FACTORS REGULATING THE REPRODUCTIVE CYCLES OF SOME UEST COAST INVERTEBRATES

by John H. Himmelman

B.Sc, Acadia University, 1967

M.Sc, Memorial University of Newfoundland, 1969

A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOB OF PHILOSOPHY in the Department cf Zoology

We accept this thesis as conforming to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA June 1976 (

an advanced degree at the University of British Columbia, I agree that

the Library shall make it freely available for reference and study.

I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.

Zoology- Department of

The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5

July 9, 1976 Date ii

ABSTRACT

Annual reproductive cycles are found in many marine invertebrates. There is a vast literature on the subject, but the mechanisms involved have seldom been demonstrated. In the present study, 8 species of chitons and one sea urchin were studied for 3-5 years in southwestern British Columbia, at Vancouver and Porteau in the Strait of Georgia estuary, and at Botanical Beach on the outer coast of Vancouver Island. Reproductive condition was assessed mainly by the gonadal index method (percentage gonadal weight). There was a distinct annual cycle in the mean gonadal index of the urchin, Strongylocentrot- us droebachiensis Miiller, and the chitons, Tonicella lineata Wood, Tonicella insignis Reeve, Mogalia hindsii Reeve, Mo£alia laevior Pilsbry, Mogalia ciliata Sowerby, and Katharina tunicata Wood. In S. droebachiensis, T. lineata, T. insignis, M. laevior, and M. ciliata an abrupt spawning occurred in the spring, usually in April, and in M. hindsii there was usually an earlier spawning. K. tunicata sometimes spawned in April but the main spawning period was June. In M. laevior, K. tunicata, and probably M. hindsii, the gonads remained small during the summer and rapid gonadal growth occurred in the autumn and winter. In contrast, in S. droebachiensis, T. lineata, T, insignis, and !• Siliata gonadal growth started shortly after spawning. The data on reproduction in Mogalia ii9.H2§§ Gould were less clear. Mature animals were found in several seasons and drops in the mean gonadal index occurred in late winter-spring as well as in the summer. In Mo£alia luscosa Gould animals in ripe and spent condition were found throughout the year.

Consideration was given to the possible factors controlling gonadal growth. In a number of species, particularly species of warm water origin, it has been clearly demonstrated that gonadal development in the spring and summer is stimulated by increased temperatures. If temperature affects gonadal development in the species in the present study, it must act in several ways, since gonadal growth occurs through 2-3 periods of steadily increasing or decreasing temperature. The initiation of gonadal growth in K. tunicata and T. lineata in California and Oregon occurred at the same time as in the present study, although temperatures in the southern localities were fluctuating due to upwelling, in contrast to the regular temperature changes which occurred in British Columbia. This would suggest that temperature was not important, at least during the early stages of gonadal growth in K. tunicata and T. lineata. There are distinct annual photoperiod changes throughout the geographical ranges of the species in the present study, and in S. droebachiensis, 1.' lineata, T. insignis, and M. ciliata most gonadal growth occurred during the period of decreasing day length. Food conditions are known to affect the number of gametes produced in a number of species, including S. droebachiensis and K. tunicata, but there is no evidence that the timing of gonadal growth in the species in my study is controlled by a change in food conditions. iii

The importance of temperature in stimulating spawning has been stressed by many authors, but I know of no instance where it has been demonstrated that a temperature change, sufficient to induce animals to spawn in the laboratory, actually occurred at the time of natural spawning. At First Narrows, there was usually a major spawning when the temperature reached 7-8 °C in the spring. However, in 1971, S. droebachiensis spawned when the temperature was about 6.3 °C» and temperature differences would not account for an abrupt spawning in 1973 In: Perspectives in Marine Biology, A. A. Buzzati-Traverso (Ed.), University of California Press, Berkeley, pp. 67-36. Compared to the prolonged spawning in 1974. At Porteau, water temperatures showed a slow rise of only 0.8 °C during a two week period in which there was a complete spawning in To nice 11a lineata, Tonicella in sign, is, and Hop_alia laevior. At Botanical Beach, temperatures were a few °C warmer than at First Narrows when T. lineata, 3. droeDachien- sis, and H. hindsii spawned, and the temperature at the time of spawning of T. lineata and K. tunicata varied several °C in different years. These observations suggest that spawning did not occur in response to a physiological threshold temperature, or to a sudden change in temperature.

In 1973, S. droebachiensis and T. lineata were collected at First Narrows in late March, prior to spawning, and maintained under various temperature and light conditions: at 5.5 and 14 °C in darkness, and at 5.5 and 14 °C in light conditions similar to those in the field. These animals did not spawn whan spawning occurred in the field. Similarly, S. drcjsbachiensis, T. lineata, and T. insig[nis collected prior to spawning in 1974 and maintained in the laboratory did not spawn. However, animals returned to the field from the laboratory did spawn. This suggested that some condition in the field, which was not present in the laboratory, stimulated spawning, and tnis factor did not appear to be light or temperature.

An abrupt spawning at First Narrows and Porteau in 1973 occurred at the time of the spring phytoplankton outburst, but in 1974 spawning at First Narrows was less abrupt corresponding to the slow development of the phytoplankton bloom in that year. In the laboratory, a large proportion of S. droebachiensis, !• and T. insianis spawned when they were exposed to natural phytoplankton collected during the bloom with a 50 u mesh net. This suggested that some substance bound to or released by phytoplankton stimulated spawning. For species with planktotrophic larvae the synchronization of spawaing with the phytoplankton bloom increases the probability of bota favourable food and temperature conditions for development or eggs, larvae, and juveniles.

Gonadal growth during the coldest part of the year and spawning at the time of the spring phytoplankton uloom was found in S. droebachiensis, T. lineata, T. insignis, M. ciiiata, and probanly K. tunicat a. This pattern is characteristic* of marine invertebrates with pelagic larvae living in cold waters. iv

TABLE OF CONTENTS

INTRODUCTION ...... 1

STUDY -&JKJEAS •*•«•••• • ^ •••*:••••••••••#••''•»••* y * • 6

RESULTS

Relation of Gonad Size to Animal Size ...... 17

Reproductive cycles

Katharina tunicata ...... ,...... 27

Mojgalia hindsii 32

Mopalia ciliata ...... , 35

Mogalia lianosa 38

MO£aiia laevior ...... 40

Mopalia muscosa 43

Tonicella lineata 46 22si£sii§ issiasis 51

Strong;y.iocentrotus droebachiensis ...... 54

Observations and Experiments on Spawning

Temperature and Spawning in the Field ...... 59

Effect of Light and Temperature on spawning

in the Laboratory 67

Phytoplankton Observations in the Field 74

Experiments on the Effect of Phytoplankton on

Spci%rni.ii•••*••••••••••••••«•**•*«••••••••'•*•• 3.2 V

DISCUSSION

The JBegulation of Reproductive Cycles , .. . . . 86

possible factors Regulating Gonadal Growth ...... 87

Temperature ...... 89

Photoperiod ...... , 92

Nutrition ...... 95

Possible Factors Regulating Spawning ...... 101

Temperature ...... ,,.,....,*.*. ,•...... 102

light 110

Chemical Factors ...... 110

Phytoplankton 112

Internal Considerations ...... 118

Evolutionary and Geographic Considerations ...... 121 fiEFERENCES ...... 127 vi

TABLES

Table I, Live weight (g) of animals at sexual maturity, and weight range collected for the study of the reproductive cycle, for each of the species in the present study...... ,y, ...... 26

Table II. Density (millions of cells/m3) of common species of phytoplankton at Stations 1, 2 and 6 before and during the bloom in 1973. (Dr John Stockner, pers. comm.) 77

Table' III., Density (millions of celis/m3) of Thalassi- sp. and Skeletonema costatum at Stations 1 and 2 from 6th March to 3rd May 1974 (Dr John Stockner,

Table IV. Density (millions ox cells/m3) of diatoms at Stations A and B in the Strait of Georgia, and for Station C in Juan de Fuca Strait, during winter and spring 1973 (Mr Jae Shim, pers. , comm.),....,.....,...,. . 79

Table V,, Density (millions of cells/m3) of diatom species at Stations A, B, and C before and during the bloom in 1973 (Mr Jae Shim, pers. comm.) .,.,...... , . 80

Table VI., Effect of natural phytoplankton and sperm suspension on St r ong v.1ocentr ot us droebachiensis,

Tonicella lineata, and Tonicella insignis. ; ...... 84

Table VII., Reported developmental times under various temperatures for the species in the present study. .... 108 vii

FIGURES

Fig. 1.. Southwestern. British Columbia, showing where the studies of reproductive cycles were made and where environmental data were collected...... 7

Fig. 2 A—H, Relation between the gonadal index percentage value (arcsin values) and the total live animal weight for (A) Katharina tunicata, (B) Mogalia hindsii, (C) Mop alia .. ciliata, (D) Mogalia Iignosa, (E) Mo £ alia laevior, (F) Tonicella lineata, (G) Tonicella insignis, and (H) Strong^locentrotus droebachiensis, using animals collected prior to the spawning period. . „ 18

Fig. 3. Mean gonadal index and 95% confidence limits for Katharina tunicata at Botanical Beach and Point No Point. The number associated with each bar in this and the following figures of gonadal index cycles is the sample size. 28

Fig.,4. Summary of spawning observations on Katha_rifia- tun• icata . 29

Fig, 5.,Mean gonadal index and 95% confidence limits for Mogalia hindsii at Botanical Beach and First Narrows.,. 33

Fig, 6. Summary of spawning observations on Sogalia hindsii; symbols defined in Figure 4, , 33

Fig. 7. Mean gonadal index and 95% confidence limits for Mogalia ciliata at First Narrows and Botanical Beach. . 36 Fig, 8., Summary of spawning observations on. Mogalia ciliata: symbols defined in Figure 4, ...... ,,,., ,,,,, . 36

Fig. 9. Mean gonadal index and 95% confidence limits for flogalia iignosa at Porteau, and mean gonadal index and gonadal indices of each animal collected at First Narrows. ,...... ,'..,, ,,y...... , 39

Fig. , 10. , Summary of spawning observations on Mojgalia Iignosa; symbols defined in Figure 4. 39

Fig, 11.. Mean gonadal index and 95% confidence limits for Mogalia laevior at Porteau and First Narrows. ...,..... 41 Fig. 12., Summary of spawning observations on MogaJLia laevior; symbols defined in Figure 4 41

Fig. 13. Gonadal indices of each individual of Mogalia museosa collected ar Botanical Beach...... ,... 44

Fig, 14, Summary of spawning observations on Mogalia viii

muscosa; symbols defined in Figure 4...... 45

Fig. 15. ... Mean gonadal index and 95% confidence limits for Tonicella lineata at Botanical Beach, First Narrows and Porteau. 47

Fig. 16., Summary of spawning observations on Tonigella

lineata; symbols defined in Figure 4. v., 48

Fig. 17. Mean gonadal index and 95% confidence limits for Tonicella .insignis at First Narrows and Porteau.,..,.,., 52

Fig. 18. Summary of spawning observations on Tonicella insignis; symbols defined in Figure 4.,.,,...,,,,«,,,,. 52 Fig. 19.,Mean gonadal index and 95% confidence limits for §irongxlocentrotus droebachiensis at First Narrows and Botanical Beach, and for Strongylocentrotus fiurjauratus at Botanical Beach...... 55

Fig, 20. Summary of spawning observations on Strongylocen- trotus drgebachiensis; symbols defined in Figure 4.,,,, 56

Fig. 21. Mean sea water temperature (± standard deviation) for the first half and second half of each month during March 1971—September 1974 at First Narrows compared to the four year mean (1971-1974) for the first half and last half of each month; mean gonadal index values for Strongylocentrotus droebachiensis, Tonicella iifieata* Tonicella iaSiailAJi/ USEJlAAii laevior. Mo£alia ciliata, Mo£alia hindsii,.and Mogalia lignosa superimposed on these data, show the relationship of these cycles to temperature. . 60

Fig. 22. A comparison of the mean gonadal index cycles for Tonicella lineata, Tonicella insignis, Mojjalia laevior and Mo£alia lignosa at Porteau during April 1971—April 1 9,73• . * • * • • -# • • • • • «• • • • * • • * •* • • • •-»»-*•'*•• •» • • ••,-*»••;•"•;'•/»".* • .. 6i

Fig,. 23. Mean sea water temperature (± standard deviation) for each month at Amphitrite Point and Saerringham Point during the period May 1970-July 1974 compared to the five year monthly mean temperature (1970—1974) for each location; mean gonadal indices of Katharlna tun- icata, Tonicella lineata, MO£aiia hindsii, Mo£alia ciliata, Strongylocentrotus droejoachiensis - and Strongylocentrotus £ur£uratus superimposed on these data show the relationship of the cycles to

Fig. 24., Mean gonadal index and 95% confidence limits for Strongylocentrotus droebachiensis at First Narrows during March-May 1973, for groups of animals transferred to four light and temperature regimes in the laboratory, and for one group wnich was ix

transferred to First Narrows on 19th April; DH (dark—warm), DC (dark—cold, 1» (light—warm) and LC (light-cold) are laboratory conditions described in til© t © xii * ./ • • * • • • • * *» • * # • • »• •» • •.•*'-•>• •••»••#,• *••#•• #'*#•••»*'.;.' 63

Fig. 25. Mean gonadal index and 95% confidence limits for S^ongj;locentrotas droebachiensis at First Narrows from March—September 1974, and for groups transferred to various temperature regimes in the laboratory.,.,.., 69

Fig, 26. Mean gonadal index and 95% confidence limits for !2ai£®Aia lineata at First Narrows from March—May 1973, and for groups transferred to various light and temperature regimes in the laboratory...... , 71

Fig. 27, Mean gonadal index and 95% confidence limits for Tonicella lineata at First Narrows during March-September 1974, and for groups placed in various temperature regimes in the laboratory, and for one group transferred to First Narrows on 15th April...... 72

Fig.,28. Phytoplankton abundance as measured by chlorophyll a for six stations in Howe Sound and one in Indian Arm during February—June 1973 (Dr John Stockner, pers. comm.): each value is the mean of five measurements made at 0-5 m; mean gonadal index values for Strongylocentrotus droebachiensis, Tonice11a lineata, and Tonieella insignis, superimposed on these data, show the synchrony of spawning with the phytoplankton bloom in 1973. . . 75

Fig.,29. Phytoplankton abundance measured by chlorophyll a for Stations in the vicinity of first Narrows during February—June 1974: values for Stations 1, 2 and 11 represent the mean of three measurements from 1—5 m (Dr John Stockner, pers. comm.) and values for First Narrows and Jericho Beach are single measurements taken near the surface: mean gonadal indices for Strojig^locentrotus droebachiensis, Tonicella lineata, and Tonicella insignis, superimposed on these data, show the prolonged spawning period corresponding to the delayed and slow growth of phytoplankton in 1974. . 76

Fig. 30. Mean gonadal index and 95% confidence limits for groups of Strongylocentrotus droebachiensis and Tonicella lineata treated with phytoplankton from 21-27th April 1973, compared with events in tanks from which these groups sere taken...... 84 X

ACKNOWLEDGEMENTS

I wish to thank John Stockner, Dave Cliff and Karen Monroe, at the Pacific Environmental Institute, Vancouver, for allowing me to use their data on phytoplankton abundance and physical conditions; and Jae Shim, Institute of Oceanography, U.B.C., for providing me with information on the diatom blooms at Juan de Fuca Strait and the Strait of Georgia. Without this information the conclusions of this paper would be poorly supported.

Dr Thomas H. Carefoot, my graduate supervisor, made detailed criticisms of several drafts of this manuscript, and of an earlier paper. .Careful consideration of this thesis was also given by my graduate committee, Drs Ian McTaggart Cowan, Gilbert Hughes and John Phillips, as well as by the external examiners, Drs Jefferson Gonor and John Lawrence. Dr Cowan also helped me with the identification of the Mopalia species,,

I am grateful to my wife, Helga Guderley, for her encouragement throughout this study, for help in collecting animals, and for frequent advice on the presentation of information in the thesis. During the phytoplankton experiments, some laboratory facilities were provided by Dr Max Taylor, and Rosemary Waters gave me technical assistance. I had many valuable discussions with Paul Falkowski. on the possible relationships between phytoplankton and spawning. Dr Norman Wilomovsky was generous in making the facilities at Botanical Beach available to me. The first few years of the Botanical Beach experience were shared with Paul Breen, Hong Woo Koo and Steve Heizer. Dolores Lauriente and Steve.Borden freguently helped me with the use of the computer facilities at O.B.C. I will always remember the encouragement and assistance of my friends, especially George Lilly, Peter Newroth, Barbara Moon, Randy Kaneen, Frank Boas, Rick Beckwitt, Fred McConnell, Joanne Druehl, Tom Munford, Peter Hochachka, Jeremy Fields, Brian Murphy, Ed Buskey and Jim Markham, Dedicated to

762 Katharina tunicata

292 Mogalia hindsii

205 Mogalia ciliata

116 Mogalia Iignosa

3 35 Mogalia laevior

146 Mogalia guscosa

1176 Tonicella lineata

373 Tonicella insignis and

940 Strongy.locentrotus droebachiensis

which were sacrificed

during the course of this study. 1

INTRODUCTION

Reproductive cycles . involve the periodic growth and development of gonads, followed by the usually abrupt release of

gametes. In the majority of marine invertebrates they follow

annual patterns, This is particularly true of shallow water

species in temperate seas, where environmental conditions show

distinct annual fluctuations, but annual reproductive cycles are

also found in some species in the tropics and in deep seas,

where conditions are nearly constant. There has been much study

CD reproduction in marine invertebrates, and the subject has

been thoroughly reviewed by Giese . and Pearse . (1974), Many

workers correlated the various phases of gonadal development and

spawning with environmental factors, particularly with

temperature. Early in this century, Appellof (1912) concluded,

from observations of the northern holothuroid, Cucumaria

frondosa, that temperature was important for two phases of the

reproductive process: (1) the development of the gonads, and (2)

the developmental stages after fertilization. The latter was

confirmed for a variety of species by Runnstrom (1927a). , He

found that the temperature tolerances of pelagic larvae were

closely related to the temperatures at the time, of spawning and

to geographical distribution., Orton (1920) correlated breeding

time with temperature for a number of European species, and

concluded that most animals breed at a definite temperature

which is a physiological constant for each species. He implied \ 2

that a certain temperature change or merely reaching a critical temperature level stimulated spawning. Many authors have accepted Orton's ideas. It has been demonstrated for a number of species that increased temperature stimulates gonadal development (eg. Loosanoff & Davis, 1963). However, the importance of temperature in the gonadal development of other species, and for spawning in general, is not clear,, Orton*s conclusions were too far reaching (Korringa, 1957). The influence of other environmental factors has also been studied.

Some researchers have considered photoperiod, which is known to coordinate reproductive activity in many mammals, birds and insects, but this has not been clearly demonstrated for any marine invertebrate., Food may be required for gonadal development, but there are few instances where it has been demonstrated that a change in the food conditions stimulates gonadal activity, flany other factors, including lunar cycles, salinity, wave action and chemical stimulation, have been related to the timing of reproductive events, but most of these considerations are speculative and not supported by environmental data. Although the literature on reproductive cycles is extensive, we still do not have a good understanding of the processes involved.

The objective of the present study was to elucidate further the factors controlling reproductive cycles. The study area was on the southern coast of British Columbia, an area characterized by a regular but moderate annual cycle of environmental conditions. Eight species of chitons and one sea urchin were examined for 3-5 years. Most of the observations were made in 3

three locations, two in the Strait of Georgia estuary, and one

on the outer coast of Vancouver Island. I hoped that

observations over different years and places would indicate that

certain environmental conditions were consistently present

during particular phases of the reproductive process. Also I

thought it would be useful to compare the reproductive patterns

in closely related and non-related species. The presence of

similar patterns would suggest that similar environmental cues

were controlling reproductive events, and the identification of

these cues in some species would aid in elucidating the cues in

species which show a similar pattern. Alternatively, different

patterns would suggest that different stimuli were used to

coordinate reproductive activity.

