill RW, Fine ML, Musick JA, Latour RJ (2008) Acoustic pressure and particle motion thresholds in six sciaenid fishes J Exp Biol 211:1504–1511 Johnston CE, Bolling MK, Holt DE, Phillips CT (2008) Production of acoustic signals during aggression in Coosa bass, Micropterus coosae Environ Biol Fish 82:17–20 Jones KA (2005) Knowing bass; the scientific approach to catching more fish First Lyons, Guilford, p 108 Environ Biol Fish (2011) 91:121–126 Kenyon TN, Ladich F, Yan HY (1998) A comparative study of hearing ability in fishes: the auditory brainstem response approach J Comp Physiol A 182:307–318 Kojima T, Ito H, Komata T, Taniuchi T, Akamatsu T (2005) Measurements of auditory sensitivity in common carp Cyprinus carpio by the auditory brainstem response technique and cardiac conditioning method Fish Sci 71:95–100 Mann DA, Higgs DM, Tavolga WN, Souza MJ, Popper AN (2001) Ultrasound detection by clupeiform fishes J Acoust Soc Am 109:3048–3054 Münz H (1985) Single unit activity in the peripheral lateral line system of the cichlid fish Sarotherodon niloticus L J Comp Physiol A 157:555–568 Myrberg AA (1981) Sound communication and interception in fishes In: Fay RR, Popper AN, Tavolga WN (eds) Hearing and sound communication in fishes SpringerVerlag, New York, pp 345–425 Popper AN, Fay RR (2010) Rethinking sound detection by fishes Hear Res doi:10.1016/j.heares.2009.12.023 Wright KJ, Higgs DM, Belanger AJ, Leis JM (2005) Auditory and olfactory abilities of pre-settlement larvae and postsettlement juveniles of a coral reef damselfish (Pisces: Pomacentridae) Mar Biol 147:1425–1434 Environ Biol Fish (2011) 91:127–132 DOI 10.1007/s10641-011-9784-4 Evaluation of a behavioural response of Mediterranean coastal fishes to novel recreational feeding situation Marco Milazzo Received: 17 March 2010 / Accepted: March 2011 / Published online: April 2011 # Springer Science+Business Media B.V 2011 Abstract Fish may learn to associate food with human presence through recreational hand-feeding, a popular tourist activity The conditional learning—e.g when an organism learns by continuous exposure to one stimulus—of different coastal fish species exposed to novel feeding situations was evaluated The latencies of learning response to the initiation of supplementary feeding were rapid and speciesspecific However differences in the learning response between different fishes decreased over time, demonstrating that associating with others might incur costs especially for small-sized species, likely due to increased competition for food Nevertheless some other fish species did not acquire any specific human oriented behavior, being naturally timid or avoiding humans Keywords Conditional learning Fish feeding Behavior Food provisioning Introduction Historically, there are many opportunities for humans to directly interact with marine wildlife both on a M Milazzo (*) Dipartimento di Scienze della Terra e del Mare, University of Palermo, Via Archirafi 28, 90123 Palermo, Italy e-mail: marmilazzo@iol.it commercial scale (i.e through fisheries) (Jackson et al 2001) and on a recreational scale (i.e through tourism) (Orams 1999) Besides the well documented responses to fishing of target and—through trophic cascading—of non-target species (Micheli et al 2005), it has been recently demonstrated that tourism activities may strongly affect the behavior of many animal species, particularly fish (Gotanda et al 2009, but see Guttridge et al 2009) As a general rule, the behavior of fish can be altered in different ways, with fish (1) avoiding humans where they are exploited (Jennings and Polunin 1995; Kulbicki 1998), (2) aggregating in specific places and approaching humans where supplementary food is provided to facilitate interaction with tourists (Cole 1994; Milazzo et al 2005, 2006), and (3) changing their behavioral strategies (e.g anti-predator responses) in order to reduce predation risk within both fished and protected areas (Guidetti et al 2008; Gotanda et al 2009) Indeed, differential response of fishes may be classically related either to an instinctive behavior, that is genetically programmed, or to the capacity of fish to adapt to novel opportunities through learning, which means that a specific behavior has been acquired or eliminated as a result of experience (Shettleworth 1984) By using food to attract animals to sites that are frequented by tourists, the recreational fish feeding— a popular activity both in tropical and temperate regions—has the potential to alter the behavior of target species (Orams 2002) Indeed, this supplemen- 128 tal food provisioning represents a novel opportunity for fishes in the wild In addition to changes in the local distribution of fishes (Cole 1994; Milazzo et al 2005) and to a dramatic alteration of predation patterns (Milazzo et al 2006), intense recreational feeding leads some fish species to change their behavior in the presence of humans (e.g acquiring a human oriented behavior); indicating that naive fish population may learn to associate food with human presence through hand-feeding (Cole 1994; Milazzo et al 2006; Ilarri et al 2008) The main objective of this study was to assess whether coastal fishes differ in their behavioral response to novel feeding situations, simulating those provided by marine tourists The conditional learning—e.g when an organism learns by continuous exposure to one stimulus—was