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Lercabeti bob di tne rt sie eeehe Wry wee / Pipe he ds . ( WIAA arheatin aaa kapha sreiu arenes eG) SALVIA Vine Medal ang) fe BANA a \ “a atey a ihw yp hat Reeve we haem wee ty 4 ‘ we a eager’ oat WV AA A tae ee bh ed UtAdi ch bractohtp phy At tae : dada amy eee ARP Cha ae SP ces be hee VAMP AA TAS LD YH owe Habs bade aed beh gk, Ct A ee bbe Oe ee ‘ Veet OOO eee ae wre LW AA a 4% be hd We nd #2406 AL | aaa ’ (Aad ater . Ted CO) HOO hA MA OH PAGE EL LT he i 7 140 Ghee aL dee wi ee) oe whi aw fat as a Be ha | c4 agahud , Lee 4 a) Cee ee ’ aan ‘ ai a4 ‘ ts PT Ce ee ee St ee | vara We BU ek ie eX eves \ GO GU seus Ww Ae ate dete hee an bn eh ahATO) Aa Oc eR pai : Veda WA Oa w a Vie brie CAP ROR AAU NN ls ty -_ ; ee 14 HOVE SOLA Oe be ei eh a rAR AAD eRe ya’ OCR Roh eee EPPO Os) ae ee LOreeyaek 1A CTA OVE Oe Ha 400 A A A A Po HR ae OA he Gg ae (Ee FPA GAD Dae a eee Ant mE shy fice Oe oak bs LE 4 fy hh ome ae wort heaved baa ee ee ee Meee Noa lect G oo PAE aL A ee ee hae en ae 7. eH he sO pws owt tid iat ede a Aieeeaa mV Ried +e A ta \e ee yt wt ewe) hy Fae wor tah ee deh tit ArecMe oe ee ye te Wed Ws Oy Hh 0s 6s Og be ay Sa cy Oe Hee 0 ait Rah AP Oe OE OOD * YN Ae ee a et hr at ee eth eh BD Ve an Hi me GN 4 AAT rer tn en a LT wd ea 4 eh ee ee re Cee 44 &41.¢6 al wo PROCEEDINGS AND TRANSACTIONS OF TH LIVERPOOL BIOLOGICAL SOCIETY. LO) See, 6 B.S SESSION 1914-1915. % tlie “* ‘i | \ ~ 4 — aly ~, LIVERPOOL: C. Tintina & Co., Lrp., PRINTERS, 53, VIcroRIA STREET. 1915. CONTENTS. I.—PROCEEDINGS. Office-bearers and Council, 1914-1915 . Report of the Council Summary of Proceedings at the Meeting List of Members : List of Societies and Academies on Hee tance Fisk Treasurer's Balance Sheet II].— Transactions. Presidential Address—‘‘ The mode of transmission of some Tropical Diseases.” By Prof. J. W. W. STEPHENS, M.D., D.P.H. Twenty-eighth Annual Report of the Liverpoo] Marine Biology Committee and their Biological Station at Port Erin. By Prof. W. A. Herpmay, D.Sc., F.R.S. : Report on the Investigations carried on during 1914, in connection with the Lancashire Sea-Fisheries Laboratory, at the University of Liverpool, and the Sea-Fish Hatchery at Piel, near Barrow. By Prof. W. A. Herpmay, D.Sc., F.R.S8., ANDREW Scott, A.L.S., and James Jonnstone, D.Sc. L.M.B.C. Memoir on “ Tubifex.” By GrrrrupeE C. Dixon, B.Sc. PAGE Vil vill 1X X11 X11 Dad 21 63 303 PROCEEDINGS OF THE LIVERPOOL BIOLOGICAL SOCIETY OFFICH-BEARERS AND COUNCIL. 1886—1887 1887—1888 1888—1889 1889—1890 1890—1891 1891—1892 1892—1893 1893—1894 1894—1895 1895—1896 1896—1897 1897—1898 1898—1899 1899—1900 1900—1901 1901—1902 1902—1903 1903—1904 1904—1905 1905—1906 1906—1907 1907—1908 1908—1909 1909—1910 1910—1911 1911—1912 1912—1913 Ex-Presiwents : Pror. W. MITCHELL BANKS, M.D., F.R.C.S. J. J. DRYSDALE, M.D. Pror. W. A. HERDMAN, D.Sc., F.B.S.E. Pror. W. A. HERDMAN, D.Sc., F.R.S.E. T. J. MOORE, C.M.Z.S. T. J. MOORE, C.M.Z.S. ALFRED O. WALKER, J.P., F.L.S. JOHN NEWTON, M.R.C.S. Pror. F. GOTCH, M.A., F.R.S. Pror. R. J. HARVEY GIBSON, HENRY O. FORBES, LL.D., F ISAAC C., THOMPSON, BE Pror. C. 8. SHERRINGTON, J. WIGLESWORTH, M.D., Pror. PATERSON, M.D., HENRY C. BEASLEY. R. CATON, M.D., F.R.C.P. Rey. T. 8. LEA, M.A. ALFRED LEICESTER. JOSEPH LOMAS, F.G.S. Pror. W. A. HERDMAN, D.Sc., F.RS. W. T. HAYDON, F.L.S. Pror. B. MOORE, M.A., D.Sc. R. NEWSTEAD, M.Sc., PES, Pror. R. NEWSTEAD, M.Sc., J. H. OCONNELL, L.R.C.P. JAMES JOHNSTONE, D.Sc. F.R.S. 1913—1914 C. J. MACALISTER, M.D., F.R.C.P. SESSION XXIX., 1914-1915. President : Pror. J. W. W. STEPHENS, M.D., D.P.H. Pror. W. A. HERDMAN, D.Sc., Vice- Presidents : F.R.S. C. J. MACALISTER, M.D., F.R.C.P. Hon. Creagurer : W. J. HALLS. HENRY C. BEASLEY. 8S. T. BURFIELD, B.A. G. ELLISON. H, B, FANTHAM, D.Sc., M.A. W. T. HAYDON, F.L.S. J. JOHNSTONE, D.Sc. Hon. Secretary: JOSEPH A, CLUBB, D.Sc. Council : W. 8S. LAVEROCK, WM. RIDDELL, M.A. KE. THOMPSON. Representative of Students’ Section : Miss M. BRADLEY, B.Sc. Hon. Librarian: MAY ALLEN, B.A, DOUGLAS LAURIE, M.A. M.A., B.Sc. J. H. O CONNELL, L.R.C.P. MAY RATHBONE, (Miss). vill _ LIVERPOOL BIOLOGICAL SOCIETY. REPORT of the COUNCIL. Durine the Session 1914-15 there have been six ordinary meetings and one field meeting of the Society. The communications made to the Society at the ordinary meetings .have been representative of many branches of Biology, and the various exhibitions and demonstrations thereon have been of great interest. By invitation of the Council, Prof. C. J. Patten, M.D., Sc.D., of Sheffield University, lectured before the Society, at the March Meeting, on “The Migration of Birds studied at Irish Light-Stations.”’ The Library continues to make satisfactory progress, and additional important exchanges have been arranged. The Treasurer’s statement and balance-sheet are appended. The members at present on the roll are as follows :— Ordinary members .. ve Ms oe a 52 Associate members... ee: ry 3 8 Student members, including Students’ Section, about 30 Total a 2. 90 SUMMARY OF PROCEEDINGS AT MEETINGS. 1x SUMMARY of PROCEEDINGS at the MEETINGS. The first meeting of the twenty-ninth session was held at the University, on Friday, November 20th, 1914. The President-elect (Prof. J. W. W. Stephens, M.D., D.P.H.) took the chair in the Zoology Theatre. 1. The Report of the Council on the Session 1913-1914 (see “ Proceedings,’ Vol. XXVIII, p. vii) was submitted and adopted. 2. The Treasurer’s Balance Sheet for the Session 1913-1914 (see “* Proceedings,” Vol. XXVIII, p. xvii) was submitted and approved. 3. The following Office-bearers and Council for the ensuing Session were elected :—Vice-Presidents, Prof. Herdman, ec! .s., and C. J. Macalister, M.D., F.R.C.P. ; Hon. Treasurer, W. J. Halls; Hon. Librarian, May Allen, B.A.; Hon. Secretary, Joseph A. Clubb, D.Sc. ; Council, H. C. Beasley, 8. T. Burfield, B.A., G. Ellison, H. B. Fantham, D.Sc., B.A.. W. T. Haydon, F.LS., J. Johnstone, D.Sc., Douglas Laurie, M.A., W. S. Daverock; M.A., BSc., J. H. O'Connell, L.R.C.P., May Rathbone (Miss), W. Riddell, M.A., and KE. Thompson. 4. Prof. J. W. W. Stephens, M.D., D.P.H., delivered the Presidential Address on ‘‘ The mode of transmission of some Tropical Diseases ’’ (see ‘‘ Transactions,”’ p. 3). A vote of thanks was carried with acclamation. The second meeting of the twenty-ninth session was held at the University, on Friday, December 11th, 1914. The President in the chair. x ~ LIVERPOOL BIOLOGICAL SOCIETY. 1. Prof. Herdman submitted the Annual Report on the work of the Liverpool Marine Biology Committee (see “ Transactions,” p. 21). 2. Prof. Dakin, D.Sc., gave an interesting account of a recent visit to the Abrollos Archipelago, off the West Coast of Australia, for the purpose of investigating the Coral formations of the Islands. The third meeting of the twenty-ninth session was held at the University, on Friday, January 22nd, 1915. The President in the chair. 1. Mr. J. W. Cutmore exhibited, with remarks, a suggested sparrow-hawk’s larder, found near Mold, N. Wales. 2. Dr. Clubb exhibited, with remarks, a series of specimens of a melanistic variety of the Water Vole, found at Leasowe, living in a colony, isolated from colonies of the ordinary species living in the same pond. 3. Mr. Heron-Allen, F.L.8., gave an address, illustrated by a beautiful series of lantern slides, on a collection of Foraminifera obtaied from marine deposits, collected by Prof. Herdman off the West Coast of Scotland. The fourth meeting of the twenty-ninth session was held at the University, on Friday, February 12th, 1915. The President in the chair. : 1. The President exhibited some old surgical instruments. 2. Mr. R. D. Laurie exhibited and described specimens from Lord Howes’ Island, off the East Coast of Australia. 3. Dr. Johnstone submitted the Annual Report of the Investigations carried on during 1914 in connection with the Lancashire Sea-Fisheries Committee (see “ Transactions,” p. 63). SUMMARY OF PROCEEDINGS AT MEETINGS. xl The fifth meeting of the twenty-ninth session was held at the University, on Friday, March 12th, 1915. The President in the chair. 1. Prof. Herdman exhibited, with remarks, the gigantic colonial Foraminifer, Ramulona, from the Indian Ocean. 2. Mr. R. D. Laurie gave an account of the distribution of crabs in the Indian Ocean. The sixth meeting of the twenty-ninth session was held at the University, on Friday, May 14th, 1915. The President in the chair. 1. Prof. C. J. Patten, M.D., Sc.D., of Sheffield University, lectured before the Society on the invitation of the Council, on “ The Migration of Birds studied at Irish Light-Stations.” The lecture was much appreciated by a large audience, and a cordial vote of thanks was accorded to the lecturer. The seventh meeting of the twenty-ninth session was the Annual Field Meeting, held on Saturday, June 19th. A very pleasant afternoon was spent in a Biological ramble through footpaths, via Meols, Newton, Frankby, and Grange Hill to _ West Kirby. At the short business meeting held after tea, on the motion of the President from the chair, Prof. EK. E. Glynn, M.A., M.D., was unanimously elected President for the ensuing session. ELECTED, 1908 X11. LIST of MEMBERS of the LIVERPOOL BIOLOGICAL SOCIETY. SESSION 1914-1915. —_————— A. ORDINARY MEMBERS. (Life Members are marked with an asterisk.) Abram, Prof. J. Hill, 74, Rodney Street, Liverpool. 1909 *Allen, May, B.A., Hon. Liprarian, University, 1888 1913 1903 1912 1886 1886 1910 1910 1902 1886 1910 1896 1912 1886 Liverpool. Beasley, Henry C., Prince Alfred Road, Wavertree. Beattie, Prof. J. M., M.A., M.D., The University, Liverpool. Booth, jun., Chas., 30, James Street, Liverpool. Burfield, 8. T., B.A., Zoology Department, University, Liverpool. Caton, R., M.D., F.R.C.P., 78, Rodney Street. Clubb, J. A., D.Se., Hon. Secretary, Free Public Museums, Liverpool. Ellison, George, 52, Serpentine Road, Wallasey. Fantham, H. B., D.Sc., M.A., School of Tropical Medicine, University, Liverpool. Glynn, Dr. Ernest, 67, Rodney Street. Halls, W. J., Hon. TREasuRER, 35, Lord Street. Hamilton, Mrs. J., 96, Huskisson Street, Liverpool. Haydon, W. T., F.L.S., 55, Grey Road, Walton. Henderson, Dr. Savile, 48, Rodney Street, Liverpool. Herdman, Prof. W. A., D.Sc., F.R.S., Vick-PRESIDENT, University, Liverpool. LIST OF MEMBERS. Xill 1893 Herdman, Mrs. W. A., Croxteth Lodge, Ullet Road, Liverpool. 1912 Hobhouse, J. R., 54, Ullet Road, Liverpool. 1902 Holt, A., Dowsefield, Allerton. 1903 Holt, George, Grove House, Knutsford. 1903 Holt, Richard D., M.P., 1, India Buildings, Liverpool. 1912 Jackson, H. G., M.Sc., Zoology Department, University, Birmingham. 1898 Johnstone, James, D.Sc., University, Liverpool. 1894 Lea, Rev. T. S., D.D., The Vicarage, St. Austell, Cornwall. 1896 Laverock, W. S., M.A., B.Sc., Free Public Museums, Liverpool. 1906 Laurie, R. Douglas, M.A., University, Liverpool. 1912 Lyon (Miss), Una, High School for Girls, Aigburth Vale, Liverpool. 1912 Macalister, C. J.. M.D., F.R.C.P., Vicz-Presipenrt, 35, Rodney Street, Liverpool. 1915 Macdonald, Prof. J.8., B.A., The University, Liverpool. 1905 Moore, Prof. B., University, Liverpool. 1913 Mottram, V. H., Physiological Department, University, Liverpool. 1904 Newstead, Prof. R., M.Sc., F.R.S., University, Liverpool. 1904 O’Connell, Dr. J. H., 38, Heathfield Road, Liverpool. 1913 Pallis, Mark, Tatoi, Aigburth Drive, Liverpool. 1903 Petrie, Sir Charles, 7, Devonshire Road, Liverpool. 1915 Prof. W. Ramsden, University, Liverpool. 1903 Rathbone, H. R., Oakwood, Aigburth. 1890 *Rathbone, Miss May, Backwood, Neston. 1910 Riddell, Wm., M.A., Zoology Department, University, Liverpool. 1897 Robinson, H. C., Malay States. 1908 Rock, W. H., 25, Lord Street, Liverpool. 1894 Scott, Andrew, A.L.S., Piel, Barrow-in-Furness. XIV 1908 1895 1886 1903 1913 1903 1905 1889 1888 1891 1905 1914 1913 1905 1910 1912 1913 1903 1910 LIVERPOOL BIOLOGICAL SOCIETY. Share-Jones, John, F.R.C.V.8., University, Liverpool. Sherrington, Prof., M.D., F.R.S., University, Liverpool. Smith, Andrew T., 21, Croxteth Road, Liverpool. Stapledon, W. C., “ Annery,” Caldy, West Kirby. Stephens, Prof. J. W. W., M.D., PresipENT, University, Liverpool. Thomas, Dr. Thelwall, 84, Rodney Street, Liverpool Thompson, Edwin, 25, Sefton Drive, Liverpool. Thornely, Miss L. R., Nunclose, Grassendale. Toll, J. M., 49, Newsham Drive, Liverpool. Wiglesworth, J.. M.D., F.R.C.P., Springfield House, Winscombe, Somerset. B. AssocrtatE MEMBERS. Carstairs, Miss, 39, Lilley Road, Fairfield Cutmore, J. W., Free Public Museum, Liverpool. Hamilton, Erik, M.Sc., 96, Huskisson Street, Liverpool. Harrison, Oulton, 18, Limedale Road, Mossley Hill. Kelley, Miss A. M., 10, Percy Street, Liverpool. Parkin, Miss A. B., 3, Cairns Street, Liverpool. Smith, Miss E. M. G., 39, Parkfield Road, Liverpool. Tattersall, W., D.Sc., The Museum, Manchester. Tozer, Miss E. N., Physiology Laboratory, University, Liverpool. | C. Universtry Srupents’ Szction. President : Miss M. Bradley, B.Sc. Secretary: J. Ronald Bruce, B.Sc. (Contains about 30 members.) ee a ” ———— if ti LIST OF MEMBERS. XV .D. Honorary MEMBERS. S.A.8., Albert I., Prince de Monaco, 10, Avenue du brocadéro, Paris. Bornet, Dr. Edouard, Quai de la Tournelle 27, Paris. Claus, Prof. Carl, University, Vienna. Fritsch, Prof. Anton, Museum, Prague, Bohemia. Haeckel, Prof. Dr. E., University, Jena. Hanitsch, R., Ph.D., Raffles Museum, Singapore. Solms-Laubach, Prof.-Dr., Botan. Instit., Strassburg. LIST OF SOCIETIES AND ACADEMIES WITH WHICH PUBLICATIONS ARE EXCHANGED. (Twenty-seven additions made since 1905 are marked with an asterisk.) ADELAIDE.—Royal Society of South Australia. Memoirs ; Transactions. AGRAM.—Societas Historico-Naturalis Croatica. Glasnik. AmstTeRDAM.—K. Akad. van Wetenschappen. Proceedings of the Section of Sciences ; Verslagen Gew. Vergaderingung ; Verhande- lingen ; Jaarboeken. Natuurkundig Tijdschrift voor Nederlandsch-Indie. Batrimore.—Johns Hopkins University. Circulars. Bastze.—Naturforschende Gesellschaft. Verhandlungen. BercEen.—Bergens Museum. Aarbog ; Meeresfauna ; Skrifter. * BERKELEY, CALir.—University.. Publications. Beriin.—Deutscher Fischerei Verein. Allgemeine Fischerei Zeitung ; Mit- teilungen ; Zeitschrift fiir Fischeret. K. preussische Akademie der Wissenschaften. Sitzwngsberichte. BrrMincHAaM.—Birmingham and Midland Institute Scientific Society. Records of Meteorological Observations. Natural History and Philosophical Society. Proceedings ; Report. Bonn.—Naturhistorischer Verein der preussische Rheinlande. Verhandlungen. Niederrheinische Gesellschaft fiir Natur- und Heilkunde. Svtzwngs- bericht. BorDEAUX.—Société Linnéenne. Procés-Verbauz. *Socicté Scientifique et Station Biologique d’Arcachon. Bulletin, *Boston, Mass.—American Academy of Arts and Sciences. Proceedings. Society of Natural History. Proceedings. Tufts College. Studies. *BRISBANE.—Queensland Museum. Annals. XVI. LIVERPOOL BIOLOGICAL SOCIETY. BRooKLyn.— Institute of Arts and Sciences. Cold Spring H. arbour Monographs ; Science Bulletin. BrussELts.—Academie Royale des Sciences, des Lettres, etc., Annuaire ; Bulletin (Classe des Sciences). *Société Royale Zoologique et Malacologique de Belgique. Annales. *Bryn Mawr.—College. Monographs. Buenos Ayres.—Museo Nacional. Annales. *BurFaLo.—American Microscopical Society. Transactions. Society of Natural Sciences. Bulletin. CaEN.—Société Linnéenne de Normandie. Bulletin. *CALCUTTA.—Indian Museum. Memoirs ; Miscellaneous Zoological Publica- tions ; Records. CAMBRIDGE, Mass.—Museum of Comparative Zoology. Bulletin. CaPE or Goop Hors.—Department of Agriculture.—Reports. Cu1caco.—Botanical Gazette. Field Museum of Natural History. Publications. CHRISTIANIA.—Videnskabs Selskabet. Forhandlinger. Crncrnnati.—Lloyd Library. Bulletin. Cotomso.—Ceylon Marine Biological Reports. *Ceylon Museum. Spolia Zeylanica. *CONCARNEAU.—Laboratoire de Zoologie et de Physiologie Maritimes. Travaua Scientifiques. CoPENHAGEN.—Conseil Permanent International pour lExploration de la Mer. Bulletin des Resultats ; Bulletin Hydrographique ; Bulletin Statistique ; Publications de Cuirconstance ; Rapports et Procés- Verbaux. Danish Biological Station. Report. Kommissionen for Havunderségelser. Meddelelser ; Skrifter. K. Dansk Videnskabernes Selskab. Oversigt ; Skrtfter. Naturhistorisk Forening Videnskahelige. Meddelelser. DusiLin.—Royal Dublin Society. Hconomic Proceedings ; Scientific Proceed- ings ; Scientific Transactions. EDINBURGH.—Royal College of Physicians. Laboratory Reports. Royal Physical Society. Proceedings. Royal Society. Proceedings. Fishery Board for Scotland. Report. * FRANKFORT-ON-THE-Mar1n.—Senckenbergische naturforschende Gesellschaft. Abhandlungen ; Bericht. FREIBURG-IN-THE- BREISGAU.—Naturforschende Gesellschaft. Bericht. GENEVA.—Société de Physique et d’ Histoire Naturelle. . Mémoires. GrEssEN.—Oberhessische Gesellschaft fiir Natur und Heilkunde. Bericht. GLascow.—Natural History Society. Glasgow Naturalist. GOrrincEN.—K. Gesellschaft der Wissenschaften. Nachrichten. *Gratz.—Naturwissenschaftlicher Verein fiir Steiermark. Mitteclungen. | GUstrow.—Verein der Freunde der Naturgeschichte in Mecklenberg. Archiv. SOCIETIES AND ACADEMIES ON EXCHANGE LIST. XVil. HAARLEM.—Museée Teyler. Archives. Société Hollandaise des Sciences. Archives Neerlandaises des Sciences Exactes et Naturelles. Hawurax, Nova Scotra.—Nova Scotian Institute of Science. Proceedings and Transactions. Hatie.—Academia Caesarea Naturae Curiosorum. Nova Acta. *HamBurc.—Naturhistorisches Museum. Mitteilungen. *TRELAND.—Department of Agriculture and Technical Instruction. Report. Kret.—Kommission zur wiss. Untersuchungen der deut. Meere in Kiel u. d. biologischen Anstalt auf Helgoland. Wiéssenschaftliche Meeres- untersuchungen. Naturwissenschaftlicher Verein. Schriften. LA Puata.—Museo. Annales ; Revista. LAWRENCE.—Kansas University. Experimental Station Reports ; Geological Survey Reports ; Science Bulletin. LEEpDs.—Yorkshire Naturalists Union. TJ'ransactions. Lerezic.—K. sichsische Gesellschaft der Wissenschaften. Berichte aber die Verhandlungen. (Math. Phys. Classe). *Lincotn, NEBRASKA.—University. Medical School Bulletin ; Zoological Laboratory Studies. LiverPoot.—Geological Society. Proceedings. Lonpony.—British Association for the Advancement of Science. Report. The Naturalist. | Royal Microscopical Society. Journal. *Mapison.—Wisconsin Academy of Sciences, Arts, etc. Transactions. Wisconsin Geological and Natural History Survey. Bulletin. Maaprsurs.—Museum fiir Natur- und Heimatkunde. Abhandlungen und Berichte. MANCHESTER.—Microscopical Society. Transactions and Report. . Victoria University. Publications. MArsEILLEs.—Musée @ Histoire Naturelle. Annales (Zoologie). MELBOURNE.—Royal Society of Victoria. Proceedings. *MILWAUKEE.—Public Museum. Annual Report ; Bulletin. Wisconsin Natural History Society. Bulletin. Monaco.—Institut Océanographique. Bulletin; Résultats des Campagnes Scientifiques. Montrvip£o.—Museo Nacional. Annales. MonTpeLiier.—Académie des Sciences et Lettres. Bulletin Mensuel. Moscow.—Société Impériale des Naturalistes. Bulletin ; Nouveaux Mémoires. Monicu.—Ornithologische Gesellschaft. Verhandlungen. Nanoy.—Société des Sciences. Bulletin des Séances. Nap.es.—Reale Accademia delle Scienze, etc. Atti ; Rendiconto. *University. Museo Zoologico. Annuario. *Nmw Haven.—Connecticut Academy of Arts and Sciences. 'ransactions. *New Yor«.—American Museum of Natural History. Bulletin. XVI. _ LIVERPOOL BIOLOGICAL SOCIETY. OBERLIN.—Oberlin College Laboratory. Bulletin. - Wilson Ornithological Club. Wilson Bulletin. Oporto.—Academia Polytechnica do Porto. Annaes Scientificos. Annaes de Sciencias Naturaes. . Paris.—Bulletin Scientifique de la France et de la Belgique. Museum d’ Histoire Naturelle. Bulletin. Société de Biologie. Comptes Rendus. Société Zoologique de France. Bulletin ; Mémoires. PHILADELPHIA.—Academy of Natural Sciences. Proceedings. *PortTicI.—Regia Scuola Superiore di Agricoltura. Bollettino. RENNES.—Société Scientifique et Medicale de Ouest. Bulletin. RomeE.—Societa Romana per gli Studi Zoologia. Bolletéino. Saint Jonn, New Brunswick.—Natural History Society. Bulletin. Saint Lovuis.—Academy of Science. Transactions. *Missouri Botanical Garden. Report. SAINT PETERSBURG.—Academia Scientiarum Imperialis. Bulletin. San Francisco.—Californian Academy of Science. Proceedings. SANTIAGO.—Société Scientifique du Chili. Actes. STAVANGER.—Museum. Aarsheff. StockHoLm.—K. Svenska Vetenskaps Academi. Archiv for Botanik ; Archiv for Zoologi. Sypnty.—Australian Museum. Catalogues ; Memoirs ; Records. Tirtis.—Kaukasisches Museum. Mitteilungen. Toxyo.—Imperial University. College of Science, Journal ; *College of Agriculture, Journal. Societas Zoologica. Annotationes Zoologicae Japonenses. Toronto.—Canadian Institute. Proceedings. TuRIN.—University. Museo di Zoologia ed Anatomia Comparata. Bollettino. UnitEeD STATES oF AMERICA.—Department of Commerce and Labour. Bureau of Fisheries. Bulletin ; Report. UpsaLa.—Regia Societas Scientiarum. Nova Acta. UrspanaA.—lIllinois State Laboratory of Natural History. Bulletin ; *Biological Monographs. *Vimnna.—K. Akademie der Wissenschaften. Bericht der Kommission fiir oceanographische Forschungen ; Mitteilungen der Erdbeben- Kommission ; Sitzwngsberichte (Mat.-Naturw. Classe). K. kk. naturhistorisches Hofmuseum. Annalen. . K. k. zoologisch-botanische Gesellschaft. Verhandlungen. | *WASHINGTON.—Carnegie Institution. Papers on Experimental Evolution. ; United States National Museum. Annual Report ; Bulletin ; Proceedings. United States National Herbarium. Contributions. WELLING TON.—New Zealand Institute. Transactions and Proceedings. *Woop’s Horu.—Marine Biological Laboratory. Biological Bulletin. Zuricu.—Naturforschende Gesellschaft. 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PRESIDENTIAL ADDRESS ON THE MODE OF TRANSMISSION OF SOME TROPICAL DISEASES. By J. W. W. STEPHENS, M.D., D.P.H., Sir Alfred Jones Professor of Tropical Medicine, University of Liverpool. [Delivered November 20th, 1914. ] In a presidential address one may, I think, expect either a consideration of the general principles underlying or on the other hand a summary of what is known about the subject. I do not feel competent to attempt the first nor perhaps is it yet time to attempt it, for the wave of discovery in tropical medicine which began about 1897 has not yet spent itself, and we are still carried along with the current of new facts. I shall endeavour, therefore, to give you simply what I am afraid must be a disconnected account of some of the more recent work on the subject. The great interest of tropical diseases lies, I think, primarily in the fact that in the most important of them one can lay one’s finger definitely onthe cause. This is often not the case in diseases of temperate climes, e.g., scarlet fever, measles, smallpox, numerous nervous disorders, etc., etc. And secondly, in many tropical diseases the transmission is by some insect. Again many are due to protozoa, an exceptional occurrence in temperate diseases. To this is due the fact that on the whole, perhaps, they are more amenable to attack by drugs, and moreover we have a second great object of attack, viz., the insect transmitter. Knowing thus in many cases the cause and the transmitter, there is the further great interest in studying the life histories of both, and finally the great probability of success in attacking one or other of the possible links in the chains of the life cycles. 4 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. MALARIA. As you know, this disease is transmitted by mosquitoes, but only by a certain sub-family, the Anophelinae, and further by only a limited number of these, though why some of these transmit while others do not, we do not know. The mode of transmission is as follows: At a certain stage of the fever the malarial parasites, which up to this time have multiplied in the blood and given rise to the fever, presumably by a toxin they secrete, become specialized into sexual forms male and female. When the blood containing these is sucked in by a mosquito, fertilization occurs im the mosquito’s stomach. That this is so, is, I may add, rather hypothetical, as it has not been actually observed, though once or twice only, fertilization has been stated to have been observed under the microscope, in an ordinary wet blood film. One observer, indeed, believes that fertilization takes place.in the blood stream, and suggests that this is the explana- tion why it has been observed so rarely on the slide under the microscope, and never actually in blood taken directly from the stomach of a mosquito immediately after it has fed. Whatever be the truth of this, the next stage is one that can readily be seen in a dissected-out stomach, viz., the encysted fertilized parasite—the oocyst. These are eventually found in numbers from one to two up to a hundred or so on the outer surface of the stomach, viz., in the muscle wall. Growth proceeds in these oocysts, and after various changes the now large oocyst is filled with large numbers of thread-like curved bodies about 12-144 long—the sporozoites. These travel—how has not been followed—to the salwary gland of the mosquito and from there they escape, during the act of biting, into the blood, say, of a healthy person. Their actual escape has not mdeed been seen, but we can safely affirm that they do escape, as healthy people bitten by infected anophelines contract malaria. eo Se Se ae ee eC mL. renee TRANSMISSION OF TROPICAL DISEASES. 5 Christophers and myself made many attempts to see the sporozoites penetrating the red cells by mixing the two together in a wet film, but always unsuccessfully. Schaudinn stated that he had seen it, using sporozoites from an oocyst in the - stomach (but I would remind you that many of Schaudinn’s observations have not been confirmed). This development in the mosquito takes about ten to fourteen days, so that it is only after this lapse of time that a mosquito that has bitten a malaria patient is capable of transmitting the infection. This cycle in the mosquito is known as the cycle of new infection. It is again only some ten days after being bitten that the attack of malaria develops. In this way the malaria parasite, when it has got into a mosquito develops and gets out again. Malaria or ague has in the past existed in many parts of England, and it is still a memory in the minds of some of that somewhat rare species “the oldest inhabitant.” Why malaria died out in England is not, I think, perfectly clear. The factors that are generally invoked to explain its disappearance are the use of quinine, drainage—which decreased the breeding areas of anophelines, the abolition of the window tax, etc. But there is a factor which though general in its action, is probably as creat if not greater than any of these—viz., the social and hygienic improvements in the condition of the people. For it has been pointed out by Christophers in India that, caeteris paribus, the ravages of malaria are greatest where the social standard is lowest. Poverty, for instance, is an important determining factor. I have spoken as if malaria had disappeared from this country, but a case has recently been reported from Romney Marsh, an old haunt of the disease, in which the source of infection could not be traced, and one if not two other cases have occurred. The occurrence of malaria at the present time in this country, then, is an extremely rare phenomenon. One, 6 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. indeed, is surprised that it is so rare, for there are stillin England abundance of Anophelines, e.g., in some of the ditches and marl pits in Wirral larvae abound, and it is the same elsewhere, and the return from the tropics to this country of cases of malaria is by no means rare. How the malaria parasite arose, whether from some form already existent in the gut of the mosquito, is a very interesting problem of. which at present we know almost nothing. : In connection with the disappearance of malane from England, I might note the peculiar fact that Christophers and myself found in India, in the midst of intensely malarial districts, villages which were completely free from malaria although Anophelines abounded. We could find no explanation of this very peculiar condition, but the same set of conditions is also reported in Italy, and the term Paludismus sine malaria is used to designate them. Though this, baldly stated, is the malaria-mosquito cycle, yet we must consider another side to the question in order to fully understand how and why it is that Huropeans contract malaria in the tropics. On examining a number of native children who appear to be in the best of health, one is surprised to find that a certain percentage, sometimes 80-90 per cent., contain parasites in their blood. These children abound in native villages, and the huts which they inhabit are infested with Anophelines, and a certain percentage of these latter are infected and infective—..e., contain sporozoites in their salivary glands. If now Kuropeans live in the vicinity of native huts, as is only too commonly the case in Africa, they soon get malaria. Itis the native children that constitute, so to speak, the reservoir of the disease in the tropics. They are the great danger. Prophylaxis is simple: remove the Kuropean quarter to a safe distance beyond the usual range of a mosquito’s flight—quarter to half a mile. This method has been carried out in many colonies with very good results. TRANSMISSION OF TROPICAL DISEASES. 7 Of course the two fundamental methods are either (1) killing the parasites in the blood by means of quinine, a method which in Italy has yielded excellent results, or (2) destruction of mosquitoes, or rather their larvae; this in some places has also given very good results, but frequently it is difficult of performance—at least with the funds available. YELLOW FEVER. Is a disease the cause of which is unknown, but we know how it is transmitted, viz., by a particularly annoying mosquito from its persistent attempts at biting—Stegomyia fasciata. We know with regard to the mode of transmission that the mosquito can only transmit if it has bitten a patient not later than the third day of the disease, and that it is able to transmit the disease only twelve days later. That is practically all we know. But there is one mystery about the mode of transmission. Yellow fever is, so far as is known, only contracted at dusk or at night, not in the day time. Yet the Stegomyia bites at all times of the day and night. Only one explanation of this peculiarity has been suggested. It is that young mosquitoes bite in the day and old mosquitoes at night. This change in habits takes place when the mosquito is about three days old, i.e. when she first lays eggs. As the mosquito is only dangerous twelve days after it has bitten a yellow fever patient, no day-biting Stegomyia is ever dangerous. This explanation, however, seems to be too good to be true. The Stegomyia is a so-called domestic mosquito, i.e. it breeds in water supplies about the house, tins, barrels, water troughs. etc., etc. It is surprising what quantities of larvae and pupae a single barrel can produce. They are as thick as tadpoles in a shallow pool. It is this fact, viz., the domestic habits of this mosquito which make it comparatively vulnerable 8 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. to attack, and it is in yellow fever that prophylactic measures have obtained their greatest success. To attack the malaria mosquito—the Anopheline—breeding in marshes, rivers, streams, wells, pools, and in fact any collection of water, 1s a much more difficult matter, but it is largely a question of finance. PHLEBOTOMUS FEVER—THREE-DAYS FEVER—OR SAND-FLY FEVER—SUMMER INFLUENZA. This is a very unpleasant fever while it lasts, viz., three days, but fortunately is never fatal, but presents, however, some resemblances to yellow fever. The disease exists along the Mediterranean littoral, im Egypt, India, and probably all over the world. It is a great cause of sickness among our troops in Malta, India, etc. The cause of the disease is unknown, but the blood is infective from the first two days of the fever. The infective matter, whatever it be, will pass through a fine filter candle. The disease is transmitted by certain species of sandflies. The facts of transmission are that a sandfly that bites a patient during the first day or second day of the fever becomes infected, but it is only about a week later that it can transmit the disease. Comparatively little is so far known about the life history of sandflies, the greater part of what we do know we owe to Newstead’s labours in Malta. They breed in caves and dark places, and lay their eggs in cracks in the soil, in old walls, etc. The egg stage lasts about eight days, the larval stage lasts two to eight weeks, pupal stage, attached to stones, two weeks. Flies in captivity live only ten days. | The flies themselves, so far as we know at pene do not survive the winter, but do so in the larval stage. The question then arises: how does the disease survive the winter? It is —_—_— TRANSMISSION OF TROPICAL DISEASES. 9 thought not to do so in man, as relapses in man are practically unknown (at least after any long period), but it is still just possible that the blood may be infective even though the patient is well, though against this is the fact that transmission experiments only succeed during the first two days of the fever. The other alternative is that the disease is transmitted through the larvae of the flies. Our knowledge is at present accordingly incomplete, and it is so, largely owing to the fact that these minute flies, which easily pass through a mosquito net, only survive captivity for about ten days, and so experimental work with them is difficult. SLEEPING SICKNESS. Is due ‘to two different species of trypanosomes, viz., Trypanosoma gambiense and T. rhodesiense. The former trypanosome is the cause of sleeping sickness in most parts of Africa where the disease exists, while 7’. rhodesiense is confined to Rhodesia, Nyasaland, and a few other adjoining territories. One peculiarity of the latter form of the disease is that the number of known cases is few, say about a hundred, but on the other hand the disease is even more deadly, or at any rate more rapidly fatal than that due to T. gambiense. The trypanosomes in each case exist in the blood and the disease is transmitted by tsetse flies, 7’. gambiense by Glossina palpalis and T. rhodesiense by Glossina morsitans. The mode of transmission is not simply mechanical, but a developmental cycle takes place in the fly, for a fly after biting a sleeping- sickness patient is only capable of transmitting the disease to a healthy person some twenty to thirty days later. The mode of transmission of 7’. gambiense is the following. The trypanosomes which are sucked into the gut disappear more or less completely in about a week, to reappear later as short stumpy crithidial forms. These eventually find their way to the 10 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. salivary glands, how is not known. I should add that the mode of development is different in the case of other trypano- somes. A further point for consideration arises. In the case of malaria the disease is purely a man to man infection, and we have the important factor that the native children, who for the most part appear to be in abundant good health, have numerous parasites in their blood, they constitute in fact the reservoir from which, for example, the European gets infected. It isamantomandisease. In sleeping sickness this is notso, Thenatives do not here constitute a permanent reservoir, for the simple reason that they all die. How then does infection spread? The answer is that the same trypanosome, at least this appears to be proved in the case of T. rhodesiense, occurs in native game. The game enjoys an immunity to the disease. The game infects the fly and so the disease is transmitted to man. The game constitutes the reservoir. What should be done in Africa to limit this spread of the disease by the game is a question into which a special Commission enquired this year. It is proposed to try an experiment to see what will be the result of exterminating or reducing the quantity of the game from a small defined . area. It is practically impossible to attack the fly. T. tullochi and T. grayi. It may be further noted that tsetse flies contain in nature these trypanosomes in their gut. So far inoculation of these into animals has produced negative results, but it would appear as if they deserved re-study. KALA-AZAR. Is an extremely fatal disease prevalent in India, Sudan, parts of China, and elsewhere. The parasites that cause this disease exist in large numbers in endothelial cells in the spleen, liver, bone marrow, etc. A disease indistinguishable TRANSMISSION OF TROPICAL DISEASES. 18 | clinically from this exists in the Mediterranean littoral, but there is this difference, that in these countries the disease is almost entirely confined to children, though very occasionally adults also contract it. Both these diseases are caused by parasites which are about the size of one-third of a red cell and have two nuclei. They can be cultivated on blood media, where they change into slender spindle-shaped flagellates. Further, in the Mediterranean countries there 1s a wasting disease in dogs which is also due to a parasite indistinguishable from that producing the disease in children and adults in India, and it is thought that there is some connection between the disease in dogs and the disease in children. The existing evidence is to the effect that this disease in children is transmitted from dogs by the agency of fleas, though others disbelieve this and claim that mosquitoes are the transmitting agents. There is no evidence however that in India this disease in dogs exists at all, so that we are puzzled by the apparent relationship of the dog disease in the Mediterranean and the non-relationship in India. The matter is further complicated by the fact that in many parts of the world a disease occurs, viz., Oriental sore. This is purely local skin affection, but in the sore, parasites are found indistinguishable from those that occur in the general infection of kala-azar. I should mention in connection with these diseases some interesting French work recently done. It is known that in the gut of many insects spindle-shaped flagellates occur. Laveran and Franchini find that the injection of these flagellates into mice gives rise to non-flagellate forms in the organs indistinguishable from kala-azar parasites, and in fact the mice die of the infection. Now in these diseases, viz., kala-azar, infantile kala-azar, dog kala-azar, and tropical sore, numerous investigators have been searching actively for the transmitting agent, e.g., fleas, bugs, mosquitoes, 12 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. sandflies, etc., and vainly experimenting to procure develop- mental cycles in these supposed transmitting agents on the analogy of malaria, sleeping sickness, etc. In the case of the dog disease successful transmission results have indeed, it is stated, been got through the agency of fleas. If we assume for the moment the accuracy of these transmission. experiments, it does not, it seems to me, follow—keeping in view Laveran and Franchini’s experiments—that they necessarily prove that the flea is the transmitting agent in the accepted sense, viz., that it transmits from dog to dog in this case. It is possible that the dog disease is due to the inoculation by fleas of their natural gut flagellates, and that each case of the dog disease arises de novo from the flagellates of a flea; the disease is in fact a flea-dog disease, but not a flea-dog-flea disease. We might, indeed, call it hemi-cyclical transmission, not cyclical, and so for all the other similar infections kala-azar, infantile kala-azar, and tropical sore. But I need hardly remind you that this is sheer hypothesis. Should it prove to be true it may also apply to diseases due to helminths and possibly bacteria. For instance, we have a Nematode disease in man and cattle characterized by the presence of nodules of worms in the skin, but. as no larvae apparently exist in the blood it is at present difficult to understand how the nematodes get out, 1.e., the disease gets transmitted. On the hypothesis I have just put forward, the explanation would be that these nematodes were derived from forms existing in the guts of insects or possibly in water. I would suggest also that Sarcosporidia form an example of hemi-cyclical infection, 1.e., they get into animals but do not get out. I must apologise for this digression into the pleasant and easily-trod path of hypothesis, but will now return to matters of fact. | ‘9 ee es Pee TRANSMISSION OF TROPICAL DISEASES. 13 SPIROCHAETES. These give rise to relapsing fever in man. The term relapsing is a very appropriate one, for after a fever lasting some days and a period of apyrexia, the whole train of symptoms recurs again. There may be more than one relapse, but the succeeding relapses are not so intense as the original fever. The disease is due to the presence of spirochaetes in the blood which, however, disappear therefrom in the interval between the attacks. There are many species of spirochaete described inman. It will suffice to mention two only, viz., Sp. duttoni, producing the African disease, and Sp. recurrentis, producing the European form. The African disease is transmitted by ticks and the European probably by lice. In the African form the tick involved is known as Ornithodorus moubata. It is a dirty-lookmg, brown, wrinkled tick, and frequents rest-houses where it is found in the walls, thatch, etc., and is found in the dust around trees where caravans halt. Unlike most ticks, after feeding it crawls away. The tick is infective probably directly after biting. The life cycle of the spirochaete is, however, believed to bethefollowing. The spirochaete in the gut of the tick breaks up into numerous ovoid granules resembling small bacili. These pass into the cells of the malpighian tubules and into the ovary. When the tick bites, these ovoid bodies are voided in the faeces and so contaminate the coxal fluid which is secreted when the tick bites, and so presumably the wound. Those that reach the ovary pass through the egg and nymph into the adult tick, so that the disease is transmitted from mother to offspring. This is said to hold good also for a second generation. Others, however, doubt the infective nature of the ovoid granules. Nothing much is known at present as to the mode of 14 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. transmission of the Kuropean and Indian relapsing fevers, but that lice and not bugs, as first thought, are the trans- mitting agent appears fairly certain. PLAGUE. Is a disease by no means exclusively tropical. It is a disease of which one of the chief signs is the great swelling of the glands, and hence the term Bubonic plague. The disease is a septicaemia, that is a blood poisoning in which the plague bacilli exist in the blood. Now it has long been known that the outbreak of plague in a place or district is preceded by a mortality among the rats—in fact the rats are dying of plague. We have then to consider the rat factor. Further, it has now been proved that the disease is transmitted from rat to man by means of a particular rat flea, viz., Pulex cheopis. How does this come about? In India we have two rats which are important in this connection, viz., Mus. decumanus, the ordinary brown sewer rat, and Mus rattus, the black or house rat. Now it is found in India that the course of plague is the following. Firstly, there is an epizootic among Mus decumanus, i.e., the rat mortality as shown by the dead rats collected is going rapidly up, say in July. It reaches a maximum ; ten days later there is an epizootic among Mus rattus, and then ten to fourteen days later the plague epidemic in man starts. The relationship of plague in man is thus more close in the case of Mus rattus than it is in the case of Mus decumanus. ‘This association of plague with M. rattus is shown by the followimg curious fact. Plague is equally distributed on all floors of a building, so is M. rattus, but M. decumanus does no go beyond the third floor. We next turn to the flea. It has been shown by numerous experiments —the experiments of Russian investigators though overlooked TRANSMISSION OF TROPICAL DISEASES. 15 were the first to prove it—that the flea transmits the disease from rat to rat, and there can be no doubt from rat to man. How does it effect this? This has only recently been explained. When the flea sucks blood containing bacilli into its stomach, the bacilli rapidly multiply and to such an extent that they actually block the oesophagus or rather the proventriculus. The result of this block is that the flea can get no more blood into its stomach and so feels thirsty. In order to relieve its thirst it goes on sucking by means of its pharyngeal pump, but as the blood cannot pass the block some of it regurgitates, owing to the increased pressure back into the wound, but it is now contaminated by the bacilli in the blocked proventriculus and so the wound gets infected. FILARIASIS. One of the most noteworthy conditions included under this heading is Elephantiasis. This is a condition, as you know, in which there is great swelling, e.g., of the legs due to obstruction in the lymphatics caused by quite delicate worms about 14-4 inches long. These worms give birth to larval forms, which are about one-third mm. long, and they find their way from the lymphatics into the blood stream where they move freely about. When a mosquito sucks blood containing these larvae, the latter pass—how has not been followed—into the thoracic muscles of the mosquito and there come to rest. After a time they become active again and are now found in the proboscis of the mosquito, not in the tube which conducts the blood up, or in the tube which conducts the saliva down, but in the muscular substance of the labium, the rod in the groove of which the tubes and cutting lancets he. They are thus in a cul-de-sac, and it is thought that they emerge by rupture of a membrane which is stretched 16. TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. over the end of the rod. They now find themselves on the skin, and in all probability they burrow their own way in through the skin and so are not injected with the blood as was naturally at first thought. Here then we have an example of a nematode worm for the transmission of which an inter- mediate insect host is necessary. What becomes of them and what happens to them from the time they enter the skin to the time at which they are found as adults in the lymphatics, is unknown. ANKYLOSTOMIASIS. This is a disease due to the presence of minute worms about one-third of an inch long that live in the gut of man. They bury their heads in the gut and produce their serious effect, in all probability, by a poison that they secrete into the wound that they make as they feed on the submucous layer of the cut. The ravages caused by this parasite in the Southern States of America are widespread, and the Americans are now making a great attempt to stamp out the disease. The mode of attack is two-fold. (1) Medicinal. Luckily the worms are amenable to treatment, and in the drug Thymol, first used by Bozzolo in 1880, we have a potent weapon. (2) Improved sanitation. In order to understand how this is applied we must first understand the life-history of the worm. The worms live in the gut of man. Their eggs pass out in the excreta. Here they hatch and become larvae. These larvae are able to penetrate the skin, so that a person walking bare-footed on contaminated soil is very likely to get mfected. The larvae as they pass through the skin cause a good deal of itching. They eventually, by a circuitous route through the lungs and trachea, get swallowed and so pass down the gullet into — TRANSMISSION OF TROPICAL DISEASES. 17 the stomach and gut. The sanitary mode of attack then is a simple one, viz., prevention of the contamination of the soil by infected patients. Where primitive hygienic methods still exist the introduction of new methods is always difficult and expensive, but the principle involved is perfectly simple. If sanitation were at a stroke made perfect, the great scourge Ankylostomiasis would be wiped out. The medicinal treat- ment is necessary for the killing of the worms in the infected patients, and except for this purpose would be unnecessary if sanitation were perfect, but as this is far from being so the medicinal mode of attack is still very necessary. It is a question of breaking one link in the chain of the life-history— the thymol kills the adult worm ; improved sanitation prevents the eggs developing in the soil. The chain is in both cases broken. It should be added that the larvae can also infect if they are swallowed, e.g., in drinking water, but whether this mode of infection or that by the skin is the more important remains to be seen. BERI-BERI. Is a disease characterized by general oedema of the body and various paralytic symptoms. It is common in China, and was responsible for much mortality in the Japanese navy. It is seen in jails, on expeditions, and a form of it occurs on board ship, e.g., a ship put into Birkenhead in 1914 with thirty- three cases on board. It has long been suspected that there was some connection between the disease and eating of rice, but this others denied. It has now been shown that it is only particular kinds of rice that give rise to beri-beri. In fact it is the kind of rice that we consume here daily in our rice puddings, i.e., what one may call a nice clean-looking white rice. Why we do not get beri-beri will appear later. The difference between this rice and the rice in its original form, 18 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. which is called paddy, is that paddy has its layers of silver skin intact, while in polished rice there is hardly a vestige of these remaining. The difference between these two rices is shown very clearly by feeding a pigeon on polished rice. In a few days most serious nerve trouble arises, and the pigeon is quickly paralysed and dies. It is further a remarkable fact that if some rice bran, i.e., the polishings of rice, be given to the pigeon when symptoms have developed, recovery takes place in a few hours, as if in a magical way. In the case of man, the different effect of the two rices is shown by dividing the inmates of a jail into two lots, one being fed on polished rice, the other on unpolished rice, i.e., paddy. Beri-beri soon breaks out among the first lot, whereas the second is exempt. This has now been shown time after time, and it is quite clear that beri-beri in many cases is due to eating polished rice, i.e., in fact a rice which is deficient in something that native rice possesses. Beri-beri is, in fact, what is called a deficiency disease. The reason we do not acquire beri-beri is that we make up the defect which is present when we eat white rice by supplying it from various articles of the mixed diet we consume as well; but where, as is often the case in the tropics, rice is the staple diet, then if this is a white rice, beri-beri breaks out unless a mixed diet is being taken in addition and in sufficient quantity. A rice capable of producing beri-beri is, or was, in use on P. and O. steamers, but the lascars do not as a rule get beri-beri because they also receive sufficient other rations. One must, however, admit that the consumption of polished rice or other cereals which are defective from the fact that they have their skin removed, e.g., a white flour, will not explam all cases of beri-beri, but that this is very often the cause there can be no doubt. : Now the importance of this work on beri-beri not only is very great in relation to the elimination of this disease, but : ; ; TRANSMISSION OF TROPICAL DISEASES. 19 it has thrown light on two other diseases not exactly tropical. The first of these is Scurvy, which we are wont to associate with Arctic expeditions. This disease also is a deficiency disease, for scurvy can readily be produced in guinea-pigs by feeding them exclusively on bread. It is not simply a case of starvation, for guinea-pigs may be starved by feeding on cabbage alone, but they show no signs of scurvy. There is an absence of something in the bread diet which they require. What it is, we do not know. This hypothetical body we call a vitamine. In all probability another deficiency disease is Rickets, a disease characterised by a failure of ossification in the bones. The disease is cured by change of diet. The ordinary process of growth is also dependent on the presence of these, at present mysterious, bodies, “growth vitamines ” as we may callthem. Rats, e.g., will not thrive on an artificially purified diet, but if a quantity of milk equal to only 1 per cent. of the artificial food be added, growth proceeds normally. This last fact raises a very important question. Growth we have seen is dependent on a vitamine. Now cancer is a pathological growth, and im all probability this growth is also a dependent on a vitamine. Now if the vitamines for ordinary and cancerous growths were different, it might be possible to construct a diet which would exclude the cancer vitamine, but this is visionary, for we are at present only at the beginning of our knowledge of vitamines. I would claim, then, that tropical medicine in this matter has not only extended its own sphere of knowledge, but has been of distinct service to medicine of temperate climes. "9109 “£ ‘Jord Aq ydvasojoyd v wt07 7] ‘QSUM UJION OY} Wlorz UOWRIS [ROISoTOTG UII Jog OUT, “T “HI ee ee ae eee 21 THE MARINE BIOLOGICAL STATION AT PORT ERIN BEING THE TWENTY-EIGHTH ANNUAL REPORT OF THE LIVERPOOL MARINE BIOLOGY COMMITTEE. In this very exceptional year, when many concerns not directly connected with the necessities of existence or the conduct of a great war must suffer more or less, it is not surprising to find that we have a less successful year than usual to record at our Biological Station. Since early in August the thoughts and energies of most of us have been diverted to other channels; and although it is right that in the interests of others we should endeavour to keep all our affairs, so far as may be possible, running normally, still until more important and pressing matters are settled it 1s well that no unnecessary time, labour and expense should be devoted to what is not essential at the moment. Consequently the Committee and our other supporters and readers will, | am sure, understand and approve if the Report this year takes a shorter form than usual, and deals with little beyond the record of routine work carried out, especially during the earlier portion of the year, at the Port Erin Biological Station and elsewhere in the L.M.B.C. District. The “Station Record” and the “Curator’s Report” which follow show that during the Easter vacation and the Spring months, when both students and senior workers frequent our marine laboratory more than at any other time of the year, the numbers were larger than ever before and the work was abundant and of high quality. Last year we recorded about seventy researchers and students; this year up to the end of July we had a total of ninety occupying work-places in the 22, TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. laboratory. It is interesting to note that of the three researchers | at work during June and July one was a Frenchman from the University of Nancy and another a Belgian from Louvain. Since then we have had no one at work except our own staff. The effect of the war upon the number of visitors to the Aquarium was most striking. During the earlier part of the Summer the numbers steadily increased beyond those of previous years, and on Saturday, August Ist, the total was 1,054 in advance of that of the same date last year, but from that time the numbers declined rapidly. Durmg August, which is usually our busiest month in the Aquarium, Port Erin was, comparatively speaking, deserted, and our total number of visitors for the year is now 12,031—a decrease of 4,576 compared with 1913. The number of “Guides” to the Aquarium sold to visitors is 657—a decrease of 383 compared with last year. _ It may be useful to students and others proposing to work at Port Erin that the ground plan of the buildings showing the laboratory and other accommodation should be inserted here (see fig. 2, p. 23). In regard to the educational work in the laboratories, the usual Haster vacation courses in Marine Biology were carried on during April under the guidance of the teachers in the Zoology and Botany departments of the University of Liverpool. Other groups of senior students came in parties or singly from at least a dozen Universities and Colleges. For example, a group of nine senior students and teachers led by Professor Cole came from University College, Reading, and another nine in charge of Dr. Stuart Thomson from the University of Manchester. A group of seven students from University College, Nottingham, attended a course under the direction of Mr. H. 8. Holden, Lecturer in Botany. We had two workers from the University of Birmingham, and other Universities represented were Cambridge (including Newnham), MARINE BIOLOGICAL STATION AT PORT ERIN. 23 é c} ~ = w Engine and Pump room HATCHERY. OfpossDy Hs a 3 Hatching Tanks. The new laboratory of Bio-Chemistry coreg ULLILULL T 4 Vestibule Plan of the Port HKrin Biological Station, showing /0 = = c < = o < Fi Director Spiral slarrcase Feet = _ 4 S f= & is ig | oS 8 fe Fig. 2. Research wing on both floors. is over the Hatchery on the right hand side of the plan. i ja ei oe Bean Tank Tank 18 el = |B) " | Ul weal Ho | Y = = ay eae) Sorlinglable Sorting table 3 | Sea? able rye Store reom Deep tank aia si — ts 5 — — NNN LABORATORY. Steps fo upper floor Ceneral work room er floor of hk Laboratory Upp seaTco Re 24 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Harvard, Melbourne, Bangor, Cardiff, Nancy, Louvain, and — the Imperial College of Science at South Kensington. Collecting expeditions as usual, both at sea and along the shore at low tide, were arranged during the Easter vacation. During the remainder of the year the Curator and his staff made periodic collections from time to time as occasion offered, and plankton samples were taken across Port Erin Bay with regularity twice in each week throughout the year. As on previous occasions, I shall first give the statistics as to the occupation of the “ Tables” during the year, then will follow the “‘ Curator’s Report,” and after that the reports that have been sent to me by various investigators on the work they have done. THE STATION RECORD. The past year has established another record as regards the number of researchers and students, no fewer than ninety having occupied our laboratories. Of this number eighty-five paid visits of varying duration from March 23rd to April 20th, while the remaining five came to conduct research at the beginning of the year and in the earlier part of the summer vacation. . List oF WORKERS. Dec. 19th, 1913 to Jan. 8th. Mr. M. C. Vyvyan.—Marine Alge. Dec. 27th, 1913 to Jan. 5th. Professor Herdman.—Oficial. 99 Mr. G. A. Herdman.—Bio-Chemistry. Feb. 27th to March 2nd. Professor Herdman.—Ofifcial. 75 Professor Moore.—Bio-Chemistry. ~ March 23rd to April llth. Mr. G. F. Sleggs.—General. a Miss G. A. Platt.—General. = Miss D. Thornton.—General. ) Miss M. M. Brew.—General. 5 Miss A. S. Meeson.—General. 2s Miss J. Price.—General. March 23rd to April 4th. Miss N. Lodge.—General. <> Miss E. Catlow.—General. a Miss C. Whittaker.—General. ‘s Miss J. Curwen.—General. “e Miss J. Lord.—General. - Miss D. Dobson.—General. a Miss M. Tesh.—General. “MARINE BIOLOGICAL STATION AT PORT ERIN. 25 March 23rd to April 11th. March 25th to April 11th. March 26th to April 9th. March 26th to April 11th. March 30th to April 21st. March 30th to April 24th. March 30th to April 13th. 39 March 31st to April 4th. March 31st to April 11th. 29 March 31st to April 13th. April 1st to 21st. April 1st to 14th. April 1st to 21st. April 2nd to 11th. April 3rd to 17th. April 4th to 18th. April 6th to 11th. 9? April 6th to 19th. 9? April 6th to 20th. April 6th to 21st. April 6th to 20th. April 6th to 28th. April 7th to 19th. April 7th to 14th. > April 10th to 14th. Mr. 8. T. Burfield.—Educational. Mr. R. D. Laurie.—Educational. Miss H. V. Davies.—General. Miss H. Clarke.—General. Professor R. J. Harvey Gibson.—Educational. Miss M. Knight.—Educational. Miss R. olden: —Marine Alge. Miss M. Jepps.—Marine Alge. Professor Herdman.—Plankton. Miss M. Latarche.—General. Miss L. Baker.—Lichens. Miss P. McKie.—Lichens. Miss E. M. Blackwell.—Marine Alge. Mr. H ‘ Mr. H. G. Jackson.—Plankton. Mr. A. J. Nicholson.—General. Miss H. M. Duvall.—General. Mr. B. Sahni.—Marine Alge. Miss E. Richmond.—General. Dr. Stuart Thomson.—Educational. Mr. G. H. Crabtree.—General. Mr. A. W. Summersgill.—General. Mr. G. Talbot.—General. Miss A. Dixon.—General. Miss F. Lea.—General. Miss C. M. Lightbown.—General. Miss Williamson.—General. Miss E. N. Cowell.—General. Mr. H. T. Cubbon.—General. Miss D. Jones.—Marine Alge. Miss J. L. Millican.—Marine Alge. Mr. H. S. Holden.—Educational. Mr. E. Holden.—General. Mr. M. Straw.—Marine Algeze. Miss C. N. M. Brett.—Marine Alge. Miss E. 8S. Hillman.—Marine Alge. Miss J. M. McClatchie.—Marine Alge. Miss D. Bexon.—Marine Alge. Miss H. C. Bowser.—Marine Alge. Miss H. H. Maguire.—Marine Alge. Mr. J. R. Bruce.—Marine Algze. Miss G. H. Wood.—Marine Alge. Miss E. M. Tate.—Marine Alge. Miss KE. Alexander.—Marine Alge. Miss E. M. Mather.—Marine Alge. Miss M. Bradley.—Marine Alge. Miss G. Hanna.—Marine Alge. Miss G. Wilkinson.—Marine Algz. Miss D. Lamble.—Marine Algez. Miss F. Tozer.—General. Miss O. V. Ellams.—General. Miss G. V. Buchanan.—Plankton. Miss R. Robbins.—Zostera. Miss R. C. Bamber.—Echinoderms. Professor J. B. Farmer.—Mosses and Liverworts. Mr. R. J. Tabor.—Lichens. Miss H. Coburn.—Marine Algz. Miss H. C. New.—Marine Alge. 26 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. April 8th to 15th. 99 April 10th to 24th. April 13th to 19th. 99 June 5th to 18th. June 5th to July 3rd. June 29th to July 11th. Professor B. Moore. = Jue Chemistry. Mr. E. Whitley.—Bio-Chemistry. Mr. J. Erik Hamilton.—General. Professor F. J. Cole-—Educational. Mr. H. L. Hawkins.—Educational. Miss N. Eales.—Educational. Miss D. Crofts.—General. Mr. Mr. Mr. Mr. Mr. W. Baker.—General. F. D. Withers.—General. McKenzie.—General. Mr. M. Smith.—General. W. Kings.—General. W. H. Evans.—General. Miss EK. L. Gleave.—Archidoris. Miss D. Hey.—Marine Alge. Miss M. Shaw.—Marine Algae. Rev. §. Frappé.—Gills of Lamellibranchs. Dr. P. Debaisieux.—Parasitology. Mr. H. 8. Holden.—Himanthalia. % The “Tables”* in the Laboratory were occupied as follows :— Liverpool. University Table :— Professor Herdman. Professor Moore. Mr. R. D. Laurie. Mr. S. T. Burfield. Mr. G. A. Herdman. Mr. H. T. Cubbon. Mr. H. Storey. Rev. 8. Frappé. Mr. A. J. Nicholson. Mr. R. J. Tabor. M anchester University Table :— Dr. Stuart Thomson. Mr. G. H. Crabtree. Mr. A. W. Summersgill. Birmingham University Table :-— Mr. H. Gordon Jackson. University College, Reading, Table :— Mr. F. D. Withers. Mr. McKenzie. Mr. M. Smith. Professor Cole. M.H. L. Hawkins. Mr. W. Baker. Mr. J. Erik Hamilton. Professor Harvey Gibson. Mr. W. H. Evans. Miss M. Knight. Miss E. M. Blackwell. Miss EK. L. Gleave. Liverpool Marine Biology Committee Tables :— Dr. Paul Debaisieux. Mr. B. Sahni. Miss EK. Richmond. Miss R. Holden. Miss M. Jepps. Mr. M. C. Vyvyan. Mr. G. Talbot. Miss A. Dixon. Miss F. Lea. Mr. A. J. Nicholson. Miss M. Latarche. Miss R. Robbins. Miss R. C. Bamber. Miss H. M. Duvall. Miss F. Tozer. Miss D. Hey. Miss L. Baker. Miss P. McKie. Miss G. V. Buchanan. Miss M. Shaw. Mr. E. Whitley. Miss C. M. Lightbown. Miss Williamson. Miss E. N. Cowell. Mr. W. Kings. Miss N. Eales. Miss D. Crofts. * Since the new research wing has been added several distinct apartments are generally available for the accommodation of the investigators assigned to any one of the University ‘‘ Tables.” MARINE BIOLOGICAL STATION AT PORT ERIN. 27 The following students of Liverpool University occupied the laboratory for periods varying from a fortnight to three weeks during the Easter vacation, and worked together under the supervision of Professor Harvey Gibson, Miss E. M. Blackwell, Miss M. Knight, Mr. R. Douglas Laurie and Mr. 8. T. Burfield :— Mr. R. J. Bruce. Miss J. Curwen. Miss E. M. Tate. Mr. G. F. Sleggs. Miss A. 8S. Meeson. Miss E. Alexander. Miss H. C. New. Miss J. Price. Miss EK. M. Mather. Miss G. A. Platt. Miss J. Lord. Miss M. Bradley. Miss D. Thornton. Miss D. Dobson. Miss G. Hanna. Miss N. Lodge. . Miss M. Tesh. Miss D. Jones. Miss M. M. Brew. Miss H. V. Davies. Miss J. L. Millican. Miss E. Catlow. Miss H. Clarke. Miss G. Wilkinson. Miss C. Whittaker. Miss G. H. Wood. Miss D. Lamble. The following students of University College, Nottingham, attended a course in Marine Botany, conducted by Mr. H. 8. Holden :— Miss M. L. Straw. Mr. E. S. Hillman. Miss H. C. Bowser. Miss C. N. M. Brett. Miss D. Bexon. Miss H. H. Maguire. Miss J. M. McClatchie. CuRATOR’S REPORT. Mr. Chadwick reports to me as follows on the various departments of the work :— The Fish Hatchery. “Hatching operations were begun this year on February 5th, an unusually early date, when a batch of 124,000 plaice eggs were put into the hatching boxes. The first batch of larvae, numbering 346,000, was set free on February 23rd. Quantities of eggs, exceeding half a million in number, were skimmed from the ponds on March 23rd and 30th respectively ; and amongst the large batches of larvae set free by Professor Herdman from his steam yacht ‘ Runa’ were three, numbering 749,700, 1,211,800 and 722,350 respectively. The spawning season closed on April 25th, the total number of eges collected being 8,895,650. 28 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. “The Hatchery Record, giving the number of eggs collected, and of larval fish set free on the various days, is as 8,895,650 Total eggs. follows :— : Eggs collected. Date. Larvae set free. Date. 210,600) at eb.) a5) te ok2 346,050 .. Feb. 23 183,750 oO. to. 20 162,750 .. Manon? 393,600 oY ee 27,900 ... een 469,350 24 to March 3 391;950-- ae 352,800 _ March 4 and 5 300,100. 4 9) eee 426,300 “e 7 and 9 384,300. 225 See 741,600 ior e all tose 615,600 eee 530,250 5 .. w4cande Ls 446,250 April 2 279,300 eee 6) 248,850" ee 4 882,000 >» 20 and 23 149,100" ae 8 1, 180,200 ie .22 24023 1,211,800. 1. See 850, DOO. dhe esc OO 122,350° 53 eee 420,000 Acoril | 364,350 .. eae 409,500 3 346,500 oo ae 525,000 z= 4 and 7 437,850 aS 252,000 a 9 210,000 3 20 508,000 jj allan aS 380,950 ee 348,600 loons 239,150. gle ee 132,300 3 20 to25 81,950 -2eMay ee 7,707,350 Total larve. ‘The early hatching season was probably due to the mild winter. The first plaice eggs were found on the surface of the pond on January 28th, and on February 3rd embryos at least a week old were obtained. In recent years the first fertilised eogs have generally been obtained on some date between the middle of February and the first week of March. So the present season is at least a fortnight earlier than usual. Lobster Culture. “The lobster hatching and rearing was this year under- taken by the Assistant Curator, Mr. T. N. Cregeen, and the increased success attained was due to his untirig and pains- taking efforts. By personally interviewing local fishermen MARINE BIOLOGICAL STATION AT PORT ERIN. 29 he succeeded in obtaining 39 berried lobsters with nearly ripe eggs, a substantially larger supply than had hitherto been attainable. As the lobsters were brought in they were at once put into the pond, in which a number of hiding places, built of bricks and rough stone, had been prepared. The first newly-hatched larvae were taken from the pond on May 28th, and from that date to September 5th constant and sometimes large supplies of larvae were obtained, the total number being 40,500, an average of about 1,039 per adult lobster. Of these, 16,000 were set free in the first and second stages, and 24,500 were placed in the hatching boxes. By daily feeding with finely-minced fish, ‘liver’ of the edible crab and plankton, and the maintenance of scrupulous cleanliness in the hatching boxes, Mr. Cregeen succeeded in rearing 1,823 larvae to the fourth or ‘ lobsterling ’ stage (see fig. 3). “ As in all our previous experience of lobster culture, there was heavy mortality at the periods of ecdysis; but the loss due to cannibalism was this year very trifling. This was probably due to the plentiful supply of fresh food supplied. Quantities of plankton were taken with a coarse tow-net almost daily, and formed a considerable proportion of the food of the larvae. One thousand seven hundred and seventy-seven of the lobsterlings were set free at suitable parts of the coast line North and South of Port Erin Bay, and 46 were retained for further experiments in rearing. Of 13 lobsterlings hatched during the season of 1913 3 still survive, but their rate of growth has been slower than that of a young lobster of a similar age found in the pond several years ago. The Aquarium. “Until the oubreak of war the Aquarium attracted increasing numbers of visitors, and on August Ist the total number was 1,054 in advance of that of the corresponding aU TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. date last year. Thenceforward the numbers decreased ; but, under the circumstances, the total for the year—12,031—is x. » 4, o 2, <) Y iw pra Y fj 8, Y SZ, rod c\ 6, 9) # 9) \ Y 9) p ee "e @, # 4 He af os, 3" Of fo a, 4 Ot oF a, e Ai tH a, 0 on a 2, 8" Of on G e Ct ir re: x \ O/-\e LJ y HY iP t) y Pee) ee Uy Aah Fic. 3. Young Lobster or ‘“‘Lobsterling”’ stage, after the fourth casting (magnified 5 times). cratifying. It shows that under normal conditions the institution would have fully maintained its popularity. The MARINE BIOLOGICAL STATION AT PORT ERIN. 31 lobster culture in the Hatchery again attracted a large amount of attention on the part of the visitors, and there can be no doubt that it favourably influenced their numbers. During the latter half of the season a small series of dissections were made from common types such as the dog-fish, cod, octopus, etc., and exhibited in jars in the Aquarium. A few additions were made to the drawings which hang on the walls. “The Curator resumed in October his lantern lectures and demonstrations to pupils of the Insular schools. Twenty- seven boys from the local Higher Education School have attended a systematic course of instruction in Nature Study on alternate Wednesday afternoons. General. “The only item of marine zoological interest which calls for notice is the extraordinary abundance of the medusa Aurelia aurita durng the past summer. Large shoals, numbering hundreds of individuals, were seen on many occasions from the middle of June onwards, and it was not until the end of September that they finally disappeared.” H. C. CHapwick. OTHER REPORTS ON WORK. Professor CoLE and his party of colleagues and students from Reading were chiefly occupied, as usual, in observing and collecting, and in making injected preparations of various invertebrata for their College Museum. Professor Cole writes to me :—‘‘ Our Easter party this year consisted of 9 persons, and the work was as usual largely educational.” Mr. Cuapwick has sent me drawings of two remarkable cases of “ twinning” in lobster larvae, which are reproduced in fig. 4. They occurred amongst the ordinary hatched larvae and were noticed while living. The figures are drawn o2 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. from the specimens preserved in formaline after their death. Both figures are magnified about 10 diameters. Somewhat similar monstrosities to these have been described by Herrick* in the case of the American lobster (Homarus americanus), and that author’s opinion is that ‘“‘We have to do here with the fusion of two embryos which ATH ANiIYy Fic. 4. Twinned Lobster larvae, hatched at Port Erin. x 10. are practically distinct from the first.’ It is possible on the other hand that the doubling may be due to an injury or some abnormal condition affecting the ovum or embryo at an early stage. A pair of perfect twins are known to have been produced from one ovum in the case of the European lobster _ (Anderton). * Bulletin of U.S. Fish Commission for 1895, p. 216. MARINE BIOLOGICAL STATION AT PORT ERIN. 33 Dr. ANNIE PorTER has just published* a full account of the new parasitic Flagellate, Herpetomonas patellae, which she discovered in the limpet while working at Port Erin last winter. Herpetomonads are known as parasites in flies and other insects, but this is the first one to be found in a mollusc. The parasite was found in the alimentary canal and liver of eight per cent. of the limpets examined at Port Erin. Dr. Porter, Dr. Fantham, and others, have shown that other species of Herpetomonads found in some fleas are of pathogenic importance and may give rise to fatal diseases in rats, mice, or dogs, that may happen to swallow the fleas and so become infected with the parasite. When reduced to eating limpets it will be well to bear in mind that eight per cent. may contain this pathogenic organism, which, by the way, is closely related to the Trypanosoma which is the cause of “ sleeping sickness.” It is to be hoped that Dr. Porter will extend her investigations to other edible Mollusca so that eventually we may have some idea of the amount of risk, if any, that exists in eating uncooked such excellent food matters as the Oyster, the Mussel and the Scallop. Miss H. M. Duvatt, B.Sc., during the Easter vacation made some observations on the methods of feeding by means of ciliary currents in the Ascidian, Clavelina lepadiformis, which is found occasionally in rock pools on the Bradda side of Port Erin Bay. It was not easy to get the animals to expand fully and feed freely in captivity, but Miss Duvall succeeded in satisfying herself that a band of accumulated food particles and mucus forms in the interior of the branchial sac, neither along the endostyle, nor yet alongside the dorsal lamina (as described by Orton), but about the middle of the lateral walls midway between dorsal and ventral edges, and from that position is drawn posteriorly straight down into the oesophageal opening which terminates the branchial sac. * Parasitology, November 9th, 1914. 34 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Particles of carmine which were added to the water and were drawn in by the animal enabled this band to be seen very clearly extending from the oesophageal opening up to the middle of the branchial sac. Naturally this interesting observation ought to be repeated, both on the same and other species, and Miss Duvall hopes to return to the investigation at the earliest opportunity. Dr. DesBatsiEvux, of Louvain, devoted his attention chiefly _ toa re-examination of Merocystis kathae, the Protozoan parasite found by Dr. Dakin in the renal organ of the whelk some years ago ; and he made a large collection of original drawings of the various stages of its life-history. He also examined various other molluscs and some fishes for Protozoan parasites. Mrx8: 2: BurFIELD, B.A., has continued his work on the remarkable pelagic Arrow-worm, Sagitta bipunctata, which he is investigating from every point of view with the object of producing an L.M.B.C. Memoir on the subject. He was mainly occupied at Port Erin in collecting material from the — plankton samples and in fixing the best specimens for sectioning with a view to histological work. Attempts were also made to keep the adult animals alive in the laboratory with a view to obtaining eggs and early stages, but without much success so far. The work will be continued in all probability durmg next Easter vacation. Miss R. C. BamBer, M.Sc., and Mr. BuRFIELD have started a series of observations on living Echinoderms with the view of ascertaining more precisely the direction of the water current through the madreporite. It is intended to repeat and extend these experiments in order to ascertain whether there are indications of an excretory function in the water-vascular system. Mr. H. G. Jackson, M.Sc., acted as my Assistant in the plankton investigation from the yacht during the KHaster vacation. The results obtained will be given in full in the next MARINE BIOLOGICAL STATION AT PORT ERIN. 35 Lancashire Sea Fisheries Report and so need not be further referred to here. The rest of Mr. Jackson’s time at Port Erin was occupied in collecting further material for his work on the larvae of Higher Crustacea—which are of importance not only for their own sake but also in relation to the feeding of fishes (see fig. 5). He examined the various plankton gatherings Fie. 5.—Larval Decapod Crustacea from the stomachs of Mackerel. [From a Photo. by Mr. A. Scott. with the object of tracing the young stages of crabs and lobsters, shrimps and prawns, and other allied animals throughout their life-histories, and also in their distribution over the district and throughout the year. Mr. Jackson published a first report on this subject in the Lancashire Sea Fisheries Report for 1912 and a second note in last year’s Report (for 1913) and has in preparation a more detailed account. BotTanicaL Norss. In addition to the courses of instruction on marine Alge referred to above, several senior students spent some time in investigating the life-histories of selected seaweeds. Apart C 36 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. from this more general work some experiments were made with a view to collecting data for an enquiry into the causes underlying certain marked features of algal distribution. Miss L. Baker and Miss E. M. Blackwell spent some time investigating the lichens of the littoral and other regions, and produced a preliminary list. The mosses and liverworts of the district were collected and classified by Professor J. B. Farmer, F.R.S., and Mr. R. J. Tabor, of London. It is hoped that several papers at present in course of preparation will be published as an outcome of the work done by the Botanical School at Port Erin during the Easter vacation, 1914. Bio-CHEMICAL RESEARCHES. The researches carried out on the Bio-Chemical side during the year 1914 have been concerned with two main problems. In the first place the nutrition and metabolism of marine animals have been studied over prolonged intervals — by means of the respiratory exchanges; and secondly the variations in alkalinity of sea-water at various periods in the year have been investigated. The second problem is related to the first, because the variations are mainly produced by changes in the balance between photo-synthetic activity of plants, and the metabolic oxidising activities of animals. The results of the metabolic experiments have been published in two papers which appeared in the Transactions of the Liverpool Biological Society, Vol. XXVIII., 1914, V1Z. :— 1. The nutrition and metabolism of marine animals: The rate of oxidation and output of carbon-dioxide in marine animals in relation to the available supply of food in sea-water, by Professor B. Moore, Edward 8. Edie and Edward Whitley. 2. The nutrition and metabolism of marine animals: The MARINE BIOLOGICAL STATION AT PORT ERIN. 37 effects in the lobster of prolonged abstention from food in captivity, by Professor B. Moore and George A. Herdman. The main results may be summarised as follows :— 1. It is, in our view, definitely settled by experiment, that sea-water does not contain any appreciable amount of organic matter capable of acting as a nutrient medium for aquatic animals. 2. We have also obtained, over longer periods, figures indicating the rates of oxidation in larger marine animals, and have definitely shown that the preponderating amount of food consumed by such animals is utilised for increases of the animal by growth and for sexual reproduction, and that but a small fraction is oxidised for the metabolic needs of the animal in other activities than growth and reproduction. 3. Lobsters provided daily with a sufficient supply of fresh sea-water can be kept alive without food during a period of over seven months. 4. The live body-weight of such lobsters does not diminish during such a prolonged period of inanition. But while the actual weight of inorganic matter remains constant, the total dry weight and total organic weight are markedly diminished, and as a result the percentage of inorganic matter in the dry weight becomes increased. 5. Asa result of the inanition the total oxidisable organic matter may fall to considerably less than one-half of the initial amount. 6. At the commencement of the period, protein, fat and carbohydrate are oxidised almost equally; later the carbohydrate becomes exhausted and, although fat is still present, nearly all the oxidation falls upon the proteins. 7. There is a satisfactory correspondence between the amount of oxygen consumed by the animals throughout the period and the amount of organic matter disappearing. The oxygen consumed corresponds very closely to that required 38 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. for oxidation of the organic matter disappéaring, so that there is no reason to suppose that the animal utilises any dissolved organic matter which might hypothetically be present in the sea-water. 8. The rate of oxidation is throughout a slow one representable by 120 to 130 milligrams per lobster of 220-300 grams at the commencement, and dropping to about half this quantity towards the end of the experiment. This amount corresponds to a little over one-tenth of a gram of protein or carbohydrate daily. The investigation of the alkalinity of sea-water has now been completed and the results will shortly appear. They show interesting chemical relationships corresponding to the known seasonal variations in the marine flora and fauna. S.Y. “ Runa,’ 1913.—FoRAMINIFERA. It will be remembered that in last year’s Report, in connection with the work done from the yacht “ Runa,” it was mentioned that a couple of dozen canvas bags of dredged sand, mud and other deposits, brought up from the bottom of the sea at various localities on the West Coast, had been sent for investigation to Mr. E. Heron-Allen and Mr. A. Earland, who are at present engaged on a monograph on the British Foraminifera. Mr. Heron-Allen has been good enough to send me the following note on the results obtained during the past year and supplementing what he enabled me to state in our last Report :— “Since the date of the last Report the examination of the samples submitted to us has been proceeded with uninterruptedly, and is now approaching completion. “The later samples have adequately fulfilled the promise — of the first four, the majority of the Stations having furnished very extensive lists and affording valuable contributions to MARINE BIOLOGICAL STATION AT PORT ERIN. 39 the list of species recorded in British waters. Over 350 species and varieties have been identified. “Station 20, ‘Between Ru Ruag and Carr Point (off Gairloch), 20 fathoms,’ has yielded remarkable quantities of fine specimens of Cornuspira foliacea (Philippi) and Jaculella acuta and obtusa, Brady, and of the rare Ammodiscus charoides (Jones and Parker). “Several species have occurred which will require very careful diagnosis, and several species new to Science may be expected. Perhaps the most interesting discovery has been that of Spiroloculina acutimargo, Brady, var. concava, Wiesner MS., of which the only hitherto recorded specimens have been sent us by the author from Hiland Pomo in the Adriatic (195 fathoms) and which we have found at Station 4 (Loch Sunart, 12 fathoms). “The last Station dredged by Professor Herdman was ‘off Bradda, Isle of Man, 20 fathoms,’ of which, at his request, we append a preliminary list of the species identified up to date. “ Station 23.—Material.—1 lb. 10 oz. of muddy shell and algal débris with large shells of bivalve Mollusca. Residue after removal of shells and stones, 10 oz. Residue after washing, 100 c.c. full of fragments of small crustacea. 112 species from the floatings.”’ List of species of Foraminifera dredged. Ott bradda Head, 20 fathoms. MPEP TEMA COPTCSSD, AO. oe... ecncocecsscesscccecscscarsecnereencs Common. 2.— os OT OS GO ET iG Neate Ale POR ee ec eee apr ere ae Very rare. 3.— THB OTERO he te Nedoe che ccisae satan «essa egyte nase swae se Frequent. 4, —Spiroloculina ieeeremmtnetiray | Waits Me casks ck issk sacra aseasarastre rss Rare. 6,— Mee Otay i tw deen dan deen daay Reans See Frequent. 6.—Miliolina PETE TSNA, 202 2 Gee pera dsstassostegpaxtencsssessceer Very rare. I CATCHES: (TS0LTL)) 053 sc .har. cc esceccecncssoscnresnecsonccs Frequent. ees, SUbrotunda (Montag) «..........ccccieecccsevescscnses Very rare. ST COMMIT (IRGUBS) i 525025 ccccnccsscccsbesacccccsccnccesee Very rare. 10.— pa SermCMaS ER CCIM DIODE Bede Sa Foes acccaspicceaseprantesscs Very rare. 11.— + Seman” RNY se coe akvacarweccasctscrsssccensecsen Common. See COMMUN (LANNE), 055. :.05eccsoosescerascccscccosvenese Frequent. Se CATIGCIATIC (CO3)- oie coccescecssccrcsccnsaccscnconcceses Very rare. 14.— i ROTOVORIOCR (AREDEL)) “s.tsacecevcdeseescnvondsvecvebasstse Rare. 40 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. 15.—Miliolina fusea, Brady <..-..s.snencsseeoe: caepssonscccee yarserent Very rare. 16.— hy. , laevigata (G'Oo) sick sence ccseseneceate ssecemenrarneteneee Rare. 17.—.- ,,. bicornis. (Wi & J.) _ eksesscsucwod.o.:0ene sateen eee Very rare. 18.— re bronentarti(@ Os) | Jose. ee cs ecordusccecseeecceaceeseenee Very rare. 19:—' -,, ' -retundata (Montagu) > 212).0u.0scccseencosssccotecceee Very rare. 20.— ss. *boseiana:(GiOD i cco. .kians reson ce-cetc ese saee oanaee see Very rare. 21.— pygmaca (Reuss) ............sseseeeseeeseseeneeeeeneees Very rare. 22. —Ophthalmidium carinabum, BO) Wisse...0s ions -ncuwes cena Very rare. 23.—Cornuspira selseyensis, H.-A. & BH. ...........csceececseceeees Very rare. 24,— Y: involvens (Reuss) ............ fe ca Sa cece nani eee Very rare. 25.—Bathysiphon argenteus, H.-A. & HB. oo... cece eees esse ne eeee Very rare. 26.—Haplophragmium pseudospirale (Will.) ............sssseeeseee Very rare. 27.— canariensé (a Q.) 54...55.0: oe adeases eee Rare. 28.—Ammodiscus pord@talis (3.965. Po) cece se daxesevecs sciesiavctpteceeeeee Very rare. 29.—Trochammina squamata J. & P. ...c..scesesceeceeenceceeeees Very rare. 30.—Textularia gramen, D’O. ........sceesseseeeeeeeeceeneeneeceeeeenne Very rare. 31.— COnICa, dO. oo oii os alten os ee Sesebac eons sk eae eeeeeeee Very rare. 32.—Verneuilina polystropha CIRGUGS)) cose5s ten caemeeares “SHadeis aaetaee Very rare. 33.—Spiroplecta biformis (P. & J.) .........ccesecsseerescesscrsesceces Very rare. 34.— 6 wrightit, Silwestrl (sh... scvcteldeksecncs anette Very rare. 35.—Bulimina. pupoides, WO... sec. dhs... .cncateceonsseen een ste eee Common. 36.— elegans, C'O.. .ccscet.ectindostes sontaesoesen tee eee Common. 37.— “a marginatay, QO... “ssinuceiptasctnestaeeeee cee eee Rare. 46.—Cassidulina laevigata, d’O. ..........sesecegeeessceceecocscereenes Very rare. 47.— Re Crassa, GO. 5 .cave.eencbacascecnare newer eee ee ree Very rare. 48,.— subglobosa, Brady. x scolsdos tit n esc teat eee Very rare. 49.—Lagena globosa (Mont.) ..........ssscescecsccecsceccecserseeceoos Very rare. 50: ,,. Tineata: (Wa) .a0.c 5. .cnsveniae eee cae tae ee Very rare. 5L—: > costbata CWA): oa Suse ce odaawetnamce cece ee eee Frequent. o2.— ,, hexagona, Will) ccc .c.csccss ounces cecesesegteetseeeneeeee Frequent. 53.— ,, squamosa (Mont.) .............00 aawvicecldeic na notecmaeemes Frequent. 54.—- ., semistriata, Wilk ..o. ccc. occ se.ncsshee case ee eee eee Very rare. BO. » 3. Striata(G@Qs) acccssteczs<.castonsssonct ee coesee er eeeee Very rare. 56.— = ,;? Suleata (Wo Gd) Secuiaaccuncotastgcweecestaseseeeeere Very rare. 57.— ,, williamsoni (Alcock) «.x.igcce eee eee eee Common. 58.— - 4, sclavatai(d’ Os). s.d.ccc.sccessasncgecccse seat eee eee Frequent. 59.— 4) laevigata (Reuss) oc. ....0.c.ncs one eaescerenesae-menseeneee Rare. 6O.——. 45. Tcidan( Wall) ic. csoecamccw sasne cawaecteatien Sone omnia eee eee Frequent. 61— ,, marginata (Walker & Boys) ...........ssceccecssssres Very rare. 62.—— > oy) ORM ab a, Wall Bias canine see abeccens ceeds cock hoes Eeeee Very rare. 63.—— ..,, , Orbigmyana (SegMenza). oi.) emavcicnepenemcaimaneemectds Very rare. 64.— 4, apiculata; Reuss ...0...2.ccdene- eiesacapn deen gen cosueeeeee Common. 65.— biearinata (Tenqitem): 2. ccseece.p cnan-rindensnioseeeeeueee Rare. 66.—Nodosaria pyrula, DO. ...........ccerseerseereccnsseceessceraseners Very rare. 67.— iS COMMUNI, CO, oh. jab vases logas cece Seca ceee eee Very rare. 68.— hs scalaris (Batsch.) Seats tive oiarcte dee aeeeaee cimeneeeee Very rare. 69.— - obliqua:(Batseh:.).« .:s00.ca0s Joss o<% «syste eeeeeeeees Very rare. 70.— i consobrina (d’O.) — .essacesécassescevaceconsmecemeee Very rare. 71.—Cristellaria crepidula (Fichtel & M.) .............cesceeeeeeeees Very rare. 72.— a PIDDA, MA Oo a, vasicwosene teseine de seals 92> caine eee Very rare. —_= ow — oar MARINE BIOLOGICAL STATION AT PORT ERIN. 41 fo ——Cristellaria Totulata (Lam.) ........0...c.sscsecesesvesccsssecense Very rare. 74.—Polymorphina lactea (W. & J.) .....ccsesecececececeeeeeneeeees Frequent. 75.— a compressa, Cd Ox: oi c.ci.2.e0c..censece ia Sacleraepiaenee Rare. 76.— ia eeAaAE Hehe COE ws «000s cae acenmecenwnaasncap asin Very rare. 17.— Ss PaO Oe ae Seattle de lignicevodeuwanhs tecaayeebretos Very rare. 78.— a3 EMSA REPEL OUSS 28 ce ce nlc vad av aslene vcieseennaia Frequent. 79.— - BV BOAIGIGES, FLCUES ......0cc0r.ensececeaecconeses Very rare. Be videring ameulosa, Will. ........0scsesesscecsccssscceceveuses Very rare. 81.—Globigerina bulloides, QO. ..........csceesecececeeeeeeereceeeees Very rare. 82.— a inflata, Fite cee, Me il dal ohan Me Very rare. $3.— spirillina vivipara, Ehrenberg ...............cscscssessesceeeeees Very rare. NID AD, DTADY .......s.0.c.cocecssnceccecsencsccsseucsns Very rare. ee Ee IAPCOMUgaba.- Will, . ......c0cccnseoeecesececscacncenccsorss Frequent. IU MCIPRRITET UL TOA, WVU. ccccnccccccccccccasvcccesecssacnuccneccces Rare. 87.— = MM RREUEE ET OUUTHILS 29 Ss crk wrosleie s Aie'inle haeiemie wislociutaisls biniek Common. 88.— = REECE PCR Seiten car Ana gas an ada seta eaiaae baeweate Common. 89.— x STB CEP EA G5 Ts sa. scisvseavenecectescsessastbvent Common. 90.— - BREA ATAE CIRO) Ye fais sain isos wis slalsin as atern'Ubard dns eme:s'auideniawea ie Frequent. 91.— = PMMIPETAS OO, - saciacneondventsandonticas sinters sinees Frequent. 92.— i RRA EPTS CRG hot Sara abies Ueiyld Sehimaaaeiene es aaatads ahs Common. 93.—Planorbulina mediterranensis, ’O. — ........ccssccesscceesecees Common. 94.—Truncatulina lobatula (W. & J.) ........c.scececcsecsccscescseees Rare. 03.— ‘- era MIIEIS ON a aieinie x densiosie sain eirncigsnn dalesinamie eee Very rare. 96.— am HEL EO Oat fececeee sere iat caese css ere ss csiees Very rare. 97. —Pulvinulina EERIE ANCE SOG NTL) kasha csiaiuncdetusienvacvoutinns eaeties Very rare. 98.— S PISMO TCV MUI Ns cattwisinsicdieais/ebesa selene apivrnncwices sess One. 99.— Bs PGA DE AES AG Es) Siicceaiachiseinetten oulaintaieee Very rare. Sse aMaNOCCCATH (LINTIC) .............0ccesessesrscoensenceneacens Very rare. EE OTIMNCTIIATISC., GO), ici cccsccscstccscdacnteccecvcccncscsscoes Common. os ETC ERs IS a dl 0 Very rare. ie —Gyvoema inhacrens (Schultze) ............ccccecscsccscenrecsones Very rare. 104.—Nonionina depressula (W. & J.) .........ccceecsecseeeeeeees ..... Common. 105.— > MamOtNeHt Ha, (MONE) ......0s00sccncssecsssdencensonss Very rare. 106.— 5 Pee T IAPR EASAE (ET GGIAVU SY» Sicleasiwcisis nis'adlvoin's beaictavionisln vars Very rare. 107.— A DMCA Oe ins stnainciansasen ses dhddscae teismeregs Very rare. 108.— pauperata, B. & Se eee Mer eat e Frequent. 109. —Polystomella striatopunctata CH Rar) he montis chute ogc aate Frequent. 110.— - PMI EE © sc eras ) 60 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. ete (ope ay Ie) eo Seve eS C1016 7S OLS ee. S322 oS oO Ke oy Sh Forward.. 42s SEOOMELe Meade- ane, R. R., Tower aay eee aa. O 10 - Mond, R., Sevenoaks, Kent.. 5 0 Monks, FE’. W., Warrington... 42 Mottram, V. H., The University, Tee earl eo. Muspratt, Dr. E. K., Seaforth Hall, Liverpool 5 0 O’Connell, Dr. J. H., rae Pe Heathfield-road, Liverpool Ly Petrie, Sir Charles, peso fone iigeronien Lae Rathbone, Miss May, Backwood, Neston ... 3 lee Rathbone, Mrs., Green Bank, Allerton, Liverpool 2 0 Roberts, Mrs. Isaac, Thomery, S. et M., France... Lert Robinson, Miss M. H., Holmfield, Aigburth, L'pool ya Smith, A. T., 43, Castle-street, Liverpool... joie)! Tate, Sir W. H., Woolton, Liverpool a 2D Thompson, Edwin, 25, Sefton Drive, Liverpool - ay: pie Thornely, Miss, Nunclose, Grassendale .. 0 10 Thornely, Miss L. R., Nunclose, Grassendale 9... 9 Toll, J. M., 49, Newsham-drive, Liverpool j death! Walker, Alfred O., Uleombe Place, Maidstone ee Ward, Dr. Francis, 20, Park Road, Ipswich 9 2 Watson, A. T., Lapton-crescent Road, Sheffield ... Pi Whitley, Edward, Oxford pie Yates, Harry, 75, Shudehill, Maneatis iin £74 1 Deduct Subscriptions still unpaid less old Subscriptions received ... us ae 6 1 £68 0 Add Subscriptions for 1915 ... att ale fio : : 1 , d } MARINE BIOLOGICAL STATION AT PORT ERIN. 61 “ SUBSCRIPTIONS FOR THE Hire OF ‘‘ WorK-TABLES.’ Victoria University, Manchester ... ee ae SED 0} -.0 University, Liverpool ee ee ee area ees ORG) University, Birmingham _... eS re S29 tO" OHO University College, London i isis ae 2 OO. Bedford College for Women, London _... re 2 DO University College, Reading Re ae oe 2 OO) £36 6 O Add old Subscriptions eae less Oe ue still unpaid ; a o- lo; 6 £42 2 0 *PIGL ‘YI9T “aquasog “IOOdMHAIT Pe ie ca a Pee ee TI6L TOF JUBIL) GL sa eeeeee bie UIC UE SCD Ot ot BOM } SI6L ‘1eque00qT oOUBl[ Cd ‘syuRyUNODDy pereqreyo —SOLIoYystq pue sIny[NoIAsy Jo prvog wWory syURIH ‘THHLVAT » MOOD 6 b LEF*ETET ‘toquteoeg 48 sv ‘oourleg—: puny Worsueyxy ‘7004409 punof pun poripny bs Sal & = © |) O2u yore ra 2 ro ’ ‘UMUNSVaAUT, ‘NOP OT €f oI | 1 | Cane Sugar. Pi lak =e eet ae ae a ema a tec Picts ++ | Lactose. oe qe Se SEA-FISHERIES LABORATORY. 125 Table I. Reactions of 197 organisms isolated from Mussels. | | = ao) =| Seen eet =| .. a8:| ¢| 2 ee | 2) 2/8] 6/4 lgesieal 8 ner 2/2) 8/312) 2) 3 Fesls 4) SHO; Al 4/4) 8 Pasiasi a ee e B. griinthal group. fe pr at 47 | 24 J & vesiculosus. e Ce) 2 = 20 | 10 2 MacConkey’s Nos. 100, 101. eee) — | —| +} — |] + — {18 | 9| 37 -B. aetds lactici. B. coli communis. ; cept | = ane a eee Nes it es 1B. cavicida. ‘4 { B. lactis aerogenes. eee jet | — | +] — |] CK + |12)] 6| 51|48B. dysenteriae. |B. capsulatus. +i +i t+}]4+])]—-!'—-] + — 9; 4, 6| B. neapolitanus. et | - | — | -—;-] + 8 |; 4) 7| B. cloacae. eer ft | + | tt | lc — | ht ce SE es eee) |-|.-| — | 7| 3] 9 2 i (el le — E canp LO +i ti+!i —-| -|]| -|] - — Givco it eet | | Fr] | Cl oe 5} 3 | 12 ES ee a a = Sh Soe als +) 4+}4+)]—-) —-| -/| + — 4 | 2 | 14 | B. coscoroba. i+) +i] +i}+/-/]- - 3 | 1 | 15 | B. rhinoscleroma. eee | — | + | — | + ae a Lei eee — | + | + | — = me eG ee | — | +) — |) — = 1 18 eee | —~ | —-|-}.+ | 1 19 eee | | | | — | — a 1 20 eer) +t} — i} —-|+| + | t 21 +i +i; —-}—-};-] -|] - aS 1 22 oo | = || — | + + 1 23 Belt i +l - | + ons Hl 24 Include also 13 organisms which do not ferment both glucose and lactose. _ ia + Table I shows that 37 distinct organisms have been separated out from the 200 isolated, that is, ‘‘ distinct ” if we are to understand that the presence or absence of the reaction with any one of the test substances used implies specific , distinctness. It is impossible to say yet whether or not this is the case. If it is regarded as essential to being Bacillus coli that an organism should ferment glucose and lactose, it can 126 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. sugars used, or to produce indole, or to produce the Vosges and Proskauer reaction, are also characters that differentiate species from species. It may be that under certain conditions the same bacillus may or may not ferment a certain substance, but in the absence of extensive research into the natural history of the organism we are not yet justified in assuming this. If we include motility of a bacillus (that is the presence of locomotory appendages, or flagella—a definite morphological character) as also a diagnostic specific feature, the number of species would be still greater. Now this character does seem to be one which has not the same value as the fermentation tests. There is some doubt as to the exact conditions in which motility ought to be looked for. MacConkey recommends that the nutrient agar culture should be examined after 4—6 hours’ incubation, while Clemesha suggests the examination of an 18 hours’ broth culture. It appears that motility may, therefore, be exhibited at one stage of a culture and not at other stages, and we cannot assume that cultures of different bacilli, in the same medium, and at the same time of incubation are strictly equivalent with regard to the morphology of the organisms. Further, motility is not always easy to observe, that is, it is not easy, at times, to be sure that the motion observed is not simply the Brownian movement of immotile particles. For these and other reasons I have not included this character among those regarded as diagnostic, though its presence or absence is recorded in the Appendix relating to the individual organisms. But it is clear that the presence or absence of the fermenta- , tion reactions, with sugars other than glucose and lactose, are not simply matters of chance; that is, the Table does not merely show the permutations of characters theoretically possible. This is what MacConkey shows, and my own results are very much the same as his. Table I shows that four combinations of reactions, Nos. 1 to 4, are exhibited by half SEA-FISHERIES LABORATORY. UAT of all the organisms studied. These, we may hold, are therefore really distinct species, or at least small groups of such. Com- paring this Table with that published by MacConkey we also see that very much the same kinds of organisms have been found in both cases. Group I, in my table, includes the species called, by MacConkey, B. griinthal, B. sulcatus gasoformans, B. castellus, and B. vesiculosus. The first three organisms differ from the latter one only in that they are motile while it is not so. No. 2 in my table appears to correspond with the organisms numbered 100 and 101 by MacConkey. No. 3, called B. acids lactici, is very rare in MacConkey’s list. B. colt communis and B. cavicuda are much more abundant in MacConkey’s list than in mine. Many of the other unnamed organisms may be found in both lists. The two groups of organisms may best be compared as follows :— MacConkey. Organisms Organisms present abundant in human in Sewage polluted faeces. Mussels. EC MOUNAIUS cov caccascerccccerscenscsvecss ) 0 ones cssccscsecseseee. | ape a ant nae tae 4% PMP IETUOSUS non ccncsscnseccsctseccascsees | a ESA eee Be. SUICALUS GASOFOFMANS .......00sececeese | DAT ]o vramsnvacetors eins 24 % B. Castes 1... eeeeeeveveeessseceseeeeeees PMOIE COMUMUNIS. 2arcacccccecssccecsecsensas ye coeocoeccceccceccece, ay an ees aats 7% NE sega ca>sssslsredes'snsecacaceens Gi rt cutisnades ats 0% REITER CCVOQCNCS —..snsc cnc sccncscscsscess CTT UC cscs ccscses couse sosncecsscecses BEM crude ag okecte eae 6% Ii. csinws pieces ccccocsevessvsaee Organisms rare in human faeces. IP TETETOCE ccc cose cece teccscscccssscesecses A. nos vomuans daha 9% ha cicescosccesssecrscsssissesees Cees Urs csssskasuitont «s 2% NEGUS gs dan sbrcencanee secede sasasaroeese DO sry ss tears Saipare 0% Re LOO GNA LOL... ..ceercevecncrsceces UO sevehixigdaas vais 10% DEMMETIORCICTOMGE «... 0c ecesscecesnsciesosesasens BS San ipatig ined don ds site | Thus we see at once that the organisms which appear normally to inhabit human faeces, and those which may be I 128 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. found in sewage polluted shellfish, are not necessarily the same. Only one group—that formed by B. grinthal and its congeners are about equally abundant in both lists. MacConkey’s organisms, Nos. 100 and 101, rare organisms in faeces, are fairly abundant in my samples of mussels, and the same is to be said of the lactic acid bacillus. Then it is very notable that B. colt communis, the third most abundant group in faeces in MacConkey’s list, 1s much rarer in mussels. Generally, one sees that a notable change has occurred in the relative proportions of the bacteria present between the time when they leave the human intestine and the time when they may be found in marine shellfish. Some of the organisms are fairly resistant and appear to withstand the change of habitat,. while others cease to multiply and die out more or less rapidly. It is also to be noted that organisms, having -a general resemblance to “ B. colz,” and which incomplete _ analyses might easily identify as “ atypical’? forms of that bacillus, are to be found abundantly in shellfish, but are either absent or very rare in human faeces. (2) The Longevity of Intestinal Bacteria in Sea-Water. What we must do, therefore, is to isolate, one by one, the most characteristic faecal organisms, and then study their natural history, that is, their rate of reproduction in fresh water, in sea-water, In sewage, in shellfish, in soil, in diffuse or bright light, and so on. Hardly any of this kind of work has been done, although it appears to be quite essential if bacteriological methods are to be employed in public health work with respect to the recognition of intestinal bacteria in open natural conditions. Not to do this investigation would be much the same as carrying on fishery regulations with only a knowledge of the morphology of fishes as it is taught in the schools, and without knowing anything about the distribution, migrations and habits of fishes in the open. No. of bacteria per SEA-FISHERIES LABORATORY. 129 Some bacteriological work of the kind I mention has indeed been carried out, notably by Clemesha in India, and by several workers in this country, with respect to the longevity of “ B. coli” and “ B. typhosus’”’ in sea-water. In these latter cases the characters of the organisms studied are, unfortunately, not given in detail, and the observations made are not very precise. I therefore give here some data relating to the longevity of intestinal bacteria in sea-water, premising that no real opportunity for a satisfactory investigation of the questions suggested above has been afforded, and that the results here given are to be regarded only as indicative of the possible methods which might be adopted. An organism was isolated from human faeces having the following characters :— Glucose +, lactose +, cane sugar +, dulcite +, adonite —, inulin —, indole +, Vosges and Proskauer’s reaction —, motility —. It was kept on nutrient agar for a month or two and was then re-cultivated in all the above media: the same series of reactions were again exhibited by it. A few drops of the dulcite-broth culture (which had been incubated for about four days) were then added to about half a litre of sea-water of normal salinity (sp. gr. = 1-024 at 18°C.), and the numbers of bacteria in this liquid were estimated. The flask containing the culture was then kept in a cupboard at ordinary laboratory temperature (about 16°C.) and in ordinary diffuse light, and the number of bacteria in it was estimated from day to day. The results were as follows :— Experiment I. Days. ee ode eee pear | ont 8. YO IO dy 1/1000 c.c. ......... (1321) |309 |106 | 45 | 29} 20/ 6 No. of bacteria per MOE O.C. .cc2005. oF Fae inemee fuer ised |P ovat, loteam Ib a On.) OR h OO | Se 130 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. The number (1,321) at the beginning of the first day was not actually observed, since the plate was so crowded as to be impossible to count. This number has been extrapolated, as will be discussed later on. At the beginning of the ninth day the plate (containing 70 organisms) was put aside and incubated for a day longer in the cold. Ten colonies were then subcultured on nutrient agar and re-cultivated in the same series of media. The same reactions were given as in the case of the original colony. A few drops of the dulcite-broth culture, after incubation for four days, were added to sterilised sea-water as before, and the number of bacteria contained in this liquid were again estimated from day to day. The results obtained were :— Experiment II. Days. i 2 3 4 | 5 No. of bacteria per 1/10000 c.c....| 21 uae doe se ae oe 1/1000: ¢.6.-..2) — 2ak 84 4 Me ae = we L/LOO ciern. (2310) 886 43 36 2 The count (2,310) for 1/100 c.c. for the first day (that is, at the beginning of the experiment) was not observed. It is the count for 1/1000 c.c., 231, multiplied by 10. The plate counted on the third day (with 43 colonies) was incubated for a day longer in the cold, and eight colonies from it were subcultured on nutrient agar. These were again subcultured in the various media, with again the same results as were given by the original colony. One of the dulcite-broth tubes was again taken, after incubation for four days, and a few drops from it were added to half a litre of sterile sea-water as before. The number of bacteria present was estimated from day to day with the following results :— SEA-FISHERIES LABORATORY. 131 Experiment III. Days. 1 2 3 4 5 6 7 No. of bacteria per 1/1000 c.c. | 30 an a e 35 H/TOO' Gc...) ... 37 5 Es 1/50 c.c Bae a 0 3 E ec. 3 1 a ; 5 Gc; 0 The experiment was now discontinued. A further experiment was made with the object of checking the original one. A similar culture to that employed above was used, butinadifferent manner. A fresh culture on nutrient agar was made from the stock culture (which had been kept for several months), and a few c.c. of sterile broth having been poured into the tube the culture was rubbed up into the liquid so as to make an emulsion of the bacteria. About 1 c.c. of this emulsion was then added, as before, to about half a litre of sterile sea-water contained in a flask, kept as before. The number of bacteria in the culture was estimated from day to day with the following results :— Experiment IY. Days. 1 Zee Ae toea tH. S F cO) lO pid No. of bacteria per 1/10,000 c.c.|279 319 |129 | 58 | 24 | 32 | 10 = 1/1,000 cc. | 1/100 ¢.c. ... oo PMA Ad Pca beep oe ten | 0 Now, considering the first three experiments, we see from these rough results that the number of bacteria per unit volume of culture first of all undergoes a very rapid reduction, and then the rate of reduction becomes very much less. It is quite clear that a graph of these ungraduated figures would show that the curve would fall asymptotically close to the axis of y, and these would approach the axis of « in the same way. 132 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. We have to deal, in fact, with the decrease of the numbers of bacteria expressed by the gas-volume law, pu = constant. So much is clear merely from the inspection of the experimental data. The law of diminution may, however, be more minutely studied from smoothed or graduated figures, and these I give in the table below. The method of graduation is explained in Appendix II. Text-figure 1 is a graph of these numbers. ‘ Days. Expt. 1 |) 2193.4). 4 5 6 | 7 | 8 | 9) 10) 41 |e Nos. of bacteria in unit I |100 | 18 | 7-41 3:5 |2-7 |2-2 |0-9| 0-6] 0-5 | 0-4 | 0-3 0-2 volume of culture as || II {100 | '7-6|1-710-5 |0-2 |, 2. | (2 | 2a) percentages of the || IIT {100 | 2-1 | 0-2 | 0-04 |0-001| 0-004 original number IV 4 days Fie. 1. Curves showing the rate of disappearance of intestinal bacteria from a sea-water culture. The points plotted represent the graduated figures of the table above. SEA-FISHERIES LABORATORY. 13a The percentages for the fourth experiment cannot be given since the mode of reduction is not quite the same as in the first three experiments. The numbers of bacteria rise from the first to the second days and then fall, and after this initial rise the mode of diminution is the same as in the earlier experiments. The difference is due to the fact that a certain quantity of food-medium was added with the bacilli inoculated, so that an initial multiplication took place. After- wards, however, the same manner of fall is to be seen from the figures. The graphs of these three experiments are rectangular hyperbolas, and are :— eye = constant. II. ya*" = constant. Ill. yax’-* = constant. The index of «, that is, the slope of the curve, the measure of the rate at which the bacteria die off during the experiment, is greatest in Land leastin III. This means that the successive cultivation of the same strain through sea-water has affected the organisms, so that their resistant power to the change medium has become less in the course of each experiment. The constant, of course, only affects the scale to which the results are plotted, or otherwise expressed. (3) The disappearance of intestinal bacteria from Mussels subjected to a cleansing process. It has been known for some time that intestinal bacteria disappear from shellfish allowed to stand in still, or running sea-water. The first. experiments of this kind were made by Klein at St. Bartholomew’s Hospital as long ago as 1905. Oysters, mussels and cockles were dosed with enormous quantities of typhoid bacilli, and allowed to stand in wooden tubs containing sterile sea-water. After several days a very marked diminution in the numbers of the contained bacilli 134 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. was experienced, and Prof. Klein came to the conclusion that a “‘quarantine”’ period of about four days duration would usually be sufficient for the cleansing of polluted shellfish. In 1908 I made several such experiments in the open at Conway and obtained similar results. In four days there was a very marked reduction in the numbers of the contained bacteria, amounting usually to well over 90 per cent. About the same time Mr. A. Scott made similar experiments by keeping mussels in the water of the tanks at Piel Hatchery, and obtained much ~ the same results. Since these first experiments others have been made, and at the beginning of 1914, when I was requested by the Chairman to report generally on the subject, some more precise observations were begun. I regret that it has simply been impossible to follow up the many obvious further questions suggested by these observations, and since the opportunity to do so is not likely soon to occur, I wish to record here the results obtained. a. Thecleansing of mussels by sterile sea-water. By “sterile sea-water’’ is to be understood sea-water taken from the flood stream, and containing no micro-organisms in 1 c.c. capable of growing on neutral-red, bile-salt, lactose agar. The object of this experiment was to ascertain the time required to wash out sewage bacteria from the alimentary canal of the mussel. Mussels, regarded as objectionably polluted, were taken from the foreshore in Barrow Channel, near the Piel Laboratory. An analysis made showed that the mean number of sewage © bacteria’ contained per shellfish was 7,400. (The individual counts of 6 plates made each from 1 c.c. of an emulsion made from the soft parts of 5 mussels were: 120, 122, 140, 150, 151, 172. Hach c.c. of this emulsion represented 1/50th part of a mussel). The mussels were put into glass aquarium tanks of about 10 litres capacity, and sea-water was run through these SEA-FISHERIES LABORATORY. 135 tanks at the rate of about 1 litre per five minutes. Samples of the mussels were taken after treatment of this kind for one, two, and four days. First Sampling. After 1 day. 1/50th mussel contained 0 intestinal bacteria, %3 . 0 7 99 9) 1 99 be) Mean per mussel = 16-6. Second Sampling. After 2 days. 1/50th mussel contained 4 intestinal bacteria. : P : 9 . ‘ 23 2» 1 >» 2» Mean per mussel = 116. Third Sampling. After 4 days. _1/50th mussel contained 6 intestinal bacteria. %» - I z » 2» » 0 » » Mean per mussel = 116. Colourless colonies appeared in the plates made from the crude mussels, but none was seen in the plates made from the treated mussels. The sewage bacteria present were therefore reduced by 98-5 per cent. This experiment was not repeated. The same results had been obtained, as on a former occasion, by Mr. Scott, and ttle more information was to be attained by laboratory trials. It might have been possible to reduce still further, the time necessary for sufficient cleansing, and it might also be desirable to observe what degree of cleansing takes place when mussels are allowed to remain in various volumes of standing sea-water, and for variable periods. But on considering the difficulties which are likely to be encountered in applying the principles of these methods on a large scale, 136 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. it was felt that further laboratory experiments would afford little additional information, and that the details of a commercial process would have actually to be studied in the working plant itself. 6. Experiments in the open sea. It was felt desirable to repeat, in new localities, the experiments in the open already made; and, in fact, a wish had been expressed by the fishermen at various musselling centres that cleansing works should be started. In September of 1914, Dr. Jenkins and I met Mr. R. W. B. Gardner at Sunderland Point, in the estuary of the Lune, and we then proceeded to visit a place which Mr. Gardner regarded as ee on suitable for relaying mussels for cleansing purposes. This place, with its immediate surroundings, is marked on the chart reproduced at the end of this paper, and described in the explanation, so that I need not further refer to it. There were already some mussels there, and a preliminary analysis showed that these were relatively unpolluted. Mr. Gardner, therefore, laid down a quantity of mussels at this spot, and I made a series of analyses. The mussels were taken from the training wall in the Lune Estuary, a place known to be highly polluted with sewage. A sample examined prior to being laid down for cleansing gave the following results :— | Plate 1, 261 red colonies, 1 colourless colony. 2, 200 3 it 5Oth 39 >) 99 pL») bi) / 3° a, 267 >) : - > 99 mussel b>) 4, 200 bP) il 99 be) bi) D, 237 bh) 2 99 99 Mean number of sewage bacteria per mussel = 11,650. First Sampling. Relaid for 2 tides. 5 Plates were made. There was no reduction in the — numbers of bacteria contained in the sample. | SEA-FISHERIES LABORATORY. 137 Second Sampling. Relaid for 4 tides. Plate 1, 16 red colonies, 0 white colonies. et 2 28 Hs Oe) 1% 3 Be oy 2D = DN ts * Mean number of sewage bacteria per mussel = 1,150. Third Sampling. Relaid for 6 tides. Plate 1, 3 red colonies, no colourless colonies. a ae i 0 an es . 1) 0 ” Mean number of sewage bacteria per mussel = 450. This experiment is exceptional in that no reduction was experienced after 1 day’s relaying. Most of the sewage bacteria were eliminated after 2 days’ relaying, and after three days the reduction amounted to 99-6 per cent. Photographs of typical cultures obtained in_ these experiments are reproduced in Plate I. At the same time a sample of mussels taken from the same place (the training wall) was laid down on the foreshore at Sunderland Point (see the chart). These mussels had not been examined before relaying, but they must have been as greatly polluted as those dealt with in the above experiment. After being relaid for two days they were sampled with the following results :— 1/50th .. 1, 7 red colonies, no colourless colonies. 2, 6 mussel » Me 2 3) 99 9) 9) 3, 9 99 99 or J The reduction experienced was, therefore, similar to that of the first experiment. Yet a further experiment was made with River Lune mussels. Some of the fishermen at Glasson Dock wished me to try relaying mussels at a point near to the railway station there (see the chart). The place did not seem to me to be at all suitable, but it was tried. A sample of mussels taken from the same source, the Training Wall, was laid down. 138 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Before relaying, five mussels from the sample were examined. Three plates were made :— 1/50th Plate 1, 1,800 red colonies, 20 colourless colonies. ne 3} The colonies were so numerous that the mussel 3 1 19 Ds plates were uncountable. These mussels therefore contained at least 90,000 sewage bacteria each. First Sampling. Relaid for four tides. Plate 1, 370 red colonies, 15 colourless colonies. a SOOO i, 23 - fs ie es ERY ie AT Bs A considerable reduction of contained bacteria was therefore experienced by these mussels. But reinfection doubtless occurred, for the results are not satisfactory. The place proposed was rejected as the result of the experiment. In these experiments the mussels were laid down on the foreshore itself, on a beach of sand and gravel. There was no enclosure of any kind. The places selected were out of the main tidal streams, and were not exposed to any sea, so that the mussels remained undisturbed—they had, in fact, previously been employed by local fishermen for stering mussels prior to sending them to market. The question then arose as to the practicability of relaying mussels in places like the Estuaries at Aberdovey and Barmouth, where the tidal streams are rapid, where there is a certain exposure to the sea, and where the shore slopes down very steeply from high-water mark. Construction of enclosures, designed to hold mussels while being cleansed, would be very difficult and expensive in such circumstances; and to meet this difficulty, Mr. G. Hazlehurst, a member of the Committee, made the very practical suggestion that a floating tank should be used for relaying. A tank boat was therefore made, big enough to hold several bags of mussels. The bow and stern were open, being guarded by strong wire gauze, and movable ‘ SEA-FISHERIES LABORATORY. 139 bulkheads were placed at each end so that the further entrance of water could be prevented after the tank had been filled. The tank could be moored at any place selected. A sample of mussels from Aberdovey Channel, near the Pier, was taken and placed in the tank, and the latter was then moored above Trevri Pomt. (See Mr. Durlacher’s Chart in the Report for 1913 for these localities.) After being relaid for three tides the mussels were sampled. The shellfish lived well in the tank, and had attached themselves to each other and to the wooden sides. First sampling. The original, un-relaid mussels. 1/50th Plate ‘ 3 red colonies, af colourless colonies. mussel ee 3 16 >»? j > i Mean number of sewage bacteria per mussel = 735. Second sampling. Relaid for three tides. 1/50th mussel 99 7 10 39 3? mA 3, 1d . %3 %3 Mean number of sewage bacteria per mussel = 600. .. 1, 13 red colonies, no colourless colonies. ‘The experiment was repeated later in the year. A sample of mussels was dredged from near Penhelyg Point, and was relaid in the tank, the latter bemg moored on the beach near the life-boat house at Aberdovey (see Mr. Durlacher’s chart). At both this place and the one in the other Aberdovey experi- ment, water circulated through the tank for about four to six hours during each tide. The tank came adry during ebb-tide, and it was expected that before the time when the number of sewage bacteria had increased notably in the water of the channel, the tank would be out of the water. First sampling. The original, un-relaid mussels. Plate 1, 13 red colonies, no colourless colonies. 99 2 ’ 8 >) 0 >) 9) 3) 3 > 8 9) 0 9) bb) Mean number of sewage bacteria per mussel = 485. 1/50th mussel 140 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Second sampling. Relaid for three tides. 1/25th ke 2 = red colonies, 2 colourless colonies. Mes ae 3, 6 29 : 0 ” ” Mean number of sewage bacteria per mussel = 200. There was very little reduction in the number of contamed bacteria in these experiments, but that some reduction did occur was quite certain. The mussels originally were not polluted to a significant extent; they were certainly quite suitable as human food, in my opinion. The object of the experiment was to test, in a preliminary way, the practicability of utilising a floating movable tank for the purpose of containing mussels undergoing cleansing, in such places where the nature of the foreshore makes it difficult or expensive to construct tanks or ponds, or other artificial enclosures. The experiments are being repeated at other places on the Welsh coast. A further experiment of the same kind was made at - Barmouth in February, 1915. The same _ difficulties presented themselves here as did .at Aberdovey; that is, the area of foreshore suitable for the construction of a tidal cleansing pond or tank is very limited. The floating tank used in the last experiment was, therefore, again used in this one. It will be seen from Mr. Durlacher’s chart, published at p. 352 of last year’s Report, that the floats used for the drift experiments passed directly in front of the inlet, called Aberamfira Harbour. But at half flood-tide we might expect the sewage coming up from the Harbour to be very greatly diluted, while the ebb-tide would contain but little contaminating matter at any state ; at all events the experiment was so arranged, and the tank so moored that it dried, that is, it came aground, for about four hours on each tide. Therefore, the first of the flood-tide, which was the water most likely to be contaminated, did not reach the mussels, while the last of the ebb-tide, water which : 1 : fl , ‘ SEA-FISHERIES LABORATORY. 141 might also be polluted from the upper part of the Estuary, did not come near them. Mussels were, therefore, collected from the bed of the River Mawddach, between Aberamfira and the Railway Bridge, the place usually fished, and these mussels were put into the floating tank. The latter was moored at about half tide level in Aberamfira Harbour. Samples were taken before treatment, and after four and six tides, that is, after two and three days. First sampling. The original, un-relaid mussels. Plate 1, 10 red colonies. wa 1/50th 3.9 Di 20 ” mussel ron aaa 23 99 4, 6 be) Mean number of sewage bacteria per mussel = 440. Second Sampling. Relaid for four tides. Plate 1, 2 red colonies. 1/50th pei ol i mussel ae, 0 03 39 4, 0 ‘ 99 Mean number of sewage bacteria per mussel = 37. Third Sampling. Relaid for six tides. Plate 1, 1 red colony. 21 .sC, mussel ” QD, 1 ” » 4, 0 ” Mean number of sewage bacteria per mussel = 37. That is, there was a reduction of sewage bacteria amounting to about 92°% of the number originally contained in the mussels. ¢& Experiments with water sterilised by means of Chlorine, These natural difficulties are so great, in some places, that the problem of naturally cleansing the mussels by exposure to clean flood-tide water is insoluble ; or the cost of so treating 142 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. the shellfish would be so great as to be prohibitive. In these circumstances the possibility of sterilismg the water used for cleansing, by some chemical means, may be considered. Karly in 1914 the Committee requested me to meet an Inspector of the Board of Agriculture and Fisheries and discuss with him, and the officials of the Conway Corperation, the methods which were in contemplation, in that district, for the cleansing of the local mussels. I did so, and made a report to the Committee, when I was requested to report further on the practicability of the method proposed to be adopted at Conway —that of the sterilisation of the water used for cleansing by means of dosage by chlorine solution. In April and May of 1914 Mr. Scott and I accordingly carried out these experiments, which I subsequently repeated. Some reference to the results obtained was made in my report to the November Meeting of the Scientific Sub-Committee, but I have been further requested to make a more complete statement. Several distinct points had to be investigated (1) The concentration of chlorine in the sea-water necessary for sterilisation ; (2) The concentration of chlorme which the mussels could withstand without interference with their normal functioning ; and (3) The length of time necessary for the removal of the bacteria contained in the cavities of the bodies of the mussels. It was already known that a very small quantity of chlorine in water sufficed for almost complete sterilisation—this was, in fact, the principle of the Candy Process of water purification, and numerous experiments had been made, to say nothing of data recorded in the text-books on Public Hygiene. It was, however, desirable to assure oneself personally of the fact of this sterilisation, so five 100 c.c. flasks were filled with normal sea-water and sterilised, and then the same volume of a culture of bacilli fermenting glucose, lactose, cane sugar, dulcite, adonite, but not mulin, forming indole and giving a positive Vosges and Proskauer reaction, SEA-FISHERIES LABORATORY. 143 and exhibiting motility, was added to each. The number of bacteria in one of the flasks was estimated, it was about 3,000. Chlorine water was then added to each of the other flasks in quantity enough to make solutions of 1, 3, 5, and 7 parts per million, and the flasks were allowed to stand at ordinary laboratory temperature for 24 hours. One c.c. of the culture was then taken from each and plated in neutral- red, bile-salt, lactose agar. All the plates were sterile. One part per million of chlorine seems, therefore, to be enough to secure the destruction of most of the ordinary bacteria present in sea-water, though there is no object in using so very dilute a solution. The next point was to determine what concentration the mussels could stand without injury. Some bleaching powder solution was first of all made, and this was added to aquaria containing mussels, but these rough trials were unsatisfactory. Finally chlorine water was made from a mixture of potassium dichromate and hydro- chloric acid, and after washing in tapwater, the gas was absorbed in distilled water. A standard solution of thio- sulphate (and one of pot. dichromate for standardisation of the thiosulphate) were made, and the strength of the chlorine water was estimated before each experiment. Several glass aquaria were filled with sea-water—each of them held about ten litres—and then dilute chlorine water was added so as to produce solutions having, very approximately, the concentra- tions of 4, 6, 8, and 10 per million. The chlorine solution and the sea-water were mixed, and mussels were added, and the behaviour of the shellfish noted from hour to hour. There was some doubt as to whether those in the solution of 10 per million opened their shells—I think they may have done so— but all the others functioned normally, even spinning byssus threads and attaching themselves to the glass of the aquaria. Even in the 4 per million solution, the smell of chlorine could be detected at the beginning of the experiment. Evidently, K 144 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. therefore, chlorine in sea-water to the extent of 5 parts per million does not interfere with the functioning of mussels— I did not expect that it would, since the chlorine throat washes used are really stronger than this. It was, however, desirable actually to make the trial. A tank with chlormated sea-water, of concentration, 5 per million, was now prepared, and mussels were placed in it. These mussels were part of the same sample used in making the experiment of cleansing by a water circulation—they - contained on the average 7,400 sewage bacteria each. The tank was allowed to stand in full daylight, though not in direct full sunlight, and samples of the mussels were taken after one and two days. First Sampling. Mussels in 5 per million chlorine sea-water for one day. 1/50th Plate : a red colonies. mussel a 3. i ie Mean number of sewage bacteria per mussel = 985. Second Sampling. After two days. 1/50th (ee x iT red colonies. mussel | et 2. 11 ” Mean number of sewage bacteria per mussel = 600. The mussels were not removed from the tank, but the water was now run off and replaced by sea-water freshly dosed with chlorine so as to make a strength of 5 per million. This water was allowed to stand another day, when the mussels were again examined. , Third Sampling. After 1 day, the water bemg changed. ]/50th mussel |3 plates, all of which were sterile. The mussels were therefore freed from sewage contanaueee tion, The experiment was repeated at Liverpool in July. Short SEA-FISHERIES LABORATORY. 145 glass specimen cylinders of about 6 litres capacity were used as tanks. It was thought, during the Piel experiments, that the mussels opened their shells more freely in the dark than in full daylight, so these glass jars were covered with black paper and were fitted with cardboard lids. A solution of chlorine im water was made as before, the strength of this was determined, and it was diluted to a convenient concentration. Titrations of the chlorine solution, and of the thiosulphate standard solution, were made immediately before each experiment. A similar jar containing only sea-water was used as a control on the chlorinated water jar. The latter contained sea-water dosed with chlorine to the concentration of 5 per million. Mussels which had been sent from Barrow Channel were placed in each jar, and then a sample of the same lot of mussels was examined. The results of this analysis were :— First Sampling. Original, untreated mussels. Plate 1, 76 red colonies. 9? 2, 78 93 i a err, 449 ~ (CC, 9) D, 138 > Mean number of sewage bacteria per mussel = 3,990. Second Sampling. After one day in chlorinated sea-water. Plate 1, 19 red colonies. ai mussel 8 4 17 st 3, 0, 42 =i Mean number of sewage bacteria per mussel = 1,300. Third Sampling. After one day in unchlorinated sea-water. ag 1, 50 red colonies. 2 1/50th ” 3 2 ” mussel é 4, 56 ® ” 5, 50 ” Mean number of sewage bacteria per mussel = 2,590. 146 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. The chlorinated sea-water in the first jar was re-dosed so as to make it of concentration 5 per million—the water itself not beimg changed. This solution was allowed to stand another day. Fourth Sampling. After two days in chlorinated sea-water. Plate 1, 8 red colonies. yoo | > 2 Bee mussel | 4, 10 : 33 5, 8 b>) Mean number of sewage bacteria per mussel = 380. The water still being unchanged, fresh chlorine solution was again added so as to bring up the strength to 5 per million. This water, with its contained mussels, was allowed to stand another day. _ Fifth Sampling. After three days in chlorinated sea-water. 1/50th Plate = ; red colonies. mussel 3 3 Mean number of sewage bacteria per mussel = 116. Fifteen mussels had been put into the jar originally. Two of them died. It is clear, therefore, that mussels may live, open their shells, and circulate water through the mantle cavity when that water contains 5 parts per million of dissolved chlorine. In such a medium mussels cleanse themselves from ingested sewage bacteria. (4) The effect of change of medium on the biological characters of intestinal bacteria. It has been suggested that micro-organisms living in the human alimentary canal, and exhibiting certain definite fermentation reactions with certain sugars, might no longer do so when they inhabit a widely different medium, say, SEA-FISHERIES LABORATORY. 147 sea-water, or the alimentary canal of a shellfish. In particular, do dulcite-fermenting organisms isolated from faeces continue to ferment dulcite after they have been living in sea-water for some time? This question was suggested to me by Dr. MacConkey as a possible subject for experiment. I regret that I have few data to give which might conceivably answer the question. : Some mussels, taken from Roosebeck Scar, were put into a clean tank in the Piel Hatchery. These mussels are very clean, so that no micro-organisms, capable of growing on neutral red, bile-salt, lactose agar, can usually be found m 1/50th part of a single animal. A culture of an organism isolated from faeces was made in dulcite broth. This organism _ had the following characters :—Glucose +, lactose +, cane sugar +, dulcite +, adonite —, mulin —, indole +, Vosges and Proskauer’s reaction —. About a dozen mussels were taken, and then, the valves of the shell being very slightly forced apart, about 1 c.c. of the culture was injected, by means of a hypodermic syringe, into the mantle cavities. The mussels were then kept out of water for about six hours, so as to allow the culture to be taken into the alimentary canal, when they were placed in a small glass aquarium through which clean sea-water was circulated at the rate of about 1 litre per five minutes. After a period of eighteen hours, five mussels were taken out and the soft parts were emulsified in 250 c.c. of sterile water. 1 c.c. of the emulsion was inoculated im each of five plates, and the latter were incubated. The colonies were so very dense that they formed a very fine haze in the medium. There must have been very many thousands of colonies on each plate. The remainder of the mussels were kept in the aquarium for another five days, when they were again sampled. Two plates were made from an emulsion of the soft parts of five mussels in 250 c.c. of water, and each plate contained 148 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. 1/50th mussel. There were 257 and 418 red colonies on these plates, thus showing a very considerable amount of reduction of the contained bacteria. Ten colonies were selected from the first plate, and pure subcultures were made. Nos. 1 to 9 gave the reactions :—Glucose, lactose and dulcite all + ; cane sugar, acid; adonite and inuline —; indole +; Vosges and Proskauer’s reaction —. No. 10 gave the same series of reactions except that dulcite was not fermented. There was thus, apparently, a slight change in the biological characters of the organism, but without further confirmatory work of the same kind one cannot positively assert that this change is a real one. There remains the series of experiments already quoted. A dulcite + organism isolated from faeces was imoculated in sea-water and cultivated there for seven days. Then it was recultivated on neutral red, bile-salt, lactose agar, and then on nutrient agar. Ten colonies gave the original reactions. The organism was again inoculated in sea-water and grown there for three days, and again passed through neutral red and nutrient agar. Hight colonies all gave the original series of reactions. Thus no change in the characters of this organism was produced by a rather long sojourn in sea-water. So far as” they go, these two experiments do not support the notion that faecal micro-organisms undergo any change of biological characters, when they enter sea-water or the alimentary canals of marine shellfish. They die out, of course, but so long as they live they exhibit their original powers of fermenting carbohydrates. Of course the number of observations made is far too few to serve as the basis for any general statement with regard to this point. (5) Summary. (1) The process of seli-cleansing of sewage polluted shellfish by placing them for some days in sea-water, which SEA-FISHERIES LABORATORY. 149 is free, or nearly so, from sewage bacteria, depends on two things. (a) The ingested bacteria are merely washed out from the mantle cavities, and internal cavities, of the animals by the stream of water which is continually being circulated through these passages. (6b) The bacteria rapidly die out in a medium which they are unable to use as a source of energy. (2) Truly intestinal bacteria are probably highly specialised organisms. They are not so much saprophytes as parasites. Their optimum temperature is about 37°C., that is, the temperature of the interior of the body of mammals, while the temperature of sea-water, and that of the interior of the body of marine shellfish, varies from about 3°C. to 15°C. It is doubtful whether there is any initial multiplication of the bacteria on entering the shellfish—to prove that there is we should have to show that the numbers of bacteria per unit volume of shellfish was generally greater than the numbers per unit volume of the surrounding water. The concentration’ of bacteria in the latter varies enormously with state of tide and other conditions, and the condition of greatest concentra- tion would be that which ought to be compared with the concentration of bacteria in the shellfish. (3) Faecal micro-organisms may disappear very rapidly a powhen introduced into sea-water, the destruction ef 90 wy (4) Mcascls may be cleansed from ingested sewage bacteria by keeping them in water sterilised by the addition of chlorine. A concentration of chlorine in. sea-water of 5 parts per million 1 . sufficient, practically, to sterilise the water, while it does not interfere with the ordinary functioning of the shellfish. ~ The action of chlorine is twofold. It may siniply render a polluted sea-water practically free from sewage bacteria, and then this sterile water washes out the ingested: bacteria from the alimentary canals and mantle cavities of the shellfish, 150 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. and does not re-infect the animals. It may also actually destroy the micro-organisms present in the bodies of the shellfish, but it does not appear likely that the action of this kind may be of importance. The trace of chlorine present in the water would immediately enter into combination with constituents of the mucus covering the body, and would not really act on ingested micro-organisms. (5) Mussels may be very quickly cleansed, to the extent of getting rid of 90% of their contained sewage organisms, by exposure to running sea-water, or to repeated changes of _ standing water dosed with chlorine. Laboratory experiments indicate that the cleansing process need not require more than one day. On a commercial scale the time required would depend on the perfection of the water circulation, and it cannot be estimated except by experiment with the actual plant suggested. : (6) Faeces, sewage, polluted estuarine sea-water, and shellfish all contain a mixture of species of glucose and lactose fermenting bacilli. All these have been called ‘“ Bacillus colt,” or “ coliform ”’ organisms, or “ typical,” or “ atypical ” coli. Some of them are of equal significance with the true B. coli communis, in that they are of exclusive intestinal origin, but others may have sources of origin of vastly less significance. This mixture of species differs in faeces and in polluted shellfish. These species probably mostly cease to multiply, and finally die out, when they enter estuarine sea-water, or the bodies of marine shellfish. But the rate at which reproduction — falls off and the rate of mortality probably differ according to the species. It 1s, therefore, of practical importance that analyses should indicate the proportions of the various species, or categories of related species, present in a sample of polluted shellfish, since this may indicate whether the pollution was recent, and therefore of possible danger, or remote, and there- fore of little significance. SEA-FISHERIES LABORATORY. é 151 (7) There is urgent need for investigation into the specific nature of the various kinds of micro-organisms present in faeces and in polluted waters and in shellfish. The natural history of these organisms also imperatively demands investiga- tion. APPENDICES. I, Reactions of micro-organisms isolated from Mussels. Mussels from Piel Shore (6.6.1913). Mean number of sewage bacteria per mussel was 1,510. The characters of 10 organisms were as follows :— mH ree | ae |)! | s ai/e¢| 8/88) 2 = Morphology. ° ~ eo 2 5 = Oo | o0'm| *a = Pees te |) ois) 3 ise ss Seo ial 418 | 48 eA se 1 Piet ft a | + | — | + | — | + | OF Baeilli. 2);+/)/+/]+)]a@)+)—);+4]—)—| O | Cocci. 3); +}+])+)]a@/+)—)|+1—!—! O | Long slender bacilli. 4)/+})/+]/+}]a@]+;—-—/+]-— | —-—| 0O| Bacilli. Sre;@)—|a@a@ia;s;—|+{—-it 0 ue Serer | + | a | +) — | —| +} —|} 0 - wt @ a|—|a a;}—/]}—-|{|—-|- 0 - Sj+)/+/+]/a})}+;,—/]+]—] +] 90 | Short bacilli. Srp a@)a@|—|ajia | —|-—j}-— | + | 0 | Long slender bacilli. Poeeniea i — | a)ia|;—}|—|]—j)}+ | O| Bacilli. | (10.7.1914). Mean number of sewage bacteria per mussel = 3,900 i | | / | } } 1) + {+ el | t ted ae le | eet te | — | t+} —-| +] —-)|: | eee) | ] Pt] 4y+it+i+) +), —-)}-([ +] - | eee |) - | tl] ltl ty 6j+i/+);—-;}+]-]-| +] -| eee | | | ee | te) +t | Ft] | + | 9); + | + ais 5 ieee acid ae el al el Die ol ul : | 152 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Mussels from Aberdovey (24.6.1913). Mean number of sewage bacteria per mussel = 20,000. The characters of 10 organisms were as follows :— | eRe | os Salers a g os | Ss 2/2 / 2) 2 eee ge ss Morphology. > | 2 |S Selec! SO | m0} tH 3 ee ee cee a ee ho eS ah So 1 Gl et eee tea a ees oe eae ea aes es en ete ee ee Peas: ca | 2 aR le |e i a ee eS eee ee eee 3/+),+)]/4+;)—-|]+]-]-—]+]—-— | 0 | Long bacilli. 4}/+/+)/)—-{]—-]—/] —| + ]-—-— ]— | O | Short bacilli. 5+} +] +)]/—-)+);—-]+1—-] -—1| g | Moderately long bacilli. 6 at a a ae a ae 5 = ata ae 22 2 7/+)+)]+]a@)]+)a{+)—|]— | g | Short baciity 8|a a Oe) = ot a ee eee 0 | Moderately long bacilli. 9/+;)/+;}+)—]+]—]+{]-—]— | 0 | Very long bacilli. | 10{ +); +/]+/]/—-/]+i]a@{—]|+]— | O | Moderately long bacilli. | “Mussels from Barmouth (22.7.1913). Mean number of sewage bacteria per mussel = (a) 2,420, (b) 6,200. The characters of 14 of these organisms are as follows :— | $1 8)/a2/S$/2) a) 6 les 2) 8 Morphology. S) ~ i) 2 3 = o | De Te 3 Slalsi2iel| ele lee ga 2 SlbHio|Al4] 4) 4 eee lS 1} +/+) —-]—-]—-]—-|]+/—-—]—J| 0} Bacilli. Ae eae cen ei mallee aera lic a 3/+i)4+]—-!|—|]—|]—]-—1{]+ | —] 0 | Long slender bacilli. 4/+)+],+)/—/+/]=—)|]+ 1-—1—]| 0 | Bacih: 5) +) +}]+]a@)—-]—|]—]|—]|—4{ O | Long slender bacilli. Go ote a ee ela (Olen lee lena eee Mower ee ee lb): (a) Nos. 1 to 7 are from Trwyn y Gwaith. ee ee cee bce eae ea ee ice ee aS ee eee Oe ok ee ht LOS chef) cag ae | ea ect loro ocean WW} +)/+}+}/—-}+}]-|]-|-|-] 0] , 2/+)/ t+) +]—-}+})-/+)]-]-] 0]. 3}+/+!}+/+]}—-;/-|+]-]|+] 0], 4a4;/+]/t+)/4+}+i+!)-l+l+i4+/ +i. (6) Nos. 8 to 14 are from Aberamffra Harbour. SEA-FISHERIES LABORATORY. 153 Mussels from Rabble Channel (8.10.1913). Mean number of 11 sewage bacteria per mussel = 21,000. Reactions of 10 of these organisms as follows :— =I oS ep oe P2 r2 a g = = o 2 =| 5 eee s | 8 |e |S jara| | eB Sepaheieaia|/eizigsisis Spe; Ai as pS ea a | et. | — | —| +| —| +] 0 mia | ti —-|:t | —| —)} 0 eet | +} — | —| —| +} 0 eae) + | +} — | —| +) +} 0 ie | + | -—| +}; -| - | O ee | to} — | —- |-—-y — | O +/+} -/}/-|;+]-/]+]-]-)| 0 ee | + | —| +} —|— | 0 eae | -| —- | +}; — | +| 0 The above are organisms from the mussels. mae | —- | + |)-— | +l —)}—{| 0 Peete. | + | | +/+ — 0 eae 1 t+ | —} —| —|—!| 0 12 The organisms 10 to 12 were isolated from the water of the Channel. Roosebeck Scar mussels kept in large tank in running sea-water (5.1914). Mean number of sewage bacteria per mussel = 590. F+HEE4++4++4+ | Glucose, — COON HSHOrwWNe Morphology. Vosges and Proskauer. ++titirgiit | Motility. Long bacilli. Very long bacilli. Long bacilli. Short bacilli. Very short bacilli, or cocci. Short bacilli. Very long bacilli. Long bacilli. Very short bacilli. Very short bacilli. No ae Ey 2) la PO A i i Seer weal (At li a Ca a TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. 