Although experimental studies are desirable, and essential

for testing hypothetical cues, thus far I have only conducted

experiments examining the stimulus for spawning. The time of

spawning is of particular importance for animals with pelagic

larvae, since it is an adaptive necessity that the larvae be

produced when conditions are favourable for their growth and

development. One would expect that a knowledge of the

requirements of the larvae would be useful in understanding the

factors controlling spawning. While the synchrony of

phytoplankton blooms and spawning has been recognized for many

years, and phytoplankton is of obvious importance to

planktotrophic larvae (Thorson, 1946), I know of no study in

which the effect of phytoplankton on spawning was examined experimentally. Presumably this is due to past emphases on

temperature as a controlling factor., In the present study, I 4

tested both the effect of temperature and of phytoplankton on spawning in three of the species studied.,

The eight species of chitons studied were: Katharina- tun• icata wood, Mogalia hindsii fieeve, Mogalia ciliata sowerby,

Mp_.ga.lia li&nosa Gould, Mogalia laevior Pilsbry, Mo£al.ia muscosa

Gould, Tonicella lineata Wood and Tonicella-insignis•Reeve. All are common intertidal and/or subtidal species on the west coast of North America. Only K. ..tunicata extends beyond this area to

Asiatic shores, In California, reproduction in K. tuaieata has been studied extensively and some reproductive studies have also been made on M. hindsii, M. . ci 1 iata, M. Iignosa and M.,muscosa

{Barnawell, 1954; Thorpe, 1962; Giese, 1969)., The geographical ranges of Mogalia laevior and T, insignis are limited primarily to the British Columbia and Washington coastline, and there are no published accounts of reproduction in these species. Except for one study on the reproductive biology of T, lineata on the

Oregon coast and in Puget Sound (Barnes, 1972), the present study is the only account of seasonal reproductive activity in the above chitons in locations north of California and closer to the center of their geographical distribution.

In contrast to the above chitons, the green sea urchin,

S£rong^locentrot u s droebachiensis Muller, has a wide geographical distribution and occurs in the North Atlantic,

Arctic and North Pacific Oceans., It is a dominant member of the

subtidal community throughout much of its range. ;, Surprisingly, its reproductive cycle has been described only on the Atlantic coast of North America (Cocanour & Allen, 1967; Himmelman, 5

1969). In the present study brief observations were also made on the northeast Pacific sea urchin, Stroagiiocentrotus fiurjDur- atus (Stimpson), primarily to compare it to S,.droebachiensis.

Both the chitons and urchins were easily found and collected, and their gonads are distinct and separate organs, thus making it easy to assess their relative size in different seasons. 6

STUDY. AREAS

Host of the field observations in the present study were made at three locations in British Columbia: at First Narrows in

Vancouver, at Porteau in Howe Sound, and at Botanical Beach on the outer coast of Vancouver Island (fig, 1),

First Harrows and Porteau,, First Narrows and Porteau are in the Strait of Georgia and are characterized by marked seasonal fluctuations in temperature and salinity,, In June and

July peak runoff occurs from the Fraser River and salinities drop below 20°/oo in the upper 3 m of the water column. During the remainder of the year salinities usually fluctuate between

23-28°/oo in the upper 40 m of the water column and rarely reach

30°/oo. At First Narrows Tonicella lineata, T o nic e ixa. ins ignis,

.aogalia laevior, Mopalia • lignosa, and Strongylocentrotus droe- bachiansis Mere collected from depths of 2—12 m below ML1S (mean low water spring tides) and at Porteau Tonicella lineata,

Tonicella insignis, Mo£alia laevior and Mo j> alia ixgnosa were collected from 3-15 m below ML8S using SCUBA. The bottom at

First Narrows is gently sloping sandstone with some areas covered by boulders. Strong tidal currents sweep across this area. , At Porteau the bottom consists of a steep boulder talus with a few areas of bedrock outcrops. Diatoms and ephemeral macrophytes are the predominant vegetation in both areas. Pig. 1. Southwestern British Coluabia, showing where the studies of reproductive cycles were made and where environmental data were collected. 8

Botanical Bea.clu Botanical Beach receives heavy wave action from the open Pacific Ocean and its hydrographic conditions are typical of outer coast areas in British Columbia. Surface temperatures fluctuate from 6-12 °C annually and salinities remain between 27—32°/oo (Green, 1968).Here 4 species of chitons and 2 urchins from the intertidal region were studied.

Illiiarina tunicata was abundant . and in order to reduce variability in gonad size due to habitat differences, all collections were made from one gently sloping bench on which the phaeophyte, HedOjghj.llum sessile, predominated. , Adult Tonicella iiagata, Mogalia hindsii and Mogalia ciliata were more difficult to find and were collected from a wider range of habitats, but mostly from vertical rock faces and undercuts. The above species were always collected from 0—1.5 m above MLSS., Mogalia lM§cosa was collected from many scattered pools at 1.8—3.0 m above MLWS tidal height. , A number of collections of Stronaylo-

£sntrotus droebachiensis and Str^ngYlgcentrotus gurguratus were made from 0-0.4 m above Ml US in one area on a sandstone bench which was protected from heavy wave action by an outer ridge of rocks,, There are mixed semidiurnal tides at Botanical Beach and the low intertidal region is usually only exposed during the lower low tide, which occurs in the early morning in the spring and summer and in the evening in the autumn and winter.,

Other collecting sites.. Several chiton species were collected at 3—9 m below MLHS on a steep rockface at Eagle

Harbour, 11 km outside of First Narrows (Fig. 1). Salinity and temperature conditions here are probably similar to those at

First Narrows, but there are no strong tidal currents. 9

Katharina tunicata was collected intertidally at three additional locations on the west coast of Vancouver Island; at

Point No Point in Juan de Fuca Strait, and at Frank Island and

Amphitrite Point, about 110 km northwest of Botanical Beach

(Fig, 1). Point No Point is somewhat protected from wave action by its location in the Strait, but Frank Island and Amphitrite

Point receive the brunt of waves from the open ocean. Tidal, temperature and salinity conditions in these areas are similar to those at Botanical Beach, although the annual range in temperature and salinity tends to decrease going from Amphitrite

Point to Point No Point, as a result of increased mixing with deeper water in the Strait, 10

METHODS

.Reproductive cycles.. The timing of reproductive events in the field was studied using the gonadal index, defined as the percentage wet weight of the gonads to the total live body weight.. This method gives a quantitative value for the reproductive state of an animal, and is particularly useful for describing reproductive cycles of species which show marked synchrony amongst all individuals (Moore, 1937; Gonor 1972;

Giese & Pearse, 1974). However, the gonadal index method does not describe gametogenic events. One does not known whether an increase in the gonadal index is due to an advancement in gametogenesis or to the accumulation of nutrient reserves.

Further, some gametogenic events may not result in a change in the overall size of the gonads. An accurate description of gametogenic events requires lengthy histological and biochemical procedures. In the present study notes were made on the colour and firmness of the gonads, and the presence or absence of young oocytes and mature ova, or the extent to which sperm oozed from the dissected testes. I emphasized the comparison of general patterns in reproductive cycles, and detailed histological events, although of interest, were not studied.

The gonadal index method is based on the assumption that the gonadal index is independent of animal size., Gonor (1972) stressed the importance of testing this assumption.„ In studying seasonal trends in reproductive activity, only adult animals 11

should be sampled, and it is desirable to limit the size range of adult animals so that variation due to size is as little as possible. In some species gonadal activity in smaller animals is not synchronous with larger animals, and sometimes, gonadal indices decrease slightly in very large animals {Gonor, 1972).

To determine a useful size range for each species in this study,

I plotted gonadal index arcsin values against body weight, using animals collected just prior to the spawning period. The angular transformation to arcsin values corrects the gonadal index percentage values so that they are normally distributed

(Sokol & Hohlf, 1969).

For each species I tried to collect a sample of 10 or more animals, within the size range determined by the above method, at monthly or bimonthly intervals, or more frequently when I anticipated spawning. flhen a species was rare, as in the case of I2££lia lignosa at First Narrows, or when wave conditions were extreme at Botanical Beach, it was not always possible to collect 10 individuals of each species. The mean gonadal index and 95% confidence limits of each sample were calculated and these values were plotted on a time scale for each species.

When spawning was imminent in the field, animals collected in the field could frequently be induced to spawn by a disturbance. To test whether spawning could be induced, I made a standard practice of returning animals to fresh seawater after allowing them to dry on paper towels for 15 minutes for weighing purposes., Spawning could only be induced in this way when it

»as about to happen in the field. 12

Environmental conditions^ Information on environmental conditions in the study areas was obtained from a number of sources. Daily temperature and salinity measurements were made of incoming water at the Vancouver Public Aquarium.. Ihe water intake was located 1 km from the collection site at First

Narrows at a depth of 6m below MLWS, and it is likely that these data provide a good record of the conditions to which the field animals were exposed. At Botanical Beach water temperatures were not recorded but daily measurements of surface temperatures were available from lighthouses at Sherringham

Point and Amphitrite Point (Fig..1; Holliser, 1971, 1972, 1974;

Giovando & Hollister, 1974; Giovando, Pacific Environmental

Institute, pers. comm.), A comparison of temperatures at or near Botanical Beach at earlier dates with those for the same periods at Sherringham Point and Amphitrite Point indicated that temperatures at Botanical Beach were closest to and slightly warmer than those at Sherringham Point, and almost always colder than those at Amphitrite Point (Green, 1968; Crean & Agnes,

1968; Hollister, 1968, 1971a, 1971b). Dr John Stockner, Pacific

Environmental Institute (pers.,comm.) provided data on temperature, salinity and the abundance and species composition of phytoplankton at several stations in the vicinity of

Vancouver and Howe Sound (Fig. 1) during 1973 and 1974. Mr Jae

Shim, Institute of Oceanography, U. B. C. (pars. comm.), provided data on phytoplankton abundance in Strait of Georgia and Juan de Fuca Strait (Fig. 1) in 1973., In addition I made some measurements of temperature and of chlorophyll a concentrations in the Vancouver area in 1973 and 1974, and a 13

thermograph record of temperature at Porteau during the spawning

period in 1973.,

Experiments on spawning.. In 1973 and 1974 laboratory

experiments on spawning were conducted. On 30th March 1973,

prior to spawning in the field, S. droebachiensis and I. lineata

were collected at First Narrows to test the effect or light and

temperature on spawning. Thirty-five to fifty individuals of

each species were kept in each of four 45 1 tanks under the

following conditions: dark-cold, light-warm, dark-warm, and

light-cold. The two tanks in the light were set up next to a

west facing window and probably received more light than animals

in the field, but with the same photoperiodicity. A continuous

incoming supply of water maintained the cold tanks at 5.0±0.5 °C and the warm tanks at 14.0±1.0 C except for 2 occasions when the

cooling system failed. During April each tank was visually

checked daily for any indication of spawning, and on 12th April,

10 individuals of each species were removed from each tank and

their gonadal indices determined. Five S.,droebachiensis were

also taken from the dark-warm tank on 20th April.J On 29th April

the experiments were terminated and gonadal indices were

determined for the remaining animals. In 1974 S. droebachien-

s±s, T. ... lineata and T. insignis were collected on several dates

prior to spawning and maintained for 1—3 months under constant

temperature conditions in the laboratory. Light conditions

during the 1974 experiments were not controlled but varied with

other research activities in the laboratory., In all of the

above experiments the chitons were not fed, except for diatom

scums which formed in some tanks, and the urchins were 14

occasionally given pieces of laraiaaria sp.

Experiments were also performed to test whether the

presence of phytoplankton would induce spawning in the

laboratory. In 1973 natural phytoplankton was collected daily at Jericho Beach with a 50 p. mesh net and added to tanks containing groups of unspawned animals. The experimental animals were from tanks which were set up on 30th March 1973 to

test the effects of temperature and light. One group of

S. droebachiensis in a 12 1 container (14 °C) and two.groups of

2* A±2.§§.ta in two 4 1 containers (8 °C) were used. , These containers were maintained without an incoming supply of water,

but were gently aerated. Prom 21th-27th April the water was

changed daily in each container and fresh phytoplankton was

added until the water was slightly murky. Spawning activity was noted and the mean gonadal index of each group of animals on

27th April was compared to its "source" tank on 12th April and

29th April.

In 1974 further laboratory experiments on the effects of

phytoplankton were conducted. A supply of mature ^ronqylocen- trotus droebachiensis, Tonicella lineata, and Tonieella insignis

was collected on 12th March and on 4th April at First Narrows, and was maintained at 5.5 °C in large tanks.. Since adeguate

quantities of phytoplankton could not be collected with a 50 p.

mesh net at Jericho Beach until 29th April, experiments on these

animals were not started until this date.,. During the experiments fresh phytoplankton was collected every 2—3 days.

Each experiment was run for 10 days. Tests were made on single 15

animals, using 1.2 1 containers for S. droebachiensis and 0.4 1

containers for T. lineata and T. insignis. Every day the water

in each container was changed and a quantity of phytoplankton

(final concentration of 10 mg chlorophyll a/m3) was added to

experimental containers, but not to the controls. After 17th

May it was not possible to collect a suitable quantity of

phytoplankton with a net, and frozen plankton from 6th May was

used after 19th May. The addition of sex products is one of the

most effective ways of inducing spawning in a variety of marine

invertebrates (Giese, 1959; Loosanoff & Davis, 1963). Thus, for

comparative purposes, for each species I also tested the effect of an addition of a small quantity of sperm suspension, using

sperm shed by animals in the plankton experiments.,,

Ide^tification of the Mopalia sjaecies^,,- The genus Mojgalia

is in need of revision and identification of the various species

is usually difficult for a person who has not spent considerable

time studying them. Therefore, I will describe identifying

characteristics which I have found useful., Mogalia muscosa is

the most distinctive species, owing to the stiff bristles which

densely cover its girdle. The plates are often worn through to

the calcareous layer, and when the integument is present it is

brown in colour and sculptured with longitudinal ridges.

Mogalia iianosa has medium coarse bristles scattered over its

girdle, and there are pink spots at the base of the bristles.

Colour streaks, of blue-grey, brown and white in varying

proportions, give a distinctive appearance to the smooth-

surfaced plates., The foot of M. Iignosa is more reddish in colour than that of the other Mopalia species. ., Mfialia- ciliata 16

usually has a wider girdle and narrower plates than the other species. The girdle has both fine glassy bristles and larger flattened bristles. There are longitudinal ridges on the central areas of the plates, and these become more oblique and irregular on the lateral areas. The plates usually have a complex colour pattern with shades of brown, grey, white, green, yellow and orange, but sometimes , one colour predominates.

Mo£alia hindsii has a moderately dense covering of fine glassy bristles on its girdle. The plates are dark brown in colour, and there are longitudinal and latteral ridges, giving a

"basketweave" appearance. The pallial groove was yellow-coloured (particularly towards the posterior end) in 95% of the animals I collected weighing over 10 g, and in a smaller proportion of smaller animals. This colouration was not seen in the other species. Mogalia laevior has a sparse covering of glassy bristles on its girdle. The surface of the plates is smooth, but there is the appearance of fine lines which laterally cross the central areas and then curve posteriorly across the lateral areas. The plates are mostly grey brown in colour, but in most animals there are some green markings and sometimes there are white streaks across the center of the plates. 17

RESULTS

Relation of Gonad Size to animal Size

Katharina tunicata. Gonadal index arcsin values plotted against body weight are presented for K. tunicata in Figure 2A, using animals collected at Botanical Beach and Point No Point prior to spawning in 1971 and 1973. At Botanical Beach no gonads were found in 12 animals weighing 3.6 g or less; the smallest animals with mature gametes were a male and a female weighing 5.3 and 7.1 g respectively; and the largest animal with an immature gonad weighed 9.6 g., Animals larger than 30 g had large ripe gonads and the variation in arcsin values remained similar in larger animals. At Point No Point animals had larger gonads, but gonad size did not appear to vary with animal size between 45—129 g.

Mopalia hindsii. Four M. hindsii weighing 1.3—2.4 g had no gonads, a male and a female, both weighing 4.6 g, contained mature gametes, and the spread of gonadal index arcsin values was similar for animals weighing 10-32 g (Fig. 2Bj.

Mopalia ciliata. Gonadal index arcsin values remained at about the same level for animals from First Narrows weighing

4-19 g (Fig. 2C). In a collection of smaller animals from Eagle

Harbour on 23rd March 1973, all animals, even a 0.6 g animal, contained mature gametes, and one male weighing 2,4 g spawned in the laboratory. 18

A 25 Katharina tunlcata +

20 A

I I e + o I ° I I « o I O 15 U < 1 . • a I O o

"8 10 C Botanical Beach o o 0 28th March - 5th May 1971 1 6th April 1973

Point No Point + 25th May 1971

04niii 0 10. 20 30 40 50 60 70 80 90 100 110 120 130 Weight (g)

Fig. 2 A-H. Relation between the gonadal index percentage value (arcsin values) and the total live animal weight for (A) Katharina tunicata, (B) Mopalia hindsii. (C) Mofialia ciliata, (D) Mopalia lignosa, (E) Mopalia laevior, (F) Tonicella lineata, (G) Tonicella insignis, and (H) Strongylocentrotus droebachien• sis. using animals collected prior to the spawning period. Mopalia hindsii

l I I 1 o I I e I I I • I I ' • I • ' I I e 1,(1 I I I o I °

First Narrows 0 22nd February 1973

Eotanical Beach 1 9th March - 6th April 1973

0+-« i—!—i 1 1 1 1 1 1 0 5 10 15 20 25 30 35

Weight (g) Mopalia ciliata 25

• •

20 • • • • •

• • CD 15 O VI •<

10 First Narrows

• 21st February - 30th March 1973

Eagle Harbour 54 • 23rd March - 4th April 1973

2 4 6 8 10 12 14~ 16 18 20

Weight (g) D 30 Mopalia lignosa

25

20 * • fa

• • 0 uo • o a. • • •' a + I 15

R-F

a 9 XM Porteau f 8th April 1971 • 16th June 1971 + 7th April 1972 • 28th December 1972 • 8th May 1973

—r— —r~ T 6 8 10 12 14 76 Weight (g) Mopalia laevior 25i

o • •

20 • • •,

• 1 T O I

2 15 •< x a* "O c

•g 10 c o First Narrows o 22nd January - 6th March 1973

Porteau 5^ • 8th February - 27th March 1973

10 15 20 25 30 ~35 Weight Cg) 35i Tonicella lineata

30 i

0 25 • • _ „ * • n 0 O _ I ' I »~ * ° B o • II o 20 I o I i I I, » 0 • ••

15 II ,'l„ First Narrows i. • i } 20th April 1971 10 i • 16th March - 20th April 1972 0 2nd February - 30th March 1973 i i 'i i Eagle Harbour i 54 1 23rd March - 4th April 1973

0-fca- -r- —i 0 2; ~6~ T 8 Weight (g) Tonicella Insignis

25 •

20H c O 4

•o 15 c M cd First Narrows 1( 5 5 f 21st January - 30th March 1973

Porteau • 8th February - 27th March 1973

Eagle Harbour I 23rd March 1973

4 6 8 10 72

Weight Cg)

Strongylocentrotus droebachiensis 40

35

30- • • • a - • 8 » O

e 25- e e o° oe e 20- o

15 First Narrows o 12th March - 7th April 1974 10

5-I

~20~ 40 60 80 100 120 140 160 180 200 220

Weight (gj 23

-Mogalia Iignosa. Gonadal index arcsin values for 5 collections of M. Iignosa from Porteau during periods when the mean gonadal index was high are shown in Figure 2D., All animals, even one .weighing 2.6 g, had large mature gonads, and gonadal size did not appear to vary with body size,, During periods when the mean gonadal index was low, five 2,3—2.6 g

animals were found and each of these contained gonadal tissue.;

Mogalia laevior. In M. laevior from First Narrows and

Porteau the variation in gonadal index arcsin values was relatively uniform for animals weighing 9—35 g (Fxg. 2£). In a collection of smaller animals from Eagle Harbour on 4th April

1973, 14 out of 17 animals weighing 2-5 g contained some mature gametes, but arcsin values were not plotted for this collection since spawning was nearly finished on this date.

