· '.'
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ABSTRACT
A study of the life history of a population of Nereis virensat Brandy Cove, St. Andrews, New Brunswick, indica-6es that these worms may live for 12-15 years, maturing at the earliest in their fourth year. The majority, however, do not mature until their fifth or sixth year. At the onset of maturity 'gonadal' clumps are found either floating free in the coelom or embedded in the parenchymal tissue at the bases of the parapodia. Eggs were observed to arise froIn these 'gonadal' clumps during every month of theyear and took 1-2 years to mature. 'Sperm plates' were only produced froID 'gonadal' clUlnps at the end of July to the beginning of' August and mature sperrn \'lere observed by the follovIing May. The ratio of males: females in the spawning population was found to be 3:1. It was also observed that this population did not undergo extensive epi tol;:al metamorphosis and that the worms "l:;herefore spawned in an atokous .condi tian. These worms did no-t; show an extensive swarming behaviour at the sea
surface p in fact only the males were observed swimrning close to
the surface of the mud p on the incomming tide, releasing a continuous stream of sperm froIn thelr pygidial papillae. What the females do in the field i8 still uncertain. Larval develop ment followed very closely that described for other nereids an0. was observed to be non-pelagic. · ",'
History of Nereis virens at Brandy Cove, St. Andrews, N.B.
Doreen Snow ',\
Some Aspects of the Life History of the Nereid Worm. Nereis virens (Sars), on an Inter tidal Mudflat at Brandy Cove, St. Andrews, N.B.
by
Doreen Rosemary Snow, B.Sc. (Hons.)
A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science
Department of Biology McGill University
lVIarch 1972
@ Doreen Rosemary Snm'l 1972 · '.'
Frontispiece 1 - Size range in the N. virens population at Brandy Cove, st Andrews, N.B. a) This worm is about O.5gm. in weight and according to this study, about one year old.
b) These worms are about 25-)Ograms. • ~. t
'.\
. 1111 ~IIIIIIII li IllIlllllllll,l, Il,llll J 111'l111~111 1111111~11 l Il.1 ~~TlMETE~S ' ., : ,', '.'
1 • 1 ' . 1 1111 1 )':NTlMETERS 2' '~, '. 4. 5,111 III 1 1III · ,.1
Frontispiece 2 - Colour differentiation in N. virens. The upper individual is immature with the typical orange-reddish-green colour, whereas the bottom individual has green eggs about 100-120u in the coelom which make the bases of the parapodia green. . ".' ..' '.' · '.'
Frontispiece 3 - Pale green 3-segment larvae of N. virens (approximately 330u) with the red pigment spots at the sides of the head. • t~ f , . ',' ",,{.
TABLE OF CONTENTS
ACKNOWLEDGElV.lENTS. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • i
LIST OF TABLES ••••••• 0 • 0 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • ii
LIST OF FIGURES ••••••••••••••••• ~ ••••••••••••••••••••••••• iii CIASSIFICATION •••••••••••••••••••••••••••••••••••••••••••• vii ll'ITRODUCTI ON •••••••••••••••••••••••••••••••••••••••••••••• 1 lV.lETHODS AND lVlATERIALS ••••••••••••••••••••••••••••••••••••• 6
Samplillg' Area •••••••.••. ., 0 •••••••••••••••••••••••••• 6 Samplillg' technique •••••••••••••••••••••••••••••••••• 6 Maintenance in the laboratory...... 13 Measurement of worm size ••••• ...... 14 Examination of Coelomic Fluid •••••••••••••••••• ..... 15 Histological treatment...... 16 Observations on Spawning...... 16 Artificial Fertilizations and Rearillg' of Larvae..... 16 Collections of Larvae in the Field...... 17
Photographs •••••.•••.••..••... 0 •••••••••••• QI • • • • • • • • 18' SIZE DISTRIBUTION
Growth Pattern••••••• •••••••••••••••••••••• 0 ...... 19
Age Cl.asses •••••••••• 0 •••••• 0 • • • • • • • • • • • • • • • • • • • • • • • 22 Age at Maturity...... 36 REPRODUCTION Development of the Coelomic Fluid ••••••••••••••••••• 47 "'~'
TABLE OF CONTENTS (Cont'd)
1) Small c oe lomocyte S·y •••••••••••••••••••••• 0 • 2) Mature trephocytes and the formation of 'parenchymal' tissue...... 48 J) Gonadal clumps...... 49 4) Phagocytic cells and later changes...... 51 Maturation and Structure of the Oocytes...... 52 Maturation and Structure of the Spermatozoan...... 62 Differentiation of the Sexes...... 70 Sex Ratio...... 71 Spawning 1) Field observations ••••••••••••••••••••••••• 7J 2) Laboratoryobservations •••••••••••••••••••• 74
J) Release of Spermatozoa ••••••••••••••••• ~ ••• 76 4) Release of oocytes ••••••••••••••••••••••••• 79 5) Conditions governing the time of spawning •• 80 URVAL DEVELOPMENT Early Development...... 85 Larvae with J-Chaetigerous Segments...... 98 Later Larval Stages...... 106 GENERAL DISCUSSION... • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 121
CONCLUSIONS •• 0 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 124 REFERANCES CITED APPENDIX · '.'
-i- ACKNOWLEDGEMENTS
l am deeply indebted to the Fisheries Research Board of Canada, Biological Station, St. Andrews, New Brunswick, who graciously provided facilities, such as laboratory space and equipment, in conjunction with the Huntsman Marine Laboratory; and to aIl members of the Biological Station for their kind co-operation and advice. l am grateful to my supervisor, Dr. Joan Marsden, for her support during this project; to Dr. Dorothy Pocock for her help and use fuI advice, especially at the beginning of this project; to John Patterson who aided in collecting the worms; to A~ Sreeharen for help with the statistics involved in this project; to Bill McMullon and Frank Cunningham of the Biological Station, F.R.B., for their help with the photographie work; to Dr. F. A. Aldrich who offered advice during the writing of this thesis; to my brother Hugh, who stuck on the stacks of photographs; to Miss Cynthia Long who helped edit the thesis and to my father who helped to type the final copy. Finally, l wish to thank the National Research Council of Canada, for their support during this project. ".~' .
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LIST OF TABLES
TABLE I. Time and duration of each collection period, showing as well the number of worms collected in each •••••..•...•.•••••..•.•...•...••...... •..•. 12 TABLE II The mean weight for each possible age class for each month, as determined from figure 5 •... showing some approximate ages, in terms of years, for worms of a certain weight •••••••••••••••••••• 32 TABLE III. Coelomic fluid categories for N. virens...... 38 TABLE IV. Ratio of males to females from January to May 1969 .•.•..••...•••..••.•.••••..•.••...••.. & • • 72 .-....
-iii-
LIST OF FIGURES
FIGURE 1. Map o~ St. Croix River and Passamaquoddy Bay showing the positions of Oak Bay, Brandy Cove, Bocabec Bay and St. Andrews Point ••••••••••• 8 FIGURE 2. Map of Brandy Cove, New Brunswick showing the collection site, Area A ••••••••••••••••••••••• 10 FIGURE 3. a) Relationship between segment number and weight in M. virens. b) Relationship between length and weight in N. virens. c) Relationship between width and weight in N. virens •...... I!' ••••••••••••••••••••••••••••••••• 21
FIGURE 4. Weight/frequency diagrams ~or Mereis virens ~rom September 1968 to September 1969 ••••••••••••• 25
FIGURE 5. Graphs o~ the logarithmic dif~erences of the class ~requencies plotted against the Iudpoint o~ the class ~or N. virens from September 1968 to September 1969 (excluding Dec. 1968) ••••••••••• 28-30 FIGURE 6. Weight/frequency distributions for the total sample of N. virens in each coelomic fluid category from September 1968 to September 1969.... 40 FIGURE 7. Coelomic fluid of M. virens with only small coelomocytes, Coelomocytes I ••••••• o...... 42 FIGURE 8. Coelomic fluid of N. virens with both small coelomocytes and large coelomocytes or parenchymal cells, Coelomocytes II.oooooooooooooo 42
FIGURE 9. Coelomic fluid o~ N. virens with small coelomocytes, parenchymal cells and gonadal clumps, Gonadal Clumps ••••••••• o...... 44 FIGURE 10. Coelomic fluid of a spent male, with sperm and phagocytic cells (cells, 15-20~. containing green crystalloid granules)...... 44
FIGURE 11. Coelomic fluid o~ N. virens with small coelomocytes, parenchymal cells, gonadal clumps and small eggs, Females I ••• o.. o...... 55 -iv- FIGURE 12. Mean oocyte diameters for each female examined over the period of September 1968 to September 1969...... 57 FIGURE 13. Coelomic fluid of N. virens during the early stages of Females II. Eggs containing numerous oil droplets and measuring 120-14o~...... 60 FIGURE 14. Coelomic fluid of N. virens during the late stage of Females II. Eggs very dense, pale green in colour and measuring 180-220~...... 60 FIGURE 15. Coelomic fluid of male N. virens in November and October, containing 'sperm plates', 50-90p.... 64 FIGURE 16. Coelomic fluid of male N. virens with 'sperm plates' breaking up into secondary spermatocytes which then undergo meiosis to produce spermatids.. 64 FIGURE 17. Coelomic fluid of male N. virens with spermatids breaking up into sperm...... 67 FIGURE 18. Coelomic fluid of male N. virens in May with mature sperm possessing an acrosome, a nucleus, mitochrondrial spheres and a tail...... 67 FIGURE 19. Average sea water temperatures for Brandy Cove, st. Andrews, N.B...... 69 FIGURE 20. Posterior end of a mature male N. virens showing the pygidial segment with the anal papillae...... 78 FIGURE 21. Longitudional section through the pygidial segment of a male N. virens showing the anal papillae packed with sperm and an opening which makes the coelomic cavity continuous with the outside .....•...... 0 ••••••••• 0 •••• 0 78 FIGURE 22. Daily heights of the low tides for the St. John, N.B. area for April and May 1969 and for May 1970, correlated with the phase of the Moon •••••••••••••• 0 •••••• 0 • • • • • • • • • • • • • • • • • • • • • • • • 82 FIGURE 23. Fertilized egg of N. virens about 10 min. after fertilization, showing the gelatinous envelope and the fertilization membrane...... 88 • f~ 1
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FIGURE 24. Ear1y c1eavage stage of N. virens - 2-ce11 stage .••. 0 ••• 0 •••••••••••••••••••••••••••••• 0 • • • • • 88 FIGURE 25. Ear1y c1eavage stage of N. virens - 4-ce11 stage •••••••••••••••••••••••••••••••••••••••••••• ~ 90
FIGURE 26. Ear1y c1eavage stage of N. virens - 8-ce1l stage ...... Il ••••••••••••• CJ • • • • • • • • • • • • • • • • • • • • • 92 FIGURE 27. Later cleavage stage of N. virens showing the macromeres on top of the micromeres...... 92 FIGURE 28. Early stage of the larvae of N. virens. a) showing the red pigment band b) showing chaetae projecting beyond the cuticle...... • . 95 FIGURE 29. Free swimming trochophore (protroch) of N. virens. a) showing the red pigment band, b) showing two pairs of chaetae projecting beyond the cuticle...... 97 FIGURE 30. Early three segment larvae of N. virens. a) just after the third pair of chaetae had projected beyond the cutic1e. b) about 2-3 days 1ater than the previous showing the beginnings of the anal cirri and the first pair of peristomial cirri ••••••••••••••• 100 c) showing the beginnings of the prostomial tentac.les ...•.•...... 0 ••••••••••• 0 • • • • • • 102 FIGURE 31. a) Late three segment 1arvae of N. virens. b) En1arged head region of the late three segment larvae of N. virens showing the one-toothed jaws •••• Q ••••••••••••••••••••••••••••• 104 FIGURE 32. Four segment larvae of N. virens. a) the fourth segment is almost formed and the anal cirri, the peristomial cirri and the prostomial tentacles are slight1y longer than in the three segment 1arvae. b) squashed four se~nent larvae showing that the jaws have two teeth ••••••••••••••••••••••••••• 108 FIGURE 33. Five segment larvae of N. virens showing the beginnings of the second pair of peristornial cirri...... 110 -vi- FIGURE 34. Six segment larvae of N. virens. a) showing the notopodia of the first pair of larval parapodia elongating to form the peristomial tentacles. b) enlarged head region showing the four toothed jaws...... 112 FIGURE 35. Eight segment larvae of N. virens. a) showing the notopodia of the first larval segment moving forward and elongating to become the third peristomial cirri and the neuropodia giving rise to the fourth. b) enlargement of the head showing the jaws wh which now possess six teeth •••••••••••••••••••••• 115 FIGURE 36. a) Ten and eleven segment larvae of N. virens. b) Enlargement of the head of an eleven segment larvae, showing that the first larval segment has now become part of the head. . . • • • • • • • • • • • • • • • • • • • . • • . . • • • . . • • • . • • • • • • • • • 117 FIGURE 37. a) Thirteen segment larvae of N. virens. b) Enlargement of the head of the thirteen segment larvae of N. virens •••••••••••••••••••••• 119 1
-vii- CLASSIFICATION
PHYLUM •••••••••• Annelida CLASS ••••••••••• Polychaeta SUBCLASS •••••••• Errantia FAMILY •••••••••• Nereidae GENUS ••••••••••• Nereis* SPECIES ••••••••• Nereis virens
*In using the designation Nereis (Linne) l am aware that this species could also be placed in the genus Neanthes (Kinberg)o The latter having the proboscis covered with paragnatha while the former has one or several groups missing. However, Turnbull (1876) and Berkeley and Berkely (1954) have described great variation to the paragnaths groupings of Nereis virens. This controversy has not yet been settled and for this study we shall use the generic designation Nereis (Linne). l
INTRODUCTION
Nereis virens (Sars), the common clam worm or sandworm, has an extensive distribution in the intertidal zone along the North Atlantic Coast. Although it can be found in many situations from high to low water mark on both rocky and sandy shores, it is especially common in sheltered areas of both sounds and estuaries, burrowing near the low water mark of sandy and Muddy shores (Pettibone, 1963). This study considers an intertidal population at Brandy Cove at the mouth of the St. Croix River. Thid river, which flows into the Bay of Fundy, has enormous intertidal mudflats which produce ideal conditions for certain polychaetes such as Clymenella torguata, Nephthys caeca and N. virens. An extremely large population of N. virens was found to occupy an area just above the extreme low water mark in a sand and mud mixture rich in organic materials and poor in clear sandy soils. The beach was stratified with large rocks at the high water mark graduating to fine muddy sand and silt at the low water mark. The worms constructed fragile, irregular, temporary burrows at depths up to 1.5ft. in the muddy sand. Although a considerable advance in our knowledge of polychaete life histories, especially within the Nereidae, has been made in the past two decades, information on many species, --....
-2- including N. virens, is still rather scanty. The most extensive study on a natural population of N. virens was carried out at Southend-on-Sea on the north side of the Thames Estuary by Brafield and Chapman (1967). Besides describing the basic patterns of oogenesis and spermatogenesis, they also observed that N. virens like N. diversicolor is not an epitokous form and that only the rnales left the sand and swarmed. They suggested that the females either discharge their eggs onto the surface of the mud without leaving their burrows or swim less actively and spend a shorter time at the surface. The~ did not observe either activity in the laboratory or in the field. They also attempted to elucidate some of the problems involved in the aging of these worms, concluding that N. virens had a two year life cycle, spawning and dying in its second year. The only other reference to longevity in this species is that of Copeland (1935) who, while keeping N. virens for physiological study, observed that isolated specimens lived under laboratory conditions for at least three years. There is no comparable account of gametogenesis and breeding in a North American population of N. virens. However, thisspecies has been of commercial importance as bait for fishermen during the last three decades, and it has received some attention, mainly from the Maine Department of Sea and Shore Fisheries in terms of worm management and conservation......