154 Kept in chlorinated Mussels from Barrow Channel (10.7.1914). water for 18 hours. “AMON | ae i Til ap | “MOTPOROYY TEINS SONG Wlse Wesce | pue soosoA Characters of 6 organisms :— ‘2IOpUT | See Nai ales “ulynUyT | eal ee ieee lear ‘agro | | aa lees Bef daalecet= -9910[ NG, | [aleealincte jleects ‘resng our | +il+i++ “280908 | Secale eae aga ty “asOonypy) | +++4++ aoe Kept wm sea-water Mussels from Barrow Channel (10.7.1914). Characters of 10 organisms :— for 12 hours. | ‘Aymow | ++++++++++4 Se "U0190 BatT _ dgoneysolg pue soSsoA -ejopar | sell se | ‘aye | sealer ‘oqquopy | Ihse ty ‘ayomar | | ++ | "980}0e'T +++14 -aviing uv | P+i+++1+++ +++4++4+4+4+44 ‘esoomp | ++++++++4++ | pies meee ee SEA-FISHERIES LABORATORY. fon Mussels from Conway taken from Bar (18.9.1914). Mean number of intestinal bacteria per mussel = 2,760. Reactions of 12 of these organisms as follows :— | a | ore és | o¢ = ma | oO tebe 5 ry a2j)a2|n) 2 #i/e|3 {3 as Morphology. ro) ~~ o a) 5 = oOo |0al] -s Eeiaeee = ia | se |S ee] & Sesio}o iat | S| 4 Al eee — |) — | +) —| — | — | + | Small bacilli. eee |) — | + | — | — | — | — | Long bacilli. Peete | | — | | | 2 eee | — | + | — | + | + | = | Small bacilli. 5 45 aie 55 al = — a = = ” “ag ee +,—|;+)}]—|--— | —4] — | Long bacilli. 7T/+;)+/}+/)—-. +/]—{+ 4-4] — | Very long bacilli. 8 == = | 25 = + = _ + — | Bacilli. 9); +i +i;t+;)—; +/] —| —| +1{ — | Small bacilli. eee | | = | — | + | Oh =| Baill. 2 | —|—j] +} — | + | Very small bacilli. 12 + a } at Spee = an ra a 99 99 eee — = Mussels from the Estuary of the Lune (26.10.1914). Re-laid at Overton for 6 tides. Mean number of sewage bacteria per mussel = 450. Reactions of 10 of these organisms as follows :— ee ) — a = = i 73 = ee i | 4 Bgl. s Oo | > 2 2 mMi/2\#\| 4/86 (38 BS Morphology. O° ~~ o 2 8 pal (e) jo) Oa) Te Sigiai/Zit|\a|3 leal & Soe | Oo }e |i sia | eS > Ay = CS a a aa Cn a ara a ee | eae 2 Wide) Roll eet i — |} +t} — | -— | -] - a Saat (2 Cc a Ul 4}+)4+/+)/-/-}-|-|+)+]_» Se] +} —| —| +] —| +] — | — | Small bacilli. eee af a | | lu] oe | | Bacilli, 7) +/+)/-/] -}| -—!|} —| +| — | + | Small bacilli. Serpe) +) +) +) —| —| +| — | Bacilli. 9)+/+)]-|+/]—-—} —| +] -—| — | Very small bacilli. myer i +) aj —| —i dj} +/+) — | Very long bacilli, — | | | ; 156 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Mussels from the Estuary of the Dovey (11.12.1914). Mean number of sewage bacteria per mussel = 200. Reactions of 16 of these organisms as follows :— 1 2 3 4 5 6 oi 8 9 10 11 12 13 14 15 16 FHEEHELE THEE +4++4 | Glucose, Fae macaial | false wiatia pel eiy aiaeme tr | Lactose. +++ yp t+tttqt 1 t+4t+4¢4t+ | Cane Sugar. HHH 4444141441 +4 | Dulcite, fetes eee | ++1 ++ | Adonite. it Pe bt ae tan Mussels from the Estuary number of sewage bacteria per mussel = 3,670. Reactions of 18 of these organisms as follows :— a BEE Somusamewe| 14 15 16 17 18 FHEFEHEFEFEH HEE LEL+ | Glucose. FHEELE LEFF EEF E+ | Lactose. ae) ese eos aries llircire tet ssa | Cane Sugar. FLLT FLT +E ELT 111 | Duleite. FFL I+1 LEH T+ +4441 | Adonite, satis Tee a eet sii vena le. Vosges and Proskauer. PHH EET +L ELI +1 +1 | Indo of FEE IPH LE LE + +444} Indole. pFH4E+4++44+ 141441 | the | Vosges and | Proskauer. Oe STR Ne la BE La sl LR BH al dk [Ea pete feat Pe J [a | Motility. [er++F Fr ts irri ii | Motility. — Morphology. Bacilli. Chains. Bacilli. 99 Chains. Very short bacilli. Chains Very short bacilli. Bacilli. Very short bacilli. Bacilli. Chains. Bacilli. Conway (12.1.1915). Mean Morphology. ee eo Small bacilli. Bacilli. Long bacilli. 29 Bacilli. Chains. Bacilli. Small bacilli. Bacilli. Small bacilli. Bacilli. SEA-FISHERIES LABORATORY. 157 Reactions of 72 organisms isolated from mussels. (These reactions are not contained in the previous tables.) | Glucose. Lactose Cane Sugar. Vosges and Proskauer Reaction. Organisms. | | +++++++4+ | Mamnite, mee bo bo O ~1 7 1 DO t++t+++++++ ++t++4+++++ I++it+4ti111 teary le liste ol | bi tti ti iti Pee elpetetealedeeleal Neslefieerl otc | 63 9 other organisms did not ferment both glucose and lactose. II. The graduation of the rough data of the Experi- ments I, II and III (see pp. 128-133) It may be well worth while to discuss the method of graduation of the rough figures obtamed in the above experiments, as they illustrate a frequent difficulty in biological work involving series of numerical values. It is quite evident, merely from a glance at the figures, that they are to be fitted to a curve, the form of which is that of the rectangular hyper- bola. But if we try to draw this curve, after plotting the points represented by the figures, we shall find that to do this by inspection is not easy ; and if we try it several times, without looking at the graphs previously made, we may find that the results are not the same. ‘This is generally true of the method of making curves representing biological statistics. The data are rough, and allow us considerable latitude in making their graphs. We can, quite unconsciously it may be, make these 158 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. graphs go as best suits our argument. We ought to have a method of making them which removes personal bias. An easy application of Pearson’s method of moments enables us to do this in the present case. Assume, then, that the general equation representing the drop in numbers of the bacteria in the cultures is :— yo =a that is, the equation to a hyperbola. Now taking logarithms and transposing we get from this equation :— log y= — n log a + log a, that is, the equation to a straight line, — n being the tangent which the line makes with the axis of x, and log a the intercept on the axis of y; we have now to find these constants n and a. If we plot these points given by the equation we find that they are irregular, and we have the same difficulty in drawing a straight line through them as we had in the original data. If the points lay very near a straight line we could find both n and a by inspection of the graph. But since this cannot be done with certainty, we have to find the constants analytically. Let us regard the logs of y and 2, not as logs, but simply as y and x co-ordinates. They form a distribution, and we have to find the mean of this, and the first moment of all the frequencies in it about the mean. The graph of the distributions shows us a series of trapezoids on unequal bases. To find the moments we must therefore calculate the areas of these trapezoids, and then the mid-ordinates. We must suppose the areas to be concentrated round the mid-ordinates. If the y- co-ordinates are y , ¥,, Ys, &c., and the «- co-ordinates, L1, L2, Xs, &c., we find the areas from :-— (Xp fe Ln) +) &e. while the corresponding mid-ordinates are given by 9 Le ar HO . a ee we ee eee ee é SEA-FISHERIES LABORATORY. 159 Fie. 2. Graduated and ungraduated data of the example on p. 160. We now take some one ordinate (not a mid-ordinate) near the mean, and we find the moments of inertia of the mid- ordinates about this arbitrary origin. This gives us »,, the first moment about the arbitrary origin. Subtracting this value from, or adding it to, the value of the arbitrary origin, according to its sign, we get the mean ; and dividing it by the area we get mw, the first moment of the whole distribution about the mean. We find that the latter is always zero, but we must calculate it in order that we may find the limits (l, and /,) of the distribution about the mean. The area of the whole distribution is, of course, simply the sum of those of the trapezoids. Now if the equation to the straight line is y= — na+a (nz + a) dx = area of the distribution. In finding the first ae what we do is to multiply each frequency by & so that ‘ts [~ (nz + a)] dx = first moment about the mean = 0. Days. x oo - Ww bd 160 We have now two equations and by solving these © TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. simultaneously we find the constants » and a. It may be worth while to give an actual example of the calculation. 2 3 4 5 6 7 8 9 1st Nos. of Values | Bases | Areas. | Distance | moments bacteria | Logs of | Logs of | of mid- of (5 x 6) from about in unit x y ordinates] trape- a arbitrary | arbitrary volume. zoids. origin. origin. y d axd 2,310 | 0-0000 | 3-3636 . 3:1555 | 0-3010 | 0-9498 | — 0-1505 | — 0-1429 886 | 0:3010 | 2-9474 ——— 2-2904 | 0-1761 | 0-4033 | + 0-088 | + 0-0355 43 | 0-4771 | 1-6335 1-5949 | 0-1250 | 0-1994 | + 0-2386 | + 0-0476 36 | 0-6021 | 1-5563 0-9286 | 0-0969 | 0-0899 | + 0-3495 | + 0-0314 2 | 0-6990 | 0-3010 as 1-6424 + 0-1145 yi — 0-1429 — 0-0284 | Area of whole distribution = 1-6424. V 1 _ — 0-0284 _ = ye 0-0172 Mean = arbitrary origin — 0-0172 = 0:3010 — 0-0172 = 0-284 Limits are therefore — 0-284 and 0-6990 — 0-284 = + 0-415 x Aven == gio ye 4 oa = 0°415 (nz + a) dx = 0-0458 n + 0-699 a — 0-284 "415 i ee [u(na + a)] da = t= — 0°284 These equations give y = — 3-7x + 2-535 We have now found the equation to the straight line which fits best the irregularly placed points which we have quoted. The area beneath this straight line, and between the first and last ordinates, is equal to that formed by the series of trapezoids. We calculate the new ordinates, — » being j= 0 0-0314 nm + 0-0458 a SEA-FISHERIES LABORATORY. 161 the tangent which the line makes with the axis of x, and a the intercept on the centroid vertical of the graph. Remembering that these are really logarithms we now find the smoothed values of the original data. We also find the equation to the hyperbola. Converting the logs back again into natural numbers we find the value of n, now the index of x and positive; a is, of course, merely a scale constant. It represents the (graduated) number of bacteria per unit volume which was present in the culture at the beginning of the experiment. We find the constants in the equation yz” = a. Ill. The Counts of Colonies on Plates. Some matters of interest with regard to methods may be noticed here. As a rule one finds different kinds of colonies on the same plate. In cultures of badly polluted or recently polluted mussels in neutral red, bile-salt, lactose agar we find both crimson and colourless colonies. The latter are always on the surface of the plates; at least, if they are present in the depth of the medium one cannot see them. They differ greatly in their ability to ferment sugars from the crimson colonies; as a rule they do not ferment glucose and lactose. They are hardly ever seen in cultures from mussels which are not badly polluted, and which have been taken from places at some considerable distance from the source of pollution. They are always absent in cultures made from mussels which have been re-laid. I attach some importance to the presence of such colourless colonies as indicating recent pollution. I have subcultured very few of them—none of these subcultures are described in this Report. As a matter of both theoretical and practical interest they ought to be closely investigated. The crimson colonies are present both on the surface 162 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. and in the depth of the plate. Those on the surface are usually flat with raised centres, and of varying shades of crimson. The central part is usually deeper in colour than the margin. Sometimes they have very little colour, but one can always distinguish them from the transparent colourless colonies. The latter are generally slightly irridescent, whereas the others (the crimson colonies) are slightly opaque even if they have very little colour. The colonies in the depths are always much smaller, and their colour is much deeper. The larger ones are egg-shaped when seen from the surface, and there is generally a slight haze round them, easily seen if the medium is clear. In cultures from mussels, made as I have described, we inoculate a complex physical mixture, a suspension, largely of broken-down solid tissue, and this often prevents the haze round the deep colonies from being visible. These deep colonies are really lenticular in shape, with their long diameters perpendicular to the surface of the plate. They assume this position, I suppose, because they grow more easily towards the surface than parallel to it. Now these differences in the naked-eye appearance of the red colonies are of little or no significance: we cannot tell, except by subculturing a colony, what micro-organism it represents. The differences are produced by the varying positions of the colonies in the medium, and by the degree of aggregation of the colonies. Consider what is the physical structure of an agar plate. It is an emulsoid consisting of two liquid phases. In a dilute solution of agar, like that employed in the preparation of “solid” nutritive media, cooling throws out of true solution a phase consisting of droplets of a relatively concentrated solution of agar in water (or water in agar). These droplets coalesce together to form a meshwork in the interstices of which is the second phase, the relatively dilute solution of agar in water. The liquids of the two phases do not separate into layers, but remain — ; j SEA-FISHERIES LABORATORY. 163 permanently in the form of a jelly, or a viscous liquid, according to the concentration. The interstices of the meshwork are, nevertheless, so fine as to prevent the transport of even such small particles as the bacilli. How these spread through the jelly is a subject for microscopical investigation by sections of the hardened substance—I do not know if this has been attempted. The agar jelly we may imagine to be like a sponge containing liquid, and the liquid consists of the dilute agar solution, and of the solutions of the nutritive and other substances entering into the composition of the medium. In a very concentrated agar jelly, the phase containmg much water would consist of droplets enclosed by the phase containing httle water, so that diffusion of electrolytes through the jelly would be very slow. But in the dilute agar jelly there would be little more resistance to diffusion than if the whole were a true molecular solution. The surface of the jelly is always moist, since it is contracting and liquid is being forced out. A colony on the surface will, therefore, grow more rapidly than one in the depth of the plate, since it will take up the dissolved nutritive substance round itself, and the concentration of the latter will be renewed by diffusion from surrounding areas of the surface as rapidly | as it is lowered. Therefore, these superficial colonies will be larger, and slightly different in appearance. Diffusion will be rather slower in the depth of the plate, and adjacent colonies will act as centres of absorption for the dissolved nutritive substances. If the colonies are numerous they ought to be smaller, since they are growing at the expense of the liquid food substances added to the jelly rather than the latter itself. And this is what we actually find. The colonies in the crowded plates are always smaller than those in plates which have been inoculated by only a few bacteria. Further, and this is a matter of considerable practical importance in methods, a crowded plate ought to indicate 164 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. a higher degree of pollution than the actual count appears to indicate. For the colony in the depth is only visible when it concentrates the stain added to the medium, either physically, or by a process of wnétra-vitam stainmg—this is a point of theoretical interest that might be investigated—and numerous colonies will deplete the jelly of the added nutritive substances. The more rapidly growing colonies will, therefore, “ starve out.” those which grow more slowly: we may safely assume that there are such individual differences among bacteria of even the same species and culture. As a matter of fact this is easily proved by making a series of decimal dilutions of the same culture. In the higher dilutions we shall find that the numbers of colonies in the plates are multiples, or sub-multiples, by 10 of some number. But the counts of the lower dilutions, if the original culture were sufficiently concentrated, are not so much as 10, 100, times those of the middle dilutions. It is important to note, there- — fore, that the estimates of the number of bacteria in the substance being investigated should be based on the higher rather than on the lower dilutions ; and that dilutions should be made so as to have few colonies on the plate, apart altogether from the question of ease in making the counts. IVY. Methods of Sampling. Precautions in taking and forwarding samples of mussels usually take the form of keeping the shellfish cool by packing the vessels containing them in ice. It is assumed that the micro-organisms contained in the shellfish may multiply if the temperature is raised, and that this may, indeed, be the case can often be shown by keeping the sample for a day or more at laboratory temperature. But if it is the case that intestinal bacteria do not continue to live and reproduce in sea-water, as the experiments described on pp. 128-133. tei ee ee — Del eh od SEA-FISHERIES LABORATORY. 165 Show, then it may also be the case that a reduction in the numbers of the micro-organisms contained in shellfish may be experienced as the result of keeping the sample for a day or two after collection. I have obtained such a result in at least one case. It is probable that micro-organisms contained in the alimentary canal of the shellfish tend to be extruded into the mantle cavity, and if the temperature rises, or the mussels are kept too long, the water in the mantle cavity may be lost by the opening of the shells. In such a case the estimate of the bacteria per mussel may be less than it ought to be. Obviously the samples ought to be stored in small sterilised vessels which hold no more than the precise number of shellfish which are to be examined, and the water in the vessel ought to be added to the mixture, which is prepared from the soft parts of the shellfish. It is as well that this possibility of a reduction in the number of bacteria should be considered in cases where apparently anomalous results are obtained. The Barmouth purification experiment of 1915, referred to on p. 140, is a case in point. Here we have to deal with an estuarine area subject to pollution, as the chart published in last year’s report shows. In July of 1913 I visited the estuary and made an inspection of the mussel beds and took samples. These gave unequivocal evidence of most undesirable pollution. A typical plate, made from 1/50th part of a mussel was photographed, and is reproduced as fig. 2 of Plate II in the present report. In February of this year (1915) the Fishery Officer at Aberdovey made the experiment to which I refer above. The samples were sent to me by parcel post. Fig. 1 of Plate II represents a typical plate made from 1/50th mussel, and the difference in the apparent pollution is very marked. Such a degree of pollution as that represented by fig. 1 I am inclined to regard as of little or no significance. The question of the extent to which the mussels underwent 166 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. self-cleansing is, of course, quite a different one, and is unaffected by any question of irregular sampling, for all the samples were taken and forwarded in the same way, and whatever applies to. the original sampling also applies to the sampling after the relaying. But we have to consider the difference in the apparent bacterial contents of two samples" of mussels sent from the same area. In summer the population of Barmouth is a holiday one (say about 5,000), whereas in winter it may be reduced to about half that number. In summer the volume of fresh water in the river is small, whereas in the months of January and February it may be considerable on account of rains. The smaller population and the increased amount of flushing of the river may perhaps account for the difference in the apparent pollution, but perhaps the difference may be due, to some extent, to the conditions of sampling. I mention this particular case as illustrating the contention that the actual conditions, as regards pollution, of a particular natural shellfish area, at a particular time, may not be accurately estimated merely from samples collected and transmitted by persons other than those making the analysis. In cases where the results of analysis are important, the collection and further history of the sample prior to analysis. is equally important. SEA-FISHERIES LABORATORY. 167 EXPLANATION OF THE CHART. Entrance to the Lune Estuary. The sketch chart has been reduced from the 6-inch ordnance map (Lancashire Sheet XXXIV). Sandbanks uncovered by ordinary tides are represented by fine stippling. Hard, stony foreshore is represented by coarser stippling and small circles. The extensive salt marshes of this district are represented by short horizontal lines. The positions of the mussel beds and the places where mussels were re-laid are indicated by coarse cross-hatching. The narrow strip of clear water is the main channel leading to Lancaster and Glasson Dock. At low water of ordinary tides it is only a few feet in depth, and it is still shallower at low water of spring tides. It is about 300 feet in width on the average. Small, shifting channels run from it towards the “ becks,”’ or brooks, ashore, and through the salt marshes. Many of these smaller channels are not shown on the sketch chart. The main course of the stream is along the deepest part of the estuary during the first part of flood tide. When the banks are covered the stream passes over them, in the main following the trend of the estuary, but with numerous irregularities in direction and velocity due to the conformation of the sea bottom. When the banks are covered by the flood tide, the water on them may be regarded as bacteriologically pure, since it comes in past Sunderland Point from the Main Lune Channel, a passage widening out into Lune Deep, practically the open sea. The flood stream runs for four to five hours, the ebb stream for seven to eight hours. The higher parts of the banks are soon uncovered, so that the water ebbing from off them has not had time to become polluted from the upper parts of the river. There is hardly 168 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. ; | any pollution of the srnaller channels on the north side of the estuary since there are only a few houses here and there along this shore. The main sources of pollution of the estuary are Lancaster and Stodday, that is, a population of over 40,000 persons. All this sewage is untreated. It enters the channel at various points higher up than the area represented on the Sketch Chart. Two sewers are shown, those at Glasson and Conder Green. These serve a population of about 1,600 persons. The sewage is untreated and discharges directly into the sea, the Glasson sewer into the main channel, and the Conder Green sewer into a brook which flows through a salt marsh into the main channel. The mussels taken from the Lune Estuary come from various places. Most are taken from the traiming wall at Bazil Point and the adjacent part of the channel. This wall is a rubble structure which comes adry at low water, so that it is washed by the water coming down the channel at the lowest states of the tide. The mussels here are thoroughly polluted. The numbers M 90,000, 11,650, and 3,700 represent the results of recent analyses—they are the mean numbers of sewage bacteria contained in a mussel. Some mussels are taken from Crook Skear, which is the area of rough foreshore opposite Sunderland Point. Others are taken from the Skears at Abbey Lighthouse, and from the bottom of the Channel near there. The water in the estuary at low water is greatly polluted. The numbers W 83 and W 82 on the sketch chart represent the numbers of sewage bacteria per cubic centimetre as found in some recent analyses. These numbers would probably vary oreatly from time to time according to the state of tide and the amount of fresh water in the river. | In the cleansing experiments the mussels were taken from the training wall, and the adjacent bottom of the channel not SEA-FISHERIES LABORATORY. 169 far from Bazil Point. They were re-laid, first of all, at the place marked a little way north-east from Bazil Point. The sand banks are very high here, so that by the time the flood-tide has reached this part of the foreshore the sewage in the lower part of the channel will have become enormously diluted. Whatever water ebbs from off this foreshore, and down the little channel passing it, will therefore be clean. As the tide ebbs out the water in the channel will become more and more foul, but it can no longer come near the re-laid mussels. The experiments were made on 7th, 8th, 9th, and 10th October, 1914. The highest spring tides were on the 4th (an 18 foot 6 inch tide at Liverpool); when they were begun the mussels covered at 11 a.m. and uncovered at 3 pm. At low water they would be covered for about two to three hours on each tide. The experiment made at Sunderland Pomt was made under much the same conditions. In these experiments the number of bacteria contained in a mussel was reduced from about 12,000 to about 400. The place selected at Glasson was, as the chart shows, much less favourable. It is very close to the outflow of sewage from the River Conder. It is well up a steep beach, so that it was covered for about three and a half hours on each tide (two to five days after lowest neaps, a 12 foot 9 inch tide at Liverpool). There was much rain. and the river was in flood. The contamination was reduced from about 90,000 to 24,000 sewage bacteria per mussel. The results might have been more favourable given better conditions, but the place is, evidently, far from suitable. 170 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. EXPLANATION OF PLATE I. Cultures of 1/50th part of a mussel in neutral-red, bile- salt, lactose agar. (See pp. 136-7.) Fig. 1. The original sample of mussels taken from the ~ training wall in the River Lune. 7 Fig. 2. The same mussels re-laid for six tides. Fig. 3. The same mussels re-laid for four tides. EXPLANATION OF PiLaTE II. Fig. 1. Culture of 1/50th part of a mussel from Barmouth, winter of 1915. (See p. 140.) The liquid inoculated was very turbid, so that the medium is greatly discoloured. There were eight red colonies in the plate. 3 Fig. 2. A similar culture made from Barmouth mussels from a neighbouring bed, taken in the summer of 1913. The red colonies are very numerous and there is a large patch of small, partially fused colourless colonies. Puate III. Lower part of the Estuary of the Lune. 7) Prare I. Cultures of Faecal Bacteria from Mussel Relaying Experiments. PLATE Cultures of Faecal Bacteria from Aberdovey Mussels. Le PearE® Ele I it Se th Mh Alt ii yi H iM re Mi ws iy NS HN te Miyjiii' Nips Ph De a } \ iil!) im; yell tty Wh min UI eed Hatt ihe ibe 5 rr ‘ HY ay Al ri i Ty Healt Sgt ' 9 7 PR ioe Zit Lower part of the Estuary of the Lune. SEA-FISHERIES LABORATORY. 172 REPORT ON HERRING MEASUREMENTS. By W. Rrippett, M.A. During the past year 19 samples of herring have been examined, amounting to 1,068 fish. Particulars of these will be found in Tables III-V. As some errors have been found in the tables published in last year’s Report, these have been checked, and the revised figures are here re-published as Tables I and II. In Tables I and II, and sample (1) of Table III, the length T is measured to the line joiming the tips of the lobes of the caudal fin when this is in its natural position; A is measured to the anus. After this, at the request of the Board of Agriculture and Fisheries, these measurements were slightly altered. 7 is now measured to the tip of the dorsal lobe of the caudal fin when extended in line with the body, and A is taken to the beginning of the anal fin. The other characters are :—_ ; T.cd. Total length to base of tail. D. Length from tip of snout to beginning of dorsal fin. V. Length from tip of snout to beginning of pelvic fin. L.cp.l. Lateral length of head. K,. Number of keeled scales between anus and pelvic fins. Ss. Sex. g. Condition of gonads on Hjort’s scale. I do not consider that the measurements recorded up to _ now present any definite evidence either for or against the question of local races. . There are differences between samples from different areas, _ but samples from the one area differ just as much among 172 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. themselves. Thus one would expect the two trawled samples to represent one race, but these two samples differ markedly. The same thing may be seen in the samples from Port Erin. At first sight this might be regarded as against any division into races, but there are two defects in the figures which make any such conclusion untrustworthy. In the first place, the sampling has, in many cases, been quite insufficient. This is especially the case in regard to the fish from the Welsh Coast. The samples for the 1913-1914 season are much too small, and while those for 1914-1915 are better, they are still, I consider, insufficient. This seems to have been a bad season on the Welsh coast ; it was impossible to get large samples, and no fish were received after the end of 1914. In the second place, many of the samples were kept im cold storage until time allowed of their being examined. This, I am now sure, renders any measurements made upon such samples quite untrustworthy. I do not believe that any correction can be applied to compensate for the distortion caused by this method of preservation. This is the only conclusion I can draw from my own results, and Williamson’s* experiments upon mackerel point to the same thing. Future samples must be examined in a fresh condition. It is possible that other modes of preservation may not be open to the same objection. Heincke applies a correction to some of his samples which were preserved in spirit. But I think that, until direct experiment has shown that such a correction gives satisfactory results, all measurements made on preserved fish must be regarded with suspicion, and not used for com- parison with measurements made on fresh material. This does not apply, of course, to the examination of skeletal characters, such as~vertebrae. So far I have not examined the vertebrae of a sufficient number of fish to be able to draw any conclusion from them. The keeled-scales, * Fishery Board, Scotland, 18th Annual Report. SEA-FISHERIES LABORATORY. tis also, are unaffected ; but here, as regards the present figures, _ we are faced with the other difficulty of insufficient sampling. Further samples from all districts must be examined before the question of local races can be discussed with any confidence. One point on which something may, perhaps, be said is the relative numbers of the sexes. MHeincke states that he has the same impression as Ewart, that the females _ come to the spawning grounds earlier than the males, and also leave earlier. He also says that he believes that among spawning herring the females are in the majority. My figures lead me to believe that males are more abundant than females. Even omitting Stages I, II and VII, so as to remove any possibility of a mistake in determining the sex, there are more males than females in my samples. This would agree with Fulton’s statement* that among fish with demersal eggs females are, as a rule, fewer than the _ males, although his figures for the herringt show practical ~ equality in numbers between the sexes, females being in a slight majority. HEwart’st account of the spawning of the herring would also seem to indicate a preponderance of males. * Fishery Board, Scotland, 9th Annual Report. + Fishery Board, Scotland, 8th Annual Report. t Fishery Board, Scotland, 2nd Annual Report. 174 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Table I. (All measurements in the tables are in millimetres.) 4 mile E. of New Quay Head, November 7th, 1913. 4’ mesh. D. V. A. L.ep.l. No. Ty T.cd. — 9; 8 g. % % % % Ted: T.cd. T.cd Ted: 1 262 230 117 | 50-87 | 131 | 56-95| 172 | 74-78 | 46 | 20-00) 14] @ iy’ 2 260 222 116 | 52-25} 122 | 54:99) 170 | 76-57} 46 | 20-72| 14] ¢ iv 3 256 221 119 | 53-84 | 125 | 56-56] 174 | 78-73) 46 | 20-81; 13 | ¢ V 4 255 223 116 | 52-01 | 123 | 55-15) 166 | 74-43} 46 | 20-62/ 14] @ Vv 5 252 221 112 | 50-67 | 123 | 55-65) 170 | 76-92 | 47 | 21-26} 15) ¢ Vv 6 250 220 111 | 50-45; 122 | 55-45| 169 | 76-81 | 47 | 21-36; 14 | 9 Vv 7 250 219 115 | 52-51 | 122 | 55-70} 165 | 75-34) 45 | 20-54] 12 | @ V 8 250 218 113 | 51-83 119 | 54:58} 164 | 75-22 | 45 | 20-64| 14 | @ V 9 250 217 112 | 51-61 | 120 | 55-29| 165 | 76-03 | 45 | 20-73| 13 | 3 Vv 10 250 213 115 | 53-99 | 119 | 55-86| 164 | 76-99} 45 | 21:12; 14) 9 V 11 249 212 109 | 51-41 | 114 | 53-77} 163 | 76-88 | 43 | 20-28; 15 | ¢ V 12 248 214 114 | 53-27| 117 | 54-67} 163 | 76-16 | 46 | 21-49| 14] @ V New Quay, November 11th, 1913. 4’ mesh. “13 {| 276 239] 123 | 51-46| 128 | 58-55| 182 | 76-15 | 53 | 22-17 | ine je) vo 14 274 242 125 | 51-65| 137 | 56-61 | 189 | 78-09 | 50 | 20-66) 14] ¢ Vv 15 268 237 124 | 52-32} 133 | 56-11 | 183 | 77-21 | 47 | 19-83) 15 | ¢ V 16 264 229 120 | 52-40 | 128 | 55:89} 178 | 77-72 | 49 | 21-39} 13 | Q ve 17 259 224 114 | 50-89} 124 | 55:34] 176 | 78-57] 51 | 22-76] 14] ¢ V 18 258 227 116 | 51:10} 120 | 52-86] 174 | 76-65 | 47 | 20-70] 14) ¢ Vv 19 257 225 111 | 49-33 119 | 52-88] 172 | 76-44] 46 | 20-44] 14] ¢ Vv 20 257 224 115 | 51-33 | 119 | 53-12} 167 | 74:55 | 45 | 20-09 | 13 Q Vv 21 256 | 223 115 | 51-56} 121 | 54-26] 171 | 76-68 | 46 | 20-62] 13] ¢ V 22 255 221 120 | 54:29; 120 | 54-29| 172 | 77-82 45 | 20-36] 15 | ¢ Vv 23 253 221 113 | 51-13 | 122 | 55-20| 174 | 78-73 | 45 | 20-36] 14 | ¢ Vv 24 250 225 113 | 50-22 | 124 | 55-11} 173 | 76-88 | 48 | 21:33] 13 | @ Vv 25 250 218 112 | 51-37| 119 | 54:58] 167 | 76-60 | 47 | 21:56} 13 | @ V @ 26 246 216 108 | 50:00; 117 | 54:16| 166 | 76-85 | 45 | 20-83) 15) ¢ Vv 27 239 210 104 | 49-52] 113 | 53-80] 158-4 75-23 | 43 | 20-47 | 13 4 | 409 | 210 | 104 | 49-52 | 113 | 538-80} 1587) 75-23 | 43 | 20-47) 18 | gi Ve Vv Penrhyn Weir, Bangor, November 17th, 1913. 1 98 | 276| 244/| 127 | 52-04) 131 | 53-68| 184 | 75-40| 50 | 20-49] 15| 9 v= 29 262 231 122 | 52-81 | 129 | 55-84] 175 | 75-75 | 50 | 21-64]; 15 | @ V ‘a 30 255 225 118 | 52-44] 126 | 56-:00| 172 | 76-44) 47 | 20-88; 15 | 9 af a 31 253 225 116 | 51-55] 128 | 56-88] 172 | 76-44] 48 | 21-33) 15 | ¢ Vv 32 253 223 117 | 52-46} 119 | 53-36| 165 | 73-99} 48 | 21-52/ 15) ¢ V : 33 249 217 114 | 52:53 | 125 | 57-60) 165 | 76-03 | 47 | 21-65) 13 2) WN. 34 248 217 114 | 52-53 | 119 | 54-83} 167 | 76-95} 50 | 2304/15 | ¢ Vv } 35 248 214 112 | 52-33 | 121 | 56-54] 164 | 76-63] 47 | 21:96; 15 | ¢ Vv & 36 246 218 118 | 54:12} 125 | 57-33 | 168 | 77-06} 49 | 22-47] 14/| ¢ V 1 : oil 242 213 108 | 50-70} 116 | 54-46] 158 | 74-17] 45 | 21-12| 14 37 | 242 | 213 | 108 | 50-70 | 116 | 54-46) 158 | 74:17 | 45 | 212 eee V | Moelfre, November 20th, 1913. t 38 272 | 239 121 | 50-62| 139 | 58-15) 184 | 76-98 | 52 | 21-75 | 15 fe) VII | 39 268 | 235 124 | 52-76] 133 | 56-59| 183 | 77-85 | 54 | 22-97) 14] @ VII 40 264 234 120 | 51-28) 131 | 55-98} 172 | 73-50.) 52°| 22-22) 14 |) Oss vewn 4] 263 230 120 | 52:17} 130 | 56-52| 172 | 74:78 | 52 | 22-60) 15! 92] V-VI 42 262 | 235 121 | 51-48! 130 | 55-31 | 175 | 74-46! 50 | 21-27) 14] 3 Vil 43 262 234 120 | 51-28] 132 | 56-41 179 | 76-49 51 | 21-79} 14; ¢ Vil y 44 259 226 120 | 53:09 | 127 | 56-:19| 175 | 77-43) 49 | 21-68/ 14] ¢ Vil 45 255 226 114 | 50-44] 128 | 56-63! 170 | 75-22) 45 |19-91} 14] g| V-VI 46 250,| 223 11] | 49-77] 122 | 54:70! 168 | 75:33 | 48 | 21:52] 15) ¢ VIT- 47 249 | 213; 114 114 | 53-52} 118 | 55-39 164 | 76-99 | 47 | 22-06 | 15 | 52. ATS oo: =e 164 | 76:99 | 47 1d tae, Vil 22-06 2) SEA-FISHERIES LABORATORY. 175 Table I—Continued. Moelfre, November 20th, 1913—Continued. D. V: A. Lep.l. g| T. | T.cd Ke.| s g % % eo 4 % en: Ted. T.ed. Ted: 48 | 246; 216] 110 | 50-92; 120 | 55-55| 162 | 75-00} 46 | 21-29) 14]; g| VII 49 | 245} 214) 114 | 53-27] 120 | 56-:07| 163 | 76-16| 48 | 22-43) 14 | 9 | V-VI mo} 245) 214) 111 | 51-87} 119 | 55-60| 164 | 76-63) 48 | 22-43; 15) g| VII Pol | 239; 209; 106 | 50-71; 114 | 54-54) 159 | 76-07| 44 | 21:05) 15 | g | V-VI fee) 209 | 209! 110 | 52-63; 118 | 56-45) 162 | 77-51 | 45 | 21-53; 13 | g | V-VI | ba | 237 | 207} 106 | 51:20) 115 | 55-55| 157 | 75-84) 46 | 22-22) 15] ¢ VII pt) 234) 207) 107 | 51-69| 115 | 55-55| 155 | 74-:87| 47 | 22-70| 14 | 9 VII Bo} 2a2 | 204; 110 | 53-:92| 114 | 55-88| 152 | 74-51 | 47 | 23:03; 15 | g | VII | ; 56-66 | 185 | 77:08 5 242 | 124 | 51-23; 130 | 53-71| 179 | 73-96 | 48 | 19-83 | 14 58 | 266) 235 | 121 | 51-48; 129 | 54-89] 179 | 76-17} 48 | 20-42) 14 1599 | 265 | 233 118 | 50-64} 130 | 55-79} 179 | 76-82} 50 | 21-45] 15 60 | 264, 236, 122 | 51-69; 130 | 55:08] 178 | 75-42) 50 | 21-14) 14 #61 | 260 229) 117 | 51-:09| 127 | 55-45; 171 | 74-67) 48 | 20-96} 14 62 | 256 | 226) 119 | 52-65) 127 | 56-19| 173 | 76-54 | 48 | 21-23 | 15 | 63 | 256, 225; 114 | 50-66] 127 | 56-44) 171 | 76-00) 48 | 21-33] 13 64) 254), 227) 118} 51-98) 123 | 54-18| 171 | 75-33 | 45 | 19-82] 14 65 | 254 | 221 116 | 52-48 | 127 | 57-46| 172 | 77-82 | 50 | 22-62] 15 66 | 253 | 220| 114] 51-81; 123 | 55-91| 168 | 76-36 | 48 | 21-81 | 15 67 | 251 | 222 | 114 {| 51-35} 119 | 53-60} 168 | 75-67 | 48 | 21-62 | 15 O3 40034003 Os 40404003 4040 54:77 | 185 | 76-76 69 | 271 239.| 123 | 51-46) 130 | 54-39; 181 | 75-73 | 48 | 20-08 | 16 70 260 | 229 | 124 | 54-14) 127 | 55-45| 175 | 76-41 | 51 | 22-27) 14 Wat | 209; 229) 115) 50-21; 122 | 53-27; 172 | 75-10 | 48 | 20-96) 14 7 | 256) 226 | 117 | 51-77| 124.| 54-86; 174 | 76-99 | 49 | 21-68 | 15 73 | 250) 221 118 | 53-39 | 124 | 56-10| 168 | 76-01 | 49 | 22-17) 14 ia} 248) 217) 114 | 52-53; 122 | 56-22| 167 | 76-:95/| 48 | 22-12/ 13 575 | 246 | 217) 114 | 52-53| 121 | 55-76| 166 | 76-49 | 48 | 22-12 | 14 76 | 241 214 | 112 | 52-33; 118 | 55-14| 157 | 73-36 | 46 | 21-49) 14 pa? | 219) 197 98 | 49-74; 105 | 53-29] 144 | 73-09 | 43 | 21-82 | 14 fae | 218 | 185 94 | 50-81 | 102 | 55-13) 138 | 74-59| 39 | 21-08} 14 p79} 203 | 177 99 | 55-93 | 101 | 57-:06| 134 | 75-70} 41 | 23-16 | 13 180 | 202 | 179 94 | 52-51 99 | 55:30 | 137 | 76-53 | 42 | 23-46 | 14 O3 404003 O3 Os 4003 404003 4040 = se,| 4 mile from New Quay Head, December 8th, 1913. 4’ mesh 282 | 252 | 130 | 51-58; 145 | 57-53| 190 | 75-39 | 53 | 21-03 | 16 | 275 | 244] 126 | 51-63) 134 | 54-91 | 182 | 74-59 | 51 | 20-90 | 14 267 | 240; 124 50-42 133 | 55:41 | 181 | 75-41 | 49 | 20-41 | 14 | ! 84 266; 236 119 | 50-42/| 133 | 56-35; 181 | 76-69) 50 | 21-14) 15 | Bo} 266, 234); 120 | 51:28; 130 | 55-55) 175 | 74-78) 49 | 20-94) 14 BO} 264) 235, 121 | 51-48; 130 | 55-31 | 179 | 76:17| 50 | 21-27/| 16 we} 208}; 229) 119 / 51-96} 129 | 56-33| 173 | 75-54| 50 | 21-83} 14 | 254 | 224) 117 | 52:23) 127 56-69 | 171 | 76-33 | 47 | 20-98 14 88 p89 | 252 | 221 119 | 53-84] 125 | 56-56| 174 | 78-73| 48 | 21-71 | 14 woo} 250) 223) 117 | 52-46| 123 | 55-15| 172 | 77-13] 47 | 21-07| 15 POL | 249) 221 | 116 | 52-48| 124 | 56-10} 171 | 77-37| 47 | 21-26 | 15 4003 05 03 05 03 0505 40404040 109 | 51-:17| 118 | 55-39| 162 | 76-05 176 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. . Table I—Continued. ‘, Moelfre, December 8th, 1913. a D Vv A. l.cp.l i No.) T. | Tied, |-~——,- = eee g % % % 9 T.cd T.cd. T.cd T.cd SS SS OS EE E> ——— EE — EE EE SSS) | | 4003 40404003 O3 03 O3 OS Penrhyn Weir, December 9th, 1913. 103 | 254 | 226 | 118 | 52-21| 124 | 54-86 | 170 | 75-22 | 48 | 21-23 | 14 104 | 254 |} 225 | 116 | 51-55} 124 | 55-11 | 170 | 75-55 | 47 | 20-88| 14 105 | 254 | 222 | 116 | 52-25; 122 | 54-99 | 170 | 76-57 | 50 | 22-51 | 14 106 | 252 |} 223 |) 115 | 51-56; 121 | 54-26] 165 | 73-99 | 49 | 21-97] 14 107 | 238] 211 107 | 50-71 | 114 | 54-03 | 155 | 73-45 | 45 | 21-32). 13 108 | 238; 210); 108 | 51-42 | 116 | 55-24 | 161 | 76-66) 45 | 21-43} 14 109 | 214); 186 97 | 52-15| 103 | 55-37] 140 | 75-26 | 44 | 23-65) 12 110 | 206; 181 96 | 53-03 | 101 | 55-80 | 136 | 75-13 | 42 | 23-20) 14 LID) 2059" Ts 93 | 51-38 | 102 | 56-35 | 133 | 73-48 | 41 | 22-65) 13 112 | 204; 183 92 | 50-27 98 | 53:55 | 134 | 73-22 | 41 | 22-40| 12 113 | 202; 174 94 | 54-02 96 | 55:17 | 129 | 74:13 | 41 | 23-56) 14 ‘114 | 201 174 90 | 51-72 97 | 55-74 | 131 | 75-28 | 40 | 22-98) 12 115 | 199] 172 88 | 51-16 94 | 54-65 | 128 | 74-41 | 40 | 23-25) 12 116). 198 |. 174: 88 | 50-57 96 | 55-17 | 130 | 74-71 | 40 | 22-98 | 14 117 | 1984 1738 91 | 52-60 96 | 55-49 | 128 | 73-98 | 41 | 23-69 | 14 118 | 195 | 174 93 | 53-44 97 | 55-74 | 1381 | 75-28 | 41 | 23-56| 13 PGS SO 2a ail 89 | 52-04 94 | 54-97 | 128 | 74-85 | 40°) 23-39 | 14 120 | 184 | 161 84 | 52-17 88 | 54-65 | 120 | 74-53 | 38 | 23-60 | 13 Tremadoc Bay, December 18th, 1913. <<< 1 = > d 4003 03 03 O3 404040404003 404003 4003 4040 bead sss a em ol) eet eet et el sh, Ft = ' 1 1 ' 1 1 1 —_ HAH HHH SSS ea a a a EEE =e Moelfre, January 6th, 1914 132 | 268 | 236 121 | 51-27| 129 | 54-66 | 177 | 75-00 | 47 | 19-91 | 14 133 | 257 | 231 119 | 51-51 | 127 | 54-97 | 174 | 75-32) 49 | 21-21) 14 121 | 261 | 230 | 118 | 51-30| 126 | 54-78 | 172 | 74-78 | 50 | 21-73]. 15 | Q Vil 122 | 255 | 227 | 118] 51-98| 123 | 54-18] 170 | 74:89] 47 | 20-70) 14] Q VI 123 | 255 | 226 | 115 | 50-88; 120 | 53-09 | 166 | 73-44| 45 | 19-91} 15] ¢ VI 124 | 250 | 222 | 114 | 51-35| 117 | 52-70 | 166 | 74-77| 45 | 20-27) 15) @ Vil 125 | 249 | 220) 115 | 52-27} 121 | 55-00 | 167 | 75-90 | 46 | 20:90; 14] ¢ VII ; 126 | 246) 218} 112 | 51-37] 121 | 55-50 | 164 | 75-22 | 47 | 21-56) 14) @ VIL 127 | 246 | 217 | 112 | 51-61 | 116 | 53-45 | 162 | 74-65 | 47 | 21-65; 15] Q VIitgg 128 | 245 | 217 | 111 | 51-15) 118 | 54-37 | 164 | 75-57] 44 | 20-27; 15) Q VE 129 | 242] 218 | 111 | 50-91) 117 | 53-67 | 162 | 74:31) 45 | 20-64; 15] Q VIG 130 | 241 | 213) 110 | 51-64) 116 | 54-46} 157 | 73-70 | 46 | 21-69; 13] ¢ VII | 131 236 | 210 | 109 | 51-90; 111 | 52-85} 155 | 73-81 | 45 | 21-43] 15 | ¢ Vil 134 | 246 | 220 | 113 | 51-36] 122 | 55-45 | 170 | 77-27 | 46 | 20-90; 15 135 | 245 | 215] 112 | 52-09] 118 | 54-88) 163 | 75-81 | 48 | 22-32] 13 136 | 244 | 214] 107 | 50-00] 116 | 54-20 | 160 | 74-76 | 46 | 21-49} 13 137 | 243 | 214] 112 | 52-33| 119 | 55-60 | 162 | 75-70 | 47 | 21-96 | 14 138 | 238 | 212 | 108 | 50-94] 116 | 54-71 | 159 | 75-00 | 45 | 21-22) 13 139 | 233 | 204] 103 | 50-49] 113 | 55-39 | 155 | 75-98 | 44 | 21-56) 13 140 | 223 | 197 | 103 | 52-28] 110 | 55-83 | 149 | 75-63) 45 | 22-84) 13 i4] 222 | 197} 101 | 51-26] 109 | 55-33 | 146 | 74-11 | 44 | 22:33) 13 142 | 222 | 193 | 101 | 52-33| 105 | 54-40] 145 | 75-13 | 43 | 22-28; 14 VII VIL VII-I VII-I fa VIEig O3 400s 4003 4003 On O2 4003 << —_ e “Mean 51-78 55-22 75:79 21-54 |14-03 | 2B Table II. ess eS Oe eS ee eS EEE ee EE 76-25 76-22 75-28 75:28 17:27 SEA-FISHERIES LABORATORY. 150 Herring : trawled off The Smalls, October 25th, 1913. (Kept in cold storage until January 12th, 1914.) 177 4093 10404093 4005 O3 03 O03 4093 40 40 4003 4095 03 05 40404005 404003 Oy 04 05 On 4905 05 40 05 4005 4040404003 OV CV 404003 OY a a a aia < S =) ~] > (e 6) bo bo ve} for) —_- w 239 |} 209) 108 | 51-67} 113 | 54:06) 157 | 75-12) 45 | 21-53 | 14 | 239 | 208 | 106 | 50-96| 112 | 53-84] 161 | 77-40 | 49 | 23-55 | 14 | 234} 203 | 106 | 52-21! 109 | 53-69 76-35 | 44 | 21-67| 14 234 | 202 | 108 | 53-46/ 112 | 55-44 44 | 21-78| 14 234 | 199] 104 | 52-26! 111 | 55-77 233 | 204] 107 | 52-45) 114 | 55-88 f ) 55:39 233 | 200 | 106 | 53-00) 110 | 55-00 232 | 209; 106 | 50-71) 111 | 53-11 bo w w bo —) — — —) f=>) Cl _ <=) ion) — _ w eet et iw) a bo bo So ~] _— © ~] OL —_ or) Je) — — ip Or Ou roan) I —_ OL ~J ~I Or CO > i Or bo _ J ow — or 3.03 05 O5 05 4905.03 Os 4904 0 5 05 05 05 O54 04 05.05 05.95 05 1219.05.03 Os 49.03 05 494040 40 As 05 49.05 49. 05 Oy O4 05 05 05 Os Os 03 40404005 404040 aaa ccd cao ogc ooo? OQ St DTW ¢ C ~! =~) co =>) — —————_ |X |] | _ ___ 22-61 21-98 22-61 22-17 21-93 21-73 22-17 22-02 22-90 21-33 21-97 21-07 21-97 21-71 22-93 23-57 22-27 22-58 22-22 22-79 21-49 21-62 21-75 21-39 21-19 22-53 20-83 20-56 21-12 21-12 21-32 22-06 20-65 21-69 22-17 21-90 184 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Table IlI—Continued. Ae A. Nore). 06 Sn cd: lee. hu a % % % T.cd. T.cd. T.cd. 86 | 232 200 105 | 52-50! 113 | 56-50} 156 | 78-00 | 46 87 2a 200 106 | 53-00 108 | 54-00 155 | 77-50 | 45 88 230 202 107 | 52-97 114 | 56-43 Lt 177-72 AS 89 230 201 LOS) 24 12 | oa 72 156 | 77-61 | 44 90 | 230 201 105 | 52-23} 109 | 54-22!) 149 | 74-12 | 44 91 230 200 105 | 52-50} 111 | 55-50} 152 | 76-00} 44 92 | 228 198 100 | 50-50 | 107 | 54:04] 150 | 75-75] 44 93 228 198 103 | 52-02 111 | 56-06 152 | 76-76 | 46 94 Pepall 197 104 | 52-79 107 | 54-31 147 | 74-61 | 45 95 226 194 102 | 52-57 106 | 54-63 147 | 75-77 | 44 96 225 196 109 | 55-61 111 | 56-63 151 | 77-04) 45 97 225 196 101 | 51-53 108 | 55-10 151 | 77-04 | 44 98 225 194 103 | 53-09 108 | 55:67 148 | 76-28 | 43 99 220 190 98 | 51-57| 104 | 54-73} 146 | 76-84] 43 100 | 214 188 99 | 52-66! 102 | 54-25| 144 | 76-59] 42 (3) Received June 25th, 1914. i 272 230 123 | 53-47 127 | 55-21 177 | 76-95 | 52: 2 270 232 123 | 53-01] 128 | 55:17-| 17 | 73-70°) Sl 3 270 230 121 | 52-60} 128 | 55-65) 182 | 79-13 | 52 4 269 230 T2105), 52-60 131 | 56-95 179 | 77-82 | 51 5 269 228 117 } 51-31 125 | 54-82 179 | 78-50 | 50 6 267 230 122 | 53:04] 130 | 56-52] 179 | 77-82] 50 af 266 230 119 | 51-73 129 | 56-08 181 | 78-69 | 51 8 263 Paya F| 118 | 51-98 124 | 54-62 175 | 77-09 | 50 9 262 226 apes atin 127 | 56-19 LID 77-43 hae 10 261 225 119 | 52-88 125 | 55:55 172 | 76-44 | 48 11 261 223 114 | 51-12] 123 | 55-15| 171 | 76-68 49 12 258 223 115 | 51-56 125 | 56-05 172. 77-13 | 47 13 257 223 117 | 52-46] 122 | 54-70) 174 | 78-02} 49 14 256 221 115 | 52-03 120 | 54-29 164 | 74-20 | 48 15 255 218 17) a3-O7 121: | 55-50 170 | 77-98 | 50 16 254 Paya | 116 | 52-48 118 53-39 170 | 76-92 | 52 17 254 220 L595) 52527 120 | 54-54 166 | 75:45 | 49 18 252 PAL) 116 | 53-45 120. | 55:29 168 | 77-41 | 49 19 252 216 112 | 51-85 121 | 56-01 166 | 76°85 | 48 20 Zae 7 U5 115 | 53-48 124 | 57-67 170 | 79-07 | 49 21 252 214 112 | 52-33 1S Sa3s4a 166 | 77-57 | 46 22 251 222 115 | 51-80 121 | 54-50 172 | 77-47 | 48 23 249 216 112 | 51-85 121 | 56-01 161 | 74-53 | 47 24 249 Pal 5) 112 | 52-09 120 | 55-81 168 | 78-14 | 46 25 248 HAL RF 112 | 51-61 120 | 55-29 169 | 77-88 | 46 26 248 213 107 | 50-23 119 55:86 162 | 76-05 | 48 Par | 247 216 112 |) S185, 116 | 53-70 166 | 76-85 | 45 935) DAT 2a 107 | 50:00} 117 | 54-67] 162 | 75-70 | 44 29 247 213 P20 118 | 55-39 166 | 77-93 | 45 307) -24742 213 110 | 51-64] 115 | 53-99} 162 | 76-05] 45 31 247 211 112 | 53-08 116 | 54-97 163 | 77-25 | 45 32 246 213 113 | 53-05. 118 | 55-39 163 | 76-52 | 47 33 246 213 111 | 52-11 116 | 54-46 168 | 78-87 | 44 34 246 212 110 | 51-88 117 | 55-18 162 | 76-41 | 46 35 246 212, 109 | 51-41 115 | 54-24 164 | 77-35 | 47 36 246 210 111 | 52-85 1B aes ore 161 | 76-66 | 46 40 C3 Os 404093 03 Oy OY Oy Oy 05 OS 49 Oy 03 0340023 4003 4003 404093 4040 Os 4003 O3 4003 40 03 40 03 40 05 Os O5 40.05 05 4005 404004 O4 =. a a SEA-FISHERIES LABORATORY. 185 Table I11—Continued. D V re Lep.l eT cd. Y K,| 8 y y ° % | Ted | Ted. Ted, Ted nf | ff Or — ve) lor) — — =) Or wo vo) bo — Ol =e a] or) lo) lor) ~ for) bo bo or re — Or bo bo © — so Le 2) — =) bo or — a | _— — — =| or or eo | o — wt — ~] on bo on) ~ tx i] bo bo bo — i to i) o bo S or) — S o on bo nw bo — — on Or Or oO bo — Or 12.2) ~] ior) for) No) yo lor) bo bo ee) w —_ a 4040 3 4003 O3 404005 05 4040 1005 05 03 05.05 40404005 4095 4003.03 40404040 10 05 O5 40404003 03 40404040 5 05 40 4040 03 40 05 40404005 OS 186 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Table [1I—Continued. Dens Vv A. l.ep.l. . No. Nea | Ted. Ky Ss. g. % % % 19% T.cd. T.cd T.cd. T.cd. 93 | 224] 193] 101 | 52-33] 105 | 54-40| 143 | 74-09 |. 43 | 22-28) 18] ¢ III 94 | 223 | 198 99 | 51-29] 104 | 53-88] 148 | 76-68| 42 | 21-76; 14] ¢ Ill 95 | 220) 191 97 | 50-78] 106 | 55-49] 148 | 77-48] 42 | 21-99| 14] 9 Til 96 | 219! 188 98 | 52-12} 106 | 56-27| 140 | 74-46| 42 | 22-34| 13 | © II O79) ya, .1S7 96 | 51-33} 100 | 53-47| 141 | 75-40] 41 | 21-92'15| 3 Til 98 | 215 | 187 96 | 51-33| 102 | 54-54] 144 | 77-00] 40 | 21-38) 14] ¢ Hl 99 | 2141) 183 95 | 51-91| 102 | 55-73 | 140 | 76-50; 40 | 21-85' 13] 3 Til 100+) 2077) - bz 92 | 51-97 99 | 55-93 | 134 7570 40 | 22-591 15 | ¢ TI (4) Received July 9th, 1914. 1| 265 | 230] 117 | 50-87) 129 | 56:08] 182 | 79-13] 49 | 21-30) 16} ¢ Ill 2| 265 | 228| 122 | 53-50] 125 | 54-82| 177 | 77-63| 53 | 23-24] 14] © IV 3 | 265 | 227| 121 | 53-30] 125 | 55-06} 171 | 75-33] 49 | 21-58 | Tso IV 4| 264} 225] 120 | 53-33) 125 | 55-55| 175 | 77-77] 50 | 22-22| 14) 6 IV 5 | 263 | 227 | .121 | 53-30} 125 | 55:06| 175 | 77-09| 52 | 22-90) 14| 9 IV 6| 263 | 225] 117 | 52-00| 125 | 55-55] 173 | 76-88 | 48 | 21-33/13 | 9 Vv 7| 260 | 226] 119 | 52-65] 125 | 55-31] 175 | 77-43| 50 | 22:12) 15] ¢ V 8 | 260] 224] 114 | 50-89] 123 | 54-91] 175 | 78-12] 53 | 23-66) 15] ¢ Vv 9 | 260 | 221 | 118 | 53-39] 121 | 54:75] 173 | 78-28| 53 | 23-98| 15] 9 IV 10 | 258 | 225] 110 | 48-88] 125 | 55-55| 170 | 75-55! 50 | 22-22| 14] 9 IV 11 | 258 | 222} 120 | 54:05! 120 | 54:05] 173 | 77-92 | 50 | 22:51 |) 145) p eens 12 | 258 | 221 | 117 | 52-94] 127 | 57-46] 173 | 78-28] 51 | 2307/15] ¢ Vv 13 | 257 | 222} 115 | 51-80] 123 | 55-40] 171 | 77-02] 49 | 22:07| 13] © V 14 | 255] 220 | 115 | 52-27] 122 | 55-45] 172 | 78-18] 48 | 21-81) 14] @ Ive 15 | 254 | 221 | 115 | 52:03] 119 | 53-84] 170 | 76-92| 48 | 21-71) 15) 9 IV 16 | 253 | 220 | 114 | 51-81| 122 | 55-45] 169 | 76-81} 49 | 22-27] 14] @ IV 17 | 253 | 220 | 114 | 51-81] 126 | 57-27| 173 |78-63.| 46-) 20-90;|eieeneee 1V=4 18 | “253 | 218} 116 | 53-21 | 118 | 54-12] . 169 |) 77-52 | 47 | 2)-a6 (eae Ill 19 | 252) 222 | 111 | 50-00] 121 | 54-50| 170 | 76-57) 46 | 20-72] 15 | 2 V 20 | 252} 220] 116 | 52-72| 122 55-45] 165-| 75-00| 46 | 20-90| 15 | © IV 21 | 252) 219; 113 | 51-59) 118 | 53-88} 166 | 75-79 | 50 | 22-83] 14] ¢ Til 22 | 252 | 215! 112 | 52:09] 119 | 55-34]. 165 | 76-74] 47 | 21-86| 14 | 9) 947 93 | 251 | 219) 113 | 51-59| 118 | 53-88| 166 | 75-79 |-49 | 22-37| 141 9 Til 24 | 251 | 218) 113 | 51-83} 121 | 55-50] 168 | 77-06 | 49 | 22-47 | 14) V 25} 251 | 216! 113 | 52-31| 116 | 53-70| 166 | 76-85| 47 | 21-75] 14] 9 Vv 26 | 250] 218 | 111 | 50-91) 115 | 52-75| 167 | 76-60| 45 | 20-64) 15) © IV 27 | 250) 217 | 113 | 52-07| 117 | 53-91) 167 | 76-95] 48 | 22-12) Ia Vv 28 | 250) 216) 119 | 55-09] 122 | 56-52| 167 | 77-31 | 50 | 23-14) 15] ¢ IV 29 | 250 | 216 116 | 53-70| 125 | 57-87| 167 | 77-31 | 49 | 22-68) 13] ¢ V 30 | 249 | 216 | 115 | 53-24] 119 | 55:09] 168 | 77-41 | 50 | 23-14/ 14] 9 IV 31 | 249 | 2141! 111 | 51-87| 119 | 55-60] 168 | 78-50| 47 | 21-96] 14| ¢ IV 32 | 249 | 214! 109 | 50-93| 112 | 52:33} 160 | 74-76| 43 | 20:09] 14] 9 TV 33 | 247 | 216) 111 | 51:38; 119 | 55-09| 164 | 75-92] 49 | 22-68] 14] 9 IV 34 | 247) 215 | 110 | 51-16) 121 | 56-27) 166 | 77-20| 48 | 22-32) 15] ¢ IV 35 | 246] 215 | 112 | 52-09) 116 | 58-95) 165 | 76-74| 47 | 21-86] 15] ¢ III 36 | 246 | 214] 109 | 50-93) 119 | 55-60) 166 | 77-57] 47 | 21-96| 14] ¢ IV 37 | 246 | 211 | 108 | 51-18| 119 | 56-39| 160 | 75-83| 47 | 22-27| 14] & Ill 38 | 245 | 215 | 148 | 50-23) 115 | 53-48| 161 | 74-88] 47 | 21-86|/ 14] ¢ IV 39 | 245 | 212) 109 | 51-41| 115 | 54-24] 165 | 77-83] 49 | 23-11/ 15] ¢ Til 40 | 245 | 212 108 | 50-94| 117 | 55-18] 162 | 76-41 | 48 | 22-64/16| ¢ Ve 41 | 245 | 212! 107 | 50-47) 115 | 54-24] 159 | 75-00] 48 | 22-641 15] ¢ IV 42| 245| 210! 111 | 52-85] 120 | 57-14] 166 | 79-04] 47 | 2238/13] ¢ V 43 | 245 | 209! 109 | 52-15} 115 | 55-02] 160 | 76-55| 46 | 2201/14] ¢ V SEA-FISHERIES LABORATORY. 187 Table IlI— Continued. 44 | 244) 211) 110 | 52-13| 116 | 54-97] 163 | 77-25| 47 | 22-27/ 14] @ IV 45 | 243 214] 111 | 51-87| 121 | 56-54| 164 | 76-63] 48 | 22-43| 141 9 IV 46) 243) 212) 111 | 52-35| 115 | 54-24| 162 | 76-41] 48 | 22-64) 14] 9 IV 47) 243) 212] 109 | 51-41} 114 | 53-77| 162 | 76-41] 48 | 22-64| 16] @ IV 48 | 243 | 209] 110 | 52-63) 117] 55-98| 167 | 79-90] 47 | 22-48] 15] @ IV 49 | 243) 208] 108 | 51-92| 116 |55-76| 164 | 78-84] 47 | 22-59| 14] 9 IV 5O | 243 | 207) 106 | 51-20| 115 | 55-55| 162 | 78-26| 45 | 21-73] 13 | @ IV Bl) 242) 212) 110/ 51-88| 116 | 54-71| 164 | 77-35] 48 | 22-64) 14] ¢ TI B2) 242) 212) 109 | 51-41| 114 | 53-77| 162 | 76-41 | 47 | 22-17/ 16 | @ V 53 | 242| 210] 107 | 50-95] 114 | 54-28| 159 | 75-71] 44 | 20-9513 | ¢ Vv 54) 241) 212) 112 | 52-83] 113 | 53-30] 159 | 75-00| 46 | 21-69| 14 | 3 Vv Bo) 241 | 211} 108 / 51-18} 113 | 53-55| 162 | 76-77| 46 | 21-80| 13] ¢ V 56 | 241 | 209) 110 | 52-63] 115 | 55-:02| 163 | 77-99| 45 | 21-53/ 13] 9@/ II 57 | 240| 211 | 109] 51-65} 110 | 52-13| 163 | 77-25] 43 | 20-38/15| ? | ?11 58 | 240} 207) 108 | 52-16| 112 | 54-10| 160 | 77-29| 44 | 21-25} 171] ¢ V 59 | 240 | 206) 108 | 52-42| 116 | 56-31} 160 | 77-67] 48 | 23-30] 16 | @ IV 60 | 240| 206! 106 | 51-45} 118 | 57-28} 165 | 80-09| 45 | 21-84/ 16] 9 IV 61 | 240} 205] 108 | 52-68] 115 | 56-09} 161 | 78-53| 45 |21-95| 16! ¢| V 62 | 240] 204] 105 | 51-47] 109 | 53-43| 156 | 76-47| 45 | 22-05] 15 | IV 63 | 240) 203] 108 | 53-20| 112 |55-17| 158 | 77-83| 47 | 23-15|16| 3 IV 64 | 239} 208) 109 | 52-40, 113 | 54:32| 160 | 76-92| 45 | 21-63/15| 3 V 65 | 239) 207| 107 | 51-69| 117 | 56-52| 160 | 77-29] 48 | 23-18) 16 | @| IV 66 | 238 | 209{ 107 | 51-19| 114 | 54-54| 160 | 76-55] 49 | 23-44/ 15 | @ ll 67 | 238) 206| 102 | 49-51] 111 | 53-88; 157 | 76-21; 46 | 22-33; 15| Q2| III 68 | 237) 208} 111 | 53-36| 113 | 54:32) 157 | 75-48] 46 | 22-11/ 15} ¢ IV 69 | 237) 205)| 105 | 51-22} 114 | 55-61| 157 | 76-58| 43 | 20-971 15 | ¢ IV 70) 237 | 202| 107 | 52-97] 110 | 54-45| 157 | 77-72| 45 | 22-27| 14| ¢@ V 71 | 236; 207| 106 | 51-20] 113 | 54-59| 162 | 78-26] 45 | 21-73|14| 6 IV 72} 236} 205) 102 | 49-75| 108 | 52-68| 155 | 75-61| 40 /19-51/ 14] ¢| V 73 | 236} 204) 106] 51-96] 114 | 55-88| 154 | 75-49] 46 | 22-54} 14] $| V 74) 235) 204| 107 | 52-45] 112 | 54-90] 160 | 78-43] 43 | 2107/15] ¢| ILI 75} 235} 203} 105 | 51-72| 108 | 53-20} 160 | 78-81] 42 | 20-891 15] ¢ IV 76 | 235| 201 | 107 | 53-23] 109 | 54-22! 157 | 78-10] 45 | 22-38} 15! ¢ IV 77| 235} 201 | 104} 51-74| 110 | 54-72| 153 | 76-11| 44 | 21-88] 14] 9 IV 78) 235) 201 | 103 | 51-24| 114] 56-71| 154 | 76-61] 46 | 22-88} 14] 9] IV 79 | 235 | 200| 100 | 50-00) 111 | 55-50) 156 | 78-00} 46 | 23-00} 14 | @ IV 80 | 234] 203] 103 | 50-73] 110 | 54-68; 153 | 75-36] 46 | 22-66|15| 3 V Sl | 234| 201 | 102 | 50-74| 111 | 55-22! 154 | 76-61| 44 | 21-88|15| @| I 82) 232] 202) 105] 51-98| 111 | 54-95| 156 | 77-22] 44 | 21-78] 15] ¢ Il 83 | 232) 200 98 | 49:00| 108 | 54:00| 152 | 76-00] 45 | 22-50} 14] @/ IV 84) 232) 199) 105 | 52-76] 109 | 54-77| 150 | 75:37] 42 | 21:10} 14) Q@| ITI 85 | 231] 200] 102] 51-:00| 104 | 52-00; 151 | 75-50| 40 | 20-:00/ 14/| ¢ IV 231 | 199| 104 | 52:26] 108 | 54-27| 149 | 74-87] 43 | 21-60] 14| ¢ V 87) 231/| 199} 103] 51-75| 110] 55-27| 152 | 76-38| 43 | 21-60/ 14] ¢ V mes) 231 | 199) 101 | 50-75| 109 | 54-77| 153 | 76-88] 43 | 2160/15} g| IV "89 } 230 |) 202/ 103 | 50-99] 108 | 53-46| 151 | 74-75| 46 | 22-771 16} ¢| III “90 | 230/ 200| 104 | 52-00} 108 | 54:00) 152 | 76-00| 45 | 22-50] 14 | Q lit 91 | 230; 200] 103 | 51-50) 106 | 53-00| 149 | 74-50] 42 | 21-00] 14| 3 IV > 92} 230) 200} 106 | 53:00) 106 | 53-00; 153 | 76-50 | 46 | 23:00| 14| @| IV meee) 230) 196; 101 | 51-53) 112 | 57-14] 152 | 77-55) 47 | 23-97/ 13 | 9} IV me4 |) 228/ 198| 101 | 51-01] 112 | 56-56| 154 | 77-77] 46 | 23-23} 16) Q II memo 227! 196} 101 | 51-53| 110 | 56-12| 150 | 76-53) 46 | 23-46/17| 2@| IV 96 | 227) 194] 103 | 53:09| 104 | 53-60!) 148 | 76-28| 42 | 21-64) 13 | &} Os mee?) 223| 193) 101 | 52-33] 106 | 54-92| 145 | 75-13] 44 | 22-79} 14) g¢| I 798) 223; 192] 101 | 52-60| 109 | 56-77| 148 | 77-08| 41 | 21-35|16| 9 lil ~ 99; 219; 187] 100 | 53-47| 109 | 58-28; 150 | losaaiier 22:99; 14) g| II 100 211 | 183 93 | 50-81 97 | 53:00 137 | 74:86] 40 | 21-85|14| 9 I 188 Table Ill —Continued. (5) Received July 31st, 1914. (In cold storage until September 23rd.) TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Nos 1 263 2 260 3 256 4 | ° 256 D 255 6 | 254 70 253 8 | 253 9| 253 10 252 11 250 12 | 250 13 | 250 14 250 15 | 250 16 | 250 17 248 18 246 19 | 246 20 245 21 244 22 243 23 | 243 24 242 25 | 242 26 241 27 | 240 28 240 29 | 240 30 | 240 31 239 32 239 33 | 238 34 | 238 35 eat 36 | 237 37 74537 38 | 236 39 | 236 40 | 236 4] 235 42 235 43 | 235 44 | 235 45 234 46 234 AT 1 233 48 | 233 AQ 4) 233 50 232 51 Zon Spal, 2B. 53 | 231 iD: Ve Med: % % T.cd. T.cd. 226 122 | 53-98 | 126 | 55-75 226 119 | 52-65| 126 | 55-75 222, 117 | 52-70 | 120 | 54-05 220 118 | 53-63} 123 | 55-91 222 116 | 52-25; 121 | 54-50 220 116 | 52-72} 121 | 55-00 222 115 | 51-80 | 124 | 55-85 219 113 | 51-59; 118 | 53-88 217 112 | 51-61 | 123 | 56-68 218 112 | 51-37} 121 | 55-50 217 112 | 51-61} 118 | 54-37 216 114 | 52:77| 117 | 54-16 216 112 | 51-85} 119 | 55-09 216 | 112 | 51-85; 119 | 55-09 216 110 | 50-92} 122 | 56-48 215 113 | 52-55| 122 | 56-74 212 108 | 50-94} 117 | 55-18 214 112 | 52-33 | 124 | 57-94 210 108 | 51-42! 116 | 55-24 212 111 | 52-35} 115 | 54-24 212 107 | 50-47, 115 | 54-24 210 115 | 54-50] 117 | 55-45 210 111 | 52-85} 116 | 55-24 211 108 | 51-18} 116 | 54-97 209 108 | 51-67} 118 | 56-45 206 | 107 | 51-94] 115 | 55-82 208°} 110 | 52-88} 114 | 54-80 207 | 106 | 51-20} 112 | 54-10 206 107 | 51-94] 113 | 54-85 206 108'| 52-42] 114 | 55-74 207 110 | 53-14| 116 | 56-03 207 108 | 52-16] 113 | 54-59 208 108 | 51-92} 116 ] 55-76 204. 106 ! 51-96} 114 | 55-88 205 110 | 53-64] 114 | 55-61 204 106 | 51-96} 116 | 56-86 203 104 | 51-23] 110 | 54-68 206 108 | 52-42] 113 | 54-85 205 103 | 50-24] 107 | 52-19 199 105 | 52-76] 110 | 55.27 205 103 | 50-24] 106 | 51-70 202 106 | 52-47; 110 | 54-45 202 104 | 51-48} 109 | 53-96 200 108 | 54:00} 114 | 57-00 201 111 | 55-22! 117 | 58-20 197 104 | 52-79! 108 | 54-82 204 106 | 51-96 | -112 | 54-90 202 105 | 51-98] 109 | 53-96 200 105 | 52-50} 111 | 55-50 202 102 | 50-49} 110 | 54-45 200 103 | 51-50} 110 | 55-00 200 104 | 52:00} 110 | 55-00 200 108 | 54:00} 110 | 55-00 A. % T.cd. 182 | 80-53 176 | 77-87 176 | 79-27 170 | 77-27 174 | 78-37 174 | 79-09 172 | 77-47 170 | 77-62 170 | 78-34 171 | 78-44 168 | 77-41 170 | 78-70 167 | 77-31 160 | 74-07 171 | 79-16 168 | 78-14 165 | 77-83 170 | 79-43 162 | 77-14 161 | 75-94 168 | 79-24 160 | 75-83 161 | 76-66 158 | 74-88 162 | 77-51 163 | 79-12 161 | 77-40 162 | 78-26 “160 | 77-67 161 | 78-15 163 | 78-74 160 | 77-29 166 | 79-80 160 | 78-43 162 | 79-02 162 | 79-40 157 | 77-34 160 | 77-67 156 | 76-09 158 | 79-39 156 | 76-09 153 | 75-74 154 | 76-43 156 | 78-00 163 | 81-09 154 | 78-17 158 | 77-45 156 | 77-22 152 | 76-00 153 | 75-74 158 | 79-00 153 | 76-50 151 | 75-50 l.epal: % T.cd. 50 | 22-12 49 | 21-68 51 | 22-97 48 | 21-81 50 | 22-51 46 | 20-90 50 | 22-51 46 | 21-00 48 | 22-12 45 | 20-64 47 | 21-65 50 | 23-14 45 | 20-83 46 | 21-29 45 | 20-83 46 | 21-39 45 | 21-22 44 | 20-56 45 | 21-43 46 | 21-69 46 | 21-69 48 | 22-74 45 | 21-43 AT | 22-27 42 | 20-09 45 | 21-84 45 | 21-63 45 | 21-73 45 | 21-84 47 | 22-81 46 | 22-22 43 | 20-77 42 | 20-19 41 | 20-09 46 | 22-43 45 | 22-05 44 | 21-67 42 | 20-38 42 | 20-48 42 | 21-10 45 | 21-95 41 | 20-29 45 | 22-27 45 | 22-50 45 | 22-38 43 | 21-82 41 | 20-09 44 | 21-78 45 | 22-50 45 | 22-27 43 | 21-50 41 | 20-50 43 | 21-50 OE CoN HSA rwone 232 230 229 229 230 © 229 | 228 | 227 | 225 226 | 225 © 224 225 SEA-FISHERIES LABORATORY. Table Il11—Continued. 189 a — ee CO He CO — eM) — i O43 4003 03 03 4003 03 4040 93404003 4003 05 03 03 4040 03 404003 404003 40 03 4003 40404005 On D Ye A Lep.l. % % | Yo Yo T.cd. Fed. T.cd ‘Ped. 104 | 52-52) 109 | 55-05| 154 | 77-77 | 43 | 21-71 107 | 52:97| 113 | 55-94] 162 | 80-19} 41 | 20-29 106 | 53:00 | 109 | 54-50| 151 | 75-50 | 46 | 23-00 100 | 50:00} 108 | 54:00} 152 | 76-00 | 42 | 21-00 107 | 53-76 | 109 | 54:77| 157 | 78-89 | 44 | 22-11 106 | 53-26| 110 | 55-27| 154 | 77-38| 43 | 21-60 102 | 51-51 | 107 | 54:04| 151 | 76-26} 40 | 20-20 101 | 51-53; 106 | 54:08} 149 | 76-02 | 44 | 22-44 100 | 50:00; 107 | 53-50) 155 | 77-50) 43 | 21-50 100 | 51-28| 104 | 53-33] 152 | 77-94} 41 | 21-02 102 | 51-51 | 106 | 53-53} 156 | 78-78 | 40 | 20-20 104 | 52-79} 107 | 54:31 | 152 | 77-15} 43 | 21-82 103 | 52:54} 105 | 53-57| 152 | 77-55} 41 | 20-91 105 | 53-57| 108 | 55:10} 149 | 76-02 | 45 | 22-95 100 | 51-28| 108 | 55-38; 156 | 80-00 | 41 | 21-02 107 | 54-59 | -109 | 55-61 | 152 | 77-55} 43 | 21-93 102 | 53:12} 106 | 54:92} 150 | 77-72 | 40 | 20-72 101 | 51-53 | 106 | 54:08| 152 | 77-55 | 43 | 21-93 100 | 51-54; 104 | 53-60} 151 | 77-83} 40 | 20-61 102 | 52-04; 107 | 54:59} 151 | 77-:04| 42 | 21-42 100 | 51-54} 108 | 55-67/| 150 | 77-31 | 42 | 21-64 99 | 51-29/| 105 | 54-40; 151 | 78-23 | 43 | 22-28 99 | 51-56| 103 | 53-64| 148 | 77-08 | 40 | 20-83 100 | 52-08| 104 | 54:16} 150 | 78-12 | 43 | 22-39 101 | 53-15 | 107 | 56-31} 149 | 78-42 | 40 | 21-05 99 | 52-38} 101 | 53-43; 147 | 77-77| 41 | 21-69 101 | 53-15) 107 | 56-31 | 151 | 79-47 | 40 | 21-05 97 | 51-05} 100 | 52-63; 149 | 78-42 | 39 | 20-52 100 | 52-08} 106 | 55-20; 148 | 77-08 | 43 | 22-39 97 | 52-15| 105 | 56-44| 145 | 77-95 | 44 | 23-65 99 | 52-94; 101 | 54:01} 142 | 75-93) 39 | 20-85 99 | 52-38; 102 | 53:97} 145 | 76-72 | 39 | 20-63 96 53-03 99 | 54-69 | 139 | 76:79 | 43 | 23-75 98 | 53-55) 100 | 54-64| 143 | 78-15) 38 | 20-76 97 | 53-00; 101 | 55-19) 144 | 78-69] 39 | 21-31 95 | 53-07 99 | 55-30; 141 | 78-77| 40 | 22-34 91 | 52-89 96 | 55-81 | 133 | 77-32 | 39 | 22-67 (6) Received August 21st, 1914. (Cold storage until September 28th.) 125 | 53-87| 131 | 56-46] 181 | 78-01 | 49 | 21-12 121 | 52-60; 130 | 56-52| 186 | 80-87) 51 | 22-17 119 | 51-96} 126 | 55:02] 178 | 77-72} 49 | 21-39 123 | 53-71 | 127 | 55-45| 177 | 77-28| 50 | 21-83 119 | 51-73 | 125 | 54:34] 181 | 78-69 | 47 | 20-43 119 | 51-96| 124 | 54:14] 177 | 77-28)| 48 | 20-96 117 | 51-31 | 123 | 538-94] 177 | 77-63] 48 | 21-05 115 | 50-66 | 122 | 53-74| 176 | 77-53 | 49 | 21-58 117 | 52-00 | 123 | 54-66] 176 | 78-22 | 46 | 20-44 116 | 51:32 | 121 | 538-53} 174 | 76-99| 48 | 21-23 119 | 52-88; 124 | 55:11} 178 | 79-11} 47 | 20-88 112 | 50:00; 118 | 52-67} 172 | 76-78) 46 | 20-53 117 | 52:00 | 123 | 54-66; 174 | 77-33) 49 | 21-77 | | _ | O35 03 03 4003 4040404093 4005 On ue) 190 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Table Ul—Continued. D. V- A. Lepl Nog) @f: 2) ed. : K,] s % % % % T.cd. ed. T.cd. T.ed. 14 | 257 | 221 115 | 52-03 | 125 | 56-56] 171 | 77-37 | 49 | 22-17} 14 15 | 256 | 222) 114 | 51-35) 120 | 54:05) 168 | 75-67) 51 | 23-42] 15 16 | 256 | 220] 117 | 53-17} 124 | 56-36; 174 | 79-09 | 44 | 20-00| 14 17 | 255 | 223) 112 | 50-22) 118 | 52-91] 170 | 76-23 | 45 | 20-17) 15 18 | 255 | 220 | 115 | 52-27} 121 | 55-00| 173 | 78-63 | 45 | 20-45) 15 19 | 255 | 218 | 112 | 51-37| 121 | 55-50| 166 | 76-14| 47 | 21-56| 14 20 | 254 |} 222 | 120 | 54:05] 123 | 55-40| 174 | 78-37) 47 | 21-17] 16 21); 254} 219; 114 | 52-05} 119 | 54-15| 170 | 77-62 | 48 | 21-91 | 15 22 | 254] 218/ 115 | 52-75| 122 | 55-96| 173 | 79-35 | 44 | 20-18) 14 23; 254; 218; 113 | 51-83} 120 | 55-04] 166 | 76-14 46 | 21-10} 13 24 | 253 | 222 | 120 | 54:05; 125 | 56-30| 175 | 78-82) 47 | 21-17} 14 25 | 253 | 221 116 | 52-48} 123 | 55-65] 171 | 77-37 | 47 | 21-26} 14 26 | 253 | 221 116 | 52-48/| 119 | 53-84! 173 | 78-28 | 45 | 20-36 | 16 27 | 253 | 221 113 | 51-13 | 122 | 55-20 |. 173 | 78-28 | 48 | 207i te 28 | 253 | 220 | 114 | 51-81} 119 | 54:09} 169 | 76-81 | 45 | 20-45) 14 29 | 253} 219) 111 | 50-68; 117 | 53-42| 169 | 77-16; 45 | 20-54) 14 30 | 252] 220 | 116 | 52-72| 121 | 55-00; 168 | 76-36 | 48 | 21-81 | 15 31 | 252} 216, 115 | 53-24| 120 | 55-55} 170 | 78-70} 45 | 20-83) 15 32} 251 | 215) 110 | 51-16] 116 | 53-95| 163 | 75-81 | 46 | 21-39 |-14 33 | 248 | 215} 111 | 51-62} 114 | 53-02} 169 | 78-60 | 46 | 21-39) 15) 34 | 248 | 214) 113 | 52-33| 118 | 55-14} 168 | 78-50} 45 | 21-02) 14 35 | 247] 217] 115 | 52-99| 120 | 55-29} 169 | 77-88} 45 | 20-73) 15 36 | 246 | 221 112 | 50-67| 117 | 52-94| 171 | 77-37| 46 | 20-81} 15 37 | 245 | 217 | 112 | 51-61} 119 | 54-83] 166 | 76-49 | 49 | 22-58; 14 38 | 245 | 213 112 | 52-58} 117 | 54-93| 162 | 76-05 | 44 | 20-65} 14 39 | 244 | 213) 110 | 51-64] 117 | 54-93} 169 | 79-34 | 44 | 20-65) 14 40 | 243; 210 | 109 | 51-90} 114 | 54-28; 162 | 77-14} 46 | 21-90) 14 Al | 235 | 217) 114 | 52-53| 122 | 56-22] 168 | 77-41 | 48 | 22-12) 14 42| 233 | 213 | 110 | 51-64) 115 |.53-99| 162 | 76-05 | 47 | 22-06) 14 43 | 233 | 203 | 102 | 50-24} 113 | 55-66| 155 | 76:35| 42 | 20-89) 14 44} 230 | 213 | 110 | 51-64! 120 | 56-33 | 165 | 77-46| 47 | 22-06) 14 45 | 230 | 198 |. 101 | 51-01 | 107 | 54-04} 150 | 75-75) 41 | 20-70) 14 46 | 225) 191 98 | 51-30| 102 | 53-40| 145 | 75-91 | 44 | 23-03} 14 47 | 222 | 207} 106 | 51-20} 113 | 54-59) 155 | 74-87 | 44 | 21-25] 15 48 | 221 190 99 | 52-10| 102 | 53-68| 146 | 76-84 | 39 | 20-52 | 14 49 | 217] 190] 100 | 52-63} 104 | 54-73) 146 | 76-84 | 41 | 21-58 | 16 50 | 215 | 203] 104 | 51-23] 111 | 54-68| 155 | 76-35 | 41 | 20-19) 14 0303 03 O3 03.03 4003 4003 04040404003 4003 104003 O3 O3 10104093 40 03 3 03 4003.03. 03 O3 +40 (7) Received September 4th, 1914.) (Cold storage until October 2nd.) 1] 273 | 233 | 121 | 51-93} 129 | 55-36| 182 | 78-11] 51 | 21-88] 14) @ 2| 271 | 235 | 127 | 54.04| 133 | 56-59] 184 | 78-29 | 53 | 22-55) 13 | Q 3 | 270) 232] 129 | 51-72] 128 | 55-17] 183 | 78-87] 51 | 21-98) Tb) @ 4 | 270 | 229 120 | 52-40} 130 | 56-76| 181 | 79-03 | 53 | 23-14) 16| ¢ 5 | 268 | 231 | 120 | 51-94| 125 | 54-11] 177 | 76-62 | 49 | 21-21] 14] ¢@ 6| 266| 229 | 121 | 52-83| 122 | 53-27] 178 | 77-72 | 49 | 21-39) 14) ¢ 7| 264 | 229 121 | 52-83} 127 | 55-45] 178 | 77-72 | 48 | 20-:96/ 15] ¢ 8 | 264] 228 | 118 | 51-75} 125 | 54-82] 177 | 77-63} 51 | 22-36) 14] ¢ 9| 263 | 226] 121 | 53-53] -126 | 55-75| 177 | 78-31 | 51 | 22-56) 13) ¢@ 10 | 263 | 225 119 | 52-88} 124 | 55-11] 172 | 76-44) 52 | 23-11/15)| 9 11 | 262} 224; 119 | 53-12] 124 | 55-35} 178 | 79-46} 47 | 20-98; 15) ¢ 12 | 261 | 226/ 121 | 53-53] 124 | 54-86] 177 | 78-31 | 49 | 21-68) 16) ¢@ OCBNIAorwondre SEA-FISHERIES LABORATORY. Table I1]—Continued. Ve A. % % T.cd. T.cd. 118 | 53-88 | 171 | 78-08 PA) | 55-00 | 175:|-'79-54 120 | 53-81 169 | 75-78 124 | 56-10| 172 | 77-82 123 | 56:16; 170 | 77-62 118 | 53-88) 172 | 78-53 120 | 55:04| 171 | 78-44 121 | 54:75 | 171 | 77-37 120 | 54-54| 171 | 77-72 125 | 56-81 | 173 | 78-63 119 | 54:15] 168 | 76-71 118 | 53-88| 169 | 77-16 120 | 55:04| 170 | 77-98 123 | 56-:16| 170 | 77-62 121 | 55-25| 169 | 77-16 124 | 56-36| 173 | 78-63 120 | 54:79 | 170 | 77-62 119 | 54-83 | 168 | 77-41 119 | 54-83 | 170 | 78-34 124 | 57-14| 168 | 77-41 119 | 55:09} 167 | 77-31 116 | 53-70| 165 | 76-38 119 | 55:09] 166 | 76-85 116 | 53-:95| 171 | 79-53 115 | 53-48] 163 | 75-81 115 | 53-73 | 165 | 77-10 115 | 54-24] 165 | 77-83 114 | 54:02| 160 | 75-83 115 | 54-50| 165 | 78-19 114 | 54-54| 160 | 76-55 110 | 52-63| 159 | 76-07 114 | 55:07| 161 | 77-77 114 | 55-61 159 | 77-56 111 | 53-88| 159 | 77-18 113 | 54-85| 160 | 77-67 109 | 53-17| 159 | 77-56 113 | 54-85| 156 | 75-72 116 nn 127 | 53-36 135 | 56-48 133 | 55-64 134 | 56-06 127 | 53-59 132 | 55-93 136 | 56-66 131 | 55-50 133 | 57-32 130 | 56-27 127 | 55-70 128 | 55-65 122 | 53-04 127 | 55-70 124 | 54-62 | 179 188 185 187 181 182 186 185 182 179 181 180 175 174 178 | 75-21 78-66 77-40 78:24 76:37 77-11 77-50 78-39 78-44 77-48 79-38 78:26 76-09 76-31 78-41 19] 0340404095 Os O54 404093 O35 4093 4095 03 40 93 4093 40 O3 03 4003 4003 40404093 03 4003 03 C3 40 O05 404095 On 4095 404095 93 95 G3 40038 192 Table [iI—Continued. TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Z & | Soe tobe 218 219 218 218 215 217 216 215 220 210 207 204 * Excluding Sample 1. D: | V. A. Lep.L. Ket o8 % % % % T.cd. T.cd. T.cd T.cd. 123 | 53-94 128 | 56-14 | 4177 |°77-63)) 51. | 22°36) Sioa 121 | 53-30 | 125 1 55-06 | 177 | 77-97 | 51) 22-46)" Sie 117 | 51-77 | 126 | (55-75 | 177 -|"78-31 | 50°) 295025 Sea 120 | 53-09| 124 | 54-86! 170 | 75-22} 50 | 22-12) 14] ¢ 115 | 51-11 126 | 56:00| 174 | 77-33] 45 | 20:00) 14] ¢ 118 | 52-67; 120 | 53-57] 176 | 78-57| 47 | 20-98} 15 | 9 115 | 50-88) 125 | 55-30| 173 | 76-54] 50 | 22-12} 14) ¢& 112 | 50-00 | 122 | 54-46! 171 | 76-33} 50 | 22-32) 15] 9 1]4 | 51-12| 120 | 53-81 |- 172 | 77-13 | 49 | 21-:97| 14] ¢ 114 | 50-89| 124 | 55-34! 173 | 77-23} 50 | 22-32) 14] 9 116 | 52-01] 121 | 54-26] 170 | 76-23. 50 | 22-42, 15}| 9Q 117 | 52-70} 120 | 54:05 168 | 75-67 | 48 | 21-62} 15] @ 118 | 53-39] 122 | 55-20) 172 | 78-28] 49 | 22:17| 14] ¢@ 115 | 51-11! 126 | 56:00) 174 | 77-33 | 50 | 22-22) Ta) 9 119 | 53:12) 125 | 55-80} 172 | 76-78 | 49 | 21-87) sie 113 | 51-83) 119 | 54-58! 166 | 76-14] 49 | 22-47) 16] ¢ 114 | 52:05| 117 | 538-42! 165 | 75-34] 50 | 22-83) 141] ¢ 111 | 50-91; 119 | 54-58! 168 | 77-06| 47 | 21-56) 14] @ 114 | 52-29] 118 | 54-12 168 | 77-06| 49 | 22-47) 14] 9 115 | 52-99| 119 | 54-83) 169 | 77-88| 49 | 22-58) 14] ¢ 114 | 52-53| 120 | 55-29! 168 | 77-41 | 48 | 22:12) 15] 9 110 | 50-22 117 | 53-42} 170 | 77-62| 46 | 21-00) 14] @Q 111 | 51-62] 121 | 56-27| 170 | 79-07| 47 | 21-86| 15] ¢ 110 | 51-16] 118 | 54:88} 167 | 77-67] 49 | 22-79; 15] 9 112 | 51-61 | 117 | 53-91 | 164 |'75-57 | 48 | 22:12) sai 110 | 50-92} 119 | 55-09! 169 | 78-24] 44 | 20:37; 14] Q@ 111 | 51-62! 116 | 53-95| 166 | 77-20] 49 | 22-79} 14] @ 113 | 53-05| 122 | 57-27! 167 | 78-40| 47 | 22-06) 15) ¢ 111 | 52-11| 118 | 55-39! 167 | 78-40| 48 | 22-53| 141] ¢ 109 | 51-41| 117 | 55-18) 161 | 75-94| 48 | 22-64) 15] ¢ HO | 52:13| LIS | 55-92 | 167 | 79-14| 47 | 22-27) 147 GZ 113 | 51-36| 117 | 53-17] 166 | 75-45) 50 | 22-72; 14]. 9 110 | 52-38| 114 | 54-28) 162 | 77-14] 45 | 21-43) 14] ¢ 111 | 53-62) 115 | 55-55| 162 | 78-26) 45 | 21-73; 14] ¢@ 105 | 51-47) 110 | 53-92] 156 | 76-47| 48 | 23-52] 14] ¢ 51-98 54-87 77-15* 21-84 (14-45 Ted. | | 22-13,| 14 Table IV. Trawled off The Smalls. Received Oc:ober 15th, 1914. ey Gie aie : | D. V. A. Lep.L. | | aM by Ko | % | Yo % hes; Ted. | ped. | T.cd. T.cd. | | 132 | 50-77| 141 | 54-22| 199 | 76-53| 55 | 2115/15] @) 136 53-12| 140 | 54-68| 196 | 76-56| 55 | 21-48, 14) Q | 140 | 54-68) 145 | 56-64) 201 | 78-51 | 58 | 22-65 | 14 | 3 133 | 52-15| 141 | 55-29; 196 | 76-86 | 55 | 21-56| 14) ¢@ 131 51:37) 142 55-68) 200 | 78-43 | 55 21-56) 15g 134 | 52-96 142 193 | 76-28.) 56 eI 56-12 |. V IV Vv Vv N ot bo A Ne > > GO He 245 249 bo bo bo bo > > > OO 243 Speier 238 237 NWNWNWNNHMNWNHMNNNNNNWNWWdY Wo Wo WW SO Go Ge Go Wo 02 Go Go Wo H OS? DS? G2 J 7 Or 1 OL WO © 232 236 233 233 232 241 236 — 233 | SEA-FISHERIES LABORATORY. 125 | 53°64 | Table 1Y—Continued. %o 70 | _% T.cd T.cd T.cd. 132 | 52-17| 142 | 56-12] 196 | 77-47 | 55 130 | 51-58| 140 | 55-55| 194 | 76-98| 55 136 | 53-33; 141 | 55-29] 194 | 76-07) 54 133 | 52-56| 142 | 56-12| 197 | 77-86| 54 132 | 51-96} 138 | 54-33 | 197 | 77-55 | 56 131 | 52-19} 141 | 56-17] 193 | 76-89| 55 131 | 52-19} 141 | 56-17]. 195 | 77-68| 54 132 | 52-58} 140 | 55-77| 194 | 77-29] 56 130 | 52-63 | 142 | 57-49| 193 | 78-13) 55 130 | 52:00} 138 | 55-20} 191 | 76-40| 54 127 | 51-21| 135 | 54-43] 192 | 77-41 | 52 126 | 51-63 | 135 | 55-32] 187 | 76-63 | 53 128 | 51-61 | 137 | 55-24] 192 | 77-41] 53 127 | 51-62} 135 | 54-87] 192 | 78-04] 55 133 | 54-28] 138 | 56-32] 194 | 79-18| 54 133 | 53-41 | 139 | 55-82] 191 | 76-70} 59 129 | 52-43| 138 | 56-09| 190 | 77-23] 55 129 | 52-43 | 135 | 54:87] 191 | 77-64| 55 130 | 52-84| 135 | 54-87] 189 | 76-82] 56 128 | 52-45| 133 | 54-50} 186 | 76-22 | 52 129 | 53:08 139 | 57-20] 189 | 77-77] 51 128 | .52-45| 137 | 56-14! 191 | 78-27] 53 129 | 52-86] 136 | 55-73} 190 | 77-86 | 53 129 | 53-08} 135 | 55:55| 183 | 75-28] 52 127 | 52-47| 135 | 55-78; 191 | 78-92] 54 123 | 51-68} 129 | 54:20} 186 | 78-15] 52 123 | 51-46) 129 | 53-97| 183 | 76-56) 53 123 | 51-46| 135 | 56-48) 187 | 78-24! 53 130 | 53-27| 133 | 54-50| 189 | 77-45 | 54 125 | 51-44] 135 | 55-55] 187 | 76-95] 51 127 | 52-69| 133 | 55-18} 186 | 77-17] 51 128 | 53-33) 132 | 55-00! 