Mogalia muscosa. Since there did not appear to be a period when all animals were in peak reproductive condition, I did not make a collection of small M, .muscosa to determine the size at sexual maturity. In each of the collections made at Botanical

Beach during 1971-1973 the gonadal index values appeared to be independent of animal size within the range 10—50 g.

Tonicella lineata. T. lineata reached sexual maturity at a smaller size than the other chitons in the present study

(Fig.,2F). In a collection of small animals from Eagle Harbour prior to spawning in 1973 all animals weighing 0.12 g or mOre contained gametes, but 4 smaller animals did not have gonads.

Low gonadal index arcsin values were not found in animals weighing more than 1.5 g and the variation in values was 24

relatively uniform after 2 g., Two animals weighing 0,4 and

0.7 g, respectively, which were collected at Botanical Beach on

21th June. 1970 spawned in the laboratory after being dried and rewetted,

Tonicella insignis,, Figure 2G shows the gonadal index arcsin values for T. insignis collected at First Narrows,

Porteau, and Eagle Harbour prior to spawning in 1973.., No gonads were found in two animals weighing 0,3 and 0,5 g respectively, and all animals weighing 0.6 g or more contained mature gametes.

Gonadal index arcsin values tended to increase slightly with increasing body weight between 3—12 g. ,,

Strongylocentrotus droebachiensis. During this study I did not make a collection of small S, droebachiensis in order to determine the size at sexual maturity.. However, I made a few observations on this species in an earlier study in Newfoundland

(Bimmelman, 1969), Animals smaller than 20 mm in test diameter

(equivalent to about 3.9 g) did not contain mature gametes during a peak reproductive period, but animals 22 mm or larger

(about 5.0 g) contained mature gametes. In Figure 2H gonadal index arcsin values are plotted against body weight for larger animals collected at First Narrows prior to spawning in 1974.

Arcsin values may decrease slightly with increasing body weight.

A summary of the observations on the size at sexual maturity for each of the species studied, as well as the size ranges which I collected for the studies on reproductive cycles, is presented in Table I. For some species a smaller size range may have been preferable to reduce further the variation in 25

gonadal index values in the samples, but excluding the extremes would have reduced the sample size too much. 26

Table I. Live weight (g) of animals at sexual maturity, and weight range collected for the study of the reproductive cycle, for each of the species in the present study.

Size when all Size range collected Size range with Smallest animal animals have for studies on no gonad with gametes "full-sized" gonads* reproductive cycles

Katharina tunicata 0-3.6 5.3 30 30-130

Mopalia hindsii 0-2.4 4.6 10 10-35

Mopalia ciliata 0.6 4 4-20

Mopalia Iignosa 2.6 2-4 4-19

Mopalia laevior 1.6 8 8-35

Mopalia muscosa 10-50

Tonicella lineata 0-0.09 0.12 2-8

Tonicella insignis 0-0.5 0.6 3-12

Strongylocentrotus 0-3.9. 5.0 20-25 30-150 droebachiensis

* Size when all animals have near maximal gonadal index arcsin values (see Fig. 2). 27

Reproductive Cycles

Katharina tunicata (Pig. 3)

The reproductive cycle of K, tunicata at Botanical Beach varied in different years in the five.year study period., During

1970 and 1971 there was a regular sequence of events; a well defined spawninq period in June, a period of gonadal inactivity during the summer, and a period of gonadal enlargement in the late autumn and winter. In late May 1970, prior to spawning, all animals had mature gonads with gonadal indices of 3.8—9,5.

During June the mean gonadal index dropped from 6,1 to 1.1, and all animals collected from late June to late October had gonadal indices of less than 2.5. Gonadal index values increased progressively from late October 1970 to March 1971., In late May

1971 one spent male and 20 ripe individuals were collected. By

10th June 30% of the animals had indices of less than 2.0, and in late June all but one animal had small gonads. It was unexpected to find a mature male with an index of 9.8 on 23rd

July, but other animals collected during that summer had small gonads.

In 1972 and 1973 K. tunicata had a markedly irregular

reproductive cycle, and there may have been two spawning periods. Gonadal growth in the autumn of 1971-72 was not as extensive as in the previous year. For example, in each of the collections from October throuqh January 401 of the animals had Gonadal Index

H> 8to m H- e IP t-" AO B |(+ >a en crisr . ID tn ID IP> M lh P 13 ac O W IP ID H) W p O 3 ifl O Irt- O H-IC 3 0> 13 P ft H- o & ID in 3 P CMP P H |rt & C IP p 3 rt- 0, BT p (6 X d) H- oat) o o & tr rt ID P K D cr H* POP H P 3 aHs & 3* 8 ID \0 (0 p m O VI la rt- tr P er B H- P O 3 O •a w D ID a. H) w •o p. o P- N H- ID 0 3 3 rt- o (D ss o Hi O B O P- H 3 rt- H rt- W O • * • H- H3 M> B tr O XJ ID M 29

J'F'M'A'M'J'J'A'S'OVD

Katharina tunicata

Botanical Beach 1970 1971 1972 1973 =1 present study 1974 Point No Point 1971 1972 Amphitrite Point 1971 Frank Island 1971

Monterey Bay, Calif. 1932 Hewatt, 1933

Central California Rickets & Puget Sound, Wash. Calvin, 1968 Monterey Area: Carmel Point 1955 1956 Yankee Point 1957 1=1 Giese, 1959b; Yankee Point 1958 Giese, Tucker & Pescadero Point 1959 Boolootian, 1959; Yankee Point 1959 Giese 6 Araki, 1962; Nimitz & Giese, 1964; Yankee Point 1960 Lawrence, Lawrence 1961 & Giese, 1965; 1962 Giese 6. Hart, 1967; Yankee Point 1963 Giese, 1969 1964 1965 Yankee Point 1966

Symbols

|- large mature gonads, spawning not started -j small depleted gonads, spawning completed t • decrease in the mean gonadal index fa beginning of spawning end of spawning approximate time of spawning ?

Fig. 4. Summary of spawning observations on Katfrari,na tunicat^. 30

gonadal indices of less than 2.0. In mid—March 1972, 9 out of

10 animals had ripe gonads (and indices greater than 3.0), and some spawning occurred by 1st May, when all but one of the animals collected contained small quantities of gametes (and had indices of 2,7 or less). There was some additional gonadal growth during May and June 1972 and on 14th June moderate quantities of gametes were found in one-half of the animals collected. A second spawning occurred sometime during the summer, but the exact time is not known since a collection was not made until September.. It may have occurred between early

May and late June, the period during which there was spawning in each of the other four years of this study.

The drop in the mean gonadal index from October to November

1972 was probably due to sampling error rather than spawning.

During this period gonadal growth in males was well in advance of that in females, and since more females were collected in

November than in October,the November index was lower..

In 1973 there was an increase in the mean gonadal index similar to that in 1971, but there were agaxn two spawning periods. The first spawning occurred between 6th April and 4th

May, and the second spawning was in June. One male from 2nd

July had not yet completed spawning but other individuals collected in July and August contained only traces of gametes

(and had indices of less than 2.5).

In late April 1974 the mean gonadal index was 9.0, surpassing the peak values in the previous years. In late May

1974 spawning was underway and it was completed by 20th June., 31

At Point No Point 10 collections of K. tunicata were made in 1971 and one was made in April 1972., The reproductive cycle here resembled closely that during the same period at Botanical

Beach. The gonads grew rapidly from February 1971 to a peak in

May. On 9th June 3 out of 10 animals were spent, and spawning

was completed on 25th June.. In 1972 there was some early spawning corresponding to the early spawning at Botanical Beach..

More than one-half of the animals from 15th April 1972 had spent or partly spawned gonads. Gonadal index values at Point No

Point were always larger than those at Botanical Beach (see

Figs. 2A S 3). I do not know whether this was due to the larger size of animals at Point No Point, to nutritional

differences in the two areas, or to other factors.

Three collections of K. tunicata were made at Amphitrite

Point and at Frank Island in 1971 (Fig. ,1). , la both areas spawning was near completion on 26th June and the mean gonadal index was 2.5±0.9 (95% confidence limits, n=10) at Amphitrite

Point and 2.8±1.1 (n=11) at Frank Island. On 10th July the

values were 1.5+0.5 (n=10) and 1.6+.Q.6 (n=11) , respectively, and

most animals did not contain a trace of gametes. On 2nd October

the gonads had started to develop and mean gonadal indices were

2.3±0. 7 (n=10) and 2. 1±0.7 (n=9) , respectively., Thus, in 1971, it appears that K. tunicata completed spawning at about the same

time from Frank Island to Point No Point on the outer coast of

Vancouver Island, and also that gonadal enlargement was started

by October at Frank Island, Amphitrite Point and Botanical

Beach, 32

A summary of spawning observations on Katharina tunicata from the present study and earlier studies is shown in Figure 4.

The main spawning season is June-July, but sometimes there is a partial spawning in the spring. Shen the latter occurred in my study it was always followed be some additional gonadal enlargement up to the time of the second spawning.

In summary, an annual reproductive cycle with spring to summer spawning appears to be characteristic of Katharina tun• icata from British Columbia to Monterey. The gonads are always depleted in late summer and gonadal growth occurs from autumn through spring.

Mop_alia hindsii {.Fig., 5)

!!• .^ifid§ii aad a distinctly annual reproductive cycle in both the intertidal population at Botanical Beach and in the subtidal population at First Narrows, At Botanical Beach in

1972, spawning was completed in June, since every animal collected contained few or no gametes.. During the summer there was little gonadal enlargement, but after September the gonads grew steadily to a peak in April 1973., A complete spawning occurred in April 1973 and all animals collected on 4th May 1973 had depleted gonads. Only three collections were made in 1974.

On 27th April 1974 the animals collected had either partly spawned or spent gonads, suggesting that spawning was nearly finished, and all animals collected in May and June had spent Mopalia hinds

Botanical Beach

Fig. 5. Mean gonadal index and 955S confidence limits for flqpalia jjindsii at Botanical Beach and First Narrows.

J'FWA'M'JVA'S'O'N'D

Mopalia hindsii

Botanical Beach 1972 H 1973 1974 First Narrows 1972 present study 1973 (- 1974 h =1

San Francisco Bay 1952 _j Barnawell, 1954 1953

Marin County, Calif. 1957 Thorpe, 1962

Monterey Bay, Calif. 1956 1957 Giese, 1959b; =1958= Giese, Tucker & Boolootian, 1959; 1959 Giese & Araki, 1960 1962

J L JL Fig. 6. Summary of spawning observations on flo.j3aJ.ia, feislsii; syabols defined in Figure U. 34

gonads.

At First Narrows spawning was completed in late April 1972.

Gonadal enlargement commenced in the spring and by August the gonads were two—thirds maximum size. Gonadal growth in the autumn and winter was slower and a peak was reached in January

1973., In marked contrast to the abrupt spawning at Botanical

Beach in 1973, spawning at First Narrows lasted 2-3 months. It was underway in early March, when 1 spent animal and 6 with intermediate—sized gonads were collected, but all animals did not have spent gonads until 10th April., In 1974 an abrupt spawning occurred., A few partly spawned animals were first collected on 4th April and spawning was completed by 15th April.

A list of spawning observations on M. hindsii in the present study and earlier studies is shown in Figure 6. In San

Francisco Bay, Barnawell (1954) observed a reproductive cycle in i* hindsii similar to that observed in my study. There was a progressive increase in the proportion of animals with large gonads from late August to November 1952, and the majority of animals collected from January-April 1953 had large gonads.

Subsequently, spawning occurred, since most or the animals collected in flay had small gonads. Egg release in fi. ...hindsii from two locations in Marin County, California, was seen in late

October by Thorpe (1962)., In the Monterey area M. hindsii spawned abruptly in March 1957, but not in the following years

(Giese, Tucker & Boolootian, 1959; Giese & Araki, 1962). There was a gradual decline in the mean gonadal index from November

1957 to July 1958, and three breeding periods occurred between 35

November 1959 and late spring 1960.,

In summary, Mopalia hindsii in British Columbia spawns annually in March-April, while in California it sometimes spawns over extended periods from early winter to late spring.„

i°£alia ciliata (Fig. 7)

The reproductive cycle of M. ciliata was studied in greatest detail at First Narrows. The data from 1972-1973 showed a regular cycle to be present, even though only a few animals were found on some collection dates. Gonadal enlargement in 1972 proceeded gradually after June reaching a peak in January 1973., All animals collected in March 1973 had mature gonads, and one male in each of the two March collections discharged some sperm suggesting that spawning was imminent.

The .. gonadal indices dropped abruptly in early April and every animal from the 14th April collection had depleted gonads.

In 1974 there may have been some summer spawning in addition to the main spring spawning. ., All animals collected on

20th April and earlier had mature gonads, but one—half of the animals collected on 24th April had spent gonads., It was unexpected to find 4 out of 5 animals on 7th May with large mature gonads. Possibly spawning was delayed in some localized areas at First Narrows and the variation in the data was due to sampling error. Most of the animals collected in June and July had mature gonadal tissues, but many of these animals had small— to intermediate-sized gonads. By 18th September 10 out of 13 Mopalia ciliata

Botanical Beach

H 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 MJ J ASONDJFMAMJ J ASONDJFMAMJ J AS 1972 I 1973 I 1974 Fig. 7. Bean gonadal index and 95% confidence limits for Mopalia ciliata at First Narrows and Botanical Beach.

JFMAMJ JASOND

Mopalia ciliata

Botanical Beach 1974 First Narrows 1971 1972 present study 1973 1974 H Eagle Harbour 1973

San Francisco Bay 1953 Barnawell, 1954

California 1956 1957 Thorpe, I960 * ° 1962 1961 J? ?

J L J L J I I I Fig. 8. Summary of spawning observations on Mopalia ciliata,: symbols defined in Figure H, 37

animals had spent gonads.,

On 20th April 1971, 2 M. ciliata with depleted gonads were collected at First Narrows. On the assumption that this species is mature during the winter, as in 1972 and 1973, spawning in

1971 probably occurred in March or April.,

At Eagle Harbour two collections of M. ciliata- were made near the time of the 1973 spawning season. The gonadal index decreased from 11.8±1. 9 (n=10) on 23rd March, to 10.-2±2. 8 (n=9) on 4th April, and all animals contained large quantities of mature gametes. Here, spawning probably occurred at about the same time as at First Narrows .

At Botanical Beach four collections of M. ciliata were made in 1974., On 27th April all animals collected had large mature gonads.. The mean gonadal index dropped abruptly in May, and all animals collected in June and July were spent.

A summary of spawning observations on M. ciliata is shown in Figure 8. In British Columbia spawning occurred from late

March through April, except in 1974 when there was some delayed spawning activity. In San Francisco Bay, Barnawell (1954) reported that animals with large gonads were found in all seasons, but his data show a decrease in the proportion of animals with large gonads from April to July, which suggests that spawning occurred in this period. From various locations in California, Thorpe (1962) reported 20 observations of individuals spawning: 12 were in the period 21th February-21th

Harch, 1 was on 15th July, and 7 were in the period 25th 38

September-20th November.

Thus, Mojgalia ciliata spawns from late winter through spring in British Columbia, but in the southern part of its range it may also spawn in the summer and autumn.

Mopalia lignosa (Fig. .9)

At Porteau M. .lignosa appeared to spawn twice in 1971.

Spawning was imminent on 8th April, when all animals had large mature gonads and 4 males spawned in the laboratory, but one month later 7 out of 10 animals had intermediate- to small-sized gonads. Subsequently there was some gonadal growth, and additional spawning occurred sometime between mid—June and late

August., In 1972 one collection of 4 animals was made on 7th

April.,, Three males had gonadal indices of 15.3-19.5, and one female had an index of 7.5. . These 4 animals appeared mature but could not be induced to spawn in the laboratory., In 1973 animals with large gonads were found in February and spawning occurred sometime prior to 10th April, when the animals contained only small quantities of gametes.

Interpretation of the reproductive cycle of M, . lignosa at

First Narrows is complicated by both the small size of many of the collections and the irregular sequence of events., In 1971 spawning may have occurred in April, since 1 animal from 20th

April released sperm in the laboratory. In 1972, 3 spent animals were found in March., Animals with larger gonads were found in April through June 1972 and some spawning may have 39

Mopalia Iignosa 16

14 10 10 12

X 14 0} 10 XJ 10 \ 1-° c \ Porteau \ \ Porteau 8 V . First Narrows 8 ro V \6 oc 6 •J : • O 4

2

0- A M j j A'S'O'N'D'J'F'M'A'M'J'J'A'S'O'N'D'J'F'M'A' 1971 I 1972 | 1973

Fig. 9. Bean gonadal index and 95X confidence limits for Mopalia lASSosa. at Porteau, and mean gonadal index and gonadal indices ot each animal collected at First Narrows.

J "F'M'A'M'J 'J 'A'S'O^NPD Mopalia Iignosa

Porteau 1971 1972 1973 present study First Narrows 1971 1972 1973

California 1957

Monterey, 1974 Watanabe & Cox, 1975 Calif. J L I I L -1 1 I I L Fig. 10. Summary of spawning observations on Mopalia Iignosa- symbols defined in Figure 4. — - —a ~' 40

occurred in the summer, since in August the animals collected contained smaller quantities of gametes. There was no evidence of spawning in November 1972 and January 1973, but by late April

1973 the gonads were notably smaller and contained only traces of gametes. The amplitude of the gonadal index cycle was much smaller at First Narrows than at Porteau., At peak periods gonadal index values at Porteau were frequently 9-13 and occasionally nearly 20, whereas at First Narrows an index of 9 occurred only twice, and peak values were usually about 6.

There are little data on spawning of fi. .lignosa in the literature (Fig. 10)., On the open coast near San Francisco Bay,

Thorpe (1962) observed spawning in 5 animals in the period 27th

February-13th March 1957, and in the Monterey area, iatanabe and

Cox (1975) induced some animals to spawn in April-May 1974 by keeping them in stale water, but spawning could not be induced by other methods.

In summary, Mopalia- lignosa appears to spawn in late winter-early spring, and a second spawning may occur in the early summer.

Mo£alia laevior (Fig., 11)

II* i^§li2£ was examined only in the sublittoral populations at Porteau and First Narrows. ,, At Porteau spawning was in progress and near completion when the first collection was made on 8th April, 1971. The mean gonadal index was low, but 4 animals released gametes in the laboratory and 3 of these had 41 Mopalia laevior

i, First Narrows 1/

Fig. 11. Mean gonadal index and 95% confidence limits for l2E§lil laevior at Porteau and First Narrows.

j 'F'M'A'M'j1 J 'A'S'O'N'D Mopalia laevior

Porteau 1971 94 1972 1973 M present study First Narrows 1972 1973 197.4 Eap.le Harbour 1973 J L J_ I L Fig. 12. Summary of spawning observations on Mopalia laevior: symbols defined in Figure 4. 42

intermediate—sized gonads., In 1972 spawning was delayed, since all animals collected on 7th April, with the exception of 1 partly spawned male, had large gonads. In 1973 there was an abrupt spawning at Porteau. On 27th March only 1 spent animal was collected, and on 10th April the population had nearly completed spawning.,

At First Narrows there was no indication of spawning in the collections from March and April 1972, but the 1 animal collected on 8th May was spent, as were all animals collected in

June and August. The gonads enlarged rapidly durng tne autumn of 1972 and reached peak size by late January 1973., One spent animal was found on 6th March and another on 30th March.„ In the latter collection 4 animals spawned in response to being dried and rewetted., There was an abrupt spawning in early April 1973 and only small quantities of gametes or none at all remained in animals collected on 14th April. In 1974 the spawning period at

First Narrows was poorly defined and there appears to have been spawning in April and May and perhaps also in the summer period.

The most striking drop in the mean gonadal index occurred between 10th—24th April, but the data were erratic and a high proportion of unspawned animals was later collected on 30th

April and 7th May. Animals collected on different dates, and probably from slightly varied localities, differed in the extent to which they had spawned, suggesting that the population at

First Narrows did not spawn in synchrony in 1974., All animals in the 18th September collection had spent gonads.

One collection of M. laevior was made at Eagle Harbour on 43

4th April 1973. The mean gonadal index was 1. 4±0. 7 (n=5) indicating that spawning was completed.