-3- Gustafson (1953), reporting observations on N. virens in Maine, clearly described the swarming of epitokous worms (with the body divided into two distinct regions) at the sea surface from the Middle of March to late in June, with the production of free swimming pelagie larva. A lot of his material was based upon general observations made by fishermen and wardens at various places along the Maine coast and no details were given concerning the larval development. In a general survey of the polychaetous annelids of the New England region, Pettibone (1963) reported numerous observations on the swarming behaviour of N. virens: they have been observed to emerge from the mud just as the tide rises; they have been observed swimming in the shallow water along the shore, in tide pools, sometimes swarming in immense numbers both in the daytime and evening; sometimes scores of spent males were observed. All swarming worms were in essentially an atokous condition. Reports have also been made of N. virens spawning in the St. Croix River and Passamoquoddy Bay by Art MacKay and his assistant (personal communications). They observed mature worms (males only) swarming as the tide was rising. Correlated with this was intense feeding activity by the gulls, which could be used as an indicator of spawning. Other general observations have been made by Mclntosh(1910), Gustafson,G.(Thorson, 1946), Mileikovskii. (1961), Khlebovich (1963) ~\
-4- and Sveshnikov (1965) on various aspects of the life history of N. virens. Mclntosh (1910) has published the following obser vations on this species; in specimens secured in May, the females discharged small eggs measuring 20-30p from the brokem posterior surfaces and in October trochophores, collected in bottom tows and assigned by him to this species, had a size of 228~. Thorson (1946) stated that Dr. Go Gustafson, at a meeting held at the Zoologista Institutionen, Lund in the autumn of 1943, gave an account of reproduction and development in this species in which he stated that the larval development was non-pelagie. There appears to be no other account of this work. Other papers present observations on N,virens in and around the White Sea. Mileikovskii (1961), Khlebovich (1963) and Sveshnikov (1965) observed swarming of epitokous worms. Khlebovich (1963) outlined a 2-year life cycle with one year-olds, length 23-85mm., width 3-7mm.,weight O.08-1.24gm., found in the littoral region. With the onset of winter these one year-olds migrated from the littoral to the sublittoral where they became mature and then swam to the surface to reproduce. Mature individuals were 190-385mm. in length,16-26mm. in width and 11.7-36.7gm. in weight. Sveshnikov (1965) captured epitokous individuals reach ing 45gm. in length. He also traced larval development to the 7-setiger stage and observed the larvae to be pelagie. It is obvious therefore, that there are cOnflicting opinions -5- on the various aspects of the life history of this worm. This is especially true of worms from the North American coast where no extensive survey has been carried out. Since biochemical work, including hormonal controlof'maturation and analysis of the lipids in the gut, body wall and coelomic, was being carried out at McGill on this species from Passamoquoddy Bay, it seemed desirable to have more conclusive information on this North American population. In an attempt to elucidate some of the aspects of the life history of N. vi~ens, we set out to determine size distribution in the natural population, age at maturity, the pattern of gametogenesis and breeding and the nature (pelagie or non-pelagie) of larval development. '.t.t
MATERIALS AND METHODS
Sampling Area All observations, unless otherwise stated, were made on a single population of N. virens at Brandy Cove, St. Andrews, N.B. (Figure 1). The sampling area chosen, Area A (Figure 2), was that of greatest density of the worms on the mudflat. Area A was very close to the lower intertidal limit making collections possible only when the tidal level was 3.0ft. or less. The maximum tides of the area are about 26-29ft ••
Sampling Technique Examinations of Area A showed a gradation in worm size, from small~r to larger worms, as one progressed toward the low water mark (Table l, Appendix). The area, therefore, was transected into 9 sections which were parallel to the low water mark; each section being two meters in length and having no limit in width. Samples of worms were collected in each section every month from areas of approximately one square meter. These samples were taken at monthly intervals from September 1968 to September 1969 (Table 1). The sampling period was 4-6 days each month, with the sampling time bei":s 1-2 hours a day. The number of worms collected on any day ranged from 50-100. Samples were taken with the aid of a cownon garden fork, which caused less -7-
Figure 1; Map of the St. Croix River and Passamaquoddy Bay, showing the positions of Oak Bay, Brandy Cove, Bocabec Bay and St. Andrews Point. . f.' ··i·
. \
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N
New Brunswick
Bocabec River:
Passamaquaddy Bay
Maine
o 2 Naut. Mil •• -9-
Figure 2; Map of Brandy Cove, New Brunswick, showing the collection site, Area A. l,' ·-·i·
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, , " , " , " , • \ , n" 1 •t .,t= 1 , , tJ , fJ' .1 .. il i 1 .. ·"i·
---\
-11- ... .,'
Table I; Time and duration of each collection period, showing as weIl the number of worms collected in each. --\
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COLLECTIONS DATE OF NUMBER OF WHOLE COLLECTIONS WORMS IN COLLECTIONS Sept. '68 Sept. 22 - Sept. 26 555 Oct. '68 Oct. 21 - Gct. 25 410 Nov. '68 Nov. 18 - Nov. 22 443 Dec. '68 Dec. 19 - Dec. 22 203 Jan. '69 Jan. 16 - Jan. 21 309 Feb. '69 Feb. 14 - Feb. 20 369 March '69 March 16 - March 21 372 April '69 April 3 - April 7 396 May '69 May 2 - May 7 402 June '69 June 1 - June 6 428 July l '69 June 30 - July 5 453 July II '69 July 28 - Aug. 2 430 Aug. '69 Aug. 26 - Aug. 31 458 Sept. '69 Sept. 24 - Sept. 28 415 TOTAL - 5643 -1)- in jury to the worms than would a shovel. The mud turned up was broken apart with the hands to insure that as many of the sInaller worms as possible were retained in the sample. The effectiveness of this method was doubtful, for the extremely small worms (less than O.5gm.), although.tediously searched for, easilyescaped notice, while larger worms escaped by burrowing rapidly as the fork was lifted. Because of the consistency of the mud it was impractical to sieve. The worms were then placed in plastic buckets which were half full of sea water (approximately 25 worms per bucket) and transported back to the laboratory.
Maintenance in the Laboratory The worms were easily maintained in running salt water which was 1-2oC warmer than the environmental temperature. On several occasions losses were sustained due to a decrease or cease in the salt water flow. On one particular occasion a considerable number of mature males and females died limiting experimentation on spawning in the laboratoryo All worms measured and used for coelomic fluid analysis were not left for more than six hours in the laboratory and in most cases they were taken directly from the plastic buckets and observed as soon as they had been anesthetized. · - ,~'
-14- Measurement of Worm Size
Whole worms, approximately 25 at a time, were ~nesthetized in 0.1-0.2% solution of MS222 (Sandos Produets Limited) in sea water. Abnormal worms sueh as those with reeent injuries, with posterior segments missing or with newly regenerating posterior segments, were disearded. After they had been in the anesthetie for 0.5-1.0hours, the worms beeame plaeid and easy to handle and the following parameters were reeorded. a) Weight The worms were dried in Kim Wipes to remove as mueh exeess water as possible and then weighed to O.OOlgm., using a Mettler P160 balance. b) Length - The worms were suspended alongside a ruler and the length measured to the nearest 0.5em. e) Width - The width of the third segment posterior to the peristomium was measured, exeluding the parapodia, to the nearest 0·5mm. d) Number of Segments
AlI the worms eolleeted from September 1968 to September 1969 were weighed. The other three parameters were only reeorded for 150-200 worms. The worms were then transferred to a beaker of refrigerated salt water until they revived and subsequently plaeed in tanks of '·,1·
-...... -15- running salt water. No after-effects were observed from the MS222.
Examination of Coelomic Fluid A monthly sample of 50-100 worms was used for the examin- -ation of the cellular elements in the coelomic fluide This was a selected sample of maturing individuals taken because of their relatively low occurrence in the total population. Ten of these were anesthetized at a time in MS222. After removal from the anesthetic they were rinsed in three changes of fresh, filtered sea water to remove debris from the outside of the animal and incisioh then placed ventral surface up on a glass plate. An incission, J-4mm. long, was made midway along the ventral body wall. Coelomic fluid which oozed out was picked up with a small syringe, and examined immediately under the compound microscope. A record was made of the various cel1 types present. A smear was made of the coelomic fluid and allowed to air dry. This was then fixed in Schaudinn's Fluid for 10-JO minutes, washed in 2-J changes oi 70% alcohol, treated with iodine alcoho1 and stained in Ehrlich's Acid Hematoxylin. The stained slides allowed a more detailed examination of the coelomic fluid and could also be kept as a reference. AlI measurements of the coelomocytes, eggs, sperm plates, etc., unless otherwise stated, were made from these smeared-dried- ... '.
1.
-16- stained slides.
Histological Treatment The worms were preserved in Bouin's Fluid and pieces of worms approximately 1 cm. in length were subsequently embedded
in paraffine Wax sections were cut at 7~ and stained in either Ehrlich's Acid Hematoxylin and Eosin or Mallory's Tripple stain.
Observations on Spawning
F~eld observations for spawning activity were made two or three times a week during April and May 1969 and from the 5-11 of May 1970, at Brandy Coye and St. Andrews Point. Activity was looked for by wading around in the water at low tide or by watching for signs of abnormally heavy feeding by sea gulls. Less frequent observations were made at Oak Bay, Chamcook and Bocabec (Figure 1) using only the latter method as an indicator of spawning. Spawning was also observed in the laboratory by placing the mature males and females in tanks and noting their behaviour. However, this study was limited due to inadequate numbers of females and because of the breakdown of the salt water system, resulting in the death of many mature worms.
Artificial Fertilizations and Rearing of Larvae "Artificial" fertilizations resulted from allowing mature .. ,.r
-17- males and females to ernit their gametes freely into the sea water in laboratory tanks. After fertilization, the eggs were either placed in a plankton net (mesh lS0~) and suspended in a tank of flowing sea water or maintained in a cold room (approximately, 7°C) in bowls of sea water. Samples were taken at least once a day, more frequently just after fertilization. The stage of development was photographed and/or drawn and the diameter of the fertilized eggs or the lengths of the larvae were measured. Whole mounts were made of some of the larvae, and stained with borax carmine.
Collections of Larvae in the Field Random samples were taken at 2-day intervals during low tidefrom the rniddle of April 1969 to the end of July 1969 in and near Area A. The larvae were obtained by stirring up the mud in shallow depressions in the beach, passing the mud through a 210u mesh sieve and then rinsing the sieve in a bucket of sea water. This method could be used for only 8 or 9 days in each month since at other low tides the area was covered with o.S-4.0ft. of
water. At such times samples were taken by stirring up the mud p dragging the sieve through the water and rinsing the sieve in a bucket of sea water. To make sure that the larvae were from the mud, a sarnple was taken through the water without stirring up the rnud, for c ornparis on. .. ,.f ···11'
-18- In the laboratory the material in the bucket was passed through the 210).1 mesh sieve again and samples of the debris were placed in petri dishes to be examined under a binocular Inicroscope. Larvae were removed from the debris with a fine pipette, placed on a slide and examined under the compound microscope. Photographs and drawings were made as before. Plankton tows were carried out at irregular intervals during April, May and June 1969 in Brandy Cove. Both horizontal and
oblique tows were Inade for approximately 10 minutes using a 150~ mesh plankton net. Plankton samples were also obtained at
approximately 2-day intervals from a 250~ mesh plankton net which had been set up on the floating pontoon by the groundfish division of the F.R.B., Biological Station. The net was hung from the side of the pontoon and it received a continuous stream of water via a small pump attached to the pontoon about a foot below the surface of the water.