186 | 77-50 | 50 125 | 52-30| 133 | 55-64) 184 | 76-98) 54 125 | 52-52) 132 | 55-46; 183 | 76-89) 54 125 | 52-75| 134 | 56-53 | 182 | 76-79 | 52 123 | 51-46| 129 | 53-97| 181 | 75-73 | 53 122 | 51-04} 128 | 53-55| 183 | 76-56] 52 123 | 51-68} 131 | 55-04; 183 | 76-89] 53 124 | 52-32| 130 | 54-85/ 183 | 77-21 | 52 123 | 52-11} 130 | 55:08; 181 | 76-69 | 53 123 | 52-34| 132 | 56-17; 182 | 77-44] 51 127 | 53-59 | 133 | 56-11 182 | 76-79 | 52 122 | 51-91) 128 | 54-46) 181 | 77-02 | 52 124 | 52:32) 131 | 55-27 180 | 75-94] 53 124 | 52-32) 131 | 55-27, 183 | 77-21 | 52 126 | 53:39| 133 | 56-35| 182 | 77-11 | 50 125 | 52:96| 131 | 55-50| 184 | 77-96 | 50 122 | 51-69] 128 | 54:23; 179 | 75-84| 52 125 | 53:19) 130 | 55-31; 182 | 77-44) 51 120 | 51-28| 129 | 55-12; 180 | 76-92] 53 124 | 53-44) 129 | 55-60) 181 | 78-01 | 52 121 | 51-27| 131 | 55-50! 182 | 77-11] 49 122 | 52-36| 127 | 54-50| 177 | 75-96 | 50 121 | 51-93 131 | 56-22 180 | 77-25) 50 124 | 58-44| 130 | 56:03) 179 | 77-15} 51 127 | 52-69| 136 | 56-43 184 | 76-34 | 51 120 | 50-84| 125 | 52:96, 177 | 75:00) 50 | 128 | 54-93 181 | 77-68 | 52 20-76 21-45 | 21-45 21-98 | 21-16 21-14 22-31 B | | et He oH 14 193 Ke D4 04 40 03 03 O53 4003 4003 O35 O35 03 03 4003 404005 03 O35 4005 4003 05 O54 O53 49 05 Os 49 05 05 40.03 40 05 O5 OY 4005 05 4040 O4 O4 O4 O43 OY OY OF 05 OL 05 OL 40 is EV. 194 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Table 1¥—Continued. iD: V. A. Lep.L. No.| T T.cd. — Ka | s g tl rey Mav % % | | —— T.c | T.cd. T.cd. | ed: 65 | 272) 231 | 119 | 51-51) 127 54-97 177 | 76-62 | 52 | 22-5115) go Tv 66 aie 231 115 | 49- 78 | 125 | 54-11 174 | 75-32 | 53 | 22-94 1 | IV 67 212 230 121 | 52-60 | 126 | 54-78 179 | 77-82) OE 22s 13 V 68 271 233 120 | 51-50 | 127 | 54-50 | 178 | 76-39 | 49 | 21:03} 14 |. ¥ 69 271 232 122 | 52-58 | 126 | 54:31 | 177 | 76-29; 50 | 21-55; 14 IV 70 | 270 | 233 | 117 | 50-21 | 126 | 54:07] 177 | 75-96 | 52 | 22-31 | 14 71 | 270 | 232; 123 | 53-01 | 127 | 54-74] 180 | 77-58 | 50 | 21-55} 15 72 | 270 | 231 | 121 | 52-38) 125 | 54-11] 176 | 76-19 | 50 | 21-64| 15 73 | 270 | 230}; 123 | 53-43) 128 | 55-65) 179 | 77-82 | 50 | 21-73; 14 74 | 270 | 229] 120 | 52-40 | 126 | 55:02) 176 | 76-85 | 50 | 21-83; 14 75 | 269 | 232 | 118 | 50-86 | 124 | 53-44| 174 | 75-00) 51 | 21-98| 15 76 | 269 | 231 120 | 51-94 | 130 | 56-27| 177 | 76-62 | 50 | 21-64; 14 77 | 269 | 230] 120 | 52-13} 124 | 53-95| 178 | 77-39) 49 | 21-30) 14 78 | 269 | 230] 115 | 50-00 | 124 | 53-95| 176 | 76-52) 50 | 21-73) 14 79 | 268 | 233 | 121 | 51-93) 125 53-64] 179 | 76-82 | 50 | 21-45) 15 80 | 268] 231 119 | 51-51 | 126 | 54-54} 178 | 77-05 | 48 | 20-77| 13 81 | 268 | 225} 117 | 52:00) 120 | 53-33] 173 | 76:88 | 50 | 22-22) 14 82 | 267 | 230 | 124 | 53-95} 131 | 56-95| 180 | 78-26) 50 | 21-73) 14 83 | 267] 230 | 120 | 52-13} 125 | 54:34| 174 | 75-65| 50 | 21-73) 14 84 | 267 | 229} 117 | 51-09} 126 | 55-02} 176 | 76-85) 47 | 20-52| 15 85 | 267) 225 | 118 | 52-44| 124 | 55-11} 173 | 76-88) 49 | 21-77| 14 86 | 266 | 227] 120 | 52-86) 125 | 55:06| 175 | 77-09] 51 | 22-46| 13 87 | 265 | 227 | 121 | 53-30} 128 | 56-34} 176 | 77-53) 51 | 22-46} 15 88 | 264 | 224 | 112 | 50-00} 123 | 54-91| 168 | 75-:00| 47 | 20-98| 13 89 | 263 | 231 | 119 | 51-51 | 124 | 53-67| 174 | 75-32 | 50 | 21-64, 14 90 | 263 | 227 | 121 | 53-30; 125 | 55-:06| 177 | 77-97] 49 | 21-58| 14 91 | 263 | 227 | 118 | 51-98} 124 | 54-62| 176 | 77-53 | 50 | 22-02| 14 | ee ee ee a _ - = 92, 263 | 227) 114 | 50-22 | 122 | 53-74| 174 | 93 | 263 | 225 | 120 | 53-33 | 126 | 56:00] 173 | 76-88 | 49 | 21-77 94 | 263 | 225 | 117 | 52-00} 120 | 53-33| 172 | 76-44| 48 | 21-33 95} 263 | 225 115 | 51-11 | 123 | 54-66] 174 | 77-33 | 49 | 21-77 96 | 262 | 226] 116 | 51-32 | 122 | 53-98] 173 | 76-54 | 48 | 21-23 97 | 262 | 225] 117 | 52-00} 126 | 56-:00| 174 | 77-33] 51 | 22-66 98 | 262 | 225) 117 | 52-00 | 127 | 56-44] 176 | 78-22 | 48 | 21-33 99 | 262 | 224] 119 | 53-12 | 123 | 54-91) 171 | 76-33 | 49 | 21-87 100 | 262 | 223 | 117 | 52-46 | 122 | 54-70| 171 | 76-68] 49 | 21-97 101 | 262) 221 114 | 51-58 | 122 | 55-20} 169 | 76-47| 49 | 22-17 102 | 261 | 228 | 117 | 51-31 | 127 | 55-70} 171 | 75:00 | 51 | 22-36 103 | 261 | 222 | 118 | 53-15 | 121 | 54-50| 173 | 77-92 | 49 | 22-07 104 | 260 | 225) 118 | 52-44 | 122 | 54-22| 172 | 76-44) 48 | 21-33 105 | 259 | 222 | 116 | 52-25] 122 | 54-99) 171 | 77-02 | 50 | 22-51 | 106 | 258 | 222 | 118 | 53-15 | 123 | 55-40| 173 | 77-92 | 48 | 21-62 107 | 258} 222 115 | 51-80 | 122 | 54-:99| 170 | 76-57 | 49 | 22-07 108 | 258 | 222 | 115 | 51-80| 122 | 54-99} 171 | 77-02 | 49 | 22-07 109 | 257 | 222 115 | 51-80; 118 | 53-15] 169 | 76-12 | 50 | 22-51 110 | 257 | 220); 112 | 50-91] 117 | 53-17] 166 | 75-45} 48 | 21-81 111 | 257 219) 118 | 53-88 | 123 | 56-16] 171 | 78-08} 46 | 21-00 112 | 257 | 219) 113 | 51-59 | 119 | 54-15| 166 | 75-79 | 48 | 21-91 113 | 256 | 220) 119 | 54-09] 123 | 55-91] 170 | 77-27} 48 | 21-81 113 | 51-13 | 117 | 52-94| 173 | 78-28) 49 | 22-17 | 116 | 52-72 | 120 | 54-54] 165 | 75-00 | 49 | 22-27 116, 254 | 219 109 | 49-77) 116 | 52-96| 166 | 75-79 | 45 | 20-54 117 | 254) 218 114 | 52-29| 120 | 55-04| 168 | 77-06| 45 | 20-64 118 | 253 | 220 109 | 49-54! 115 | 52-27| 165 | 75-00] 46 | 20-90 119 | 253 | 215 | 113 | 52-55 | 115 | 53-48] 164 | 76-26) 49 120 | 239 | 206 | 101 | 49-02 | 110 | 53:39) 157 | 76-21} 43 46493996 999499<5<9S9449<<4<<- _ A — —_ i bo OL Cr Or bo bo —_ SEA-FISHERIES LABORATORY. Table Y. Herring from Welsh Coast. (1) From Aberdovey. Received October 22nd, 1914. iD. | V. A. Lep.1. | o.| T. | T.cd. | | Re fia: | % | | % % % T.cd. | | T.cd. T.cd. Teds a 277 | 240 126 | 52-50 | 130 | 54-16; 182 | 75-83| 51 | 21-25; 15| ¢ my 272 | 240 122 | 50-83 | 129 | 53-75! 183 | 76-25| 49 | 20-41| 15/|- 3 am, 265) 231 Peaemer-os | 125 | 54-11). 175 | 75-75. | 47 | 20-34) 15 | 9° | =} 265] 230 118 51-30, 126 | 54-78 | 180 | 78-26 | 49 | 21-30) 14/ 9. 5 | 258 | 223 113 | 50-67! 121 | 54-26; 171 | 76-68] 49 | 21-97; 14| 9 mee 202) 219) 110 | 50-22| 120 | 54-79| 163 | 74-42| 49 | 22-37| 14] 3 | or} 250 | 216 109 | 50-46! 119 | 55-09, 163 | 75-46 | 49 | 22-68/ 13} Q | 8| 244] 214)| 109 | 50-93; 120 | 56:07, 160 | 74-76 | 47 | 21-96| 13 | 9 | 9; 240; 211 106 | 50-23} 114 | 5402) 157 | 74-40 | 45 | 21-32! 12 Q 10; 237; 206; 104 | 50-48; 111 | 53-88) 158 | 76-69 | 44 | 21-36; 15 | Q ll eames) 104) 51-48) 110 | 54:45) 149 | 73-76 |.44 | 21-78| 138 | 3} 12 | 232) 200 100 | 50:00; 110 | 55-:00| 154 | 77-00 | 43 | 21-50) 13 | 3 13 | 229; 199; 101 | 50-75| 107 | 53:76; 149 | 74-87) 45 | 22-61) 15 | 9 | ie) 223) 194) 101 | 52-06; 107 | 55-15| 145 | 74-74| 45 | 23-19); 12 | @ 5 | 221 193 98 |-50-77| 102 | 53-12| 142 | 73-57| 42 | 21-761 — | ¢ | 16 | 221 191 97 | 50-78| 103 | 53-92| 142 | 74-34] 43 | 22-51) 14 | 9 oa | = 221 189 95 | 50-26| 103 | 54-49; 141 | 74-60| 41 | 21.69/15) ¢ 18; 219 |- 189 Sriiolea2 | 104 | 55:02| 142 | '75-13.| 42 | 22-22) 14] ¢ 19 | 219; 188 96 | 51-06| 104 | 55-32| 143 | 76-06 | 42 | 22-34) 14] ¢ (2) New Quay Bay, 4 in. mesh. Received October 28th, 1914. ig | 284 | 246 125 | 50-81| 136 | 55-28) 189 | 76-82] 52 | 21-13| 14) @Q eae) aoe | 123 | 52-11; 132 | 55-93) 183 | 77-54) 53 | 22-45) 14) @ | 270 | 234) 120 | 51-28; 126 | 53-84] 180 | 76-92] 48 | 20-51| 14] @ 4; 264) 228 | 120 | 52-63 | 126 | 55-26| 179 | 78-50| 49 | 21-49) 15 | @2 5} 261 229 | 116 | 50-65) 124 | 54-14| 175 | 76-41/| 49 | 21:39) 16] ¢ me} 260; 228; 117 | 51-31| 123 | 53-94| 173 | 75-87) 50 | 21:92} 14] 3 >7| 260) 224, 113 | 50-44) 118 | 52-67; 171 | 76-33) 47 | 20-98; 16 | 9 meee 2oe | 225) 112 | 49-77| 121 | 53-77|. 169 | 75-11 | 47 | 20-88; 13] 9} 259) 223 118 | 52-91) 122 | 54-70; 175 | 78-47) 51 | 22-87; 15 | 3 ‘10 | 251 217 | 113 | 52:07; 117 | 53-91 | 169 | 77-88] 44 | 20-27; 14) Q men | 250 | 216 | 111 | 51-38) 115 | 53-24| 165 | 76-38| 44 | 20:37) 15 | ¢ (12 245 212 | 109 | 51-41 | 114 | 53-77; 160 | 75-47) 46 | 21-69) 16) 92 We (3) Moelfre. Received November 19th, 1914. 1 Sia | 234) 122 | 52-13 | .125 | 53-41] 179 | 76-49| 51 | 21-79) 15 | Q =| 265) 230 118 | 51-30 | 122 | 53:04] 177 | 76-95) 48 | 20-87; 15| 3 3| 264) 228 116 | 50-87| 124 | 54:38} 176 | 77-19] 48 | 21-05) 14] @ | 4) 264 226 116 | 51-32 120 | 53:09} 176 | 77-87| 48 | 21-23) 14] ¢ | 5 258 223 113 | 50-67 119 53-36 170 | 76-23 48 | 21-52 | 14 3 | 6 258 219 115 | 52-51 116 52-96 170 | 77-62 | 48 | 21-91 | 14 3 | 7 256 224 119 53-12 122 54-46 172 | 76:78 | 49 | 21:87} 13 as 8 253 228 117 | 61-31 123 53-94 171 | 75-00 | 48 | 21:05) 13 2 9 250 213 112 : 52-58 118 55-39 165 | 77-46| 46 | 21-69); 13 3 WO | 246 | 212 107 | 50-47; 115 | 54-24] 162 | 76-41] 45 | 21-22) 13] ¢ ll 245 212 110 | 51-88 116 (54-71 161 | 75-94) 45 21-22 | 14 Q 12 244 210 106 50-47 114 | 54-28 160 | 76:19 | 46 | 21-90 | 15 3 13} 234! 202) 103 | 50:99 109 53-96] 150 | 74-25] 42 | 20-79| 14), ¢ 14 228 194 100 51-54. 104 53-60 150 | 77-31 | 40 | 20-61 | 14 ) 3 gg ( \ fx di en 196 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Table Y—Continued. (4) Aberdovey. Received November 19th, 1914. D. V.- Lepl INo;) "00. "Bred: Ke Rye % % | % T.ed. Tied. T.ed, ) Tied. 1} 270 | 235; 122 | 51-91! 127 | 54:04; 177 | 75-31 | 50 | 21-27) 15 2| 270) 234] 124 | 52-99; 130 | 55-55} 180 | 76-92] 52 | 22-22 | 14 3 | 270 | 234) 121 | 51-70; 126 | 53-84] 176 | 75-21 | 52 | 22-22 | 14 4} 268 -230°} 118 | 51-30) 124 | 53-:95| 178 | 77-39) 50 | 21-7315 5 | 265 | 230 116 | 50-43 | 124 | 53:95} 174 | 75-65} 52 | 22-60} 13 6 | 262 | 226) 115 | 50-88; 120 | 53-:09| 170 | 75-22) 50 | 22-12) 14 TA 208 224 | 115 | 51-33 | 122 | 54-46) 170 | 75-89] 50 | 22-32) 15 8 | 258 | 222 116 | 52-25) 121 | 54-50; 170 | 76-57] 48 | 21-62] 14 9} 256 | 223 115 | 51-56) 123 | 55-15| 171 | 76-68) 49 | 21-97] 14 10 | 255 | 220 | 115 | 52-27) 122 | 55-45| 165 | 75-00} 48 | 21-81 | 13 11 | 254 | 220] 113 | 51-39; 118 | 53-63| 166 | 75-45] 47 | 21-36 | 14 12 | 252 217 111 | 51-15) 118 | 54:37) 164 | 75-57) 45 | 20-73 | 14 13 | 252 217 114 | 52-53 | 122 | 56-22| 167 | 76-95} 48 | 22-12/ 13 14 | 252) 215) 113 | 52-55) 119 | 55-34) 167 | 77-67} 46 | 21-39 | 13 15 | 251 | 220] 112 | 50-91) 117 | 53-17| 168 | 76-36 | 45 | 20-45) 15 16 | 245 | 214 | 106 | 49-53) 110 | 51-40) 160 | 74:76 | 44 | 20-56 | 15 17 | 243 | 213) 106 | 49-76| 114 | 53-52} 159 | 74-64] 45 | 21-12) 15 18 |; 234; 198 | 102 | 51-51) 108 | 54-54| 152 | 76-76] 43 | 21-71} 15 19 | 231 200 103 | 51-50} 109 | 54:50; 150 | 75-00 42 | 21-00 | 14 20 | 230 199 | 101 | 50-75; 109 | 54-77| 151 | 75-87 | 42 | 21-10) 13 21 | 222 | 194 96 | 49-48 | 103 | 53:09) 142 | 78-71 | 41 | 21-13 | 14 22 \/ 219 | 189 97 | 51-32) 102 | 53-97] 144 | 76-13 | 42 | 22-22) 14 234 BLT) U87 97 | 51-87} 100 | 53-47) 140 | 74-86) 41 | 21-92) 14 24} 210 183 94 | 51-36 99 | 54:09 | 138 | 75-40 | 39 | 21-31 | 13. \ | (5) New Quay Bay, 4 in. mesh. Received November 19th, 1914. 1 | -282 | 242 | 125 | 51-65; 133 | 54-95] 182 | 75-20 | 51 | 21-07} 14 2 268 | 234] 119 | 50-85} 129 | 55-12)-175 | 74:78} 51 | 21-79 | 14 3 | 267 | 235 | 123 | 52:34) 125 | 53-19| 179 | 76:17) 48 | 20-42) 16 4 | 267 | 230 120 | 52-13 | 125 | 54:34] 176 | 76-52 | 48 | 20-87} 14 5 | 262 | 228 118 | 51-75 | . 126 | 55:26] 174 | 76:31 | 49 | 21-49| 14 6 | 262 |) 226) 117 | 51-77] 123 | 54:42} 171 | 76-54} 49 | 21-68} 14 7 | 262 | 223 116 | 52-01) 118 | 52-91) 171 | 76-68) 51 | 22-87) 15 8 | 260 | 225 117 | 52-00 | 122 | 54-22) 169 | 75-11 | 50 | 22-22 | 15 9| 260 | 221°) 115 | 52-03). 121 | 54-75] 171 | 77-37) 47 | 20-26) "iS 10.| 257 | 223 | 114-1 51-12) 123 | 55-15| 172 | 77-13) 47°| 2-07 eee 11 254 | 222 113 | 50-90 | 121 | 54-50) 172 | 77-47| 44 | 19-82 | 16 12 | 254 | 217 115 | 52-99} 120 | 55-29} 169 | 77-88} 46 | 21-19) 14 13 | 252)| - 219 111 | 50-68} 116 | 52:96) 164 | 74-88] 47 | 21-46] 15 14] 251 217 112 | 51-61 | 118 | 54:37) 163 | 75-06 | 46 | 21-19} 15 15 | 251 216 110 | 50-92} 113 | 52:31 | 165 | 76-38] 47 | 21-75] 14 16 | 247); 214 108 | 50-46) 115 | 53-73 | 162 | 75-70} 45 | 21-02 | 14 17 | 246; 214; 109 | 50-93; 113 | 52-80} 161 | 75-23 | 44 | 20-56 | 14_ 18 | 243; 211 106 | 50-23] 112 | 53:08) 160 | 75-83 | 43 | 20-38 | 14 (6) Moelfre. Received November 23rd, 1914. 1| 259) 223) 116 | 52-01) 122 | 54-70| 172 | 77-13| 47 | 21-07} 14 2 | °255,| | 220 112 | 50-91 | 120 | 54-54] 166 | 75-45} 46 | 20-90 | 15 3 255 | 220 113 | 51-36| 117 | 53-:17| 165 | 75-00} 46 | 20-90 | 14 4} 253) 215] 110 | 51-16) 118 | 54:88) 166 | 77-20) 49 | 22-79 to 5 | 248] 211 108 | 51-18) 112 | 53-08} 162 | 76-77} 43 | 20-38 | 16 6 | 246] 213 109 | 51-17} 115 | 53-99} 163 | 76-52 | 46 | 21-69) 14 Os 4040 Os 40 40 Os 40 On C8 FO 04 O4 Oy OK 05 04 404003 03 4003 40 Os 03 05 4003 4040 05 Oy 04 OS 0 05 494003 4005 404003 03 03 O5 eet et te — — — Ne} — pond Ne} cn) — J a] 1. | V: A. epi ae ene. A oe rn ied: | acd? F.cd. | ied. 107 | 51-69| 113 | 54-59 | 160 | 77-29) 45 | 21-73 106 | 52:73 | 108 | 53-73) 154 | 76-61 | 44 | 21-88 104 | 51-74| 109 | 54-22| 153 | 76-11| 45 | 22-38 102 | 50-74} 108 | 53-73| 154 | 76-61 | 42 | 20-89 | 102 | 51-25| 111 | 55-77| 150 | 75-37| 43 | 21-60 99 | 50-00} 106 | 53-53) 145 -73-23) 42 | 21-21 99 | 50-00| 106 | 53-53| 149 | 75-25 | 42 | 21-21 98 | 50-25) 104 | 53-33) 150 | 76-92'| 43 | 22-05 98 | 50-25| 107 | 54-87) 146 | 74-87| 40 | 20-51 100 | 51-02, 106 | 54-08) 148 75-51| 41 | 20-91 99 | 51-29! 101 | 52-33) 144 | 74-61 | 41 | 21-24 98 | 51-30; 106 | 55-49| 146 76-43 | 42 | 21-99 95 | 50:00} 106 | 55:78) 145 76-31 | 40 | 21-05 98 | 51-30| 102 | 53-40} 144 | 75-39! 40 | 20-94 98 | 51-57| 102 | 53-68| 143 | 75-26| 42 | 22-10 97 | 51-05| 101 | 53-15| 142 | 74-73 | 41 | 21-58 98 | 51-57| 100 | 59-63) 142 74-73| 41 | 21-58 95 | 50-00| 103 | 54-21| 141 | 74-21] 41 | 21-58 94 | 50-26| 104 | 55-61] 144 | 77-00| 40 | 21-38 98 | 52:12 102 | 54:25} 142 | 75-53 | 42 | 22-34 97 | 51-87) 101 | 54-01) 141 | 75-40| 41 | 21-92 92 | 49-72} 101 | 54:59| 139 | 75-13] 40 | 21-62 93 | 50-00} 99 | 53-22] 139 | 74-73| 41 | 22-04 95 | 51-35| 102 | 55-13| 142 | 76-75| 42 | 22-70 94 | 50-81| 97 | 52-43| 138 | 74-59}-41 | 22-16 95 | 51-63| 98 | 53-26| 137 | 75-54 | 40 | 21-73 91 | 50-00! 96 | 52-74| 185 | 74-17| 42 | 23-07 91 | 50-27} 100 | 55-24] 136 | 75-13] 41 | 22-65 92 | 51-68} 95 | 53-37) 133 | 74-71 | 40 | 22-47 89 | 50-28} 96 | 54-23| 134 | 75-70! 40 | 22-59 (7) Pwllheli. Received November 23rd, 1914. 122 | 52-13 128 | 54-70| 178 | 76-06/ 51 | 21-79 121 | 51-93| 126 | 54:07| 179 | 76-82] 51 | 21-88 118 | 51-08) 128 | 55-45| 180 | 77-92] 50 | 21-64 116 | 50-21 125 | 54-11 172 | 74-45| 51 | 22-48 115 | 50-21} 120 | 52-40| 169 | 73-80} 48 | 20-96 118 | 52-44| 127 | 56-44! 169 | 75-11] 50 | 22-22 117 | 50-64| 124 | 53-68| 178 | 77-05] 48 | 20-77 119 | 52-42) 121 | 53-30] 173 | 76-21] 49 | 21-58 117 | 51-54| 126 | 55-50] 174 | 76-65] 51 | 22-46 115 | 50-88} 123 | 54-42| 172 | 76-10] 48 | 21-23 115 | 51-11} 121 | 53-77] 169 | 75-11| 51 | 22-66 119 | 52:19} 122 | 53-50| 172 | 75-43| 51 | 22-36 113 | 50-44| 121 | 54-01} 171 | 76-33} 50 | 22-32 114 | 50-00} 122 | 53-50} 173 | 75-87) 47 | 20-61 118 | 52-44] 125 | 55-55! 174 | 77-33| 49 | 21-77 118 | 52-44/ 121 53-77 | 174 | 77-33 | 48 | 21-33 113 | 50-22) 122 | 54-22) 170 | 75-55| 47 | 20-88 vate 124 | 55-11| 173 | 76-88| 47 | 20-88 114 | 51-12} 123 | 55:15] 171 | 76-68| 49 | 21-97 112 | 50-45] 121 | 54:50|} 169 | 76-12 | 47 | 21-17 113 | 51-36 | 123 | 55-91) 168 76-36 | 47 | 21-36 114 | 51-12) 120 | 53-81) 168 | 75-33) 46 | 20-62 SEA-FISHERIES LABORATORY. Table VY—Continued. O3 40 O3 Os O3 03 4040404093 40 03 4003 40 93 O3 404040 O53 O35 03 05 05 O03 49005 O35 | D3 05 03 Cs 03 404093 40935 404095 O35 404005 03 4005 05 40 VII 198 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Table Y¥—Continued. | 1D Vv. A. Lepal: INO. oO sledge ly ah tae Bee |) ee | % | % % | % | Ted: | Exed: T.cd. | Eee 23} 255 |) 221 114 | 51-58) 120 | 54:29} 168 | 76:01 | 49 | 22-:17| 14] ¢ 24 | 255) 220 115 | 52-27| 120 | 54-54| 168 | 76-:36| 46 | 20-90| 14; 92 25 | 254 | 222 112 | 50-45} 118 | 53-15] 168 | 75-67] 47 | 21-17| 14 | 2 26 | 254 |) 221 111 | 50-22 | 121 | 54-75| 171 | 77-37| 47 | 21-26) 15) 3 27 | 254] 220] 111 | 50-45; 119 | 54:09) 164 | 74-54] 48 21-81) 14) ¢ 28 | 254) 220 111 | 50-45! 115 | 52-27| 165 | 75-00 | 47 | 21-36; 15 | 9Q 29 |. 252.) 216 109 | 50-46| 119 | 55-09| 167 | 77-31 | 46 | 21-29) 15) ¢ 30 | 250) 218 112 | 51-37 | 115 | 52-75| 164 | 75-22) 47 | 21-56] 15) ¢ 31 250 | 217 109 | 50-23 | 116 | 53-45| 164 | 75:57 | 47 | 21-65| 15) ¢ 32 | 249 | 218 113 | 51-83 | 117 | 53-67] 165 | 75-68] 48 | 22:01) 15) ¢ 33 | 249 | 210 110 | 52-38 | 116 | 55-24} 159 | 75-71 | 46 | 21-90] 14 | 3 | 34 | 248 213 111 | 52-11 | 118 | 55-39} 166 | 77-93 | 45 | 21-12) 15) @ 35 | 247 | 217 114 | 52-53 | 114 | 52-53 | 162 , 74-65] 46 | 21:19; 14] ¢ 36 | 247 | 212 108 | 50-94; 111 | 52-35] 159 | 75-00] 45 | 21-22) 14 | 9Q 37 | 246 | 213 109 | 51-17| 117 | 54-93 | 164 | 76:99 | 46 | 21-59; 15| ¢ 38 246 210 107 | 50-95} 115 | 54-76| 160 | 76-19] 46 | 21:90| 14) ¢ 39 243 212 108 | 50-94] 115 | 54-24| 159 | 75:00 | 45 | 21-22} 14] ¢ 40 | 241 206 103 | 50-00 | 108 | 52-42| 154 | 74:75 | 44 | 21-36| 13 | 92 4] 240 | 209 106 | 50-71 | 114 | 54-54] 159 | 76-07) 45 | 21-53| 14| ¢ 42 | 237 | 207 104 | 50-24; 110 | 53:14] 155 | 74-88] 43 | 20-77| 14 | 3 43 | 235 | 203 102 | 50-24; 108 | 53-20] 153 | 75-36) 45 | 22-16] 14 | 9 44} 231 203 103 | 50-73 | 108 | 53-20} 152 | 74-87] 44 | 21-67| 14 | ¢ 45 | 220 191 96 | 50-26| 101 | 52-88| 141 | 73-82} 43 | 22-51; 13| 2 46 | 210 182 90 | 49-78 93 | 51:09 | 136 | 74:72 | 38 | 20-87| 14 | ¢ (8) Moelfre. Received December 9th, 1914. 1 | 271 | 233 |) 120 | 51-50| 126 | 54:07} 180 | 77-25) 47 | 20-17) 15] ¢ 2| 268 | 233 116 | 49-78 | 123 | 52-78; 177 | 75-96 | 49 | 21:03; 15] ¢ 3 | 268 | 233 120 | 51-50 | 127 | 54-50} 178 | 76-39 | 49 | 21:03| 16°] ¢ 4; 267; 231 | #116 | 50-21) 121 | 52-38; 171 | 74:02 51 | 22-48/15| ¢ 5 265 | 230 119 | 51-73 | 125 | 54-34 | 178 | 77-39 | 47 | 20-43| 16 | 3g 6 | 265 | 229 116 | 50-65 | 125 | 54:-58| 174 | 75-98 | 50 | 21-83) 15 | ¢ 7 | 262 | 226 115 | 50-88 | 121 | 53-53 | 172 | 76-10 | 50 | 22-12) 15| ¢ 8 | 262 | 224 117 | 52-23] 124 | 55-34] 174 | 77-67) 50 | 22-32) 14] 3 9; 261 225 | 113 | 50-22} 123 | 54-66| 168 | 74-66 | 47 | 20-88] 14] 9@ 10 |-261 224 | 115 | 51-33 | 123 | 54-91| 169 | 75-44/ 51 | 22-76) 14| ¢ 11 260 | 226 116 | 51-32 | 124 | 54-86| 172 | 76-10 | 47 | 20-79; 14) ¢@ 12 | 260 | 225) 114 | 50-66) 125 | 55-55| 174 | 77-33 | 49 | 21-77) 14 | @ 13 | 260 | 224) 114 | 50-89} 124 | 55-34] 173 | 77-23 49 | 21-87) 15 | ¢ 14 260 222 114 | 51-35] 123 | 55-40| 168 | 75-67! 50 | 22-51 | 14) 9 15 | 259 | 225] 114 | 50-66] 120 | 53-33 | 172 | 76-44} 48 | 21:33) 15) ¢ 16 | 259 | 224 113 | 50-44] 120 | 53-57| 169 | 75-44) 49 | 21-87) 15] ¢g 17 258 222 110 | 49-54 | 123 | 55-40| 170 | 76-57 | 48 | 21-62|; 16; ¢ 18 | 257 | 224) 113 | 50-44) 119 | 53-12| 170 | 75-89 | 48 | 21-42 | 14 | -9 19%) 257 cn 220 111 | 50-45 | 120 | 54-54] 168 | 76-36} 50 | 22-72) 14) @ 20 | 255} 221 110 | 49-77} 119 | 53-84] 165 | 74-66) 49 | 22:17) 14] ¢g 21 255 | 221 114 | 51-58 | 122 | 55-20] 173 | 78-28) 48 | 21-71); 14] ¢ 22 | 255 | 220 114 | 51-81 | 122 | 55-45 | 167 | 75-90 | 48 | 21-81; 14] ¢ 23 | 254 | 221 114 | 51-58 | 120 | 54-29) 169 | 76-47 | 46 | 20-81 | 14] 3 24 | 254 | 217 114 | 52-53] 119 | 54-83) 165 | 76:03 | 48 | 22-12) 15 | 9 25 | 252) 219 113 | 51-59 | 120 | 54-79| 167 | 76-25/| 48 | 21-91) 14] 3 26 | 252 217 112 | 51-61 | -116 | 53-45 | 165 | 76-03 | 49 | 22-58; 14] ¢g 27 | 249 | 213 109 | 51:17} 118 | 55-39; 164 | 76-99 | 47 | 22:06) 15) ¢g 28 | 247 211 110 | 52-13 | 119 | 56:39 167 | 79-14 | 48 ey LB" as CwBIMSHK Pode — Eee —— —_—_——————. (9) New Quay, 4 in. mesh. Received December 10th, 1914. 232 227 227 225 225 226 225 222 219 219 221 221 235 229 227 123 117 119 116 118 113 lll 116 113 112 113 112 —_ QO) Moetfre. Received Dever Moelfre. 118 120 120 116 113 112 116 115 117 113 120 113 105 113 SEA-FISHERIES LABORATORY. \ 53-01 51-54 52-42 51-55 52-44 50-00 49-33 52-25 51-59 51-14 51-13 50-67 Table Y—Continued. -—————S |§ — | —_- ——_—_——— 108 106 103 103 105 104 104 103 101 100 100 96 127 125 127 12] 123 121 120 124 118 121 116 119 128 130 128 121 123 121 122 120 122 122 122 122 117 120 54-74 55-06 55-94 53°77 54-66 53-53 53-33 55-85 53-88 55-25 52-48 53-84 54-46 55-79 55-89 53°30 54-42 55-25 54-99 54-54 54-46 54-99 55-45 55-96 54-92 54-29 | A. 158 157 162 156 156 159 157 153 153 153 153 146 145 145 144 142 144 142 141 143 138 136 178 174 175 173 171 172 173 171 168 169 166 168 | 184 179 180 170 174 170 170 170 169 172 170 169 165 168 76-72 76-65 77-09 76-88 76-00 76-10 76-88 77-02 76-71 77-16 75-11 76-01 78-29 76-82 78-60 74:89 76-99 77-62 76-57 77:27 75-44 77-47 77-27 77-52 77-46 76-01 21-55 21-58 21-58 20-00 21-33 20-35 20-00 21-17 21-91 21-00 20-81 20-36 Received December 17th, 1914. 199 | | 14 O03 03 O3 4003 03 03 05 O03 02 4O Os 4OO3 40.04 C3 4004 05 40404005. 05 4040 4005 0 0405 ON. O4 Pmt rad ed rem TY et ted ed ed <“didicicicidiicicicid $$ $$$ $$ O3 03 05 0303 404003 05 03 40 OK O11 OD } 200 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Table VY—Continued. | | Li ae V. . A. Lep.1. No.) T T.cd. | oC ee eee eee K, | 8s g. | % Yo. {| | % % T.cd. T.cdue| | T.cd. T.cd. ‘ 15 | 250 |. 211 | 111 | 52-60) 117 | 55-45) 163, | 77:25 | 47") 22-27) eee Vil 16} 249; 213 ; 111 | 52-11) 119 | 55:86 168 | 78-87 | 47 | 22:06; 14] Q VIL. 17 | 244 | 207 | 107 | 51-69; 116 | 56-03; 160 | 77-29 | 45 | 21-73): 13) 6 VII 18 | 244} :207 | 108 | 52-16) 114 | 55:07: 158 | 76-32 | 44 | 21-45) 147) ¢& VII 19 | 242; 206 | 105 | 50-97; 115 | 55-82 158 | 76-69/ 46 | 22:33; 13] ¢ VII 20 | . 241 208 | 105 | 50-48 | 112 53-84: 157 | 75-48] 47 | 22-59} 14] ¢ VIT 21 234 | 199 | 100 | 50-25} 109 | 54:77 156 | 78-39| 43 | 21-60). 15 | ¢ A 22 | 232) 203 | 105 |'51-72| 112 | 55-17) 157 | 77-34) 45 |:22-16|) 16) ¢ VIL 23 | 232 | 199 | 102 | 51-25) 107 | 53-76. 151 75-87} 44 | 22-11|- 14 | Q I 24.0) 232 197 | 103 | 52-28 | 107 | 54-31 | 154 , 78-17| 45 | 22-84). 15 | 2 ie 2a Zab 199 | 100 | 50-25) 108 | 54:27) 150 75-37] 44 | 22-11| 14] ¢ ig 26 | 228 198 | 100 | 50-50] 106 | 53-53, 149 75-25) 43 | 21-71) 14] Q le Die oad 196 | 99 | 50-51] 105 | 53-57) 146 , 74-49] 44 | 22-44] 14] ¢ I 28 | 225] 193 | 96 | 49-74] 103 53-36| 144 | 74-61 | 43 | 22-28] 14 Q 1% 29 | 225 | 193 | 98 | 50-77| 105 | 54:40} 147 | 76-16 | 42 | 21-76; 14) ¢ is 30 | 225] 192 | 99 | 51-56) 107 | 55-72) 146 | 76-04 43 | 22-39) 14] Q 1a 31 224 | 193 | 99 | 51-29] 104 | 53-88 149 | 77-20 | 43 | 22-28; 14] ¢ I 32 | 224) 192.) 97! 50-52) 102! 53-12) 145 | 75:52)) 419) esa eee i | ao) 224a OU 99 | 51-83] 105 | 54-97| 145 | 75-91 | 44 | 23-:03|} 14) ¢ 1 34 | 224), 190-| 98 | 51-57) 103 | 54-21; 144 | 75-78 | 43 | 22-63) 16) 9 Ill 30. 22eal LOR 97 | 50-78| 106 | 55-49! 147 | 76-96 , 42 | 21-99| 14] Q. I 36 |” 223 191 96 | 50-26] 105 | 54:97) 148 | 77-48 | 42 | 21:99| 14] ¢ le 37 | -223 191 98 |-51-30| 103 | 53-92 | 142.|.74-34 | 41 | 21-46) ieee = 38 | 220) 188] 94 | 50-00} 104 | 55-32) 142 | 75-53) 43 | 22-87) 13) @ i 39.1. .220.| 187.|..98..| 52-40 |...103. | 55-08 |. ..143_.|-76-47.| 43.) 22-995). 155 ee 40 | 219); 188 | 97 | 51-59] 104+ 55-32; 141 | 75-00} 42 | 22-34) 14) ¢ I 41 219 | 186 | 96 | 51-61] 104 | 55-91 | 142 | 76-34) 43 | 23-11; 12] @ I 42 | 218 190 | 95 | 50-00} 103 | 54-21| 143 | 75-26 | 41 | 21-57; 13] @ | = 43 | 218 | 188} 99 | 52-66| 104 | 55-32| 145 | 77:12) 41 | 21-80; 14]; 9 I 44 | 218 188 | 96 | 51-:06| 102 | 54-25| 142 | 75-53 | 43 | 22-87) -14 | @ I4 45} 216; 185 |. 96 | 51-89| 101 | 54-59 | 142 | 76-75 | 41 |.22-16) 15) ¢@ 14 46 | 215 | 183] 94°| 51-36} 100 | 54-64| 141 | 77-04] 40 |:21-85| 14| Q 1% 47 | 214] 183] 99 | 54:09] 107 | 58-47) 139 | 75-95/| 41 | 22-40) 14; ¢ Ils 48 | 213 185 | 95 | 51-35} 100 | 54-:05| 139 | 75-13 | 42 |:22-70| 14) ¢ 1g | 49 | 213 182 | 93 | 51-:09| 100 | 54:94] 139 | 76-37| 40 | 21:97; 15 | @ I 50 196 168 | 85 | 50-59 93 | 55-35 | 128 | 76-19} 38 |'22-61; 14! 2 | I @ | | | | Ay are i en en cee einen Ens “vias oS ae Mean | 51-22 | 54-31 | | 76-09 | | 21-69 14-19 | SEA-FISHERIES LABORATORY. 201 ON “WHITEBAIT” COLLECTED IN MENAI STRAIT. | By AnpDREw Scott, A.L.S. A number of samples of ‘‘ whitebait’ caught in the weir at Gorad Coch, near the Swillies in Menai Strait, between Anglesey and the mainland, were sent to me for examination in 1914 by Captain Robert Jones, the head fishery officer for that district. The collection lasted for seven months, from March to September, and samples were taken as the fish made their appearance. In some months two samples were taken, in others only one. Altogether ten samples were investigated. It was anticipated that some useful information would be obtained relating to the species of fish included under the general name “‘whitebait,’’ and would settle the question whether young herring occur amongst the mixture. The results are satisfactory in so far that they show that the whitebait from Gorad Coch are young clupeoid fish, such as sprats and herring, and that young herring 35 to 67 millimetres in length are frequently present. In all probability the herring are hatched in spawning areas at the sea-bottom in some of the bays adjacent to the openings into the Strait. Many more samples will require to be dealt with to determine the frequency of their occurrence, and a careful investigation of the sea-bottom in Carnarvon and Beaumaris Bays would have to be made to find out if herring spawning takes place in these areas. There is considerable difficulty in determining whether the smaller fish, of about 43 millimetres in length and under, are young herring or some other young clupeoid. They are scaleless and almost transparent. The position of the dorsal fin in relation to the pelvics is 202 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. no guide, as it has not taken up its final position. The relative differences in the beginning of the dorsal and pelvic fins in the adult sprat and herring are quite different from what is found in young stages under 43 millimetres. The beginning of the pelvic fins in adult sprats is usually distinctly in front of the dorsal fin. In the case of the herring, the dorsal fin starts in front of the pelvics. Ehrenbaum’s figures in ‘‘ Nordisches Plankton ’’ show that young herring up to 34 millimetres in length have the pelvic fins well in front of the dorsal. It is evident, therefore, that the dorsal fin must change its position and gradually advance nearer the head as the fish grows and the scales make their appearance. This advance of the dorsal fin may not be completed till the young herring reaches a length of 47 millimetres, as shown by the table giving the measurements of the fish collected on May 28th. The only way to distinguish young herring from young sprats before the transforma- tion is complete is by examining considerable numbers of fish. Young scaleless herring can be readily separated from young sprats when mixed up with them, by their bodies being much narrower and ribbon-like. The following tables give the size of the fish, measured from the tip of the snout to the end of the caudal fin, the distance of the dorsal and pelvic fins from the snout, and the difference in the position of the beginning of the pelvic fins from the snout compared with the dorsal fin. The photographic illustrations 1-10 represent a typical fish from each sample, 11 and 12 the two extreme sizes from a sample collected on May 28th, 13-16 fish of nearly the same length but different character in a collection taken on July 17th. ye The illustrations of fish of nearly the same size but different character are useful in showing the marked SEA-FISHERIES LABORATORY. 203 distinctions that occur. The two lower fish are young sprats measuring 37'4 and 373 millimetres in length. The dorsal fin of the slightly larger one is 0°5 millimetre further away from the tip of the snout than the pelvics. The pelvic fins in the smaller one are 0°2 millimetre further away from the snout than the dorsal. The fish are moderately deep and the scales are developing. I regard the two upper fish as young herring. The first one is 346 millimetres long. The second is 389 milli- metres in length. The dorsal fin of the former is 2°5 millimetres further away from the snout than the pelvies. In the latter it is 2°3 millimetres further away from the snout. Both fish are narrow and ribbon-like, and were probably almost transparent when alive. The scales are not developed. These characters agree well with the description of young herring of this length given by Ehbrenbaum. If HEhrenbaum’s identification of the young fish is correct, it is quite clear that considerable change takes place in the position of the dorsal fin in relation to the pelvics before the final characters are visible. The dorsal fin represents part of the remains of the embryonic fin which extended along the whole of the back, round the tail and along the ventral margin. The embryonic fin gradually disappears as the larva grows till only the persistent parts known as the dorsal, caudal and ventral fins are left. The pelvic fins are appendages developed after the transformation of the embryonic fin is completed, but before the dorsal part becomes a fixture, and are distinct outgrowths from the body. Their position is probably nearly permanent all through the life of the fish. As the fish grows in length and depth the dorsal fin is gradually pushed forward, and finally becomes attached to some of the dorsal spines of the back- bone just above the pelvics. 204 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. If we compare the young herring (figs. 18 and 14) and the two young sprats (figs. 15 and 16) with the illus- trations representing the first three samples, it can easily be seen that the fish captured on March 11th and 31st and on April 16th are young herring. The fish caught on March 11th measured 34°77 to 45°7 millimetres in length. The next lot taken on March 3lst were probably part of the same shoal that visited the strait nearly a fortnight earlier, as the measurements are practically the same. Those caught on April 16th probably belonged to a later hatching than the first two samples, as they are distinctly smaller fish. Their size ranged from 33°7 to 407 millimetres. It is scarcely lhkely that they belonged to the same shoal represented on March 11th, as we would naturally expect larger fish after fully a month’s longer time to grow. It will be noticed on looking over the column giving the difference in the pelvic fins that the variation is by no means uniform. Generally, however, the variation in the pelvic fins is greater in the smaller fish than in the larger ones. This ought to be the case if the dorsal fin moves forward as the fish grows. The sample taken on May 14th consisted of a mixture of young herring and sprats. Fig. 4shows one of the herring. The whole of the fish captured on May 28th were young herring from 42°6 to 673 millimetres in length. Figs. 11 and 12 show the largest and smallest fish in this catch. The small one is nearly the same size as the largest fish caught in April, and it is evident that the collection contains at least two distinct generations of herring. The fish caught on June 29th were apparently all sprats, measuring from 236 to 43°6 millimetres in length. The sample taken on July 17th contained ninety-seven fish. Hight of them were young herring from 33°7 to 38°9 millimetres. The remainder were sprats from 37°38 to 53°6 SEA-FISHERIES LABORATORY. 205 millimetres long, with the keeled scales well developed. Only one herring was found in the sample taken on August 8th. The fish caught on August 24th were all sprats. The pelvic and dorsal fins in nearly all the sprats examined in August were exactly the same distance from the snout. ‘The dtfference in the few exceptions was very slight. The sample taken on September 9th consisted wholly of sprats with well-developed keeled scales. The fish in this collection were typically sprats, but there is considerable variation in the relative positions of the dorsal and pelvic fins. This variation ranged from 0 to 23 millimetres, as shown in the table giving the results of the measurements of the September fish. 206 Total Snout to | Snout to length of | beginning | beginning fish. of dorsal | of pelvic fin. fins. mm. mm. mm, 45-7 22-2 21-5 44-9 22-1 21-7 44-5 22-0 20-8 44-2 22-1 20-7 43-7 22-1 20-9 43-4 21-7 20-5 43-1 21-9 20-5 43-1 21-4 19-6 42-7 21-9 20-5 42-7 21:9 20-2 42-7 21-9 20-2 42-5 21-5 19-8 42-5 20-9 19-3 42-2 21:3 19-7 41-8 21-4 20-1 41-6 21:3 19-4 41-6 21-1 19-7. 41-6 21:3 19-7 41-5 20-9 18-9 41-5 21:0 19-9 41-4 21-3 19-6 41-3 21:5 20-1 40-9 21-4 19-2 40-9 21:0 19-2 40-7 21-2 19-3 40-7 21-0 19-7 40-6 21-6 19-7 [ilies ea Lees pa bl em eeeleel emda fet fame NS et et tet NS et ee et ed eet ee el ee ee ee et et et OO CWOMNWKRUIHOHRhOWARGUNGAROKRYDPRHORY | March 11th, 1944. TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Total Snout to | Snout to length of | beginning | beginning fish. | of dorsal | of pelvic fin. . fins. mm, mm. mm. 40-6 21:1 19-0 40-4 21-2 20:3 40-1 20-5 19-3 40-1 21:1 19-3 40-0 20-9 19-0 39-7 20-4 18-4 39-4 20-1 18-4 39-3 20-3 19-2 39-2 20-2 18-6 39-2 20-4 18-6 38-8 19-9 18-2 38-4 19-9 17:3 38-4 19-9 17:7 38-2 20-1 18-1 38-0 19-6 17-6 38-0 20-3 17:8 37-9 20-0 17-6 378 19-5 17:3 37°38 20-1 17-7 37-5 19-6 17-9 37-2 19-8 17-6 37:1 19-5 17-2 36:9 19-6 17:8 36-9 19-8 17-1 36-6 19-5 16-9 35-6 19-1] 16-5 34:7 18-7 16-2 | Pelvic 4 fins, + or — KHAMWDMDWNOUIARDWKRAMAOOHGUBDAHHOCSHHOE ested RR TIE oR es FIST | Cae) PR fe cect eet 9H a (oT ea DD DDH DDH DDD DNDN DH HEHE Dee On SEA-FISHERIES LABORATORY. 207 March 31st, 1914. Total Snout to | Snout to Pelvic || Total Snout to | Snout to Pelvic length of | beginning | beginning fins. length of | beginning | beginning fins. f >) of dorsal | of pelvic | + or — fish. of dorsal | of pelvie | + or — fin. fins, fin. fins. 1m mm, mm. mm. mm. mm. 15-7 23-2 22-3 0-9 — 40-8 21-5 20-3 1-2 — 44-7 22-3 22-0 0-3 — 40-8 21-1 19-2 1-9 — 4-4 21-8 21-2 0-6 — 40-6 21-1 19-3 1-8 — 2 21-9 — =—-20-8 1-1 — 40-5 20-8 19-2 1-6 — 9 21:5 20-3 12— || 40-5 20-5 18:5 2-0 — 43-7 — 20-7 20-7 aap 40-2 21-1 19-2 1-9— 43-5 22-3 20-1 1-2— 40-2 20-9 19-5 1-4— — 43-0 21-6 20-8 0-8— | 40-1 21-0 19-4 1-6— — «42-8 21-2 20-8 0-4 — 40-0 20-6 18-7 1-9— 42-7 21-0 20-5 0-5 — 39-9 20-9 19-1 1:8 — 42-2 20-8 19-3 15— 39-9 20-9 19-4 15—- 41-9 21-2 20-0 1-2— 39-9 21-1 18-6 2-5 — 41-90 21:6 19-9 1-7 — 39-8 20:8 19-0 1:3 — 41-9 20-9 20-0 0-9 — 39-6 20-7 19-3 1-4 — 41-9 21-4 20:3 1-1 — 39-5 19-7 18:3 1-4— 41-9 212 .| 19-8 1-4— 39-5 20-1 18-4 1-7 — 41-8 20-5 19-5 1-0 — 39-5 20-0 18-2 1-8 — L1-7— 21-7 20-1 1-6 — 39-4 20-2 18-5 1-7— 41-4 21-1 20-0 1-1 — 39-4 20-2 18:3 1-9— 41-2 21:7 20-2 15 — 39:3 20-1 18-7 Das 41-3 21-3 19-6 1-7— 39-3 20-0 17:7 2-3 — 21-0 19-6 1-4 — 38-6 19-5 18-2 1:3 — 21-1 19-8 1:3 —- 38-6 20:5 18-6 1-9 — 20-7 18-9 ee | 2s 19-3 17-5 1-8 — 20-9 19-3 16— | 37:9 19-8 17-7 2-1 — 20-8 19-9 0-9 — 36:7 19-3 17-5 1-8 — 20-8 19-9 0-9 — 35:9 19:3 16-8 2-5 — 20:7 19-2 1-5— ) | | i 208 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. April 16th, 1914. Total Snout to | Snout to | Pelvic Total Snout to | Snout to Pelvic length of | beginning | beginning fins. length of | beginning | beginning fins. fish. of dorsal | of pelvic | + or — fish. of dorsal | of pelvic | + or — fin. fins. fin. fins. mm. mm. mm. mm. mm. mm. 40-7 21-2 19-2 2-0 — 36-9 18-9 17-0 2-9 — 40-6 21-1 19-2 1-9— 36-8 19-7 16-6 3-1— 40-6 21-1 19-2 1-9 — 36:3 be oe) 17:3 1-8 — 39-5 20-6 183 | 23— 36-1 19:0 | 16-6 2-4 — 39-5 21-0 18-6 2-4 — 36-0 18°74 16-1 26— — 39-3 19-4 17-5 1-9 — 36-0 19-5 17:3 22-— — 38-8 19-9 © 17-8 ON 35-9 18-8 16-8 20-5 38-7 20-1 17-7 2-4 — 35-7 ‘19-1 16-4 7— — 38-5 19-3 17-4 1-9 — 35-7 19-1 16-9 22— 3 38:3 20-7 17-9 2-8 — 35°6 18-7 16-6 21— 38:3 19:8 18-2 1-6 — 35-6 18-7 16-4 2:3-— 38-2 20-3 17-2 3-1 — 35-5 19-8 17-1 2-7-— 37-9 19-9 17-9 2-0 — 35-5 19-3 16:3 30-— 37:8 19-8 17-4 2:4 — 35:5 19-0 16-6 24— 37-6 20-1 17:7 2-4 — 35-4 _ 19-4 16-4 30-— | 37:6 19-8 17-7 2-1 — 35-4 19-0 16-4 2-6 — 37:5 19-1>".. 16-6 2-5 — oD 19-0 16:3 2-7 — 37°5 19-8 17-4 2-4 — 35-2 18-7 16-9 1-:8— 37-4 19-2 17:3 1-9 — — 35:0 18-8 16-0 2-8 — 37-4 19°6 FF | 2:5 — 34:8 18-4 15-9 2:5— 37-4 19-4 17-5 1-9 — 34-7 18-7 16-2 2-5 — 37-4 19-8 17:8 2-0 — 34-7 18-7 15:9 2-8 - 37:3 19-7 2. 2-5 — 34-7 18-5 16-2 2 37:3 19-5 17-1 2-4 — 34-6 18-7 16-2 2 37-2 19-4 17-4 2-0 — 34:3 18-1 15-6 2-57, 37-1 19-4 17-4 2-0 — 34:3 18:7 16-1 2-6 — 37:1 19-9 16-9 3-0 — 33°8 18-1 16-6 1-5 — 37:1 19-9 17-4 2-5 — 33-7 17-8 15-4 2-4 — 37-0 aos 17:8 2-0 — SEA-FISHERIES LABORATORY. 209 May 14th, 1944. Total Snout to | Snout to Pelvic Total Snout to | Snout to Pelvic length of | beginning | beginning fins. length of | beginning | beginning fins. fish. of dorsal | of pelvic | + or — fish. of dorsal | of pelvic | + or — fin. fins. fin. fins. mm. mm. mm. mm. mm. mm, 59-8 27-2 28-2 EO-- 46-8 22-6 21:5 1 52-9 24-4 24-4 fps 46-7 22-3 22-3 Bare 52-4 24-1 24-7 0-6+ 46-6 22-3 21-8 0-5 — 52-2 23-9 25-0 1-1+ 46-6 22-0 21-6 0-4 — 51-9 23-5 24-2 0-7+ 46-5 22-8 21-1 1-7 — 51-8 23-4 24:5 1-1 +- 46-5 22-3 21-0 1-3 — 51:3 23-4 23-9 0-5+ 46-5 22-5 21-6 0-9 — 51-2 23-7 24:1 0-4+ 46-5 22-1 22-1 38 51-1 23-4 24-0 0-6+ 46-3 22-0 21-2 0-8 — 50-1 24:3 25-4 | et 46.2 22-3 21-7 0-6 — 50-1 23-2 23-5 0-3+ 46-1 22-0 22:0 ae 49-9 23-2 24-0 0-8+ 45-9 22-8 21-9 0-9 — 49-4 23-0 22-5 0-5 — 45-7 22-5 21-4 1-1 — 49-4 22-4 23-2 0-8+ 45-4 22-2 21-5 0-7 — 48-8 23-3 22-8 0-5 — 45-4 20-5 20-5 aes 48-8 23-5 23-5 oe 45-2 22-6 21-2 1-4 — 48-4 22-0 22-0 are 44-9 21-2 20-8 0-4 — 48:3 22-5 22:5 os 44-6 21-4 20-2 1-2— 47-9 23-1 22-6 0-5 — 44-5 22-2 20-6 1-6 — 57 24:2 23-0 1-2 — 44:3 22:3 21-2 1-1 — 47 22-6 22-5 0-1 — 44-1 22-2 21-1 1-1 — At 4 22-8 22-2 0-6 — 43-9 22-1 20-8 1-3 — 47-4 22-3 22-3 is 43-3 21-8 20-4 1-4 — 47-2 22-7 22-7 sie 43-2 21:3 20-1 1-2 — 47-2 22-5 22-5 oe 42-7 21-7 19-7 2-0 — 46-8 22-2 22-2 ae | 210 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. | May 28th, 1914. | Total Snout to | Snout to Pelvic || Total | Snoutto | Snoutto | Pelvic} length of | beginning | beginning fins. length of , beginning | beginning fins. — fish. of dorsal | of pelvic | + or — |, fish. | of dorsal | of pelvic | + or =§ fin. HIBS pe fin. fins. : : mm, mm. mm. mm. mm. mm. *67°3 29-7 32-2 2:3 ae 23-5 24:3 65-7 30-7 Doe ami eek ica ell Lee ke 23-0 24-1 : 64-5 28-7 30-6 1-0 50-9 22-8 24-5 : 58-6 27-2 28-6 1-4+ 50-9 23-3 24-0 ; 57-9 25-9 27-6 re 50:7 22-5 24-5 : 56-6 Zd-3 > 26-6 ae 50:5 23:5 24-0 | : 56-6 25-9 26-8 Oo. 50-5 22-2 24-0 7 56-5 20. |, C20 1-6 50-5 23-0 23:8 ; 56:5 24-3 26-2 Loess 50:3 23-1 23:8 : 56-4 26-0 27-6 ICG 50-2 23-4 24-9 : 56:3 25-2 26-0 0-8+ 50-2 22:7 24-3 ‘ 56-1 25-7 26-7 iO 50-2 23-4 23-8 : . 55-7 25-5 26-3 0-8+ 50-1 23-0 * 23-3 i) 55-7 25-6 27-0 1-4, 50-0 _ 22-6 23-5 0-05-49 54:8 24-6 25:5 0-355 49-8 | 22-4 23-7 1-3 53-4 24-4 25-4 IQ. 49-7 22-9 24-0 Thee 53-2 24-1 - 25-7 16+ 49-4 22-9 23:3 0-4-4 52-8 24-2 26-0 1-3 = 49°33 22-6 23-8. 1-2 52-5 24-7 ‘Pooh 0-44 48-8 22:5 23-6 lt 52-0 24-4 25:8 1-44 48-7 22:5 23-1 0-6+ 51:8 23-7 24-8 iTS 48-0 22-2 23-0 0-348 51:8 24-0 24-5 0-5+ 47-7 22:8 23-2 0-4-5 51-7 24-0 25-5 1-5+ 47-3 23-0 22-8 0-29 51-6 23-4 25-3 BCE 47-1 21-9 22-3 04+. — 51-6 23-3 24-4 (Ie) 25 44-8 20-7 21-0 03+ — 51-6 23-8 25:3 io 44-3 20-5 21-2 O-7+ 51-6 23:3 24-8 1-o5r 44-3 21:0 20-5 0:5.— 51-5 23-6 24-3 OFT 742-6 18-7 17-2 aT cc | * Fig. 11, Plate III. + Fig. 12, Plate III. SEA-FISHERIES LABORATORY. 211 June 29th, 1914. OE eee Total Snout to | Snout to Pelvic || Total Snout to | Snout to Pelvic length of | beginning | beginning fins. ~__ length of | beginning | beginning fins. __ fish. of dorsal | of pelvic | + or — | fish. of dorsal | of pelvic | + or — ‘ fin. fins. | | fin. fins. mm. mm. mm. | mm. mm. mm. 43-6 20-5 20-3 | ue | 37-0 Vi-1 16-5 0-6 — 43-2 19-8 neg th. OLS | / 87-0 Ria « | 16-8 0-7 — 41-9 19-5 19-5 | 36-9 18-0 16-8 1-2 — 41-3 18-3 18-3 re | «636-6 16-6 15-7 0-9 — 40-7 18-7 18-4 03-— || 36-6 17-0 17-0 “o 40-1 18-6 18-0 0-6 — 36-3 16:8 16-2 0-6 — 39-9 19-0 17-8 Ea" | 36-3 17-5 16-5 1-0 — 39-5 18-5 18-3 0-2— || 36-2 17:6 16-8 0-8 — 39-5 £7-9 17-9 ote | 36-0 17-8 16-6 1-2 — 39-3 18-4 17-5 0-9-— || 360 174 | 165 0-9 — 39-2 18-2 17-3 09- | 35:9 17-4 16-6 0-8 — 39-0 19-0 17-6 14— | 34-9 16-8 15-9 0-9 — 39-0 18-2 _ 178 0-4 — 34-2 iy? a 16-9 0-2 — 38:9 | 18-5 17-6 0-9 — 33°7 I@b 7} 15-5 1-0 — 38-8 18-3 18-3 a 3371 165 | 152 | I3= 38-7 | 18-3 17:9 0-4 — 32-9 Ui | een oh ES 38-6 18-5 17-6 0-9 — 31-8 las 14-7 1-1 — 38-4 | 17:8 17:8 ee 3) We, weeded 154 | 140 1-4 — 38-2 18-4 17-5 09-— || 31-0 143 | 130 1-3 — s84 | 170 17-0 ue ‘|| --308 167 | 142 1-5- 38:0 17-4 17-4 ns | 30-6 15-9 13-0 2-9 — 38:0 18-0 16-9 li- | 29-7 15-0 13-4 1-6 — 7 o/ 17:8 16-8 1-0 — 29-2 14-8 12-8 2-0 — 37-5 178 | 169 | O9- | 23-9 15-5 13:3 2-2 — 375 «18:3 17-7 | (06 1-6 — 6— || 236 149 | 133 | | 212 Total length of fish. 48-8 48-7 48-3 48-2 47-9 47-8 47-7 47-6 47-5 47-5 47-5 47-5 47-5 47-3 47-2 47-0 46-6 46-6 46-3 46-3 45-8 45-7 45-6 45-5 45-5 Snout to beginning of dorsal fin. mm. 25-5 25-6 23-9 24-6 23-7 24-8 24-4 23-9 23-8 24-3 24-8 24-2 23-9 24-1 23-2 22-2 23-2 21-7 22-5 22-7 24-0 22-0 23-7 23-7 22-1 22-8 23-8 22-5 23-2. 23-1 22-7 22-3 21-4 22-3 21-8 21-7 22-9 21-5 22-0 22-4 21-7 22-3 21-0 21-1 21-7 21-9 = 20'5 fe alles 21-3 | July 17th, 1944. Snout to beginning of pelvic fins. mm. | 25:5 | 25-6 23-9 24-6 23-7 24-8 24-4 23-6 23-8 23°5 24-8 24-2 23-0 24-1 23-2 22-2 23-2 21-7 22-3 22-7 24-0 22-0 22-8 23-7 22-1 22:8 . 23-8 21-7 23-2 23-1 22-7 22-3 21-1 22-3 21-8 21-7 22-9 21-5 22-0 22-4 PALGT| 22-3 21-0 21-1 21-7 21-9 20-5 21:5 21:3 | Pelvic fins. + or — 0-2 — 00 0-8 — 03— Total length of fish. mm. 45-4 45-0 44-8 44-7 44-3 44-3 44-2 44-0 43-8 43-8 43-7 43-7 43-7 43-7 43°5 43-4 43-3 43-3 43-1 43-1 43-1 43-0 42-7 42-7 42-5 42-5 42-0 41-6 41-4 41-1 40-8 40-5 40-5 40-1 *38-9 38-7 38-3 38-3 37-5 437-4 37-3 36-7 36:3 36-0 35:9 34:8 **34-6 33°7 Snout to beginning of dorsal fin. mm. 21-7 rae) 20-7 20-5 20-1 20-1 21-8 19-9 20-2 20-5 20-9 20-9 19-4 20-2 20-3 20-5 20-8 20-1 19-9 21-7 19-2 20-0 19-6 20-8 20-3 19-0 19-1 19-5 18-9 18-5 18-5 19-5 19-2 19-0 18-2 18-4 17:0 18-0 17-0 17:5 17-2 17-4 17-4 17-4 18-0 17:0 17-1 16-2 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Snout to beginning of pelvic fins. mm. 21-7 21-7 20-7 20:5. 20-1 20-1 21-8 19-9 20-2 20-5 20-9 20-9 19-4 20-2 20:3 20-5 20:8 20-1 19-9 21-7 19-2 20-0 19-6 20:8 19-4 19-0 19-1. 19-5 18-9 18-5 18-5 19-5 19-2 19-0 15-9 18-4 17-0 18-0 17-0 17-0 17-4 14.9 — 15-2 14-5 15-3 14-6 14-6 13-8 Pelvic | - fins. + OF — TP |e oa Bora O8 NW ONwo: 3: : ** Fig. 13, Plate III. * Fig. 14, Plate III. + Fig. 16, Plate III. + Fig. 15, Plate III. ‘ SEA-FISHERIES LABORATORY. Total Snout to length of | beginning fish. of dorsal fin. mm. mm. 52-1 23-2 51-5 23-1 50-9 25-7 50-7 22-8 50-6 25-0 50°3 21-9 50-2 24-4 49-9 22-4 48-8 22-3 48-8 22-5 48-8 21-7 48-5 21-9 48-5 22-2 48-1 21-4 48-1 21-8 48:0 | 20-8 47-4 | 22-3 473 21-9 470 | 21-5 468 | 21-5 46-7 21-0 46-5 | 21-5 463 21-4 46-2 | 20-7 Snout to beginning of pelvic fins. mm. 23-2 23-1 25:7 22-8 25-0 21:9 24-4 22-4 22-1 22:5 21-7 21:9 22-2 21-4 21:8 20-8 22:3 21-9 21-5 21:5 21-0 21-5 21-4 20-7 August 8th, 1914, Pelvic ! fins. 4 Or — Total length of fish. 0-2—. | 213 Snout to beginning of dorsal fin. Snout to beginning of pelvic fins. 2-0 + 0-2 — 214 Total length of fish. mm. 58-2 57-2 56:5 56-1 55-7 55-4 55:0 54:5 53-7 53:0 52-7 52-7 52-6 52-6 52-5 52:5 52-4 52-3 51-7 51-1 50-3 50-3 50-3 49-8 49-4 49-3 49-2 Total length of fish. August 24th, 1914. Snout to beginning of dorsal fin. mm. 25-9 25:8 26-3 25-5 25:5 25-8 25-6 24-8 24-9 25-0 23-7 25-0 23-4 23°9 24-0 23-0 24-4 24-0 24-0 23-7 23-0 23-1 22-4 22-3 22-5 23-4 22-4 Snout to beginning of pelvic fins. mm. 25-7 25:8 26-3 25:5 25-5 25:8 25-6 24-6 24-9 24-2 23-7 24-8 23-4 23-9 24-0 23:0 24-4 24-0 24-0 23-6 23-0 23-1 22-4 22-3 22-5 23-4 22-4 Pelvic fins. + or — 0-2 — 0-2 — ASCE 0-2 — Orle= TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Total length of fish. i mm. 48-8 48-8 48-7 | 48-3 47:8 47-2 46-7 46-4 46-3 45-8 45-8 44-8 44-8 44.7 44-5 44-4 44.3 44-] 44-0 44-0 43-5 43-5 42-5 42-3 41-5 4] -2 740-5 Snout to Snout to beginning | beginning of dorsal fin. mm. 21-8 22-0 22-0 22-6 20-7 22:0 21-3 21-0 21-3 21-2 22-7 22-8 21-7 20-2 20-5 21-0 20-3 19-4 20-0 21-0 19-5 20°3 19-3 19-1 20-5 18-3 ie-9 September 7th, 1914. Snout to beginning of dorsal fin. mm. 37-4 34-6 35:5 35:2 35:5 32-3 31-6 31-6 32-0 313 31-1 31-6 30:8 32-3 Snout to beginning of pelvic fins. mm. 36-8 33-0 34:8 33:2 33-2 31-4 31-4 31-0 31-3 30-7 29-9 31-5 30-2 30-6 Pelvic fins. Ot Pei We lal MOSH SOSOONNOHS AOE NVWHDASNWowo+Itnca | Total length of fish. Snout to beginning of dorsal fin. mm. 31-8 32-9 31-4 29-7 29-0 28-7 29-5 27-8 26-7 26:7 26-4 25-4 24-1 24:0 of pelvic fins. mm. 21-8 22-0 21-8 22-6 20-7 218 21-3 21-0 21:3 21-2 23k 22-8 21-7 20-2 20-5 21-0 20:3 19-4 19-8 21-0 19-5 20-3 19-3 19-1 20:5 18-3 Snout to beginning of pelvic fins. mm. 31-4 32-4 2979: 29:3 28:8 28-1 27°8 PAs elt 26-0 25-4 26-4 24:5 23:7 23-1 og- Pelvic fins. a + or ~ . e . . ORS: WYK UANWKRAGA oz chee Sel I tf eee FP ie a fe lee a eee eee ee ee Prate [. wN d Typical Herring from each sample, March to May. Typical Sprat from each sample, June to September. Rianne aioe . a ere DP yy Les > Wek: tte Sabie A Bh Mo! fe ie : = : ie and 12. Largest and smallest fish, May 2 and 14. Young Herring, July 17th. and 16. Young Sprats, July 17th. ee: «I. Big, 2. Pie 3. Fig. 4. Bag. .5. Big. 6. Pie. 1. Fig. 8. Fig. 9. Fig. 10. or SEA-FISHERIES LABORATORY. yak EXPLANATION OF PLATES. Prare I. Young Herring. March 11th, 1914. b>) 99 39) dl1st, 7) me a April “6th, :;, oe) V5) May 14th, 9 i 41) 39) 28th, 13 ° Prate IT. Young Sprat. June 29th, 1914. - 2: dialliver delle, mu He * Aue? Sth, i; bi] 33 - 24th, 2} ss Sete Cte. =. 5, Prate, ITf. Figs. lland12. Largest and smallest fish. May 28th. Figs. ldand14. Young Herring. July 17th. Figs.l5and16. Young Sprats. July 17th. All the figures are magnified about one-third. 216 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. THE FAT-CONTENT OF IRISH SEA HERRING. By Jas. Jonnstone, D.Sc. Throughout the period when biometric investigations on Irish Sea herrings were made by Mr. W. Riddell, fat-analyses of the muscle substance of the fish were also made. Samples of herring from the summer fishery near the south end of Isle of Man, from the winter fishery in Carnarvon and Cardigan Bays, and from the sprat fisheries in Morecambe Bay, and in the Estuary of the Mersey have been examined. Several interesting points suggesting further investigation have arisen in connection with this research, but so far time has not been available for their proper treatment. I hope, however, to return to this subject when the summer fishery of 1915 begins. The investigation may be of some importance in relation to methods of food preservation, a subject which indeed suggested it in the first instance. The analyses were made by means of the ordinary Soxhlet apparatus, using carbon tetrachloride as a solvent instead of ether. The extreme oiliness of some of the fish made the sampling a matter demanding some care. The muscle substance could not easily be chopped up, because much oil would have been lost in the process. The herring to be sampled was, therefore, lightly scrubbed with a test-tube brush so as to remove the scales. The skin was then dried with a towel, and, by means of a sharp razor, a series of cuts were made through the flesh as close together as possible. A tangential cut was then made so as to free these thin sections of muscle substance, which were then lifted by clean forceps and put into a paper thimble contained in a weighing bottle. Both thimble and bottle had previously been dried in the water oven and weighed. The whole was then weighed and the weight of the sample of muscle substance obtained by SEA-FISHERIES LABORATORY. AW difference. The bottle with its contents was now dried in the water oven until the weight was constant, a matter usually of about 20 hours, or more. The fat was then extracted. In the richest samples the oil oozed out through the thimble in the process of drying, and it was necessary to rinse out the weighing bottle, adding the solvent to the extraction flask. It was also necessary to plug the opening of the thimble lightly with dry cotton wool to prevent the breaking away of small fragments of the dried tissue and the entrance of these into the extraction flask through the siphon tube. In extraction the flask and Soxhlet were wrapped round with a towel so as to keep the temperature of the solvent as high as possible—it could almost be made to boil in the Soxhlet by this method. Extraction was usually carried on for two or three hours by which time all the fat was removed. Indeed after the third siphoning all the colour had usually disappeared from the solvent in the Soxhlet. The flask containing the solution was then detached and the solvent distilled away. The flask was then dried in the steam oven till its weight was constant. This process was hastened considerably by frequent blowing into the flask by means of a small hand bellows. The heavy vapour of carbon tetrachloride was thus removed. As a rule constancy of weight was attained in about 8 to 10 hours. Checks on the accuracy of the analyses were made by drying and weighing the residues. The latter were also stored in order that Kjeldahl nitrogen analyses might be made, but no time was available for this latter work. The error in the analyses is, I think, fairly small; and when the individual differences of fish and fish are considered it may be concluded that further refinements in the methods of estimation would be futile. The results are given in tables I to III. From the dates of the analyses the samples can be identified, and a comparison with Mr. Riddell’s results (published in this report) can be made. The degree of ripeness of the fish and the relation of the sample 218 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. to the time of spawning can thus be ascertained. At the beginning of the series of analyses the herrings were mostly ripening fish in the condition denoted by Hjort’s Nos. II and III in his scale of ripeness. At the end of the analyses the herrings were mostly spent fish. Table I. Manx Herrings (Mature). Weight of se | Date. Sex, &. muscle Dry Weight of | Percentage} Percentage substance.| weight. oil. of oil. of water. ) 1914 June 3/ Unripe ¢ 7-195 es 0-383 5:3 ie “ @| 4-790 Wy 0-173 3-6 June 25; Unripe ¢ 7-225 be 2-008 27:8 ss 93 2 8-264 She 2-205 26-8 Ss July 2/| Unripe ¢ 7-793 3-309 1-901 24-4 57-5 5 6 & 6-383 =e, 1-882. 29-5 ee July 9) Unrige’ “¢ 5-737 2-958 1-863 32:5 48-4 Ls 96 Q|- 6-688 3-644 2-339. 34:9 46-6 July 31 | Full 3 6-140 | 2-380 1-038 16-9 61-2 > “ 2 37077 TiS) 0-950 26-5 - 42:3 Aug. 22 | Full 3 5-258 2-442 1-246 23-7 53-5) ae : .; 2 5-453 - 2-162 0-964 17-6 62-3 Sept. 4 | Spawning g| 4-869 1-782 0-791 16:3 63-4 ? 3 4-972 1-980 0-902 18-1 60-2 Sept. 30 | Spent 5-669 2043 | 0-512 90 - 63-9 3 i Q 5-635 1-897 0-503 8-9 66-3 Table II. Welsh Herrings (Mature). Weight of | Date. Sex, &c. muscle Dry Weight of | Percentage| Percentage e substance.| weight. oil. |< of onl of water. 1914 Oct. 27 | Full 3 4-846 1 755 0-901 18-6 63-8 : Q| 6-175 1-904 0-698 11-3 69-1 Nov. 19 | Full 3 4-823 | 1-911 1-086 22-5 60-4 35 Q 6-399) | 2 1-993 0-758 11-8 68-9 Nov. 20 | Full 36 | 5-506 2-015 1-054 LOM 63+4 i Q] 5394 | 1-886 0-891 16-5 65-0 Dec Gy erull SO | SATA = e978 1-019 18-6 63-8 = 5 Q@;. 3-946 | 1-186 0-462 olde 69-9 Dec. 18 | Virgin ¢ 4-038 1-252 0-416 10-3 68-9 ‘s Spent @] 4-517 1-296 0-349 77 1913 Dec. 10 2-866 0-117 4-1 — J Full 3. 2-887 0-255 8-8 s 4-485 0-556 1-2 Dec. 19 | Spent 2 4-580 0-127 - 2-8 te us Q 3-594 0-313 8-7 SEA-FISHERIES LABORATORY. 219 Table III. Sprats and Immature Herrings. Weight of Date. Sex, &c. muscle Dry Weight of | Percentage| Percentage substance.| weight. oil. of oil. of water. Morecambe Immature Herrings. 1914 | May 24 | 2-766 ae 0-085 3-0 May 30. 4-857 | 0-202 4-] » 3108 0-101 3-2 | Morecambe Sprats, Immature. : May 29° Olea | ke | 0-205 9-5 | _ Mersey Estuary Sprats, Mature. Jan. 27 | | 5-230 1566 | 0-624 11-9 70-0 | ' | Table I gives the results of analysis of the summer-caught herrings sent from Isle of Man. In the earlier analysis the tissue was dried so as to facilitate the fat extraction, but weighings were not made. Male and female fish were usually taken from each sample, and the separate analytical results are given. But, on looking at this and the other tables, one sees no significant sexual differences, so that the means of the separate estimations of male and female fish have been taken in making the graph of the results. Table II deals with winter-caught herrings from the Welsh Bays, and includes a few results obtained in December of 1913. Table III gives only a few results of analysis of small herrings and sprats from Morecambe Bay and the Mersey Estuary. The herrings were all immature. The Morecambe Bay sprats (almost “whitebait ”) were also very small. The Mersey sprats were large and mature fish with gonads fast approaching maturity. They would probably have spawned in the spring of this year. The results of the tables, as regards the Manx and the Welsh fish, are represented in the graph which follows this. 220 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. ; ae ‘yosseA UST Avg WOAIeUIeD 4e Bos O49 Jo oinqvsoduIe4 UI UOTYVIIeA oY} syuosetded OUT] USxOIq ey], ‘ssuTIIEY Ys pues XUBPY JO sofOSNUr oY} UI Joye pUB Ye} UT SUOTIeIIeA O44 Jo UOTyeIUSeAdos [worydeixy) “T ‘OTT JOPVWUIIPC | J2QWAACN/ 1 4290420 _ saguiaydas | ysNBnHW LSM Ss _2uar ~4 ey, | oO f YS/\oM | ata Je IS YD{LIY2) 49g 05 OF 109 OF Tye 8HO % % SEA-FISHERIES LABORATORY. yp AN _We see, at once, some interesting relationships. It is clear, in the first place, that the variations of fat and water in the muscle substance of the fish are very closely complementary to each other.* The scale for the water contents has been reversed so as to show this relationship the more clearly. It is very apparent in the case of the Manx herrings, and much more so in the case of the Welsh herrings. As the fat-content increases, the water-content decreases, and wice versa. Now this is what one would expect. Carbohydrates are practically absent in most sea fishes; not even in the liver of well-fed herrings can these substances be detected.t The proportion of protein we may suppose, without evidence to the contrary, will tend to remain constant, so that it must be the water in the flesh which undergoes seasonal variations. This is not quite the case, as the graph shows, and there must be an appreciable. variation in the percentage of protein in the muscle substance. Nevertheless the variation is_ slight compared with that of the water and fat. To a certain extent the latter variation is similar, in its progress, to that of the sea-temperature. The latter cannot be exactly estimated, since the positions of the herring shoals were not ascertained when the samples were forwarded to us. Further these positions were, no doubt, variable, being nearer to the land at one time than at others, and this would make an appreciable difference in the temperature curve. The sea-temperature is, therefore, that at Carnarvon Bay Light Vessel, a position where the land hardly influences the annual variation of temperature, and which may be taken as generally representative of the water in the open sea round the south end of Isle of Man. There is a general relationship between * This confirms Milroy’s results of analysis of herrings made for the Scottish Fishery Board (see 24th Ann. Rept. Fishery Board for Scotland, 1905 (1906), Pt. III, pp. 83-107. Also 25th Ann. Rept., 1906 (1908), Pt. III, pp. 197-208. {See Stirling, 2nd Ann. Rept. Fishery Board for Scotland. Appdx. F., No. I. pp. 31-46, Plates I and IJ, 1883 (1884). ID, TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. fat-contents and sea-temperature, that is, metabolism is more intense the higher the temperature of the medium. Still the correspondence is not very close and we must find some other factor governing the variation in fat contents. This latter factor is the reproductive cycle. The period preceding the final maturation of the genital products is marked by a large increase in fat-contents of the tissues of the fish, and the act of spawning is accompanied by a fairly rapid decrease in this fat-contents. We might generalise these facts by saying that the fish accumulates fat in its tissues in order that this stored nutritive matter might be drawn upon for the maturation of the eggs and spermatozoa (in the herring the masses of these products are about equal): the protein ~ and fat present in the reproductive glands must come from somewhere. Now this generalisation cannot be quite true. The ratio of proteim to fat in the ovaries increases greatly as the genital organs ripen, and the increase in mass of these organs is, therefore, not altogether due to the transference of fat from the muscular tissues to the genital organs. Further the greatest decrease in fat-contents occurs after spawning, when this stored nutritive material is no longer required in the reproductive process. Yet again there must be some relation between the spawning operation and the general metabolism of the animal in order that this change may occur. Evidently there are two independent factors which influence the metabolism of the fish (1) the variations of sea temperature, and (2) the reproductive cycle. It is certain that even virgin herrings would show as well marked a rise and fall of fat- contents as do sexually mature fish, thus virgin plaice do certainly show marked variations in “condition” which corresponded with seasonal changes in the sea. But, all the same, the maturation of the ovaries and testes and act of spawning do certainly influence the general metabolism of the animals. The rapid decrease of fat at the time of spawning SEA-FISHERIES LABORATORY. 293 cannot be explained satisfactorily, for the final stage of matura- tion of the ova probably consists mainly in the imbibition of water from the general circulating fluids of the body. We have also to notice the very high proportion of fat in these herrings. In July over one-third of the wet weight of muscle from a female fish consisted of fat. The percentage of fat was 34-9, that of water was 46-6, and if we take Milroy’s average value of protein in the muscles of female herrings as 18% these numbers will add up to 99:5%. Comparing Table I with other published analyses of herrings we find the fat percentages very high, and this statement applies, to a less extent, also to the Welsh winter-caught herrings. Even the famous Loch Fyne summer herrings are less rich in fat than are these Manx ones, and one’s own experience in merely handling the fish agrees with these analytical results. The reason that the Loch Fyne herrings are less oily than the Manx ones is not that there is a corresponding difference in the planktonic food present in these sea areas. The summer crustacean plankton of Loch Fyne appears to be much denser than that round the south end of Isle of Man. The difference appears to depend on the fact that the spawning period of the Manx herrings occurs very shortly after the maximum of sea-temperature, so that the two factors, the increase of metabolism due to rise of temperature, and that due to the ripening genital products, work together in the same direction. Herring do spawn in Loch Fyne, but this appears to be rather exceptional—the shoals migrate out from the Loch in order to spawn. Only the temperature factor therefore operates in augmenting the fat-contents, and the temperature of Loch Fyne is, throughout the summer and autumn, lower than that of the sea to the south of Isle of Man. Some other points of interest in relation to the morphology and bio-chemistry of the fat tissues are being investigated, but the opportunity has not yet occurred to complete this work. P 224 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. REPORT ON THE PERIODIC SAMPLES OF SHRIMPS FROM THE MERSHEY ESTUARY. ‘ By T. Monacuan. Assistant Naturalist in the Fisheries Laboratory. These investigations were first carried out during 1912 and part of 1913, but a definite result could not be obtained on account of the insufficiency and irregularity of the samples sent to the laboratory. The result of the observations for 1912-13 were published in the Annual Report for 1913. This year the samples have been sent in with greater regularity so that the observations are more complete. The samples were taken from the Rock Channel and Crosby Channel, both of which may be included in the term ‘Mersey Estuary.”’ It will be noticed from the data (Table I) that, with a few exceptions, two samples were sent for examina- tion each month, thus enabling me to compare the variation in numbers of the sexes more closely than in 1913. As each sample was sent the shrimps were first sorted out in their sexes and then counted. They were measured (from the tips of the antennules to the extreme end of the tail) im millimetres, and averages were determined for each group. At the same time a microscopic examination was made of the eggs on the berried females, and four different stages of development were recorded, viz. :—(1) Segmentation com- mencing. (2) The eye forming. (3) Eye fully developed and the appendages just showing. (4) Yellow and black pigment formed, the larvae ready to hatch. A number of eggs in each stage were then measured. These results are, however, not yet completed, and are consequently not given in this Report. Table I shows the data as to samples obtained, and the average lengths of the berried females, non-berried females and males in each sample. Table II gives the percentages of berried females, non- berried females and males for each month. SEA-FISHERIES LABORATORY. 225 TABLE I. Percentage of Average Date, 1914. Females Number. Length carrying in mm. eggs. January 9th ...... Berried Females ......... 69-6 394 74-2 Non-berried Females ... 172 65-5 566 PCIE 8 vce 5 cialcsivenin's» 234 53-7 800 February 3rd_...| Berried Females ......... 50:8 230 63-0 Non-berried Females 222 53-5 452 WEEE Me oir ilu ns Sisenejeces 823 45-6 1,275 Mareh Sth ......... Berried Females ......... 50-2 204 65-3 Non-berried Females ... 202 54:4 406 SUT GS 9 fe 1,770 53-7 2,176 March 23rd _...... Berried Females ......... 82-3 234 67-5 Non-berried Females 50 63:5 284 RNY Ain cickenasanbsdecensne 253 52:0 537 Pepe LGR: .....0... Berried Females ......... 68-2 206 70-0 Non-berried Females ... 96 64-2 302 MMTEE HI bl iiy vo uitens ¢haarke 752 52-7 1,054 April 30th ......... Berried Females ......... 50-9 208 71-2 Non-berried Females 200 58-0 408 NAAIBAL .S evuse vac dnatiian ss 1,150 54:5 226 Date, 1914. | May 12th May 27th June 12th June 26th | ' July 9th July 27th TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. @eesecesen eeceosceccerve TABLE I.—Continued. Berried Females ......... Non-berried Females ... Berried Females ......... Non-berried Females ... Berried Females ........ Non-berried Females on Males Berried Females ........ Non-berried Females ... eee vescecoereecseereuse eeosesacveseereseeae eeereeccereesesseses eeeeeesrrereereesens Percentage of Females carrying ii-2 52-5 Average Number. Length in mm. 428 69-7 95 61-7 523 384 53-0 907 370 65-3 178 66-0 548 1,152 55:7 1,700 428 68-5 135 58-0 563 131 52°5 694 420 68-0 119 59.5 «539 47 51-0 586 238 71-0 215 66-7 453 170 56-5 623 426 72-2 132 65-2 558 30 50-2 SEA-FISHERIES LABORATORY. DoT TABLE J.—Continued. Percentage of Average Date, 1914. Females Number. Length carrying in mm. eggs. August 7th ...... Berried Females ......... 36-4 310 67-7 Non-berried Females ... 540 59-0 850 LA GS) Ue re 1,000 48-75 1,850 September 9th ...| Berried Females ......... 38-6 242 72-0 Non-berried Females 384 61-7 626 | Lb L32 2 700 52-0 1,326 September 30th...| Berried Females ......... 28-7 17 ai 73-5 Non-berried Females ... 300 66-7 421 RI in as ¥sikina bu deaa 355 55:5 776 October 15th ...| Berried Females ......... 24-1 74 77-0 Non-berried Females ... o72 69-7 346 oo 312 57-7 658 October 30th ...| Berried Females ......... 5-2 14 74-6 Non-berried Females ... 252 72°5 266 Re Beis: icietindeolain’ 700 56:5 966 November 17th ...| Berried Females ......... 1-8 8 76-2 Non-berried Females 434 68-5 442 CMLEY Ya suslosaneutedeaieny 94 55-2 228 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. TABLE I.—Continued. Percentage of Date, 1914. Females carrying eggs. December Ist...... Berried Females ......... 75 Non-berried Females ... Males gaat ee means December 16th ...| Berried Females ......... 24-6 Non-berried Females ...| — | | Milles f saxatenectucseeteccae 1915 January 26th ...| Berried Females ......... 69-2 Non-berried Females ... Moailes?) =. 2sfecuxsat acces al February 9th...... Berried Females ......... 63-6 Non-berried Females ... NEG sit 357) Scho asesteceece February 23rd ...| Berried Females ......... 73-9 Non-berried Females ... Number. 305 461 242 Average Length in mm. SEA-FISHERIES LABORATORY. 229 TaBLeE II. Percentage of Percentage of 1914. Percentage of Berried Non-berried Males. Females. Females. santary (1) ............ 29-2 49-2 21-5 Hebrnary (1) ............ 64-5 18-0 17-4 Lg ae 74:5 16-1 9-2 (1) a 72-8 15-8 11-3 OC) ae 58-9 30-6 10-4 OS eee 13-9 66-2 19-8 OO ee 16-5 54:8 28-6 een). -............| 54-0 16-7 29-1 September (2) ......... 50.1 17-2 32-5 Seer {2)......-........ 62-3 5-4 32-2 November (1) aa 17-5 1-4 80-9 Mecember (2) ......... 38-1 7:0 54-7 eer Considering now the data given in the tables we may make some conclusions with respect to the spawning period of shrimps in the Mersey Estuary. MALES BERRIED FEMALES JAN IFEB | MART APRILIMAY IJUNE JULY |AUG | SEPTI OCT | Nov I DEC Fic. 1. Variations in the relative numbers of males, non-berried females, and berried females in the samples examined during 1914, 230 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Fig. 1 is constructed. from the data of Table II. The percentages plotted are calculated on the whole sample, and these figures may be necessary for a detailed consideration of the life-history of the shrimp in local waters. I do not consider it further in the meantime. It will be obvious, however, that the variation in the percentage of berried females in the whole catch may not give us accurate information as to the period of maximum spawning, for it is based on the males and females. We have, therefore, to calculate the percentage of females that carry eggs and regard this (pro- visionally) as giving information as to the period of the year during which spawning is in progress. The data of 1913 and 1914 have therefore been combined, and some additional samples obtained in 1912 and 1915 are also included. The average percentages of female shrimps carrying abdominal eggs (berried females) have been calculated for each month. These new average percentages are as follows :— Nov. | Dec. | Jan. | Feb. | Mar. | Apr. | May. Fare July. | Aug. | Sept. | Oct. —_——<—<———_ | — | ———_———|\qe~_- me — qq _—| qe ~ wq“— so “€—e sic“ somo |—-—q— 3-2 | 23-4 | 66-3 | 62-7 | 70-7 | 68-9 | 74-9 | 69-0 | 73-4 | 41-9 | 33-7 7-6 Percentage of females Carrying abdominal ova 80 10 XI X Months Fic. 2. Variation in the percentage of female shrimps carrying abdominal eggs | during the years 1912-1915. SEA-FISHERIES LABORATORY. 231 We may consider these data more closely. The columns represent the data of the above table. The +’s represent the rough data smoothed by the method described by Dr. Johnstone in last year’s Report.* The mode, or time of maximal spawn- ing, is about 10th April, when about 75 % of all the female shrimps’ sample were found to be carrying abdominal eggs. The minimum proportion of berried females was observed in the samples taken during November, and this was the case in each of the years (1912, 1914 and 1915) in which samples were taken in that month. The proportion of berried female shrimps rises from the minimum towards the maximum rather more rapidly than it drops from the maximum towards the minimum. The spawning period seems, therefore, to be a prolonged one, and indeed it is common experience everywhere off the _ Lancashire Coasts to find berried females throughout the year. But it does not follow that it is so prolonged as the figure indicates for, having extruded its eggs, the female carries them for a considerable time attached to her swimmerets. It is not known how long a period the eggs require for incuba- tion, but according to EKhrenbaum* it may be four weeks (or even less) in summer, and four to five months in the winter. This is for the German North Sea Coast, and since the variation in the length of the incubation period depends on the temperature of the sea Hhrenbaum’s estimates will probably be approximately true also for the Mersey Estuary shrimps. During the spring and summer when the sea temperature is rising rapidly the proportion of female shrimps carrying abdominal eggs will, therefore, tend to be diminished by the hatching out of the larvae. This is, of course, also the case during the latter half of the year when the sea temperature * Rept. Lancashire Sea-Fish. Laby. for 1913, p. 83. *“ Zur Naturgeschichte von Crangon Vulgaris Fabr.” Mitth. f. Sektion f. Kusten-und Hochsee fischerei, Deutschen Fischerei-Vereins, Jahrgang, 1890. Berlin, 1890. 232 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. is falling, but the proportion will diminish far less rapidly than in the spring and summer, for the incubation period ‘during the month of minimum sea-temperature is at least four times greater than during the month of maximum sea- temperature. If we could allow for this variability the curve of spawning of fig. 2 would fall more rapidly than it appears to do, that is to say, the spawning period would not be so prolonged as the figure indicates. With the exception of Ehrenbaum’s well-known work no good investigation of the life-history of the North European shrimp has been made, so that we do not know how long the incubation period is, nor how it varies with the temperature. — Further, the stages of development are not so well-known that we can say exactly at what time, prior to the time of observation, an egg was spawned. By counting only new-laid ova we could, of course, make an estimate of the variations in the spawning time, and it was with this object that observa- tions of the stages of embryonic development were made. But it will be necessary to carry on these observations, first of all, in an aquarium. According to Ehrenbaum there are two spawning periods in the year with respect to the shrimps in the Heligoland Bight, the first from the middle of April to the beginning of June, and the second during October and November. It is not impossible that there may also be two chief spawning periods in Liverpool Bay, but the observations made so far do not appear to show that this is the case. SEA-FISHERIES LABORATORY. 233 [Percy SLADEN MemoriaL TRust RESEARCH. | STUDIES OF CERTAIN PHOTO-SYNTHETIC PHENOMENA IN SEA-WATER. I—SEASONAL VARIATIONS IN THE REACTION OF SEA-WATER IN RELATION TO THE ACTIVITIES OF VEGETABLE AND ANIMAL PLANKTON. Il—THE LIMITATIONS OF PHOTO-SYNTHESIS BY ALGAE IN SEA-WATER. By BENJAMIN Moore, EpMuND BrypGES RUDHALL PRIDEAUX, and GEORGE ANDREW HERDMAN. (From the Marine Biological Station, Port Erin, Isle of Man, and the Laboratories of Bio-Chemistry and Physical Chemistry, | University of Liverpool.) Our attention was first drawn to the seasonal variations in the alkalinity of sea-water recorded in this paper, and to the remarkable degree to which green algae can reduce the hydrogen-ion concentration of sea-water in which they are grown, by preliminary observations made by Moore, Edie and Whitley at Port Erin in the Spring of 1912. A severe epidemic of a disease, characterised by large areas or rounded spots of ulceration on the skin, had killed a considerable number of the plaice used for spawning purposes in the pond attached to the Fish Hatchery, although this pond had an abundant daily supply of fresh sea-water. At the request of Professor Herdman the water of the pond was examined chemically, and the only important fact discovered was that it was much more alkaline than the water of the Bay. At the same time the pond-water was green in colour from the presence of floating mono-cellular algae, and a minute green flagellate Infusorian in great profusion, while, 234 TRANSACTIONS. LIVERPOOL BIOLOGICAL SOCIETY. as usual at this time of year, the sea-water in the Bay was almost free from green organisms. It was also observed that the water of the Bay was less alkaline to phenol-phthaléin than - it had been on previous occasions when titrated in connection with other work. For these reasons, it was determined to follow up the reaction of the sea-water at intervals throughout the round of the year, and also to make a series of observations as to the speed with which green organisms added to a confined volume of sea-water increased the alkalinity, and the limit to which the alkalinity could be so raised before photo-synthetic action ceased, or the organism perished. [.