There are no spawning data on Mogalia laevior in the literature. The data from Porteau and First Narrows show a distinct annual cycle with usually an abrupt spawning in the spring (Fig. 12).

H2£dlia museosa (Fig. 13)

Midway in the study of M. muscosa, I realized that the reproductive condition of animals varied greatly in different pools at any one time, and since animals from different pools on the same collection dates were not separated, mean gonadal indices were not calculated. Figure 13 shows the gonadal index values of each animal collected on 15 dates at Botanical Beach during 1971-1973. At all times in the year mature and spent animals were found at Botanical Beach and no peak period of reproductive activity was evident., Animals within any one pool tended to have similar gonadal indices. For example, on 4th

August, 5 animals from a pool at 2.6 m above MLSS had indices ranging from 1,9—4.2, whereas 5 animals from a pool at 1.8 m had values of 5.0—8.6. This chiton species lives higher in the intertidal region than the other chitons studied, and the variation in the reproductive condition of animals in different pools was probably related to such factors as pool size, intertidal height and food conditions. To determine seasonal trends in .M. muscosa it would be necessary to make collections 44

Mopalia muscosa

Botanical Beach

H 1 1 1 1 1 1 1 0 I i I I l 1 1 1 1 1 1 1 1 1 1 1 1—7~*~T 01 1 ASONDJFMAMJ JASON -F' •M • A• M• J• J• A• s O N D J F M A M J J 1971 1972 I 1973

Pig. 13. Gonadal indices of each individual of flooalia flascosa collected at Botanical Beach. 45

J'F'M'A'M'J'J'A'S'O'N'D Mopalia muscosa

Botanical Beach iQ7i 1Q7-3 Animals with mature gonads „„„ . _ , 1971-1973 f„„„j f. , ^ t, present study round throughout the year 3

Monterey Bay, Calif. 1932 ? Hewatt, 1933

Pug e t S ound, Wa sh. Rickets & Calvin, 1968 — —

Corona del Mar, Calif. MacGinitie & MacGinitie, 1949

San Francisco Eay 1953 Animals with mature gonads found throughout the year Barnawell, 1954

Santa Monica Bay: Flat Rock 1960 1961 Boolootian, 1964b; Monroe & Sunset Point 1960 Boolootian, 1965 1961 Latigo Point [i960 1961

Monterey, Calif. 1974 Watanabe & Cox, 1975

-I L ± Pig. 14. Summary of spawning observations on flopalja muscosa: symbols defined in Figure 4. 46

throughout the year from a specific pool which contained enough animals to sustain this amount of sampling.

In the literature there are reports of observed spawnings or of gonadal index decreases in M. muscosa in all seasons of the year (Fig. 14). Barnawell (1954) found a high proportion of individuals with large gonads throughout the year in San

Francisco Bay. In Santa Monica Bay, California, Boolootian

(1964b) and Monroe and Eoolootian (1965) indicated that major drops in the mean gonadal index in three different populations occurred during the winter and spring, although their data also showed that animals with mature gametes were found even when the mean gonadal index was low. It would appear that Mopalia

1useosa spawns throughout the year, and that the extent of reproductive activity is related to environmental conditions in the immediate vicinity where the animals are found.

Tonicella lineata (Fig. 15)

Observations on the reproductive cycle of T., lineata were made at three locations over four breeding seasons. At

Botanical Beach in late May 1971, 2 out of 10 animals were spent, indicating that spawning had begun., One month later all animals were spent., In 1972 there was a complete spawning between mid-March and mid-April. Two spawning periods occurred in 1973., The major drop in the mean gonadal index occurred during the period 6th April-4th May, and ail animals from the latter date contained small quantities of gametes., Animals from 47

24 Tonicella lineata 20 12 15 15| jFirst Narrows 16 14

12

8

Botanical Beach 0 •H—-H 1 I • I 1 1 1 1 1 1 1 1 1 1 (-

A M J J 1 A71 S O N D J FM A'M'J'J'A'S'O'NVJ'F'M'A'MVJ'A'S'O'N'DVF'M'A'MVJ'A'S 9 I 1972 | 1973 | 1974 Fig. 15. Mean gonadal index and 9531 confidence limits for Sgsicella lineata at Botanical Beach, First Narrows and Porteau. 48

II I I 1 1 1 1 1 1 ~1 J FMAMJ J ASO ND Tonicella lineata

Botanical Beach 1970 o"? 1971 1972 1973 1974 Porteau 1971 =4 present study 1972 1973 First Karrows 1971 . 1972 1973 1974 o"9 Eagle Harbour 1973

San Pedro Point, Calif. 1957 Thorpe, 1962

San Juan Island, Wash. 1955 1958 o*S> 1959 1968 1969 1970 Oregon: Boiler Bay 1968 Barnes, 1972 Yaquina Head 1967 Yaquina Head 1968 Yaquina Head 1969 Strawberry Hill 1968 Cape Argo 1968 Cape Argo \- 1969

Fig. 16. Summary of spanning observations on Tonicella lineata- symbols defined in Figure 4. ~ * 49

21st May and 4th June contained medium to large . quantities of gametes, suggesting that there was some additional gamete production. The second spawning occurred after 4th June and all animals in July and August had depleted gonads. ,, In 1974 the mean gonadal index dropped in late April-early May, and animals collected on 23rd May and later had spent gonads.

At Porteau, spawning in 1971 was probably in progress on

8th April when 1 animal in 5 collected had a small gonad, and was nearly finished on 8th May when 9 animals in 10 had depleted gonads. In 1972 only one collection was made and this was on

7th April. One animal had a depleted gonad and 10 others had large.ripe gonads, suggesting that spawning had begun.. An early spawning occurred in 1973. On 27th March 1973 spawning was imminent, since 1 male spawned in the laboratory from being handled, and on 10th April spawning was nearly finished.

At First Narrows spawning occurred at about the same time in both 1971 and 1972. On 20th April 1971 the 22 animals collected had mature gonads and two-thirds of these spawned after being dried and rewetted. On 17th May all animals had depleted gonads. In 1972 spawning could not be induced in the laboratory in animals from 20th April, but 2 animals had depleted gonads,, Following this the mean gonadal index dropped abruptly and all animals from 8th May were spent.,. In 1973

%* lineata spawned several weeks earlier. The events were followed closely. On 30th March the mean gonadal index was

14.5, and all animals had large gonads. By 14th April the index had dropped to 10,1 and one-half of the animals had partly 50

spawned gonads. The index on 17th April was 5.1 with all animals having small- to medium-sized gonads, and on 28th April several animals contained barely a trace of gametes. In 1974

!• lineata spawned over a much longer period than in the previous years., The animals had large ripe gonads on 10th April and earlier, but there was a decrease in the mean gonadal index after this date until 7th May, as the proportion of animals with depleted gonads increased._ Spawning activity continued into the summer. Nearly half of the animals collected in June and July contained relatively large quantities of gametes and animals from July spawned from being handled. By 18th September all but

1 animal in 13 had depleted gonads.

At Eagle Harbour one collection of T. lineata was made on

4th April 1973. One animal in 18 (of the adult animals collected weighing over 1.5 g) had spawned, suggesting that spawning had barely begun (mean gonadal index 14.0±2.8).

Figure 16 summarizes spawning observations on T_.ylij3ea.ta,

The major drop in gonadal indices occurred in April—early . May, except for the June spawning at Botanical Beach in 1971, and the late May—early June spawning which occurred at San Juan Island,

Washington, in 1968 and 1969 (Barnes, 1972). At Botanical Beach in 1973 and at First Narrows in 1974, there was some additional gonadal development and spawning after the major April spawning.

This did not occur when there was a complete spawning in the spring, which left all animals with small depleted gonads. The observations of spawning in late June and early August at San

Juan Island in 1955, 1968 and 1969 (Barnes, 1972) were probably 51

made during summer embryology courses at the Friday Harbor

Laboratories so that earlier events in these years were not followed. Spawning was not always synchronous in different locations. For example, Barnes (1972) found that spawning occurred a month earlier in Oregon than at San Juan Island in both 1968 and 1969, In the present study spawning at Botanical

Beach was always earlier or later than at First Narrows and

Porteau, and the population at First Narrows always tended to lag behind the population at Porteau.

In summary, Tonicella lineata has a distinctly annual reproductive cycle, The gonads are small in mid-summer and gonadal growth starts in the autumn and reaches a peak in mid-winter to spring. , The major spawning period is April but spawning may continue into May and June.

Tonicella insignis (Fig. 17)

T..insignis displayed a distinctly annual . reproductive cycle with spawning usually confined to a relatively short period in the spring. For example, the interval between the last collection before spawning and the first collection after spawning was only 14 days at Porteau in 1973, and at First

Narrows it was 18 days in 1972 and 15 days in 1973. It is likely that the actual period of spawning was even shorter than these collection intervals. All animals collected before spawning had gonadal indices of 8.0 or greater, and shortly after spawning all animals had indices of 3.0 or less, with most 20i 52

Tonicella insignis 106 16f 11 x N ,10 o •o i16 S 12 f „. First Narrows

"O c 14 13 o O 8 First Narrows 9 Porteau s 9 First Narrows «/ 13 6

0- H 1 1 1 1 h 1 1 1 1 1 1 1- H 1 1 1- -) 1 1 1 1 1 1——I 1- M J • J A SONDJ FMAMJ J A SO NDJ FMAMJ J A SON DJ FMAMJ j AS 1971 I 1972 I 1973 i 1974

Fig. 17. Hean gonadal index and 95% confidence limits for Tonicella insitjnis at First Narrows and Porteau.

J FMAMJ JASOND

Tonicella inslflnls

Porteau 1971 1972 1973 present study First Marrows 1972 1973 1974 Eagle Harbour 1973

_J J I L Fig. 18. Summary of spawning observations on Tonicella insignis: symbols defined in Figure <». 53

individuals containing no trace of gametes. ... The exact time of spawning varied in 1972 and 1973 but occurred at about the same time at both Porteau and First Narrows in any one year. In 1972 spawning occurred sometime after 7th May at Porteau, and between

20th April—8th May at First Narrows., In 1973 both populations spawned in the first week of April.

In 1974 collections were only made at First Narrows. , In marked contrast to the abrupt spawnings in 1972 and 1973, the spawning period in 1974 was poorly defined, A decrease in the mean gonadal index occurred during April but spawning was not completed in April. Forty percent of the animals collected on

7th April had large mature gonads., As in Mogalia ciliata,

Mogalia laevior and Tonicella lineata, this probably represents sampling error due to varying degrees of spawning - activity in slightly different locations at First Narrows. Two to four animals with ripe gonads were found in each of tne collections from late May through July. In September no anxmais contained mature gametes and unpigmented oocytes were seen in the females.

Two collections of T.., insignis were made at Eagle Harbour during the 1973 spawning period. The mean gonadal index (of adult animals, weighing 2 g or greater ) dropped from 10.3±1.5

(n=8) on 23rd March to 3.8±1.3 (n=16) on 4th April. On the later date few animals were yet in spent condition.

There are no spawning data on Tonicella insiflnis in the literature. Figure 18 summarizes the results of the present study. It is clear that this species has a distinct annual cycle with gonadal growth beginning in early summer and usually 54

an abrupt spring spawning.

Strongyj-oceutrotus droebachiensis (Pig. 19)

Most of the observations on S. droebachiensis- were made on a sublittoral population at First Narrows. In 1971 there was an abrupt and complete spawning during the period 23rd March-20th

April. , This was followed by a long period of gonadal growth until a peak was reached in March 1972. Another abrupt spawning followed, but a few weeks later than in 1971. On 20th April only 1 spent animal was collected, and on 8th May only 1 animal had not completed spawning. Gonadal growth ia the summer of

1972 was slight compared to that in the summer of the previous year, but more rapid growth followed in the autumn and winter.

In 1973 spawning again occurred in early Apr_l. ,.• The mean gonadal index dropped from 23.7 on 30th March to 2.4 on 14th

April. In 1974 spawning was less abrupt than in 1972 and 1973,

On 10th April, 2 partly spawned animals were found and none were found on 15th April. There was a marked decrease in the mean gonadal index by 20th April and all animals collected on 24th

April had completed spawning. Unexpectedly, some animals which had not completed spawning were found on 30th April, and this must be a reflection of "patchiness" in spawning activity.

In 1973 and 1974 observations were made on the time of spawning of S, droebachiensis in the low intertidal at Botanical

Beach. On 6th April 1973, 1 animal in 10 was spent, and on 4th

May every animal was spent. Thus spawning was at about the same 55

Strongylocentrotus droebachiensis 30 15 13 25 14

15] First Narrows ST 20 I*** to

75 15 •o 20 ro \ Botanical Beach c S. purpuratus, o O 10 , / Botanical Beach 10 S. purpuratus, " / Botanical Beach Botanical Beach 10

16' M A M J J ASONDJ FMAMJ J ASONDJ FMAMJ J ' A ' S'o ' N ' D ' J ' F'M ' A'M ' J ' J ' A ' S ' 1971 I 1972 i 1973 I 1974

Fig. 19. Mean gonadal index and 95% confidence Units for §iE2Sailocentrotus droebachiensis at First Narrows and Botanical Beach, and for Strongylocentrotus £urj>uratus at Botanical Beach. 56

1 1 J'F M A M 'J'J'A'S'O'N 'D Strongylocentrotus droebachiensis

First Narrows 1971 =i 1972 1973 M present study 1974 1=1 Botanical Beach 1973 1974 =i

Bergen, Norway — Rdnnstrom, 1927b

Tromso, Norway 1943 H Vasseur, 1951 1951 . _

Drobak, Trondheim & _ Vasseur, 1952 Tromso, Norway Salisbury Cove, Maine -—1 Harvey, 1956

1965 Tucker & Boolootian (in Boolootian, 1966)

Lamoine, Maine 1965 Cocanour & Allen, 1967 j- 1966

—, i Portugal Cove, Nfld. 1968 | 1 — -1 Himmelman, 1969 ==1969 =1 Bellevue, Nfld. 1965 H Acreman, 1966 Cape Cod Bay, Mass. & Boothbay, Maine Stephens, 1972

Margaret's Bay, N. S. Miller & Mann, 1973

1 1 1 1 1 1 1 1 1 1 1 Pig. 20. Sumoary of spanning observations on Strongylocentrotus droebachiensis: symbols defined in Figure 4. ~ ~ 57

time as at First Narrows in 1973. In 1974 spawning was nearly completed when the first collection was made on 27th April.

A number of collections of a closely related sea urchin,

Stronaylocentrotus purp. uratus, were also made at Botanical Beach from the same site where S. droebachiensis was collected. ,. In

1972 gonadal growth in the autumn coincided with that in

S..droebachiensis, but the spawning pattern in 1973 was strikingly different.. In S. fiurguratus there was a very gradual spawning over 2-3 months. It started in early Bar eh and was not completed until early May. A similar slow . spawning may also have occurred in 1974.,, It was in progress at the time of the first collection on 27th April, and all animals were not spent until June.

The present study, and reports by Cocanour and Allen (1967) and Himmelman (1969), provide information on reproductive activity of S, droebachiensis in different seasons. The gonads were always small in the late spring, and gonadal growth started in the summer and reached a peak in late winter. Many authors have reported the time of spawning of S. ...droebachiensis

(Fig, 20), In agreement with the present study, Stephens (1972) indicated that S, droebachiensis spawns abruptly and that its precise timing may vary in different years and locations., For example, at Boothbay Harbor, Maine, spawning was sometimes two

weeks earlier than at Cape Cod, Massachusetts. ;, In Nova Scotia,

Miller and Mann (1973) indicated that an earlier spawning period may have occurred in November. This was apparently based on the appearance of animals with large ripe gonads at this time. 58

However, this can hardly be considered as evidence that gametes were released. In Newfoundland, I found some animals with small' gonads in December—January but not in the previous November

(Himmelman, 1969). It was not certain whether spawning occurred or whether the small gonads were characteristic of a new collection site.

In summary, the majority of species in the present study have a distinctly annual reproductive cycle, , The main spawning period is spring, usually April, when there is an abrupt and synchronous spawning in Strongylocentrotus droebachiensis.

Tonicella lineata, Tonicella insignis, Mojaalia ciliata and

Mopalia laevior. Katharina tunicata sometimes spawns partly at this time, but the main spawning period is June.,, At First

Narrows spawning in Mopalia hindsii is earlier than in the above species.. In K. tunicata, M. laevior, and possibly Mopalia iliUdsii, the gonads are' small for several months following spawning and rapid gonadal growth occurs in the autumn and winter.. In contrast, gonadal growth in T. ., linea, ta, T. insignis,

M. ciliata and S. droebachiensis starts shortly after spawning.

The data on Mo£alia lignosa and Mopalia muscosa do not indicate that there is an annual reproductive cycle.. Mature animals were found in several seasons of the year, and in M. lignosa spawning was seen in late winter-spring as well as in the summer. 59

Observations and Experiments on Spawning

Temperature and Spawning in the Field.

In the literature there is much emphasis on temperature as a cue for spawning in marine invertebrates. Orton (1920) stated that most animals breed at a definite temperature, and implied that merely reaching a critical physiological temperature level stimulated spawning. ,. This hypothesis can now be examined for the species in the present study.

first Narrows., Figure 21. shows the bimonthly mean and standard deviation of daily temperatures of incoming water at the Vancouver Public Aquarium during the period March

1971—September 1974. The four year monthly mean values,

1971-1974, are also shown so that periods which were warmer or colder than normal can easily be recognized. Superimposed on these data are the gonadal index cycles for the species studied at First Narrows, Stronqylocentrotus dr o e ba. c hie n sis and

Tonicella lineata were the only species studied at First Narrows in 1971. The winter and spring of 1971 was the coldest period during the present study. , In that year 3. .,droebachiensis spawned in early April when temperatures averaged 6.3 °c, and

lineata spawned in late April-early May when the temperature was over 7 °C. If a critical temperature level induced spawning in these species the critical level must have been colder for

Strongylocentrotus droebachiensis than for I, lineata. However, in the three following years S. droebachiensis and I. lineata 60

FIRST NARROWS

24

20-

16

c . o O

8:

W9 M. laevior .^-M. laevior . >^ 1 M hindsi| M. hindsii^ H 1 . t MAMJ J ASO N DJ FMAMJ J A ONDJFMAMJJA S O N D J F MAMJ J A 1971 I 1972 I 1973 1974

Fig. 21. Mean sea water temperature [± standard deviation) for the first half and second half of each month during March 1971-September 1974 at First Narrows compared to the four year mean (1971-1974) for the first half and last half of each month; mean gonadal index values for St£oncjil,oc.entr.otu.s d.r.oe.ba.c.h.ie.n,sis, lonicella lineata, ToQisglla. icsisnis, Hop.a,li§. lagvisE* iSojoalia. Sili§.ta., Mo£alia hindsii, and flSfiliia Iignosa superimposed on these data, show the relationship of these cycles to temperature. (To make the lines more visible, a number of mean gonadal index values for April, Hay, and June, 1974, have been shifted slightly.) 61

20 T PORTEAU

16 +

T. lineata x -o 12 > / T. lineata- / ! M. lignosa // / c ..-•'"li // / - -"1 03 /V / \ ru // c 8 v / \ M. laevior-*// \ t o CD T. insignis-*/ \ i

/*-M. laevior A L _/ M. lignosa \/| •y*<-l. insignis K

0 -i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i AMJ JASONDJ. FMAMJ JASONDJFMA 1971 I 1972 I 1973

Pig. 22. A comparison of the mean gonadal index cycles for lonicella lineata, Tonicella insignis, fiopalia. laevior and Mpj^alia lig_nosa at Porteau during April 1971-April 1973.