Photographs Photographs were taken using a Nikon microscope attachment on a Zeiss compound microscope. SOIne photographs were taken Growth Pattern The worms in eaeh monthly sample were weighed as deseribed in the materials and Methods and the weight of eaeh worm tabulated as in Table II (Appendix), using a elass interval of' 0.5gm. Worms ranged between 0.0-30.0gm. in weight, with about 98% of the worms weighing less than 15.0gm. Length, width and segment number were determined for only a small proportion of the worIns weighed, in order to establish their relationship to weight. The relation ships between weight and eaeh of the other three parameters are shown in the seattergrams in Figure 3a,b,e. Figure 3a shows that the rate of' segment proliferation is high in worms weighing less than 1.Ogrn. Thereàfter, the rate of addition of new segments tapers off slowly and f'inally reaehes a plateau at 4.0gm. when the worms have attained 120-150 segments. Length also inereases rapidly as the worms grow to a weight of 3.0-4.0gm. (Figure 3b). However, no plateau is attained at this time; for in worrns weighing more than 4.0gm. there does appear to be a small but signif'ieant inerease in length as weight inereases, probably due to the enlargement of' existing segments. Width (Figure 3e) on the other hand, inereases markedly in worms up to about 2.0gm. and more slowly thereafter. Although measurements are searee f'or worms greater than 6.0gm., data available does -20- Figure 3: a) Relationship between segment number and weight in Nereis virens. b) Relationship between length and weight in Nereis virens. c) Relationship between width and weight in Nereis virens. · - ,~' .-.~'. -21- 160 a " " ... Z III 60 ~III ut 40 20 0 40 b É 30 "1 :c ;; .. Z III ...... : ... 0: 20 10 i-· 10 c 5 O~--~--~2~--3~·--~4--~5~--.~--~7~~.~--9~~1~0~~,1~~1~2--~1~3~~14~~'5 WRIGHT -sma, -22- suggest a continuous increase in width as weight increases. It can easily be seen that growth in young worms up to 60-70 segments is rnainly by segment proliferation with little increase in length, width or weight. Thereafter, segment proliferation slows do'll'ffi and growth appears to become increasingly due to the enlargernent of existing segments. A sirnilar basic pattern of growth was worked out by Clark and Scully (1964) for N. diversi- eelor. In that species, however, growth by segrnent proliferation occurs only up to 40-50 segments. This is reasonable since N. diversicolor attains a total of only 100-110 segments in cornparison to the 120-150 segments characteristic of N. virens. Clark and Scully (1964) also made similar observations on Nef'htys !JDrnber31 Nephthys lombergi and concluded that this pattern of growth .may be common to a number of polychaetes. These relationships also enable us to predict the approxirnate segment nmnber, length and width of a worm with a given weight. Age Classes W'eight/frequency diagrams were constructed to illustrate the monthly weight distributions throughout the period of September 1968 to Septernber1969 (Figure 4). From February 1969 to September 1969 there was a steady increase in the number of worms in the weight classes 0.0-0.5grn. and 0.5-1.0gm. The rise in February and March 1969 could indicate recruitment of young worms into the • • {~f ···l· -23- population. However, recruitment itself' would not account f'or the rnuch larger number of' worms within these size groups in September 1969 as compared to September 1968. Inf'iltration of' the beach by other worms was possible but it was highly unlikely that the sampling was extensive enough to disturb the popuiation; for Ganoros (1951) has reported that a commercial area may be dug as many as 20 times during one season (spring and summer) without altering the spawning population and Dow and Wallace (1955) have pointed out that the worms are so mobile that many of' them are not even exposed to commercial diggers. It is more probable to assume that as recruitment was taking place the author was becoming more and more ef'f'icient at detecting the small worms and that the increase is, theref'ore, due to a combination of' both of' these peaks f'actors. In the weight distributions over 1.0gm. many paeks are apparent but the f'ailure to trace them f'rom one month to another discredits their signif'icance. In an attempt to extract signif'icant trends, the data in Table II (Appendix) was anaIyzed according to a technique developed by Bhattacharya (1967) to separate polyrnodal distributions into their components. Based on the assumption that weight was distributed normally within an age group and that the polyrnodality observed in a sample was the resultant of' several age groups mixing in a sample; we could let y(x) denote the observed f'requency in the class with x as its midpoint and let h denote the class --l, -24- Figure 4: Weight/frequency diagrams for Nereis virens from September 1968 to September 1969- -25- Jan. '69 Juil" J '69 \ Juil" lE '69 100 70' \ A.~: '69 Sept. '69 15 20 25 30 15 20 25 30 WEIGHT -gm•• · . f.' -26- interval: then plot y(x+h)/y(x) against x on semi log paper or ~ log y = log y(x+h)/log y(x) against x on ordinary graph paper and look for regions where the graph looks like a straight line with negative slope. The number of such regions would be the number of components and the mean of each component could be calculated from the equation ur =). r + h/2 (where '" = x intercept). These components could then be designated as age classes. Two other commonly used methods for the analysis of polymodal frequency distributions, Harding (1949) and Taylor (1966), did not give any results. The graphs (Figure 5) of the logarithmic differences of the class frequency plotted against the midpoint of the classes for each monthly sample shows five to six approximately straight regions with negative slope, indicating five or six possible components or age classes. The relative means of these possible age classes were calculated using the equation given and are tabulated in Table II. To designate these as age classes, however, we wou Id have to assume that size and age are directly related. There are two possible reasons for the lack of correlation between age and size. One is the fact that normal growth may be hampered by frequent regeneration of lost posterior segments. However, the work of Clark and Scully (1964) on young N. diversicolor indicated that although their worms lacked the growth promoting hormone in the -27- Figure 5= Graphs of the logarithmic differences of the c1ass frequencies (ie. weight frequencies for each c1asss in Table II. Appendix) p10tted against the midpoint of the c1ass for N.virens from SepteInber 1968 to September 1969 (exc1uding December 1968). . ~.' -28- Oct.'.. Nev. ' .. ~St J ~ ~ ~ ··20 III :::) g •... ··40 ut ut C .-60 ...u !... ••• ... 0 lOI ~ III 11& Jen. ' .. III '.Il ...... ë5 u j 1... MIDIIOINT o. CLASS -29- -80 -60 ..rch ' •• April ' .. -40 -20 ;: ~ S! ~ 0 .!.. ...u Z III May '69 June '69 ...•III G u i :le ~ •CC ..0" MIDPOINT OF CLASS "'-:"1 -JO- '.0 u~ -040 Z... ;:) CS ...IIIa= '" ~... -'10 u... 0 III ~ IIIa= Au•• '69 Sept. '69 ...III ë u iz ...;; c CI \ ...0 \ \ \ . \ \ ~ \ MIDPOINT OF CLASS .. ,.f '-c' . -~t 1 -31- ganglion when they were regenerating they still conformed to the normal pattern of growth. Thus, growth did not appear to be curtailed by regeneration. The other reason is the possibility that less than optimuln growing conditions in certain areas on the beach may result in two size groups for one year. The growing conditions probably depend chiefly on the availability of food. In the absence of any information at aIl on the natural variation and significance of this factor, it will have to remain an unknown feature deserving further exaznination. Then too, the means appeared to be influenced by the sampling. In the April and May 1969 collections, larger samples were deliberately taken in the lower regions of Area A in order to ob tain a larger number of mature animaIs. Although April 1969 was not affected, the means of the first two age classes in May 1969 (Table II) were much higher than those of the adjacent months, indicating that the data could be influenced by the sampling technique. However, assuming that each age class in the population has been sampled satisfactorily, we could consider these means to be a close approximation of the means of the respective age classes. Further examination of Table II showed that a new class appeared in June 1969 with a mean weight of O.4gm. From Figure J a&b, respectively, it was possible to deduce that these worms had approximately 95 segments and a length of 8-12cm. However, larvae which resulted from fertilizations which occurred during the third -)2- MEAN AGE Wt. Yrs. JlllI A (M) (A) M A M A M A A MM 1 "'- - '68 Sep 0.68 2.72 4.41 7.48 10.8.5 12.60 Oct 1.20 2.04 4.6.5 7.1) 9 • .57 11.9.5 Nov 1.00 li ).20 2i .5.7.5 )i 8.07 4i 11.60 .5~ Dec '69 Jan 1.4.5 2.66 .5.71 8 • .5.5 11.00 Feb 0.6.5 ).8) .5.).5 7 • .5.5 10.88 Mar 0.78 2.80 6.0.5 7.4.5 10.9.5 Apr 1.10 2.70 4.55 6.7.5 9.)7 May 2,,10 2 ).6.5 ) .5.8) 4 7.7.5 .5 9.9.5 6 Jun 0.40 1.22 4.20 ? 7 • .50 10.26 Jul 0.80 2.1.5 4.20 7.00 9·8.5 120.50 Jul 0.34 2 • .50 4.35 7.7.5 ? 12 • .50 Aug 0.3.5 li 2 • .58 2i .5.38 3* 8 • .50 4* 10.0.5 .5i 12.00 6* Sept TABLE II The mean weight for each possible age class for each month, as determined from Figure V, showing some approxirnate ages, in terms of years, for worms of a certain weight. ···i· -33- week of May 1969 had attained only 5 segments by the end of July 1969 (see section on larvae). Also, larvae reared in the lab ....; oratory for as long as three months had achieved a maximum of only 10-13 segments. Thus this new age class of June 1969 was not from May's spawning and probably represented worms from the spawning of the previous year, making this class at least one year old. If we consider this first age class in June 1969 to be one year old, then the second age class with a mean weight of 1.22gm. is two years old, the third age class with a mean weight of 4.20gm. is three years old and so forth up to the sixth age class with a mean weight of 10.26gm. which is six years old. The respective ages, in terms of years, for each age class is also shown in Table II. Because of the scarcity of data on worms between 15.0- 30.0gm. it was impossible to establish age classes within this range, however, the longevity of these worms could possibly be somewhere around 12-15 years. Such longevity has never been reported for any polychaete, let alone any nereid. N. diversicolor, a close relative, exhibits a life span of eighteen months (Dales, 1961) which seems to conform to the 1-2 year life span exhibited by the majority of polychaetes. A few exceptions are Arenicola Inarina, which lives for at least six years (Thandrup, 1935); Glycera dibranchiata, which has a life span of up to 5 years (Klawe and Dickie, 1957); and Cirriformia .. , -34- tentaculata with a life span of 5-7 years (George, 1964). Since Copeland (1935) kept isolated specimens of N. virens living under laboratory conditions for at least three years and since Dow and Wallace (1951) have expressed the opinion that this species spawns towards the end of their third year, it is possible that N. virens does not conform to the normal 1-2 year life span and could possibly exhibit a life span well over three years as outlined in this thesis. However, exactly how long is not known for sure. This interpretation of the life span of N. virens is not in agreement with the results of Brafield and Chapman (1967), who based their conclusions on two samples; one of 728 worms taken just prior to spawning and another of 706 worms taken 5 Inonths after spawning. The weight distributions they obtained suggested that N. virens had a two year life cycle, with the mean weight of 1 year old worms being 2-3gm., 2 year old worms being 17-t8gm., and older worms 35gm. They suggested that N. virens normally matures during its second year and than spawns and dies. Apart from the fact that Brafield and Chapman's study was based on a smaller number of worms weighed (1.434 vs, 5.640) and fewer samples (2 vs. 14), there are some aspects of their study which could perhaps be reinter preted in view of this study of N. virens in New Brunswick. Brafield and Chapman (1967) stated that worms derived froID spawn at the beginning of May were still so considerably less than a gram in weight in September that they escaped notice. But .. f.' "./ -35- in our study worms of 0.1-0.2gm. had an average length of 2-9cm. and 73-93 segments and were relatively easy to find. Thus, if the worms of Brafield and Chapman were so small as to escape notice at the end of September, it would seem unlikely that they would have grown over the winter and attained a maximum of 120-150 segments and a mean weight of 2-3gm. by the beginning of May the following year. It is indeed possible that there may be more than one age class represented in these young worms. It is also hard to believe that mature worms, 7-28gm., are only two years old. Brafield and Chapman also found two mature animaIs in their first age class of 0-7gm. The only explanation they gave for this was that the single male and female might be unusually small "near 2 year old" worms rather than one year olds and thus wou Id spawn with the other two year olds. Other aspects of the growth of N. virens throughout the period of this study can also be observed from Table II. The mean weight for each age class increased from September 1968 to January 1969- Throughout the winter Inonths of February, March and April growth slowed dom~ and some age classes showed a decrease in mean body weight. It is possible, since food is not readily available during these months, that a partial reabsorption of some of their own body tissues or of fat may occur. Growth resumed again in late April and early May, with the increase in temperature (see Figure 19, section on reproduction) and food supply, and the ... ' -36- mean body weight increased considerably during the summer months. Table II also shows that growth by increase in weight appears to reach a maximum in the 3-4 year old worms, who gained approx imately 4.09gm. during 1968-1969. Thereafter increase in weight declined, with 4-.5 and .5-6 year olds gaining 2 • .57gm. and 1.1.5gm. respectively, showing that growth rate does decrease in worms 4 years and older. However, 1-2 and 2-3 year old worms also show a srnaller increase in weight than 3-4 year olds. This is probably due to the fact that growth in worms less than 4.0gm. (3 years and under) is mainly by segment proliferation, increase in length, and increase in width rather than increase in weight alone. As a cqn no"" result the actual growth rate in these young worms c~t be determined unless the interactions of these four factors are studied experimentally. Age at Maturity The cellular contents of the coelomic fluid of the .50-70 worms which had been weighed. were examined every month from September 1968 to September 1969 and classified into one of the following categories: Ceolomocytes l, Coelomocytes II, Gonadal Clumps, Females I. Females II and Males. The cellular elements contained in each category are listed in Table III. Weight/ frequency distributions for the worms in each of the above categories are shown in Figure 6. Most of the sarnples tended to ...... -37- be from worms over 2.0gm. because it was hard to extract any coelornic fluid from those below 1.0gm. and none less than 2.0gm. were observed to be mature. As shown in Figure 6, only small coelomocytes occurred in worms from 0-10gm., large coelomocytes in worms from 2-25gm., gonadal clumps in worms from 3-23 gm., and the stages of Females l, Females II and Males were observed in worms weighing 4-25gm. It does appear that the presence of only small coelomocytes represents the youngest stage in the development of the coelomic fluide The next stage, containing both small coelomocytes and large coelomocytes, being first observed in worms weighing 2&3gm., whereas gonadal clumps and other sexual stages first appeared in 3-4gm. worms. This indicates that N. virens probably begins sexual development when it is 3 years old (Table II) and does not reach maturity until the following year. It is also interesting to note that in the weight distributions for aIl mature worms (Females II and Males) collected from September 1968 to September 1969 (Figure 6), three peaks can be distinguished; one at 4-5gm., one at 8-9gm., and the third at 10-11gm. These weights coincide remarkably with the fourth, fifth and sixth components in Table II and thus we could deduce that these peaks represent 4, 5 and 6 year old worms, respectively. Although a very few worms probably attain maturity by the end of their third year, it does appear that norrnally the youngest worms to become mature are 4 years old. · . t~' "',f' -38- CATEGORIES CONTENTS OF THE COELOMIC FLUID COELOMOCYTES l Smal1 Coe1omocytes (roun4, ovoid or el1ip -soid cells ranging in size from 3-10u (Figure 7)) COELOMOCYTES II Small Coelomocytes Large Coelomocytes or Mature Trephocytes (round cel1s, 30-59u in diameter, containing most1y fat (Figure 8)) GONADAL CLUMPS Smal1 Coelomocytes Large Coelomocytes Gonada1 Clumps ( dense clumps of cells staining dark1y with hematoxy1in (Figure 9)) FEMALES l Sma1l Coelomocytes Large Coelomocytes Small Eggs (10-90u in diameter) Gonada1 Clumps in those with average egg diameters less than 60u. FEMALES II Sma11 Coe1omocytes Large Coe1omocytes Eggs 100-240u in diameter MALES Small Coe1omocytes Large Coe1omocytes Various stages of spermatogenesis Table III; Coe1omic f1uid categories for Nereis Virens. .' '.' -39- .) Figure 6: Weight/frequency distributions for the total samp1e of N. virens in each coe1omic f1uid category from September 1968 to September 1969- .