—SEASONAL VARIATIONS IN THE ALKALINITY OF SEA- WATER. Certain of the observations were carried out by Moore, Edie, and Whitley in August, 1912; the remainder were made by the present authors since that time. Three types of observation were carried out :— 1. Titrations were made of a measured volume of sea- water to two coloured indicators, viz., phenol-phthaléin and methyl-orange using centi-normal solutions of hydrochloric acid. These titrations give the required amount of alkali to alter the hydrogen-ion concentration from approximately P,,, 10-* to P,, 10-*’ and indicate quantitatively what has been termed qualitatively by Sérensen? the “ Buffer” effect of the dissolved alkali-acid, or amphoteric, salts, and by Moore and Wilson® the “‘ Reactivity ” of the solution due to these same amphoteric solutes. This determination is one of great importance in estimating the characteristics of a physiological solution, since upon it depends the protective action against large variations in hydrogen-ion concentration. 2. The hydrogen-ion concentration was determined by the colorimetric method introduced by Friedenthal and Salm! and amplified and improved by Sérensen?:> and Palitzsch.* SEA-FISHERIES LABORATORY. 235 3. The hydrogen-ion concentration was also determined electrometrically on several occasions, although this is a difficult estimation with sea-water on account of nearly all the “ Re- activity’ or “‘ Buffer”’ action being due to bi-carbonate of magnesium. - This was done to give a control to the colorimetric method, and on the whole the agreement was found to be excellent, when Sérensen’s correction for the salt error had been applied. A somewhat exclusive importance has been attached in recent years to a determination of the hydrogen-ion concentra- tion of a physiological fluid such as blood-serum or sea-water. Important as this figure may be in indicating the actual reaction of the fluid, it gives no idea of the resistance of that reaction to change when alkali or acid is added, and from the point of view of the protective action of the physiological fluid to any living organisms of which it may form the environment or “external medium.” This resistance to change is an all-important factor. It.is fashionable to-day to look askance at earlier methods of titration to coloured indicators, and to say that these only give the amount of acid or alkali necessary to bring the solution to an arbitrary level of hydrogen-ion concentration at which the indicator employed changes colour, and so titrations can furnish no indication of the hydrogen-ion concentration of the solution in its original condition. While this is true, and while we may now smile at the old-time results with coloured indicators which led to the same solution being written down as both acid and alkaline, and gave rise to such phrases as the “acidity ’ and “ alkalinity ”’ of the blood, it does not follow by any means that titration of physiological solutions is based on error and should be abandoned. In order to appreciate the properties as to alkalinity and acidity of a solution two things are essential, (1) to know the actual hydrogen-ion concentration and (2) the amount of alkali or acid required to be added to 236 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. shift the hydrogen-ion concentration between two definite values. The first of these figures can be determined either electro- metrically, or by the more rapid and convenient colorimetric method with indicators, which is based on the electrode method. The second may be obtained by titrating to the change points of colour of two different indicators with a sufficient range between them. The second figure is no less important than the first, for while the first gives the hydrogen-ion concentration with which the living organism is actually living in equilibrium, the second — shows the amount of production of acid or alkali by the organism which is compatible with a given change in hydrogen- ion concentration, and hence demonstrates the degree of protection that the medium is capable of affording to the organism. All living organisms are extremely sensitive, both in their actual viability and also in the degree of their physiological activity to small changes in hydrogen-ion concentration, the growth and development and metabolic activity being all profoundly affected by comparatively slight changes. aay? The resistance to change in ionic concentration has been referred to as the “Buffer” effect by Sérensen, and was called the “ Reactivity ” of the solution by Moore and Wilson to distinguish it from the “ Reaction,” which is quite a different thing, Although the expression “ Buffer Effect ’” has been used a great deal in late years, few attempts have been made to obtain a quantitative expression for the “ Buffer Effect ” or ‘* Reactivity ’’ although it was determined for blood-serum in 1906 by Moore and Wilson,® when the interesting result was obtained that it corresponds here to the total isotonicity figure of the serum as determined by the freezing-point method. It was shown by these authors that the “ Reactivity ” can SEA-FISHERIES LABORATORY. 237 ‘be obtained by taking the algebraic differences in the titration figures to phenol-phthaléin, and to methyl-orange, or di-methy1- amido-azo-benzol. When a solution containing acid-salts of carbonates or phosphates, or amphoteric electrolytes, such as proteins, has acid or alkali added to it there is a middle zone throughout which the hydrogen and hydroxyl-ionic concentrations vary very slowly, and outside this on either side there is abrupt rise and fall. By choosing two coloured indicators, one near each end of the range of such indicators, the breadth of this zone, or in other words, the “ Buffer Effect’ or “‘ Reactivity,” can be measured with fair accuracy. This is the figure which is determined by our titrations. Another important effect which is given by measuring the variations in titration to a given indicator (suitably chosen so as to be sensitive to the varying factor) is the amount of variation in a metabolic product such as carbon-dioxide from one time or condition to another. Such an activity cannot be followed by determining hydrogen-ion concentration alone, although it is undoubtedly interesting to observe (as has been done below) the variation in the hydrogen-ion concentration corresponding to a given change in carbonic-acid concentration, arising from photo-synthesis or respiration. But in a solution such as blood-serum or sea-water quite an appreciable variation in titration to phenol-phthaléin due to change in concentration of dissolved carbon-dioxide may occur, with scarcely a detectable change in hydrogen-ion concentration. A ten-thousandth normal solution of a caustic alkali is much more alkaline than blood serum or sea-water, but one e.c. of centi-normal acid added to 100 c.c. of this caustic alkali solution already suffices to remove the difference and makes the solution neutral, while such an addition scarcely appreciably affects the reaction of the serum or sea-water. This important effect was pointed out by Moore, Roaf and ‘i 238 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Whitley> in 1905, when investigating the effects of acid and alkali added to sea-water upon growth and development of Hchinus eggs. These authors state that “‘a solution of the mixed phosphates or carbonates in which there is an approximate balance between the concentration of hydrogen- and hydroxyl- ions such that these concentrations are nearly equal, cannot, however, be regarded as neutral in the same sense as distilled water is neutral, or as being acid or alkaline in the same sense as a solution containing only free acid or free alkali can be regarded as being acid or alkaline. | ‘Nor will such a solution of phosphates and carbonates, as is present in blood plasma, or sea-water, have a similar action upon living cells to either distilled water or a neutral solution of such salts as sodium chloride of equal osmotic concentration. | “Therefore blood plasma, and to a less extent sea-water, possess, on account of the mixed phosphates and carbonates which they contain, a steadying action upon variations m the concentration of the hydrogen- and hydroxyl-ions. When acid or alkali is added to the plasma, instead of there occurring a corresponding swing in the concentration of the hydrogen- and hydroxyl-ions, there takes place an alteration in the equilibrium of the ions of the phosphates and carbonates, which neutralises, in great part, the hydrogen- or hydroxyl-ions added, and prevents the plasma becoming markedly acid or alkaline. Without such a controlling action the life of the cells would be rendered impossible, for, as our experiments show, the living cell is most sensitive to even small variations in either hydrogen- or hydroxyl-ion.”’ The action of small variations in the hydrogen- or hydroxyl- ionic concentrations of plasma or other “ external media ”’ is only to-day gradually becoming recognised in the government of respiratory activity and other fundamental physiological functions, and hence attention may perhaps be drawn to the SEA-FISHERIES LABORATORY. 239 fact that the above views were expressed in the Proceedings of the Royal Society nearly ten years ago, and that shortly thereafter the “‘ Buffer” effect in the plasma was not only recognised, but estimated by Moore and Wilson, and published in 1906 in the first volume of the Bio-chemical Journal. The following tabulated statement gives a record of the titration to phenol-phthaléin and to methyl-orange of the sea-water freshly drawn in the neighbourhood of Port Erin, Isle of Man, and in the Irish Sea, at various seasonal periods during the years 1912-14. The titrations were made by adding four drops of 0-5 per cent. solution of phenol-phthaléin (by measure about 0-13 c.c.) to 100 c.c. of the sea-water, and then titrating with N/100 hydrochloric acid till the pink colour was just on the point of disappearing. Two points are noteworthy, first that throughout the long series of observations the fresh sea-water was invariably alkaline to phenol-phthaléin indicating a value of hydrogen-ion concentration lying above P,, 10~*, or considerably higher than the values obtained by other recent observers such as Sdérensen and Palitzsch*-4 in sea-water from other regions, and, secondly, that there is a seasonal variation, the hydrogen-ion concentra- tion being higher in winter, and decreasing in spring and summer. Although the latter variation is small, the differences in the amount of acid required to reduce the level to the neutral point of the phenol-phthaléin indicator show, when applied to the vast volumes of sea-water affected, great photo-synthetic activities of plants, and corresponding animal activities in oxidation. The results indicate a great crop, or annual variation in the water, corresponding to the seasonal variations on the land in the round of the year. The photo-synthetic crops in the sea-water, reckoned as carbohydrate produced, are similar to those on a land surface, and amount to several tons of carbohydrate per acre; this then forms the food for the floating animal-life of the sea. Q itt Py — 4 i i vi . hdl . tt 240 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Titration Values of Sea-Water at different Seasons of the Year. ‘Buffer ’’ or | No. of c.c. of N/1O0 HCl to. |Rffect between | N/100 HCl i : required to pate, | Pimeg,ef Collection | petri 100 Pgs 10-* tand| nents “phthalem. |e 10-4 x =| C:e teas 10-4N. orange. SuMMER 1912. Aug. 8—|Life-boat Slip ..................08. 2-25 c.c — | — », 12—Aquarium Tank ...........:...... 2-2 22:3 24:5 c.c. » 19—jAquartum Tank .....0.......<0+.- 2:0 = — » 24— Aquarium Tank ............c..00. 2-2 — = »» 90—Aquarium Tank ...............64: 2-2 — = Sept. 3—j|Aquarium Tank .................. 1-8 = aE » %—=|Aquarium Tank .2,2.0:2.A5RRER JAN. FEB. ROLL Lb echnick aie! teem ited Lior SSR EERO ONALO RAR Rees BISSRRRRKRARABRLSS SLIT ITSSSHSVSee Decrees Fanrenueir. a SEA-FISHERIES LABORATORY. 247 lay below 1-0, while in February it had risen to 1:2 to 1:3, on April 8th it had reached 1-6, while the sea-water temperature had not moved till April 19th when it had only risen by 1° F. If the alkalinity at Life-boat Slip be taken from April 13th till April 20th, 1913, the average is 2-4. Thus, at the point where the temperature is only beginning to rise, the alkalinity has nearly reached a maximum. The alkalinity of April 20th is 3-0, compared with the figures 3:2 and 3°3 on June 14th and 15th, 1913. On the former day the temperature is only 1°F. above the average March temperature, on the latter days the temperature is 7° F. higher. The observations in June, July and August, 1914, show that there is no increase in alkalinity with temperature only. It is also to be noted that the great Spring outburst of diatom activity appears before there would be time for any effect of rise of temperature. Although the first upward movement in temperature of.the sea-water and the enormous increase in vegetable plankton lie close together in time, it is to be remembered that the physical cause must have a latent period ahead of the biological effect. The first movement of temperature is very slight, and when it is remembered that it must first stimulate growth, and then many succeeding generations arise before there is a massive increase in the vegetable plankton, it may probably be said that if the vegetable plankton (and the increased alkalinity) were due to the increased warmth of the water, then these would be found at least a week later in time than the first rise in temperature. There is, in fact, no such accordance observable, and it is far more probable that the increased length of day and altitude of sun, supplying a rapidly-increasing amount of solar light energy, are the potent causes in producing the diatom outburst and accompanying rise in alkalinity of sea-water. If the actual variation in alkalinity observed be taken as a rough index of photo-synthetic activity, and as 248 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. an approximation the carbon be assumed to be all converted into carbohydrate, then some calculation may be given to demonstrate what such figures mean. : The amount of 8-8 milligrams per litre of carbon-dioxide changed corresponds to 2-4 milligrams of organic carbon, and this is 6 milligrams per litre of carbohydrate, synthesised, or 6 parts in a million of the sea-water—at first sight a ridiculously small quantity, but it is spread out in the sea. Six milligrams in a litre gives 6 grams in a cubic metre of sea- water. If the change by photo-synthesis occurred to only a depth of one metre, this would mean on a square kilometre of sea surface 6 millions of grams, or 6,000 kilograms of carbo- hydrate synthesised. But the change occurs for a distance down in all probability of some hundreds of metres instead of one metre. Convection currents, winds and tides, mix the water thoroughly up for many metres deep. The careful observations of Sven Palitzscht have proved that the alkalinity of the sea-water does decrease as the depth from the surface increases, but there is scarcely any appreciable decrease at 100 metres, and even at 400 metres the decline is usually still small. ’ - This decline in alkalinity at greater depths is of interest as an evidence of photo-synthetic activity and its relationship to alkalinity of the water. Its cause is probably three-fold, first the photo-synthetic activity decreases with depth as the intensity of the light diminishes in traversing the water ; secondly, with increasing depth there is less admixture by currents with the more alkaline water due to photo-synthesis of the upper layers; and thirdly, organic débris of plant and animal in descending becomes oxidised, and these oxidations again set free carbon-dioxide which lowers in turn the alkalinity of the water. © If it be supposed, for the moment, that photo-synthesis SEA-FISHERIES LABORATORY. 249 did not exist, then the whole depth of the sea in process of ages of time would come into complete equilibrium with the air, and the dissolved carbon-dioxide would be the same in the upper layers and in the depths, and accordingly there would be no drop in alkalinity in the deeper zones. | The slow drop in alkalinity demonstrates an effect of photo-synthesis, but it is so small within the upper portions that it may unquestionably be taken that photo-synthesis, aided by convection currents, has the full alkalising effect for at least 100 metres from the surface. This supposition leads to a photo-synthetic effect of 300,000 kilograms of dry organic matter such as carbohydrate per square kilometre. Expressed in English measurement this amounts to about two tons of dry organic matter per acre, and since vegetable crops do not contain on an average more than 20 per cent. of dry organic matter, this ocean crop corresponds to at least ten tons of moist plant organisms per acre. The factors entering into such a computation, in the present fragmentary state of our knowledge, are of course vague, but the above is certainly a minimum, and it becomes obvious that the sea has annually a vast crop of green-plant organic matter comparable to that growing in the fields on land. Another interesting point arising from these alkalinity determinations is that the degree of photo-synthesis and the corresponding weight of ocean crop is probably much more abundant nearer to the littoral. It is frequently observable in the table, that water taken along shore is more alkaline than that taken from on board a vessel three to five miles from shore. Observations at great distances out at sea during the various seasons are most desirable but difficult to obtain. 250 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. DETERMINATIONS OF THE POTENTIAL OF HyprRoGEn-Ion CONCENTRATION. This is the modern method of expressing the state of acidity or alkalinity of a solution at any given moment as distinct from the amount of added acid or alkali necessary to move the reaction to a given position. It is a static thing while the “ Buffer Effect’ or “ Reactivity” is kinetic or dynamic. It might be termed the reaction of the fluid as distinct from its reactivity. The one expresses present position, the other ease or difficulty of moving into another determined position. It has been known for some time that sea-water is an alkaline solution: this is stated by earlier observers such as V. Bibra,® Guignet and Telles’? and Tornoe. Later Natterer® observed that water from the eastern part of the Mediterranean was coloured red by phenol-phthaléin, and, unconsciously foreshadowing qualitatively Sdrensen’s later colorimetric method, was able to note by comparing the depths of shade of pink that the deep sea-water was less alkaline than the surface water. xt | Ruppin!® was unable to note any pink coloration in water from the North Sea and Baltic, but as the water was drawn in 1909 from points near Kiel, the water may well have been contaminated sufficiently artificially to reduce slightly the alkalinity and throw it below the value necessary to give any — colour reaction with phenol-phthaléin. _ Loeb?! found the water of the Atlantic Ocean at Wood’s Hole to give a pink colour with phenol-phthalém, while the water of the Pacific Ocean at Pacific Grove was less alkaline and gave no pink colour with this indicator. ; Correlated with this, Loeb found the interesting fact that unfertilised ova of Strongylocentrotus developed partheno- genetically to a much greater extent in the more alkaline water SEA-FISHERIES LABORATORY. 251 of the Atlantic than on the Pacific Coast. Addition of small amounts of alkali to the water of Pacific Grove, so as to bring its alkalinity up approximately to the level of the water of _ Wood’s Hole, caused the parthenogenetic cleavage to increase. This is in agreement with the much earlier discovery of Loeb? that slight additions of alkali aided parthenogenesis and hastened cell-division. The same result on increased rate of cell-division was obtained by Moore, Roaf and Whitley® in fertilised eggs of Echinus esculentus. This suggests an interesting thought as to the chemical causation of the outburst of animal life in the spring. It may be that it is the slow increase in alkalinity of the sea-water caused by the photo-synthetic activity of the increasing daylight upon the algae which reacts upon the animal life and causes increased cell division, as also that sexual activity (at least im the lower forms) which produces the shedding of ova and sperms. In any case it is quite clear that the increased alkalinity of the spring will be favourable to parthenogenesis, as also to cell-division after fertilisation. The earliest observations by the hydrogen-electrode method upon the hydrogen-ion concentration of sea-water appear to be those made by Cottrell upon the suggestion of Loeb}? in the water of San Francisco Bay. The experimental difficulties of such an observation as pointed out lately by Palitzsch are very great and were apparently not overcome by Cottrell, who found sea-water less alkaline than the neutral point (P,, 10-™). The Danish observer, W. E. Ringer!*, was the first to overcome substantially most of the difficulties of the task lying in the way of obtaining hydrogen saturation of the electrodes and fluid without displacing carbon-dioxide, and so in the process of determination altering the alkalinity. This observer made a long series of observations of the water of the North Sea, the Zuidersee, and Bémmel-fiord, and found it to 252 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. vary slightly but to lie within the limits P,, 10~"* and P,, 10-4. The latter value lies well within the phenol-phthaléin range. It is to the Carlsberg school of workers, and notably to Sérensen and Palitzsch?:3:4, that we owe, first, extensive and careful determinations in various seas of the hydrogen-ion concentration of sea-water, secondly, a painstakmg and elaborate investigation of the so-called “ Salt Error ” introduced into colorimetric observations by the action of the neutral salts of sea-water and chiefly the sodium chloride upon the coloured indicators. Thanks to the labours of these observers, the deviation caused by the salt is now accurately known: it has been established that under given conditions this deflection in the colorimetric results 1s constant, and hence can be allowed for by deductions placed on record in the Sdérensen-Palitzsch tables. Accordingly, a rapid and accurate method is placed in the hands of observers for estimating the hydrogen-ion concentration of sea-water. This colorimetric method of Sérensen has therefore been mainly used by us, but we have on certain occasions controlled it, by using the electrometric method with the electrodes. We have found concordance good when allowance is made for the “Salt Error” by use of the tables. The Sérensen method has now become so well known that it need not be described in detail,* so its principle only will be pointed out. Mixtures in varying proportions of two solutions, one with a higher the other with a lower hydrogen-ion concentration, are prepared in a series of test-tubes. The hydrogen-ion concentrations of these mixtures have been directly determined once for all by Sérensen and tabulated so that they can be referred to, and have also been plotted in * See Walpole, Bio-Chemical Journal, Vol. V, 1911, p. 207, and Vol. VIII, 1915, p. 628. SEA-FISHERIES LABORATORY. 253 eurves by Walpole,!® which may be utilised instead of the tables. Minute directions are given by Sorensen for the preparation of the stock solutions from which the mixtures are made up at the time of each experiment. In making up the mixtures matters are so arranged that the volume of the comparison mixture is always 10 c.c. For example, calling the solutions A and B, a series of mixtures is made containing, say—(1) Nine of A and one of B, (2) Hight of A and two of B, (3) Seven of A and three of B, and so on. In an additional tube 10 c.c. of the sea-water is taken of which the hydrogen-ion concentration is to be determined. All the tubes are placed in a special test tube rack so that they slope at an angle of 45° to the horizontal and rest on a white surface such as milk- glass. An equal and definite volume of the coloured indicator to be used is now added to each tube and the tube containing the sea-water to be analysed for its hydrogen-ion concentration is carefully compared as to tint of colour with each tube, and that one selected with which it most closely matches. After a rough approximation a series of tubes lying closer in their steps of variation can be chosen. That one which matches closest is taken as having the same hydrogen-ion concentration as the sample to be tested. In the case of sea-water, after identification and establishment of the hydrogen-ion potential by table or chart, the salt error deduction must be made. Naturally, different coloured indicators, and different comparison mixtures, are selected according to the level of hydrogen-ion concentration of the solutions to be tested. In our experiments on sea-water about Port Erin we have always found phenol-phthaléin appropriate as a coloured indicator, and the most useful comparative solution-mixture that known as “borate mixture”’ and hydrochloric acid. The “ borate mixture ”’ contains 12-404 grams of boric acid and 100 e.c. of normal caustic soda in one litre, and was carefully prepared according to Sorensen’s directions. The hydrochloric acid 254 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. solution added in varying proportion to this is a deci-normal solution of hydrochloric acid. On one or two occasions the phosphate combination was utilised, one ingredient of this is one-fifteenth normal primary potassium phosphate (acid phosphate), the other one-fifteenth normal secondary sodium phosphate (alkaline phosphate), mixed as above described. The following table gives the results of the observations :— 7 Seasonal Variations in Hydrogen Ion Concentration of Period of Year. November. 1O12y sos-cee a. December, VOR2n eves scence Hebruary, TOUS cece eeeaee April, 1913 “ck. ocean eae May. L918 ae05 seccenmeaeenes June; 19S sehr eae July, LOLS Aisin eeaeen cere Sea- Water. Potential of Hydrogen Ion Concentration colorimetrically determined with Borate and Hydrochloric Acid mixture, and corrected for Salt Effect (Sorensen). — 8-13 — 8-10 — 8-20 — 8°37 — 8-31 — 8-30 Potential of Hydrogen Ion Concentration, electro- metrically determined by hydrogen electrode method. — 8-20 — 8-16 — 8-20 — 8-40 ~ 8:35 The results of the experiments demonstrate two things, first that the hydrogen-ion concentration for the Irish Sea in the neighbourhoods tested is fairly low indicating a corre- spondingly higher alkalinity ; secondly that the alkalinity is ancreased in the spring and summer months. The variations are too small to warrant any further deduction than this. Such observations are excessively difficult to carry out, but their trend is sufficient to confirm the results of the titration experiments, namely that the alkalinity of sea-water is lowest in winter and increases in the spring. SEA-FISHERIES LABORATORY. 255 I].—Tue Limits or Puoto-SyntHesis By ALGAE AS THE ALKALINITY Duk To THEIR ACTION INCREASES. As was stated at the outset, our attention was first drawn to the alkalinity question in relationship to photo-synthesis, by the plaice disease in the spawning tank, and the presence in this pond-water of an immense number of floating mono- cellular algae with which minute green flagellata were also present in great abundance. It was found then, in April 1912, that the alkalinity of the pond-water was very considerably higher than natural sea-water. The diatom outburst of the Spring had not yet appeared and the alkalinity of the Bay water was low; on the other hand it was found that the alkalinity of one portion of the pond (which is separated into two parts by a wall and sluice) was such that it required 3-3 c.c. of centi-normal acid to neutralise 100 ¢.c. to phenol-phthaléin, and the other portion of the pond- water required 3-8 c.c. of centi-normal acid in a similar titration. As may be seen by comparing with the table given in Section I, these figures are above normal for fresh sea-water, and the pond-water was therefore in a pathological condition with regard to alkalinity. The experiments of Loeb, and of Moore, Roaf and Whitley mentioned in Section I., indicate that such alkaline water would have a stimulating and in-co-ordinating action upon cell-division. In fact the amount of increased alkalinity would be just that which increases cellular activity. Given a provocative cause of any kind such as a bacterial infection, the conditions therefore were just’ those which would aid such an ulcerative disease as that from which the plaice were dying. The pond-water was examined again for alkalinity and contrasted with the alkalinity of freshly taken water from Port Erin Bay in November, 1912; the results are shown in the following statement :— R 256 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. November 17th, 1912. Took three samples of water (A) from Pond I. (large spawning pond), (B) from Pond II. (smaller pond at West end beyond sluice), (C) from open sea at Breakwater. A sample of 100 c.c. in each case was titrated with centi-normal hydrochloric acid, with phenol-phthaléin as indicator (4 drops of 0-5 per cent. phenol-phthaléin in the 100 c.c.) with the following results :— (A) Pond I. oe a Required 1-9 c.c. (B) Pond IT. oe re = 1-9 ere: (C) Breakwater ... ae a 0-9 c.c. Thus, the water from the two ponds was approaching the spring alkalinity while the Bay water was at winter level. The. ponds were shortly afterwards emptied, the walls disinfected and algae removed as far as possible, and then refilled. Examined again in February, 1913, the alkalinity — was found to be the same as that of the “ Bay” water. Samples of ‘‘ Pond” water and of “ Bay” water taken then and titrated alongside each other as before, gave in each case an alkalinity represented by 1-3 c.c. of centi-normal acid. It hence became obvious that the increased alkalinity was caused by the algal growth causing photo-synthesis and the conversion of bi-carbonate into alkaline normal carbonate. The same effect was demonstrated in the determination of the hydrogen-ion concentrations in the “ Pond ” and “ Bay ” waters by Sérensen’s method. At this earlier period the sea- waters were contrasted with mixtures of the two phosphatic solutions, because the alkalinity was not expected to run so high. The two pond-waters in November, 1912, matched at 9-8 c.c. of alkaline phosphate to 0-2 c.c. of acid phosphate, while the ‘‘ Bay” water matched it at 9-7 c.c. alkaline phos- phate to 0:3 c.c. of acid phosphate, After correction for salt effect these values correspond to Py, 10°" for the “ Pond” water and P,, 10~** for the “ Bay ” water. SEA-FISHERIES LABORATORY. 257 It thus became of interest to determine the limits to which, under the most favourable circumstances of a smaller volume of sea-water exposed to light in presence of green algae, such increase of alkalinity and depression of hydrogen-ion concentration could be carried. The fact that sea-water is usually alkaline to phenol- phthaléin is sufficient to show that, save for an infinitesimal amount of dissolved carbon-dioxide requisite to keep up potentially the partial pressure of carbonic acid anions in the water, all the carbonic acid is present as a bi-carbonate along with a small fraction as normal carbonate. When photo-synthesis commences this small potential amount of carbon-dioxide is synthesised into organic carbon compounds, this change upsets the equilibrium and so the bi-carbonate (of magnesium) breaks up yielding a supply which restores the tension in solution of carbonic acid. The question is, how far can this process go before the rising alkalinity destroys the algae, and the answer is beautifully given by the following experiments. It has already been shown by Nathansohn"‘ that aquatic plants can assimilate perfectly in water containing bi-carbonates, but not in water containing only normal carbonates; but as far as we are aware, the exact point of stoppage in a natural mixture of carbonates and bi-carbonates such as is present in sea-water, or the value of the hydrogen-ion concentration at the end of the process, have not hitherto been determined. The result is imteresting—the photo-synthetic action stops at the precise point where all the available bi-carbonate has been converted into carbonate, and the hydrogen-ion concentration has then reached the surprisingly low value indicated by the potential P,, 10~°". Reperiment 1. August 29th, 1912. A sample of sea-water was taken at the “ Life-boat Slip” and this was carefully neutralised by adding the calculated amount of centi-normal “7 258 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. hydrochloric acid as estimated from a titration of its alkalinity. A volume of 2,000 c.c. was measured off and to this a quantity of green algal confervae found growing in a vessel in the laboratory was added. The amount of moist algal matter added was not estimated, but it certainly did not exceed half a gramme. The algae were added at 12.15 p.m. on August 29th and the whole was placed in a wide-mouthed bottle, which the mixture just filled. The bottle was stoppered and left m the open air exposed to daylight. A sample was taken off and tested as to alkalinity on August 30th at 9.15 p.m.; the alkalinity had risen so that 3-8 ¢c.c. of centi-normal acid were required to neutralise 100 c.c. to phenol-phthaléin. Thus the alkalinity as a result of photo-synthesis in such a restricted volume had already risen above anything naturally found in sea-water. 7 A second titration was carried out on September Ist at 10.20 p.m., that is about 82 hours from the commencement of the experiment, when the alkalinity was found to have increased enormously, 9-7 c.c. of N/100 acid being required to neutralise 100 c.c. of the solution to phenol-phthaléin. A third titration was made at 6 p.m. on September 2nd, the value of the alkalinity to phenol-phthaléin had now reached 11-1 cc. A fourth titration at 9-20 p.m. on September 3rd gave 11-7 c.c. ; a fifth, on September 4th at 10.20 p.m. gave 12-3 c.c.; a sixth, on September 5th at 8.40 p.m. gave 12-2 c.c.; and a seventh, at 6 p.m. on September 6th gave 11-4 c.c. A naked-eye examination during the experiment showed that the algae remained green, and apparently the growth was healthy until September 4th when the alkalinity had reached itsmaximum. From this point onward the growth commenced to turn brown and die, and on September 6th, when the alkalinity had commenced to fall off again, the green organisms were evidently dead. The drop in the figure was hence probably due to bacterial decomposition. -SEA-FISHERIES LABORATORY. 259 The figure obtained at the point of maximum alkalinity in the above experiment bears an interesting relationship to the figure for the total alkalinity of sea-water, as shown by the titrations to methyl-orange in the first table of the preceding section of the paper. Since the turning point in colour of methyl-orange lies above the hydrogen-ion concentration of carbonate mixtures, it gives the total content of the sea-water in all bases, including magnesium and calcium oxides, and the value for such bases lies between 24 and 25 c.c. of centi-normal acid for 100 c.c. of sea-water. Now the maximum value of the alkalinity at which the algae cease to photo-synthesise, and die, corresponds to 12-3 c.c. of centi-normal acid or exactly one half of the total alkaline bases. The algal cells behave like a sensitive colour-indicator and cease to functionate precisely at the point where all the bi-carbonates have been converted into normal carbonate. Up to this point, they have, in presence of light actively converted carbon-dioxide into organic carbon compounds and have flourished ; at this point the gradient of alkalinity begins more rapidly to rise and they are killed off. Experiment 2. At 3 p.m. on November 17th, 1912, a wide-mouthed bottle holding approximately two litres was filled with sea-water at the Life-boat Shp, about three grams moist weight of Ulva enteroides was added, and the bottle, stoppered, was exposed to the daylight on the wall of the Fish- spawning pond. A sample of the water was titrated at 10.45 a.m. on November 18th, and required 2-4 c.c. of centi-normal acid to neutralise to phenol-phthaléin, the neutralisation figure of the “ Bay ”’ water being at the same date 0-75 c.c. Thus, in a winter day when the exposure to daylight could not have exceeded four hours of diffuse daylight, the photo- synthetic activity had brought the alkalinity of this confined volume of sea-water from the mid-winter to the spring level. Titrated again at 4 p.m. the alkalinity had increased to 5-9 e.e. ; ? 260 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. the day was bright but without much sunshine, so that on a winter day in about five hours interval the alkalinity had increased, as a result of photo-synthesis, from 2-4 c.c. to 5-9 c.c. The bottle and the contained algae was carried over to Liverpool, being exposed on board the steamer, and later on the roof of the laboratory, to such illumination as was available. It was titrated for a third time on November 22nd at noon. The alkalinity had reached 7-4 c.c. of centi-normal acid per 100 c.c. or more than double the maximum alkalinity in late spring or summer in the sea. | The hydrogen-ion concentration was also determined in this experiment after about four hours’ exposure in November daylight ; it matched at 9-9 c.c. of alkaline phosphate in the phosphatic mixture, the normal sea-water standing at 9-7. Next day at 4 p.m. it was more alkaline than the full strength of the alkaline phosphate, so that the hydrogen-ion concentration was below P,, 107°". Experiment 3. February 13th, 1913. A sample of sea- water was taken at 12-40 p.m. and a small quantity of green seaweed (Ulva enteroides) was placed in it. A titration of the water as then taken gave 1:3 c¢.c. of centi-normal acid per 100 c.c. The bottle containing about 2,000 c.c. of sea-water, and being quite full and stoppered, was exposed to the daylight. February 14th, at 11.30 a.m., the seaweed was floating at the top and showed bubbles of gas entangled. Unstoppered, stirred up, allowed to come to rest, and took 100 c.c. for titration. The sample required 5-9 c.c. of centi-normal acid to neutralise it to phenol-phthaléin. A Sérensen determination gives a match at 7-6 Borate to 2-4 HCl, equivalent to P,, 10~**. The water removed for the determinations was replaced by fresh sea-water so as to fill the bottle, which was re-stoppered and left exposed to the light. On February 15th at 11.30 a.m. the water was titrated again, the weather having been bright — in the interval, but with little direct sunlight. The seaweed SEA-FISHERIES LABORATORY. 261 had floated to the top and contained a good many bubbles of gas. The contents were well stirred up and a sample taken for titration ; the result was 8-5 c.c. A Sorensen determination gave a match at 9-1 Borate and 0-9 c.c. HCl, equivalent to B10: *. The next titration made in this experiment was carried out at 11.30 a.m. on February 16th. There had been a little bright sunlight on the afternoon of February 15th, and a dullish morning on the 16th. The titration gave ll-lce.c. The Sorensen determination gave an alkalinity exceeding the full strength of the Borate solution, that is to say above P,,10~*”. This shows that in an interval of three days in February, the small quantity of seaweed used in the experiment had been able to increase the alkalinity of two litres of sea-water almost to the maximum point. The bottle containmg the sea-water and green seaweed was taken over to Liverpool and exposed on the roof of the Bio-chemical Laboratory. The weather was fairly bright, and the bottle was exposed to diffuse light during the day, but was not in any direct sunlight. Analysed on February 17th, that is, four days from the commencement of the experiment, it gave an alkalinity corresponding to 12-4 c.c. per 100 c.c. of centi-normal acid. A titration against methyl-orange for total alkali gave 24-0 c.c. of centi-normal acid. Here again the full point of alkalinity is reached at almost exactly one half of the total alkali available. The quantity of seaweed in the water was separated and analysed. Dried from adherent moisture and weighed in the moist condition, it amounted to 3-05 grams. After drying at 105°C, this yielded 0-568 gram of dried matter. Incinerated this left 0-180 gram of ash, or about 30 per cent. of the dried weight. The ash contained Na, Mg, Fe, Cl, and SO,, and P,O;. There was a considerable amount of iron in the ash. 262 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Experiment 4. This experiment was made to test the rapidity of action in an early summer period and was not carried to the end as the others. June 14th, 1913, at 10 a.m., seaweed placed in water, about 2,000 c.c., in fully-stoppered bottle. Titration value of water, at commencement, 3-7 C.c. alkaline. Again titrated at 10 a.m. next day, after bright sunshine meanwhile, titration had increased to 8-7 alkaline. Sérensen had gone up from 6-1: 3-9, Borate and Hydrochloric acid to 9-4: 0-6, equivalent to P,,10~°°. SUMMARY AND CONCLUSIONS. 1. Photo-synthesis by green algae causes a marked diminution in hydrogen-ion concentration in sea-water, and in confined volumes of water this variation in the direction of increased alkalinity may act as the inducing or favouring cause for pathological conditions and disease. 2. Certain salts of the sea-water (notably carbonate and bi-carbonate of magnesium) act as a steadying agency in preventing too rapid variations in the ionic concentrations of hydrogen and hydroxyl, and so safeguard life in the ocean. Within viable limits, physiological activity may be stimulated at certain seasons in which the alkalinity of the sea-water is increased, or, in other words its hydrogen-ion concentration diminished. Thus a rise in alkalinity in Spring would aid, along with temperature and sunlight, in producing increased cell-division. 3. The “ Buffer” effect or “ Reactivity’ of sea-water has been estimated between two fixed points, viz., the turning point to methyl-orange and the turning poimt to phenol- phthaléin, and found to correspond to about 22 x 10% N. This range of ‘ Reactivity ’’ does not show seasonal variation, and the hydrogen and hydroxyl ionic concentrations simply vary within its limits. SEA-FISHERIES LABORATORY. 263 4. The “Reactivity” effect is mainly produced by dissolved magnesium bi-carbonate, and not calcium bi-carbonate as usually stated. 5. In all cases the normal fresh sea-water gave a pink colour with phenol-phthaléin indicating a potential of hydrogen- ion concentration lying below Py, 10-°, the average being about P,,, 10~ *”. 6. The seasonal variations in P, have been followed out by the colorimetric method of Sorensen and by the Hydrogen Electrode method, and a small but distinct increase in alkalinity in Spring has been detected. 7. This vernal increase in alkalinity is not due to increasing temperature disturbing the equilibrium between the carbon-dioxide of sea-water and atmosphere, for the rise in alkalinity clearly precedes in time the rise in temperature. It is caused by photo-synthesis as is shown by its coincidence in its occurrence with the rapid lengthening of the day in March and the increasing sun’s altitude, as also by the great changes. in alkalinity which may be produced by exposure of sea-water containing algae to sunlight. 8. Algae continue abstracting carbon-dioxide and _ so increasing alkalinity until all the bi-carbonates have become changed into normal carbonates, and then definitely cease to functionate and rapidly die at this latter point. The potential of hydrogen-ion concentration falls to Py, 10° before synthesis ceases. At this point the sea-water gives an intense pink to phenol-phthaléin, and titration gives a figure almost exactly half that of the total reactivity effect. We desire to express our indebtedness to the Percy Sladen Memorial Trust for the necessary funds for this Research which were allotted to one of us by the Trustees. 264 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. LITERATURE. 1. FRIEDENTHAL, AND FRIEDENTHAL AND SALM. Arch. f. Anat. u. Physiol., 1903, 8. 550. Zeitsch. f. Electrochem. Bd. X, 1904, S. 113, 8. 341; Ibid., Bd. XIII, 1907, S. 125. 2. SORENSEN. Biochem. Zeitsch. Bd. X XI, 1909, S. 131, and other papers in Comptes rendus du Lab. de Carlsberg, notably T. VIII, pp. 1, 396. 3. SORENSEN AND PaLirzscH. Comptes rendus du Lab. de Carlsberg, T. IX, 1910. Biochem. Zeitsch. Bd. X XIV, 1910, S. 387. 4. PawitzscH. Report on the Danish Oceanographical Expeditions, 1908-10. Carlsberg Fund, ed. by Joh. Schmidt, Copenhagen, 1912. Vol. I, p- 239. Biochem. Zeitsch. Bd. XX XVII, 1911, 8. 116. Comptes rendus du Lab. de Carlsberg, T. X, 78, 1911. 5. Moorz, Roar anD WuittEy. Proc. Roy. Soc. B. Vol. 77, 1905, p. 102. | Moore and Witson. Biochemical Journal, Vol. I, p. 297, 1906. 6. V. Brera. Liebig’s Annalen, Bd. 77, 1851, S. 90. 7. Guianet ET TELLES. Comptes rendus de Acad. des Sciences, T. 83, 1876, p. 920. 8. Tornor. Den norske Nordhavsekspedition, 1876-8, 27, 1880. 9. NatreRER. Denksch. de kais. Akad. d. Wissensch. math-natur- wissen. Klasse, Bd. 59. Ber. d. Kom. f. Erforschung d. ostl. Mittelmers, 83, 1892, und 60, 54, 1893. 10. Ruprrs. Wissensch. Meeresuntersuch. Kiel, neue Folge, 11, 281, 1909. 11. Los. Arch. f. d. ges. Physiol. Bd. 118, 186, 1907. 12. Logs. Arch. f. Entwickelungsmechanik d. Organismen, 1898, Bd. VII, 631. Arch. f. d. ges. Physiol, 1903, Bd. 99, 637. Ibid., 1904, Bd. 101, 340. Ibid., Bd. 103, 1904, 506. University of California Publica- tions, Physiology, 1903-4, Vol. 39, 139. 13. Rincer. Verhandlingen u. h. Rijksinst. v. h. ondersook. der zee, 1908. 14. Narsansonn. Ber. u. d. Verhandl. d. k. Sachs. Gesellsch. d. Wissensch. zu Leipzig. math-naturwissen. Klasse, Bd. 59, 1907. 15. BrOnsTED UND WESENBERG-LuND. Internationale Revue d. ges. Hydrobiologie u. Hydrographie, 1912, 8. 251. 146. Watrote. Biochemical Journal, Vol. V, 1911, p. 207, and Vol. VIII, 1915, p. 628. ; SEA-FISHERIES LABORATORY. 265 HYDROGRAPHIC OBSERVATIONS MADE IN THE IRISH SEA DURING 1914. By Henry Bassett, D.Sc., Professor of Chemistry, University College, Reading. The hydrographic observations in the eastern portion of the Irish Sea which were begun in 1907 were continued during 1914, but owing to various circumstances they were very incomplete during the past year. A certain amount of data has, however, been collected, and it has been thought worth while to record it. Observations were made during the months of February, April, May, June, July, and September. Various causes had prevented hydrographic cruises being made durmg January, March, and August, and at the end of September the observation steamer was taken over by the Admiralty. The 24 stations at which water samples were collected were the same as during 1913 and are indicated on the chart given in last year’s Report on p. 191. February 3 to 7, 1914. Stations I to IV, 3/2/14. Surface observations only. | . Station. Time. saga a al ot I 54°N. ; 3°30’ W. 9.25 a.m.| 5-1 | 17-82 | 32-20 | 25-47 II 54°N. ; 3°47'W. 10.30 a.m.| 6:3 | 18-63 | 33-66 | 26:47 mil ODA NN. ; 4°4'W. 11-40 a.m.} 6-65} 18-74 | 33-86 | 26-58 IV 54°N.; 4°20’ W. 12.55 p.m.| 7:9 | 18-98 | 34-29 | 26-75 266 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Station V, 53°53’N. ; 4°46’W. 5/2/14 (9.35 a.m.) Depth (metres) i ley 6. Sas Ct 0 8-35 18-99 34-31 26-71 30 8-15 18-98 34-29 26-71 60 8-15 18-98 34-29 26-71 | Station VI, 53°43’N.; 4°44’W. 5/2/14 (10.55 a.m.) Depth (metres) WUE Clie. Solas ot 0 8-65 18-97 34:27 26-62 30 8:5 18-96 34-25 26-63 70 8-45 13:97 34-27 26-64 Station VII, 53°33’N. ; 4°41’W. 5/2/14 (12.15 p.m.) ile | gle S°/ Depth (metres) 43 ot 0 | 8-1 | 18-96 34-95 26-69 30 7-96 18-95 34-93 26-70 83 7-95 | 18-95 34-23 26-70 Stations VIII to XIX. Surface observations only. Stations XX to XXIV were not visited owing to the very bad weather experienced during this cruise. Station. Date and Time. | T° Cl’/o0 St/sa Ct p-m. VIII 53°27'N. ; 4°5’W. 5.2.14 3.0 68 18-69 | 33-77 | 26-49 a.m. IX 53°31/N. ; 3°31’W. 6.2.14 9.25 | 6-2 18-47 | 33-37 | 26-26 x 53°37'N. ; 3°45’ W. 6.2.14 10.25 | 6-7 18-70 | 33-78 | 26-53 XI 53°43’N. ; 3°58’ W. 6.2.14 11.20 | 7:15 | .18-88 34-11 26-72 .m. XII 53°48’N. ; 4°12’W. 6.2.14 12.15 | 7-4 18-92 | 3418 | 26-74 XIIt 53°54’N. ; 4°27’ W. 6.2.14 L10 se 7:6 18-96 34-25 26-76 XIV 54°32’N. ; 4°37’ W. 6.2.14 5.10 | 8:3 18-91 34-16 26-59 XV 54°37'N. ; 4°45’°W. 6.2.14 6.10 | 8-3 19:00 | 34:33 | 26-72 a.m. XVI 54°35'N. ; 4°27'W. 7.2.14 8.0 6-6 18-70 | 33-78 | 26-54 XVII 54°34’N.; 4°12’W. 7.2.14 9.0 6-2 18-59 33-58 26-43 XVIII = 54°32’N. ; 3°55’W. 7.2.14 10.0 6-3 18-67 | 33-73 | 26-53 XIX 54°29’N. ; 3°43’W. 7.2.14 11.0 5:3 18-04 32-59 25-76 SEA-FISHERIES April 8, 1914. Stations I to VII. LABORATORY. 267 Owing to bad weather only surface observations were made. Station. Time. Be Ch aot /cauk ot I 54°N.; 3°30'W. 10:10 a.m.| 7-3 | 18-10; 32-70 | 25-59 II 54°N.; 3°47’ W. 11.15 a.m.} 7-55] 18-75 | 33-87 | 26-48 Me bEN.; 4°4'W. 12.20 p.m.| 7-8 | 18-96 | 34-25 | 26-73 IV 54°N. ; 4°20'W. 1.25 p.m.| 8-0 | 19-04 | 54-40 | 26-82 V 53°53 N.; 4°46'W.| 3.0 p.m 7-9 | 19-07 | 34-45 | 26-87 VI 53°43'N. ; 4°44°W.| 4.0 p.m] 7-9 | 19-06 | 34-43 | 26-86 7VIL 53°33'N. ; 4°41’W.|. 5.0 p.m. 7-8 | 18-96 | 34-25 | 26-73 May 5 to 7, 1914. Stations I to IV, 5/5/14. Surface observations only. / Station. Time. fa oe a Ot I 54°N. ; 3°30’ W. 8.40 a.m. 9-35 | 18-19 | 32°86 | 25-41 II 54°N. ; 3°47'W. 9.45 a.m. 8-95 | 18-75 | 33-87 | 26-26 TIT 54°N.; 4°4’W. | 10.45 a.m. 9-05 | 18-82 | 34-00 | 26-35 IV -54°N.; 4°20'W. | 11.45 a.m.| 9-15 | 19-02 | 34-36 | 26-61 Station V, 53°53’N.; 4°46’W. 5/5/14 (1.30 p.m.) Depth (metres) TL tay | my ot 0 9-05 19-06 34-43 26-69 30 8-9 19-06 34-43 26°71 60 8-85 —* —- —— * Bottle broken in transit. 268 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Station VI, 53°43’N. ; 4°44’W. 5/5/14 (2.45 p.m.) Depth (metres) aha Cle Sis Ct Oe 9-1 19-10 34-51 26-74 30 8-95 - 19-09 34-49 26-74 . 65 8-9 19-09 34-49 26-75 Station VIT, 53°33’N. ; 4°41’W. 5/5/14 (4 p.m.) Depth (metres) ES Clene S77 a ot 0 9-3 19-06 34-43 26-65 30 9-2 19-06 34-43 26-66 se 9-05 19-05 34-42 26-67 Stations VIII to XXIV. Surface observations only. Station. Date and, Time. | ‘T° -,| :Cl°/,, as ee Ot a.m. Vill Ba ot N. 3 4-5 W. 6.5.14 11.15 9-15 18-94 34-22 26-50 : .m. IX Da obeNe saa ola. 6.5.14 P30 9-80 18-50 33-42 25:78 x 53°37'N. ; 3°45’ W. 6.5.14 4.0 9-45 18-71 33-80 26-13 XI 53°43/N. ; 3°58 W. 6.5.14 5.0 9-15 18-91 34-16 26-46 XIt 53°48’N. ; 4°12’W. 6.5.14 5.55 | 8:95 | 19-04 34-40 26-67 XIII 53°54’N. ; 4°27'°W. 6.5.14 6.55 | 8-95 19-05 34-42 26-68 XIV 54°32’N. ; 4°37 W. 7.5.14 5.35 | 9-45 18-78 33-93 26-22 XV 54°37'N. ; 4°45’ W. 7.5.14 4.55 | 9-30 18-41 33-26 25-72 XVI 54°35'N. ; 4°27'W. 7.5.14 3.45 | 9:45 18-26 32-99 25-50 XVII 54°34’/N. ; 4°12’W. 7.5.14 2.50 | 9-45 18-49 33-40 25-82 XVIII 54°32°N.; 3°55'’W, 7.5.14 1.55 | 9°55 18-21 32-90 | - 25-40 XIX 54°29/N. ; 3°43’W. 7.5.14 12.45 | 9-85 17-92 32°38 24-95 a.m. xX 5A°D4/N. ; 3°S7'°W. 7.5.14 9.20 | 9-20 18-15 32-79 25:38 XXI 54°20’N. ; 4°13’W. 7.5.14 8.25 | 9-25 18-58 33°57 25-97 m. XXII 54° SIN. SOTA. 7.5.14 P30 9-55 18-42 33:28 25-70 XXIII 54°10’N. ; 3°42’W. 7.5.14 8.50 | 8-95 18-43 33-30 25-81 XXIV. 54°S’N.; 3°27°W. 7.5.14 9.45 | 9-70 18-10 32-70 25:23 SEA-FISHERIES LABORATORY. June 2 and 3, 1914. Stations I to IV, 2/6/14. | | Surface observations only. Station. Time. dae #1 Gi a g°/ fete) OE I 54°N.; 3°30’W. | 5.55 pm 11-6 | 18-43 | 33-30 | 25-36 II 54°N. ; 3°47'W. 6.55 p.m.) 11-9 | 18-32 | 33-10 | 25-15 TIT 54°N.; 4°4’W. 9.20 p.m.| 11-8 | 18-42 | 33-28 | 25-31 IV 54°N.; 4°20'W. | 10-20 p.m.| 11-1 | 18-79 | 33-95 | 25-96 Station V, 53°53’N.; 4°46’W. 3/6/14 (9.40 a.m.) | ze Depth (metres) sh Cle i«. tyes Ot 0 102 | 19-01 34-34 26-43 30 9-9 19-08 34:47 26-58 65 ae) 19-07 34-45 26-56 Station VI, 53°43/N.; 4°44’W. 3/6/14 (10.50 a.m.) Depth (metres) a” | Cl°/ 50 | Shen | ot 0 10-25 19-09 34-49 26-53 30 10-1 19-09 34-49 26-55 56 10-15 19-10 34-51 26-56 Station VII, 53°33’N.; 4°41’W. 3/6/14 (12 noon). Depth (metres) i hs e/a. ot 0 10-5 19-04. 34-40 26-41 30 10-28 19-05 34-42 26-46 62 10-24 19-06 34:43 26-49 270 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. July 9 to 10, 1914. Stations I to IV, 9/7/14. Surface observations only. Station. Time. T°. Cee ot |- | __f I 54°N.; 3°30°W. | 11.0 a.m. 15-5 | 18-48 | 33-39 | 24-64 II 54°N.; 3°47'W. 1.30 p.m.| 13-6 | 18-58 | 33-57 | 25-18 TIT = 54°N.; 4°4’W. 2.35 p.m.) 13-6 | 18-72 | 33-82 | 25-38 IV 54°N.; 4°20’W. 3.30 p.m.| 13-2 | 18-86 | 34-07 | 25-66 Station V, 53°53’N.; 4°46’W. 10/7/14 (11.50 a.m.) Depth (metres) af Chey Soiee oe 0 13-5* 18-98 34-29 25-76 30 12-1 19-02 34-36 26-09 83 11-95 19-02 34-36 26-11 * Possibly this should have been 12-5°. Station VI, 53°43’N. ; 4°44’W. 10/7/14 (1 p.m.) — Depth (metres) dg Cle Shiis. ot 0 12-65 19-05 34-42 26-02 30 12-4 19-05 34-42 26-07 68 12-35 19-05 34-42 26-08 Station VII, 53°33’N. ; 4°41’/W. 10/7/14 (2.5 p.m.) Depth (metres) ,° CRs. Spee ot 0 F 13-4 19th 34:16 25-68 30 ee 13-23 18-91 34-16 25-76 58 13-2 18-9) 34:16 25-76 SEA-FISHERIES LABORATORY. raga September 7 to 8, 1914. Stations I to IV, 7/9/14. Surface observations only. Station. — Time. 2 AOI SPY ereeeeseeeneneenenneterieet emanate I 54°N. ; 3°30’ W. 2.25 p.m.| 16-9 | 18-41 | 33-26 | 24-22 TI 54°N.; 3°47'W. | 3.20pm 16-2 | 18-68 | 33-75 | 24-76 Tl 54°N.; 4°4’W. | 4.15pm) 15-4 | 18-94 | 34-29 | 95-29 IV 54°N.; 4°20'W. | 5.15pm 15-2 | 18-96 | 34-25 | 25-37 Station V, 53°53’N.; 4°46’W. 8/9/14 (9 a.m.) Depth a - wl a Sige ot 0 14-6 19-02 34-36 25-58 30 14-5 19-00 34:33 25-58 91 14-4 19-00 34-33 25-60 Station VI, 53°43’N. ; 4°44’W. 8/9/14 (10.10 a.m.) Depth (metres) a hig ae Sta aes os ) 14-8 19-01 34-34 25-53 30 14-72 19-01 34-34 25-54 Depth (metres) Avy 0. he ~ | ot 0 15-6 18-95 34:23 25:27 30 15-55 18-94 34-22 25-26 272 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. During the past year an important paper on the seasonal changes of salinity in the Irish Sea has been published by D. J. Matthews (Fosheries, Ireland, Sci. Invest., 1913, IV [1914]). It is based on the hydrographic work carried out by the Irish Department of Agriculture and Fisheries. Matthews shows that there is a general northward flow of water through the Irish Sea, thus confirming the conclusions drawn by myself in a paper on “ The flow of water through the Irish Sea,” published in the Lancs. Sea-Fish. Lab. Report for 1909 (also Trans. Biological Soc. Liverpool, Vol. 24, 1910). As regards the hydrographic conditions in the mouth of the Bristol Channel, Matthews holds different views from those expressed by myself in the above-mentioned paper. Perhaps he is right, but, as he practically admits himself, further work is necessary to settle the point. I must, however, protest against the manner in which Matthews has referred to a paper by Nielsen (Meddelelser fra Kommissionen for Havunderségelser : Hydrografi, Vol. 1, No. 9, Copenhagen, 1907), in such a way that it appears to give support to his own assumption of an eddy-like circulation of the water in the mouth of the Bristol Channel. Nielsen originally concluded that “all around Ireland there flows a current in an anticyclonic direction.” He says also (loc. cit., p. 25) that “the north-going current, west of Ireland, sends a branch around the north coast of the island and down through the Irish Channel, so that this island has a coast current in an anticyclonic direction like Iceland, Scotland, etc.” He came to this conclusion largely owing to a paper by Matthews (Report on the Physical Conditions in the English Channel, 1903. First Report of the North Sea Fisheries Investigation Committee [southern area] London, 1905), in which hydrographic results obtained off the south of Ireland and in the English Channel were interpreted as indicating a southerly flow of water from the Irish Sea. SEA-FISHERIES LABORATORY. 273 Now that this southward flow of water can no longer be assumed, Matthews, forgetting apparently why Nielsen postulated the anticyclonic circulation all around Ireland, quotes him as postulating an anticyclonic circulation of the water only off the south of Ireland (an eddy, that is to say) and uses this as some sort of support for his own assumption of an eddy in a cyclonic direction in this same Bristol Channel area. The diagram I published, in the paper on the flow of water through the Irish Sea already referred to, indicated an anticyclonic eddy in the Bristol Channel which carried some less saline water southward. ; T would like to point out that in the charts on which it was based, which were published in the same paper, all the results obtained by the Plymouth observers were included and the actual salinities marked on the charts. The course of the isohalines depended almost entirely on these results, and not in any essential manner upon the few “ liner ” observa- tions which were also indicated on the charts. Matthews, no doubt correctly, objects to the inclusion of these latter results as being less trustworthy ; but if they are left out the course of the isotherms is not affected and Matthews’ criticism of the manner in which the charts have been drawn is not really justified. 274 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. ON THE PELAGIC FISH-EGGS COLLECTED OFF THE SOUTH-WEST OF THE ISLE OF MAN IN 1914. By ANDREW Scott, A.L.S. I hoped it would have been possible to continue the report, commenced last year, on the pelagic fish-eggs taken in the plankton collected by the Lancashire and Western Sea-Fisheries steamer, but this has not been possible. An accident to the ship in February laid her up for repairs during the most important month of the spring. By the time the repairs were completed, and the ship ready for sea again, the maximum spawning period in 1914 was past. Later on in the year the ship was taken over on Government. Service, and the sea work brought to an abrupt conclusion for the time being. Only thirteen of the collections taken while the vessel was carrying on investigations contained pelagic fish-eggs, compared with eighty-three in 1913. 7 The samples of plankton taken throughout the year in Port Erin Bay, and on occasions outside, around the south-west of the Isle of Man in connection with the ‘Intensive Study ”’ investigations, have been the only means of obtaining continuous information regarding the occurrence of pelagic fish-eggs in 1914. This in-shore area, especially Port Erin Bay, is more liable to be influenced by the action of winds and currents than the open sea. The appearance of eggs in the plankton taken in the Bay, then, will probably only give an approximate idea of the spawning periods compared with the results that might be obtained in the open water of the central area. The central area may be regarded as the portion of the Irish Sea extending from between Cumberland SEA-FISHERIES LABORATORY. 275 and the Isle of Man to off the coast of North Wales. This includes one or two fairly well defined spawning grounds. The investigations carried on at the South- West of the Isle of Man in 1914 do not alter the approxi- mate duration of the spawning period that is given in the XXI Annual Report for the species dealt with below. There is a well marked difference in the number of eggs of the valuable food fishes present in the Bay compared with the area outside, as shown by the tables given. It is evident, therefore, that spawning takes place outside, and the eggs are carried into the Bay by winds and currents. The arrangement here is the same as that adopted in the XXI Annual Report, page 233. Clupea sprattus, Linn.—Sprat. Eggs of the sprat were observed in the plankton collected in Port Erin Bay on May 25th. They appeared to be continually floating about this area during June and July, but none were found later than July 30th. A collection taken off Kilan Head in Cardigan Bay, by the Fisheries steamer on April 5th, was estimated to contain 805 sprat eggs. Two were found in a haul 8 miles S.E. of Point Lynus on May 6th. Our records extending over eight years show that sprat eggs may occur in the plankton from the beginning of April until the middle of September. It is possible that the actual spawning period in some parts of the Irish Sea may be even earlier than is indicated by the presence of the eggs in the plankton. The reproductive organs of sprats sent to me from Morecambe for investigation on February 5th, 1915, were nearly all well advanced towards maturity. In one or two cases the ovaries and testes were quite mature. He! i 276 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Gadus callarius, Linn.—Cod. The pelagic eggs of the cod are generally to be found in small numbers in the plankton taken inside the break- water at Port Erin between the end of February and April. They are no doubt carried into the Bay by winds and currents. It is scarcely likely that spawning will take place in the shallow water of the Bay. Cod eggs are moderately abundant at times outside the Bay from a few miles West of Bradda Head to South of Calf Island. Some of the collections taken at Station III have been estimated to contain from 865 to 1,590 cod eggs. The first cod eggs appeared in the Bay on February 26th. Throughout the next two months very few of the bi-weekly hauls in the Bay, and outside of it, were without the eggs of this fish. Plankton collected 10 miles off Point Lynus on February 5th contained five cod eggs. Another sample from 11 miles N. by E. of the Liverpool North-West Lightship, on February 17th, contained three eggs. The hauls made in the central area of the Irish Sea and in Carnarvon Bay in April showed cod eggs to be generally distributed. The following is a list of the cod eggs observed at the South-West of the Isle of Man in 1914:— No. of Eggs. No. of Bags. Feb. 26—Port Erin Bay ... 31 Apr. 4—Station I ............ 974 Mar. 4—Port Erin Bay ... 19 » 4—Station III ......... 1,590 », 11—Port Erin Bay ... 4 ,» 8—Off Spanish Head 44 ,, 18—Port Erin Bay ... 19 » 9—Port Erm Bay ee. 5 », 21—Port Erin Bay ... 2 » ll—Off Spanish Head Bal », 24—Port Erin Bay ... 15 » l1—Port Erin Bay ... vi .s5 27—Port Erin Bay ... 6 », 14—Off Spanish Head.. 17 », 30—Port Erin Bay ... 1 », 14—Port Erin Bay ... 8 Apr. 1—2 miles W. of ,, 15—Station III ......... 280 Bradda Head ... 282 » 16—Station III ......... 753 » 1—2 miles off Perwick 16 ,», 17—Station ITI ......... “6 » 1—Off Spanish Head 32 , 17—Off Bradda Head... 162 po. OE Etaoin te eee 32 , 18—Station III ......... 2 » 2—sStation III ......... 160 , 18—Off Bradda Head... 10 » 2—S. of Calf Island... 5 ,» 18—Off Calf Island ... 1 »» 2—Port Erin Bay ... 4 , 20—Station IIT ......... 4 » o&—Station I ............ 533 » 20—N. of Calf Island... 3 » &—Station III ......... 865 » 20—S.W. of Calf Island 6 Station I lies 5 miles West of Bradda Head. Station III lies 3 miles North-West of Bradda Head. SEA-FISHERIES LABORATORY. pA bi Gadus aeglefinus, Linn.—Haddock. There has been a marked scarcity of this fish in the Irish Sea during the past five or six years. No haddock eggs were obtained in 1911 and 1912. Only two were seen in 1913. They were from a haul taken five miles N.W. from Peel, Isle of Man, on May 7th. The investi- gations carried on in 1914 give no indication that there will be an early recovery of this fishery. A migration of the adults into the Irish Sea, however, may take place quite unexpectedly at any time. Herdman and Dawson, in the Lancashire Sea-Fisheries Memoir No. II, ‘‘ Fish and Fisheries of the Irish Sea,’’ page 47, state that the value of the Irish Sea fishery for haddock in 1900 was £35,000. The only eggs observed during 1914 were obtained from plankton collected in Port Erin Bay on May 12th and 18th. The haul on May 12th contained two eggs, and that on the 18th one only. Gadus merlangus, Linn.—Whiting. The eggs of the whiting appeared in the Bay plankton on March 4th, and were generally distributed there and in the open area outside during March and April. The open sea collections taken in connection with the “Intensive Study ”’ investigations usually contained much larger numbers of eggs than those taken in the Bay. It was estimated that fully eight thousand were present in a haul from Station III on March 3rd. The collections taken in the central area of the Irish Sea in April showed the eggs to be fairly plentiful everywhere. A haul taken on April 14th, 8 miles N. } W. from Liverpool North- West Lightship, was estimated to contain 4,900 whiting eggs. 278 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. The following list gives the distribution of the eggs off the South-West of the Isle of Man in 1914:— Fane No. of Eggs. No. of Eggs. Mar. 4—Port Erin Bay ... 8 Apr. 4—Station I ...... caper 1,369 » %7—Port Erin Bay .... 23 » 4—Station III ......... 1,120 »» 11—Port Erin Bay .... 16 ,» 8—OffSpanish Head... 134 », 18—Port Erin Bay ... 21 » 9—Port Erin Bay ... 291 »» 2l—Port Erin Bay ... 4 » ll—Off Spanish Head.. 485 » 24—Port Erin Bay ... 24 », 1ll—Port Erin Bay ... 93 », 27—Port Erin Bay ... 6 », 14—Off Spanish Head.. 178 », 380—Port Erin Bay ... 2 », 14—Port Erin Bay ... 51 » ol—3-4 miles W. of ,». 15—Station III ......... 850 Bradda Head ... 32 ,» 16—Station III ......... 1,451 Apr. 1—2 miles W. of » 17—Station ITI ......... 84 Bradda Head ... 773 ,» 17—Off Bradda Head... 85 » 1—2 miles off Perwick 26 ,» 18—Station III ......... 36 » 1—Off Spanish Head.. 4] ,. 18—Off Bradda Head... 67 » 2—Station I ............ 218 », 18—Off Calf Island...... 51 » 2—Station III ......... 1,040 » 20—Station III ......... 94 » 2—S. of Calf Island... 22 » 20—N. of Calf Island... 4 », 2—Port Erin Bay ... 69 » 20—S.W. of Calf Island 20 » . -o— station ID v.3.c.c.e0 2,113 », 24—Port Erin Bay ... 10 of o Station TID. t.2.3.... 8,145 May 4—Port Erin Bay ... | Gadus virens, Linn.—Green Cod. Eggs which were probably identical with those of the green cod were first noticed in the Bay plankton in 1914 on January 28th. They occurred throughout the whole of February, and once in March, the 31st. A haul taken on February 26th contained 114 eggs identified as Gadus virens. Green cod eggs were present in only one of the collections taken outside the Bay, but this is probably due to the spawning period of the fish being over before the ‘‘ Intensive Study ’’ investigation of the outside area commenced. None were found in the plankton from the central area of the Irish Sea. Gadus luscus, Will.—Bib. Gadus minutus, Linn.—Poor Cod. Eggs of both these species of fish occur in the plankton of the Irish Sea, but the difference in size is so very slight that it is almost impossible to separate SEA-FISHERIES LABORATORY. 279 them with certainty. They were observed in the central area nearly a fortnight earlier than in Port Erin Bay in 1914. The eggs were found for the first time in the Bay plankton on March 4th, and were of frequent occurrence both inside the Bay and at the observation stations outside during March, April and May. As usual, the largest numbers were taken at the open sea stations. It was estimated that 2,660 bib or poor cod eggs, probably both species, were present in a haul taken at Station III on April 3rd. The maximum spawning period of the valuable gadoids frequenting the area at the South-West of the Isle of Man in 1914 appears to have been at the beginning of April. The largest numbers of eggs were found on April 3rd and 4th. Onos spp., Risso.—The Rocklings. The eggs of one or more species of rockling are amongst the first of the pelagic fish eggs to make their appearance in the plankton of the Irish Sea, and are usually the last to disappear. Their occurrence in the Bay in 1914 extended from January 19th right on through the spring, summer and autumn until October 9th. This is almost identical with the distribution found in 1910. There is a distinct variation to be found amongst the eggs, both in the size of the egg itself and of the oil-globule. It is almost certain that two species of rocklings are occasion- ally represented in the same haul. Rockling eggs collected in September measured 0°832 mm. in diameter with an oil-globule 0°176 mm., and 0°67 mm. in diameter with an oil-globule 0°15 mm. Scomber scomber, Linn.—Mackerel. Eggs identified as those of the mackerel only occurred twice during 1914. Two were found in Port 280 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Erin Bay plankton collected on July 27th and again on July 30th. Drepanopsetta platessoides, Fabr.—Long Rough Dab. The well-defined eggs of the long rough dab, after being apparently absent from the area round the South- West of the Isle of Man for three years, occurred in thirteen hauls taken in the spring of 1914. All the records except one are from outside Port Erin Bay, between Bradda Head and Calf Island. They were present in numbers varying from one to nine in nearly every sample of plankton taken between April 2nd and 20th. The fish no doubt spawns in the soft muddy area in the vicinity of Bradda Head. The eggs were not observed in any of the collections from the central area of the Irish Sea. The following is a list of the positions where the plankton containing these eggs was collected :— — No. of Eggs. No. of Eggs. Apr. 2—Station I ............ 1 Apr. 11—Off Spanish Head.. 9 »» o2—Station Tis aecene. 3 », 1l1—Port Erin Bay 1 55 do Station WT Getencces-c 2 » 15—Station Hil ......... 1 » 9—Station III ......... 3 » 16—Station III ......... 5 37 4—Station I scecsisere 2 » 17—Station III ......... 1 po) Ae Statione lire ees 2 ,, 20—S.W. of Calf Island 1 » 8—Off Spanish Head.. 1 Zeugopterus punctatus, Bl.—Mauller’s Top-knot. The eggs of a species of top-knot, which is probably the above, appeared to be generally distributed in the Bay from April 24th to the end of June in 1914, and in the adjoining area outside from March 31st to April 20th. There does not appear to be the same marked difference in the numbers generally present in the Bay compared with those found in the area outside. That is clearly the case with the valuable food fishes such as the cod, whiting, plaice, &c. The following table of distribution is given for the sake of comparison with the food fishes : — SEA-FISHERIES LABORATORY. 281 No. of Eggs. No. of Eggs. Mar. 31—3-4 miles W. of May 4—Port Erin Bay ... 10 Bradda Head ... 2 », 12—Port Erin Bay 6 Apr. 4—Station ITI ......... 6 ,, 14—Port Erin Bay 9 » 8—Off Spanish Head 1 ,, 18—Port Erin Bay 5 » 11—Off Spanish Head 2 May 21—Port Erin Bay 52 » 14—Off Spanish Head 3 ,, 25—Port Erin Bay pore tse —ohation PLE ......... 2 ,, 28—Port Erin Bay 40 » 16—Station III ......... 9 June 1—Port Erin Bay ll » 18—Station IT] ......... 1 »» . 11l—Port Erin Bay 5 » 20—Station III ......... 