I 62

BOTANICAL BEACH

0 -I H-rH 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 H 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1—1 MJ J ASON DJ FMAMJ J A SONDJ FMAMJ J A SONDJ FMAMJ J A SO N DJFMAMJJ 1970 I 1971 I 1972 I 1973 I 1974

Fig. 23. Mean sea water temperature (± standard deviation) for each month at amphitrite Point and Sherringham Point during the period May 1970-July 1974 compared to the five year monthly mean temperature (1970-1974) for each location; mean gonadal indices of Katharina tunicata, Jonicella. lineata, flo.Ea.lia. hifiliii, fl2E<=I±a ciliata. Strongylocentjotus d.roeba chiefs is and Strong, y.- l2£e.atrotus £ur£ura.tus superimposed on these data show the relationship of the cycles to temperature. spawned at about the same time. In the spring of 1972 there was an abrupt and synchronous spawning by S. droebachiensis,

T. lineata, Tonicella insignis and Mojaalia laevior in late

April. The water temperature at this time had reached 7,6 °C and was near the mean late April temperatures for 1971—1974,

The warmest spring was 1973 and in this year the above species and Mogalia ciliata again spawned abruptly and in synchrony, but in early April rather than late April. Temperatures at this time were between 7.4-8.0 °C and were thus similar to temperatures at the time of spawning in 1972., In 1974 winter and spring temperatures were again above the four year mean temperatures for winter and spring, but the spawning pattern was strikingly different than in the previous years. For most

species the period of spawning was poorly defined.J A major drop in mean gonadal indices of S. droebachiensis, X..lineata,

!• -ifisiaiiS* fi».iat©li2£» aQd a. .ciliata occurred in mid—April when temperatures were 7.6-8.1 °C, but T. lineata, T.,insignis,

S* laevior and fl. ciliata were not depleted of gametes until several months later. Mogalia hindsii was only studied in 1973 and 1974 and in these years it spawned earlier than the above species, during the period when temperatures were just starting to rise. The scanty data on Mogalia Iignosa suggest that there may have been winter as well as spring—summer spawning, which would imply that a critical temperature was not important for this species.

Thus, the hypothesis that a given temperature level acts as a cue for spawning would account for the delayed spawning which was observed in most species in 1972 compared to 1973, and for 64

the delayed spawning of f. lineata in 1.971.., Also the major drop in gonadal indices in 1974 occurred at about • the same temperature as in the preceeding years, namely 7.6 °C. On the basis of the earlier spawning time in H. hindsii, presumably this species would have a lower temperature threshold for spawning., The data were inconsistent for S. droebachiensis. since it spawned at a colder temperature in 1970 than in the following years. The temperature hypothesis would not account for the gradual spawning which occurred in most species in 1974, compared to the abrupt spawning in the previous years.

It may be that an increase in the rate of warming, rather than a particular temperature level, induced spawning. In 1971 when T. ,.lineata spawned temperatures were increasing at an accelerated rate compared to the prespawning period, but in

S..droebachiensis spawning occurred before the acceleration in the rate of warming began. There was an increase in the rate of warming when most species spawned in 1972, 1973 and 1974.

Porteau^ Temperature measurements were made at Stations 5 and 6 in the vicinity of Porteau (Fig. 1) during February—April by Dr John Stockner, Pacific Environmental Institute. There was an increase of 1.4 °C or less at depths of 2-10 m during the period 27th February-4th April, and a faster rate of warming occurred between 4-24th April. A thermograph placed at 8 m below MLIS in the collection site at Porteau showed a rise of

0.8 °C between 27th March-IOth April 1973, and it was during this period that there was an abrupt spawning by I. lineata,

S« iSSiaii3. aQd JH. laevior (Fig. 22) . Temperatures were 7.5—8.3 65

°C when this spawning took place. If a critical temperature stimulated this abrupt spawning, the response to this stimulus must be very sensitive indeed.

Botanical Beach,. Figure 23 shows the mean monthly temperatures calculated from daily surface temperatures for the period May 1970-July 1974, and the five year mean for each month of the year (1970-1974) at both Sherringham Point and Amphitrite

Point lighthouses., The mean gonadal index values for animals at

Botanical Beach are superimposed on these data. Except for l°£alia muscosa, the animals at Botanical Beach were collected from 0-1.5 m above MLWS, and Green (1967) calculated that animals from this region of the shore at Botanical Beach are submerged more than 801 of the time., Hedgpeth and Gonor (1969) stressed that correlations between surface temperature measurements and biological activities in intertidal animals are not • very reliable, since the actual temperatures to which the animals are exposed varies greatly depending on the exact location of the animals on the shore. Nevertheless, there are some striking differences in reproductive activity of animals at

Botanical Beach in different years which did not appear to be related to temperature., In 1970, K. tunicata spawned in early

June when the water temperature was 9 °C or warmer.,,. In 1971,

K. tunicata again spawned in June and when a similar temperature was reached. In 1971 Tonicella - lineata spawned in synchrony

with K. tunicata. It should also be noted that K.:tunicata spawned at about the same time at Point No Point, and that spawning by K. tunicata was completed at about the same time

(late June) at Frank Island and Amphitrite Point as at Botanical 66

Beach and Point Ho Point. It is known that temperatures tend to decrease going from Amphitrite Point to Sherringham Point due to mixing with deeper water in the Juan de Fuca Strait.. In 1972, winter and spring temperatures were well below the mean for

1970-1974, but spawning at Botanical Beach occurred earlier than in the previous years and probably before temperatures reached 8

°C. In late March-April 1972, there was a complete spawning occurred in T,,lineata and a partial spawning in K..tunicata.

Spring temperatures in 1973 were above normal but again a major spawning occurred in April when the temperature was approximately 9 °C., At this time Strongylocentrotus droebach• iensis and Mogalia hindsii spawned completely and there was a partial spawning in T. lineata and K. tunicata. , In 1974, I only observed the latter part of the spawning period but spawning occurred when temperatures were about 8 °C., In contrast to the above species, spawning in Strongylocentrotus purguratus in 1973 was a slow process, starting when temperatures were near the annual minimum of 7.0-7.5 °C and continuing until they reached

9.0 °C. Thus at Botanical Beach spawning did not appear to be correlated with a particular temperature. In the coldest year,

1972, spawning by K. tunicata and T. lineata occurred earlier or as early as in the other years and at a lower temperature, less than 8 °C. In 1970 and 1971 spawning was late, when temperatures were above 9 °C, and in 1973 and 1974 there was spawning in April when temperatures were approaching 9 °C.

In summary, at First Narrows, T. .lineata, I..insignis,

M. laevior and M. ciliata spawn when the water is 7-8 °C.

S. droebachiensis usually spawns at this temperature but 67

sometimes spawns at a lower temperature. M. hindsii spawns near the annual minimum or when temperatures are beginning to increase. In contrast, at Botanical Beach there is no consistent relationship between spawning and temperature in

S« droebachiensis, T. lineata, K. tunicata and M. hindsii, except that the temperature is always above 8 °C. S. purpuratus has a prolonged spawning period and therefore does not appear to be induced by a critical temperature.

The Effect of Temperature and Light on Spawning in the Laboratory,

Strongylocentrotus droebachiensis. , The animals maintained under the various light and temperature regimes showed no indication of spawning at the time it occurred in the field

(Fig. 24). It is notable that on the two occasions when the cooling system failed (on 6th and 10th April) the temperature increased about 5 °C in 2 h in all four experimental tanks, but no spawning resulted. The gonadal indices of animals in the light-warm, light-cold and dark-cold tanks remained high from the start of the experiments on 30th March until the experiments were terminated on 29th April, but there was a fall in the index for animals in the dark-warm tank,, The latter was due to spawning which started on 23rd April, when an air bubbler was inadvertently turned on "high" for a few hours causing a great deal of agitation. In an additional experiment, 9 animals from the light-cold tank were transferred to a wire cage at below low tide level at First Narrows on April 14., When these animals 68

30 DC LW Stronqylocentrotus droebachiensis

20

C

~o co c o 10

group moved to field from April 19-28 10 field (First Narrows) 0 —i i March April May

Fig. 24. Bean gonadal index and 95% confidence liaits for Strongylocentrotus droebachiensis at First Narrows during March—May 1973, for groups of animals transferred to four light and temperature regimes in the laboratory, and for one group which was transferred to First Narrows on 19th April: Dl (dark-warm), DC (dark-cold, LH (light-warm) and LC (light-cold) are laboratory conditions described in the text.

I 69

30 Strongvlocentrotus droebachiensis 13

1974

14 1 18 15 9 15 20, S.5°C laboratory c? 20 — ~ " 5.5°C CO T> 13 CO c o CD 10 field (First Narrows)

8

13 r <

161 ^ 0 March M

Fig. 25. Mean gonadal index and 95% confidence limits for Strongylocentrotus droebachiensis at First Narrows from March-September 1974, and for groups transferred to various temperature regimes in the laboratory. 70

were retrieved on 28th April all had small depleted gonads, and their mean gonadal index was significantly smaller (P<0.01)* than for animals in the light-cold tank on 29th April (Fig. 24).

In 1974, S. droebachiensis collected on 12th March and 4th April and kept at 5.5 °C in the laboratory until 12th June, and animals collected on 10th April and kept at 10.5 C until 21st

May, showed little change in gonadal indices (Fig. ,25).,

Tonicella lineata. The experiments on T. ,,lineata yielded results similar to those for S. droebachiensis. In 1973 there was no spawning in experimental tanks at the time of spawning in the field, but some spawning did occur in the dark-warm tank, probably starting on 23rd April when S. droebachiensis in the same tank started to spawn {Fig. 4). Two groups of animals were collected at First Narrows on 14th April, midway through the natural spawning period, and were maintained in two 10 1 containers with an incoming supply of water at 5.5 °C and 14.0

°C respectively; there was no evidence that spawning continued.

On 30th April the mean gonadal index of these groups was similar to the value for 14th April, when they were collected, and larger than the field animals on 28th April, although significantly larger (P<0.05) only for the group at 5.5 °C.

Similarly, animals collected prior to spawning in 1974 and maintained in the laboratory at 5.5 °C or 10,5 °C did not spawn

(Fig. 27), On 15th April, 1974, 15 animals, which were

•Throughout this thesis when two groups were compared statistically, arcsin transformations were made of gonadal index values, followed by a t-test. 71

DW DC Tonicella lineata 20 10T10 1973

13 x 16 -c8

CO 12 laboratory "8c - O O

8

14

field (First Narrows) 0 March April May

Fig. 26. Mean gonadal index and 9555 confidence limits for Tonicella lineata at First Narrows from March—May 1973, and for groups transferred to various light and temperature regimes in the laboratory. 72

20 Tonicella lineata 1974

x 16

group moved to / field (First Narrows) field from April ^ 15-30 0 March A M

Fig. 27. Mean gonadal index and 95% confidence limits for Isaiseiil iiaeata at First Narrows during March-September 1974, and for groups placed in various temperature regimes in the laboratory, and for one group transferred to First Narrows on 15th April. 73

originally collected on March 12 and had been kept at 5.5 °C in the laboratory, were returned to First Narrows where they were kept in a plastic container perforated with holes,, At the same time a control group was transferred to 10.5 °C in the laboratory, 1 °C warmer than the temperature in the field. On

30th April the mean gonadal index of the group in the field was

9.5/ similar to the value for the natural population (Fig. 27).

The control group had a mean gonadal index of 15.3 on 21st May, close to the value for 12th March when these animals were collected, although not significantly larger (P<0.05) than the group transferred to the field.

In summary, in 1973 and 1974 S. droebachiensis- and

lifieata were collected prior to spawning and maintained in the laboratory under various light and temperature conditions.

Spawning did not occur in these animals when spawning occurred in the field animals,, When groups of unspawned animals were returned to the field their gonads decreased in size and became similar to the field animals., Some condition in the.field, which was not present in the laboratory, apparently stimulated spawning, and this factor did not appear to be light or temperature. 74

Phytoplankton Observations in the Field,

Measurements of the abundance and species composition of phytoplankton were made at six stations in Howe Sound and one in

Indian Arm during 1973 by Dr John Stockner, Pacific

Environmental Institute. In 1973 the spring phytoplankton bloom developed rapidly in early April. Chlorophyll a- measurements were less than 1 mg/m3 until the end of March, but high values were recorded in the first week of April (Fig. 28). At stations

1 and 2, about 20 km from First Narrows (Fig. 1), values of

8.9—9.3 ffig chlorophyll a/m3 were recorded on 6xh April, and at

Station 6, 2 km from Porteau, there was 5.0 mg/m3 on 4th April.,

This phytoplankton outburst coincided with spawning in most of the species studied., At First Narrows there was a complete spawning of I. lineata, T. ifisignis, S. .droebachiensis,

M. laevior and M..ciliata between 30th March and 14-17th April, and at Porteau T. lineata, T. insignis and M. laevior spawned completely between 27th March and 10th April.,

The synchrony between the onset of the phytoplankton bloom and the release of gametes in S. ,droebachiensis and the two

Tonicella species suggests that some factor associated with the presence of phytoplankton was the cue for spawning., Data on the abundance of the more common species of phytoplankton found during the bloom at Stations 1, 2, and 6, compared to their abundance before the bloom, are shown in Table II., Thalassi- osira sp. (T, pacifica Gran & Angst and/or T. nordeaskioeldii

Cleve) was absent or in small guantities before the bloom and dominant during the bloom, with densities of 36 9-586 million 75

February M AM J

Fig. 28.* Phytoplankton abundance as measured by chlorophyll a for six stations in Hove Sound and one in Indian Arm during February—June 1973 (Dr John Stockner, pers. comm.): each value is the mean of five measurements made at 0—5 m; mean gonadal index values for Strongylocentrotus droebachiensis. Tonicella lineata, and Tonicella insignis, superimposed on these data, show the synchrony of spawning with the phytoplankton bloom in 1973. 76

February MA M J

Fig. 29. Phytoplankton abundance measured by chlorophyll a for Stations in the vicinity of First Narrows during February—June 1974: values for Stations 1, 2 and 11 represent the mean of three measurements from 1—5 m (Dr John Stockner, pers. comm.) and values for First Narrows and Jericho Beach are single measurements taken near the surface: mean gonadal indices for Strongylocentrotus droebachiensis. Tonicella lineata. and Tonicella insignis. superimposed on these data, show the prolonged spawning period corresponding to the delayed and slow growth of phytoplankton in 1974. 77

Table II. Density (millions of cells/m3) of common species of phytoplankton at Stations 1, 2 and 6 before and during the blooa in 1973. (Dr John Stockner, pers. comm.)

Station 1973 27th Feb.-6lh Mar. 2nd-6th April

Bacillariophyccae Thalassiosira sp. 1 0.0 398 2 11.8 369 6 0.0 586 Skeletonema costatum 1 16.5 13.4 2 11.0 7.06 6 0.0 7.85

Nilzschia seriata 1 2.35 11.8 2 0.78 0.0 6 0.0 3.14

Nacicula sp. 1 0.0 0.0 2 1.57 6.29 6 6.28 4.71 Dinophyceae Gymnodimium sp. 1 0.0 7.85 2 0.0 3.92 6 0.0 1.57

Phytoflagellates 1 0.0 15.7 2 91.2 12.6 3 0.0 33.0

Table III. Density (millions of cells/m3) of Thalassiosira sp. and Skeletonema costatum at Stations 1 and 2 from 6th Harch to 3rd May 1974 (Dr John Stockner, pers. comm.).

Station 1974 6th March 2nd April 3rd May

Bacillariophyceae Thalassiosira sp. 1 0.0 2.94 530 2 0.0 8.82 1070

Skeletonema costatum 1 32.3 84.3 6210 2 13.7 42.1 12100 78

cells/la3. There was little change in the guantity of Ske let ci• nema costatum (Grev.) Cleve, Mitzschia seriata• Cleve,

Saiisaia SP« and phytoflagellates before and during the bloom.

JSilSSdinium sp.: was not found before the bloom and was in small guantities during the bloom.

In 1974 the spring phytoplankton bloom did not develop as early or as rapidly as in 1973. .. Figure 29 shows chlorophyll a measurements for five Stations in the vicinity of First Narrows during the period when spawning occurred. There was less than 1 mg chlorophyll a/m3 at all Stations until the end of April. At

Jericho Beach, net plankton could not be collected until April

29 when a value of 6.2 mg chlorophyll a/m3 was found; and at

Stations 1 and 2, values of 4.0 and 5.8 mg/m3 were found on May

3. Samples collected at the surface at First.Narrows only approached 1.0 mg/m3 from late April through May. The low values in this area may have been due to the mixing effect of the strong tidal rapids, and higher values may have occurred during other parts of the tidal cycle. It is clear that there was no sharp increase in phytoplankton in 1974 compared to that in 1973, and these events correspond well to the delayed and poorly defined spawning in S. .droebachiensis, I, lineata,

iJBsianis, M. laevior, and M. ciliata-in 1974. At Stations 1 and 2 the density of Thalassiosira sp. was low on 2nd April but high on 3rd May, when its density was second to that of Skeleto- nema costatum (Table III) .

Information on the spring phytoplankton bloom in 1973 was also collected by Mr Jae Shim, Institute of Oceanography 79

Table IV. Density (millions of cells/m3) of diatoms at Stations A and B in the Strait of Georgia, and for Station C in Juan de Fuca Strait, during winter and spring 1973 (Mr Jae Shia, pers. comm.) ...

17th 13-14th 13-14th 17th 15-16th 17th 19th Station Depth January February March April May June July

lm 3.0 32.9 2.7 1570 25.8 3580 2.0

25m 0.6 32.0 3.0 710 10.3 1.6 0.8

lm 6.3 10.6 11.2 2730 125 23.8 57.7

25m 11.4 16.6 0.4 146 3.6 0.4 14.7

lm 1.6 6.7 8.8 109 37.9 134 78.3

25m 18.2 7.9 18.6 72.0 29.3 12.5 185 80

Table V. Density (millions of cells/m3) of diatom species at Stations A, B, and C before and during the bloom in 1973 (Mr Jae Shim, pers. comm.)

1973

Station A Station B Station C

14th 17th. 13th 17th 14th 17th 16th 17th Depth March April March April March April May June

Bacillar io phyc eae

Thalassiosira pacifica lm 0.0 153 0.0 524 0.0 61.0 0.0 0.0 25m 0.0 58.9 0.0 54.6 0.0 33.7 1.9 0.0

Thalassiosira nordenskioeldii lm 0.0 112 0.0 173 0.0 4.7 12.5 0.0 25m 0.0 108 0.0 5.7 0.0 2.0 10.6 1.9

Thalassiosira aestivalis lm 0.5 0.0 3.0 0.0 0.0 0.0 5.3 1.3 25m 0.0 . 0.0 0.1 0.0 0.0 . 0.0 3.3 0.7

Thalassiosira eccentricus lm 0.0 2.1 0.0 0.8 0.0 1.3 0.6 0.0 25m 0.0 0.0 0.0 0.0 0.0 10.6 0.0 0.0

Skeletonema costatum lm 0.0 481 0.5 1160 0.0 4.1 1.9 116 25m 0.0 228 0.0 92.2 0.0 8.4 0.0 0.0

Nitzschia delicatula lm 0.1 221 1.4 96.3 0.4 . 7.9 1.9 0.0 25m 0.0 38.0 0.0 9.6 1.0 0.7 7.7 0.0

Nitzschia longissima lm 0.1 12.5 0.0 13.0 0.6 1.3 1.3 0.0 25m 0.0 0.0 0.0 0.0 1.0 1.3 0.2 0.7

Nitzschia closterium lm 0.0 4.2 0.4 2.3 0.2 0.7 0.0 0.0 25m 0.6 0.0 0.0 0.0 0.2 0.0 0.0 0.0

Corethron criophilum lm 0.0 200 0.0 82.2 0.0 0.7 0.0 0.0 25m 0.0 41.8 0.0 5.7 0.0 0.3 0.0 0.0

Chaetocerus debilis lm 0.2 247 0.0 357 0.0 2.7 0.0 0.0 25m 0.0 7.7 0.0 0.4 0.0 1.2 0.4 0.0

Thalassionema nitzschioides lm 0.1 0.7 0.0 5.4 0.1 0.2 1.9 3.3 25m 0.2 1.4 0.1 0.4 0.8 2.7 0.8 3.9

Paralia sulcata lm 0.0 0.0 0.0 0.0 5.4 9.2 12.5 9.2 25m 0.0 0.0 0.0 0.0 11.2 6.8 3.2 2.6

Schroederella delicatula lm 0.4 1.4 0.0 0.0 0.0 0.2 0.0 0.0 25m 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 81

0. B. C. (Table IV). At stations A and B in the Strait of

Georgia (Fig. 1) there was a marked increase in the abundance of diatoms from mid-March to mid—April, from 2.7—11.2 to 1570-2730 million cells/m3. This coincided with the sharp phytoplankton increase and spawning of S. droebachiensis, T. lineata,

H« insignis, M. laevior and M. ciliata observed in early April

1973 in the vicinity of Vancouver and Howe Sound., At Station C in Juan da Fuca Strait, 36 km from Botanical Beach {Fig. 1), this bloom was less intense with only a density of 108 million cells/m3 found on 17th April. At Botanical Beach M. hindsii and

S. droebachiensis spawned completely in April 1973, but

K. tunicata and T, lineata only spawned partly.. The latter two species completed spawning in June, and this .coincided with a second bloom in Juan de Fuca Strait (Table IV).,

The species composition of diatoms at Stations A, B, and C before and during the bloom in 1973 is shown in Table V, The sharp increase in Thalassiosira £acifica and T. nordenskeoldii at Stations A and B in the Strait of Georgia was comparable to that observed at Stations 1, 2, and 6 near Howe Sound and

Burrard Inlet (Table II). However, the most striking increase at Stations A and B was in Skeletonema costatum, with densities of 481-1160 million cells/m3 recorded at 1 m depth on 17th

April. Notable increases were also seen in Nitzschia longissima

(Berb.) Holfs, Corethron criophilum Castracane, and Chaetocerus debilus Cleve. At Station C, the April bloom was not heavy, and

!• M£ifi£a was tiie most common species with 61 million cells/m3 found at 1 m depth on 17th April. Other species which showed a marked increase from March to April at Station C were 82

1" nordenskeoldii, and I. eccentricus (Ehr.) Cleve, and Skeleto• nema costatum., During the June bloom at Station C, Skeletonema costatum was the predominate species, and other species which were important in the other blooms were uncommon. In these phytoplankton observations (Table IV"), Skeletonema costatum showed increases which most closely corresponded to the spawning times of S. ...droebachiensis, T. lineata, T. insiynis, M. laevior,

I' ciliata, and K. ..tunicata at First Narrows, Porteau, and

Botanical Beach.

in both 1973 and 1974 there was a close correlation between the spring phytoplankton outburst and spawning in the field.