-.~' . -40- 30 COELOMOCYTU J: . 20 \ COILOMOCYTES lE 10 ~\ V\ '"~ ..o ~20 oII...... GONADAL III CLUM" ~ Z 10 20 FEMALUlt 10 1 2S _ li 10 15 20 WEIGHT-gms. ..•. -41- Figure 7: Coelolrlc fluid of N. virens with only small coelomocytes. Coelomocytes I. (smear fixed in Schaudinn'sFluid) Figure 8: Coelomic fluid of N. v.irens with both small coelomocytes (sc) and large coelomocytes or parenchyma cells (pc), Coelomocytes II. (smear fixed in Schaudinn's Fluid) -42- ~ \\ .•... -42- '",~" '" -. ~""" . :.,., '. C ., .- .'. f".~ '-~ \ .. ... l ~:~ ~, ., i< ~"t_ :.1.4 -43- Figure 9: Coelomic fluid of N. virens with small coelomocytes (sc), parenchyma cells (pc) and gonadal clumps (gc), Gonadal Clumps. (smear fixed in Schaudinn's Fluid) Figure 10: Coelomic fluid of a spent rnale, with sperm (sp) and phagocytic cells (cells, l5-20~, containing green crystalloid granules). (fresh coelomic fluid) . '.' -44- • ~... ,...... ,.;I.\'~" ,-. -44- • · '.' -4.5- On the basis of this, the pattern of growth and sexual maturation in the st. Andrews population of N. virens may be envisaged as follows; they reach maximum segment number in two years, attaining 80-90 segments by the end of their first year and acquiring most of the remaining segments by the end of their second year. During their first year the coelomic fluid contains only small coelornocytes. Towards the end of their second year, however, when they are 2-3grn., they begin to produce large coel omocytes, which increase conciderably in number during their third year. Gonadal clumps then begin to appear in these 3-4gm. worms, whereupon they mature during the following year and spawn in rlIay when they are just 4 years old. Growth pattern also appears to be affected by the onset of seslnent sexual developmentsince sefment proliferation more or less stop- ped at about 4.0gm., although growth by the enlargement of existing segments continued. We may regard the attainment of maximum segment number as a crucial point in the life history of this worm, one that appears to coincide with the onset of sexual maturation in 3 year old worms. However, most worms do not become mature until they are Inuch older for only a small proportion of the worms weighing 4.0gm. or more were mature at any one time. Why sorne worms mature much later than others is not known. It could be that they have to lay down a certain amount of large coelomocytes (mature trephocytes or parench~nal cells) before -46- they can mature or it is possible that certain environmental or hormonal influences affect the maturation cycle. How long they actually survive after spawning is also uncertain. Crowther (1923 & 1928), stated that "all mature males died but that some of the females at least, burrowed into the mud. presumably to pass through another cycle of development". Our observations however, on spawned-out worms in both the laboratory and the field (page 51) suggests that survival after spawning is highly unlikely. Further rearing studies, especially the rearing of worms to sexual maturity in the laboratory, as well as more field studies are needed to test these hypotheses. The assumption that size classes represent age classes needs further support and the whole question of the relationship.between nutritive and/or hormonal factors and the time required to lay down sufficiently large numbers of coelomocytes deserves further analysis. At present it is clear that this study of N. virens in St. Andrews suggests a life history significantly different from that portrayed in the only comparable study on this species, that of Brafield and Chaprnan (1967) on a population in England. · ".f REPRODUCTION The cellular contents of the coelomic fluid of 50-70 worms were also examined every month from September 1968 to September 1969 to follow the stages in the development of the coelomic fluid and to follow oogenesis and spermatogenesis. Observations on spawning were made both in the field and in the laboratory, as described in the Materials and Methods. Development of the Coelomic Fluid From Table III, four basic stages in the development of the coelomic fluid can be observed. 1) only small coelomocytes 2) mature trephocytes and the formation of parenchymal tissue 3) 'gonadal' clumps and the presence of sex products in the coelomic fluid 4) spent worms with phagocytic cells in the coelomic fluid and a few remaining eggs or sperm 1) Small coelomocytes Although small coelomocytes (Figure 7) were found in aIl worms, they were the only constituent of aIl those below 2.0gm. · i.' -48- and most of those between 2 & 3gm. From 3-10gIn. the number of worms with on1y sma11 coe1omocytes decreased marked1y. Although it was not the point of this study to identify and de scribe the various small coelomocytes, it is interesting to note that there may be two types present. Observations using Wright's stain showed some small coelomocytes to be basophilie while others were acidophilic (Dr. Singh, personal communications). According to Liebman (1946) annelids possess two types of small coe1omocytes, lymphoidocytes and immature trephocytes. A1though Liebman's conclusions were drawn from his work on oligochaetes, it was confirmed by Dales (1950) for polychaetes. Thus, the two types of small coelornocytes indicated by Wright's stain could be lymphoidocytes (leucocytes) and immature trephocytes. 2) Mature trephocytes and the formation of • parenchymal , tissue Mature trephocytes (Figure 8), termed coelomic corpuscles by Dales (1950), were first observed in worms ranging between 2.0 &3.0gm, in weight. These trephocytes were assumed to contain rnost1y fat since they showed the sarne staining properties as adipose tissue when stained with Ehrlich's Acid Hematoxylin and Eosin (fat does not stain with dyes used for nuclei and cytoplasIn, such as H & E, and is usua1ly removed during the preparation). The trephocytes increased in number and eventua1ly came to form a 'loose parenchyma' which adhered to the gut, blood vessels and also packed the bases of the parapodia, giving them a distinct • f.f -49- yellow colour (Pocock (1970) and author's observations). This parenchyma appeared to be equally well developed in both male and female animals. Such an accumulation of adipose tissue or 'parenchymal' tissue has been described for N. diversicolor (Dales, 1957; Clark, 1960) as well as for other polychaetes (Clark, 1960). Parenchymal tissue has been observed in N. virens by Pocock (1970). However, Brafield and Chapman (1967) mentioned numerous coelomic corpuscles which gradually disappeared as the oocytes approached maturity, but made no mention of the formation of a 'loose parenchymal' tissue. Dales (1950), as well as many earlier authors (Kukenthal,1885; Schroder, 1886; Mclntosh, 1907, 1910; Herpin, 1925 and Romieu, 1921 a&b) , considered this tissue to be a source of nutrients for the developing female germ cells. The situation in the males is not as clear, although Dales (1950) stated that in N. diversicolor the formation of a 'loose parenchyma' was transitory. This did not appear to be the case in our population of N. virens where this tissue was observed te be as abundant in the male as in the female. 3) Gonadal clumps It appears that when the coelom and bases of the parapodia had become packed with 'parenchymal' tissue, the formation or proliferation of 'gonadal' cells begins. Histological sections .\ -50- of worms, stained with Ehrlich's Acid Hematoxylin and Eosin, did not reveal the site of proliferation. However, although histo logical observations were not conclusive, it is believed that 'gonadal' cells originate either from the coelomic epithelium or from specific coelomocytes in the coelomic fluide The question remains unresolved. The 'gonadal' cells were released into the coelomic cavity where they underwent massive mitotic divisions to produce what are termed here, 'gonadal' clumps (Figure 9). The clumps varied in size considerably and were nothing more than dense masses of cells staining darkly with hematoxylin. They could be found at aIl times of the year, floating free in the coelom, although some were embedded in the 'parenchymal' tissue at the bases of the parapodia. This type of free-floating gonad was produced in every se~nent except the first few of the pharynge al region and no region of the body appeared more prolific than others. At this stage it was impossible to differentiate between male and female 'gonadal' clumps. The germ cells arose in these 'gonadal' clumps; oogenesis and spermatogenesis taking place directly in the coelomic fluide "Gonadal' clumps have not been described before in N. virens, and no similar phenomenon is characteristic of N. diversicolor as described by Dales (1950). He observed that female sex cells were proliferated from the germinal epithelium in the ventral part of the coelom in the septal region of the worm, and that these sex • ".f -51- cells broke loose into the coelomic fluid at a very early stage. The sperm rnother cells he observed to break away into the coelomic fluid in the form of 'sperm plates'. 4) Phagocytic cells and later changes At the first of May 1969, just before spawning, the coelomic corpuscles were replaced by phagocytic cells (Figure 10), which were much the same shape and size as the 'parenchyrnal' cells or coelomic corpuscles. They were, however, distinctly green in colour in both males and females and contained numerous green crystalloid granules. In spent worms these phagocytic cells packed the bases of the parapodia, giving the worms a green colour. Besides phagocytic cells, some rnuscle cells were also found in the coelomic fluide However, there was no consistent pattern of microscopie muscle degeneration since in both males and females the degree varied from partial to apparently none at all. No worrns were observed to have undergone extreme muscular degeneration. Spawned males and females were kept alive in the laboratory for 1-2 rnonths without indication of further metamorphosis and spent females, collected in the field up to two months after spawning, showed no extreme muscular degeneration. In a closely related species, N. diversicolor, the degree of muscular degeneration was also found to be rather variable (Dales, 1950). However, worms distended with ripe gaInetes were extremely • ".1 -52- fragile. This fragility appears to be characteristic of the Nereidae and is presumably due to histolysis of the body wall muscles (Dales, 1950; Clark, 1960). Apparently, the body wall muscles break down at this time and make possible lesions through which the gametes can escape (Dehorne, 1924). However, although Pocock (1970), in a lipid analysis of the gut, body wall and coelomic fluid of N. virens, observed that most of the structural lipids were withdrawn from the gut and body wall during maturation, the actual degree of muscular degeneration of the worms in our study was found to be rather variable, and the males were observed to release sperm via pygidial papillae. Fragility is not only characteristic of mature worms, for immatures when handled will form lesions in the body wall and often will split into two pieces. However, mature worms do appear to be more fragile, and this could possibly be due to a combination of sensitivity to mechanical stimuli, withdrawl of structural lipids from the body wall and the distention of the body with ripe gametes. It is not really possible at this time to de duce that breakdown in body wall lnuscles allows for the formation of lesions through which the gametes can escape. Maturation and Structure of the Oocytes Females were first distinguished when the 'gonadal' clumps began to form eggs. Figure lla shows a 'gonadal' clump squash and -5)- it is quite obvious that these cells had undergone meiosis, each cell having produced 4 cells 2-)p in diameter. These cells increased in size. producing recognizable ova. The smallest eggs found free in the coelomic fluid were about 10u in diameter, but usually they remained in clumps until they reached 25~ in diameter (Figure llb). Eggs of more than 50p always occurred singly. The diameters of 50 eggs, chosen at random. were measured from the coelomic fluid sample of each fe'male examined over the period of September 1968 to September 1969. Mean diameters of the oocytes for each female over this period are shown in Figure 12. It can easily be seen that we have two distinct groups of females, as previously mentioned; Females land Females II. Females l, with small eggs 10-90~ in diameter, persisted throughout the year. Their eggs were not dense and contained mostly oil droplets (Figure lla&b). Those females having egg diameters of 10-60~ were still producing a considerable number of new eggs froID 'gonadal' clumps, whereas those with egg diameters of 60-90~ produced relatively few eggs from 'gonadal' clumps. In late July and August worms with egg diameters of 90-110~ were first seen. In these worms the production of new eggs had ceased and by September the fast growing process had already begun and two distinct groups of females were present. Females II, with large eggs 100-240p in diameter, were found from Septelnber to May. During the fall and early winter these eggs · ".f -54- Figure 11: Coelomic fluid of N. virens with small coelomocytes (sc), parenchymal cells (pc), gonadal clumps (gc) and srnall eggs Ceg), Females I. (smear fixed in Schaudinn's fluid) a) showing a gonadal clump squashed that appears to have undergone rneiosis producing oogonia. b) showing a clump of eggs, each around lOu, and one which is about 40u. . (.' -55- -sc .. .., : -55- -sc •.....- . ':.\,::70S' L\ .~,~...... : .. ~ .. : -56- Figure 12: Mean oocyte diameters for each female examined over the period of September 1968 to September 1969. -57- 2,'0 220 .i. 200 110 of ..c I!.. 160 -! 