2 ,, 15—Port Erin Bay 3 »» 20—N. of Calf Island... 4 ,, 18—Port Erin Bay 1, » 20—S.W. of Calf Island 57 ,, 22—Port Erin Bay 3 » 24—Port Erin Bay 16 ,, 20—Port Erin Bay 1 » 27—Port Erin Bay 12 », 29—Port Erin Bay 4 » 30—Port Erin Bay ... 8 Lepidorhombus megastoma, Donov.—Megrim or Sail Fluke. The pelagic eggs of the megrim or sail fluke occur frequently outside Port Erin Bay, but are only captured irregularly inside the breakwater. They first made their appearance in 1914 in a haul taken in the Bay on March 21st, and again on the 30th. present in nearly every haul taken at the observation stations outside the Bay from April Ist to 20th. In one of these hauls from outside the Bay it was estimated that there were 494 megrim eggs. The eggs were They were generally distributed in the central area of the Irish Sea throughout the month of April. The following table gives the distribution of megrim eggs off the South-West of the Isle of Man in the plankton collected in 1914 :— No. of Eggs. No. of Eggs. Mar. 21—Port Erin Bay ... | Apr. 11—Off Spanish Head.. 104 » 30—Port Erin Bay .... 1 », ll1—Port Erin Bay a7 Apr. 1—2 miles off Perwick 1 ., 14—Off Spanish Head.. 52 » 1L—Off Spanish Head.. 1 », 14—Port Erin Bay .. 35 » 2—tation I ............ 24 ,» 15—Station ITI ......... 42 » 2—station ITI ......... 70 » 16—Station III ......... 244 » 2—S. of Calf Island... 4 » 17—Station III ......... 5 Se —URCION Lo... seccccee 494 , 17—Off Bradda Head... 8 » o—station ITI ......... 189 », 18—Station ITI ......... 7 Se el 207 » 18—Off Bradda Head... 7 » 4—Station III ......... 76 » 18—Off Calf Island...... 11 » 8—Off Spanish Head.. 18 ,, 20—Station ITI ......... 26 » 9—Port Erin Bay 9 » 20—S.W. of Calf Island 3 282 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Pleuronectes platessa, Linn.—Plaice. The pelagic eggs of this important food ee were observed in the Bay collections as early as February 26th, 1914. They appeared to be fairly plentiful at times in the area extending from Bradda Head to Calf Island during the first two weeks of April while the ‘‘ Intensive Study ’’ investigations were being conducted. One of the hauls taken at Station III was estimated to contain 461 plaice eggs. The egg is easily recognised by its large size, corrugated shell, and absence of oil-globule. The following table gives the records of plaice eggs from the Bay plankton and from the open sea :— No. of Eggs. No. of Eggs. Feb. 26—Port Erin Bay .... 3 Apr. 11—Off Spanish Head.. Mar. 7—Port Erin Bay .... 1 » 11—Port Erin Bay : ,, 30—Port Erin Bay ... 1 », 14—Off Spanish Head | 3 Apr. 2—Station I ............ 5 ,, 15—Station ITI ......... 21 » 2-—Station III ......... 15 », 16—Station III ......... 93 » 2—S8. of Calf Island... 2 » 17—Off Bradda Head .. 2 » o—Station III ......... 4] » 18—Off Bradda Head... 4 ay - » A Station ys cece 58 », 20—Station III ......... 1 » 4 Station III ......... 461 » 20—N. of Calf Island... 1 » 8—Off Spanish Head.. 3 » 20—S.W. of Calf Island . 2 Pleuronectes limanda, Linn .—_Dab- ‘The eggs of the dab were only observed three times in the area at the South-West of the Isle of Man in 1914. Four were found in a haul taken at Station III on April 4th, one off Spanish Head on April 8th, and three at Station III on April 17th. They were generally distributed, and at times fairly plentiful, in the central area of the Irish Sea during the whole of April. Pleuronectes microcephalus, Donov.—Lemon Sole. - Pelagic eggs identified as those of the lemon sole do not appear to occur very often in the plankton collected at the South-West of the Isle of Man. None are recorded during the first six years of the ‘‘ Intensive Study” SEA-FISHERIES LABORATORY. 283 investigations. They were only observed once in the Bay in 1914, when three were found in a haul taken inside the breakwater on April 14th. Lemon sole eggs were also identified once in the plankton collected in the central area. A gathering taken ten miles S.W. from More- cambe Bay Light Vessel on April 5th contained 150 eggs of this fish. Callionymus lyra, Linn.—Dragonet. The very characteristic eggs of this fish were first taken in the Bay plankton in 1914 on February 10th. They appeared to be generally distributed in the South- West area until the end of June. Dragonet eggs are not so abundant there as in the central area of the Irish Sea, where a single haul may contain as many as 10,000. The following table gives the distribution of dragonet eggs at the South-West of the Isle of Man in 1914:— No. of Eggs. No. of Eggs. Feb. 10—Port Erin Bay ... 1 Apr. 11—Port Erin Bay ... 13 » 17—Port Erin Bay ... 1 » 14—Off Spanish Head 92 ,» 20—Port Erin Bay 1 , 14—Port Erin Bay ... 8 » 26—Port Erin Bay ... 5 ,» 15—Station III ......... 88 Mar. 4—Port Erin Bay ... 4 » 16—Station III ......... 96 » 7%—Port Erin Bay ... 4 » 17—Station ITI ......... 5 ,» 11—Port Erin Bay 4 ,» L7—Off Bradda Head.. 14 , 18—Port Erin Bay 4 »,5 i17—Port Erin Bay ..., 5 ,, 2l—Port Erin Bay ... 1 » 18—Station ITI ......... i ,» 24—Port Erin Bay ... 2 ,» 18—Off Bradda Head.. 12 ,» 27—Port Erin Bay ... 4 5, « LS—OR Call vo. icb bee 2 ,» 30—Port Erin Bay ... 2 ,» 20—Station ITI ......... 34 » sl—3-4 miles W. of » 20—N. of Calf Island.. l Bradda Head . 3 » 20—S.W. of Calf Island 5 Apr. 1—2 miles W. of ,, 24—Port Erin Bay 7 Bradda Head . 8 », 27—Port Erin Bay 3 » 1—2 miles off Perwick 2 May 10—Port Erin Bay 1 ,» 1—Off Spanish Head.. 8 » 14—Port Erin Bay 9 » 2—S. of Calf Island... 4 », 18—Port Erin Bay 2 » 2—Port Erin Bay ... 1 », 2l—Port Erin Bay 20 »» o—Sstation ITI ......... 6 », 25—Port Erin Bay 3 SO 8 ,, 28—Port Erin Bay 9 » 4—Station IIL ......... 27 June 11—Port Erin Bay 10 ,» 8—Off Spanish Head.. d ,» 15—Port Erin Bay 1 » ll—Off Spanish Head.. 62 , 29—-Port Erin Bay 3 284 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. AN INTENSIVE STUDY OF THE ~ MARINE PLANKTON AROUND THE SOUTH END OF THE ISLE OF MAN.—PART VIII. By W. A. Herpman, F.R.S., ANDREW Scott, A.L.S., and H. Manet Lewis, B.A. [Inrropuctory Notr.—This is now the eighth year (1914) of our detailed analysis of the plankton collected week by week at Port Erin. Whether we shall now be able to complete our contemplated ten years of continuous observations seems a little doubtful. The taking of periodic samples in Port Erin Bay is still going on, and will probably continue to go on as before, but the observa- tions in the open sea at three and five miles west of Bradda Head, which have been carried out every year during the time of the great vernal maximum, will not be possible during the present ninth year of the work. The plan of work in collecting the samples, in working them up and in preparing this report, has been practically the same as in previous years. Mr. H. G. Jackson, M.Sc., again acted as my very efficient scientific assistant on the yacht ‘‘Runa’”’ during the work at sea in April, 1914, and carried out the preservation of the material and the preliminary examination under my direction at the Port Erin Biological Station; while the six weekly Bay samples throughout the year were taken by Mr. T. N. Cregeen, and were carefully preserved by Mr. Chadwick. | Furthermore, the three joint authors have divided between them the rest of the work on the usual plan. Mr. Scott has carried on the further and more detailed examination of the samples; Miss Lewis has done the statistics, calculating the totals and averages, and drawing curves for all the groups and many of the individual organisms; while I have been responsible for supervising SEA-FISHERIES LABORATORY. 285 the whole, and for the form in which the report is now presented. I feel that Mr. Scott and Miss Lewis have done the major part of the hard work, and that I deserve little credit except for planning the work, enabling it to be carried out, and expressing my opinion on details of the investigation and on the general conclusions. As in the case of the last few years, we do not consider the present report to be an exhaustive statement of the results to be obtained from a study of the collections. It is again only an interim report to record the progress of the investigation. We look-forward to giving a fuller discussion of the ten years’ material when the series of observations is completed. We have now in hand a considerable bulk of unpublished figures, curves and other data. We may refer readers to the previous parts of this report (from 1907 onwards) for any desired details as to the apparatus and methods of work and the results so far obtained.—W. A. HERpMAN. | MATERIAL AVAILABLE. The collections made during 1914 have amounted to 393—all taken within the same limited sea-area off the Isle of Man as in former years. Our table for the whole series of samples taken during the eight years of the investigation is now as follows :— Ar SEA, FROM YACHT. In Bay | Year. $< —_—___—__ —————————__ throughout | Totals. Spring. Autumn. Year. | 1907 218 279 138 | 635 1908 156 242 157 | 555 1909 329 147 231+49 | 756 1910 107 249 296 652 1911 120 84 314 | 518 1912 87 0 299 / 386 1913 82 41 282 ) 405 1914 102 0 291 393 Totals ......... 1,201 1,042 2,057 4,300 286 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. On account of the meeting of the British Association in Australia in the summer of 1914, the yacht ‘‘ Runa”’ was not put in commission that autumn, and consequently no samples were obtained outside Port Erin Bay at that time. Nor have we any supplementary material this year from any other parts of the district. As Mr. Riddell has stated on an earlier page of this report, the Sea- Fisheries steamer ‘‘ James Fletcher’’ ceased to make her periodic cruises after September, and the plankton samples, although worked up, are not reported on, as they form an incomplete series. They remain available, if it seems desirable, for inclusion with those of the following year. No change has been made in the nets employed, or in the method of using them (see former reports). PLANKTON oF Port Erin Bay rn 1914. As before, the plan of work within the Bay has been to take two horizontal hauls (coarse and fine nets) and one vertical haul twice each week throughout the year— that is, about 24 hauls per month. The twelve months of 1914 are represented by these hauls as follows :— Month... /J6000 20. I | If pir); IV; V | Vij Vill|-VIll | 1X) Xe —— ee ee ee No. of Hauls ...... 24 | 21 | 24 | 27 | 24 | 27 | 27] 27 | 24 | 27 | 18 | 21 A glance shows that only in one month (November) has weather interfered much with the work. As a rule the collections were made with regularity, and the monthly average is high. We are much indebted to — Mr. Cregeen, of the Biological Station, for his successful endeavours to keep us supplied with the necessary statistical data. a _=—s © eho gt eG ee SEA-FISHERIES LABORATORY. 287 On the whole, the agreement between the evidence given by the smaller vertical hauls at the mouth of the Bay and that of the much larger surface hauls obtained on the same occasions is satisfactory, but naturally the vertical hauls sometimes yield additional information. Our total plankton curve for the Bay did not this year rise to quite such a high point in the vernal maximum as that reached in the spring of 1913, but on the other hand the autumnal maximum was greater in 1914, and, in fact, the curve remains at a higher level from August onwards to the end of the year (fig. 1). Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Fic. 1.—Curves for the total plankton in the years 1914 (whole line) and 1913 (dotted line). The following table shows the monthly averages of the total catch, and of the chief groups of the plankton per haul of the standard net (coarse and fine nets together forming one standard ‘‘ double haul ’’) :— T 288 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. 1914. Double | Average | Diatoms. | Dinoflag- | Copepoda. | Copepod. hauls. catch. ellates. } nauplii. c.c. January ... 8 2-5 29,725 2,179 4,923 1,524 February... 7 3:2 70,486 1,973 7,032 3,380 March ...... 8 6-5 759,072 5,701 7,276 7,385 April $f 6cc 9 34:7 3,257,761| 15,278 26,121 66,601 May? ae 8 34-8 22,370,831] 37,365 12,042 40,920 June ry e.23 9 24-5 2,364,992) 13,911 27,281 43,978 July cis 9 12-7— 1,259,516 5,654 40,949 44,089 August 9 10:8 380 2,443. 37,889 |. 52,372 September —«88 12-7 206,177 1,375 69,702 |. 32,711 October ... 9 11-3 908,827; 20,658 27,411 51,022 November 6 7-9 569,727| 22,503 54,549 18,905 December a 3:7 77,220} 16,343 18,546 6,984 From this table we see that the spring maximum was again in May, agreeing in this respect with that of 1911 and of 1913—though really earlier in the month than in the latter year. The actual largest haul was 88'5 c.c. on May 4th. On the whole, the curve of the monthly averages of the total catch agrees fairly well with that for 1913, but while the averages for May, June, and July are lower, those from August to the end of the year are higher. We noticed last year (1918) that the autumn maximum was an unusually low one; this ' year it is somewhat higher, but not so high as we have sometimes recorded. | It will be noticed from the table that although the Copepoda, as usual, attain their highest numbers much later in the year than the Diatom maximum, the Dinoflagellates this year attain to their maximum in the same month, May, with the Diatoms. Last year (1913) ‘the Dinoflagellate maximum—a much greater one than this year—was as late as July. We consider that, however, to be unusually late. We have drawn curves for these various groups in the two years, but consider it unnecessary to reproduce them, as the changes from month to month can be readily SEA-FISHERIES LABORATORY. . 289. followed with the eye on the table above and the corresponding one in our last report. | The summer minimum of Diatoms was unusually well marked in 1914, when the average standard haul fell in August to less than 400. In 1913 it was over 94,000. The autumnal increase, in 1914, to over 900,000 in October was much more marked than in the previous year, when the maximum was 187,000, in September. But, in fact, all the groups show larger numbers in the autumn and winter months of 1914 than in the corresponding months of 1913. There was a rather unusual temporary increase of Copepoda in April (26,000, while May showed only 12,000), and apparently a sudden change from this zooplankton to the phytoplankton took place at the end of April. There was a large zooplankton haul (62°65 c.c.) on April 27th, consisting chiefly of Copepoda, and an equally large (70 c.c.) haul on April 30th, which was almost a pure phytoplankton (Chaetoceras, Lauderia, and Hucampia). DIATOMS. There is nothing very remarkable to record in connection with the occurrence of Diatoms in the Bay plankton of 1914. ‘The numbers first rose to over a million on March 24th, had increased to over 15 millions by April 30th, attained their maximum of 155,288,000 on May 4th, then dropped to 114 millions on May 7th, and remained fairly high till the second week of July. Then came the summer minimum. They began to increase again at the end of September, and reached to between one and two millions on October 9th, 15th, 18th, and November 3rd. The maximum of over 155 millions on May 4th is mainly composed of a single species, namely, 290 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Chaetoceras debile, of which there were 148 millions present in the double haul. It may be recalled that the previous year (1913) it was another species, Asterionella japonica, that was present in great abundance, running — up to nearly 200 millions per haul on May 16th. In 19138 the vernal phytoplankton at Port Erin was an ** Asterionella-plankton,’’ in 1914 it was a “* Chaetoceras- plankton.’’ We pointed out last year the probability that amongst the competing common spring Diatoms some slight advantage enables sometimes one form and sometimes another to gain the mastery and become for a short time enormously abundant. THE More Important GENERA OF DIATOMS. We give here our usual short summary of the distribution throughout the year of the more important Diatoms. Biddulphia.—The spring maximum (78,100 on March 380th) was again low, as in the two previous years, but the autumn figures resemble closely those of 1911 when there was the unusually high maximum of 660,600 on November 24th; this year we have 800,400 on November 3rd. These are the only two years of our investigation in which the autumn maximum has been higher than the spring maximum. This includes the two forms B. mobiliensis and B. sinensis, of which sometimes the one and sometimes the other is the more abundant. Chaetoceras.—This was again by far the most abundant Diatom in the plankton, and is represented in our gatherings throughout the year. The spring increase began early in March, the numbers reaching to over a million before the end of the month. On April 30th we had 10,443,500, and on May 7th 10,207,200, while the maximum occurred between those dates with the | SEA-FISHERIES LABORATORY. 291 enormous haul of 151,220,000 on May 4th. As pointed out above, 148 millions of this total belong to the species C. debile. After the middle of May the numbers rapidly fell off to a few tens or hundreds in August. They rose again in September, and reached 1,792,200 on October 9th. - Coscinodiscus.—This genus was more abundant both at the time of the spring and the autumn maximum than in any previous recorded year. The maximum was on April 30th, with 930,000 in the standard haul, and in October we had 102,000, on the 18th, and on November ard 83,000. The genus was practically unrepresented in our nets from the middle of June till the beginning of October. : Rhizosolena.—The highest monthly average (2,013,916) was, as usual, in June (in 1913 it was in July, but in every other year in June), but the actual maximum was on July 2nd with 6,726,000. The genus was entirely absent from our gatherings after July 16th until October 12th, from which date till the end of the year the largest haul was only 6,000 (October 18th). Only in 1908 have the autumn numbers for Rhizosolenia been lower than in this year. Thalassiosira.—This genus was less abundant in 1914 than in the previous four years, but the numbers were higher than in 1907-9. It appeared in our nets for the first time in the middle of March (with the exception of 20 specimens in one haul in January), and the numbers increased to the maximum of 750,600 on May 7th, and then fell to zero by the end of the month. It was represented again from October 9th to 23rd (15,000 on October 15th), and on two occasions in November (1,000 on the 9th, 300 on the 23rd), and was then absent for the rest of the year. 992 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Guinardia.~-The numbers for 1914 are lower than usual, the maximum being only 1,740,000 on June 11th. This year agrees with every other recorded year in having the Guinardia maximum in June. The highest numbers for the autumn were only 8,400 on October 12th, and 8,500 on the 18th. Lauderia.—The maximum of this genus has been sometimes in April and sometimes in May. This year it — was in the latter month, with 3,600,000 on May 4th, and the high monthly average of 831,800. Absent after June Ist until September 28th, the numbers then rose again to 88,000 on October 15th, and fell to zero by November 19th. : We give here, for comparison with those published in other years, the table showing the monthly averages of the above seven genera of Diatoms. 1914. | Biddul- | Chaeto- | Coscino- | Rhizoso- | Thalassi-| Guin- Laud- phia. ceras. discus. lenia. | osira. ardia. eria. Jan. ...) 10,075 8,195 7,397 55 2 132 Heb: ...|. 22,230| 36,124 8,257 7 0 560 Mar. ...| 45,100) 625,606) 49,839} 2,642 2,969 4,331 162 April ... 9,972) 2,355,146) 329,406 93,167 8,114 55,100} 373,193 May ... 2,652|20,832,575 17,285) 417,112) 108,131 89,762} 831,800 June ... — 26 15,731 430] 2,013,916 0| 333,356 378 July ... 26) 15,085 42) 1,240,334 0 3,996 Aug. ... 6 374 0 0 0 0 Sept. ... 85) 206,032 25 0 0 0 Oct. ...| 26,140} 820,602} 38,002 1,638 3,329 3,113) 12,579 Nov. ...| 406,100} 110,187) 37,292 420 217) 3,462 275 Dec, ... 33,000 29,500 8,584 17 0 10 The increase and diminution of the several forms, month by month, is shown almost as clearly as it would be by a curve. ; DINOFLAGELLATA. The monthly averages for Ceratuwm and Peridinvum in 1914 were as follows :— - | - SEA-FISHERIES LABORATORY. © ~~. 293 f1914. Ceratium Peridiniunt- “4914. | Ceratium | Peridinium tripos. spp. | tripos. spp. January ...... 2,102 Pee Fuaby"s.is ra 3,987 1,582 February ...... 1,734 0 | August ...... 1,003 1,422 Lu 3,835 84 | September 1,335 20 UP rien ds ces: 7,993 3,519 October ...... 19,522 0 RF hase 12,947 17,322 November ...| 21,917 . 333 OP esata - 5,744 8,027 December ...| 15,571 0 This year agrees with 1912 in having the spring maximum of both groups in May, but the distribution in 1914 was more regular than in the former year. It will be seen from the figures given above that the curve of the monthly averages is very regular indeed, rising in the case of Ceratium from February to May, then falling to a minimum in August and rising again to the autumn (and this year higher) maximum in November. A similar regularity is to be noticed in the case of Perndimum. The actual maximal hauls are, for Ceratium, 30,000 on May 12th and 32,000 on October 15th, and, for Peridinium, 48,000 on May 14th, and 2,000 on November 3rd, this last figure being the sole record we have for the last three months of the year, and evidently, therefore, quite an exceptional occurrence. NOcTILUCA. Noctiluca miliaris was rather more abundant in 1914 than in the previous year, and the maximum appears to have been in May (10,200 on the 21st, 10,800 on the 28th). This is unlike 1912, when we stated that, ‘‘on the whole, Noctiluca seems to be least plentiful in spring and early summer, and to become more abundant in autumn.’’ Last year we recorded as characteristic ‘‘ the constant presence of the organism in small quantity.’’ But on the whole, as we then pointed out, it is more usual for ‘* Voctiluca to be either totally absent, or present in great profusion.”’ 294 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. CoPpEPOoDA. We give here, as usual, a short summary of the occurrence of the commonest species of Copepoda. Calanus finmarchicus.—In previous years the summer maximum has usually been in July, with a second climax in October (September in 1913); but in 1914 the maxi- mum was in August (7,320 on the 6th), and the numbers were then low till the end of the year. Pseudocalanus elongatus.—The maximum haul this year was 41,000 on June 22nd, but the monthly average for April was higher than that for June. In the curve of the monthly averages we have three high peaks, in April, June and August, with depressions in May and July. © Oithona similis.—The maxima this year were in July and November, with a smaller one in September. In July the largest hauls were 54,180 on the 2nd, and 73,600 on the 24th; while in November there was an unusually large haul of 199,300 on the 9th. This late autumn maximum was much higher than in any previous recorded year. ASE Vins Temora longicornis.—The highest monthly average was this year in June, but the two largest individual hauls were one in April (23,200 on the 24th), and one in July (18,190 on the 27th). From this summer maximum the numbers fall off gradually to the end of the year. _ Paracalanus parvus.—The maximum was, as usual, in September, following the summer minimum, and this year it was very much higher than we have had to record before, the largest haul being 138,300 on September 10th. Acartia clausi.—The numbers were, on the whole, lower than in most recent years, the largest haul being only 17,400, on July 13th. There were only six hauls with numbers exceeding 10,000, two in June, one in July, SEA-FISHERIES LABORATORY. 295 | and three in August, so that the maximum, though low, | appears to be spread over these three months. Anomalocera patersoni.—This species was present in eleven hauls taken during April, May and June, the largest being 4,800 on April 24th, and the others ranging from 340 to 2. The large April haul probably represents an unusual invasion of this oceanic species. Centropages hamatus.—Centropages is only entirely absent from our nets in two months during 1914, namely, January and December, but the numbers are, as usual, not very high. The maximum was 500, on August 20th. We need give no further records of Microcalanus pusillus. It is apparently no longer of importance in our district, and there are several other forms, such as /sias clavipes, which are quantitatively more worthy of record. The monthly average hauls for the eight more important species of Copepoda in 1914 are as follows :— 1914. F alanus. Pseudo- | Temora.| Centro- | Anomal-| Acartia.| Oithona.| Para- | calanus. pages. | ocera. calanus. January . 13 1,175 6 0 0 131 3,302 257 February . | 3 855 4 1 0 50 5,520 555 |) March ...... 2 1,091 197 6 0 35 5,755 91 i April ......... 613 | 13,332 | 3,369 15 611 643 | 7,392 67 Eee 144 | 2,119 2,160 73 28 2,192 5,200 48 ED. dy r02s cn 610 | 11,003 | 4,229 21 4 5,501 5,891 0 Be ibis oan 1,540 | 4,998 3,722 87 0 4,720 | 23,860 2,019 August ...... 1,687 | 10,679 2,018 103 0 5,444 | 11,932 5,857 | September 74 | 3,731 853 98 0 2,707 | 16,937 | 44,207 | October 47 4,020 92 69 0 4,409 8,987 9,746 November... 19 1,664 12 1 0 921 | 45,845 6,013 December | 2 1,456 0 0 0 303 | 14,874 1,817 Again we find there are slight differences in detail between this and other years, but on the whole the curves are in general much the same, and the history throughout the year can be readily followed by the eye. For example, 7emora is obviously a summer species with its maximum in June and a minimum in mid-winter, while 296 TRANSACTIONS. LIVERPOOL BIOLOGICAL SOCIETY. Paracalanus shows a minimum in spring and. early summer and a maximum_later in the autumn. CLADOCERA. The numbers for this group were again low,, as they were in 1913. It is obvious from the following histories that our two species of Cladocera maintain their character as a summer group. | | Podon intermedium first appeared in the Bay gatherings on April 27th, and was usually present from that time onwards to October 18th. It was most abundant at the end of May and beginning of June (850 on May 28th was the maximum), and again in ae (e.g., 560 on the 13th). Evadne nordmanni made its first appearance in the Bay on April 9th, reached its maximum of 1,370 on May 28th, was present in varying quantities up to August 6th, and again from September 3rd to 19th, and on October 28rd. SAGITTA. Sagitta bipunctata was again represented in practi- cally every haul of our nets, but the numbers were lower than usual. On only twelve occasions throughout the year in our “‘ official ’’ hauls did the numbers rise to over 100, and the maximum (on May 28th) was only 400. It ought to be noted, however, that while the ordinary surface gatherings taken across Port Erin Bay in spring contained only small numbers of Sagitta, we found that by using weighted nets in a deeper zone of water outside larger hauls of Sagitta were obtained. We — had found the same on previous occasions, and it is evident that sometimes most of the Sagztta are below the surface. SEA-FISHERIES LABORATORY. 297 OIKOPLEURA. Oikopleura dioica was, as usual, present in the Bay gatherings throughout the year. The maximum haul was 30,060 on June Ist, but the highest monthly average was in April (10,403 as against 10,099 in June), while there ‘was a depression between these two peaks in May, when the monthly average was only 2,387. The numbers fell in July and August, but were fairly high again in September and October (10,910 on September 28th; 9,500 on October 18th; 8,550 on November 3rd). On the whole this record is more like that of 1912 than of 1913; but the differences between the three are not very great. Various LARVAE. ECHINODERM LARVAE were again, as in 1913, most abundant in February and in March, but the numbers reached were not quite so great—20,000 on February 26th being the maximum haul at that time of year. A few were present in other months, and there was a quite exceptional haul of over 25,000 on June 22nd, 1914. POLYCHAET LARVAE were present throughout most of the year, and the record was very much like that of 1913, the maximum being again in spring. The largest haul in 1913 was 115,000 on March 3rd, and that of 1914 was 150,200 on March 7th. The “‘ Mrrrarta’’ Polychaet larva is again seen by this year’s record to be a cold water form, having its maximum in spring and being rare or absent in the warm months of summer. The largest hauls were 2,580 on January 13th, 8,200 on February 26th, 4,100 on March 18th, and 10,000 on April 9th. Although February and April show the greatest individual hauls, March has the highest monthly average (1,875 against 1,431 in February 298 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. and 1,444 in April). The lowest monthly average is 9, in July. Cras ZoEAS were present in very small quantities in 1914, in most months the numbers recorded being only two or three in a haul. The largest haul, and, in fact, the only one which runs to three figures, is 320 on April 24th. A considerable haul of the ‘‘ Mysis’’ -stage of Crangon, amounting to about 560 individuals, was obtained on April 8th, at a mile off Spanish Head, with a net weighted so as to tow at about 5 fathoms. Crangon larvae, although frequently present, usually occur in much smaller numbers per haul. The Nave of BaLanus ranged in 1914 from January to May inclusive, and during these months the average haul and the greatest single haul were as follows :— NAvpP.Lius STAGE. | Cypris STAGE. 1914.00 |} J Average. Greatest haul. Average. Greatest haul. January ... 17 90 a == — February... 6,504 39,800, on 26th — — March ...... 20,330 126,800, on 24th — = Avaril yoo oce 14,509 76,600, on 24th 16 140, on 30th May s.coce ~ 3,600 28,000, on 4th 343 1,920, on 21st ume: 1220.8 —_ — 7 61, on 1st After this large haul early in May the numbers fell off rapidly, and not a single Nauplius was captured in June. In the previous year the range was from the end of January to late in June, and the largest haul was over 450,000 on March 26th, a good deal larger than anything obtained in 1914. The Cypris stages made their appearance (two individuals) on April 20th, and increased slowly up to 1,920 on May 21st, and then, after a haul of 61 on June Ist, rapidly disappeared, the last captured being a- single specimen on June 25th. The great reduction in SEA-FISHERIES LABORATORY. 299 numbers in passing from the Nauplius to the Cypris stages is always interesting. GASTROPOD LARVAE Were again present in considerable quantity in every month of the year. The largest individual hauls were on February 26th (5,200), October 28th (5,800), and November 9th (13,800). In the previous year the largest hauls were in March and November. It looks as if there were two different sets of Gastropods, the one reproducing in early spring, and the other in late autumn. These larvae obviously require closer study and identification. LAMELLIBRANCH LARVAE were also present in quantity throughout the year, and were more abundant than the Gastropods in every month. The highest records were on February 26th (8,800), May 12th (6,050), September 15th (15,200), October 31st (16,000), November 9th (13,000), and December 9th (15,800). These numbers, though not quite so high, correspond fairly well with those for 1913. MEDUSAE AND CTENOPHORA. Although Medusae were present in small numbers throughout the year, they only reached a hundred or over per haul in April, May, June, and October (500, the maximum, on October 9th). The numbers are a good deal smaller than those present in June, July, and September of 1913. Ctenophora do not seem to have ever been present in any quantity in 1914. Nothing corresponding to the vast swarms of Plewrobrachia which we have occasionally met with in the past occurred during this year. Fisu Haas. Mr. Andrew Scott has dealt with the fish eggs in more detail in a separate article* in this report, so we * See p. 274. 300 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. need only note here that the Rockling eggs ranged in occurrence from January 19th to October 9th, and attained their highest monthly average (80 per haul) in May; while all other fish eggs together ranged from the end of January to the end of July, and the highest monthly average was 106, in April. The greatest haul of Rockling eggs was 301 on May 21st, and of all other fish eggs taken together 445 on April 9th. It must be remembered that these numbers are for Port Erin Bay, and may be greatly exceeded by hauls in the open sea outside. Eggs belonging to as many as eight species of food-fishes occurred in some of the hauls in the open sea during April. FuRTHER REMARKS. We have no special occurrences of rare or oceanic forms to record this year; but we may note the presence of two interesting organisms which have appeared frequently in the plankton from the Lancashire coast (Barrow Channel), and which we figure, from living specimens, asa help to the identification. Figures 1 and 2 show an organism which is not uncommon in our local plankton. Seen from above (fig. 1) it is quite circular in outline, and with the excep- tion of the protoplasmic bodies in the centre, it is perfectly transparent, so that the periphery is difficult to make out. The central area inside the outer rim seems curved so as to have a biconvex shape in profile view (fig. 2). It always contains one, frequently two, and rarely three or four, of the circular protoplasmic bodies. This seems to be the organism named by Hensen (who recorded it from the Western Baltic) the ‘“ Barbierbeckenstatoblast.” 7 SEA-FISHERIES LABORATORY. 301 Figures 3 and 4 show an organism which is less commonly met with here. In this form the outer rim curves upwards, and we have never seen more than one protoplasmic body in the centre. The central cavity, containing the oval-shaped protoplasmic body, is some- what like a spinning-top with a bluntly rounded point. This form appears to be the organism described by Cleve as Pacillina (fungella) arctica, from plankton collected by the Swedish Expedition to Spitzbergen in 1898. It has also been recorded from the North Sea, the Skagerrak and the Kattegat. A form described by Vanhéffen under the name ‘‘Chinesenhut’’ closely resembles Cleve’s Pacillina, but differs in the outer rim being turned down- wards and in having the produced part of the capsule containing the protoplasmic body quite pointed. 302 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Both these forms, or very closely related organisms, are figured by Lohmann in his “‘ Kier und Cysten des Nordischen Planktons,’’* and he is of opinion that the two are closely related and that Bergh’s view that they are Molluscan egg-capsules is correct. 'Two or three other observers have expressed similar opinions since. We are certainly inclined to regard both of these as Gastropod eggs, and the form shown in figs. 1 and 2 has a close resemblance to the egg-capsule of the common Littorina littorea. Dr. W. M. Tattersall has kindly sent a sketch of the egg-capsule of L. littorea, on which he has been making observations,+ and this shows the closest resemblance to our fig. 1 above. We give here the usual temperature chart of Port Erin Bay, drawn from Mr. Chadwick’s daily records. JAN. FEB. MAR. APR. MAY JUNE. JULY. AUC. SEPT OCT NOV_ DEC. 310172431 7 14 2128 7 1421284 11 18252 916233061320274 II 1825 1 8 152229 5121926 310172431 7 14 21285121926 es( TTT TT TUTE eee ee oF tT TT TP et EEE CEE es eS ee es( tT TTT TPCT EEE EE ES SSS See e2; ETT TTT TTT errr Pee a Ss Gti} TTT TT TTT PE ee See 6OLT TTT TTT Perr ee ET EE a i aa sort TTP TLE ARE eS eee eee SS(TT LPP PERE ee 87 CCE eT ee os ae SsetLTT Er PERERA Ee eee 55 Cee aa a @ eet tt ee m S317 TPIT asetitt Ti tr Pr ee @ Oli tT Tet ty TT Pee ae ee ee #50( 177 TELE EIT TIA Cees ECE ee Se ~“49[ [TUT EPS eee a 4S sielat TTT CPT 247 et Ae eae ee ee Pa 5 70 AS eee Ee eee S49 lel ba epee tee IV TTT Sah a a @ 441 IAT RYT AT TY Oe SESS SEES SERS RR Se eee ease eeeees SSUsE +435 UT UT TY [4 as SESE SERRE RRSRERRERREER ee Biesih — a 2 aie HERES REPRE ERR RRERDE 41) oie aS [ey as aha ERRERREDRRERRRERRESAeees LL ERE® 40 Tia tt ft | BEER SEARS PERRET 59 dias Ts hg Be [| 38 Cee | | YAuniva # pope 36 [ IY] | al aia 35 aul | | * Nordisches Plankton, Bd. I., Lief. 10, p. 17. +t Which will be shortly published in Irish Fishery Bd. Sci. Investig. i 11. 12. Por ee ee Pe L.M.B.C. MEMOIRS. 303 Mo. XXII. -LUBIFEX BY GERTRUDE C. DIXON, B5Sc., LECTURER IN BIOLOGY, KING’S COLLEGE FOR WOMEN, LONDON TABLE OF CONTENTS. Introduction le Methods ... Il. Historical Survey III. General Remarks External Characters Body Wall Coelom and Coelomic Fluid ... Alimentary Canal Circulatory System Nervous System Excretory Organs Reproductive Organs ... Parasites Gonads wae ee ate ‘ Development and Structure of Spermatozoa Sperm Sac ate the ate Sperm Ducts or Vasa Deferentia Development and Structure of Ova ... Egg Sac ... Oviducts ... Spermathece Spermatophores ... Cocoon Bibliography Explanation of Plates [From the Zoological Department, King’s College, London.] PAGE 304 304 305 308 309 315 318 320 326 333 341 347 349 351 356 358 371 376 377 378 382 387 388 392 395 304 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. INTRODUCTION. I. MetHops. My investigations have been carried out on the living worm by teasing out certain parts of the body, and by means of a large number of series of transverse and longitudinal sections. I have found the method of teasing out certain parts of the worm, either in water or salt solution, quite invaluable. By this means all the reproductive organs can be liberated and examined in the living condition, and very pretty staining effects have been obtained by the addition of methylene blue. Certain parts of the nephridia can also be obtained in this way, especially — the vesicle referred to later. The vesicle is always removed with portions of the nephridial tubes, but it is difficult to remove all the nephridium because the tubes are much coiled, and the nephrostome lies in the next segment in front as in all other Oligochaeta. In preparing specimens for section cutting, various preserving fluids and staining reagents were employed. The killing process is a difficult one, as the contortions of the worm on the addition of any chemicals are very violent. The worm, if left to itself during the process, becomes so bent and shrunken that it is useless for sectioning. It is advisable, therefore, to hold the worm at both ends while it is being immersed in the killing fluid, and thus any serious contraction of the internal organs may be prevented. The great disadvantage of this method is that the brain and anterior part of the nerve cord are usually pressed out of shape, but it is easy to hold a few specimens nearer the middle, while killing them, when the anterior end can be obtained in fairly good condition. TUBIFEX. 305 For killing and preserving, Tellyeoniczky’s aceto- bichromate was first used, but the tissues shrank badly in this reagent. J then tried Perenyi’s fluid and Bles’ fluid,* and these, while killing the worm very quickly, do not cause the tissues to shrink. Although, later on, other killing reagents were used, I found none so generally satisfactory as Bles’ and Perenyi’s fluids. Zenker’s fluid was used, especially for nephridia, with good results. The staining reagents most commonly used were borax carmine with picro-indigo-carmine as a counter stain, and brazilin,t which is a very delicate and effective stain. Iron haematoxylin was also used, and_ this rendered the muscle fibres very distinct. Il. Huzisroricat SuRVEY. Before entering upon a description of the minute anatomy of Tubifex rivulorum, which forms the subject of this Memoir, it will be interesting to notice to whom we are indebted for the present condition of our knowledge of this form. As early as 1745 Bonnet referred to it, but was content with describing certain peculiarities in the habits and general form of the worm, and with referring to its method of regeneration after artificial fission. He did not attempt to give any details of its structure. Schoeffer in 1764 gave a figure and description of Tubifex rivulorum, which he called ‘‘ Kleinen Wasseraal.”’ * Bles’ fluid ine oe 90 parts 70% alcohol. 7 ,, strong Formol. 3 ,, Glacial Acetic Acid. + Brazilin vis oa 1% Iron Alum in 70% alcohol. 0-5 % Brazilin; 306 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. O. F. Miller in 1773 classed it under Lumbricus as L. tubifez, but his description is very imperfect, and is largely corrected by D’Udekem. Lamarck (1816) separated Lumbricus tubifex and L. lineatus of Miiller’s Lumbricus and formed a new genus, Tubifex: the first he called Tubifex rivulorum, the second Tubifexr marinus. Owing to apparent similarities in the structure of these worms and that of the Naiads, Lamarck united them in a class Hespides. In 1842 Hoffmeister recalled the attention of naturalists to the old “‘ genus Lombric ”’ of Miller, which he divided into three new genera, Lumbricus, Enchytreus and Senurus, and he placed Tubtfex rivulorum in his new genus Senurus. It differed from the ‘‘ genus Lombric’’ by the absence of a gizzard and by the fact that the setae are of unequal length. His observations on Tubifea rivulorum are very incomplete and inaccurate- Grube in 1851 classed Tubifex among the Naiads under the name given to it by Hoffmeister—(Senurus variegatus), and he assigned to it the following characters: ‘‘Ohne Kiemen, Borsten Bundelchen zwei- zelich, obere Borsten haar und hakenformig, selten obere und untere hakenformig, Blut lebhast roth oder roth gelb.”’ Budge (1850-51) has provided us with descriptions of the genital and respiratory organs. These descriptions are more accurate than those of Hoffmeister, but still very incomplete. D’Udekem in 1855 published an account of the anatomy of the worm, dealing with all the systems in order, but studying them only in the living animal and in preparations made by teasing out parts of the body and then subjecting them to examination. His observations are certainly more detailed and accurate than those of F TUBIFEX. 307 earlier observers, but there are several inaccuracies, most of which have been noticed and corrected by still more recent observers. Many of the terms then in common use have since been discarded, and to some of these Beddard in his Monograph of the Oligochaeta refers, viz., the capsulogenous gland is now known as the spermatheca, the cloaca as the spermiducal gland, the vesicula seminalis as the sperm sac and the matrice as the egg sac. j In 1871 McIntosh published a paper in which he gives short accounts of certain of the organs, but no complete description of any system except the circulatory, as he concentrated attention on certain minor points such as the perivisceral fluid and corpuscles and the granular glands which form a complete investment around the intestine and dorsal vessel. His results will be considered in detail later. Vejdovsky in 1884 published his well-known work “System und Morphologie der Oligochaeten.’’ In this he makes frequent reference to Tubifex, and increases a good deal our knowledge of the structure of this worm. But in a work which embraces the whole of a large group it is impossible to describe in any great detail the structure of one member of that group. The same may be said of Beddard’s Monograph of the Oligochaeta, published in 1895. Several other papers have been inserted in the Bibliography at the end of this work. Some of these deal only with one small part of the subject, and reference will be made to them in the right places. Others have been mentioned in the list because they have some more distant bearing on the work, but yet are sufficiently allied to it to be of interest. It has seemed advisable to revise all! this scattered work, and, by a careful and detailed investigation of the SSS ser: —— 308 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. worm, to build up a Memoir which shall give, as far as possible, a full account of the structure of this form, together with any other points of interest which may arise during the investigation. This paper, previous to its publication, was presented as a thesis for the D.Sc. degree of the University of London, and in this connection I wish now to express my gratitude to Professor Jackson who kindly photo- graphed the drawings for me. It is with pleasure also that I acknowledge my indebtedness to Professor Dendy for valuable suggestions made while the work was in progress and for careful criticism of the finished work, which was carried out under his supervision in the Zoological Laboratory at King’s College. Ill. Generat REMARKS. Tubifex rivulorum is found in large numbers in the mud of the estuarine Thames, where, at low water, they may often be seen as bright red masses. In every consignment of mud which was examined this species was always found associated with another worm—Limnodrilus udekemianus—both belonging to the family Tubificidae. The two worms can easily be distinguished from one another if they are examined under a low power of the microscope; but, with practice, it is also possible to separate them when in the mass. One notices that the anterior segments of all of them are reddish in colour, but there is a marked difference in the posterior segments of the two forms. In the case of T'ubifex rivulorum the posterior segments also are red in colour, but in ~ Limnodrilus udekemianus the red colour is masked by the presence of pigment in the body wall. The pigment is yellow or orange in colour, and is confined to a narrow TUBIFEX. 309 band in each segment, forming an incomplete ring around it and giving the worm a striped appearance. If specimens of both worms are examined under the microscope, it will be seen that the setae are different, Tubifex showing two kinds of setae, capilliform and sigmoid, while Limnodrilus has only sigmoid. If the receptacle in which the worms are being kept is suddenly jarred with a sharp knock, they all simultaneously contract, hiding themselves almost completely in the mud. Twubifex rivulorum is also sensitive to light, for, if a bright beam from an arc lamp be projected through the water on to the mass, all the worms belonging to this species contract instantly and disappear. Limnodrilus udekemianus appears to be unaffected by the light, for the posterior end of the body still continues to wave about in the water. If individuals of both species be placed on a slide, it is possible to distinguish them by their movements. Tubifex rwulorum is much more rapid in its movements, and retains its activity much longer than Limnodrilus udekemianus after being deprived of the mud in which it lives. | EXTERNAL CHARACTERS. Tubifex rivulorum is a small worm varying from 30-50 mm. in length, and is of a bright red colour. The colour is due to the presence of red blood, which is clearly visible through the transparent body wall. The worm is bluntly pointed at both ends, the widest part of the body being in the anterior third. The body is divided throughout its length into a number of segments, all quite clearly marked off from one another, and decreasing in size towards the posterior end. The number of segments 310 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. in a fully-developed worm varies from 112 to 180. The 3) worm never exhibits any ‘‘ secondary’ annulation of the segments, such as occurs so generally in Limnodrilus. The Prostomium: This appears as an outgrowth or process of the first segment. It is conical in shape with a slightly blunted apex and overhangs the mouth on the dorsal surface. It is separated from the first segment by a transverse furrow. It is extremely sensitive, and is undoubtedly used as an organ of touch, and consequently is plentifully supplied with nerves which arise from the brain. Setae: The setae, as in all Oligochaeta, are the organs of locomotion. They consist of chitinous rods derived from specialised cells of the epidermis. Part of each seta is buried in the body wall, and may project into the body cavity, but the rest protrudes beyond the surface of the body. 26 The development of these organs has been carefully observed and described by earlier writers, especially Vejdovsky, who first established the fact that all setae are ectodermal in origin. A varying number of setae are ‘ implanted in “‘setigerous sacs or follicles’’ which arise as invaginations of the epidermis. The arrangement of the setae in a sac is like that of the sticks of an open fan (Pl. II, fig. 5). Hach seta has its origin at the base of the sac, but it is lodged in a separate cavity and divided from its fellows by a small piece of the body wall. The cuticle, which forms the outermost layer of the body wall, is continued into each of the cavities, and forms a lining to it for the greater part of its length. Near the blind end of the sac the cuticle is absent, and is replaced by a large TUBIFEX. 311 group of ectodermal cells whose boundaries are indistinct, but whose nuclei are large, round and nucleolated. According to Vejdovsky, any of these cells are capable of giving rise to new setae to replace those which may have been lost. The free ends of the setae are seen constantly to change their position as the worm rapidly coils and uncoils itself. Sometimes they are directed forwards, then backwards, and often, especially when the worm is quiet for a moment, they are placed almost at right angles to the body. When the worm is crawling forwards the setae are always pointing somewhat backwards, but a sudden twist of the body is sufficient to change their position completely. There are two sets of muscles by which the setae are moved : — (a) The parieto-vaginal muscles, which are attached to the base of the setigerous follicle and to the body wall on all sides of it. These muscles regulate the movements of the whole follicle. If those lying on the posterior side of the sac contract, the inner ends of the setae are drawn back, and the free ends are pushed forward, whereas, if those muscles on the anterior side contract, the setae are directed backwards (PI. II, fig. 5, m.p.). (b) The intrafollicular muscles (fig. 5, mjf.). These are attached to the body wall and to the individual setae, and thus the movements of the setae can be regulated. The development of the setae in this worm resembles, in all essentials, that of the setae of other Oligochaeta, and therefore it is only necessary to refer to it briefly. The setae appear as small cones of chitinous substance at the bottom of the sac. The apex of the seta is first developed and then growth in length proceeds from the inner end until the seta has pierced the body wall and attained its full length. 58) WF TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. The setae are arranged in four bundles in every segment of the body except the first and the last. Two of these bundles are ventral in position, and lie one on either side of the mid-ventral line. These are spoken of as the ventral bundles (Pl. ITI, fig. 11, v.bl.). The other two are much more dorsal in position, and are referred to as the dorsal bundles (fig. 11, d.bl.). Three kinds of setae are present, viz., capilliform, uncinate, and pectinate (Pl. IT, fig. 6). The distribution of the capilliform setae is limited, for they are confined to the dorsal bundles, and do not usually extend further back than the 30th segment. The uncinate setae are common to both dorsal and ventral bundles throughout — the body, while the pectinate form is limited to the dorsal bundles of the anterior segments, and is absent from some of these. The capilliform setae are long, straight, hair-like in form, and narrowing to an extremely fine point at the free end (Pl. II, fig. 6, d). By far the greater part of the seta is exposed, only about a quarter of it being embedded in the setigerous sac. These setae are quite smooth and devoid of barbs, such as are described by Beddard (1895) as occurring in Lophochaeta ignota. The longest ones occur in segments 6 to 9; behind the latter segment they become smaller and smaller, until, at last, they completely disappear. It is very unusual for there to be more than three capilliform setae in a bundle, and very commonly only one or two are present. In those cases where three setae are present, they vary consider- ably in length, and as a rule two of them are long while one is much shorter. Beddard (1895), in his classification of the setae of Oligochaeta, divides them into two main groups :— (a) Long, slender setae gradually diminishing in diameter towards the pointed extremity—capilliform. TUBIFEX. ais (6) Shorter setae of a curved form, something like an elongated S with a thickening at about the middle— sigmoid setae. The uncinate and pectinate setae of Tubifex rivulorum belong to this latter class. Of these, the uncinate form is much more common than the pectinate. They are both of the same general shape, but the thickening referred to by Beddard is not very strongly developed. Neither does it occur at the middle of the seta, but is always nearer the outer free end and approxi- mately divides the seta into a distal third and a proximal two-thirds (Pl. II, fig. 6, a) In the uncinate setae the distal end is bifid, but the two prongs vary somewhat in size and shape in different setae. The uncinate setae in the dorsal bundles have the two prongs equal in size and of the same shape, that is, somewhat narrow throughout and tapering gradually to a very fine point at the distal end (Pl. II, fig. 6, b). In the ventral bundles, where the uncinate setae only are present, the two prongs are of slightly different size and shape. The dorsal one is somewhat slender, and comes to a sharp point at the tip. The ventral one is shorter than the dorsal, and of a blunter nature, its apex being somewhat more rounded (Pl. II, fig. 6, a). There is never, however, such a marked difference in the size of the prongs as in the case of Limnodrilus, where the ventral one is always much shorter than the dorsal. The number of uncinate setae which may be found in a bundle differs somewhat in different regions of the body. For instance, in both dorsal and ventral bundles of the first two setae-bearing segments there are never more than two setae, and these are very small compared with those which come close behind. In segments 3 to 9 or 10 they reach their maximum size, and there are often four or five present in the ventral bundles, but rarely more than 314 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. three in the dorsal ones, where they are accompanied by capilliform and sometimes by pectinate setae. More posteriorly they decrease in size and number, soon dwindling down to two in a bundle, and near the posterior end of the body it is usual to find only one small seta in each bundle. The pectinate setae are, as already mentioned, less numerous than the uncinate type, and are confined to the dorsal bundles of the anterior segments. There, there- fore, it is possible to find in the same bundle setae of three different kinds: capilliform, uncinate, and pecti- nate, the total number rarely exceeding six: The pectinate setae resemble the uncinate of the dorsal bundles in general form. The only difference between them is the presence, in the pectinate form, of subsidiary prongs between the two main divisions of the seta (Pl. II, fig. 6, c). These subsidiary prongs are figured and described by McIntosh, Lankester and Beddard. Their number varies from 1 to 4, the latter number occurring most rarely. iy I have not yet been able to identity the webbed setae described by Lankester (1871). He discusses at some length the form of the sigmoid setae in this worm. He states that he has often seen sigmoid setae, in the dorsal bundles of the first ten segments, which are provided with a web between the two prongs. Benham, who also investigated this species, could not identify the web. During my observations on the setae I have particularly looked for the appearance of this web, but have always failed to find it. A possible explanation of this difference of opinion is that Lankester was looking at the pectinate setae which do occur in the dorsal bundles of these anterior segments only, and mistook the subsidiary prongs for a continuous web stretching across between the two main prongs. Again, Lankester recounts how he has seen a TUBIFEX. 315 a number of hairs (6 or 7) surrounding certain of the setae near the apex. He states that they result from the splitting up of the horny substance of the seta. He goes on to describe how a number of small dark particles are placed at intervals along the hairs. It is quite easy to recognise the condition that he describes, but it seems to me that his observations may bear another interpretation. When the worms are kept in the laboratory for a time, even though they are placed under running water, they become infested with fungus growths. These have the form usually of long, delicate filaments which appear to have their origin between the prongs of the setae, and present exactly the appearance described by Lankester. THE BODY WALL. As in most Oligochaeta the body wall consists of the following layers :—(1) Cuticle, (2) Epidermis, (8) Circular Muscles, (4) Longitudinal Muscles, and (5) Peritoneal Epithelium. 1. The Cuticle is a delicate layer of non-cellular nature which lies outside the epidermis, and is formed as a secretion from the epidermal cells. It completely invests the body, and is about 3 u thick. 2. The Epidermis consists of a single layer of cells throughout, but varying somewhat in thickness in different parts of the body. For purposes of description we may divide the epidermis into :—(a) Ordinary or extra- clitellar epidermis; and (b) Clitellar epidermis. (a) Ordinary or Extra-Clitellar Epidermis. The ordinary epidermis consists of two well-marked types of cells, viz., gland cells and columnar supporting cells, and is from 6m to 84 thick. The gland cells are large, somewhat irregular in outline, and with the nucleus, as a rule, situated in the lower half of the cell. The | Hi 4 5 i 316 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. cytoplasm of these cells is densely granular. The nuclei are rounded or slightly oval in shape, and each contains a nucleolus (Pl. II, fig. 5, hyp.). The gland cells are separated from one another by the columnar epidermal cells, or ‘‘supporting cells”’ as Atheston (1898) calls them. They are narrower than the gland cells, generally columnar in outline with the broader end lying next to the cuticle. The nuclei are oval in shape, quite conspicuous, and situated near the middle of the cell. , (6) Clitellar Epidermis. The clitellum does not develop until the worm has nearly reached sexual maturity. It then becomes differentiated from the ordinary epidermal cells, and is confined to the principal reproductive segments, viz., segments 11 and 12. It has the form of a complete girdle, but is less well developed on the ventral than dorsal surface. It merges gradually into the ordinary epidermis anteriorly and posteriorly. It is discontinuous, of course, at the openings of the setigerous follicles, the spermathecal pore and penis. It is composed of a single layer of cells throughout. At first, the cells are little different from the ordinary gland cells of the epidermis. The nucleus is a con- spicuous rounded body, situated at the base of the cell, — and exhibits a very well-marked reticulum with a nucleolus. The cytoplasmic contents of the cell are finely granular. This is the usual structure of the clitellum, even in individuals which appear to be quite mature, and, consequently, this is the condition which has been described by most observers. Atheston (1898), for example, says of the gland cells of the clitellum that they are smaller and more numerous than those of the ordinary epidermis, otherwise they are similar. TUBIFEX. 317 Ekitaro Nomura (1913), in his description — of Limnodrilus gotot, gives a short account of the clitellar gland cells as they occur in that worm. He says that the gland cells are 20-23 long, that is, four times the length of the ordinary clitellar cells, and 8-10 u across. “Three well-marked stages can generally be observed in mature specimens: a highly vacuolated condition, a more or less granulated condition, and one in which the cells contain many globules.”’ In most of the specimens of 7. rivulorwm that I examined the clitellar gland cells were only a little larger than the ordinary gland cells, and the contents were granular. In a few cases, however, the clitellum was enormously enlarged, and I can only suppose that the maximum development of the clitellum is not reached until a very short time previous to the forma- tion of the cocoon. The cells at this time may be as much as 40 to 45y long, and ll» to 144 across (Pl. III, fig. 12). They are almost oblong in shape, tapering just a little at the inner end. The nuclei are still visible, but are much less distinct. They remain near the inner end of the cell. The increase in size of the cells at this time is accompanied by the deposition in them of a large number of globules of the secretion which forms the cocoon. Many of the cells are quite full of the secretion, which masks, almost completely, the cyto- plasmic contents. In others, where the secretion is present in smaller quantities, it 1s arranged fairly regularly in the form of rounded masses, 2 # in diameter, which congregate principally near the lateral walls of the cell, where they are arranged in longitudinal rows (Pl. III, fig. 12, se.c.). The cytoplasm is somewhat vacuolated. Between these enlarged and modified gland cells are supporting cells, which are as long as, but much 318 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. narrower than, the gland cells. The nuclei of these cells are oval in shape, and may be situated in any part of the cell (fig. 12, an. c.). Certain fibres from the longitudinal muscular layer may turn outwards and come into close contact with the inner ends of the gland cells. 3. The Circular Muscle Layer lies just within the epidermis. It is an extremely delicate layer, and is with difficulty recognised in transverse and longitudinal sections. Occasionally it can be fairly clearly seen in an oblique section, when the individual fibres appear as incomplete bands surrounding the body (fig. 5, c. m.). 4. The Longitudinal Muscles are better developed than the circular, and can be seen in longitudinal sections of the body wall as elongate fibres. Seen in transverse section they have the appearance of flat plates or lamellae embedded in a granular substance. There is no axial core, as is so characteristic of the longitudinal muscles of Lumbricus, neither are they divided into dorsal and ventral regions by lateral lines, as is described by Nomura (1913) for Limnodrilus gotov (fig. 5, 1. m.). 6. The Peritoneal Epithelium consists of a single layer of somewhat flattened cells with prominent rounded nuclei. COELOM AND COELOMIC FLUID. The coelom of Tubifex rivulorum is, as is usual in the Oligochaeta, well marked and spacious, and divided into a series of compartments by septa. The number and arrangement of the septa correspond to the external segmentation of the body. The septa between segments 1 to 4 are incomplete. The coelom only communicates indirectly with the exterior—by means of the nephridia and genital ducts. There are no dorsal pores such as are a TUBIFEX. 319 found in earthworms. The coelom shows no division into different cavities, other than that due to segmenta- tion, with the exception of the egg sac and sperm sac, which are simply portions of the coelom bounded by special walls and arising as simple outgrowths of the coelom of certain segments. These organs will be described in detail with the rest of the reproductive system. The coelom is lined throughout by the delicate peritoneal epithelium which is also reflected over the other organs in the body cavity. The shape and size of the cells forming this epithelium vary considerably in different parts of the body, but for the present these modifications in its structure will only be mentioned briefly. The parietal and visceral layers are those which form the innermost layer of the body wall and the outer- most layer of the intestinal wall respectively. The cells of the parietal layer have already been described. The visceral layer is formed chiefly of much enlarged pear- _ shaped cells—the ‘‘chloragogen cells’? of Claparéde. This applies to the visceral layer around the intestine. In the region of the buccal cavity, pharynx and oesophagus, the cells of this layer very nearly resemble those of the parietal layer. In certain regions of the nephridial tubes the peritoneum is composed of very large, vesicular cells which are very easily detached from the tube and from each other. In other parts of the tube, chiefly those nearer the nephrostome and nephridiopore, the ordinary, flattened epithelial cells are present. : The coelom is always filled with a colourless coelomic _ or perivisceral fluid, by which all the organs of the body are constantly bathed. Suspended in the fluid are a number of colourless corpuscles which may be described Vv 320 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. as coelomic or perivisceral corpuscles. The number of these corpuscles in different individuals varies enormously, but two kinds are usually recognisable—amoeboid and spherical. In some individuals the spherical corpuscles are much more numerous than the amoeboid, while in others they are more evenly distributed. It is often rather difficult to decide whether a particular corpuscle is in the amoeboid or spherical condition. It is not really actively amoeboid, yet it is irregular in outline and with no clearly defined contour. It would seem, therefore, that transitional stages may occur at such times when an amoeboid corpuscle, having, as Beddard suggests, become loaded with granules of an excretory nature, ceases to be amoeboid and gradually becomes spherical in outline. Both kinds of corpuscles are granular, the granules sometimes being of a yellowish hue. THE ALIMENTARY CANAL. The general arrangement of the various regions of the alimentary canal can be clearly seen if the living worm be examined under the microscope, owing to the transparency of the body wall. If examined in this way, it will be seen that the wall of the alimentary canal in the first five segments is almost colourless, or only slightly yellowish in colour. In segment 6, however, the appearance changes very suddenly, for the wall has now a blackish or brownish hue, due to the presence of specialised cells of a glandular nature known as chlora- gogen cells, which will be described in detail later. More posteriorly the colour again changes, becoming gradually lighter and lighter until in the region of the anus the wall of the alimentary canal has again a pale yellow colour, As in all Oligochaeta, the alimentary canal has J - TUBIFEX. 321 the form of-a straight tube extending from the mouth, situated on the ventral surface of the first segment, to the anus, which is surrounded by the last segment of the body, or the anal segment. The alimentary canal can be divided into the following regions:—(1) mouth, (2) buccal cavity, (3) pharynx, (4) oesophagus, (5) intestine, and (6) anus. 1. The Mouth. The mouth is ventral in position, surrounded by the first segment of the body or peristomium, and overhung by the prostomium. When closed it appears as a transverse slit bounded by a slightly puckered wall, but when it is open the aperture is rounded (PI. II, fig. 4, mo.). 2. Buccal Cavity. The mouth leads into the buccal cavity, which is short—only extending through the first segment of the body. It is partly covered by the cerebral ganglia, dorsally. The buccal cavity is capable of extrusion, but this does not seem to take place very often under ordinary conditions. I have, however, frequently observed it when ether has been added to the water in order to quiet the worm when it is under observation. The buccal cavity can then be seen as a somewhat frilled organ protruding from the mouth. When once the cavity has been extruded, it is not readily drawn in again. A transverse section of the buccal cavity, when not _ extruded, shows it to be of wide calibre, with a straight, unfolded wall. The latter is very thin, and is composed of a single layer of epithelial cells, somewhat cubical in shape, with well-marked nuclei and numerous short cilia. Outside this layer of epithelial cells a few muscle fibres are scattered, but they are not sufficiently numerous to form definite muscular layers (PI. LI, figs. 4 and 7, bu.c.). 3. The Pharyne. Leading out of the buccal cavity is the pharynx, which extends through segments 2 and 3, §22 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. It is surrounded by the peripharyngeal connectives which connect the cerebral ganglia, lying dorsally, with. the sub-oesophageal ganglion or anterior end of the ventral nerve cord. The pharynx can never be everted, and is provided with exceedingly muscular walls, the musculature being chiefly dorsal in position. The lumen is folded and ciliated, the cilia being considerably longer than those of the buccal cavity (PI. II, fig. 4, ph.). 4. The Oesophagus. The pharynx opens into the oesophagus, which also extends through two segments (4 and 5), and communicates at its posterior end with the intestine. The oesophagus is a narrow tube, its lumen somewhat folded and ciliated. The wall is thin, consisting of a ciliated epithelium internally and a few muscle fibres externally, arranged in circular and longi- tudinal directions, the circular muscles inside (fig. 4, oe.). Situated in those segments occupied by the oesophagus, and, to a less extent, those occupied by the pharynx, are organs of a glandular nature known as septal glands. These organs occur in many Oligochaeta, but are specially well developed in aquatic forms, although they have been described by Vejdovsky as being very well marked in Allolobophora foetida. These glands are attached primarily to the septa, but may extend on to the walls of the oesophagus, and to a large extent lie freely in the body cavity. Each gland is a mass of pear- shaped cells, the narrower part of each being prolonged into a duct which opens into the oesophageal region of the alimentary canal. The cells possess very distinct nuclei, situated in the broader part of the cell (Pl. II, fig. 4, s.gl.). 