Temperatures only increased slightly during the spawning period and there was no apparent trend in salinity., These observations suggest that phytoplankton itself, or some factor associated with the phytoplankton bloom, stimulated spawning.

Effect of Phytoplankton on Spawning in the Laboratory

In 1973, following the observed synchrony of the spring phytoplankton bloom and spawning in the field, laboratory experiments were conducted to test the hypothesis that the presence of phytoplankton would induce spawning. Groups of unspawned animals, from the tanks which were set up on 30th

March to test the effect of light and temperature, were treated with natural phytoplankton from Jericho Beach (see Methods).

These experiments were run from 21-27th April. In all three treated groups spawning started on the first or second day of 83

the experiment and heavy spawning followed. Since there were not enough animals to establish proper controls, the mean gonadal index of each treated group on 27th April was compared to its "source" tank on 12th April and 29th April (Fig.,30). In every case the animals exposed to phytoplankton had significantly smaller gonads (P<0.01).

The results of the 1974 experiments on the effect of phytoplanktom on spawning are shown in Table VI, In experiments performed at 5.5 °C, 62% of the treated Strongylocentrotus droe• bachiensis from 12th March spawned in 10 days of plankton treatment (Expt. 1) ,compared to 44S of the animals from 4th

April (Expt. 2) . At 7.0 °C, 7 out of 10 animals treated with sperm spawned (Expt. 3, Table VI) . ,

At 5.5 °C, 64% of the Tonicella lineata from 12th March spawned in 10 days of plankton treatment (Expt.,4), compared to

1% of the animals from 4th April (Expt. 5). In Experiment 6 performed at 10 °C, all 9 animals from 12th March spawned, and in Experiment 7, which was started a few days later, 67$ of the animals from 12th March spawned. In the latter experiment 2 of the treated animals died and were not counted in the results.

The 13 animals which did not spawn at 5.5 °C in Experiment 5 were transferred to 10.0 °C on 15th May, No spawning resulted during the next three days of warmer conditions. , From 18-28th

May frozen plankton was added to these animals and 9 out of the

13 animals spawned (Expt. 8). The 7 control animals in this experiment were also from Experiment 5 and none spawned, but 2 died., When sperm suspension was added to 12 T..lineata from 84

30 10

14 3. droebachiensis, DC

| 20 10 10 I lineata, DC 10 "O CO c 15 o I lineata, LC f \\ \J CD 10 7

star t of plankton treatments 0 H h- 12 21 27 29 April 1973 Fig. 30. Mean gonadal index and 9536 confidence limits for groups of Stronqylocentrqtus droebachiensis and Tonicella lineata treated with phytoplankton from 21-27th April 1973, compared with events in tanks from which these groups were taken.

Table VI. Effect of natural phytoplankton and sperm suspension on StrongyJ.ocentrotus droebachiensis. Tonicella lineata. and Tonicella insignis. 10-7/1

Expt no. and date Date Exp. T Total Total % No. of collected (°C) spawned unspawned spawned controls*

Strongylocentrotus droebachiensis Plankton treated 1. 3rd-13th May 12th March 5.5 8 5 62 6 2. lst-llth May 4th April 5.5 4 5 44 5 Sperm treated 3. 30th April-lOth May 12th March 7.0 7 4 64 5

Tonicella lineata Plankton treated 4. 4th-14th May 12th March 5.5 7 4 64 ' 9 5. 6th-16th May 4th April 5.5 1 13 7 7 6. 15th-25th May** 12th March 10.0 9 0 100 8 7. 19th-29th May** 12th March 10.0 4 2 67 8 8. 18th-28th May** 4th April 10.0 9 4 69 7 Sperm treated 9. 4th-14th May 12th March 5.5 8 4 67 9

Tonicella insignis Plankton treated 10. 4th-14thMay 12th March 5.5 12 4 75 7 Sperm treated 11. 6th-16thMay 12th March 5.5 6 3 67 4

* No control animals spawned in any of the experiments. ** Frozen plankton was used after 19th May (see text). 85

12th March, 8 animals spawned (Expt. 9, Table VI).,

A larger proportion of Tonicella insignis spawned at low temperatures than did T. lineata. At 5.5 °C, 12 out of 16 animals spawned within six days of plankton treatment, and 3 of the unspawned animals were later found to have undeveloped gonads (Expt. 10). Six out of 9 animals treated with sperm spawned (Expt. 11, Table VI).

These laboratory experiments suggest that the presence of phytoplankton stimulates spawning in S. droebachiensis,

T. lineata, and T. insignis, and that sperm suspensions are also effective in inducing them to spawn. 86

DISCUSSION

The Regulation of Reproductive Cycles

Reproductive cycles are not 'independent events but are coordinated with other biological activities, for example, in the urchin, Strongylocentrotus intermedins, the annual cycle in gonadal development is coordinated with annual cycles in'the rate of food consumption and of body growth (Fuji, 1967).

Cycles in the levels of various biochemical constituents of various body organs are known for many invertebrates (eg,

Giese, 1966, 1969) and presumably are brought about by metabolic shifts, changing the proportion of incoming energy (or stored energy) channeled into somatic or gonadal growth. It is likely that neuroendocrine mechanisms, as are known for many vertebrates, are important in coordinating reproduction with other events in invertebrates, but they are not well understood.

At certain points in these internal physiological cycles the external environment may affect these internal processes, thereby acting as a cue. Such external cues may be of particular importance during reproductive phases, - since the young must be produced when environmental conditions are favourable, and to ensure the coordination of gamete release in animals which spawn. The fact that most organisms show some degree of seasonality in their reproductive cycles indicates that they are affected by external factors. In trying to elucidate environmental cues several approaches are useful. 87

Examination of reproductive cycles in different locations and in different years, if coupled with good data on the physical and biotic environment, may suggest the importance of some factors and make others less likely. If the cycles vary in different locations, local factors are probably important, whereas if events occur in synchrony over distinct geographical areas, the cycles may be controlled by some condition which is uniform over widely separated areas or may have started at some distant reference point and be maintained internally without additional external stimulation., In the latter instance, the rhythm should continue if animals are brought into the laboratory from the field. It must be emphasized that environmental factors which are closely correlated with certain reproductive events can only be considered as possible cues. ,, Some environmental conditions may be a prerequisite for certain events, but may not directly initiate the process., Experiments, preferably under a wide variety of conditions, should be performed to test any hypothetical cue.

Possible Factors fiegulating Gonadal Growth

The interpretation of the results of the present study is limited by a lack of information on internal events in the animals studied. Some gametogenic phases, such as the initiation of gametogenesis or vitellogenesis, may be stimulated by environmental cues, but the timing of these events was not examined. . 88

Si§£o^gical Events..., Some information on gametogenesis of the species considered in the present study is available from earlier papers. Nimitz and Giese (1964) report: that in

Katharina tunicata the active period of gametogenesis starts in the winter when the gonads are growing rapidly, and there is evidence of nutrient transfer from the gonadal epithelium (and possibly other body organs) to the developing gametes,, They state that oogenesis in K. tunicata takes two years, with the oocytes reaching 35-50 ju in diameter in the first season, and

175 p. in the second season when they are mature ova. , Anderson

(1969) describes the subcellular differentiation of oogonia into mature ova in Mopalia muscosa, using animals collected in the winter. He does not indicate the proportion of various stages found at this time of the year,, Barnes (1972) reports a distinctly annual gametogenic cycle in Tonicella lineata. In males, after spawning the testicular lumens are empty of sperm and the germinal lamellar epithelium is thin, with areas of sperm production being rare. In the summer and autumn the germinal lamellae thicken and there is a gradual increase in sperm production along their surface,, By February there is sperm production along the entire lamellar surface and the lumens are filled with spermatozoa. The lamellae become thinner before spawning occurs, indicating that new spermatids are no longer being produced. In females, young oocytes appear just before or soon after spawning, and early stages of oogenis predominate throughout the summer. The oocytes enlarge during the autumn. By January the ovarian lumens are beginning to fill with mature ova and early oogenic stages are uncommon, .,• Prior to 89

spawning the lumens are bulging with mature ova and other stages are uncommon.

In the present study it was observed that the gonads in

Katharina tunicata, Mopalia laevior and Mopalia hindsri usually remained small during the summer and grew rapidly in the autumn and winter, whereas in Strongylocentrotus droebachiensis,

Tonicella lineata, Tonicella insignis and Mopalia ciliata the gonads started growing shortly after the spring spawning., The possible relationship between these observed patterns in gonadal development and temperature, photoperiod and nutrition will now be examined.

Temperature

The influence of temperature on gonadal development has been stressed more than any other factor. In some species, for example mussels and oysters (Clipperfield, 1953; Korringa,

1957), the gonads are inactive during the winter but become active when the temperature reaches a certain level in the spring. In a variety of invertebrates including lamellibranchs, opisthobranchs, echinoids and shrimp, which normally reproduce in the spring or summer, it has been shown that animals can be brought into ripe condition in the winter by exposing them to increased temperatures in the laboratory (Townsend, 1940; loosanoff £ Davis, 1963; Smith & Carefoot, 1967; Little, 1968).,

In the bay scallop, Aeguipecten irradians, the initiation of gametogenesis reguires both an increase in temperature to 15—20 90

°C and a supply of food (Sastry, 1966, 1968), Once a certain amount of nutrients have accumulated the completion of oogenesis requires a further increase in temperature to over 20 °C, but a food supply is no longer necessary., Temperature appears to act directly on the gonads of the scallop, Pecten yessoensrs, since in experiments by Yamamoto (1951) a rise in temperature caused maturation of primary oocytes and passage of the eggs into the oviducts in ovaries which had been removed from the animals.

Leonard (1969) found that gonadal development started earlier in the oyster, Ostrea edulis, in animals which were transferred to

Southern California than in the parental population in Maine.

The clam. Mya arenaria, on the Atlantic coast of North America, usually has one breeding period during the warmer months of the year, but in the southern part of its range there may be two breeding periods as a result of an interruption of gametogenesis during the period of highest temperatures in mid-summer (Hopes &

Stickney, 1965; Pfitzenmeyer, 1965). , Also, Cochran and Engelman

(1972) found that Strongylocentrotus £U££urajus in southern

California lost its ability to produce gametes when temperatures rose to 17 °C. Other species reguire low temperatures for gonadal development. For example, Crisp and Patel (1969) demonstrated that three boreo—arctic barnacle species reguire conditioning for several weeks below a certain temperature before the gonads will mature. Thus, for numerous species it is clear that temperature is the major stimulus for gonadal development.

In the present study, gonadal growth of Katharina- tunicata and Mogalia laevior began in the autumn when temperatures were 91

declining (Figs. .1.3 & 16). Webber and Giese (1969), from observations on the reproductive cycle of K. tunicata over a ten year period in the Monterey area, reported that there was no consistent relationship between gonadal growth and temperature.

In some years gonadal growth occurred while temperatures were steadily declining, while in other years temperatures fluctuated, , In the present study, Tonicella lineata, ••Tonicella insignis, Mogalia ciliata and Strongylocentrotus droebachiensis started gonadal growth in June (except when spawning was delayed) and during the first 3—4 months of gonadal growth temperatures were increasing. However, Barnes (1972) observed the same pattern of gonadal growth in a population of T, lineata in Oregon where summer temperatures fluctuated due to upwelling.

In all of the above species the gonads continued enlarging as temperatures dropped to the winter minimum and as warming began in late winter and spring.

It is possible that different gametogenic activities are stimulated by different temperature conditions. , In Barnes1

(1972) description of oogenesis in T. ..lineata early stages were common in the summer and autumn but not in January. January through March appeared to be a period when oocytes grew and matured, rather than a period when new oocytes were formed.

This change in gametogenic activity could be related to the change from decreasing to increasing temperature. , Thus, in

T. lineata and other species which spawn in the spring, a slight increase in temperature may be required for gonadal maturation.

This would not be the case for Mogalia hindsii- and another chiton. Crygtoehiton stelleri, since in these species peak 92

gonadal indices and spawning occur in the coldest period of the year (Fig, .5;Tucker & Giese, 1962; Lawrence, Lawrence & Giese,

1965)

Photoperiod

The hypothesis that photoperiod controls gonadal development in some species seems plausible. The annual cycle in daylength, the time between sunrise and sunset, becomes increasingly pronounced going from the aguator to the poles.

This trend is even more accentuated when the morning and evening twilight periods are included in the calculation of effective daylength (Sadleir, 1969). For example, at 50° latitude there is a very rapid change in effective daylength in June-July and again in December-January, while during the rest of the year there is nearly a steady increase or decrease in daylength.

Photoperiod control of reproduction is well known in many vertebrates, and the mechanism involves photoreceptors and nervous and hormonal pathways (Bullough, 1961). The hypothesis that photoperiod controls reproduction is appealing in that it would account for synchrony amongst widely separated populations.

In marine invertebrates only a few researchers have examined the possible control of reproduction i»y photoperiod, and a few others have alluded to this possibility, While Barnes

(1963) and Crisp and Patel (1969) indicated that decreased temperatures are the most important influence on gonadal 93

maturation in boreal cirripedes, they also demonstrated that long—day photoperiod strongly inhibited this process. Gonor

(1973) noted that gonadal growth in several populations of

Strongylocentrotus purpuratus in Oregon did not start until late

June, and gonadal enlargement occurred between the summer and winter solstices (although the maturation phase continued into the winter). Boolootian (1963) periodically took biopsies of the gonads of the same male Strongyloc^entrotus purpuratus and found that under a 14 h light : 10 h dark photoperiod mainly spermatogonia were produced, but when the photoperiod was changed to 6 h light : 18 h dark, spermatogonia decreased in numbers and spermatids and spermatozoa were produced. , In recent laboratory studies on S, purpuratus, Cochran and Engelman (1975) reported that the onset of reproductive activity was not affected by photoperiod. These experiments were conducted in the autumn and early winter when one would expect rapid gonadal growth to be occurring coincidental with decreasing photoperiod.

However, their experiments were poorly designed in that their criteria for the onset of reproductive activity was the appearance of mature gametes oozing from the gonads. They would not have detected a proliferation and development of earlier stages by this method, ,

In the present study, during the period of decreasing photoperiod, there was gonadal growth in Strongylocentrotus•• droebachiensis, Tonicella lineata, Tonicella insignis- and

Mopalia ciliata, but gonadal growth continued into the winter and spring. As described earlier, in T. lineata in Oregon only the latter stages of oogenesis were common after December 94

(Barnes, 1972), It is possible that the change from decreasing to increasing daylength may have stimulated gonadal maturation in the species which spawn in the spring.

For a few marine invertebrates there is some evidence that photoperiod affects gonadal development via a neuroendocrine mechanism,For example, in Octopus, Hells and Malls (1959), and

Hells (1960) reported that in October a secretion from the optic gland stimulated gonadal development, and the secretory activity of this gland was in turn controlled by inhibitory nerves from the brain. Also, in some Crustacea, production of gonadotrophic hormones by the androgenic gland in males and by the ovary in females is inhibited by a hormone produced in the X—organ sinus gland system located in the eyestalk (Adiyodi & Adiyody, 1970).

This organ in turn may be controlled by the central nervous system. These internal pathways are conceivably influenced by photoperiod. Mi Hot t (1966) suggested that in echiaoids light passing through the test might stimulate the gonads directly, or there may be an intermediate neuroendocrine pathway., Similarly, in chitons the gonads lie directly under the dorsal plates and in most species light passing through the plates could affect the gonads in some direct way. Alternatively light could affect gonadal development by a neurosecretory pathway involving the light sensing organs, the aesthetes, which are located in the plates. 95

Nutrition

Large quantities of nutrients are required for gonadal growth and it is plausible that the timing of gonadal growth is influenced by nutrient availability. Several authors indicated that external food conditions might directly stimulate gonadal development, Pearse (1965, 1966) observed a marked synchrony in gametogenesis in different populations of the seastar,

Odontaster validus, in the Antarctic, where the annual temperature range is only 0.8 °C, and suggested that this synchrony starts with the accumulation of nutrients during the summer period of phytoplankton abundance.. Similarly, Holland

(1967) speculated that the well defined reproductive cycle of the urchin, Stylocidaris affinus, living in the Mediterranean under almost constant temperature, oxygen concentration and salinity conditions, was related to the annual photoperiod cycle or consequent changes in the availability of algal food. In some barnacles and copepods in temperate waters, there are several breeding periods during spring through autumn, and it has been indicated that regeneration of the gonads is dependent on the availability of food (Marshall. & Orr, 1955.; Crisp &

Davis, 1955; Corkett & McLaren, 1969; Barnes 6 Barnes, 1975).

In contrast, in some boreo—arctic barnacles, food availability is not the major cue for gonadal development, although it has been demonstrated that the decrease in the food sypply in the autumn hastens the latter stages of gametogenesis (Crisp. &

Patel, 1969). Thus, for some species food supply may influence gonadal development. , 96

k number of studies on marine invertebrates have been concerned with the sources of nutrients for gonadal development, and some contrasting strategies have been described. , In the seastar, Pisaster ochraceus, most feeding occurs in the summer when the gonads are small and little occurs in the Winter when the gonads are enlarging (Farmanfarmaian, Giese, Boolootian &

Bennett, 1958; Grenfield, Giese, Farmanfarmaian, & Boolootian,

1958; fiauzey, 1966; Nimitz, 1971) . There is a reciprocal decrease in the size of the hepatic caecae as the gonads grow, suggesting that reserves in the hepatic caecae are used for gamete production. In contrast, the urchin, Stronqyiocentrotus eurpuratus, does not appear to have a major storage organ apart from the gonad itself, and to a large extent nutrients for gonadal growth probably come directly from ingested food (Giese,

1959b, 1966).,,. Giese suggests that the differences in these two animals may have evolved in response to differences in the availability of their foods, since the food supply of the omnivorous urchin may be more continuous than that of the seastar. However, Hauzey (1966) suggested that for Pisaster the efficiency of feeding may be greatest in the summer, and that reduced feeding in the winter would increase the space in the arms, thus allowing greater gamete production.