'Ë 1 DI 140 ...III ~ C 120 ëi c:> 100 c:> III '1 ., 1 00 60 40 20 s.pt. Oct. Nov. Dec:...... Feb. Mar. ,.... May Jun. Ju'·l JuI·2 Aue- Sept. lft& ..... · ,.t ".' -58- grew rapidly, increasing from 110-16o~ (Figure 13) in September to 16o-240~ in February, When the eggs attained 210-240~ (Figure 14) growth slowed down and soon stopped, for no eggs exceeding 240~ were observed in May. However, mature females were found in May with egg diameters of 180-200~ and these spawned in the spring with females whose egg diameters were 210-240~. This variability in diameters of mature eggs is presumably a result of the wide variability observed at the onset of the fast growing process. Thus, while eggs of 150-16op attained 210-240~, those of 100-120~ attained only 180-200~. No differences were noted in the larval development from both sizes of eggs. These mature eggs were pale green in colour with a definite limiting membrane, a clear cortical layer and the granular material confined to the central core (Figure 14)" From Figure 12 it can be seen that it takes the oocytes at least one year to mature. The oocytes produced in May and June could grow sufficiently over the summer to reach a size of 90p by the end of August. However, it is obvious from Figure 12 that egg production in females is a randoIn process, occurring at any time during the year and that entry into the rapid growth phase is dependant upon production of a sufficient quantity of eggs of the required size. Thus eggs produced in August and September, 1968 would not enter the rapid growth phase until the following September, taking over a year to reach maturity; whereas those -59- Figure 13: Coelomic fluid of N. virens during the early stage of Females II. Eggs containing numerous oil droplets and measuring 120-140\1. Figure 14: Coelomic fluid of N. virens during the late stage of Females II. Eggs very dense, pale green in colour and measuring 180-220~. ~ ",1 -60- ..... ~ .. ~ • -...... : ... '..:', • o • '. • . ,).: .... ,r:-J - .' ~ .. " ·0 f! .. ~. ,..c>'" .. o· ~ •"J • ..~ -60- -1" • • o. '. :',.~, ,. (1 "~. .. ., .~ -~ - ' :.' • • '" ,\ ~, ( ~\. • · ,.t - 1 -61- produced in April 1969 could enter the rapid growth phase in Septelnber 1969 and be mature within a year. Oocytes therefore, appear to mature in 1-2 years depending upon the time of year when they were produced. There are many similarities between this account of oogenesis in N ,Tirens and that of Brafield and Chapman (1967). They observed the oocytes of March to May to be mature by the fallowing April, a period of little over a year with the rapid growth phase occurring in August and September. However, they did not find two distinct classes of felnales, although they did find a few worms between September and December with unusually small oocytes. They suggested that this might be due to neurohormonal abnormal ities in the worms which had prevented the oocytes from entering -~, the rapid growth phase. They did not mention the possibility that these worms could be the ones to enter the rapid growth phase the followir~ August and September. The small size of their samples (20-30 fortnightly) might weIl account for the small number of such females in their collections. On the other hand it is perhaps not surprising to find such differences between two populations of N. virens, especially when they are separated by the Atlantic Ocean. rAaf N. diversicolor is quite different in ~ oocytes were only observed in the coelom from September to early May (Dales, 1950). The oocytes, however, do undergo a rapid growth phase and do show , , " -62- great variability in size when mature (200-270~ in diameter). Maturation and structure of the Spermatozoan As previously mentioned, 'gonadal' clumps were observed in thEl coelomic fluid all year round. However, they only gave rise to spermatocytes at the end of July or the beginning of August. At this time the 'gonadal' clumps began to break up into 'sperm plates', which are characteristic flattened groups of cells found in many polychaetes, although differing in size and appearance in different species. By the end of August the coelom contained mostly 'sperm plates' with relatively few 'gonadal' clumps and by the end of Septembêr no 'gonadal' clumps were observed in male animaIs. At this time the coelom was packed with 'sperm plates' ranging in size from 15-50~. These 'sperm plates' increased in size and by the end of November had reached diameters of 50-90~ (Figure 15). At the end of December the 'sperm plates' had started to break up into primary spermatocytes (Figure 16). Each primary spermatocyte then under went meiosis to form 4 spermatids (Figure 16 and Figure 17). Since 'sperm plates', primary spermatocytes and spermatids were aIl present in the coelomic fluid at the same tirne (Figure 16), it was assumed that the transition from primary spermatocytes to spermatids was of short duration. Such characteristic quartets were observed again in January, although at this time Inost had separated into · ,.t -6)- Figure 15: Coelomic fluid of male N. virens in November and October, containing "sperm plates;' (spp) 50-90",. Figure 16: Coelomic fluid of male N. virens wi th "sperm plates~' (spp) breaking up into secondary apermatocytes (ss), which then undergo meiosis to produce spermatids (s). · ,.t -64- -6L~- -65- individual sperme By the end of February only individual sperm, possessing a tail, an acrosome, a large nucleus and four mito chrondrial spheres were present (Figure 18). Although, these sperm appeared to be fully mature, they were not active at this time and did not show any activity in the laboratory until April, a month prior to spawning. In April only a portion of the sperm were active in Inaturing males, whereas all the sperm were active at the beginning of May. Temperature May be relate4. to spermatogenesis, for the production of 'sperm plates' from 'gonadal' clumps occurred at the end of July, beginning of August, when the maximum sea temp eratures for the area were observed (Figure 19). This allowed for synchronization from the very beginning and explains why spermato genesis proceeded synchronously in all animals, with spawning taking place in May after a subsequent rise in temperature. By the beginning of June all mature males had disappeared from the population at Brandy Cove, and no spent males were found in the field. The various stages of spermatogenesis described here are characteristic of polychaetes and agree with the observations of Brafield and Chapman (1967) on this species. They also observed increasing activity in sperm during the two months prior to spawning, with spawning being in May. Although they first observed recogniz able 'sperm plates' in September and October, it did appear as if # I~' -66- Figure 17: Coelomic fluid of male N. virens with spermatids (s) breaking up into sperm (sp). Figure 18: Coelomic fluid of male N. virens in May with mature sperm possessing an acrosome (a), a nucleus (n), mitochrondrial spheres (ms) and a tail (t). • r. t -67- -67- · '.' ""c'" -68- Figure 19: Average sea water tempe rature for Brandy Cove, St. Andrews, N.B. · '.' -69- ,,."1 .... , , '\ ,, "\ "'1,'. " \ 12'0 ~" \ :Ut-,. \, '1\1, ,,:'!,,;;\ \ " \ 1~0 '\ \ ,. '''23 \ \ \ '-'u .~,''' ~\ . .. \ a_II Hlgh, " a.A -,~ \ _~ ..... Year,-~I ,_1 \"\, ~ .... \" le 'V' ,0"23 \, , ~ \ \, 1 \ • 1 \ ,13 1:'. 1 \ \ ' III \ \\ " l '\! ! H '22\' '~. 1 "' ~ 1 0 ; \'.\ \ "'''67 \\'., \ 1 1 lU '~ ," Record L_ \ '0:} 1 1 .. ,,~,~' 'IllY :..,.....- and Veor --...... -, '",,.' : :: ,,~ , 1 \ '.' , '31\ i' : \ 1 2'0 ,- , \ 1 \ ~3 \ i \ 1 \ 1 \' " \ " \ 1 '2a1' ... _.. _-"u 1 \...... __ _1 -2'Ol~~~~~~~~'2~3~~~~~~~~~;-~~,-~-j-1~~~~~ SON D J""f - A M J1969 JASON D J F 197'0M A M 1968 -70- they missed the actual production of 'sperm plates' in August. Differentiation of the Sexes Males could not be distinguished froID immatures in the late summer and early fall, for both were an orange-reddish-green in colour. However, by November, the coelomic fluid of the males was becoming whitish in colour giving the bases of the parapodia a milky green hue. By December aIl the males possessèd this colouring. Females l were indistinguishable from immatures. Females II, which were first observed in late August and early September, contained pale green ova 100-160p in diameter. These were easily seen through the body wall and gave the bases of the parapodia a green tinge. As the egg size increased, the colour at the bases of the parapodia deepened to an eme~ald green, in contrast to the milky green hue of the males. Thus, the sexes could be disting uished externally from November to May. Apart from colour differences, Females II could easily be distinguished by observing the eggs through the body wall at the bases of the parapodia. Males also possessed another distinguish ing characteristic which was the appearance of an anal rosette just prior to spawning. This anal rosette was lacking in the females. The colour differences observed here were also observed by ," Pocock (1970) in the same population of N. virens, and Brafield and 1 ...• '. -71- Chapman (1967) found similar colour differences in their mature worms. However, their females with large oocytes were lime green in colour whereas ours were emerald green. Similar colour diff erences were also noticed in the closely related species, ~ diversicolor (Dales, 1950). Sex Ratio In the population of N. virens studied here, there was a definite 3:1 ratio of males to females (Table IV). Only maturing worms collected from January to May were used in this assessment. The ratio of males to females for the other months was indeter- minable beeause in December the sample eontained no females, in spawning ~ales and females occurred in equal numbers. It may, however, be typical of many of the Nereidae sinee Clark (1961), in a review of the literature on sex ratios in nereids, eoncluded that there is often a preponderance of males over females in breeding swarms. N. diversieolor, a non-swarming species, is repDrted to have an excess of females (Dales, 1950; Bogucki, 1954). -72- TABLE IV l\'lONTH TOTAL WORlVIS NUMBER OF NUMBER OF RATIO OF COLLECTED MALES FEMALES M:F January 309 20 7 3 1 February 369 22 7 3 1 March 372 28 10 3 1 April* 396 May 402 12 3 : 1 *April values are misleading since we were looking for a specifie number of females and as a result disregarded the total nurnber of males. Thus, a true ratio for April is not possible. Table IV; Ratio of males to females from January to May, 1969- ! -73- \. Spawning Spawning of atokous worms was observed both in the field and in the laboratory. Observations were made as described in the Materials and Methods. 1) Field observations Although numerous observations were made at Brandy Coye, St. Andrews Point, Bocabec and Chamcook during April and May 1969, spawning activity was only noticed by the author at Brandy Coye in the middle of the afternoon, just after low tide on May 21st, 22nd and 23rd, 1969. On each occasion only males were observed, swiwning close to the surface of the mud emitting a continuous stream of sperm in the region of their anus. No females were seen either swarming or sWiwning during these periods. However, ova in various stages of early development were obtained the following day by sampling the uppermost detritus layer of the mud; so fertilization did take place and females must have been present. It is indeed possible that the presence of sperm released by the males swimming close to the surface of the mud p may have stimulated ~he females to appear later during the advancing tide. Although observations were made again by the author during the new moon from May 5-11, 1970, no activity was noticed in the four areas mentioned previously. However, spawning most likely had not occurred for mature males and females were still present -74- and no larval stages could be found. Spawning probably took place the end of May, since Art MacKay collected swarming worms in this area at that time. An examination of 30 of these worms spipped to McGill University showed them all to be spent males. Pettibone (1963) cites various records of scores of spent males observed, but no mention is made of whether or not the swarms were composed of only males or both males and females. Although Brafield and Chapman (1967) did not observe swarming activity, they examined the coelomic fluid of swarming worms that had been washed ashore and found them all to be spent males. Whether the females emerge from their burrows and swarm later than the males or just remain in their burrows and undulate their posterior segments, as Brafield and Chapman suggested, is still uncertain. However, it is surprising, even with a 3:1 ratio of males to femals, to find no females in samples of swarming worms. 2) Laboratory observations Attempts were made to induce spawning in the laboratory. In May 1969 activity under observation was miminal and the attempt apparently a failure. However, the worms spawned sometime during the night and successful fertilizations resulted. These fertilized eggs were attached to the sides and bottom of the tank and micro scopie examination showed them to be in very early stages of development. Successful observations on spawning were made later -75- in May 1969 and repeated again in May 1970. Shortly after both sexes were placed together the males started to swim. While swimming, they emitted a cORtinuous stream of sperm in the region of their anus. Sometimes the males started swimming as soon as they were placed in the tank, probably due to the mechanical stimulation caused by moving them from one tank to another. The presence of sperm then appeared to stimulate the females, for they began to swim along the bottom of the tank in an undulatory pattern characteristic of nereids. Activity increased until the posterior portion of the body was undulating vigorously, the eggs being ex~ruded in the region of the anus at the completion of each undulatory stroke. The anterior part of the body remained more or less rigide The swimming of the males then increased in wi/d vigour, both sexes becoming engaged in a wid frenzy which continued until all were comp:etely eXhausted. However, if during this process either sex was removed from the presence of the other emission stopped. This may suggest that the emission of gametes is a controlled process. Animals interrupted in this manner could be induced to spawn again. It was interesting to note that when the males were first brought into the laboratory in May, they would swim around the tank emi tting sperme.and then gradually settle to the bottom if females were absent. The males could easily be induced to spawn by handling or disturbing them, however, if females were not -76- present they soon settled to the bottom. Similar behaviour was not observed in the females. This is the first account of spawning of N. virens in the laboratory. It does show that the males are the first to spawn, stimulating the females to become active later. However, although the females remained close to the bottom of the tank, the tank was only 7 inches deep and these observations clearly do not indicate whether or not the animaIs normally remain in their burrows. Then too there were not enough females available to attempt such exper iments. However, these observations are consistent with the fact that females either spawn in their burrows or spawn close to the surface of the mud (Copeland, 1935) and thus could explain why samples of swarming worms consist only of males. 