5. The Intestine. The intestine commences in segment 6, and extends throughout the entire remaining length of the body to the anus, which, as already stated, TUBIFEX. Baa opens to the exterior on the last segment (Pl. II, fig. 4, im.). It is overlaid in the anterior segments by the supra-intestinal blood vessel, and more posteriorly by the dorsal vessel, and itself covers the ventral vessel which lies freely in the body cavity. The intestine is kept in place by the septa, which constrict it slightly at intervals, and by special muscles which pass from it to the body wall. Its lumen is larger than that of the oesophagus, and is unfolded. It is ciliated throughout, the cilia being specially long and abundant in the posterior segments of the body, especially the last six or seven segments. The structure of the intestinal wall from within outwards is as follows :— (a) A single layer of somewhat elongated cells forming the ciliated epithelium. The cilia are not very conspicuous except at the anterior and posterior parts of the intestine (Pl. III, fig. 13, ct.ep.). (6) A very thin muscular layer composed of a number of isolated circular (on the inside) and longi- tudinal (on the outside) muscle fibres (fig. 13, c.m.; l.m.). The wall of the intestine is very vascular, and the blood yessels are situated between the ciliated epithelium and the layer of circular muscles (fig. 13, b.v.7.). These vessels form what is known as the intestinal network (see circulatory system). (c) A glandular layer composed of a very large number of unicellular glands which cover the entire surface of the anterior half of the intestine, and also cover the dorsal blood vessel (fig. 18, c.c.). It is to the presence of these glands that the dark colour of the intestinal wall is due. They were called chloragogen cells by Claparéde, and this name is still applied to them. When examined in section, they are seen to be somewhat 324 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. club-shaped cells with a fine external membrane enclosing a large number of granules of two kinds, some being much larger than the others, and of a dark brownish hue. Both kinds of granules are very inert, and stains and other reagents have little or no effect upon them. If the body wall of the worm be ruptured, these cells, which are very easily dislodged from the wall of the intestine, can be liberated into the water. In the water they swell considerably, and the two kinds of granules which they contain can be clearly seen (Pl. III, fig. 14). The larger ones tend to congregate near the centre of the cell, the outline of which becomes rather indistinct and shadowy when the cell is immersed in water. The smaller granules can be more clearly seen near the periphery of the cell where the larger granules are not so abundant. All the granules of a cell which has been freshly liberated from the body of a living worm exhibit active molecular — movements, and this movement is kept up for some time if the wall of the cell is not broken. Gradually, however, this molecular movement becomes slower and slower until it finally ceases. The granules then appear to congregate at one part of the cell, sometimes at the side, sometimes nearer the middle, the rest of the cell being quite clear and transparent. Probably this massing together of the granules heralds the breaking down of the cell. All these points in the structure of the chloragogen cells can be made out in the fresh material. This amount of investigation was carried out with considerable care by McIntosh, and his results were published in his paper entitled ‘‘On some points in the structure of Tubifex’”’ (1871). In quoting Dr. Buchholz’ paper “ Beitrége zur Anat: der Gattung Enchytreus’’ (1862), McIntosh mentions that this author has put a distinct nucleus in = TUBIFEX. 325 all his figures of the chloragogen cells of Enchytreus. McIntosh appears to have made efforts to demonstrate the presence of a nucleus in the fresh, unstained chlora- gogen cells of Tubifex, but owing, as he says, to the large number of granules always present in the cell, he was not able to do this. It certainly does need considerable care in observation in order to see the nucleus, but it is comparatively easy to do so if the cells are floated on to a large quantity of water on a slide and a cover-glass put on. The cells then roll about in the water, and during their movements it is often possible to see the nucleus. McIntosh also states that even after careful examination of transverse and longitudinal sections of the worm he arrived at the same result. I find, however, that in any section of the intestinal part of the worm, which has been appropriately stained, viz., with borax-carmine, the nuclei can be very plainly seen, as they are large and each has a distinct nucleolus (Pl. III, fig. 18). The true shape of the cells is better seen in sections than by examining them in the fresh condition. In sections they are seen to be truly pear-shaped, the broader end lying freely in the body cavity. In such preparations the nucleus, which is oval in shape, is usually situated in the narrower part of the cell. It is quite useful to make permanent smears of these cells and stain them on the slide. A stain which differentiates the nucleus particularly well is Brazilin. In these smears the granules stain very slightly: not nearly so heavily as does the nucleus (PI. III, fig. 15). Rice (1902) has published a paper on the chloragogen cells of Lumbricus herculeus, and he enters in some detail into their origin, growth and structure, and gives some suggestions as to their function. It is evident from his descriptions that their growth and structure are very 326 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. similar to those of the chloragogen cells of Tubifex. Rice carried out a series of feeding experiments, and found that neither excess of food, nor starvation, nor a varied diet, had any effect upon these cells. He also pointed out that the granular contents are apparently lifeless, for no stain appreciably affects them, nor are they altered in any way by the addition of strong acids. The lifeless condition of the adult cell suggested to Rice the possibility that these cells have performed their function in the young worm—presumably they would have some connection with the elaboration of food, as they. cover the dorsal vessel as well as the intestine—and have become functionless in the adult. This theory is supported by the fact that these cells in a very young worm exactly resemble those of the ordinary peritoneum. In the larger worms a gradual development into typical chloragogen cells can be traced. The cells increase in length, the characteristic granules appear, and they become less and less responsive to stains, with the exception of the nucleus which stains throughout. Earlier observers believed these cells to be secretory in function, but, judging from their lifeless condition in the adult, their function, if any, would surely be one of excretion rather than secretion. CIRCULATORY SYSTEM. The circulatory system, as in all other Oligochaeta, is a closed one, having no communication with the coelom. It consists of a series of main vessels having a longitudinal direction, and united with one another by lateral vessels in each segment. The blood vessels are well-developed tubular structures, and their walls are very delicate. In none of the vessels does there seem to be an epithelial TUBIFEX. oat lining. Its place in these lower forms is taken by a delicate membrane, destitute, apparently, of any cellular structure. Beddard described the same condition in other aquatic worms, viz., Naiads and Enchytreidae. It is only in the true earthworms that this membrane is replaced by an epithelium consisting of large and conspicuous cells. In the smaller vessels, such as the periviscerals and intestinals, the wall consists of nothing more than this membrane covered externally by a single layer of flattened, peritoneal cells. Such branches must necessarily be non-contractile. The walls of the main. blood-vessels show a rather more complicated structure, but still remain very thin and delicate. The membrane, mentioned above, is supported externally by a few muscle fibres. The circular muscles are very few, and are situated just outside the membrane. The longitudinal fibres are better developed, and lie just underneath the peritoneal epithelium, which forms the outermost covering of the wall. Owing to the rapid movements of the worm, the dorsal and ventral vessels are constantly thrown into a number of zig-zags or S-shaped portions, which are rendered much more apparent by the addition of chloroform or ether to the water in which the worm is placed, when the move- ments of the worm become very violent. The main trunks which have a longitudinal direction are the dorsal, supra-intestinal, and ventral vessels. 1. The Dorsal Vessel extends through the entire length of the body from the anal segment to the prostomium (Pl. I, fig. 1, d.v.). It lies dorsal to the alimentary canal for the greater part of its length, but in the region of the reproductive organs it changes its position and comes to lie nearer the ventral vessel. Further back it reassumes its original position. | f ( i | h t i w i al uM Hi Pa ¥) | A ii 328 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. 2. The Supra-intestinal Vessel is always attached to the dorsal wall of the intestine, and is invested by a layer of chloragogen cells continuous with those which form the outermost layer of the intestinal wall (Pl. III, fig. 9, st.v.). It originates in segment 5 as an offshoot of the dorsal vessel which lies above it, and it extends through the body to a short distance behind the segments containing the reproductive organs. Nomura (1913) states that in Jamnodrilus gotor this vessel opens into the dorsal vessel again at the posterior part of the body. Although I have carefully examined many serial sections, I have not been able to find this second opening of the supra-intestinal into the dorsal vessel in Tubifer rivulorum. The vessel seems rather to diminish gradually in size, and finally to disappear. In those segments of the mature worm which contain the reproductive organs, the supra-intestinal vessel is slightly displaced and lies a little to one side of the mid-dorsal line. This is probably due to the pressure exerted by the reproductive organs on the other organs in the body. | ty 7 ‘ ‘ Are ht. inv, PV. Vv, Trxt-Fic. 1. Diagram of segments V—IX showing the positions of the principal blood vessels with their connections in these segments. d.v. dorsal vessel, si.v. supra-intestinal vessel, in. intestine, v.v. ventral vessel, pv.v. perivisceral vessel, in.v. intestinal vessel, At. heart. 3. The Ventral Vessel extends through the whole length of the body, and lies beneath the alimentary canal between it and the nerve cord (Pl. I, fig. 1, v.v.). It 7 ! TUBIFEX. 329 commences in segment 1, where it is paired, its two parts uniting with the two branches into which the dorsal vessel divides, also in segment 1. These two parts of the ventral vessel pass backwards as two converging trunks as far as segment 3, where they unite. It is attached to the intestine throughout the greater part of its length, but occasionally appears to le freely in the body cavity. It should be noticed that the nephridia of the posterior segments of the body are situated close to the ventral vessel with the walls of the nephridial tubes closely pressed againstit. In the last segment of the body the dorsal and ventral vessels unite and are slightly coiled. These longitudinal trunks are connected with one another by a series of commissural vessels. These are of two kinds: (a) intestinal, (b) perivisceral or coelomic. (a) Intestinal Vessels. The principal intestinal vessels, of which there is a pair to each segment, connect the supra-intestinal with the ventral vessel and are well developed in all segments behind the fifth. They have their origin from the supra-intestinal vessel near the middle of the segment and well in front of the peri- visceral trunks (Pl. VI, fig. 42, in. v.). These intestinal vessels do not lie freely in the body cavity but pass beneath the layer of chloragogen cells, and thus encircle the intestinal wall. In the posterior segments they are connected with the dorsal instead of the supra-intestinal vessel. In addition to this principal intestinal trunk, there are accessory vessels, which are, however, much less con- spicuous (fig. 42, 2.v.1). These are best seen by treating the living worm withetheronaslide. At first, this has the effect of causing strong contractions of the intestine and blood vessels, which rather hinder than aid the observer. But if the worm is allowed to remain on the slide for 330 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. some time these contractions become less violent, as the animal becomes exhausted, until, finally, all movement ceases except the occasional contraction of the intestine. The body of the worm becomes somewhat flattened, and the chloragogen cells, which at first completely hide the underlying tissues, become clearer. It is-then possible to investigate the blood vessels which pass to the intestine wall. These branches become filled with blood, and, if the worm is lying on its side, are comparatively easy to see. The number and arrangement of these smaller intestinal vessels vary in different parts of the body. The largest vessel, which has already been described, is constant. The variable ones are the accessory intestinal branches which he between the intestinal proper and the perivisceral trunk (Pl. VI, fig. 42). In the anterior segments of the intestinal region of the body there may be as many as five pairs of accessory intestinal vessels, all branched and forming a vascular network over the intestinal wall, but they do not appear to have any connection with the ventral vessel. In the posterior segments these vessels gradually decrease in size and number, and become very difficult to follow. (6) The Perwisceral Vessels are present in all segments of the body except segment 8. They arise in pairs from the dorsal vessel near the posterior border of each segment, and are connected with the ventral vessel below. These are quite large trunks, passing out almost at right angles to the dorsal vessel, and form in most segments a series of complex coils, often extending from one end of the segment to the other (Pl. VI, fig. 42, pv.v.). This coiling of the perivisceral vessels allows of ample freedom of motion for the worm—a very necessary precaution in view of its rapid and sudden movements. They do not branch, and, therefore, there is no integu- a y= | lat a - TUBIFEX. 331 mental network in this worm, such as is described by Beddard as occurring in Ilyodrilus and Bothrioneuron. In the anterior segments of the body the perivisceral trunks lie freely in the body cavity. More posteriorly, particularly in that part of the body which is waving about in the water, these vessels, while not branching, are always pressed closely against the body wall for a good part of their length, and remain in this position Text-Fic. 2. Diagrammatic tranverse section through segment 1X to show the relative positions of the principal blood vessels. hyp. epidermis, lm. longitudinal muscles, c.c. chloragogen cells, in. intestine, si.v. supra- intestinal vessel, d.v. dorsal vessel, v.v. ventral vessel, n. nerve cord. permanently. This would suggest that aeration of the blood takes place through the body wall,which is very thin near the posterior end of the body. At its anterior end the dorsal vessel divides into two branches, which become slightly coiled and then dip down ventrally to become continuous with the anterior ends of the ventral vessels. In segment 2 the perivisceral vessels are given off from the dorsal vessel near the posterior border of the segment. 332 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Instead of passing out at right angles to the dorsal vessel, as is the rule, they have a somewhat more forward direction and may extend into the segment in front, the septum between segments 1 and 2 being absent (Pl. VI, fig. 41, pv. v.2). They become slightly coiled, and then dip down to open into the ventral vessel a short distance behind their origin from the dorsal vessel. The perivisceral vessels of segments 3-7 have a similar distribution to that described above for segment 2, but in segments 4-7 they lie entirely in the segment to which they properly belong, owing to the presence of septa between adjacent segments, thus preventing the passage of the vessels from one segment to the other. It should be noted also that the right and left periviscerals of segments 2 and 3 open into the right and left branches of the ventral vessel respectively. In segment 8 the perivisceral vessels are absent. In segments 9 and 10 they become coiled around the anterior and posterior sperm sacs respectively. In segments 11-17 of a mature worm which contain the ovisac the periviscerals are always coiled around this organ. In all the segments behind those containing the reproductive organs these vessels pass simply from the dorsal to the ventral vessel. The ‘‘hearts’’ are situated in segment 8. They originate from the supra-intestinal vessel above, in the posterior part of the segment, and open into the ventral vessel below (Pl. I, fig. 2, ht.). They are much enlarged, especially near their origin from the supra-intestinal trunk, and are contractile. The blood is a red, non-corpusculated fluid, the haemoglobin being dissolved in the blood plasma. TUBIFEX. 339 THE NERVOUS SYSTEM. The nervous system of Tubifex rivulorum is formed upon the same plan as that of all Oligochaeta. It consists of cerebral ganglia united to a ventral chain of ganglia or nerve cord by peripharyngeal connectives. As in most other Oligochaeta the whole nervous system lies com- pletely in the body cavity. It is possible to examine the arrangement of the ganglionated chain and peripheral nerves in the living worm, especially after the addition of ether to the water in which the worm is lying. The ether increases the transparency of the body wall so that the internal organs can be seen more easily, and in some cases the alimentary canal becomes so contorted that the ventral nerve cord is left quite freely exposed for a considerable distance. This is a very useful method of investigation, as it enables one to compare the form and size of the ganglia, and the proportion of connective to ganglion in the segment in different parts of the body, and these proportions vary a good deal. It is, however, of very little use for a detailed examination of the brain. In the living worm it is usually very difficult to define the outlines of the brain, even after the addition of ether. It has happened, however, very occasionally, that I have been able to see the brain quite clearly by this method. The difficulty which is usually experienced in . examining the brain in the living condition is due partly to the greater transparency of the brain compared with that of the body wall, and partly to the contractions of the pharynx over which the brain is situated. The great drawback to the use of ether is that the blood vessels in the anterior region of the body become somewhat distended and quite full of blood. This, of course, only serves to obscure the brain more. 334 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Even though one may be fortunate enough occasionally to get a good idea of the shape of the brain, it is better to complete the investigation by means of serial sections, which are, of course, necessary for a proper examination of histological details, both in the brain and ventral nerve cord. Many attempts were made to make use of zntra-vitam methods of staining, as it was hoped that the nervous system would be rendered more distinct when the tissues were stained in the living condition. These attempts, however, were not successful, as the stain (methylene blue) penetrated the body wall very slowly and incompletely, and did not reach the nervous system at all. ? The Gerebral Ganglia, as in most aquatic Oligo- chaeta, lie far forwards, just behind the prostomium. Their posterior border lies at the level of the boundary between the buccal cavity and pharynx. They are dorsal in position, and form a comparatively large brain, the structure of which is complicated by the presence of several lobes. The brain is concave behind and in front, and consists of a solid mass of nerve fibres and nerve cells. Anteriorly it is produced into three horns or lobes, two lateral and one median: the former may be described as the antero-lateral lobes, and the latter as the anterior median lobe (PI. I, fig. 1, an.l.; m.l.). From these, nerves arise which will be considered later. The anterior median lobe is characteristic of the family Tubificide, but is not always present as a simple lobe. In a few more highly specialised forms, e.g., Bothrioneuron, it consists of a median nerve communicating with a small ganglion placed a little way in front of the brain (Beddard). The antero-lateral lobes described above are not figured by Vejdovsky (1884) in his drawing of the brain . | TUBLFEX. 335 of Tubifex rivulorum, and he definitely states that the brain in this region is devoid of processes or lobes. He figures, however, postero-lateral lobes from which well- developed muscles pass to the body wall, and these lobes can be clearly seen (Pl. I, fig. 1, ps.l.), but I have never been able to find the muscles. Beddard described, in his definition of this species, a median, smaller, posterior lobe corresponding to the anterior one; but this I have never been able to see. It is particularly easy to see the outline of the brain just at this point, as the dorsal vessel runs under it here, and the red colour of the blood in this vessel causes the brain to show sharply outlined against it. In addition to the lobes already mentioned, there are yet two more to be noted, and these are very important, as it is from them that the peripharyngeal connectives arise. ‘They are situated between the antero-lateral lobes and the postero-lateral lobes, and are slightly more ventral in position than either of these. They pass obliquely outwards, downwards and backwards, and finally bend sharply inwards, terminating in the nervous bands known as peripharyngeal connectives which finally unite, in the median line ventrally, with the first ganglion of the nerve cord known as the sub-pharyngeal ganglion Se 11) fig. 8 sp.g.). Cerebral Nerves. I have been able to recognise three pairs of these, two pairs arising from the brain proper and one pair from the peripharyngeal connectives. Of these three pairs of cerebral nerves, one pair arises from the median anterior lobe of the cerebral ganglia. They really originate as one nerve forming a continua- tion of the median lobe, but this condition only obtains for a short distance, as the single nerve soon divides into two branches which diverge from one another. Both branches, however, pass forwards towards the tip of W SE St a a ee eras ee Gas a Ee a 336 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. the prostomium, with which they ultimately become connected. Each nerve branches several times, and gives rise to a large number of extremely fine fibres, which terminate in the sensory cells with which the prostomium is amply provided. It is the presence of these sensory cells which renders the prostomium such an important tactile organ. The second pair of nerves arising from the cerebral ganglia form continuations of the antero-lateral lobes of the brain, which have already been described. They are short, and pass directly to the lateral walls of the prostomium and first segment. Here they branch, forming a number of fine fibres which spread themselves over and into the body wall of this region. The third pair arise as branches of the peripharyngeal commissure, and pass to the body wall of the first segment, where they also branch (Pl. II, figs. 7 and 8). The Ventral Nerve Cord originates at that point where the two connectives arising from the cerebral ganglia unite below the gut, having first encircled it. The nerve cord is a ganglionated chain extending from the second segment to the last segment of the body, and it lies freely through all its length in the body cavity. It is surrounded by a connective tissue sheath, from which, at intervals, branches arise and pass to the body wall. By this means the nerve cord is kept in position in the body cavity. It is necessary to exercise care in identifying nerves, for these branches of the connective tissue sheath closely resemble the nerves in appearance, though not, of course, in structure. The cord is also enclosed in a muscular sheath, which, however, does not completely encircle it, but is confined principally to the dorsal surface. The sheath ig very delicate, and is composed of a few muscle fibres 4 , TUBIFEX. Bo placed longitudinally. There is a single ganglion situated near the posterior border of each segment, and these are connected into a continuous chain by a series of connectives which unite adjacent ganglia. In _ the anterior segments the ganglion and connective are about equal in length, the connective, if anything, being a little longer. More posteriorly, however, where the segments become shorter, the proportions of these two parts of the nerve cord also change. The connectives become shorter and shorter, but the ganglia change very little in size, so that, finally, the connectives are extremely short, and the ganglion occupies almost the whole length of the segment. But throughout the length of the nerve cord the two parts are sharply marked off from one another. This is very clearly seen if one examines the living worm, but when the nerve cord is seen in section the difference between the two is even more apparent. The connective is of about the same diameter throughout its length, but the diameter of the ganglion varies in different parts. In most cases it can clearly be divided into three lobes placed end to end and separated from one another by constric- tions. Of these lobes, the first is the largest, while the other two are of nearly the same size, the posterior one being very slightly smaller. The ventral nerve cord gives off branches or peripheral nerves in each segment. There has been a considerable difference of opinion amongst earlier authorities as to the number of these branches in each segment. Vejdovsky (1884) figures no fewer than five pairs; D’Udekem (1855) states that there are three pairs, but Nasse (1882) was only able to find two. After careful examination of the living worm and sections, I have come to the conclusion that there are three pairs of these lateral nerves in each segment. Their places of 338 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. origin from the nerve cord are approximately the same in all the segments. There is always one pair arising from the connective and two pairs from the ganglion. The first pair comes off from the connective just behind the septum, while of the two pairs originating from the ganglion one springs from the most anterior of the three lobes, just at that point where the connective merges into the ganglion. The third pair is to be found more posteriorly, and usually originates at or near the constriction which separates the first and second lobes of the ganglion. hese three pairs of nerves all have a similar distribution. They are true lateral nerves, and do not, as in some forms, e.g., most of the Lumbriculidae, arise from the mid-ventral line asa single nerve, and after entering the body wall divide into two branches. They arise, on the contrary, from the lateral part of the cord, but slightly nearer the ventral than the dorsal surface. They pass out at right angles to it, and extend for some distance in the body cavity before plunging into the body wall. HisToLoGy oF THE NERVOUS SYSTEM. In order to get a clear idea of the histological details of the structure of the brain and nerve cord, it is necessary to examine transverse and longitudinal sections of these organs. If the sections are appropriately preserved and stained, the various elements become fairly well differentiated. The two outermost coverings of the nerve cord have already been mentioned, but in sections it can be seen that both the connective tissue sheath and the muscular layer are extremely delicate. The most conspicuous part of the former are the nuclei of the connective tissue cells, and, indeed, in places it is very difficult to identify any other structural details. The TUBIFEX. 339 nuclei are oval in shape, and the cells to which they belong are considerably flattened. The muscular layer has already been sufficiently described. The brain is composed of both nerve cells and nerve fibres, the cells being disposed dorsally and laterally, while the fibres occupy the ventral and central parts of the brain. The nerve cells, in section, appear as almost rounded bodies with clearly marked rounded nuclei. The nerve fibres are embedded in a transparent matrix, and when cut transversely give the brain, in the region in which they are, a finely granular appearance. The nerve cord, as already mentioned, can be divided into connectives and ganglia, the ganglia being represented on the surface of the cord as swellings between the connectives. In sections it can be seen that the difference in size of the cord in different parts is due to the presence or absence of nerve cells. The connectives are formed only of nerve fibres, whereas the ganglia possess in addition a large number of nerve cells, their lateral and ventral position being different from that which they occupy in the brain (Pl. VII, fig. 46, n.c.; 7.). The mass of nerve fibres, particularly in the ganglia, is divided up into different regions by the interposition between the nerve fibres of a delicate fibrous layer, which appears to be a continuation of the connective tissue sheath which completely surrounds the cord (fig. 46). As in most Oligochaeta, giant fibres are to be seen in the nerve cord throughout its length. These occupy the same position in the cord as do those of Lumbricus, that is, they lie close to its dorsal surface. The number of fibres present, however, varies in different parts of the body. Anteriorly, just behind the brain and for several segments, the cord contains only one giant fibre, and this hes in the mid-dorsal line. More posteriorly there are 340 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. three giant fibres, the middle one being the largest. They are still situated dorsally, and lie side by side. The structure and function, and the history of our knowledge, of the giant fibres in certain Polychaet worms, especially Halla parthenopeva, has been dealt with in great detail by Ashworth (1908), and it therefore seems unnecessary to refer to the subject in detail here. It may, however, be advisable to include a brief account of the structure of a giant fibre as described by Ashworth, as follows :— Each giant fibre consists of a sheath composed of interlacing glia fibres of different diameters and embedded in a finely granular protoplasm. The centre of each fibre is occupied by a bundle of fibrillae, also embedded in a matrix known as the interfibrillar substance. The number of these fibrillae in a fibre differs in different Annelids. The space which is always present between the bundle of fibrillae and the sheath of the fibre is filled with a semi- fluid perifibrillar substance, which is colourless, hyaline and contains very fine granules. In my preparations of the nerve cord of Tubifex rivulorum only the sheath of the giant fibres has been clearly marked, the fibrillae and the _perifibrillar substance not staining at all well, thus giving the fibres the appearance of empty tubes. This is due to the extreme difficulty which is always experienced in attempting to differentiate clearly the fibrillae of the giant fibres. All ordinary methods of preserving and | staining the material, with which I was familiar, were unsuccessful, and as Ashworth’s paper did not come into “my hands until this work was ready for publication, I have not yet had an opportunity of testing whether his methods of preserving and staining Halla parthenopeia are equally successful in the case of Tubifex rivulorum, — TUBIFEX. 341 THE EXCRETORY ORGANS. The excretory organs or nephridia consist of a system of paired tubes, which are present in most segments of the body. They are absent from a few of the anterior segments, but as in most aquatic Oligochaeta they begin well before the genital segments, from which, however, they are absent. I have been able to trace these nephridia as far forward as segment 7, but not in front of that. Behind the genital segments they are to be found through all the remaining segments of the body. The nephridia are coiled tubes occupying two segments, and, typically, two only, but in a few cases I have seen the coils of a nephridium of one segment lying in the segment behind, but this must be considered as an abnormal condition. Each nephridium is provided with an internal and an external aperture, the former opening into the body cavity, the latter to the exterior. The nephridia lie nearer the ventral surface of the body than the dorsal, and are situated one on either side of the ventral vessel and very close to it. In the posterior segments of the body certain of the coils of the tube appear to be very intimately connected with this vessel, so that even though the movements of the worm may be extremely violent, these tubes do not become displaced. Nasse (1882), in his work on the family Tubificidae, described the presence of branches of the ventral vessel which arise near the nephridia and pass into close connection with these organs. That is, he claims that there are special blood vessels conveying blood from the ventral vessel to the nephridia, in which organs, pre- sumably, the blood is purified. Nasse does not explain how the blood is returned to the main circulation, but he suggests, at any rate, an arrangement of the vessels and 342 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. nephridia which is closely allied to that found in higher Oligochaeta and typically in Lumbricus. Vejdovsky (1884) contradicts this, and definitely states that there is no connection between the ventral vessel and the nephridia. I am disposed to agree with Vejdovsky that there are no special branches passing from the ventral vessel to the nephridia, but at the same time we ought not to lose sight of the fact that, as has already been stated, the nephridia are always in close connection with the ventral vessel. This suggests the possibility that the excretory products which collect in the blood during its passage round the body are passed out from it, while in the ventral vessel, to the nephridia, the walls of which are ‘very thin and would form no barrier. to the diffusion through them of these waste products. 3 Since the nephridium occupies two segments, it follows that the tube must pierce the intervening septum. We may, therefore, divide the whole nephridium into pre-septal and post-septal portions. The pre-septal part consists of the nephrostome, or funnel, and a very short, uncoiled portion of the nephridial tube. By far the greater part of the nephridium, therefore, lies behind the septum, and consists of a much coiled tube which can be divided into certain regions according to the structure of its walls. This tube terminates in the external aperture or nephridiopore, which is situated at the apex of an enlarged vesicle forming the terminal portion of the nephridial tube. It is possible by careful teasing out of part of the intestinal region of the living worm to separate portions, at any rate, of the nephridium. It is extremely difficult, however, to obtain a good view of the nephro- stome and nephridiopore by this means. In some cases the nephridium can be examined zn sztu if the body wall be sufficiently transparent, and in this way the cilia can © TUBIFEX. 343 often be seen in motion. If the tube be removed from the body, the cilia very quickly cease to move, and it is impossible then to decide whether any particular portion of the tube is ciliated or not (Pl. VI, fig. 43). The nephrostome is small and very simple in structure. Its diameter in the widest place is only slightly greater than that of the nephridial tube. Its lumen is a little larger than that of the tube, and its walls are somewhat thicker, making it funnel-shaped. It is composed of a very few cells, the nuclei of which are large, round and nucleolated (Pl. VII, fig. 47). The inner borders of the cells, those abutting on the lumen, are ciliated, the cilia being long and pointing chiefly in one direction, namely, from the free end of the funnel down the tube. Some of the cilia, however, fringe the free edge of the funnel, and these are particularly long and very active. By their rapid movements they create a current in the direction of the funnel, into which the excretory products are drawn. The pre-septal part of the tube is extremely short and uncoiled, and its cavity is directly continuous with that of the funnel. Its walls are thin, and the nuclei of the cells are flattened in a direction parallel to that of the tube. The tube is ciliated in this part. ‘ The post-septal portion of the nephridium can be sub-divided into three regions:—(1) A delicate, much- coiled tube with extremely thin walls. (2) This passes into a slightly thicker walled tube of a yellowish colour, and decked with specially modified peritoneal cells. (3) This again passes into a somewhat thinner walled tube, which is covered with specialised peritoneal cells for part of its length, and which finally opens into a small vesicle communicating with the exterior at the nephridio- pore (Pl. VI, fig. 43, ¢.). 344 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. (1) The first portion of the tube has thin, transparent walls, and is profusely coiled, the coils being quite irregular in their arrangement and forming no definite loops such as are so characteristic of the nephridia of Lumbricus and other types. Its cavity is intra- cellular and ciliated throughout. It is invested by an extremely delicate peritoneal covering, the cells of which are much flattened and possess oval nuclei. The first portion of the tube is separated from the second by a curious structure, which, as far as I can tell, has never been described by any other observer as occurring in Tubifex. It has been figured by Hisen (1885), however, in the nephridia of Spirosperma ferox. He calls it an ampulla, and this name will be used for the corresponding structure as found in Tubifex. Hisen describes the ampulla as an enlargement of the tube of the nephridium, but he does not attempt to give any account of its structure. He does not even venture to express an opinion as to whether it is a permanent or only a temporary structure. Beddard (1895) in his monograph refers to the ampulla in his account of the characters of the family Tubificidae, as occurring in Limnodrilus, Spirosperma ferox and many earthworms. He a however, no account of its structure. In Tubifex the ampulla is undoubtedly a constant organ, for I have never failed to find it in any of the specimens that I have examined, whether in sections or by teasing out the nephridium from the living worm. Further, it is present in the nephridium of all segments from the anterior to the posterior end of the body. ‘The ampulla.always occurs between the first portion of the ~ nephridial tube and the second, which is characterised by walls of a yellowish colour. The ampulla is usually pear-shaped, although in a few cases it is somewhat more TUBIFEX. 345 circular in .outline. The first part of the tube opens directly into it at its broader end, while the second portion of the tube leaves it at the opposite or pointed end (Pl. VII, fig. 48). The structure of the ampulla is always the same. It has the form of a swollen bladder bounded by a delicate wall composed of a single layer of cells with prominent oval nuclei. The junction of the first part of the tube with the bladder is marked by a circlet of specially large rounded cells, with prominent nuclei, which by their arrangement form a sort of collar round the tube. In the cavity of each ampulla there is, as a rule, a brownish, granular mass of irregular shape lying somewhat nearer to its broader end (Pl. VII, fig. 48, gr. m.). In many cases this mass appears to have no connection with the wall of the bladder, but in others I have been able to see exceedingly fine processes extending from it to the wall. In an ampulla removed from the living worm this mass appears to be solid, but in section it is seen to be hollow, its cavity being continuous with that of the first part of the nephridial tube. It is very difficult to decide whether this central eanal of the granular mass ends blindly, or whether it communicates with the cavity of the ampulla. I am inclined to think that the latter is the case. There is no suggestion of branching of this canal, nor the formation of fine nephridial tubules within the mass itself. In fact, the latter seems to be composed of a large number of inert granules which are affected by neither killing reagents, preserving fluids nor stains. As already mentioned, the nephridial tube is ciliated throughout, but just at the point where it enters the ampulla it is provided internally with cilia which are particularly long and which project into the ampulla, or more accurately into the canal of the granular mass 346 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. (Pl. VII, fig. 48, cz.1). If one liberates .an ampulla with a portion of the tube from the living worm, these cilia exhibit lively movements even for some time after those in the nephridial tubes have become motionless. The wall of the ampulla is not ciliated. (2) The second portion of the nephridial tube opens out from the pointed end of the ampulla, and directly after its origin it is bent back sharply so that it runs for a short distance parallel to the ampulla and the first part of the tube. This part of the tube is characterised by the fact that its walls are somewhat thicker than those of the first part, that they are yellowish in colour, and the cells of which they are formed are very granular. The tube is not much coiled, but is bent on itself two or three times to form well-marked loops which lie approximately parallel to each other and appear to be bound together in a common investing membrane. For the first part of its length this tube is provided with a layer of peritoneum, composed of flattened cells similar to those investing the first part of the tube. These gradually give place to specially modified cells known as vesicular peritoneal cells (Pl. VI, fig. 44, v. pt.). These are large, rounded, bladder-like cells with very thin walls. These cells are particularly well seen in the fresh material, but are liable to undergo considerable shrinkage during the processes of killing and fixing. When examined in the living condition, these cells may be clear and devoid of any special solid contents, or they may be filled with minute brownish granules which exhibit active molecular move- ments even after the cell has been dislodged from the wall of the nephridium. (3) The third part of the tube is ciliated, is of some- what wider calibre than the first part, and has thin walls. The proximal portion is covered with vesicular peritoneal TUBIFEX. 347 cells similar to those described above, but distally the peritoneum is again composed of flattened cells. The distal end of this tube expands rather suddenly to form a small, contractile vesicle, which opens to the exterior at the nephridiopore. The tube is ciliated to its distal end, and a tuft of specially long cilia projects into the vesicle. The latter is somewhat pear-shaped when fully expanded, narrowing considerably as it approaches the nephridiopore, which is situated on the ventral surface of the body, a short distance in front of the ventral setae on either side. REPRODUCTIVE ORGANS. Tubitfez rivulorum is hermaphrodite, the ova and spermatozoa maturing at the same time. The worms exhibit fully-developed reproductive organs during the autumn and early winter, that is, from October to December. The first cocoons are laid about the beginning of November, and can be found in large numbers in the mud for the next two months, after which time their formation ceases. The mature worms show well marked differences in external appearance from those in which the reproductive organs are not well developed, especially in the anterior segments. * The sexual organs occupy segments 9-16 or 17 in quite mature worms, and these segments are considerably swollen and of a dull whitish colour, due mainly to the large size of the sperm sac and to the large ova which are present. During May, June and July it is comparatively common to find worms which present somewhat this appearance, and it would be easy to make an error and suppose that such worms were mature if they were not more minutely examined. Further investigation shows that this whiteness and 348 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. opacity are due to the presence of parasites belonging to the species Urospora saenuridis, which will be described later. The reproductive system is a complex affair, as is usually the case in hermaphrodite forms, and it will be well, first of all, to enumerate the various organs which ! | assist in the process of reproduction and then enter into lH their structure and arrangement in detail afterwards. | First, then, there are the ovaries (Pl. I, fig. 2, ov.) and testes in which the ova and spermatozoa respectively undergo the earlier stages of their development. They are not, however, permitted to come to maturity in these organs, but are transferred quite soon to special sacs known as the egg sacs (Pl. I, fig. 8, ovs.) and sperm sacs (Pl. I, fig. 2, sp.s.), which, at first, oceupy only one segment each, but in the mature worm may extend through several segments. When the ova and spermatozoa are fully developed, they are transferred to the cocoon by means of special ducts, which are provided with external apertures perforating the body wall. These ducts are known as the oviducts (female) and the sperm ducts or vasa deferentia (male) (Pl. I, fig. 2, v. d.). The sperma- tozoa do not reach the cocoon directly, by means of the vasa deferentia, but are transferred, during copulation, to special organs set apart for their reception. These | organs are known as the spermathecae (Pl. I, fig. 2, sp.). j The terminal portion of each vas deferens is dilated to | form an elongated chamber known as the spermiducal gland (Pl. I, fig. 2, at.), which opens to the exterior by the penis, a chitinous organ, capable of protrusion (Pl. I, fig. 2, pe.). The penis aids in the transference - of the sperm from the sperm sac of one worm to the spermathecae of the other during copulation. In con- nection with the spermiducal gland, and formed as a re ee ee a 5 opts ee big Mal Se. eter rere A inn SSE tor > x. oe Bienes iy Se TUBIFEX. 349 proliferation of some of its cells, is an irregularly- shaped mass known as the prostate gland. The cells of this gland secrete a cementing substance which is passed into the spermatheca with the sperm, and is used for moulding the spermatozoa into a solid mass, of characteristic shape, known as a spermatophore. The position which these organs occupy in the body is ‘constant, and will be noticed in the description of the various organs. I. THe Gonaps. Both ovaries and testes are present in the same worm, there being only a single pair of each. As is usual in the Oligochaeta, the testes lie in front of the ovaries, and in Tubifex they are situated in adjacent segments, the testes in segment 10 and the ovaries in segment 11. In their relative segments they occupy exactly the same position in relation to the other organs present in that segment; that is, they he one on either side of the intestine and slightly nearer the ventral than the dorsal surface of the body. Not only do they correspond in their position in the segment, but, in the young condition at any rate, they are exactly similar in appearance and structure. Both ovaries and testes are derived from peritoneal epithelium, and appear in the earliest stages of their development as small masses of a few undifferentiated cells forming the germinal epithelium. In such a condition as this it is only by noticing the segment which they occupy that one can distinguish between the gonads. They are attached to the septum forming the anterior boundary of the segment which they occupy and hang freely into the body cavity. Ata little later stage, but when the cells of the germinal epithelium are still undifferentiated, the 350 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. shape of the mature gonad is indicated. They are both somewhat pear-shaped, the broader end being attached to the septum (Pl. I, fig. 2, ov.). In a mature worm, however, the ovaries often attain so great a size that they are somewhat bent round in the segment and the original shape is lost: this is also partly due to the presence of the oldest ova at one side of the ovary. The ovaries persist throughout the reproductive season, and only attain their full size when the rest of the reproductive organs are developed. If, however, in a mature worm one seeks for the testes, one will not be able to find them. This is due to the fact that they have been completely enclosed in the sperm sac, which is of large size and in which the spermatozoa complete their development. It is necessary, therefore, to examine much younger worms in order to find the testes. In fact, they are quite well developed in those individuals in which there is no trace of any other part of the reproductive system except the ovaries. The testes develop somewhat earlier than the ovaries, and, therefore, in a young worm it will be easy to identify them without verifying their position, as they will be larger than the ovaries. When the testis has attained its full size it consists of a mass of rounded cells with clearly marked nuclei, but without any specially characteristic features. These may be called the spermatogonia, or sperm mother cells, and here the development of the spermatozoa in the testis ceases. The spermatogonia must be transferred to the sperm sac before further develop- ment can take place. Many of the earlier writers outed the testis with the sperm sac or with part of it. D’Udekem (1855), for example, speaks of the testis as occupying segment 8 where it appears as an unpaired organ below the intestine a. ae — ¥ TUBIFEX. 351 and having the form of a voluminous gland, greyish in colour. What he has described as the testis is really a portion of the sperm sac, for the latter in a mature worm not only extends backwards through several segments, but may pass forwards in front of that segment which originally contained the testis. McIntosh (1871), too, has confused the sperm sac with the testis in stating that the testis of one side remains in segment 9, while its fellow extends back as far as segment 16. The ovaries are still small when the testes have attained their full size, but while the latter soon disappear the ovaries gradually increase in size until they occupy a large proportion of segment 11. In a fully developed ovary it is easy to recognise ova in several stages of development. II. DEVELOPMENT AND STRUCTURE OF THE SPERMATOZOA. The germinal epithelium in the testis gives rise by ordinary cell division to a number of spermatogonia, which, at an early stage in their development, are separated from the testis and pass into the sperm sac, which, at first, is a simple, undivided sac. The spermato- gonia, when they leave the testis, are uninucleate, but very soon the nucleus divides several times, and, as its division is not at once accompanied by division of the cytoplasm, each spermatogonium or sperm morula, as it is called, becomes multinucleate (Pl. V, figs. 28 and 29). Calkins (18954), who has described in detail the spermatogenesis of Lumbricus, states that the spermato- gonia, while still in the testis, are multinucleate. Careful examination of many preparations of the spermatogonia of Tubifex has led me to decide that in this form, at any rate, the spermatogonia only become multinucleate on leaving the testis. x re ee = ee SS SE aa SE 4 = a ef. | * “ = 352 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. The nuclei of the sperm morula, which, when first formed, are scattered irregularly through the cell, gradually become arranged more regularly around the periphery of the cell; the central portion of which has no nucleus and is, therefore, entirely cytoplasmic in nature. The cytoplasm surrounding the nuclei at the periphery of the sperm morula exhibits slight cleavage marks around each nucleus, and these cleavages deepen until the nuclei are completely constricted off from one another, while still remaining in connection with the cytoplasmic mass occupying the centre of the morula, which remains undivided throughout and is known as the sperm blasto- phore (Pl. V, figs. 830 and 31). During this protoplasmic cleavage the sperm morula or sperm polyplast increases considerably in size, and the nuclei are large, rounded, and possess nucleoli. Each of the elements making up the fully formed sperm morula is called a spermatocyte, and these are at first large and comparatively few in number (Pl. V, fig. 31). The spermatocytes are arranged extremely regularly around the sperm blastophore, but this arrangement can only be fully appreciated when the sperm morula is viewed in section (Pl. V, fig. 32). The nucleus of each of the spermatocytes now under- goes karyokinetic division, probably twice, and the cells then divide so that the number of elements comprising a sperm morula in its later stages of development is very much increased. The cells thus formed are much smaller than the spermatocytes, as there is no appreciable increase in the size of the whole morula at this stage (Pl. V, fig. 33). The cells resulting from the division of the spermatocytes are known as spermatids, and from these the mature spermatozoa are derived by a simple meta- morphosis. The spermatids are at first rounded in shape, and the nucleus forms the greater part of the cell, the ' Se ee ee i ‘. TUBIFEX. 353 cytoplasm being much reduced in quantity. Gradually, however, the spermatids become oval in shape. The nucleus of the spermatid then elongates considerably to form the filiform head of the spermatozoon so characteristic of the group (Pl. V, fig. 34). The middle piece of the spermatozoon is not very conspicuous, but appears as a direct continuation of the head. The distal extremity of the spermatid now becomes much drawn out, forming a long tail which is, in the normal sperma- tozoon, considerably longer and thinner than the head. It stains but faintly with nuclear stains, as it is composed of cytoplasm only, derived in all probability from the delicate layer of protoplasm surrounding the nucleus of the spermatid. At first, the fully formed spermatozoa are arranged very regularly around the blastophore, as regularly, in fact, as were the spermatids from which they were derived, each one with the anterior end of the head buried in the substance of the blastophore. The tails of the spermatozoa are at first quite straight, and while still in this condition become motile (Pl. V, fig. 35). When the spermatozoon is quite matured and ready to leave the blastophore, the tail usually exhibits a simple coil at the free end. The spermatozoa now become much less regularly arranged on the blastophore and by degrees they separate completely from it. When they are set free they remain for a time in the cavities of the sperm sac, but finally they find their way to the ciliated funnel of the vas deferens, around which, in the mature worm, they congregate in great numbers. In most of the individuals I have examined, I have found developing in the sperm sac, side by side with the normal spermatozoa, other structures which resemble them to a certain extent, but which can easily be distinguished from them. At first I was inclined to look ayes 354 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. upon these structures as parasites, but a closer examina- tion of their development has led me to suggest that two kinds of spermatozoa are present. On this understanding the same terminology will be adopted in the following description as was used above in describing the develop- ment of the normal spermatozoa. The central blastophore from which the spermato- cytes are constricted off is larger, and more irregular in outline, than that on which the normal spermatozoa are formed. The spermatocytes themselves, too, are less regularly arranged, often tending to be formed in groups of four or five together (Pl. V, fig. 36). The nuclei of the spermatocytes stain very deeply. The changes taking place during metamorphosis can be easily followed. The spermatocytes, which are at first rounded, become oval in shape, the outer free end being considerably more pointed _ than the opposite end, which is embedded in the blasto- phore (Pl. V, fig. 387). The spermatids formed by division of the spermatocytes are much larger and less numerous than those developed from a normal sperm polyplast, and their arrangement on the blastophore is as irregular as that of the spermatocytes from which they are derived (Pl. VI, fig. 38). Im the earlier stage of metamorphosis there is no semblance of a true tail, but when the head has lost its oval form and becomes much elongated, the tail is gradually differentiated. At first it is quite short, but it increases in length as the head of the spermatozoon becomes fully developed, and in the final stages the tail is as long as, or a little longer than, the head (Pl. VI, fig. 39). The mature spermatozoa are quite - irregularly arranged, but they tend to lie together on the blastophore in bundles of five or six, just as the © spermatocytes have been described as doing, and from it they finally separate. In some cases I have been able to TUBIFEX. 355 find in the same sperm smear most of the stages in the development of these spermatozoa, but in other cases they seem to occur much more rarely. At first I tried to incorporate the stages of develop- ment described above in my description of the development of the normal spermatozoa, but when I was able to get such a complete series of each I gave up the attempt. I now suggest that this worm possesses dimorphic sperma- tozoa, a condition which has been described as occurring in Paludina amongst the Mollusca and in certain Amphibia, Birds and Mammals. In Paludina the two kinds of spermatozoa are different in shape, the normal one being divisible into a spirally coiled head and an extremely long tail. The larger kind is vermiform, and bears a tuft of cilia at one end. In Birds and Amphibia the two kinds of spermatozoa are of the same form, but differ in size, the smaller one being functional. In Tubifex rivulorum both kinds of spermatozoa are elongated structures, and each is divisible into head, middle piece, and tail, but the proportions of these parts to each other in the two forms are rather different (Pl. VI, fig. 40). The head of the normal spermatozoon is small, forming about one-sixth of the total length (Pl. VI, fig. 40a); it is oval in shape with a sharply pointed anterior extremity, and measures about 4m in length. It is composed almost entirely of nuclear substance, there being such a small quantity of cytoplasm present as to be almost negligible. The middle piece, which is shorter than the head, lies behind that structure, and gives attachment to the tail. It is composed of cytoplasm and stains only slightly with nuclear stains, such as haematoxylin. The tail is very long and delicate, and is also composed of cytoplasm. The other spermatozoa are much larger, and the head forms about half the whole —— ee 356 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. structure. The head is elongated, but less sharply pointed at the anterior end than is that of the normal sperma- tozoon (Pl. VI, fig. 408). The middle piece is less distinctly marked off from the head than in the first case, and the tail is straight throughout. Ill. THe Srerm-sac. The sperm-sac is developed, as in all Oligochaeta, for the reception of the spermatogonia, which are early removed from the testis and transferred to this organ. In it the mature spermatozoa are formed. In a worm in which the reproductive organs as a whole are immature, the sperm-sac can be seen in its simplest condition. Itis an unpaired structure, and arises as a pouch-like outgrowth of the septum between segments 10 and 11. This outgrowth is directed backwards and projects into segment 11. The cavity thus formed is at first quite simple, and is bounded by a layer of flattened peritoneal epithelium which is derived from the tissue of the septum. As the number of spermatozoa increases, the sperm-sac also enlarges, and it soon becomes too big for the segment in which it developed, and projects still more posteriorly into segment 12. In a mature worm the number of spermatozoa is very large, and these are present in the sperm-sac in all stages of development. The sperm-sac increases tremendously in size, extending further and further back until it may occupy as many as seven or eight segments (Pl. I, figs. 2 and 3, sp.s.). It is the presence of this large sperm-sac which gives the mature worm a swollen and opaque appearance in the region of the reproductive segments. It is very common also to find that the sperm-sac has encroached upon the segments in front of that occupied by the testis, and z { 5 4 , c in tell eet iti i i ei te i i | il ti ee, on -~ = ie Po —_— Tae a ee TUBIFEX. 357 segment 9 at least, and occasionally segment 8, also enclose a portion of the sac. The development of this sac both anteriorly and posteriorly to the segment in which it first appears may be due to simple pressure exerted by the developing spermatozoa. It has already been said that the sac arises as a simple outgrowth of the septum between segments 10 and 1l. As it increases in size, however, its structure becomes more complicated. The cavity becomes divided up into a series of much smaller spaces by the growth inwards of its walls. These small spaces remain in communication with one another, and are filled with spermatozoa in all stages of development. It is difficult to believe that the tremendous number of sperm morulae present in the sac have all been derived from the small and inconspicuous testes. Some writers have suggested that the peritoneal epithelium lining these coelomic spaces of the sperm-sac is capable of forming germinal tissue. If all the spermatozoa were derived from the spermatogonia of the testes which are transferred, as they are formed, to the sperm-sac, one would expect to find the youngest sperm morulae nearest the segment in which the testes lie. But this is not the ease, for it is common to find spermatogonia and fully developed spermatozoa lying side by side in any part of the sperm-sac, and there is no suggestion of regularity in their arrangement. This irregular arrangement is what one would expect if the epithelium of any part of the sperm-sac were capable of forming germinal tissue. Throughout the life of the worm the ciliated funnel continues to hang freely into the cavity of segment 10, and never becomes enclosed in the sperm-sac as is the case in Lumbricus. It does not seem quite clear, perhaps, at first sight, how the mature spermatozoa from the sperm- sac reach the funnel. If, however, we bear in mind the SET ee EE eo z= - ~ pam aa SESE car = sable an le ag Eee oe eS 358 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. development of the sperm-sac, there is little difficulty in understanding this. The sperm-sac extends forwards into segment 9, and backwards as far as segment 16, in both cases ending blindly. There remains, however, an opening from the sperm-sac into segment 10, from which it was derived as simple, pouch-like outgrowths of the anterior and posterior septa, respectively. Although the structure of the sperm-sac becomes much more com- plicated as the worm matures, the sac retains its connection with segment 10, and so the spermatozoa can pass into the coelom, and thence to the surface of the expanded funnel of the vas deferens which lies in the same segment. IV. Tuer Sperm Ducts or VASA DEFERENTIA. The spermatozoa when mature are conveyed to the exterior by special ducts known as the sperm ducts or vasa deferentia. As is always the case in the Oligochaeta, the number of these ducts corresponds to the number of the testes, therefore in Tubifex rivulorum there is a single pair. Each duct consists of a much coiled tube with its origin in segment 10—the segment which contains the testes; and its external aperture in segment 11. The whole duct, however, does not lie in these two segments—its length is so great and it exhibits such complex coiling that it extends posteriorly through several segments, sometimes reaching as far back as segment 15. For the purpose of description we can divide the sperm: duct into the following regions :—(1) The ciliated funnel (Pl. I, fig. 2, cv. f.), (2) the coiled tube (Pl. I, fig. 2, v.d. 1, v.d. 2), (3) the spermiducal gland with the prostate (Pl. I, fig. 2, at.), and (4) the penis (Pl. I, fig. 2, pe.). ee ee ee eee TUBIFEX. 359 These can all be studied in detail by means of transverse and longitudinal sections, but it is interesting and instructive to liberate the sperm duct from the body in the living condition. This is possible by appropriate and careful teasing out of the reproductive segments of a mature worm. During this teasing-out process, the duct can be recognised as a small, whitish, shining mass, formed of a number of coils, which, with care, can be unravelled on the slide. It is comparatively easy to get the tube with the spermiducal gland and penis still attached, but the structure of the penis cannot be seen well by this means, the only feature which is clearly brought out being the chitinous nature of the penis sheath. It is a much more delicate operation, however, to get the extreme anterior portion of the tubular part with the ciliated funnel still attached. The difficulty is due to the fact that the funnel is situated in a different segment from that occupied by the tube, and the latter is usually broken off at the septum. If one is fortunate enough to get the funnel also, the shape of the latter can be fairly clearly seen, and its appearance will be described later. Although this method of investigation is invaluable for obtaining a correct idea of the relation of the various parts of the sperm-duct to one another, it is of no use for histological details. 1. The Giliated Funnels of the sperm ducts are situated in the segment in front of that which contains the tubular part of the duct, that is, they lie in segment 10, or the segment that contains the testes, and they open directly into the body cavity. When seen in the living condition the funnel has the form of a flat, plate-like expansion, but when sections are examined it will be seen that its shape and appearance vary a good ET eo Se 360 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. deal in worms of different ages. In a very young worm in which both testes and ovaries are present, but before the development, to any great extent, of the sperm sac, the ciliated funnel is visible. It is the first part of the vas deferens to be formed, and appears as a _ local proliferation of the cells of the septum between segments 10 and 11. It rests at first directly on the septum, and it is only at a later stage, after the formation of the tube itself, that it hangs freely into the body cavity. Even at such an early stage as this, it is slightly hollowed out, with its convex side attached to the septum and its concavity opening into segment 10. As the vasa deferentia develop the ciliated funnels increase in size, lose their direct connection with the septum 10/11, and take up their normal position in the segment, being situated one on either side of the intestine and a little ventral in position. Hach funnel forms a markedly cup- shaped expansion of the anterior end of the vas deferens. It has a circular outline in transverse section, and opens into the body cavity by a wide aperture (Pl. IV, fig. 18s). The funnel is composed of a single layer of cells which, when seen in section, are quadrangular in shape, those nearer the centre of the funnel being squarish in outline, while those nearer the edge are somewhat longer than broad (Pl. IV, fig. 17, ct. ep.).. They are all provided with large nuclei, those in the squarish cells being almost spherical, while the others are oval in shape, but all possess a very distinct nucleolus. This epithelium is ciliated, the cilia being very conspicuous both in stained preparations and in the living specimen. In the latter, the cilia do not seem to be disposed equally over the inner surface of the funnel, but rather to be confined to certain tracts. There is nothing in the sections, however, to suggest this arrangement, and it is very likely that the TUBIFEX. 361 cilia are not all equally active at the same time, but that some cease entirely to vibrate for a time, and then resume their action again. Outside the ciliated epithelium is a single layer of very much flattened peritoneal cells, which is continuous with the peritoneum, completely investing the rest of the vas deferens. This layer is extremely delicate, but is rendered more conspicuous by the somewhat swollen, oval nuclei which the cells contain Sei LV, fic. 17, pt.). The ciliated funnel of a mature worm has a very characteristic appearance. It is no longer cup-shaped, but the edge is so sharply recurved towards the vas deferens that the funnel becomes much more shallow and is almost turned inside out (PI. IV, fig. 17). The surface of the funnel which bears the cilia is thus exposed as fully as possible. There is not the least doubt that this interesting change in the shape of the funnel is intimately connected with the transference of mature spermatozoa from the sperm sac to the vas deferens. But it is difficult to say whether this change in shape is completed before the spermatozoa actually reach the funnel, in order to expose as much surface as possible for their reception, or whether it is due entirely to the pressure exerted by the enormous number of spermatozoa which congregate upon it. In a mature worm the whole of the ciliated surface of the funnel is covered with a dense mass of sperma- tozoa, not arranged irregularly, but with their heads entangled amongst the cilia of the funnel, and their tails » lying parallel to the cilia (Pl. IV, fig. 17, sm. t.). At first sight, the funnel appears to be provided with enormously long cilia, but a closer examination of the stained preparations reveals the true state of things. Just outside the boundary of the cells composing the funnel is a narrow zone which is clearly differentiated 362 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. from the rest of the apparent cilia. This differentiation is due to the staining properties of this region, for the stain which is absorbed is a nuclear stain such as haematoxylin or borax carmine, whereas the cilia stain only with a plasma stain. This deeply-stained zone just outside the cells of the funnel is caused by the presence of the heads of the spermatozoa, which always stain heavily with a nuclear dye (Pl. IV, fig. 17, sm. h.). In many of the preparations which I examined the cilia of the funnel were not visible at all owing to the large number of spermatozoa present, but m a few cases the spermatozoa have slightly shrunk away from the funnel, and in these the true cilia can be seen clearly. Their length is not more than one-third or one-fourth that of the spermatozoon tails. It seems to me that we have here an example of the phenomenon of stereotropism, so well known already in the case of spermatozoa, which appear to be always attracted by any surface. If one examines ~ a funnel in the living condition one often sees a very large number of spermatozoa hovering around and on the funnel, and it is very possible that in the processes of killing and fixing the worm they are maintained in this position. 2. The Coiled Tube. The ciliated funnel opens into the tubular part of the sperm-duct, just in front of the septum between segments 10 and 11. The tube, therefore, perforates the septum. It belongs typically to segment 11, but owing to its great length it extends posteriorly through several segments, although its external aperture remains in segment 11. If the tube be examined under the microscope directly it has been removed from the body, it will be seen as a long, delicate, transparent structure with a very clearly marked cavity. If one watches it for a short time it will be seen that the i¢ —_— a a — TUBIFEX. 