Several studies deal with nutrient supply for gonadal development in Katharina tunicata. Nimitz and Giese (1964), from histological observations, report that nutrients for gamete production are derived primarily from reserves in the gonad itself or from ingested food. , When they starved animals for 5 months in a period prior to spawning, normal but fewer gametes 97

were produced., The gonads, however, did not increase in size, whereas a control group fed Pelyetia and Ijid^ghycus did show gonadal growth, although not as great as in the field animals.,

Lawrence, Lawrence and Giese (1965) found that the size of the digestive gland of K.,tunicata had a seasonal cycle which was inverse to that of the gonad, and suggested that it had a storage function. In a later study, Lawrence and Giese (1969) kept animals at 13 °C without a supply of food. after four and a half months there was a marked drop in lipid levels in the foot and mantle, while the gonads showed a slight increase in lipid content and the gonadal index rose from 1,0 to 2.7.

Meanwhile, the gonadal index in field animals rose to 8.0, but there was a drop in lipid levels in the foot, mantle and digestive gland comparable to that in the starved animals.,

Lawrence and Giese interpret this to mean that the possible nutrient reserves in somatic body organs are not enough for normal gonadal growth and that additional nutrients must come from ingested food., Giese and Hart (1967) studied changes in the biochemical constituents of various body organs of

K. tunicata throughout the year and concluded that during gamete

production, carbohydrate in the gonad is converted to protein in the testes and to protein and lipid in the ovary. Thus, it appears that K. tunicata relies on both stored and ingested nutrients for gamete production.

For a variety of invertebrates, both food quantity and quality are reported to influence the amount of gametes produced

(Moore, 1937; Vevers, 1949; Vadas, 1968; Ebert, 1968)., From numerous observations on the feeding of K. tunicata in the 98

field, as well as observations on gut contents, I have concluded that the main food of K. tunicata is the phaeophyte Hedophyllum sessile, the alga it is most commonly associated with. In

Nimitz and . Giese's (1964) experiment the control animals were fed Pelyetia and Iridpphycus and this diet may not have been as nutritious as the diet available to field animals; this could possibly have caused the reduced gonadal growth in the laboratory animals. The presence of gametes after 5 months of starvation in Nimitz and Giese's experiment, and the slight increase in the mean gonadal index of animals starved by

Lawrence and Giese (1969) [they do not indicate whether mature gametes were produced] suggests that after November the gametogenic process in K. tunicata will continue irrespective of laboratory and food conditions. However, in K. tunicata, as in other species, food does appear to affect the amount of gametes produced.

In some marine invertebrates it has been demonstrated that the rate of food consumption decreases at the time when the gonads are increasing in size, even when excess food is available., This has been reported for Strongylocentrotus ii}£ermedius» Strongylocentrotus droebachiensis and Strongylpcen- trotus purpuratus (Fuji, 1962, 1967; Ebert, 1966; Himmelman,

1969). Ebert (1966) suggested that this may be due to the size of the gonads, since there is not enough room in the test for a large gut and large gonads, although he also indicated that food may be less abundant in the field when gonadal growth is occurring. Fuji (1962) considered that the reduction in the feeding rate was a physiological response to the gonadal 99

development. A seasonal cycle in the . feeding rate of

K. tunicata at Botanical Beach Mas described by Himmelman and

Carefoot (1975), During August-early September 1971 about 0.9 g of fledoghyllum sessile were consumed daily. K. tunicata at this time had small gonads. In October and December only 0.3-0.6 g were consumed daily and at this time the gonads were enlarging.

The highest feeding rates occurred in May-July 1972, about 1.2 g daily, when spawning was not yet completed, and tne rate again dropped in the late summer when the animals were spent., Thus, the feeding rate of K. tunicata was low when the gonads were beginning to grow, but in contrast to the urchxns, feeding increased before spawning was completed. In all of the above species the feeding rate decreased as the gonads were enlarging, but there was no evidence that a change in the food supply stimulated gonadal growth.

Many other factors may influence the nutrition of an animal

(eg. see review on echinoid nutrition by Lawrence, 1975). The amount of nutrients available to an animal may not decrease in proportion to the decrease in feeding rate at the time of gonadal growth. For example, in Strongylocentrotus intermedins and Strongylocentrotus droebachiensis the weight of food absorbed per weight ingested increases as less food is passing through the gut (Fuji, 1962, 1967; Himmelman, 1969). Also the guality of food in the field may change in different seasons.

For example, at Botanical Beach, Himmelman and Carefoot (1975) found that there is an increase in the calorific content of the phaeophytes, Hedophyllum sessile and Lessonigpsis littoraiis, and of the rhodophyte, Iridaea cordata, during the late summer 100

to early winter and a decrease in the spring. Thus, at

Botanical Beach during the period when gonadal growth occurs in

Katharina tunicata, Strongylocentrotus . droebachiensis and

Stro n gylocentrotus £J!££uratus the caloric intake of these animals may not have decreased in proportion to the reduction in their feeding rates.

The reverse situation appears to be the case for SJtronjjylo- centrotus droebachiensis populations feeding on Laminaria longicruris in St. Margaret's Bay, Nova Scotia. Data presented by Mann (1972) indicate that the caloric content of this alga on a live weight basis would have a seasonal cycle which is the reverse to that observed for the three west coast algae

(Himmelman and Carefoot, 1975)., Consequently, Strongylocentrqt- us droebachiensis feeding on this alga would have a marked seasonal fluctuation in caloric intake, This was confirmed by

Miller and Mann (1973). They reported that the number of calories consumed, as well as the number of calories absorbed,

•by S. droebachiensis feeding on Laminaria longicrusis decreases from June to March, but from August to March there was an increase in the number or calories channelled into gonadal growth and little channelled into somatic growth.,, In their experiments, the animals were provided with excess food. Thus it would not appear that food availability caused the change in energy flow from somatic to gonadal . growth. , Temperature . and

photoperiod approximated that in the field.„ It is interesting that Miller and Mann (1973) report there was a decrease in the calorific content of the gonad/g wet weight from June to March,

•when the gonads were increasing in size. , This is primarily due 101

to an increase in water content, thus indicating that some, of the enlargement as measured by the gonadal index is not due to the addition of organic materials. An increase in water content as the gonads increase in size has also been noted in Strongy- i°£S3.t£2.tus purpuratus (A. L. Lawrence & J. M. ,.Lawrence in

Giese, 1 966) .

In summary, for growth in some species there must be an incoming supply of nutrients from ingested food during the period when the gonads are growing, while in others, provided that there is soma storage of materials, gonadal development will proceed even when there is no food available. For most species poor food guality or a scarcity of food will reduce the numbers of gametes produced. There is no evidence suggesting that gonadal development in the species considered. in the present study is stimulated by changes in food guality or availability.

Possible Factors Regulating Spawning

It is likely that some mechanism has evolved to ensure that spawning will occur when conditions are favourable for both fertilization and larval development. , For animals which shed their gametes directly into the sea, the probability of fertilization will be directly, related to. the degree of synchronization of spawning amongst animals in a population; and when synchronous spawning occurs it is probably in response to external cues (Giese & Pearse, 1974), In examining the stimulus 102

for spawning, both physical and biotic conditions required for the success of larval stages should be considered., One would expect a change in one or more environmental factors at time of, or just prior to, spawning. Alternatively, in some species the stimulus for spawning may preceed the favourable period for larval growth, so that the larvae have developed to a stage where they can take full advantage of the favourable period

(Pearse, 1965) .

Many researchers have investigated the stimulus for spawning. However, there are relatively few organisms for which the spawning cue (s) has been elucidated. , Orton's (1920) work has been freguently referred to, but it should be noted that he did not distinguish between the conditions which stimulated gonadal development and those responsible for spawning. Many reports are purely descriptive and spawning factors may be suggested but usually without experimental proof., A surprising number of harsh treatments, for example electrical shock, scraping the animals, exposure to Kraft pulp mill effluent and other chemicals, are known to cause spawning in the laboratory

(Young, 1945; Breese, Millemann & Dimick, 1963), but it is difficult to imagine their role in natural spawning.

Temperature

Temperature more than any other factor has been considered to control spawning. There is a distinct annual temperature cycle in most shallow water habitats and many workers consider 0 103

that spawning starts when a physiological threshold temperature is reached, or when there is a sudden change in temperature.,

For example, Clipperfield (1953) reports that in a number of locations in Great Britain Mytilus edulis spawns when the mean sea temperature rises from 9.5 to 11-12.5 °C. He suggests that spawning is stimulated by this temperature rise, although he also indicates that laboratory studies produced conflicting results. Mytilus edulis is probably exposed to at least a 3 °C change during every daytime low tide. Loosanoff and Davis

(1963) report that they could not induce this species to spawn by thermal shock. Orton (1920) reports that Qstrea-edulis spawns when the temperature reaches the critical level of 15-16

°C, but later studies on this species indicate that although a certain temperature is reguired for gonadal maturation, temperature does not act as a "trigger" for spawning (Korringa,

1957), This appears to be true of many species. Loosanoff and

Davis (1963) studied 19 species of lamellibranchs and report that most of them ripened out of season when subjected to warmer temperatures, but temperature does not always stimulate spawning. The standard technigue Loosanoff and Davis used to induce spawning was a sudden rise in temperature of 5-10 °C and the addition of gonadal tissues. This treatment was only reliable for about half of the species, and only a small number could be induced to spawn by a temperature change alone. It is unlikely that M. edulis, Qstrea edulis, and the lamellibranchs studied by Loosanoff and Davis naturally encounter sudden temperature changes sufficient to stimulate spawning.. Thus, while temperature is often referred to as the stimulus for 104

spawning, this has usually not been adeguately demonstrated.

Field observations made during the present study indicated that there was usually a slight increase in the rate of warming during the spawning period, compared to the period prior to spawning, but the total temperature increase was still small., at First Harrows, Stronqylocentrotus droebachiensis, Tonicella lineata, Tonicella insignis, Mopalia laevior and Mopalia ciliata had a major spawning when the temperature reached 7—8 °C, except for S. droebachiensis in the cold spring of 1970 when spawning occurred at 6,3 °C. The temperature was 7.5—8.3 °C at Porteau when T. lineata, T. insignis and M. laevior spawned in 1973. At

Botanical Beach in some years K. tunicata spawned when temperatures were at least 9 °C and in other years spawned when temperatures were 1—2 °C colder,, T, lineata, Mogalia hindsii,

M..ciliata and S, droebachiensis probably spawned at higher temperatures at Botanical Beach than at First Narrows, and there was considerable warming over the prolonged spawning period of

Strongy.locentrotus ^urpuratus at Botanical Beach. The abrupt spawning in most of the species in the present study suggests that spawning happens as a sensitive response of the animals to an external stimulus. , At Porteau in 1973 there was a gradual rise of not more than 0.8 °C over the two week period when the complete spawning occurred. In this case, if temperature was the spawning stimulus, the animals must have had a very precise temperature threshold. Such a precise temperature threshold was not indicated by the variable correlation of temperature with spawning in different years at Botanical Beach, or by the differences in temperature at the time of spawning between First 105

Harrows and Botanical Beach.

In contrast to Orton's (1920) hypothesis of a physiologically constant spawning, it is clear for the species in the present study that temperatures at the time of spawning vary considerably in widely separated geographical locations.

Most of the species range at least from central California to

Alaska, and while the annual temperature minimum in Central

California is about 11 °C, the same level is near the annual maximum for Alaska. Barnes (1972) showed that the temperature was about 11 °C when Tonicella lineata spawned in Oregon, whereas at First Harrows and Porteau it was 7—8 °C., Mean temperatures were always above 12 °C when Katharinatunicata spawned in the Monterey area, and in the present study, were probably always less than 11 °C. In Newfoundland, Strongylocen- t£otus droebachiensis spawned when temperatures were less than 3

°C (Himmelman, 1969) while in the present study this species spawned at temperatures above 6 °C.

In my laboratory studies, animals collected prior to spawning and kept in conditions warmer and colder and lighter and darker than in the field did not spawn when spawning occurred in the field. Further, in the 1973 experiments, no spawning occurred when the cooling system failed, causing a temperature increase of 5 °C in two hours. Barnes (1972) also reported that he could not induce spawning in Tonicella lineata by temperature and other stimuli.. He tried alternating heat and cold, alternating light and dark periods, guiet water conditions and electrical shock with no success. Matanabe and Cox (1975) 106

tried to induce Mopalia Iignosa and Mogalia muscosa to spawn by- thermal shock, electrical, chemical and mechanical stimulation.

The only situation in which animals spawned was when they were placed in sea water and left until the water became stale, , It seems unlikely that the slight temperature changes which occurred in the field would have induced the sudden spawning observed in most of the species in my study.

There is sufficient evidence that in some species a certain temperature level is necessary for spawning to occur. For example, spawning in Mytilus edulls, Crassostrea virginica, and

Hercenaria mercenaria is inhibited when animals are exposed to lowered temperatures (Galtsolf, 1938; Loosanoff & Davis, 1951,

1963: Bayne, 1965), These critical temperature levels for gametogenesis and spawning are not constant, but may vary in different populations throughout their geographical range

(Loosanoff, 1968) .

Temperatures required for spawning are probably related to those reguired for normal development of the embryos and larvae.

For a number of bivalves and for some other invertebrates it has been demonstrated that the larvae have narrower temperature tolerances than do the adults, and that early cleavage stages may have even narrower temperature tolerances than the larvae

(Pelseneer, 1901; fiunnstrom, 1927a; Loosanoff & Davis, 1963;

Kennedy, Boosenburg, Zion & Castanga, 1974; Kennedy, fioosenburg,

Castagna and Mihursky, 1974). Sastry (1966) states that the lower temperature limit for spawning in the bay scallop,

Aeguipecten irridians, corresponds to the lower temperature 107

limit for cleavage., Galtsolf (1938) reports that the American oyster, Crassostrea yirginica, can he induced to spawn at 20.5

°C but not at 18.0 °C [although Loosanoff and Nomejko (1951) report that this species sometimes spawns at temperatures as low as 16.5—18.5 °C] and Davis and Calabresse (1964) report that

Crassostrea ylrginica larvae show a progressive increase in growth rate from 17.5—30.0 °C. The larvae of the European oyster, Qstrea edulis can survive temperatures of 12.5—27.5 °C and grows best above 17.5 °C (Davis & Calabresse, 1969). This corresponds to the reguired spawning temperature reported by

Orton (1920) for this species. Experiments by Bayne (1965) on the mussel, Mytilus edulis, in North Sales and Denmark .indicate that normal cleavage occurs between 8—18 °C. , There is a marked increase in larval growth rate from 10—13 °C and a decrease above 18 °C in larvae from Denmark, but not in larvae from North

Hales. As noted earlier, Mytilus edulis in British waters spawns as the mean temperature rises from 9.5 to 11—12.5 °C

(Clipperfield, 1953).... Thus, for these and other species natural spawning appears to occur when temperatures are favourable for the temperature sensitive larval stages,

For a number of the species in the present study, developmental rates under various temperatures have been reported (Table VII). Temperature tolerances have only been determined for Strongylocentrotus droebachiensis, and the temperatures at which the other species were studied were probably considered by the respective researchers to be within a natural or favourable range for development., The embryos and larvae of S, droebachiensis has normal development from ~1 to 108

Table VII. Beported developmental times under various temperatures for the species in the present study.

Length of Time of hatching free-swimming Chours after fertilization) period Reference

Strongylocentrotus droe normal development between -1 and 11 C RunnstrOm, 1927a droebachiensis Runnstrem, 1927b 2.5 months Turner, 1965 9 weeks

normal development between -1 and 9 °C

80 h (0 °C) > Stephens, 1972

50 h (4 °C)

30 h (8 °C)

Mopalia ciliata 36-42 h (normal sea temp• 7-14 days erature, probably 10 C) I Thorpe, 1962 12-24 h (12-15 °C)

Mopalia lignosa 19-25 h (13 C) 4.5 days (13 C) Watanabe & Cox, 1975

Mopalia muscosa 20-24 h (13 C) 10 days (13 C) Watanabe & Cox, 1975

Tonicella lineata 48 h (10 C) 4.5 days (10 C) Barnes, 1972 43-44 h (12-13 °C) 2.7 days (12 13 °C) J 109

9—11 °C and its growth rate within this range increases with increasing temperature* The studies on Mogalia muscosa, Mopalia

AiSLSSsa, Mogalia ciliata and Tonicella lineata -were made in

California and Oregon and there was normal development in these species at 10—15 °C. In the latter two species growth increased with increasing temperature (Table VII). , During the present study spawning in the above chiton species probably occurred within a temperature range suitable for larval development.,

However, in S,.droebachiensis, it is possible that in some areas of southern British Columbia, especially in the Strait of

Georgia, lethal temperatures may sometimes be reached before larval development is completed.

In summary, although temperature is known to be an important stimulus for gonadal growth and maturation in numerous species, there are few substantiated instances of temperature acting as a stimulus for natural spawning. In Strongylocentrot- us droebachiensis, Tonicella lineata, Tonicella insignis and probably other species in my study, it appears unlikely that temperature acted as a cue for spawning. For a number of marine invertebrates it is clear that natural spawning occurs when temperatures are favourable for larval development, and for some species reduced temperatures will inhibit spawning, thereby acting as a safeguard against the waste of gametes. 110

Light

Light has been shown to act as a cue for synchronous release of gametes in several marine invertebrates. During the breeding seasons of several species of hydroids spawning occurs in the early morning., Ballard (1942) demonstrated that this was solely in response to light after a period of darkness.

Re—illumination activates the germ cells to undergo the final stages of maturation (even in oocytes dissected from the animals) and after a period of 55 minutes in light, mature gametes rupture from the walls of the gonophores. Also, some ascidians are known to spawn when exposed to light after a period of darkness. Lambert and Brant (1967) suggests that spawning in Cigna intestinalis is initiated when a nervous pathway is stimulated by heme-proteins which absorb light.

Korringa (1947) suggests that the presence or absence of moonlight may be a factor in animals with lunar spawning rhythms. Conceivably photoperiod could act as a signal for spawning in chitons and urchins, but the variable spawning time in different places and in different years during the present study do not support this hypothesis.,

Chemical Factors

Many chemicals, for example adrenalin, thyroxin, KC1 and ammoninated sea water, are known to cause ripe animals to spawn

(Galtsolf, 1940; Sagara, 1958; Breese, Millemann Dimick, 1963).

However, it is not known that they are functional in nature. 111

Sax products, particularly sperm, causes spawning in many invertebrates (Heath, 1905; Fox, 1924), and these have obvious value in increasing the degree of synchrony in spawning amongst individuals in a population. In the present study sperm suspension stimulated spawning in about two-thirds of the

Stronqylocentrgtus droebachiensis, Tonicella lineata and

Tonicella insignis tested. This type of stimulation has been studied most in Crassostrea virginica (Galtsolf, 1938; Nelson,. £

Allison, 1940). Sperm suspension is usually far more effective than physical stimuli (Young, 1945), and the response to sperm is sometimes used as a criteria for whether an animal is in condition to spawn (Galtsolf, 1938, 1940). Galtsolf (1940) reported a substance in egg suspension which stimulated spawning in male Crassostrea vir^inica., This substance was thermostable and water soluble and thus resembled a substance which Miyazaki

(1938) extracted from the green algae, Ulya and Entermorgha, which induced spawning in the Japanese oyster, Crassostrea gigas. A variety of plant substances (maltose, arabinose, starch and yeast) are also reported to stimulate spawning in

Crassostrea virginica. Many extracellular products are released by algae (Hellebust, 1974) and it is conceivable that, some may cause natural spawning. The high effectiveness of chemical stimulation by sperm and other substances suggests that the natural spawning stimulus is chemical. 112

Phytoplankton

The close correlation of spawning with the . development of the spring phytoplankton bloom in several locations in 1973 and

1974, and the experimental observation that natural phytoplankton stimulated spawning in the laboratory, suggests that the phytoplankton bloom acts as the cue for natural spawning. As mentioned earlier, the addition of sperm is reported to be one of the most effective methods for inducing spawning in marine invertebrates, but in the present study phytoplankton was as effective as sperm in causing spawning in

Strongylocentrotus droebachiensis. Tonicella lineata and

Tonicella insignis. It is possible that phytoplankton and sperm act through a similar mechanism involving chemoreceptors and a neuroendocrine pathway. Certainly this presents an interesting physiological problem, and the specificity of the receptor to different phytoplankton species is of ecological interest.