3) Release of spermatozoa The males of N. virens were observed to have an anal rosette (Figure 20), which is a terminal disc with the anus in the center and bordered by a number of papillae. Histological studies, using Ehrlich's Acid Hematoxylin and Eosin, showed the anus to he free of sperm while the pygidial papillae were packed (Figure 21). A duct could be seen (Figure 21) at the end of each pygidial papillae, making the coelomic cavity continuous with the outside. The sperm of N. virens may therefore be presumed to be released via ducts in the pygidial papillae. Sperm were not released from any other ," .. , 1 -77- -~ Figure 20: Posterior end of a mature male N. virens showing the pygidial segment (pys) with the anal papillae (ap). X10 anus (an) anal cirri Cac) Figure- 21: Longitudional section through the pygidial segment of a male N. virens showing the anal papillae (ap) packed with sperm (sp) and an opening (d) making the coelomic cavity continuous with the outside. .•. /. S'. • ,".1 -\ -79- region of the body and lesions were not observed in the body wall. The anal rosette, consisting of a ring of pygidial papillae, is characteristic of many mature male nereids (Gravier, 1923). Although Gravier thought that the sperm were discharged through the anus after histolysis of the gut, Defretin (1939) showed that the sperm were actually emitted via the pygidial papillae in ~ pelagica and N. irrorat~. No anal rosette was found in N. diver sicolor (Dales, 1950), and Brafield and Chapman (1967) did not observed one in their population of N. virens. 4) Release of oocytes Observations on spawning females showed that the eggs are released only in the region of the anus. The pygidial segment was distended with ripe gametes (eggs) during spawning but no anal rosette was present. Histological sections did not indicate the presence of coelolnoducts in any region of the body and no lesions were visible. However, although no lesions were seen, it is probable that the eggs are released either through lesions in the posterior segments or through the anus after lesions have been formed in the gut wall. According to Clark (1961) the eggs of nereids are generally shed by dehiscence. Dales (1950) observed this for N. diversicolor and it is extremely likely that N. virens conforms to this general pattern as welle Mclntosh (1910) also observed eggs being released · ,.t -80- from the posterior portion of the body in N. virens, although he did not de scribe the mechanism. 5) Conditions governing the time of spawning The first spawning observed during this period was by Art MacKay at St. Andrews Point on April 13, 1969, three days before the new moon (Figure 22a). Observations by the author for three days fOllowing proved fruitless, although spawning was not com pletely fini shed since mature males and females could still be collected from the beach. The next spawning was observed by the author in May, 1969 during the last three days of the new moon (Figure 22b). In 1970 spawning worms were searched for unsuccess fully during the new moon from May 5th_11th (Figure 22c) and the first observed spawning that year occurred at the end of this month (collection of spent males received at McGill from Art MacKay). This was during the last quarter, or 4 days before the new moon. These observations seem to indicate that spawning is centered around the new moon, which agrees in part with Pettibone (1963), who reported observations of spawning of N. virens on the New England coast to be centered around the full and new moons in the early spring. However, it is just as valid to say that there is a bimonthly spawning centered around the first and last quarters, depending upon where you set your limits. If the latter supposition # 1.' -81- Figure 22: Daily heights of the low tides for the St. John, N.·B~ area for April and May 1969 and for May 1970, correlated with the phases of the moon. -\ , ,, \ .. May '69 of• ... \ ...e .. \ , , " '" ~ ,- '" 0 , _ ...... " ... \ 0 ~ \ \ , ... /"\ 1 \ :z: "'-' iii ":z: 8 May '70 .... 7 - \ ,- 6 ,- 5 1 1 4 , , \ , 1 ~, , , 3 1 , , 1 ..... _, " 2 , , '-" DAYS • new moon œ -flret quarter --- evonlna tldes a-fUll moon ----- mornlng tld•• C> - la.t quarter -83- is right, then either there is no lunar effect in N. virens or different populations have different lunar cycles. Assuming the latter, the the St. Andrews Point population could be assumed to spawn around the first quarter, the Brandy Cove population around the last quarter and various populations along the New England coast at either the new or full moon. However, more observations are needed on the spawning populations in these areas to determine if there is a lunar periodicity in spawning and a difference between populations. It is perhaps significant that spawning took place in Passamoquoddy Bay in April and May, 1969 as temperature increased r (Figure 19), even though males and females appeared to be mature as early as February. The attainment of a definite tempe rature Wq~ threshold or temperature change os considered by Orton (1920) and Thorson (1946) to be the actual stimulus required to initiate spawning. Thorson (1946) also points out that there is a vertical distribution of tempe rature on the shore so that 9ptimum spawning temperatures will be reached progressively later at greater depths. This may be relevant here since the population at St, Andrews Point is much higher on the beach than the population at Brandy Cove and spawned a Jnonth earlier. This does not rule out lunar periodicity in spawning for as Korringa (1947) points out," ••••• telnperature conditions preponderate to establish the breeding -84- season. Within this breeding season a monthly rhythm may come into operation". In other words there may be a dual effect, with a definite temperature threshold being required before the worms could assume a lunar rhythm. Whether this is in fact true for N. virens is not known and can only be determined by more extensive studies of spawning behaviour. LARVAL DEVELOp~mNT "Artif'icial' f'ertilizations were carried out in the laboratory by placing mature males and f'emales in laboratory tanks and allowing them to emit their gametes f'reely into the water, as described in the Materials and l\~ethods. Successf'ul f'ertilizations were not obtained by opening the body cavity of' mature males and f'emales, obtaining the gametes and mixing them in beakers of' salt water. Larval development was f'ollowed in the laboratory using these 'artif'icially' f'ertilized eggs. Most of' the larvae were maintained in a cold room (7°C) in bowls of' sea water. Sarnples of' larvae were also obtained f'rom the f'ield and compared to those reared in the lab orat ory. No dif'ferences were observed in development. Early Development Early development appeared to f'ollow the basic pattern already described f'or other nereids. Typical spiral cleavage, as described by Wilson (1892) for N. limbata and N. megalops, was observed. Although cleavage times were slower than f'or most species of' nereids, they were approximately the same as those observed by Dales (1950) f'or N. diversicolor. A gelatinous envelope began to extrude about 10min. af'ter f'ertilization and within two hours it was f'ully developed (150~ thick) (Figure 23). This thick gelatinous envelope has been · .~ ( -86- described for other species of nereids (Herpin, 1925), although Dales (1950) found that it was not always present in N. diversicolor. Both 'artificially' fertilized eggs and those obtained in the field possessed gelatinous envelopes. These envelopes were apparently sticky, for the eggs adhered to one another and to the sides and bottom of the tanks in the laboratory. Neither single eggs nor clumps of eggs were observed floating freely at the surface and those that were loosened frOID the sides of the tank gradually settled to the bottom. In the field several clumps were found attached to rocks but most were single and anchored to particles in the upperrnost detritus layer of the mud. The gelatinous envelope became more noticable a few hours after fertilization, due to the adherence of debris. The first cleavage occurred 13-16 hours after fertilization (Figure 24). Cleavage was unequal, as it is in most large yolky polychaete eggs (Anderson, 1966). The second cleavage (Figure 25) occurred about 3 hours later and by 22 hours most of the eggs were well into and some were past the third cleavage (Figure 26). Later cleavage stages were extremely hard to follow due to the yolky nature of the eggs and to the thick gelatinous envelopes, which were by then covered with debris. However, the large yolky macro meres could be seen on top of the microlneres (Figure 27). These yolk rich cells eventually became enclosed in the digestive tract, where they were utilized for food. # (~t ... ' 00\ -87- Figure 23: Fertilized egg of N. virens about lOmin. after fertilization, showing the gelatinous envelope (ge) and the fertilization membrane (frn). Figure 24: Early cleavage stages of N. virens- 2-cell stage. -88- } r -83- ,l, •. , .... • cr:'.' , "' '" " .: . ~.',' .. ~ ,. • : t ,..... • l"~ ~~ •. -) r ; ... :,. ge .' ." , -0' .. ô,.1- :"" '~.:~ .... !. ~ :. ~,:~~,:; • . :.4/ - .' .>~~ " '0 ." "0 ,0'-" ,0 .. f ' "'1· -89- Figure 25: Early cleavage stage of N. virens - 4--cell stage. -90- .~ ., ..~ ...• ". " '~.. " , ~ -.. ..ç , .. . " ~. ." . " ' ,il-,·,'.2·,:' . ~ ~ . .. ;'.:- . '.( -90- : . ' :.l ,,'" -, '.,.,., '. -91--- Figure 26: Early cleavage stage of N. virens - 8-cell stage. Figure 27: Later cleavage stage of N. virens showing the macromeres (m) on top of the micromeres. O·'l· -92 - f.:~ <- : -::. .~'. .:? ge .•:' '1:.--', ge .~.~~. l~ ~''f., II" .. ... ::i.- -;: . -92 - .. ~ .; ) .<,. g e ~ '. g e ". · ".' -93- At 96 hours the protrochal band of cilia was just beginning to forrn and associated with this was the beginning of a red pigment band (Figure 28a). The larvae then elongated and became more or less conical in shape. A second band of cilia, the telotroch, was developing at this time and the first and second pairs of chaetal rudiments appeared simultaneously. By 120 hours therefore, the larvae, still inside the gelatinous envelope, possessed a well developed band of protrochal cilia, a red pigment band that was continuous in sorne and not in others, a well developed telotroch and the first two pairs of chaetal rudiments. The larvae contracted periodically, these contractions becoming lnore and more vigorous, and by 144 hours sorne of the larvae were rotating within their gelatinous envelope. By this time the chaetal sacs had undergone further development and the chaetae now projected beyond the cuticle (Figure 28b). They were observed to twitch and it could, therefore, be assumed that some parapodial muscles had formed. By 168 hours sorne of the larvae had hatched but most were still rotating inside their gelatinous envelopes. Although all larvae were free-swimming by 192 hours (Figure 29a&b), they were always close to the bottom and some even atternpted to crawl. At this stage they possessed the protrochal ciliary band, two pairs of chaetae projecting tu-FfS beyond the cuticle (Figure 29b) with tuffs of cilia just behind thern, the beginnings of a third chaetal sac and a well developed telotroch. • ,-.f -94- Figure 28: Early stage of the larvae of N. virens. a) showing the red pigment band (rpb). b) showing chaetae (ch) projecting beyond the cuticle. -95- " . . '.' 'i...'~~Î"~,. ::~ :.~ t. .', -96- Figure 29: Free swimming trochophore (prototroch) of N. virens. a) showing red pigment band (rpb). b) showing two pairs of chaetae projecting beyond the cuticle. .. ,~, --\. -97- -97- -98- Larvae with 3-Chaetigerous Segments One to two days after the larvae hatched, further elongation had taken place and the third pair of chaetal sacs had projected beyond the cuticle. This three segment stage lasted for three weeks, the larvae undergoing great morphological change before the fourth segment was added. The youngest 3-segment larvae observed is shown in Figure 30a and measures 235-245\1 in length. In these young larvae the red pigment band began to condense at the sides of the head and two pairs of red cup-shaped eyes had formed. The developing larval gut surrounded themacromeres: anteriorly the p~arynx.could be IOh3' ruc!/r;q/ seen as a clear oval region with a median longitudional_cavity and posteriorly an extension of the gut toward the anus could be distinguished. These Iarvae were normally observed crawling through the debris, however they still possessed aIl the cilia previously mentioned and ocassionally would draw their parapodia to the sides of their body and swirn near the surface of the debris for 2-3cm. In the 3-segment Iarvae, 10-12 days old (Figure 30b) and measuring 310-330~, a more distinctive head region was observed. The red pigment band had condensed at the sides of the head and it was interesting to note that in some larvae it was present on both sides while in others it occurred only on one side of the head. A pair of peristomial tentacles were present just posterior to the '.1 -99- Figure JO: Early three segment larvae of N. virens. a) just after the third pair of chaetae had projectec beyond the cuticle. b) about 2-J days later than the previous, showing the beginnings of the anal cirri (ac) and the first pair of peristomial cirri (pc). ...•. • l~ 1 -100- ... " -100- . Il! '*"-""'.'- -"" , ...",'. ~ . (~ , -101- Figure 30: cont. c) showing the beginnings,.of the prostomial tentacles (pt). --.... -102- · '.' -102- -10)- Figure )1: a) late three segment larvae of N. virens. b) enlarged head region of the late three seglnent larvae of N. virens showing the one-toothed jaws. ...• ' -104- \ -104- · ,.1 -\ -105- pigment spots and the anal cirri had begun to forme The chaetae had become more prominant and were now borne on recognizable parapodia. (In this study the development of the parapodia was not followed in detail.) The larval gut had extended to form the anus, the macrorneres were still present and no food was observed in the gut. A day or two later the larvae resembled the one shown in Figure 30c. These larvae differed little from those of Figure 30b, except that they possessed the beginnings of prostolùal tentacles and a Inore developed pharyngeal region with simple jaws. Through out the later three segment larval stages the peristomial and prostomial tentacles of the head region and the anal cirri grad ually elongated (Figure 31a). The tips of these carne to bear hair-like processes, silnilar to those in N. diversicolor (Dales, 1950), which are probably sensory in function. Palps were not yet visible. The larval gut, which was easily seen through the body wall, appeared to be completely formed, although the larvae still possessed a large quantity of yolk and were not feeding. The jaws, which were more pronounced, possessed only one tooth (Figure 31b). The chaetae had increased in number and length by the late 3- chaetiger stage; aIl bands of cilia were still active and locolnotion was much the same as in the early 3-chaetiger larvae. However, the larvae now appeared to swim only when disturbed. These larvae lneasured 390-410~ in length. It must be mentioned here that the · (,' ....' -106- lengths of larvae do not include either the lengths of the prost- omial tentacles or the anal cirri. Later Larval Stages About 4-5 weeks after fertilization the fourth segment was added (Figure 32a), the larvae measuring 460-480p in length. The prostomial and peristornial tentacles of the head region and the anal cirri had elongated further and the palps were beginning to forme The jaws possessed another tooth by this stage (Figure 32b) and the red pigment spots had disappeared from some individuals. The macromeres, in most larvae, had broken down into an amorphous mass of yolk and some particles of debris were present in the gut. In some cases the macromeres had begun to break down during the late 3-chaetiger stage and aIl were broken down by the fifth. When the fifth segment was added the larvae were approximately 5-6 weeks old and 570-580~ in length. At this time a second pair of peristomial tentacles had begun to form (Figur~ 33). The prost omial and peristomial tentacles, already present, had elongated, the red pigment spots had finally disappeared, the palps had enlarged, the anal cirri had elongated and the larval cilia had almost disappeared. Although debris was present in the gut, remnants of yolk were still appc.rent. S'IX -.s.e..