the behavioral response considered In particular, fishes were experimentally fed with bread by means of handfeeding snorkelling in the water, and their learning consisted of an acquired human oriented behavior even when food was not presented After humans and food have been memorized as associated by a fish, the human presence triggers the food-related behavior Materials and methods To estimate the latency with which fishes begin to exhibit a human-positive behavior, experimental hand-feeding was performed in a manipulation location (MANIP) in the Ustica Island marine protected area (MPA; Southern Italy) on 33 separate days from 09:00 to 11:00 h, between early July and early September 2001 In this location two snorkellers fed fish with kg of bread for 20 min, simulating the activity of a large number of tourists that several times a day feed the fishes bread in another feeding site inside the no-take zone of the MPA (Milazzo, pers obs.) To determine whether fishes learned to associate humans with food, they were also fed at a second artifact location (ART) In this case, supplementary food was provided to them directly from a row boat, when no humans were in the water, and thus no association human/food was possible for them Relative human oriented behavior (e.g approachability, AC) between locations was measured from Environ Biol Fish (2011) 91:127–132 counts of fishes using an inverse fixed point technique (Milazzo et al 2006) In each sampling location, the number of fishes approaching the snorkeller was counted at intervals by identifying and counting the number of individuals within a cylinder of m radius and height (total volume 25 m3) having the sea surface as the base and the observer as the central axis These counts were also carried out in four control locations (CTL1-4), where food provisioning was not allowed Three out of these four control locations were popular sites for snorkellers and swimmers (CTL2–CTL4); the 4th was situated inside the no-entry area of the MPA where human access is strictly forbidden (CTL1) At each location, 33 replicated approachability counts were performed on different days early in the morning (07:30–08:30 h), to avoid biased sampling when other people were in the water If higher fish density increases the likelihood that fishes pass close to the observer by chance, differences between locations in the behavioral response of fishes to feeding disturbance may be confounded by between-locations differences in the density and species composition of fishes Since higher natural densities of fish species at each location could lead to higher densities of fish responding to the feeding stimuli and actively learning, at the beginning of the study density estimates of fishes (e.g background density, BD) were collected at all locations using 12 replicate 50×5 m benthic transects conducted by scuba divers at 5–10 m depth (Harmelin-Vivien et al 1985) Differences in the composition and structure of the fish assemblages among the six locations considered (MANIP, ART and CTL1,2,3,4) were tested using 1-way permutational multivariate analysis of variance (PERMANOVA, Anderson 2001) In addition to between-locations comparisons at the beginning of the experiment, these average background density estimates (BD) were compared with each approachability count (AC) repeated in time as:  ACi LRi ¼ ln BDi  where LR is the log learning ratio for a given species i When LR≥0 (e.g., the density of fish approaching humans was equal or higher than their background density) it is assumed that the fish species learned Environ Biol Fish (2011) 91:127–132 Thus latency with which fishes begin to exhibit approachability towards humans was expressed as the number of days after which a given species showed a continuous LR≥0 Results and discussion Before the experiment initiation, there were no statistically significant differences in the composition and structure of the fish assemblages among the six locations considered (PERMANOVA, F 5,66 = 0.61104; P>0.05), and a total of 26 fish species were identified and counted for background density estimates Among these, 12 fish species—in addition to participating to feeding—exhibited a clear human oriented behavior, with the rainbow wrasse Thalassoma pavo (Linnaeus, 1758), the damselfish Chromis chromis (Linnaeus, 1758), the saddled bream Oblada melanura (Linnaeus, 1758) and the goldline Sarpa salpa (Linnaeus, 1758) being the most reacting and abundant [average background density (±SE): T pavo, 47.86±3.54 ind.·250 m−2; C chromis, 14.21± 4.41; O melanura, 19.76±2.85; S salpa, 17.01± 3.47] Although participating to supplementary feeding and consequently acquiring a human oriented behavior, the abundance of different fish taxa [e.g the labrids Coris julis (Linnaeus, 1758) and Symphodus ocellatus (Forsskål, 1775), the sparids Diplodus spp., Boops boops (Linnaeus, 1758) and Spondyliosoma cantharus (Linnaeus, 1758), the serranid Epinephelus marginatus (Lowe 1834), the carangid Seriola dumerili (Risso 1810), and the Mugilidae] approaching humans never exceeded their background density in the hand-feeding location (LR[...]... stage (56.344.67% of the time; Fig 1a) In examination of the total time that each parent is spending with the offspring, we find a significant effect of offspring stage (F1, 49 =8.34; p=0.006), a significant effect of the sex of the parent (F1, 49 = 72.36; p