The various species in the present study can be compared according to the degree to which their spawning coincided with the phytoplankton blooms,,, S. droebachiensis and Tonicella insignis usually spawned abruptly early in the bloom, while

!• Aill®ata spawned more .slowly during the bloom.,, T.. ...lineata probably reguires continuous phytoplankton stimulation since animals transferred from First Narrows to the laboratory in the middle of the spawning period in 1973, appeared to stop spawning

(Fig. 26). Mopalia laevior and Mopalia ciliata spawned abruptly at the onset of the phytoplankton outburst in 1973, and more gradually in .1974, corresponding to the slow increase in 113

phytoplankton in that year. Spawning of Katharina tunicata at

Botanical Beach was closely timed with that of T. lineata in each year during 1971—197 4, and in 1973 both species only

spawned partially in April and completed spawning in June.

Spawning in Strongylocentrotus droebachiensis at Botanical Beach in 1973 was completed during April. I indicated earlier that the April 1973 phytoplankton bloom in Juan de Fuca Strait was

not as intense as in Strait of Georgia (Table IV).,, The partial

spawning in T. lineata and K. tunicata and the complete spawning

in S. droebachiensis coincided with this April phytoplankton

bloom, and spawning in T. lineata and K. tunicata was completed in June when a second and more intense bloom occurred,,, Although

phytoplankton observations were not made during other years at

Botanical Beach, the synchrony of spawning in X. .lineata . and

K. tunicata suggests that these two species were responding to

the same stimulus. The synchrony of spawning by Mopalia laevior, Mogalia ciliata and Katharina tunicata with

phytoplankton blooms in the years when.there were data on the timing of the bloom, and the close timing of spawning in these

species with spawning in Strongyiocentrotus droebachiensis.

Tonicella lineata and Tonicella insignis in years when there

were no data available on phytoplankton, suggests that the

phytoplankton bloom was also the cue for spawning in M. laevior,

£iAi§.ta and K. tunicata. However, this assumption reguires

experimental evidence,,

Spawning by the other species in the present study did not

appear to coincide with the spring phytoplankton blooms,, At

First Marrows Mo£alia hindsii spawned in the winter in 1973 and 114

just prior to the bloom in 1973. In Mopalia muscosa, spawning was not synchronous for animals in different pools at Botanical

Beach and therefore was probably not related to a widespread factor like the phytoplankton bloom. In Mopalia lignosa there appeared to be spawning before the bloom in 19 73 and another spawning during the summer., The slow spawning in Strongylocen• trotus purpuratus was in marked contrast to the other species.

It seems unlikely that it uses phytoplankton as a cue for spawning. Thus, Mopalia hisgsii, Mopalia muscosa, Mopalia lignosa and Strongylocentrotus purpuratus probably do not use phytoplankton as a stimulus for spawning.,

While phytoplankton appears to stimulate spawning in some of the species in the present study, the influence of other factors cannot be excluded.. In the 1974 experiments, when phytoplankton was added to Strongylocentrotus droebachiensis and

Tonicella lineata at a temperature of 5.5 °C, a smaller proportion of animals collected on 4th April spawned than those collected on 12th March. However, when the . experimental temperature was 10 °C, a similar proportion of T.,lineata from

12th March and 4th April spawned., Field temperatures at First

Harrows (as measured at the Vancouver Public Aguarium) at 6 m

below MLWS increased 1 °C or less between 12th March and 4th

April. It would appear that lowering the temperature inhibited

spawning, and that once animals have adapted to warmer conditions, even just a slight temperature increase, the

temperature level at which spawning is inhibited is increased.

During the experiments on S. droebachiensis it was frequently

observed that spawning did not begin when an animal was exposed 115

to phytoplankton, hut started when the water was being changed.,

[Animals which spawned in this way were counted as positive results in Table V, since controls handled in the same way did not spawn.] Changing the water caused a slight temperature change and mechanical agitation. In three instances it was found that the spawning began even when fresh sea water was added very slowly, lowering the temperature only 2 °C. , Animals

exposed to sperm responded in the same way. Thus, in S. droe-

bachiensis phytoplankton and sperm seemed to sensitize the animals so that spawning is provoked by a slight, disturbance.,

fthen determining the gonadal index of animals from the field, a

wet weight was recorded after allowing animals to drain on paper towelling for 15 minutes, and the animals were then returned to cold water., Handling S. droebachiensis, •T.,lineata, and

2- insignis in this way caused some individuals to spawn, but only when spawning was imminent in the field. In early June

1974 a few animals spawned without previous exposure to

phytoplankton. These animals had been held in the laboratory at

10 °C for a prolonged period.

The synchrony of spawning with phytoplankton blooms has

been known for many years, Thorson (1936, 1946, 1951) emphasized that pelagic larvae of benthic species are usually

most abundant during phytoplankton blooms, whether it be in the

Arctic where a single bloom period occurs, or in temperate seas

where there may be spring, summer and autumn blooms., In

Thorson*s time there was much emphasis on the importance of

temperature in controlling biological processes, including

spawning. However, Thorson (1946) clearly stated that it cannot 116

be excluded that phytoplankton acts to stimulate spawning. In

Greenland, spawning and phytoplankton blooms may occur before there is a rise in temperature (Thorson, 1936)., This synchrony is not limited to benthic species for it has also been observed in pelagic animals, for example euphausids (Einarsson, 1945) and calanoid copepods (Marshall, Nicholls & Orr, • 1934)., At

Igloolik, in the Canadian Arctic, Grainger (1959) notes that the young of herbivorous zooplank'ton species become abundant at the time of the summer phytoplankton increase, and in some species this may occur before there is warming of even the surface

waters. Barnes (1957), and Barnes and Stone (197 3) report that

the release of nauplii by some barnacles is stimulated by the

spring diatom outburst, and emphasize the importance of this

synchrony for all animals with planktotrophic larvae, whether

the eggs and sperm are released directly into the sea or the

earlier stages are nurtured by the adults and then released.

The advantages to spawning at the same time as the

phytoplankton bloom may vary according to the species. For

animals with planktotrophic larvae the availability of food is

of obvious importance. The larvae of S. droebachiensis are

pelagic for 2.5 months (fiunnstrom, 1927b) to 6 weeks (Strathman,

pers. comm.), but feeding cannot begin until the pluteus

develops, which takes more than a week (Stephens, 1972).

Strathmann (1971) offered natural phytoplankton to 8-armed

plutei and found that they could ingest particles as large as

armoured dinof lagellates measuring SO x 60 x 40 jx. „ Short chains

°f Skeletonema costatum and Thallassiosira sp., were found in

the stomachs of larvae, and there was evidence that diatoms were 117

easily digested, Skeletonema costatum and Thailassiosira spp, were important elements of the blooms in 1973 and 1974

{Table II, III, and V). Loosanoff and Davis {1963) indicated that food and temperature were very important factors in the growth of a number of bivalve larvae., The food requirements varied for different bivalve species and for different developmental stages within a single species,, They stated that dinoflagellate blooms cause abnormal development and high mortality to bivalve larvae., In chitons, the egg is large and usually develops into a free swimming trochophore, which after a short period settles and metamorphoses directly into the adult form (see descriptions of larval development by Barnes, 1972;

Barnes & Gonor, 1973; Watanabe & Cox, 1975). The chiton species which use phytoplankton as a spawning cue almost certainly spend their entire pelagic existence under bloom conditions, but they

probably do not eat phytoplankton. Possibly other factors are advantageous for chiton larvae. The spring phytoplankton bloom is one of the most sharply defined events in temperate and polar

seas and this alone would make it a reliable spawning cue to ensure fertilization. The eggs of T. lineata and I. insignis can easily be dispersed by water currents and Barnes (1972) reported that T. ...lineata eggs do not deteriorate for at least

14 h.

Hydrographic conditions at the time of the spring

phytoplankton bloom are described by Sverdrup {1953). During the winter there is a mixing of the water column to a great

depth, but as spring approaches the depth of this mixed layer

decreases due to heating at the surface or to the formation of a 118

less saline top layer. , The bloom occurs when the depth of the mixed surface layer is less than the critical depth where

production due to photosynthesis exceeds destruction due to respiration., Light penetration and intensity influence the

depth of this critical level. Larvae released during the bloom are likely to stay in this surface layer., Here temperatures are

usually warmer than at greater depths and in most species this increases the growth rate of the larvae., Since so many species

spawn during the phytoplankton bloom, the total amount of

predation may be less per species than if different species were

to alternate their spawning. Even though phytoplankton may not

be used as food for some pelagic larvae, the increase in

production of benthic algae, at about the same time as the

bloom, may be important as food for recruiting benthic

organisms., Also organic matter settling from the bloom may be a

useful food source for young animals.

Internal Considerations

In the past decade there have been numerous studies on

endocrine mechanisms in invertebrates, and it is becoming

increasingly clear that they play an important role in the

reproductive biology of marine invertebrates (see reviews by

Highman £ Hill, 1969; Goulding, 1972; fingerman, 1974). For

example, some endocrine control mechanisms have been worked out in echinoderms. Chaet and McConnaughy (1959) first reported

that spawning in starfish could be induced when a substance from

the radial nerve is injected into the coelom. Numerous other 119

studies have further elucidated the mechanism of starfish spawning (Kantani £ Skirai, 1972), A polypeptide produced by nervous tissue, when released into the coelom, causes the ovaries to produce a second substance, 1-methyiadenine. , The latter substance induces oocyte maturation, dissolves the attachment of the oocytes to the follicles, and causes the ovarian wall to contract, thereby resulting in the release of gametes. Other substances have been found which block meiosis and inhibit spawning, and thus prevent the release of gametes before the appropriate time (Ikegami & Tamura, 1973) . ... In the urchin, Strongylocentrptus purguratus, it has been demonstrated that the concentration of a neural substance which induces spawning fluctuates with the reproductive cycle (Cochran &

Engelmann, 1972)., Thus, if external factors affect reproduction, they are likely mediated by endocrine pathways.,

Unfortunately, the majority of endocrinological studies have not considered the influence of external factors on these internal control mechanisms.

As mentioned earlier, in some vertebrates it has been shown that photoperiod acting through a neuroendocrine pathway coordinates seasonal reproductive activity. A number of researchers have proposed that photoperiod may also influence endocrine systems which regulate reproductive cycles in marine invertebrates (Sells £ Hells, 1959; Hauenschild, 1964; Adiyodi £

Adiyodi, 1970; Golding, 1972). This should be further

investigated.

Some internal events, once initiated, might proceed without 120

farther coordination from the external environment. .. This was suggested for portions of the gametogenic seguence in barnacles

(Crisp & Patel, 1969). Also endogenous sequences have been suggested to play a role in reproductive cycles of animals living under nearly constant environmental conditions (Pearse,

1965: Holland, 1967), In some lamellibranchs, spawning may not require an external stimulus, but may happen spontaneously when gametogenic events, which were initiated by increased water temperature, are completed (Loosanoff and Davis, 1963; Sastry,

1966). Here spawning may not require an external stimulus, but may happen spontaneously when the animal has reached a certain point in development. Alternatively, internal mechanisms may change the condition of the animal so that any slight disturbance will cause spawning. Once gametes are in the water other animals will be stimulated to spawn (KecJces, Ozretic &

Lucu, 1966).. Boolootian (1964a, 1966) described an experiment in which a group of §tronqxlocent£otus purpurafeus were maintained in a circulating seawater system, and yet spawning occurred at the normal time over two consecutive years. It

appears that external factors may coordinate internal events at

specific points, and that once initiated some reproductive seguences will proceed without further coordination by external factors. 121

Evolutionary and Geographic Considerations

Reproductive cycles have evolved in response to selective

pressures for increased reproductive success., In marine invertebrates the early developmental and larval stages are most sensitive to adverse physical conditions (Runnstrom, 1927a), and there would be a distinct advantage to coordinating these phases with favourable conditions. Further, the availability of food

is important for species with planktotrophic larvae. ., Thorson

(1946) demonstrated the increased tendency of species in polar regions to brood their young and related this to the harsh

physical conditions and short periods when food is available to

the larvae. The species in cold waters which have pelagic larvae usually synchronize this phase with phytoplankton blooms,

which also may coincide with slightly increased temperatures.

These species typically develop their gonads during the winter

so that the gametes are ready for release - when this short period of favourable conditions arrives. Alternatively, if early

development under these low temperatures is very slow, spawning

may occur before the bloom so that the larvae have reached the

feeding stage and can make immediate use of the phytoplankton

when the bloom occurs (Pearse, 1965). In warm seas, the

majority of species have pelagic larvae and the different

species spawn at various (and sometimes several) times during

the year. Here, plankton production is more or less constant

and the scattered spawning periods probably decrease competition

for food among planktotrophic larvae (Thorson, 1946, 1950).

In temperate seas there are species of both warm and cold 122

water origin. ,, Species of cold water origin spawn when the phytoplankton first become abundant in the spring.. Their larval

phase is often intolerant to warm conditions, as in the case of

Strongylocentrotus droebachiensis, and must be completed before

warm summer conditions set in {fiunnstrom, 1927a)., Species of warm water origin usually develop their gonads only when temperatures reach a certain level in the spring, or gonadal

development may be started in the autumn, arrested in the winter

and resumed in the spring. The larvae of these species cannot

tolerate cold temperatures (fiunnstrom, 1927a), and spawning

occurs in the summer and autumn. Spawning may be coordinated

with the presence of certain phytoplankton food species for the

larvae, or with reduced feeding activities of predatory animals

(Thorson, 1946, 1958).. The species of cold water origin usually

have short spawning periods, while species of warm water origin

often have prolonged periods of gametogenesis and spawning.

Also since a certain temperature may be required for

reproduction in warm water species the reproductive periods are

usually delayed and shortened in the colder parts of their

geographic range (Orton, 1920; Hopes & Stickney, 1965).

Similarly, a number of species which evolved in boreal seas may

have shortened reproductive periods in the warmer parts of their

range, since gonadal activity may cease when temperatures are

too warm (Runnstrom, 1927a; Cochran & Engelmann, 1975). For

cold water species the advantages of spawning at the time of the

phytoplankton bloom (and increased temperatures) are understood

at least in part, but the advantages associated with the

variable spawning periods in warm water species are not clear. , 123

The waters along the Pacific coast of North America, from

Point Conception, California, northward to the Gulf of Alaska and westward along the Alaskan Peninsula, are cool and do not

fluctuate greatly throughout the year {less than 10 °C annual

variation). Strongylocentrotus droebachiensis is a circumpolar

species which extends from the Arctic into the North Altantic

and North Pacific. Throughout this range it displays the cold

water reproductive pattern, with gonadal growth in the winter

and an abrupt spawning at the time of the first increase in

phytoplankton. Its larvae cannot tolerate temperatures above

9-11 °C {fiunnstrom, 1927a; Stephens, 1972) and the southern

geographical limit of this species is probably determined by the

temperatures reached during the bloom period and several weeks

later. Strongylocentrotus purpuratus is endemic to the

northwest Pacific and while the timing of its gonadal growth

coincides with that in S. droebachiensis, its spawning pattern

is strikingly different. Reproduction has been studied in

S. EM££M£§l=iiI> more than any other species on the Pacific coast,

but the factors controlling its winter—spring spawning are not

known. ,

The genus Tonicella also occurs in the Arctic and extends

into the North Pacific and North Atlantic. Dall (1878, 1921)

reported that Tonicella lineata ranges from northern Japan and

the Okhotsk Sea northward to the Bering Sea, throughout the

Aleutians, along the Alaskan Peninsula and southward along the

North American coast to San Diego. However, Sirenko (1973)

considered the form on Asiatic shores to be a separate species. 124

Tonicella insignis has a narrow range, being reported only from

Alaska to Puget Sound (Burghardts, 1969). Both T. lineata and

2* insignis showed an increase in gonadal size from the summer through the winter and spawning was stimulated by the spring phytoplankton bloom. In T. lineata there was evidence that spawning was inhibited if temperatures of 7-8 °C are not reached at the time of the bloom. Thus, T. lineata and T, insignis also display the reproductive pattern which is characteristic of cold

water species.

The genus Mopalia occurs only in the North Pacific and there has been extensive speciation in this genus on North

American shores. Mopalia muscosa and Mopalia ciliata are the

most wide spread, occurring along the Alaskan Peninsula and southward to Baja California (Ball, 1878, 1921; Berry, 1922).

Mopalia lignosa and Mopalia hijidsii extend from Sitka, Alaska,

to the southern part of Baja California peninsula (Ball, 1921),

while Mopalia laevior is known only from Sitka {Burghardts,

1969) to central California (Rice, 1971). These species

exhibited various reproductive patterns., M. hindsii spawned

when temperatures were near the annual minimum or had just

warmed slightly, and M..ciliata and M. laevior spawned at the

time of the spring phytoplankton increase.,. In these three

species gonadal growth usually started in the summer and

continued through the autumn and early winter. The data for

Mopalia lignosa were less clear but gonadal growth and spawning

may have occurred in the late winter as well as in the spring

and summer., Reproductive activity in M. luseosa probably

occurred throughout the year and seemed to vary in the different 125

pools where the animals were found, rather than with more general environmental conditions. , Unlike the other species in this study M. muscosa occurs primarily in the mid-intertidal region, and is thus exposed to more erratic changes in environmental conditions. Thus, several patterns of reproductive activity are found in the genus Mopalia.

Katharina tunicata and another chiton. Cryptochiton stelleri, are known from the Pliocene, and have a longer geologic history than the other chitons in this study . (Berry,

1922). Both genera are monospecific and show little morphological variation throughout their extensive geographical ranges., They are thus matured species (or euspecies) as defined by Dillon (1966). Cryptqchitgn stelleri occurs from Hokodate in northern Japan, on the Kuril and Sakhalin Islands around the

Okhotsk Sea, at the southern extremity of Kamchatka, in the

Aleutians, and along the Alaskan Peninsula and south to southern

California; and K. tunicata has a similar distribution except

that it is found only from Kamchatka on Asiatic shores (Dall,

1878, 1921). Both species showed little gonadal growth during

the summer and rapid development in the autumn and winter, but

their spawning times are separate. In the Monterey area, even

in the same years, Cryptqchitgn stelleri spawned in late winter

to early spring and K. tunicata spawned in the late spring to

early summer (Fig.,4; Tucker and Giese, 1962; Lawrence, Lawrence

& Giese, 1965). However, in Japan Cryjetochiton stelleri was

reported to spawn in May (Okuda, 1947).

In summary, all of the species in the present study show 126

gonadal growth during the colder part of the year. The ones

with strong affinities to Arctic forms spawn at the time of the spring phytoplankton bloom, and the phytoplankton itself is probably the spawning stimulus. Some of the species which occur

only in the temperate waters of the northeast Pacific spawn at

various times from winter to early summer, and spawning is

probably controlled by other factors. None of the animals in

this study require increased temperatures for gonadal growth as

is the pattern in warm water species.

The present study has described the pattern of reproductive

events in eight species of chitons and two sea urchins in

British Columbia waters. While no experimental observations

were made on the factors controlling gonadal growth, the

correlative observations on gonadal growth and environmental

conditions provide a basis on which hypotheses for experimental

studies can be made. The effect of photoperiod and temperature

on gonadal development should be investigated., Strongylocen•

trotus droebachiensis, Tonicella lineata, and Tonicella insignis

would be useful species for such studies since they can be

easily maintained in the laboratory, and the latter two species

reguire only a small amount of laboratory space, while the

synchrony of spawning and phytoplankton blooms has been

recognized for many years, the present study on S.. drojsbjiciden-

iiS' T. lineata. and T. insijjnis is the first demonstration that

phytoplankton, itself, is the cue for the release of gametes.

The species of phytoplankton which induce spawning, and the

physiological mechanism involved, remains to be elucidated. , 127

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