[j/YIen-t /ql"Vq The sixth segment larvae (Figure 34a), 640-680).1. in length, differed little from the fifth except for the further elongation • .'~ 1 -107- Figure 32: Four segment larvae of N. virens. a) The fourth segment is almost formed and the anal cirri, the peristomial cirri and the prostomial tentacles are slightly longer than in the three segment larvae. b) Squashed four segment larvae showing that the jaws have two teeth. · ,.t -108- 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 -109- ; Figure 33: Five segment larvae of N. virens showing the beginnings of the second pair of peristomial cirri. -110- -110- ~\ r -111- ) Figure 34: Six segment larvae of N.virens. a) Showing the notopodia of the first pair of larval elongating to form the peristomial cirri. b) Enlargelnent of the head region showing the four toothed jaws. -112- • '(~ o "" . 200J.!.. , '" / . 'o·' -112- .(~ • o . . 200P · ~.. ' -113- of the prostomial and peristomial tentacles and the anal cirri p the increased prominance of the palps and the fact "that the jaws now possessed four teeth (Figure 34b). Towards the end of the sixth segment stage, just as the chaetal sacs of the seventh segment appeared, the notopodia of the first pair of larval parapodia began to elongate (Figure 34a). Larvae with 8, 10, 11 and 13 segments (Figures 35a, 36a, 36a and 37a), respectively measure 940-980~, 1350-1460~, 1600-1800~ and 2000~ in length. Besides the addition of more segments these larvae underwent major changes in the anterior region. During the seven and eight segment stages the notopodia of the first pair of parapodia elon~ated further and began to move ante ri orly, and the neuropodia were beginning to elongate (Figure 35b). These gave rise to the third and fourth pair of peristomial tentacles~ since they continued to elongate and move anteriorly until they reached the first and second pair. These 11 and 13 segment larvae now possessed a fully developed head and were considered to be miniature adults. They were first observed in the laboratory about 2* - 3 months after fertilization. Changes in the jaws were also noticed; those of 8-segment larvae possessed six teeth on each jaw while 13-segment larvae had eight. No paragnaths were seen. Although 13-segment larvae were reared in the laboratory, those older than 6-segments could not be found in the field. However, mud samples were inconclusive since the larvae were most -114- Figure 35: Eight segment larvae of N. virens. a) Showing the notopodia of the first larval segment moving forward and elongating to bec Olne the third peristolnial cirri and. the neuropodia giving rise to the fourth. b) Enlargement of the head showing the jaws which now possess six'teeth. -115- 200P -115- 200J,l -116- Figure 36: a) Ten and eleven segment larvae of N. virens. b) Enlargement of the head of an eleven segment larvae, showing that the first larval segment has now become part of the head. "',l' '\ -117- -117- -118- Figure 37: a) Thirteen (or twelve, if you disregard the first larval segment) segment larvae of N. virens. b) Enlargement of the head of the thirteen segment larvae of N. virens. "'l '. -\ -119- · ,." -119- -120- likely maeerated while sieving the fine,silty mud, but it seems likely that they begin to burrow into the mud at this stage. All the larvae less than 6-segments were found in the uppermost detritus layer of the mud. Considering as well the faet that plankton samples in this area ineluded no nereid larvae, it was eoneluded that development in N. virens was non-pelagie. This aeeount of the larval development does not agree with the only other aeeount of larval development in Nereis virens (Sveshnikov, 1965). He found development to be pelagie with the larvae settling during the 7 setiger stage and none of the stages he diagrammed resemble any stage deseribed here. · '.)' GENERAL DISCUSSION This study o~ the li~e history o~ N. virens indicates the population at Brandy Cove does not undergo epitokal metarnorphosis, does not have an extensive swarming behaviour at the sea sur~ace and has a no'n-pelagic larval development. This agrees with the usual breeding biology o~ brackish and ~reshwater nereids (Clark, 1961) in which epitokal metamorphosis and swarming is rare and in which the pelagie larval phase is abreviated or suppressed. However, N. virens has been previously described as both epitokous (Gustafson, 1953; Sveshnikov, 1955; Khlebovich, 1963; and Mileikovskii, 1961) and atokous (Brafield and Chapman, 1967). It has been described as swarrning in enormous numbers at the sea sur~ace (Gustafson, 1953; Clark, 1960) and as swimming close to the surface o~ the rnud (Pettibone, 1963) as was observed in this study. The larvae have also been described as non-pelagie (Gusta~son,G. (Thorson, 1946» and as being pelagie (Sveshnikov, 1955) • Thorson (1936) states that among the Polychaeta, individual species show an astonishing lability in their mode o~ reproduction and developrnent, ~rom asexual to sexual, from viviparity and brood protection to pelagie larvae. For exarnple, Thorson (1936) points out that Nereis zonata has been observed to be epitokous and to produce pelagie larvae (verbal in~ormation ~rom H. Ditlevsen), · f,·' -122- whereas he observed this speeies to be atokous and to undergo a non-pelagie larval development in the inner fjords of East Green land. Other species also exhibit different rnodes of reproduction, such as Nereis pelagiea whieh undergoes ~on-pelagie development at 1 8' - d ~)' / a r v'CI Cherbourg (Herpin, 1925) and has an 18 day pelagie larvae at Pi'lmoufh -- pl~nouth (Wilson, 1932). Johnson (1943) observed Nereis vexillosa spawning both in the nereis phase (atokous) and in the heteronereis phase (epitokous) and Hemplemann (1911) described a population of sexually mature Nereis dumerilli in whieh some individuals, called 'nereidogenie', gave rise to heavily yolked eggs and non-pelagie larvae while other heteronereid forms, ealled 'planktogenie', produeed less heavily yolked eggs whieh developed into pelagie larvae. Henee, as Thorson (1936) has suggested, nereids may have the ability to vary the type of egg produeed aeeording to the eeologieal conditions under whieh they are living, thus enabling the larvae to survive in ecologically different areas. Thus N. virens may possess two modes of reproduction, one with epitokous individuals swarrning at the sea surface and giving rise to pelagie larvae and the other with atokous individuals swimming close to the surface of the rnud and giving rise to non pelagie larvae. Thorson (1936) suggests that in Nereis zonata the latter mode oeeurs in the inner fjords whereas the former oeeurs on the outer coast of Greenland. This may also be true of N~ virens sinee Sveshnikov (1965) and Khlebovieh (1963) observed -123- the former rnode in the \Vhite Sea whereas the latter mode was found in this study on a brackish, intertidal beach population, at the Inouth of the St. Croix River. The whole problem requires further examination, especially the study of populations of N. virens in different environmental situations in relation to egg size, larval type and way of life. Meanwhile the possibility that N. virens is a species that does vary its rnode of reproduction with environmental conditions rernains. • ".1' CONCLUSIONS 1. The growth pattern in young worms up to 60-70 segments is mainly by segment proliferation. Thereafter segment proliferation slows down and growth appears to become more and more due to the enlargement of existing segments. N. virens exhibits a maximum of 120-150 segments 0 2. In worms below 12gms. we could differentiate five to six possible age classes, while in worms from 12-25gms. insufficient nUlnbers prevented analysis of the components age classes. 3. Longevity of these worms could possibly be somewhere around 12-15 years. 4. The youngest worms to become mature are 4 years old;Jal.nd most do not become mature until they are 5 and even 6 years old. S. They were definitely atokous and extensive metamorphosis into a free-swimming epitokous or 'heteronereis' phase was not observed. 6 Germ cells proliferate either from the coelomic epithelium or arise from specifie coelomocytes and undergo massive mitotic divisions to produce what was termed here 'gonadal' clumps. They were observed floating free in the coelom or embedded in the parench~nal tissue at the bases of the parapodia. 7. 'Gonadal' clumps were found at all times of the year and were indistinguisable in male and female animals. -125- 8. Males of N. virens at Brandy Coye were observed from August until May. Sperm plates were produced from 'gonadal' clumps in August, spermatids froID the sperm plates in November and December and single sperm from spermatids in January and February. 9. Eggs were observed to arise from 'gonadal' clumps at all times of the year. Two distinct groups of females were observed; Females l with small eggs 10-90u and Females II with eggs 100-240u. Eggs around 100-120u would enter the rapid growth phase in September and increase to 220-240u b~ February. Eggs took anywhere froID 1-2 years to mature. 10. A ratio of 3:1, Males:Females was observed. 11. Spawning of atokous males was observed in the field in May. Males first appear on the rising tide, swimming close to the surface of the mud. No females were observed swarming in the field. 12e In the laboratory the males started to swim first, stimulating the females to start undulating their posterior segments. What the females do in the field is still uncertain. 13. Sperm are released via a duct in the pygidial papillae 14. Eggs are believed to be released via lessions in the last few posterior segments or via the anus after histolysis of the gut. 15. Spawning appears to be centered around the new moon, · ",l' -126- from about 6 days before to 6 days after. Temperature is probably the initial stimulant in that they need a definite tempe rature threshold before the lunnar cycle is instigated. 16. Spent worms were kept alive for 1-2 months without indication of any extensive Inetamorphosis. How long they survive after spawning and whether or not they live to go through another reproductive cycle is still uncertain. 17. Larval development was followed in the field to the 6- setiger stage and in the laboratory to the 13-setiger stage and was found to follow the basic pattern described for other nereids. 18. Larval development was non-pelagie, and the larvae were observed in the uppermost detritus layer of the mud to the 6-setiger stage. · 1." -127- REFERENCES ClTED. Anderson, D. T. (1966). The comparative Embryology of the Polychaeta, Act. Zool. Stockholm, Vol. 47 (1). pp. 1-42. Berkeley, E. & Berkeley C. (1954). Additions to the Polychaete fauna of Canada, with comments on some older records. Jour. Fisheries Res. Board of Canada, Vol. 11 (4). pp. 454-471- Bhatlacharya, C. G. (1967). A simple method of resolution of a distribution of Gaussian components. Biometries, Vol. 10. March 1967. Bogucki, M. (1954). Adaptacja Nereis diversicolor (o. F. Muller) do rozcienzonej wody morskiej i wad slodskiej. Polsk. Arch. Hydrobial, Vol. 2, pp. 237-50 (Clark). Brafield, A. E. & Chapman, G. (1967). Gametogenesis and breeding in a natural population of Nereis virens. Jour. Mar. Biol. Assoc. U.K., Vol. 47, pp. 619-627. Clark, R. B. (1960). Fauna of the Clyde Sea Area: Polychaeta. Millport: Scot. Mar. Biol. Ass. (Clark. 1961). Clark, R. B. (1961). The origin & formation of the heteronereis. Biological, Reviews, Vol. 36, ppo 199-236. Clark, R. B. & Scully, U. (1964). The hermonal control of growth in Nereis diversicolor (O. F. Muller). General comparative Endocrinology, Vol. 4, pp. 82-90. Copeland, M. (1935). Keeping Nereis for physiological study. (Dow & Wallace, 1951). Crowder, W. (1923). Dwellers of the Sea Shore. MacMillan. Crowder, W. (1928) • A Naturalist at the Sea Shore Century Company. Dales, R. P. (1950). The reproduction & larval development of Nereis diversicolor (O. F. Muller). Jour. Mar. Biol. Assoc. U.K., Vol. 29 (2), pp. 321-361. · .... ' -128- Dales, R. B. (1957). Preliminary observations on the role of the coelomic cells in food st orage & transport in certain polychaetes. Jour. Mar. Biol. Assoc., U.K., Vol. 36, pp. 91-110. Dales, R. P. (1961). An annual history of a population of Nereis diversicolor (O. F. Muller), Biol. Bull., Vol. 101, pp. 131-137. Defretin, R. (1939). Emission des spermatozoides chez quelques Ner~idiens. Bull. Societ~ Zoologique de France, Vol. 64 pp. 316-321. Dehorne, A. (1924). Marche Gén~rale des phénomènes de myelyse chez Hediste diversicolor pendant la maturation des ovocytes. C. R. Soc. Biole~ Paris, Vol. 91, pp. 303-4. Dow, R. L. & Wallace, D. E. (1955). Marine worm management & observations. Fisheries Circular #16, Maine Department of Sea & Shore Fisheries, Augusta, Maine. Ganoros, A. (Sept. 1951). Cownercial worm digging report for the Depart. of Sea & Shore Fisheries, Mimeographed (Gustafson, 1953). George, J. D. (1964). The life history of the cirratulid worm, Cirriformia tentaculata, on an intertidal mudflat. Jour. Biol. Assoc. U.K., Vol. 44, pp. 47-65. Gravier, C. (1923). La Ponte et l'incubation chez les Ann~lides. Polychaetes. Anneles de (Science Nat.) Zoologie, T6. pp. 153-247. Gustafson, A. H. (1953). Some observations on the dispersions of the marine worms Nereis & Glycera. Depart. of Sea & Shore Fisheries, Fisheries Circular, No. 12, June 1953. Harding, J. F. (1949). The use of probability paper for the graphical analysis of polymodal frequency distributions Jour. Mar. Biol. Assoc. U.K., Vol. 28, pp. 141-153. Hempelmann, F. (1911). Zur Naturgeschichte von Nereis dumerillii. Aud &Edw Zoolog~cal Stuttgart, Bd. 25, pp. 1-135. 2.oo JDj/CC{ Herpin, R. (1925). Recherches biologiques sur la reproduction et la development de quelque. Annélides Polychaetes. Bull. Soc. Sci. Nat. Quest (II), 5. pp. 1 250 ( ) _ ~L)esf F/'Clnce~Nqnfes) '1 ,S;;>?_ /-~50 · .,." -129- Johnson, M. w. (1943). Studies on the life history of the marine annelid Nereis vexillosa. Biol. Bull., Woods Hole, Vol. 84. pp. 106-114. Khlebovich, V. V. (1963). Biology of N. virens (Sars) in the Kandalakska Bay. of the White Sea. Trudy Kandalakshkogo Gasudarstvennago. Zapovednika. Vol. 4, pp. 250-257. Klawe, W. L. & Dickie, L. M. (1957). Biology of the bloodworm Glycera dibranchiata (Ehlers) & its relationship to the bloodworm fishery of the maritime provinces. Bull. Fish. Res. Bd. Canada, No. 115, 37 pp. Korringa, P. (1947). Relations between the moon & periodicity in the breeding of marine animals. Ecological Monographs, Vol. 17, pp. 347-381. KÜkenthal, W•• (1885). Ueber die lymphoiden Zellen der Anneliden. ~ena Z. Naturw. 18, pp. 319-64. :fend Liebman, E. (1946). On trephocytes & trephocytosis; a study on the role of leucocutes in nutrition and growth. Growth, Vol. 10, pp. 291-330. Mclntosh, W. C. (1907). Notes froID the Gatt y Marine Laboratory, St. Andrews. Ann. Mag. Nat. Hist. (7), 20, 169-85. (Clark, 1961). Mclntosh, W. C. (1910). British Marine Annelids, London, Roy. Soc. 2, pt. 2. Mileikovskii, S. A. (1961). The Polychaete N. virens in the Kola Gulf. Zool. Zhur. 40 (9); 1421-1423, 1961. Referet Zhur., Bid., 1961, No. 5, Zh. 40 (Translation). Orton, J. H. (1920). Se~ temperature, breeding & distribution in Marine Animals. Jouro Mar. Biol. AssocG, U.K. Vol. 12, pp. 339-366. Pettibone, M. H. (1963. Marine Polychaete worms of th~ New England Region 1. Aphroditidae through Trochochaetidae. U.S. National Museum, Bulletin 227, part 1. pp.160-183 on Nereidae. Pocock, D. M. (1970). A comparison of lipids & fatty acids in mature & immature Nereis virens, a marine worm (Annelida., Polychaeta) PhD. Thesis, Dept. of Zoology, McGill University. ·1.1' ..