363 cavity of one part of the tube is surrounded on all sides by a wall of equal thickness. This part is nearer to the ciliated funnel, and its walls are thin. On the other hand, the part nearer to the spermiducal gland has much thicker walls, and, moreover, these walls contract a little and the cavity of the tube is thrown out of position so that it no longer lies centrally, but takes on the form of a very open spiral, the whole tube meanwhile becoming slightly shorter. If transverse sections of the sperm duct were cut after its removal from the body, the canal would be seen to be lying nearer the outer wall, first on one side, then on the other. In sections of the sperm duct cut when it is in position in the body, however, the canal lies centrally for all its length, which seems to indicate that the spiral curve of, the canal in the first case was due to contraction on its removal from the body. Beddard states in his Monograph on the Oligochaeta that the coiled tube in all the members of the Tubificidae is ciliated throughout, but this statement is not entirely correct. In the immature form of Tubifex rivulorum cilia are present throughout the whole length of the duct, but the coiled tube of the mature worm can be divided into two parts, one ciliated, the other non-ciliated. The former lies nearer to and in connection with the ciliated funnel, while the latter opens into the spermiducal gland. These ciliated and non-ciliated parts of the tube can be easily recognised, both in the living condition and in sections. We will first confine our attention to the vas deferens of the mature worm, though it will be necessary later on to refer to the immature form for the purpose of comparison. (a) The ciliated portion of the _ tube occupies about half its total length, and _ follows immediately upon the ciliated funnel (Pl. I, fig. 2, 364 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. v.d.1). Ciliary action does not occur by any means regularly in this part, for it is often completely in abeyance, but the cilia are so long that they can be easily seen, even when they are motionless. In the wall of the ciliated portion of the tube we can recognise the following layers: —(1) Ciliated epithelium, (2) Muscular layer, and (3) Peritoneum. The ciliated epithelium forms the innermost layer of the wall of the duct and is composed of a large number of flat, annular or ring-like cells piled very regularly on one another. Each cell is perforated in the centre by a large, rounded lumen, about 42 » in diameter, the wall around it being comparatively thin. A continuous tube is formed by the regular arrangement of the cells and by the fact that the cavity is pierced in exactly the same position in all the cells (Pl. IV, fig. 204). Hach cell possesses a single, elongated, somewhat spindle-shaped nucleus which may extend almost half way round the cell (Pl. IV, fig. 194). It is pointed at both ends and somewhat broader near the middle, where it exhibits a well-marked, rounded nucleolus. The inner edge of each cell is plentifully provided with cilia which are rather longer than the radius of the cavity, and have a somewhat spiral arrangement (Pl. IV, fig. 194, cz). The cells themselves appear to be embedded in a_ structureless matrix which stains with plasma stains and forms an extremely delicate layer right round this part of the duct. The centre of the tube is usually occupied by a dense mass of spermatozoa, which in the living condition can be seen travelling down the tube. The muscular layer is but feebly developed in this part of the tube. It consists of a single layer of longitudinal muscle fibres which are disposed at equal distances around the tube (Pl. IV, fig. 20a; l.m.). They do not pass straight down the wall ; | : / { TUBIFEX. 365 | of the tube, but have a somewhat spiral arrangement. The peritoneum, which completely invests the tube, is composed of a single layer of very much flattened cells with rather large, oval, nucleolated nuclei (Pl. IV, fig. 20, pt.). (6b) The non-ciliated portion. The ciliated part of the tube gives place gradually to the second part, which is characterised by the absence of cilia and the presence of a much thicker wall. Consequently, this part of the canal has a diameter almost twice as great as that of the first part (Pl. I, fig. 2, v.d.2.). The lumen of the tube is of exactly the same diameter throughout, so its increase in size is due entirely to the greater thickness of its wall. The epithelial cells which form the innermost layer of the wall of this second part of the tube have the same general form as those described above. That is, they are ring-shaped, piled one on top of the other extremely regularly, and each one is perforated in the centre by a rounded lumen. Fach cell has its greatest thickness around the lumen of the tube, and tapers off considerably at its outer edge (Pl. V, fig. 21). The nuclei of these cells resemble very closely those of the first part of the tube, and are situated near the inner edges of the cells (Pl. IV, fig. 198). The greater thickness of the wall of this part of the tube is due to a considerable increase in the quantity of the matrix in which the cells are embedded, and by which they are surrounded (Pl. V, fig. 21, ma.). This matrix still stains only with plasma stains, but it is no longer structureless, for it is characterised by the formation in it of a large number of exceedingly fine fibrillae. These are not visible in the fresh material, but are rendered very distinct by the action upon them of fixing reagents and by the use of appropriate stains, 366 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. They are arranged very regularly, originating at the inner edge of the cell and radiating out to the periphery. These fibrillae are so numerous that there is but little of the normal cytoplasm remaining. Owing to the large number of these fibrillae which are present, and to the consequent disappearance of the cytoplasm of the cells, the cell boundaries become very indistinct, and are barely recognisable. The nuclei, however, persist in their original position, and indicate, sufficiently clearly, what was the arrangement of the cells (Pl. IV, fig. 203). In the mature worm these cells are not ciliated, but the inner edge of each is considerably thickened and strengthened by the deposition of a secretion resembling cuticle in appearance and staining reactions. Since the inner edge of each cell is thus thickened, the secretion has the form of a series of circular bands arranged extremely regularly throughout the length of the tube (Pl. IV, fig. 20B, an.r.). It is extremely difficult to decide what is the exact nature of the substance composing these rings. Its position in relation to the cell and to the lumen of the duct suggests a chitinous or cuticular secretion, but there is no doubt that it is capable of contraction, which suggests that it is muscular in nature. These circular bands convert this part of the vas deferens into a much more solid structure than it would otherwise be, and also help to keep the lumen open. Outside the epithelial layer is a single layer of longitudinal muscle fibres arranged similarly to those of the first part of the tube. They are continuous, at the end of the vas deferens, with those which surround the spermiducal gland. A peritoneal covering invests the vas deferens in this part also, and its cells are flattened, forming a single layer, and provided | with oval nuclei. The change from the narrower to the wider part of —=~ = = Oe = TUBIFEX. 367 the vas deferens is a gradual one, and this transitional part of the tube is worth further examination. As has been already noticed, the lumen remains of the same size throughout. The wall, however, thickens gradually. The cells of which it is composed become somewhat larger, and are a little further apart from one another, but their boundaries are quite distinct (Pl. V, fig. 21). The matrix increases in quantity, it is developed between adjacent cells and also in a thick layer outside them. The fibrillae are quite distinct, and very numerous even in this region. For a short distance the cells of the second part of the tube are ciliated, but there are no annular rings. As soon as the rings appear the cilia are lost, and at this stage also the cell boundaries become less distinct. The vas deferens of quite a young worm is ciliated throughout, and the wall of the second part of the tube is only very slightly thicker than that of the first. That is. the matrix is only present in small quantities at first, and there are but slight indications of the annular rings. It seems strange that so little notice has been taken of the complicated structure of the vas deferens in this worm, and, possibly, in many others. Beddard, as well as stating that the tube is ciliated throughout, adds in another place that the wall in Oligochaeta as a whole is usually composed of an inner ciliated epithelium and an outer peritoneal covering. He seems to consider it a very exceptional condition for there to be any muscular elements at all, and he quotes Eudrilus (an earthworm) as his only example. Nasse (1882) seems to have been the first and only observer to note the essential points in the _ structure of the vas deferens of Tubifex, but his descrip- tion gives one the impression that he was uncertain on - some points, such as the actual form of the cells, whether they were annular or only spindle-shaped, and whether an 368 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. external peritoneal covering is present or not. In a paper recently published in the Zoologischer Anzevger, Dr. Bohumil Cejka (1918) gives a short account of the structure of Litorea krumbachi, in which he deals with the vas deferens. From his description of the histological structure of this tube, I am inclined to think he has seen something of the structure which I have attempted to describe. He says, for instance, that two regions are recognisable. In a transverse section of the first part of the duct he describes the tube as circular and the lumen as being provided with strong cilia. At the periphery he has seen black fibrils, which he believes play an important part in contraction. The second part of the tube, he says, is not ciliated, and has thicker walls which are composed entirely of gland cells—a structure which is quite different from that just described for Tubzifex rivulorum. 3. The Spermiducal Gland with the Prostate. This gland has the form of an elongated, somewhat pear- shaped and curved sac, into the broader end of which opens the second part of the vas deferens, while the narrower end communicates with the exterior by the penis, which will be described later. The cavity of the spermiducal gland is a direct continuation of the cavity of the vas deferens, and is also continuous with that of the penis (Pl. VII, fig. 45). We may, therefore, consider the gland and penis as being modified portions of the sperm duct. Attached to the gland at one side, and near its swollen extremity, is a glandular, irregularly- shaped, lobate mass forming what is known as the prostate (Pl. VII, fig. 45, pr.). The cells of which the prostate is composed are large and pear-shaped, with © prominent rounded nuclei situated usually in the swollen part of the cell. The narrower part of each cell forms its a | q . % y w———= =. ws, So” ‘ TUBIFEX. 369 duct, and in sections these are all seen to converge _ towards one point, that point being where the prostate is in communication with the spermiducal gland. At this point, the muscular and peritoneal layers of the wall of the gland are interrupted so that the cells of the prostate and those of the innermost layer of the gland are intimately connected with one another. In fact, it has been said by those observers, e.g., Vejdovsky, who have studied the development of the spermiducal gland and the prostate, that the latter is simply formed as a prolifera- tion of the cells of the lining epithelium of the gland. The wall of the spermiducal gland is composed of the following layers:—(a) An inner epithelium, (6) A muscular layer, and (c) Peritoneum. Beddard states that the lining epithelium of the spermiducal gland in Limnodrilus is ciliated, and this suggests the possibility that a similar condition obtains in the other members of the family Tubificide. Vejdovsky certainly believed it to be ciliated in Tubifex, as he states that the prostate is derived from cells of the ciliated lining epithelium. In the mature worm, at any rate, I have never been able to identify cilia in the gland, and as the second portion of the vas deferens is not ciliated this is not surprising. The cells of which this epithelium is composed differ a good deal in structure and appearance at different stages of their development. In a young worm, whose reproductive organs are all developed but not yet matured, the lining epithelium of the spermiducal gland is composed of a single layer of rather low cells, cubical in shape, with oval nuclei which are large and conspicuous and situated near the outer end of the cell. The cell is filled with a dense, granular cytoplasm, which stains deeply with nuclear stains such as borax carmine. Just at the 370 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. junction of the prostate with the gland, this single layer of cells gives place to a mass of irregularly shaped, some- what elongated cells which encroach a good deal upon the cavity of the gland in this region. In a mature worm the appearance of these cells is very different. They become much enlarged, so that they tend to obliterate the cavity of the gland. Their outlines become less distinct, and the nuclei, which remain near the outer end of the cells, are very difficult to identify. This increase in the size of the cells is accompanied by a change in their contents. The cytoplasm becomes less granular and very vacuolated, and its appearance suggests the presence of some secretion which stains readily with ‘‘ plasma” stains, such as picro-indigo-carmine (Pl. VII, fig. 45). Outside the epithelium is a layer of longitudinal muscle fibres placed somewhat obliquely, and outside this again a delicate peritoneal covering of flattened cells. 4. The Penis. The terminal portion of the spermi- ducal gland is modified to form a protrusible penis which opens to the exterior on the ventral surface of segment 11. The structure of the penis is somewhat complicated. Its cavity is continuous with that of the gland, and it is surrounded by a penis sheath, which, when the penis is retracted, is double, the outer layer being continuous with the epidermis of the body wall (Pl. VII, fig. 45, pe.s.). We may, therefore, speak of the outer and inner penial sheaths, both consisting of a single layer of epithelial cells, somewhat cubical in shape and provided with conspicuous, rounded, nucleolated nuclei (Pl. V, fig. 22, pe.s. and pe.s.!). The cuticular secretion, which forms a delicate layer outside the cells of the epidermis, is continued over the cells of the penial sheath, and in the J retracted condition of the penis may completely fill.the — space between the outer and inner penial sheaths. The © 1 ; 4 = Sh eee eh .hCU ee TUBIFEX. 371 outer sheath is provided with muscles externally, these being disposed mainly in two ways (Pl. V, fig. 22). There is a comparatively delicate layer of circular muscles completely investing the sheath, and, from this layer, oblique muscles pass, in a somewhat irregular fashion, to the body wall. Certain of these fibres branch, and the branches anastomose with the longitudinal muscles of the body wall. | The cavity of the penis proper is surrounded by a single layer of elongated cells (Pl. VII, fig. 45, le.) which encroach considerably on the lumen. Near the apex of the penis they are so large that they almost obliterate the lumen altogether. V. Tur DEVELOPMENT AND STRUCTURE OF THE OVA. The ovary of a young worm consists of a solid mass of undifferentiated cells, of which those which are nearest to the septum to which the ovary is attached are the youngest. The nuclei of these cells are often to be seen undergoing mitotic division, and by this means the number of the cells making up the ovary is increased. _ The first formed cells are gradually pushed further away from the septum, and these give rise to potential ova or _ oogonia, which can be recognised by the large size of _ their nuclei as compared with the amount of cytoplasm surrounding them. Vejdovsky suggests that in such a form as Rhynchelmis the oocytes are at first amoeboid, and that their subsequent rapid increase in size is due to the ingestion of the ova around them. I have never been able _to identify amoeboid oocytes in Tubifex, but some sections have revealed what appears to be an equally effective mode of nutrition. The young ovary is, as has already _ cells result in the formation of a central, non-nucleated, B12 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. been stated, composed of a solid mass of cells, all of which seem to have equal chances of developing into mature ova. In many cases, however, development only continues in those cells around the periphery of the ovary. The central cells do not develop any further, but gradually lose their individuality, the cell membranes disappear, and the nuclei are absorbed. These changes in the central cytoplasmic core surrounded by developing oocytes. It is interesting to notice that these oocytes are distinctly pear- shaped: the narrower part of the cell being embedded in the central mass, while the nucleus is lodged in the broad part of the cell (Pl. V, fig. 26, oc.). Certain of these cells develop much more rapidly than the others, and increase.so much in size that they protrude considerably beyond the surface of the ovary, and are only enclosed by a fine membrane composed of a single layer of much- flattened cells. It is very easy to conceive of these oocytes being dislodged from the ovary, and this is what actually does happen, for when they have reached a certain size they are transferred to the egg sac, while still immature, and here they complete their growth. In the course of my investigations of the living worm, I have several times succeeded in teasing out and freeing from the body the ovary, in a more or less complete condition. In most cases, its appearance very nearly resembles that seen in sections, and although the oocytes in this condition are very transparent, it is possible, by — the addition of a little methylene blue, to stain them — sufficiently to examine them before disintegration sets in, which, by the way, takes place very rapidly. Asa rule, © there is no special blood supply to the ovaries. It has already been mentioned that the perivisceral vessels of segments 10, 11 and 12 are much enlarged in the mature ~ TUBIFEX. a fh worms, and that these vessels give off important branches which are directed backwards over the reproductive organs. There are, however, no special branches from these vessels to the ovaries. In one case I found quite a different state of affairs on teasing out the ovary from the living worm (Pl. V, fig. 25). Only a portion of the ovary was obtained on this occasion, but the oocytes present were situated on all sides of a central “‘ stem’’ or rod in which a blood vessel was lying—this could easily be identified by the red colour of the blood which was still present in it (Pl. V, fig. 25, st.). The oocytes were arranged quite irregularly on the stem. Large and small ones lay side by side, sometimes grouped together into clusters, sometimes occurring singly. Each oocyte was attached to a short branch of the main stem terminating in a tiny swollen head, the oocyte itself being spherical in outline, or nearly so. Opposite to each stalk a short branch was given off from the blood vessel—this branch entered the stalk and terminated in the swollen head already mentioned. It was not possible, of course, to decide what was the connection of this ‘‘ ovarian vessel ”’ with the rest of the circulatory system, as the ovary was completely isolated. Although I examined many more ovaries I never saw this interesting condition again. As the oocytes contain no yolk-granules as long as they remain part of the ovary, they are then most suitable for purposes of examination, as the yolk-granules tend to obscure the other structures in the egg (Pl. V, fig. 27). The structure of such an oocyte of Rhynchelmis has been described in detail by Vejdovsky (1884), but the deserip- tion does not entirely agree with the conditions obtaining in an oocyte of Tubifex, at the same age. The oocyte is spherical in outline, and surrounded by a delicate investing membrane. The cytoplasm forms 374 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. a fine network, the density of which is only slightly | greater at the periphery than nearer the centre of the cell. In Rhynchelmis, Vejdovsky figures a much denser portion of the cytoplasm around the periphery with a more delicate network in the interior. The nucleus is large and rounded, and at this stage is situated near the centre of the cell, but as the ovum matures it comes to lie nearer the periphery. The nucleus is surrounded by a well- marked nuclear membrane, but I have not been able to identify any perforations through which the nucleoplasm is in communication with the cytoplasm, such as are figured by Vejdovsky as occurring in Rhynchelmis. The nucleus is composed of a large number of chromatin granules embedded in the usual linin network—the linin stains only faintly, but the chromatin stains much more deeply, and, therefore, shows up well in good preparations. The nucleus contains one or more nucleoli, which appear to have a different structure in different ova. In all cases there is one large nucleolus which is spherical in outline, and which we may describe as the “‘ principal ”’ nucleolus.* In some cases this nucleolus has a vacuolated appearance, showing one or two fairly large vacuoles, in other cases it stains uniformly and appears to be an undifferentiated mass of chromatin, while in yet other ova it has a granular appearance (Pl. V, fig. 27, p.nul.). In addition to this principal nucleolus there is usually a number of smaller masses which we may call “‘ accessory ”’ nucleoli.* These are not always present, but in those oocytes in which they do occur their number varies from 2 to 5. These accessory nucleoli very often have no connection with the principal one, but sometimes one large accessory nucleolus is in conjunction with the principal one, forming a compound body. In this case pak * Compare Wilson’s ‘‘The Cell in Development and Inheritance,” fle ————— Cr lee TUBIFEX. : 375: the accessory nucleolus usually stains more deeply with a nuclear stain than does its companion (PI. V, fig. 27, ac. nul.). Very soon after the oocyte has been transferred to the egg sac, yolk granules appear in the spaces of the cytoplasmic network, the cytoplasm meanwhile shrinking away from the wall a little. Later on the yolk granules become so numerous that the protoplasm cannot be seen at all. The formation of the granules seems to have no connection with the position of the nucleus, that is, they are scattered irregularly through the cytoplasm. The granules are spherical bodies of comparatively large size, varying from 2m to 4m in diameter. They stain very deeply with both nuclear stains such as haematoxylin and plasma stains, such as picro-indigo-carmine (Pl. V, fig. 24, ¥.'gr.). It is interesting to notice that maturation of the egg- cell actually begins while the latter is still enclosed in the egg sac, but I have not been able to decide exactly how nearly the process is completed before the egg is extruded, as all the specimens I have obtained which have been undergoing maturation have been at about the same stage. The oldest oocytes within the egg sac often exhibit a fully-formed nuclear spindle, the nuclear membrane having already disappeared (Pl. V, fig. 24). The spindle is somewhat dumbbell-shaped, and exhibits the normal structure. The two centrosomes are far apart from one another, but are connected by a large number of spindle threads. The star or aster (Pl. V, fig. 24, as.) is formed by a number of delicate threads which, surrounding the centrosome, radiate out into the adjacent cytoplasm. The chromatin has grouped itself into a number, about 24, of small rounded masses, which are arranged on the equator of the spindle (fig. 24, chr.). The nucleus at this time is situated near the periphery of the cell. = = at = iy ee Se ee 376 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. VI. Tue Eae Sac. The egg sac is set apart for the reception of immature ova which have become separated from the ovary. In this organ the ova complete their growth and undergo the first stage of their maturation process. The egg sac is an unpaired structure, and arises as a single, pouch-like outgrowth of the septum between segments 11 and 12. It is directed backwards, and in its simplest condition occupies only segment 12. But it soon enlarges considerably, and extends into the segments behind the twelfth. The sperm sac in the course of its development becomes pushed into the egg sac, so that we have here the curious condition of one sac lying actually within the other (Pl. III, fig. 11, sp.s., ovs.). The cavity is, of course, coelomic in origin, and remains undivided throughout, that is, there is no division of its cavity into smaller ones, such as occurs in the sperm sac. The wall of the egg sac is thin and composed of a single layer of flattened peritoneal cells, with no muscular elements at all (Pl. I, fig. 3, ovs.). Kgg-cells of different ages can be found in a ege sac, and their position in this organ suggests very forcibly that they have all been derived from the ovary. The youngest oocytes are situated in the most anterior segments: that is, in those nearest the ovary, and many of these have few or no yolk granules. The oocytes, when liberated from the ovary, fall into the cavity of segment 11, in which the ovary lies. As the egg sac is an outgrowth of the posterior septum of this segment, it is a very simple matter for the oocytes to find their way from the segment into the egg sac. TUBIFEX. aT VII. Tue Ovipwucts. There has been a considerable difference of opinion amongst earlier observers as to the position of the oviducts in most members of the Tubificide. Without going into the whole history of the question, we may notice that D’Udekem (1855) believed that in Tubifex rivulorum the oviducts were connected with the male ducts, indeed that they actually surrounded the termmal portion of the vasa deferentia. His views were supported by later observers, such as Claparede (1861) and Hisen (1885). Vejdovsky at first was inclined to agree with D’Udekem’s description of the relations between these two ducts; but later he changed his opinions, and came to the conclusion that the oviducts are situated between segments 11 and 12. This conclusion was based upon certain experiments which he performed. For example, he kept worms in certain chemical reagents, and observed the extrusion of the eggs between these two segments. For some time I was unable to identify the oviducts in my sections, but Mr. E. S. Goodrich, F.R.S., was kind enough to lend me some of his slides in which they were shown quite plainly. Since then, on a re-examina- tion of my own preparations, I have been able to distinguish these structures, though much less clearly than in those of Mr. Goodrich—to whom I wish to express my indebtedness. There can be no doubt that, while the oviducts are small and comparatively inconspicuous, they are normally present, at any rate in the fully mature worm. There is a single pair lying in the intersegmental line 11, 12 (Pl. I, fig. 2, ovi.). The duct is very short, and opens internally by a wide, funnel-shaped opening into the coelomic cavity of segment 11, while the external opening, which is small and inconspicuous, lies in the same longi- tudinal line as the male openings. 378. TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. VIII. Tue SSG RSE. During copulation the spermatozoa of one worm are transferred to the other, and stored in special organs known as the spermathecae (Pl. I, fig. 2, sp.). In Tubifex rivulorum there is one pair of these organs situated in the same segment as the testes, viz., In segment 10. Their general form is best seen by liberating them from the living worm. This can easily be done if a mature worm be placed on a slide and a cover-glass put upon it. The slightest pressure on the cover-glass is usually sufficient to rupture the body wall in the region of the reproductive segments, which are much distended owing to the large number of spermatozoa and ova developed. If the body wall be ruptured, the spermathecae, which for the greater part of their length lie freely in the body cavity, are usually forced out, but remain attached to the worm at their external apertures (Pl. VII, fig. 49). The ease with which this operation can be performed is due to the fact that the spermathecae are of considerable length and are bent round in the segment. They are also resistant to pressure, to some extent, and as soon as the body wall of the worm is _ ruptured they spring free. They are visible to the naked eye in this condition as small, pear-shaped, opaque, but glistening bodies, but it is necessary, of course, to examine them under the microscope in order accurately to describe their form. When examined thus, the whole organ can be divided into two regions :—(a) a pouch or sac-like portion (Pl. VII, fig. 49, sp. p!.) which narrows considerably to form (6) the duct opening to the exterior near the ventral setae of segment 10 (Pl. VII, fig. 49, © ; sp. d.). a The proportions of these two parts to one another HH tyes TUBIFEX. “4519 vary considerably according to the condition of the worm— in a mature worm the duct is usually somewhat longer than the pouch, but in the younger condition they are more nearly equal. Typically, the spermatheca lies entirely in segment 10, but about the time of copulation, and certainly when the spermatozoa are to be found in these organs, they enlarge considerably and may be either coiled round in segment 10 or extend into segment II, or even more posteriorly into segment 12 (Pl. IV, fig. 16, sp.). In the latter case there is a sharp bend in the spermatheca, and the duct runs forwards again parallel to the pouch in order to open to the exterior on segment 10. The pouch is quite simple, although it is so voluminous, and is devoid of diverticula; neither has it any connection with the alimentary canal, as is the case in certain Enchytreeidae. The structure of the wall of the spermatheca difters considerably in different parts. The wall of the pouch is thin, but three layers are recognisable. Internally there is a single layer of epithelial cells which seem to be of two kinds. Certain of the cells are squarish in shape and have very large nuclei, which are oval or rounded in shape, and usually include one principal and one or more accessory nucleoli. These cells are probably glandular in nature. Between these are smaller, narrow cells, whose nuclei are much less conspicuous, and which we may term interstitial cells. The wall of the pouch is strengthened by the presence of a few scattered muscle fibres placed parallel to the longitudinal axis of the spermatheca and outside the layer of epithelial cells. Outside this again, the wall is covered by a single layer of flattened peritoneal cells, the nuclei of which are oval in shape and form the most conspicuous part of the cells. The duct when seen in transverse section is circular 380 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. yo in outline, and its wall shows the same layers as are to. be seen in the wall of the pouch, but their structure and arrangement are somewhat different (Pl. V, fig. 23). There is a very marked change in the character of the | & cells forming the inner epithelium. There has been | considerable discussion amongst the earlier observers as ~ to whether the epithelium of the duct is ciliated or not in the spermathecae of species belonging to the family Tubificidae. Beddard, although he mentions the views of different authors upon the subject, does not venture any opinion himself. He states in his general account of the % | f ‘ 1 4 4 spermathecae of the Oligochaeta that ciliation does not, as a rule, occur, but that it has been described in a few forms, for example, Tubifex. As no reference was given, I have been unable definitely to trace the paper from which he obtained this information. It seems likely, however, that he is referring to a paper by Nasse (1882), which he mentions in his general description of the family Tubificidae. Nasse definitely states that there are cilia in the duct of the spermathecae of Tubifex, ‘In Ausfiihrungsgange trigt das Epithel eine Cuticula und flimmert.’’ Vejdovsky (1884), careful observer though he was, has denied the presence of cilia, and Stole in his monograph on the Tubificidae. (1888) does not figure them. I am now in a position to confirm the statement made by Nasse, for in sections of the spermathecal duct, both transverse and longitudinal, the cilia are particularly obvious (Pl. V, fig. 23, cz.). They are not visible when one examines the spermatheca in the living condition, for the wall of the duct is rendered opaque by a thick, muscular covering. The epithelium of the duct is 4 composed of a single layer of cells almost columnar in shape, but considerably longer than broad, and with large oval nuclei. The cilia are rather short, shorter hE) apt Lt op ewe. eh TUBIFEX. . 381 considerably than the radius of the cavity of the duct, so that they do not obstruct the passage much. The muscular layer is much more _ powerfully developed around the duct than on the pouch. The muscles form a double layer, those on the inside being arranged circularly, while outside this is a very definite sheath of longitudinal muscles. These appear to be continuous with those on the pouch, the circular muscles being interposed between them and the _ ciliated epithelium. The cells of the peritoneum covering the duct are somewhat different from those investing the pouch. They are no longer flattened, but squarish in outline, and form a rather more conspicuous layer than that of the pouch (Pl. V, fig. 23, pt.). The nuclei are usually situated near the middle of the cell, the contents of which are very granular. The terminal portion of the duct of the spermatheca is protrusible, and when it is protruded the duct narrows considerably near the end and leads to the exterior by a very narrow but straight passage. Near the sperma- thecal pore (Pl. IV, fig. 16, sp. p.) the ciliated epithelium ceases, and that which lines the narrow passage leading to the exterior is similar in structure to that forming the epidermis of the body wall, with which it is directly continuous. The muscular layer extends to the end of the duct, where the muscles become connected with those of the body wall. When the terminal portion of the duct is retracted, the passage to the exterior is no _ longer straight, but its wall is folded back in the form of a cone or arrow-head. 382 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. IX. THe SPERMATOPHORES. ~ A spermatophore consists of a large number of spermatozoa cemented together by a substance derived from the prostate, the cells of which open into the spermiducal gland as already described. These spermato- phores are comparatively solid and resistant structures, and can always be found in the spermathecae of a mature worm. When properly formed, they have a _ very characteristic shape and structure, but it is quite common to find ill-formed ones which are not at all typical in shape or structure. This seems to be due to a lack of the cementing substance. ae The number of these structures which may be found in one spermatheca varies considerably. I have often found only one, more commonly two or three, and occasionally as many as five or six. They vary, too, very much in size, but, as a rule, the perfectly formed ones are nearly or quite as long as the spermatheca in which they are found. They are arranged quite irregularly in the spermatheca, sometimes lying coiled up entirely in the pouch, and in other cases occupying the duct and whole length of the pouch, and yet lying coiled up in it. Their arrangement can best be seen when the sperma- thecae are liberated from the living worm, as described above (Pl. VII, fig. 49). They can then be examined within the spermatheca, or, with care, it is possible to liberate them by rupturing the wall of this organ, when those which are coiled up in it break free and may be removed in a perfect condition. They stain well in a very dilute solution of methylene blue, but permanent prepara- tions cannot be made in this way. They can, however, be fixed to the slide, and stained first with borax-carmine ~ and counter-stained with picro-indigo-carmine, when the ae of TUBIFEX. 383 various parts will be well differentiated, the heads of the spermatozoa staining well with the borax-carmine, and _ the tails with the picro-indigo-carmine. The spermatophores are visible to the naked eye, and when first liberated from the spermatheca appear as small, fine, white, glistening bodies, the largest being about 1-2 mm. long. When examined under the microscope they will be seen to have quite a complicated structure, but before describing this it will be well, perhaps, to say a word or two about their formation. } During copulation, the spermatozoa, together with the _ secretion of the gland cells of the prostate, are transferred from one worm to the spermathecae of the other. It seems certain that the moulding, which must take place before the spermatophores attain their final shape, is carried out entirely in the spermatheca, and moreover in one part _ only of the spermatheca, viz., its duct. The duct leading from the external aperture to the pouch is so narrow, and the quantity of sperm forced in so great, that the mass “must necessarily conform very closely to the shape of the duct. And this we find is the case even to the minutest detail. We will first describe the general form and minute structure of the spermatophore, and then show how this agrees with the general shape of the sperma- _ thecal duct. | The fully-formed spermatophore is a narrow, much elongated structure, many times longer than broad, and somewhat worm-like, its widest part being near the middle and tapering off at both ends. The anterior end is always simple in structure, but the posterior end may be ‘similar to it or may terminate in a beautifully moulded and curved conical head (Pl. VII, fig. 50). It has been suggested that the presence or absence of a conical head is characteristic of two different species, but this is 384 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. impossible, as I have found spermatophores with and without the head in the same spermatheca or in the two spermathecae of the same individual. I shall refer to this again later. | The minute structure of these spermatophores has been described by Vejdovsky (1884), and by Lankester (1871, a). When examined in transverse section, the spermatophore is seen to be circular in outline. In the centre is a cavity which we will call the axial cylinder (Pl. VII, figs. 51 and 52, az.c.). It extends from end to end of the spermatophore, and even into the conical head. It is widest in the centre, and tapers off at both ends. This cavity is filled with a substance which, when the spermatophore has just been liberated from the worm, is granular in appearance. This central mass stains deeply with borax-carmine, and in permanent preparations has the appearance of a mass of longitudinal fibres, which usually become somewhat shrunken by the action on them of reagents. Outside the axial cylinder is a very narrow dark band, which stains much more deeply than the substance of the axial cylinder (Pl. VII, figs. 51 and 52, sm.h.). For this reason it is very conspicuous. When highly magnified it is seen to consist of a very large number of minute oval masses, which, owing to their staining properties, I conclude are the heads of the spermatozoa. The rest of the spermatophore is made up — of a cementing material in which the tails of the sperma- tozoa are embedded (Pl. VII, figs. 51 and 52, sm.t.). As — was pointed out both by Vejdovsky and Lankester, these — are placed parallel to one another, but obliquely to the — axis of the spermatophore. The tails of the spermatozoa — appear as striations in the homogeneous substance in which they are embedded, and when viewed from the surface are seen to pass obliquely round from left to right. — o , , TUBIFEX. 385 Both Vejdovsky and Lankester state that not all the tail is embedded in the cementing material, but that a small portion lies outside, so that the whole spermatophore is surrounded by the free ends of the spermatozoa. The spermatophores which I have examined show a certain amount of variation in this respect. In many cases the tails are undoubtedly partly free from the cementing material, and the free ends are placed at right angles to the longitudinal axis of the spermatophore, and not obliquely to it. In some cases, however, the layer of cementing material is thicker than in others, and when that is so there is no portion of the spermatozoon lying free. Whether this is an abnormal condition, or merely due to the amount of cementing material present, is very difficult to decide. | Lankester’s description differs from this in several details. He states that there is a narrow, highly refringent band outside the axial cylinder and another of similar nature outside the layer of cementing material and spermatozoa, between it and the free ends of the tails of the spermatozoa, the latter being in constant vibratile motion. He also says that the whole spermato- phore exhibits movement when liberated from the spermatheca into a dilute salt solution. Although I have performed this operation many times, I have never succeeded in persuading the spermatophore to exhibit the active movements which have been described. Further, Lankester believes that the head of the spermatozoon is much elongated, of almost the same length as the tail, and that the head as well as part of the tail is buried in the cementing material towards the outside of the spermato- phore. I think there is no doubt that the heads of the spermatozoa lie just outside the axial cylinder, but very close to it, and are confined to the deeply staining zone 386 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. described above, and that the oblique striations already referred to are caused by the tails of the spermatozoa alone. It 1s very evident that the very characteristic form of the spermatophores is due entirely to the shape of the spermathecal duct and its external aperture. The duct is circular in section, so also is the spermatophore. During the rapid movements of the worm it is very natural that the spermatophore should be forced through the duct into the wider pouch by a slightly spiral motion, and this explains the oblique arrangement of the tails. The muscular walls of the duct may also assist in forcing the spermatophore into the pouch. As a rule, the first- formed spermatophores, those which are forced into the pouch soon after or during copulation, are pointed at both ends or without the arrow-shaped head described above. On the other hand, it is very usual to find that the spermatophores which are formed last, that is, those which lie partly in the duct and partly in the pouch, are provided with the conical extremity. There can be little doubt that the arrow-shaped head is moulded in that part of the duct following directly on the external aperture, for the shape of the two are exactly similar when the ~ spermathecal duct is retracted. I conclude, therefore, that the spermatophores, moulded first in the spermatheca, are forced through the duct and into the pouch quite rapidly—partly by pressure from behind exerted by other masses of spermatozoa and cementing material, and partly by the contractions of the worm. At any rate, the process would seem to be such a rapid one that the spermatophore does not remain any length of time in the duct. On the other hand, towards the close of copula- — tion, the formation of the spermatophores would take place more slowly, and the posterior ends of the last TUBIFEX. 387 one or two could remain in the terminal portion of the spermathecal duct a sufficient time to allow of their being moulded to its shape. It is true that I have never observed the posterior end of the spermatophore lying in this position, but I have found it several times only a short way along the duct and with the conical head perfectly moulded. X. THe Cocoon. It is common amongst all Oligochaeta for the ova and spermatozoa to be deposited in cocoons, and Tubifexr rivulorum is no exception to the rule. The worms are sexually mature in the autumn, and the cocoons are first seen in November. They are deposited on the mud in which the worms live, and very soon are completely buried in it. The cocoons are made of a fibrous substance secreted by the glandular cells of the clitellum, and have a characteristic form. They are usually whitish or greyish in colour and semi-transparent, but when viewed with the naked eye they appear opaque, but this is due to the eggs which they contain. They are usually oval in shape, but sometimes become more nearly spherical, and at either end they are drawn out into a short neck through which, when the young worms are ready to hatch out, they emerge (Pl. VII, fig. 54). The neck is filled with a plug of the same substance of which the cocoon is formed, but it is probable that it is of a less resistant nature. The number of eggs which may be present in a cocoon is very variable. Sometimes there is only one, more commonly from four to nine, but occasionally the number is greater, and in a very few cases I have seen as many as thirteen or fourteen. Although I have not been able to find any spermatozoa even in a freshly-laid 388 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. cocoon, there can be no doubt that in Tubifezr, as in other Oligochaeta, the spermatophores are passed into the cocoon with the eggs, and are there dissolved to set the sperma- tozoa free. The rest of the cocoon is filled with a clear, colourless fluid, probably albuminous in nature. It seems to depend largely on the number of eggs in a cocoon as to whether they all develop into embryos or not. I have been able to count as many as nine young worms in a cocoon, all of which hatched out, but never more than that number ; so it is probable that, as a rule, all the eggs in a cocoon develop, but when the number is unusually large certain only of them develop, the others breaking down. The young worm when hatched exactly resembles the adult in external form, and, with the exception of the reproduc- tive organs, all the organs of the body are well represented. The newly-hatched worm is about a quarter of an inch long, and consists of 30 to 35 segments. The alimentary canal is complete with the exception of the anus, which does not develop until later. The whole canal contains a large number of yolk granules, the remains of those found in the egg, and there are some also in the coelomic cavity. The dorsal and ventral blood ’ vessels are visible, the former already slightly contractile. The setae are perfectly developed, but small in size, and there are not more than two or three in each bundle in the anterior segments, and one more posteriorly. PARASITES. Tubifex rivulorum is a host for several internal parasites, and in addition to these it often has attached to the body wall externally Vorticellae and Fungi. The Vorticellae are not true parasites, for they all possess an active circlet of cilia at the distal end and a TUBIFEX. 389 mouth. The worm is simply made use of in this case as a substratum to which the Vorticellid becomes attached. These Protozoa are very abundant on some individuals, and completely absent from others, but, as a rule, when present, they are confined to the posterior segments of the body, those which wave about freely in the water. The fact that Fungi often attack these worms was recognised by McIntosh (1871), who speaks of Fungi growing on the dis-organised anterior segments, while the posterior ones are in full activity. I have often observed fully active worms which are infected with Fungus growths, and it seems very probable that the Fungus increases so much in quantity that it actually causes the disintegration of the segments which are attacked—the anterior segments are usually affected first, but the growth may spread throughout the entire length of the body. These Fungi appear, as a rule, to attach themselves in or near to the setigerous sacs, for they are usually to be seen emerging from between the two prongs of the sigmoid setae, forming long, delicate filaments, several of which may originate from the same seta-bundle. The internal parasites may be found in the alimentary canal, sperm sac and body cavity. McIntosh states (1871) that he has found numerous examples of Opalina amongst the sandy mud in the intestinal canal. He gives figures of some of these specimens, which have very diverse forms. These worms were captured from the margin of the River Tay. It is interesting to note that, although I have examined numerous worms from the Thames both in sections and in the living condition, I have never once found Opalina in the alimentary canal. I have been more successful, however, in finding parasites in the sperm sac and body cavity. During the summer months of 1912, when the worm was really immature, 390 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. several specimens were found which at first sight had the appearance of maturity, for, in the region of the repro- ductive organs, the body was white, swollen and opaque. When the body wall in this region was punctured, parasites escaped in large numbers. At first they appeared as small, rounded, bodies, white and glistening, | each of which proved to be a cyst bounded by a fairly thick wall (Pl. VII, fig. 534). If a cover-glass be put on, or the cyst be subjected to slight pressure, the wall bursts and its contents are liberated. These consist of an enormous number of extremely minute spores, each one somewhat awl-shaped, and provided with a caudal filament at oneend. At the opposite end is a rounded, clear space, and between the two what may be described as the body of the spore, consisting of granular protoplasm and NE DOL TALL OO embedded in it an elongated nucleus, apparently broken into two parts (Pl. VII, fig. 533, nu.). This parasite was first described by Kolli, and was named by him Urospora saenuridis. It is a Gregarine of the sub-order Eugregarinae, and tribe Acephalina.* | McIntosh (1871) described these parasites as awl- shaped bodies, but he did not appreciate their real significance, as he interpreted them “ as stages in the development of the spermatozoa.’’ Nasse also saw them (1882), and interpreted them correctly as parasites occurring in the sperm sac. Muinchin* mentions another Gregarine parasite (Synactinomyzxon tubificis) as occurring in the sperm sac of T'ubifex rivulerum, but I have not been fortunate enough to find it. The last parasite noticed as occurring in Tubifex during my observation of the worm was Caryophyllaeus, ‘ a Cestode belonging to the family Caryophyllacea of the 1 * Vide ‘‘ A Treatise on Zoology.’’—Hd. by HE. Ray Lankester. Part I, i pp. 194 and 298. ; TUBIFEX. 391 Cestoidea Monozoa. The parasite occupies the coelomic cavity, and has only been found during the summer months, when the reproductive organs are undeveloped. The mature form is to be found in the intestine of certain fishes, and the young in the anterior segments of Tubifex rivulorum, usually in segments 10 to 17. Each one is provided at the anterior end with a characteristic mobile organ capable of being thrown into a series of undulating folds. At the opposite end the cylindrical body is produced into a tail provided with three pairs of hooklets. Bes ci 392 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. BIBLIOGRAPHY. 1908. AsHwortTH, J. H. The Giant nerve cells and fibres of Halla parthenopeia. Philos. Trans. Royal Society of London, Vol. 200, p. 427. 1898. ArHEston, L. The epidermis of T. rivulorum with especial reference to its nervous structure. Anat. Anz., XVI, pp. 497-509. 1893. Brpparp, F. E. On the Atrium and Prostate in the Oligochaeta, P.Z.8., pp. 475-487. 1895. A Monograph of the Order Oligochaeta, Oxford. 1888. ——— _ The Nephridia of Earthworms. Nature, Vol. XX XVIII, p. 221. 1911. Spermatophores of Earthworms. P.Z.S., p. 412-420. 1890. Brnyuam, W. B. “ Atrium” or “ Prostate.” Zool. Anz., XIII, pp. 368-372. 1880. BuoomrieLtp, J. The general features of the Development of Spermatozoa in the Vermes. Zool. Anz., III, pp. 65-67. 1745. Bonnet, C. Traité d’Insectologie, ou Observations sur quelque espéces de Vers d’ Eau douce, qui, coupés par morceaux, devinnent autant d’animaux compléts. Paris. 1850. BupeeE, J. Ueber die Geschlechtsorgane von Tubifex rivulorum. Arch. f. Nat., XVI, pp. 1-8, Pl. 1. 1851. Ueber Respirationsorgane von Tubifexrivulorum. Verh. Nat. Ver. Preus. Rheinl., VII, pp. 258-9, Pl. V, Fig. 5. 1862. BucunHouz, W. H. Beitrage zur Anat. der Gattung Enchytraeus. Schriften der Physik. 6konom. Gesellschaft in Konigsberg, p. 108. 1881. Burscuii, O. Beitrage zur kenntniss der Fisch-Psorospermien. Zeit. f. wiss. Zool., Vol. 35, pp. 629-651. 1895. CALKINs, G. Observations on the Yolk nucleus of Lumbricus. XIV. ‘Trans. New York Acad. Sciences. 1895a. Spermatogenesis of Lumbricus. Journ. of Morph., Vol. 1913. Czzka Bouumi. Litorea krumbachi—Ein Beitrag zur System- atik der Enchytraeiden. Zool. Anz., XIII, Band Nr. 4. 1861. CLAPAREDE, E. Recherches Anatomique sur les Oligochaetes. Mem. Soc. Phys. Geneve, XVI, p. 217-291. 1905. Deppotia, Po. Untersuchungen uber die Spermatogenese von Lumbricus terrestris. Zool. Anz., Bd. XXVIII. 1886. DeEwitz, J. Pfluger’s Archiv, Vol. 37, p. 219, 1885; Vol. 38, p. 358, 1886. 1886. DirrENBAcH, O. Anatomische und Systematische Studien an- Oligochaeta limicolae. Inaugural Dissertation, Giessen. From Ber. Oberhess. Ges., XXIV, pp. 65-109. 1856. Dovére, M.P. Essai sur l’ Anatomie d. 1. Nais sanguinea. Mem. Soc. Linn. Normandie, Vol. X, pp. 306-321. 1855. D’Uprxem, J. Histoire naturelle du Tubifex des ruisseaux. Mem. Cour. et des Sav. Etr. Belg., XX VI, 38 pp. 1879. Eisen, G. Preliminary Report on Genera and Species of Tubi- ficidae. Bih. K. Vet. Ak. Handl., V, No. 16, 26 pp. 1885. Oligochaetological researches. Ann. Report Commissioner of Fish and Fisheries, Washington, pp. 879-964. TUBIFEX. 393 1894. Foot, K. Preliminary note on the Maturation and Fertilisation of the egg of Allolobophora foetida. Journ. of Morph., IX, No. 3. 1879. FRraisse, P. Ueber Spermatophoren bei Regenwurmern. Arbeit. Zool. Inst. of Wuerzburg, Bd. V, pp. 38-54, Pl. 4. 1895. Goopricu, E. S. On the structure of Vermiculus pilosus. Q.J.M.S., XXXVII (n.s.), p. 253. 1851. Groupe, A. E. Die Familien der Anneliden mit Angabe ihrer Gattungen und Arten. Berlin, 164 pp. (Also in Arch. f. Nat., XVI.) 1884. Hammonp, A. Some further researches on Tubifex. Journ. Micr. and Nat. Sci., III, pp. 147-155, Pl. XVI. 1906. Harcirt, CoHas. W. Experiments on the behaviour of tubicolous Annelids. Jour. Exp. Zool., Vol. 3, pp. 295-320. 1899. Hartar,S. On Limnodrilus gotoi. Annot. Z. Japon, III, pp. 5-11. 1889. Hermann, F. Beitrage zur Histologie des Hodens. Arch. f. mikr. Anat., XXXIV. 1909. HzesszE, EpMonp. Quelques particularités de la spermatogenése chez Oligochaeta. Arch. Zool. exper. (4), T. 10, pp. 441-446. 1842. HorrmeristerR, W. De Vermibus quibusdam ad Genus Lumbri- corum pertinentibus Dissertatio Berolini, 28 pp., 2 plates. 1909. JosepH, H. Die Amdbocyten von Lumbricus. Ein Beitrag zur Naturgeschichte der celluliren centren. Arb. Z. Inst. Wien, XVIII, pp. 1-60. 1906. KammerreR, P. Der Bachrohrenwurm Tubifex rivulorum (Lamarck). Wochenschr. Aquar. Terrar. Kunde. Jahrg. 3, pp. 467-468, 480-482, 491-492. 1816. Lamarck, J. Histoire naturelle des animaux sans vertébres. Ist edition, Vol. III, p. 225. . 1871. Lanxester, E. R. Outlines of observations on the organisation of Oligochaetous Annelids. Ann. and Mag. Nat. Hist., Ser. IV, Vol. VII, pp. 90-101. 1869. A Contribution to the knowledge of the lower Annelids. Trans. Linn. Soc., XX VI, pp. 631-646. 1871. On the structure and origin of the Spermatophores or Sperm ropes of two species of Tubifex. Q.J.M.S. (.s.), XI, pp. 180-187, Pl. X and XI. 1906. Loxrs, J. Dynamics of living matter. New York, p. 156. 1895. Matruews, A. Maturation, fertilisation and polarity in the Echinoderm egg. Journ. Morph., X. 1871. McInrosu, W. C. On some points in the structure of Tubifex. Tr. Roy. Soc. Edin., XX VI, pp. 253-267. 1908. MicuarE“sEN, W. Zur Kenntniss der Tubificiden. Arch. Nat. Jahrg., 74 Bd., 129-162. 1908. MuUxier, C. Regenerationsversuche an Lumbriculus variegatus und Tubifex rivulorum. Arch. Entw. Meck. Bd. 26, pp. 209-277. 1773. MUtuier, O. F. Vermium Terrestrum et Fluviatilium ace. Historia Hafn et Lips. 1882. Nassz, D. Beitriige zur Anatomie der Tubificiden. Diss. Inaug. Bonn, 32 pp., 2 plates. 1913. Nomura, Exiraro. On two species of aquatic Oligochaeta (Limnodrilus rag and L. willeyi). Journ. of Coll. of Science, Imperial Univ. of Tokyo, Vol. XXXYV, Art. 4. 394 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. 1875. Prrrier, E. Sur le Tubifex umbellifer. Arch. Zool. Exp., IV, Notes pp. VI-VIII. | 1903. PIERANTONI, UMBERTO. L’Ovidutto e la emissione delle uova | nei Tubificidi (Contributo alta biologia degli Oligochaete marini). Arch. i Zool. Napoli, Vol. 1, pp. 108-119. : 1893. Ranpoutpo, H. Ein Beitrag zur Kenntniss der Tubificiden. Vierteljahrschr. Nat. Ges. Zurich, XX XVII, pp. 145-147. 4 1869. Ratze., F. Beitrige zur anat. und systematisch Kenntniss der ; Oligochaeten. Zeitschrift f. wiss. Zoologie, Bd. XVIII, pp. 563-591, Pl. 42. 1867. Beitriige zur anat. von Enchytraeus vermicularis. Zeit. f. wiss. Zool., Bd. XVIII, pp. 105-109, Taf. 6, 7. 1902. Rice, Wa. J. Studies in Earthworm chloragogues. Biol. Bull. Woods Holl, III, pp. 88-94. 1878. Rotueston, G. The blood corpuscles of the Annelids. Journ. of Anat. and Physiol., Vol. XII, Pl. III, pp. 401-418. 1764. ScHorrrer, J. Abhandlungen von Insecten Regenberg. _1906.. SoutHERN, R. Notes on the genus Enchytraeus with description of new species. Irish Naturalist, Vol. 15, pp. 179-185. 1909. Contributions towards a Monograph of the British and Trish Oligochaeta. Proc. Roy. Irish Acad., Vol. X XVII, p. 119. 1913. StepHENson, J. On Intestinal Respiration in Annelids, with considerations on the origin and evolution of the Vascular System in that Group. Trans. Roy. Soc., Edin., Vol. XLIX, Part III (No. 14). 1909. Report on a Collection of the smaller Oligochaeta made by Capt. F. H. Stewart, I.M.S., in Tibet. Rec. Ind. Mus., Calcutta, IIT, pp. 105-114. 1910. On some littoral Oligochaeta of the Clyde. Trans. Roy. Soc. Ed., Vol. XLVII, Pt. 1 (No. 2). 1910a. Studies on the aquatic Oligochaeta of the Punjab. Rec. Indian Mus., Vol. V, Part I. 1912. — On a collection of Oligochaeta mainly from Ceylon. . Spolia Zeylanica, Vol. VIII, Pt. XXXII, p. 251. 1888. Srotc, A. Monographie Ceskych Tubificidii Morfologicka a systematicka studie. Abk. Bohm. Ges., Series VII, Vol. 2, No. 11, 45 pp. 1907. Sztiro, Anpor. Adatok az édesvizi csévagé féreg. (Tubifex tubifex). Mull kivalaszté szerveinck ismeretéhez. Allatt Kozlem. Kot. 6, pp. 31-36. 1884. Vespovsky, F. System u. Morphologie der Oligochaeten. Prag., 166 pp., 16 plates. 1891. — Note sur une Tubifex d’Algerie. Mem. Soc. Zool. Fr., IV, pp. 596-603, Pl. XV. 1907. Watson, A. T. The habits of tube-building worms. Brit. Assoc. Rep., 76th meeting, p. 599. 1906. Wacner, F. Zur Oecologie des Tubifex und Lumbriculus. Zool. Jahrb. Abt. Syst., Bd. 23, p. 295. 1852. Wux1ams, T. Blood proper and chylaqueous Fluid of Inyerte- brate animals. Philos. Trans., Pt. II. 1904. Wuitson, E. The cell in Development and Inheritance. TUBIFEX. | 395 EXPLANATION OF PLATES. List oF REFERENCE LETTERS. a. = Uncinate setae from ventral bundle. ac. nul. = Accessory nucleolus attached to principal one. ac. nult. = Accessory nucleolus. am. = Ampulla. an. |. = Antero-lateral lobe. an. r. = Annular ring of vas deferens. ar. h. = ‘“‘ Arrow-head ” of spermatophore. as, = Aster. at. = Spermiducal gland. ax. c. = Axial cylinder. b. = Uncinate setae from dorsal bundle. bl. = Blastophore. br. = Brain. bu. c. = Buccal cavity. b. v. = Blood vessel. b. v. 7. = Blood vessel in intestinal wall. b. w. = Body wall. c. = Pectinate seta. ca. f. = Caudal filament. ca. 8s. = Capilliform setae. c. ¢. = Chloragogen cells. ce. = Cells of vas deferens. chr. = Chromosomes. cht. = Chromatin. ct. = Cilia. ci. f. = Ciliated funnel. cl. = Clitellum. cle. = Cleavage marks. c. m. = Circular muscles, co, 8. = Connective tissue sheath. ct. ep. = Ciliated epithelium. cu. = Cuticle. cul. = Prolongation of cuticle into setigerous follicle. cy. = Cytoplasm. extremity cy. m. = Undivided cytoplasmic mass to which oocytes are attached. d. = Capilliform setae. d. bl. = Dorsal seta bundle. d. t. = Dorsal tubes or neurochord. d. v. = Dorsal vessel. e. = Subsidiary prongs. eg. = Eggs. e. m. = Opening of prostate into spermiducal gland. ept. = Epispore. f. = Fibres of nerve cord. fb. s. = Fibrillar substance. fl. = Flagellum. g. c. = Gland cells of epidermis. gr. m. = Granular mass. h. = Large blackish-brown granules. ht. = Heart. hyp. = Epidermis. 7. = Smaller granules of chloragogen cells. in. = Intestine. in. c. = Interstitial cell of epidermis. in. v. and in. vi. = Intestinal ves- sels. le. = Large cells surrounding the lumen of the penis. l. m. = Longitudinal muscles. m. = Muscles from pharynx to body wall. ma. = Matrix. m. f. = Intrafollicular muscles. mi. = Middle piece. m. 1, = Anterior median lobe. mo. = Mouth. m. p. = Parieto-vaginal muscles, mu. = Muscular layer. mu. 1, = Muscular layer, circular inside, longitudinal outside. n. = Nerve cord, n. b. = Nerves to body wall. 396 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. n. Cc. = Nerve cells. n. cd. = Non-ganglionated part of nerve cord. n. cd', = Ganglionated part of nerve cord. ne. = Nephridium. n. f. c. = Nuclei of young follicle cells np. = Nephridiopore. n. pr. = Nerve to prostomium. ns. = Nephrostome. n. 8. f. = Nuclei of setigerous follicle. nu. = Nucleus. nut, = Nuclei in mitotic division. nul. = Nucleolus. nu. mem. = Nuclear membrane. nu. sp. = Nuclear spindle. oa. = Ovum. ob. m. = Oblique muscles. oc. = Oocyte (pear-shaped). oc. y., oc. yl. = Young and older oocytes respectively. oe. = Oesophagus. ov. = Ovary. ovi. = Position of oviduct. ovs. = Ovisac. pe. = Penis. pe. s. = Outer penis sheath. _ pe. st. = Inner penis sheath. ph. = Pharynx. p. nul. = Principal nucleolus, pp. c. = Peripharyngeal commissure. pr. = Prostate. pro. = Prostomium. pr. t. = Preseptal part of nephridial tube. ps. l. = Postero-lateral lobe. pt. = Peritoneum. pv. v. = Perivisceral vessel. pv. v. 2 = Perivisceral vessel of seg- ment 2. pv. v. 3 = Perivisceral vessel of seg- ment 3. r. = Wall of nephrostome. s. = Setae. se. = Septum. se. c. = Secretion of gland cells. s. gl. = Septal glands. s. f. = Setigerous follicle. si. v. = Supra-intestinal vessel. sm. t. = Tails of spermatozoa. sm. h. = Heads of spermatozoa. sp. = Spermatheca. spc. = Spermatocyte. sp. d. = Spermathecal duct. sp. g. = Sub-pharyngeal ganglion. sph. = Spermatophore. spm. = Spermatids. spo. = Spores. sp. p. = Spermathecal pore. sp. p\. = Spermathecal pouch. sp. Ss. = Sperm sac. s. S. = Setigerous sac. st. = Stalk. t. = Contractile nephridial tube. te. = Position of the testes in the immature - worm before’ the enka of ~~ development of the sperm sac. u. = Wall of ampulla. va. ep. = Vacuolated epithelium. v. bl. = Ventral seta bundle. v. d. = Vas deferens. v.d.1=Ciliated part of vas deferens. v. d. 2 = Non-ciliated part of vas deferens. pt. = Vesicular peritoneal cells. v. = Ventral vessel. cy. = Wall of cyst. ; = Thin-walled coiled part of tube. = Wider part of tube with vesi-. cular peritoneal cells. gr. = Yolk granules. . = Thick-walled tube leading to nephridiopore. x < TUBIFEX. 397 Prats I. Figs. 1 to 3. Diagrammatic representation of the first Fig. 4. Fig. 95. Fig. 6 Pie. 7. Fig. 8 Fig. 9 Fig. 10 eighteen segments of the body with the principal organs in each segment, as they appear in the living worm. Puate II. Longitudinal section through the prostomium and first six segments of the body to show the anterior part of the alimentary canal and nervous system. x 160. Section through one of the dorsal setigerous pages. x, O10. Isolated setae from dorsal and ventral bundles. x 300. Transverse section through the first segment of the body, showing the buccal cavity and brain. x 200. Transverse section through the first segment, showing brain, peripharyngeal connectives and sub-pharyngeal ganglion. x 200. Puate III. Transverse section through segment 8 in the region of the hearts. x 160. Transverse section through segment 11, showing the ovaries and one spermatheca. x 100. 398 Fig. Fig. Fig. 16. Fig. Fig. TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Fig. 11. i: 13. 14. 15: Transverse section through segment 14, showing the ovisac and sperm sac. x 90. Longitudinal section through the body wall of a mature worm in the region of the clitellum. x 620. Longitudinal section through the wall of the intestine, showing the details of its structure. x 370. Chloragogen cells in the living condition, drawn directly after their separation from the wall of the intestine. The nucleus is not shown, as it is very difficult to see in the fresh cells. x 1000. Chloragogen cells: the cells were separated from the wall of the intestine, fixed in Perenyi’s fluid and stained with Brazilin on the slide. The irregularity in shape is due to the shrinkage. x 1000. Pruate LY: Longitudinal section through the reproductive seoments 10-13 of a mature worm. One of the spermathecae (sp.) only is shown cut twice. It is much enlarged and extends into segment 12. The septa between the segments were not visible in the preparation. x 120. Fig. 17. Longitudinal section through the ciliated funnel of a mature worm covered with spermatozoa, showing its structure and con- nection with the vas deferens. x 230. TUBIFEX. 399 (a) Longitudinal section through the ciliated funnel of an immature worm. x 2950. (B) Transverse section of the same. x 250. (A) Transverse section of ciliated portion of vas deferens. x 800. (Bs) Transverse section of non-ciliated portion of vas deferens. x 800. (4) Longitudinal section through ciliated portion of vas deferens. x 800. (Bs) Longitudinal section through non-ciliated portion of vas deferens. x 800. Pruate V. Longitudinal section of vas deferens. The section shows the transition from the ciliated to the non-ciliated part of the duct. x 800. Transverse section of the retracted penis. x. O10. Transverse section of the spermathecal duct. x 360. An oocyte from the ovisac, undergoing matura- tion. x 160. An unusual form of the ovary which was liberated from the body and drawn in the living condition. Longitudinal section through a portion of the ovary, showing pear-shaped oocytes. ~ 480. Longitudinal section through a portion of the ovary, showing typical young and older oocytes, the latter just before their separa- tion from the ovary. x 280. 400. TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. Figs. 28-35. Stages in the development of the normal spermatozoa. Fig. 28. Uninucleate spermatogonium from testis. x 820. Fig. 29. Multinucleate spermatogonium from sperm sae. § x W720: Fig. 30. Harly stage in the formation of the spermatosphere, showing the first cleavage of the cytoplasm. x 1720. Fig. 31. Formation of spermatosphere complete. Surface view of spermatosphere with spermatocytes. x 470. Fig. 382. Section of spermatosphere with spermato- cytes. x 470. Fig. 33. A rather later stage. x 670. Fig. 34. Formation of spermatids which are just beginning to elongate. x 610. Fig. 35. Spermatozoa fully formed, but still attached to the blastophore. x 350. Figs, 36-40. Stages in che” - development of “Ginnie spermatozoa. Fig. 36. Formation of spermatocytes. x 660. Fig. 37. Spermatids just beginning to elongate. x 660. Prats VI. Fig. 38. Later stage in the elongation of- the spermatids. x 660. ce Fig. 39. Immature “‘ giant’’ spermatozoa. x 660. Fig. 40. (a) Ordinary form of the spermatozoa. x VTL0. (2) eee form of the spermatozoa. TUBIFEX. 401 A figure drawn from a living specimen to show the arrangement of the blood vessels in the prostomium and first five segments of the body. The coiling of the perivisceral vessels is slightly simplified. x about 220. A figure drawn from a living specimen to show the arrangement of the blood vessels in an intestinal segment. x 180. A diagrammatic representation of a complete nephridium. A small portion of the nephridial tube covered with vesicular, peritoneal cells—drawn in the living condition. Puiare VII. Longitudinal section through the spermiducal gland, prostate and penis. ~x 110. Transverse section of the nerve cord. x 630. Longitudinal section of a nephrostome with the preseptal part of the nephridial tube, part of the body wall and septum. x 520. Longitudinal section of an ampulla of a nephridium. x 790. Spermathecae liberated from the body through a rupture in the body wall caused by pressure. x 50. External form of a very large spermatophore. x 80. 402 Fig. - TRANSACTIONS LIVERPOOL BIOLOGICAL | ay 52. 08. —~B4, Lanstnadital section of part of a Pere: x 620. oo Praasverse section of a spermato v aes (a) Section of a complete cyst saenuridis. x 390. . (B) Detailed drawings of two of the x 1900. i 6 General view of a cocoon mi ripe eggs. ee C. TINLING AND CO., LTD., PRINTERS, 53 VICTORIA STREET, ; 5 B. a MEMOIR XXIIL M' Ferlane & Erskine. Lith. Edi * (1) 1, aa a = 3 is) ' —_—— Pe -anencccneenaaa Sls at MPH veg eo an iy Miss ~igees Bee Gey M' Farlene & Erskine, Lith. Edin Pe a) UZ) spun mor ait Gibtbere 00! MH iy g E AW peaugauapad Di wit oogg7a08 einen ww Cotta OH ‘Ops Les > ’ arn crt Ar AAA NSE SEES Lol eleloolayecelsiekuatlels eis|eieleletesel * ie psasy et ee . cur i : pale ~~ aa) 7 AQ = = ae ane Hit eines Einee Nit acy atti ess BEB v CouguuuooUubil ‘£ LM. B.C. Memorr XXIIL. i : . ‘ A : i S : ’ P : , = % ’ , - < 4 * f 2 f ~ rt ¥ “f ' 5 ri | - \ al f 3 ¥ de LM.B.C. Memoir XXIIL | Plate IE 1: << oes G 4 OM S> a5 ey ; SS vy Ct Mei aR eG ON SS CET aneern Tene ‘ sawed Sy ass Wn ce ge LT) > WO vara yittneXisuo™s Var: rs We. s 7. AML Sion MNyIPA AG) THE ; a J AS Sart —— ney), ATT Poe” - c —_ een UP - “iy A Lag it 5 5 ® a r cl. ep.- q Pe me. 7 Fig. 15. wv. G.C.D. del. TU BIFEX. M' Farlene & Erskine, Lith. Edin. Puate IV. M' Farlene & Erskine, Lith. Edin. f a Vuwelew woye ( > aa fy ae — KH LM. B.C. Memoir XXIII. G.C.D. del. on 7h. *y it % B.C. Memorr XXIIL Spc. Fig, 35. Fig. 34. Fig. 35. Fig. 37. ~G.C.D. del. T U B I F EX. M'Ferlane & Erskine. Lith. Edin. Ss S g 5; S o ss 5 2) y is 1 oemeemmerernste de Se iit i saa aaa ; ers ma ETRE M a ye a Pears M'Farlane & Erskine, Lith. Edin. : Fig. 40 ates ~ Sane ee eA Fig. 42 ! 3 ‘ 3 2a, A li ag SNE nama 3 ie TITS : : a i sg Pe ls een eee _— ‘i ao << _ geen" ¢ < 1p a © — A, _wK Jt 1 ama Af a ae Y wat - cae *s, ~~ AS eee \ A ae, ¢ a fh we “) \ Ke Vf woe ‘ . ’ Fig. 59. a am Sco INTRONS SF Tp qty ANTEATER eeTE TMT PUN : mi mt Fes a l pot i ~ iti? Was nN oe jg 4 ty 4 \ i, Y = ‘ tC } ‘3 ' = % 5 4 : ' “ea —. ‘ Ss 1 EEX, ens ™ ‘ i ™ Rin oie: a ee oer SVigpii aR are y og 5 os Fig. 41. —— SF ce eee ae al — So B.C. Memoir XXIIL. Se DT me oe G.C.D. del. Ls SS CUCU ™M.B.C. Memorr XXIIL. Puate VIL. Pe. a te OORE! Pan M' Farlene & Erskine, Lith. Edin. PE EE. mG.C.D. del. wl MO 00905 3331 90