•. -130- Romieu, M. (1921a). Sur les éléocytes de Perinereis cultrifera (Grube). C.R. Acad. Sei., Paris 173, 446-9. Romieu, M. (1921b). Observations cytologiques sur les leucocytes de Perinereis cultriferao C.R. Ass, Anat., Paris, 82, 187-93. Schr~der, Go (1886). Anatomisch-histologische Untersuchungen ," von Nereis diversicolor (O. F. Muller) Kiel. IhS=t. 200 . 1r,s,t. ilJien . .J f.(clfhe.now .. Carl /(oppel) rp, /- Thamdrup, H. M. (1935). Beitr~ge Zur Okologie der Wattenfauna auf experimenteller Grundlage. I\1edd. KOInI11. Danrnarks Fisk. Havund Kjobenhavn, Sere Fiskeri. Bd. 10, 125 pp. Thorson, G. (1936)0 The larval development , growth and metabolism of arctic marine bottom invertebrates. IVIeddelelser OIn. Gronland, Bd. 100, Na. 6, pp. 1-155. Thorson, G. (1946). Reproduction and larval development of Danish marine bottom invertebrates. Medd. Komm. Danrnarks Fisk. Havund. Kjobenhavn, Sere Plankton Bd. 4, No. 1. 523 pp. Turnbull, F. M. (1876). On the anatomy & habits of Nereis virens. Trans. Conn. Acad. Arts. Sei. Vol. 3, 265-80. Wilson, D. P. (1932). The development of Nereis pelagica (Linnaeus). Jour. Mar. Biol. Assoc. Vo~ XVIII, pp. 203-17. Wilson, E. B. (1892). The cell- lineage of Nereis. Jour. Morph., Vol. 6, pp. 361-480. • .~ l' -1)1- APPENDIX - Table 1; Frequency distribution of N. virens in weight classes shown, using an O.5gm. interval, for the worms collected in each section every month from September 1968 to September 1969. Sections #0 and #1 were near the low water mark, whereas section #9 was higher up on the beach. -132- APPENDIX - Table l September 1968 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0- 0.5 1 1 1 6 1 3 10 .5- 1.0 2 2 1 3 3 6 3 1.0- 1.5 1 1 1 2 3 15 10 1.5- 2.0 1 1 2 4 2 4 6 10 2.0- 2.5 3 2 1 7 4 9 6 2.5- 3.0 5 3 3 4 11 3.0- 3.5 3 2 2 6 2 1 4 8 3.5- 4.0 3 2 3 3 4 4 4.0- 4.5 3 3 2 1 1 4 9 4.5- 5.0 1 6 1 3 4 1 5 5.0- 5.5 1 1 4 3 2 2 3 5.5- 6.0 2 3 2 1 2 4 6.0- 6.5 1 5 2 6 1 3 1 6.5- 7.0 1 3 2 4 2 3 1 7.0- 7.5 1 1 2 1 1 1 7.5- 8.0 1 1 2 1 1 1 3 8.0- 8.5 1 1 8.5- 9.0 1 1 2 2 3 1 9.0- 9.5 1 2 1 2 1 9.5-10.0 1 1 2 1 10.0-10.5 1 3 1 1 10.5-11.0 3 2 1 2 1 . ,." -133- Appendix - Table l - continued September 1968 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11.0-11.5 2 2 1 1 1 11.5-12.0 2 2 1 12.0-12.5 1 2 1 2 12.5-13.0 2 1 1 1 13.0-13.5 2 1 13.5-14.0 1 2 14.0-14.5 1 14.5-15.0 1 15.0-15.5 1 15.5-16.0 1 16.0-16.5 2 16.5-17.0 1 17.0-17.5 17.5-18.0 18.0-18.5 2 18.5-19.0 1 1 · "." -134- Appendix - Table l - continued Octo1?er 1968 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0- 0.5 3 3 1 3 6 .5- 1.0 1 5 3 4 11 1.0- 1.5 2 8 11 10 1.5- 2.0 1 2 10 6 8 2.0- 2.5 1 4 9 3 3 8 2.5- 3.0 1 1 4 5 5 7 3.0- 3.5 1 4 2 7 4 3.5- 4.0 2 6 1 8 5 4.0- 4.5 1 2 1 2 4 4 4.5- 5.0 2 1 8 3 5.0- 5.5 1 2 2 5 5 5.5- 6.0 1 1 1 2 1 3 6.0- 6.5 2 4 5 2 3 6.5- 7.0 2 1 3 1 1 2 7.0- 7.5 2 1 2 1 1 7.5- 8.0 2 2 5 1 8.0- 8.5 5 2 1 1 8.5- 9.0 2 1 2 9.0- 9.5 2 2 9.5-10.0 1 10.0-10.5 6 1 1 10.5-11.0 3 2 3 · .-", -135- Appendix - Table l - continued October 1968 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11.0-11.5 2 11.5-12.0 1 1 1 12.0-12.5 2 2 12.5-13.0 2 1 13.0-13.5 1 13.5-14.0 1 2 2 14.0-14.5 1 1 14.5-15.0 2 2 1 15.0-15.5 1 15.5-16.0 1 1 16.0-16.5 16.5-17.0 17.0-17.5 1 17 .5-18.0 18.0-18.5 18.5-19.0 19.0-19.5 1 · .... ' '\ -136- Appendix - Table l - continued November 1968 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0-:- 0.5 1 2 2 1 3 1 13 12 .5- 1.0 1 4 4 8 8 1.0- 1.5 2 2 2 3 9 8 1.5- 2.0 3 1 3 3 7 4 2.0- 2.5 2 2 3 4 1 2 12 10 2.5- 3.0 1 3 6 2 1 6 5 3.0- 3.5 1 5 1 6 2 10 3 3.5- 4.0 2 4 3 3 1 9 7 4.0- 4.5 8 2 4 1 4 3 7 4.5- 5.0 1 2 1 3 2 1 4 2 5.0- 5.5 1 4 1 1 3 3 6 3 5.5- 6.0 2 1 2 2 1 5 6 6.0- 6.5 3 3 2 1 1 3 6.5- 7.0 3 2 1 3 1 7.0- 7.5 1 4 2 3 1 1 1 1 7.5- 8.0 2 2 2 2 1 8.0- 8.5 1 3 8.5- 9.0 1 5 2 1 3 9.0- 9.5 2 1 1 9.5-10.0 2 4 1 2 10.0-10.5 1 2 1 1 10.5-11.0 1 3 2 1 • {.f' -137- Appendix - Table l - continued November 1968 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11.0-11.5 1 2 2 Il. 5-12.0 3 2 3 1 12.0-12.5 1 2 12.5-13.0 2 1 2 l3.0-13.5 1 2 l3. 5-14. 0 1 1 14.0-14.5 2 1 14.5-15.0 1 15.0-15.5 3 1 1 15.5-16.0 16.0-16.5 1 16.5-17.0 1 1 17.0-l7.5 17 .5-18.0 18.0-18.5 1 18.5-19.0 1 1 19.0-19.5 19.5-20.0 1 · ,... ' -138- Appendix - Table l - continued January 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0- 0.5 5 1 3 5 4 9 0.5- 1.0 2 2 4 2 2 7 4 12 LO- loS 1 5 3 1 5 1.5- 2.0 1 2 4 5 3 2.0- 2.5 1 2 2 4 9 4 2 2.5- 3.0 2 1 1 6 3 4 3.0- 3.5 1 1 1 4 2 5 3.5- 4.0 1 1 2 1 1 1 4.0- 4 .. 5 3 1 3 5 2 4 3 4.5- 5.0 1 1 1 4 1 2 5.0- 5.5 1 4 2 3 3 5.5- 6.0 2 2 2 4 1 2 3 6.0- 6.5 3 2 1 2 6.5- 7.0 1 3 2 7.0- 7.5 1 2 2 1 5 1 7.5- 8.0 1 2 3 8.0- 8.5 2 2 1 1 4 8.5- 9.0 3 5 1 1 9.0- 9.5 1 2 9.5-10.0 1 1 3 10.0-10.5 3 2 1 10.5-11.0 3 2 1 1 2 • f,I' -139- Appendix - Table l - continued January 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11. 0-11. 5 2 1 1 11.5-12.0 1 1 2 12.0-12.5 12.5-13.0 13 .0-13.5 1 1 2 13.5-14.0 1 1 14.0-14.5 1 1 14.5-15.0 1 1 15.0-15.5 15.5-16.0 1 16.0-16.5 16.5-17.0 17.0-17.5 17.5-18.0 18.0-18.5 1 18.5-19.0 -140- Appendix - Table l - continued February 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0- 0.5 5 3 2 5 5 13 20 3 0.5- 1.0 1 1 2 3 8 11 5 1.0- 1.5 1 4 3 4 2 4 6 5 1.5- 2.0 2 4 1 1 5 1 2.0- 2.5 1 2 5 4 3 6 4 2.5- 3.0 3 2 3 5 3 3 3.0- 3.5 3 4 1 4 1 3.5- 4.0 1 4 1 2 4 1 2 4.0- 4.5 1 4 3 3 5 4.5- 5.0 1 2 2 2 1 2 2 5.0- 5.5 1 2 3 1 5.5- 6.0 3 2 1 1 6.0- 6.5 3 1 3 1 3 3 6.5- 7.0 2 1 1 6 3 2 7.0- 7.5 1 1 2 2 7.5- 8.0 2 1 3 3 3 1 1 8.0- 8.5 1 5 4 1 1 1 8.5- 9.0 3 1 2 1 2 9.0- 9.5 2 2 1 2 9.5-10.0 1 2 1 1 10.0-10.5 3 2 1 1 10.5-11.0 2 1 1 1 1 1 1 · ",.' -141- Appendix - Table l - continued February 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11.0-ll.5 1 1 1 11.5-12.0 1 1 1 12.0-12.5 2 12.5-13-0 1 1 13.0-13.5 2 1 13.5-14.0 1 1 14.0-14.5 1 14.5-15.0 1 1 15.0-15.5 1 2 15.5-16.0 16.0-16.5 1 16.5-17.0 17.0-17 .5 17.5-18.0 1 18.0-18.5 18.5-19.0 1 · ,.1' -142- Appendix - Table l - continued M.arch 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0- 0.5 7 2 5 3 9 8 26 0.5- 1.0 3 1 1 10 7 9 10 1.0- 1.5 1 2 2 2 4 3 16 7 1.5- 2.0 2 1 1 2 5 4 9 4 2.0- 2.5 1 2 2 1 8 5 2.5- 3.0 3 1 2 8 8 5 4 3.0- 3.5 1 6 5 2 5 3 3.5- 4.0 1 4 1 1 4.0- 4.5 1 1 2 4 4 5 4.5- 5.0 1 4 3 1 5.0- 5.5 1 1 6 5.5- 6.0 1 1 1 4 2 6.0- 6.5 2 2 3 1 6.5- 7.0 3 2 3 2 7.0- 7.5 1 1 2 4 7.5- 8.0 1 1 3 1 8.0- 8.5 1 1 1 1 8.5- 9.0 1 2 9.0- 9.5 1 2 1 1 9.5-10.0 2 2 1 1 10.0-10.5 1 1 1 10.5-11. 0 1 • .. l' -143- Appendix - Table I - continued March 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11.0-11.5 1 11.5-12.0 1 12.0-12.5 1 1 2 12.5-13 .0 1 13.0-13.5 1 1 13.5-14.0 1 14.0-14.5 1 14.5-15.0 15.0-15.5 1 2 15.5-16.0 1 16.0-16.5 1 16.5-17.0 1 17.0-17.5 1 1 17.5-18.0 1 18.0-18.5 1 18.5-19.0 1 · '.1' -144- Appendix - Table l - continued April 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0- 0.5 3 3 1 7 1 19 0.5- 1.0 1 4 4 1 2 8 7 9 1.0- 1.5 1 5 4 6 3 4 6 1.5- 2.0 3 1 3 2 3 6 5 2.0- 2.5 2 3 2 2 5 4 3 6 2.5- 3.0 2 3 4 3 2 1 6 2 3.0- 3.5 2 2 1 7 2 2 6 3.5- 4.0 1 3 3 4 2 2 4.0- 4.5 5 1 2 1 3 4.5- 5.0 1 3 2 1 1 3 1 5.0- 5.5 2 2 3 3 1 3 1 5.5- 6.0 1 4 1 1 1 1 2 1 6.0- 6.5 9 5 3 5 6.5- 7.0 1 5 4 2 7.0- 7.5 4 2 1 3 2 7.5- 8.0 4 2 2 6 3 8.0- 8.5 5 3 1 1 1 8.5- 9.0 2 2 3 3 1 2 1 9.0- 9.5 4 1 3 9.5-10.0 1 1 1 10.0-10.5 2 1 1 10.5-11.0 1 2 3 1 3 -145- Appendix - Table l - continued April 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11.0-11.5 2 2 2 11.5-12.0 2 1 1 12.0-12.5 1 1 12.5-13.0 2 13.0-13.5 1 13 .5-14. 0 14.0-14.5 1 14.5-15.0 15.0-15.5 1 15.5-16.0 16.0-16.5 1 1 16.5-17.0 17.0-17.5 17.5-18.0 1 18.0-18.5 18.5-19.0 -146- Appendix - Table l - continued May 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0- 0.5 4 7 6 10 2 0.5- 1.0 4 8 4 8 10 6 1.0- 1.5 1 2 5 4 6 7 4 1.5- 2.0 1 1 1 6 3 4 5 5 2 .. 0- 2.5 1 2 4 4 3 6 3 9 2.5- 3.0 1 2 10 10 9 7 3.0- 3.5 1 4 1 2 2 1 3 8 3.5- 4.0 6 4 2 4 3 5 4.0- 4.5 2 3 1 5 2 4 4.5- 5.0 1 2 1 2 l l 3 5.0- 5.5 2 1 3 l l 7 5.5- 6.0 1 3 2 1 5 2 4 6.0- 6.5 2 3 l 2 l 4 6.5- 7.0 4 1 2 1 2 7.0- 7.5 1 1 1 2 l 7.5- 8.0 l 2 8.0- 8.5 1 3 l l 8.5- 9.0 1 2 l l 9.0- 9.5 l 1 1 l 2 l 9.5-10.0 2 2 l 10.0-10.5 1 1 1 l 1 10.5-11.0 l 1 1 1 · ' .. ' -147- Appendix - Table l - continued May 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11.0-11.5 2 2 1 1 11.5-12.0 1 1 12.0-12.5 12.5-13 .0 1 1 1 13.0-13.5 2 2 1 13.5-14.0 14.0-14.5 1 1 14.5-15.0 15.0-15.5 1 1 1 1 15.5-16.0 16.0-16.5 1 1 16.5-17.0 2 17.0-17 .5 3 17 .5-18.0 18.0-18.5 18.5-19.0 1 19.0-19.5 1 • f,l' ·z 1 -148- Appendix - Table l - continued June 1969 Sections Intervals No. 1 No. 2 No. 3 No. 4 }TO. 5 No. 6 No. 7 No. 8 No. 9 0- 0.5 3 8 9 3 11 2 4 17 J 0.5- 1.0 6 4 3 4 2 9 7 6 12 LO- loS 1 1 1 7 1 10 1 5 7 1.5- 2.0 2 4 5 6 8 5 2 2.0- 2.5 2 3 1 1 4 6 7 3 2.5- 3.0 2 3 1 3 3 2 4 1 3.0- 3.5 1 2 4 4 2 7 3 2 3.5- 4.0 2 2 1 2 2 4 2 4.0- 4.5 2 1 1 2 7 2 2 4.5- 5.0 1 2 2 3 1 8 1 5.0- 5.5 3 1 2 2 3 5.5- 6.0 3 2 1 2 2 2 6.0- 6.5 1 1 1 2 3 6.5- 7.0 3 1 2 1 3 2 1 7.0- 7.5 1 2 2 4 1 1 4 7.5- 8.0 2 1 1 1 8.0- 8.5 1 2 1 8.5- 9.0 1 1 1 2 1 3 9.0- 9.5 1 2 1 9.5-10.0 2 1 2 2 2 10.0-10.5 2 2 1 1 1 1 10.5-11.0 2 1 1 1 1 1 -149- T Appendix - Table ..L - continued June 1969 Sections Intervals No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 11.0-1L5 1 1 1 1 1 11.5-12.0 1 12.0-12.5 1 12. 5-l3.0 1 2 13 .0-l3. 5 1 l3 .5-14.0 1 1 1 14.0-14.5 14.5-15.0 1 15.0-15.5 1 15.5-16.0 1 1 16.0-16.5 1 16.5-17 .0 1 17.0-17.5 1 1 17.5-18.0 18.0-18.5 18.5-19.0 1 -150- T Appendix - Table .1. - continued Ju1y l 1969 Sections Interva1s No. 1 No·. 2 No. 3 No. 4 No. 5 No. 6 No. 7 t~o. 8 No. 9 0- 0.5 4 2 17 4 22 6 8 18 12 0.5- 1.0 16 8 18 4 7 5 5 8 9 1.0- 1.5 4 3 5 3 7 3 3 13 11 1.5- 2.0 2 2 2 3 7 4 2 4 6 2.0- 2.5 1 1 2 1 3 3 3 7 7 2.5- 3.0 1 2 1 4 2 4 6 3.0- 3.5 2 1 3 3 2 3 3 3.5- 4.0 1 2 3 3 3 3 4.0- 4.5 4 3 1 2 3 3 4.5- 5.0 1 4 1 2 1 5.0- 5.5 3 2 2 5.5- 6.0 2 l 3 1 1 6.0- 6.5 1 1 3 1 1 2 6.5- 7.0 1 2 2 1 1 1 7.0- 7.5 1 1 2 3 7.5- 8.0 3 1 1 1 1 8.0- 8.5 3 1 1 2 1 8.5- 9.0 1 1 9.0- 9.5 2 1 9.5-10.0 10.0-10.5 1 1 1 1 10.5-11.0 2 -151- Appendix - Table l - continued Ju1y l 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 11.0-11.5 1 1 1 2 11.5-12.0 1 1 2 12.0-12.5 1 12.5-13 .0 2 1 13.0-13 .5 1 1 13 .5-14.0 1 14.0-14.5 1 14.5-15.0 1 15.0-15.5 15.5-16.0 16.0-16.5 1 16.5-17.0 1 17.0-17.5 1 17 .5-18.0 18.0-18.5 18.5-19.0 1 · "", -152- Appendix - Table l - continued. Ju1y II 1969 Sections Interva1s No. 1 Noo 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 0- 0.5 5 25 7 4 13 23 16 0.5- 1.0 6 2 12 11 3 11 9 10 LO- loS 2 2 11 6 1 6 8 7 1.5- 2.0 2 1 12 6 7 3 5 2.0- 2.5 1 1 4 1 4 1 7 2.5- 3.0 1 1 3 2 2 4 2 4 3.0- 3.5 1 2 2 3 2 2 3.5- 4.0 1 6 1 4 1 2 1 2 4 4.0- 4.5 5 2 3 2 2 4 2 4.5- 5.0 1 3 2 2 3 1 2 4 5.0- 5.5 5 3 2 1 5.5- 6.0 1 3 1 2 1 1 6.0- 6.5 1 2 1 3 3 1 6.5- 7.0 1 1 1 7.0- 7.5 1 1 1 1 7.5- 8.0 2 4 1 1 1 1 1 8.0-· 8.5 1 8.5- 9.0 1 1 3 9.0- 9.5 1 1 1 9.5-10.0 1 2 10.0-10.5 1 1 10.5-11. 0 2 1 -153- Appendix - Table l - continued Ju1y II 1969 Sections Intervals No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 11.0-11.5 11.5-12.0 1 1 2 12.0-12.5 12.5-13.0 1 13.0-13.5 3 13 .5-14. 0 2 14.0-14.5 1 1 1 14.5-15.0 1 15.0-15.5 1 15.5-16.0 16.0-16.5 16.5-17.0 l 17.0-17.5 17.5-18.0 1 18.0-18.5 18.5-19.0 · "," -154- Appendix - Table l - continued August 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0- 0.5 5 2 15 3 15 9 20 16 0.5- 1.0 3 9 8 11 8 10 6 29 1.0- 1.5 3 5 3 11 5 4 12 1.5- 2.0 2 1 4 4 5 1 6 10 2.0- 2.5 1 1 2 1 3 1 4 7 2.5- 3.0 1 2 3 2 4 1 5 4 3.0- 3.5 1 5 1 4 4 1 6 3.5- 4.0 3 1 3 2 4 4.0- 4.5 1 1 1 6 3 3 4.5- 5.0 1 2 1 2 1 3 5.0- 5.5 1 2 l 4 5 5.5- 6.0 1 1 3 3 2 4 6.0- 6.5 2 1 3 1 6.5- 7.0 1 1 7.0- 7.5 2 1 2 2 2 2 2 7.5- 8.0 1 1 2 1 1 2 8.0- 8.5 1 2 8.5- 9.0 1 1 1 9.0- 9.5 1 1 1 1 9.5-10.0 1 1 2 10.0-10.5 1 2 1 10.5-11.0 1 · '.1' -155- Appendix - Table l - continued August 1969 Sections Intervals No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11. 0-11.5 1 1 1 1 11.5-12.0 2 12.0-12.5 1 1 12.5-13.0 1 13.0-13.5 13 .5-14.0 14.0-14.5 1 1 14.5-15.0 15.0-15.5 15.5-16.0 1 16.0-16.5 1 16.5-17.0 17.0-17.5 17.5-18.0 18.0-18.5 1 18.5 -19.0 · ',.' -156- Appendix - Table l - continue September 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 0- 0.5 1 1 11 7 16 24 37 0.5- 1.0 5 7 10 4 13 15 16 1.0- 1.5 1 2 7 3 15 7 8 1.5- 2.0 1 5 3 5 7 8 2.0- 2.5 1 4 2 6 8 3 2.5- 3.0 1 2 4 3 6 3 3.0- 3.5 2 1 4 4 2 8 3 3.5- 4.0 1 2 2 6 2 1 4.0- 4.5 3 3 4 2 4.5- 5.0 1 3 3 3 3 5.0- 5.5 2 1 2 2 2 1 5.5- 6.0 2 2 2 2 6.0- 6.5 3 2 1 4 1 6.5- 7.0 1 1 2 3 7.0- 7.5 1 6 7.5- 8.0 1 1 1 1 2 8.0- 8.5 1 1 8.5- 9.0 1 9.0- 9.5 1 1 1 9.5-10.0 1 1 10.0-10.5 2 1 10.5-11.0 · '.. ' -157- Appendix - Table l - continued September 1969 Sections Interva1s No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 11.0-11.5 11.5-12.0 1 12.0-12.5 1 12.5-13 .0 1 13.0-13.5 1 13.5-14.0 14.0-14.5 1 14.5-15.0 1 15.0-15.5 l 15.5-16.0 1 16.0-16.5 16.5-17.0 17.0-17.5 17 .5-18.0 18.0-18.5 1 18.5-19.0 1 APPENDIX - Table II. Frequency distribution of N. virens in weight c1ass ranges shown, using 0.5 gm. interva1s, for the worms 1 ~ V\ 0:> weighed from September 1968 to September 1969. 1 APPENDIX - Table II C1ass Range 1968 1969 (gros.) Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May Jun. Ju1-1 Jul-2 Aug. Sep. 0- 0.5 41 25 35 34 26 57 60 34 29 57 93 93 91 97 .5- 1. 0 30 29 25 22 35 31 41 36 40 54 80 64 88 70 1.0- 1.5 48 40 26 29 15 29 37 29 29 34 53 43 44 43 1.5- 2.0 44 38 21 23 15 14 27 23 26 32 32 36 33 29 2.0- 2.5 38 33 36 21 24 25 20 27 32 27 28 19 21 24 2.5- 3.0 34 25 24 16 17 19 30 23 38 19 20 19 22 19 3.0- 3.5 32 19 28 9 14 13 23 22 23 25 17 12 24 24 3.5- 4.0 26 26 29 10 7 15 7 16 24 15 15 22 13 14 4.0- 4.5 28 15 29 3 21 16 18 12 17 16 16 20 19 12 4.5- 5.0 31 15 16 6 10 12 9 12 11 19 9 18 10 13 -- 5.0- 5.5 20 16 21 2 13 7 8 15 15 11 7 11 13 10 5.5- 6.0 25 11 19 3 16 7 9 12 18 12 8 9 15 8 6.0- 6.5 23 16 13 3 8 14 9 22 13 8 9 11 7 11 6.5- 7.0 20 10 10 4 6 15 10 13 10 13 8 3 2 7 7.0- 7.5 12 8 14 4 12 6 8 12 6 15 8 4 13 6 7.5- 8.0 12 11 9 2 6 14 6 17 3 5 7 11 8 7 8.0- 8.5 6 9 4 1 10 13 4 11 6 4 8 1 3 2 8.5- 9.0 11 6 12 4 10 9 3 14 5 9 2 5 3 1 9.0- 9.5 8 4 4 0 3 7 5 8 7 4 3 3 5 3 Appendix - Table II - continued C1ass Range 1968 1969 (gms. ) Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May Jun. Ju1-1 Jul-2 Aug. Sep. 9.5-10.0 6 1 9 0 5 5 6 3 5 9 0 3 4 2 10.0-10.5 7 8 5 1 6 7 3 4 5 8 4 2 4 3 10.5-11.0 9 7 7 3 9 8 1 9 4 7 3 3 l 0 11.0-11.5 6 2 5 0 4 3 1 6 6 5 5 0 4 0 11.5-12.0 5 4 9 1 4 3 2 4 2 1 3 4 2 1 12.0-12.5 8 4 3 1 0 2 4 2 0 1 1 0 2 1 12.5-13.0 4 3 5 0 0 2 1 2 3 3 3 1 1 1 13.0-13 .5 4 1 3 0 3 3 2 1 5 1 2 3 0 1 13.5-14.0 2 4 2 0 2 2 1 0 0 3 1 2 0 0 14.0-14.5 1 2 3 0 2 1 1 1 2 0 1 3 2 1 14.5-15.0 2 5 1 0 2 2 1 0 0 1 1 1 0 1 15.0-15.5 1 1 5 1 0 3 3 1 4 1 0 1 0 1 -- 15.5-16.0 1 4 0 1 0 1 0 0 2 0 0 1 1 16.0-16.5 2 0 1 0 0 1 2 2 1 1 0 1 0 16.5-17.0 1 3 1 1 0 2 1 1 1 17.0-17.5 0 1 2 3 2 1 17.5-18.0 0 1 1 1 1 1 18.0-18.5 2 1 1 1 1 1 18.5-19.0 2 2 1 1 1 1 1 1 19.0-19.5 1 1 1 1 19.5-20.0 1 1 1 20.0-20.5 1 1 Appendix - Table II - continued C1ass Range 1968 1969 (gms. ) Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May Jun. Ju1-1 Jul-2 Aug. Sep. 20.5-21.0 1 1 1 21.0-21.5 1 1 1 2 21.5-22.0 1 1 1 22.0-22.5 1 22.5-23.0 1 23.0-23.5 23.5-24.0 24.0-24.5 1 24.5-25.0 1 1 ovel' 25.0 1 2 1 1 2 1 3 .--. TOTAL 555 410 443 203 309 369 372 396 402 428 453 430 458 415 Specific weight of worms over 25.0 gms. September 1968 27.953 gms. October 1968 31. 700 and 35.008 gms. November 1968 28.848 gms. January 1969 25.874 gms. Mar ch 1969 30.664 and 31.332 gms. April 1969 32.290 gms. May 1969 28.115, 28.450 and 31.711 gms.