J. mar. biol. Ass. U.K.

(1977) 57, 859-867 859

Printed in Great Britain

A MORPHOLOGICAL STAGING SYSTEM

FOR THE LARVAL DEVELOPMENT OF THE HERRING,
CLUPEA HARENGUS L.
M.J.DOYLE*
Dunstaffnage Marine Research Laboratory, Oban, Scotland

(Figs. 1-5)

A staging system for the larval development of the herring {Clupea harengus), based
upon external morphology is described. The principal criterion of each stage is as follows:
stage 1, yolksac morphology; stage 2, differentiation of the dorsal fin; stage 3, dorsal
upturn of the posterior tip of the notochord, and stage 4, relative shortening of the gut.
Larvae were reared to metamorphosis from artificially fertilized eggs and regularly
sampled. A typical specimen in each substage was drawn and the durations of the sub-
stages were estimated at laboratory water and sea-surface temperatures. Rapid staging of
lightly anaesthetized larvae, which could then be used in experiments, was possible.

INTRODUCTION
It has been possible for some years to rear artificially-fertilized herring eggs through
to metamorphosis making experimental studies on the larval phase of the life history
practicable (Blaxter, 1968). However, a comprehensive larval staging system has not
previously been published.
Early workers (e.g. Masterman, 1896; Ehrenbaum, 1909; Fage, 1920; Lebour, 1921)
produced figures of individual wild-caught larvae but were unable to study the whole
pre-metamorphic period. Incomplete systems, designed solely to meet the needs of their
investigations, were produced by Blaxter & Hempel (1961) and Blaxter (1962).
The present study was intended to produce a complete series of stages, from hatching
to metamorphosis, which would be of general use. The following desiderata were con-
sidered to be minimal:
(a) successive stages should represent the normal sequence of development;
(b) the stages should be based upon stable morphological characters visible under low-
power magnification in living or preserved specimens;
(c) if possible fin ray and myomere counts should be avoided to facilitate rapid staging
of lightly anaesthetized larvae;
(d) the main stages should be divisible into substages when more accurate selection
of larvae is necessary.
This paper describes 4 main- and 13 substages of larval development and lists their
approximate durations at laboratory water and sea-surface temperatures.
* Present address: Department of Biology, Southampton University, Medical & Biological Sciences
Building, Bassett Crescent East, Southampton, SO9 3TU.

28 MBI 57
86o M. J. DOYLE
M A T E R I A L S AND M E T H O D S
The herring larvae used were obtained by artificially fertilizing eggs taken from Spring-
spawned Firth of Clyde females, with sperm taken from males of the same population, according
to the method of Blaxter (1968). Antibiotics were not used.
The staging system was produced provisionally by studying small samples of preserved larvae.
Then large numbers of larvae were reared enabling the system to be checked, modified and illu-
strated with living and preserved material. Details of the staging system are shown in Fig. 1.

Fig. 1. Diagram showing details of staging, (A) stage 1 and, (B) stage 4 herring larvae, (A) The
depth of the yolksac, a is 2 J or more times the depth of the adjacent myotomes, b. Therefore this
larva is in substage la. dpf: dorsal primordial fin; vpf: ventral primordial fin. (B) Counting the
number of myomeres between the posterior point of insertion of the pelvic fin (1) and the anus
(2). This larva is in substage 4a.

Table 1. Larval sampling

Days post-hatching Maternal stock Sample size
0 Female No. 1 5°
2 Female No. 1 50
2 1st Mixed female population 5°
4-14 (on 1st Mixed female population 5°
alternate days)
16-68 (on 1st Mixed female population 25
alternate days)
70 Female No. 1 25
72-78 (on Female No. 1 12
alternate days)
80-90 (on 2nd Mixed female population 12
alternate days)
96-114 (on 2nd Mixed female population 12
each 6th. day)
120 2nd Mixed female population 3
126 2nd Mixed female population 2

Notes: Female No. 1 larvae and 2nd Mixed female larvae hatched within a 36 h period. 1st Mixed
female larvae hatched within a 12 h period. Day 2 samples from both populations contained larvae of
similar substages. Shortage of larvae made it necessary to change populations on days 70 and 80.
 LARVAL DEVELOPMENT OF HERRING 861

Eggs from 8 females were fertilized separately but to obtain a high percentage fertilization the
sperm from 4 males were mixed. For most of the work described here larvae from the fertilized
eggs were pooled in two aquaria. Regular random samples were taken to obtain a developmental
series and to enable the approximate duration of each substage to be calculated. A summary of
sample sizes, sampling frequency and maternal stocks is given in Table 1.
Circulating sea water, used only once, prevented wide temperature fluctuations but the animals
were not reared in a constant temperature room. A daily temperature record was kept (Table 2).
Typical mid-substage and borderline preserved individuals were selected for camera lucida
drawings.
Table 2. Durations of larval substages and water temperatures
Durations of
substages and
main stages Mean Estimated durations
Substage at labora- laboratory Mean sea of substages and
(with tory water Calendar water surface main stages at sea
stage temperature dates of temperature temperature surface temperature
totals) (days) substage (°C) (°C) (Days)
la 3-2 12-3-14-3 89 7 3-8
lb 3-7 15-3-18-3 8-5 7 43
1C 39 19-3-22-3 8-7 7 4-6
1 10-8 12-7
2a i6-6 23-3-8-4 10-5 7 22-9
2b 6-9 9-4-15-4 113 7-5 9-8
2C 13-2 16-4-28-4 n-8 7-5 19-6
2 36-7 52-3
3a 9-7 29-4-8-5 10-9 9 n-5
3b 7-5 9-5-16-5 n-4 9 9-4
3c 20-3 17-5-5-6 129 10 26-5
3 37S 4T4
4a io-8 6-6-16-6 12-4 11 12-3
4b 24-7 17-6-11-7 13-3 12 27-8
Insufficient data

Total time to beginning of metamorphosis: Laboratory temperatures, 120-5 days; Sea temperatures
152-5 days.

Herring larvae are too sensitive to survive repeated anaesthesia and handling and so it was not
possible to discover the duration of each substage by observing the development of a number of
known individuals. An alternative is to sample at intervals and to calculate the proportion of the
total developmental time spent in each substage (Crisp, 1954). If the total development time is
known the actual duration of the separate substages can be calculated. If samples vary in size the
number of larvae in each substage within a sample must be expressed as a percentage of the total
number in that sample. When sampling does not occur at regular intervals each percentage must
be multiplied by A;,, which is half the time interval between the (t — 1) and the (»'+1) sample.
The time interval tn occupied by a given substage is thus,
2 {SnjS)i 100 At;
T £ 2 y\SnlS)i 100 At,-3
n %

where T is the total time of development and (Sn/S)i is the fraction of the total number in the i.
sample lying between stages n and n +1. The present calculations were based upon the period from
50 % larvae hatched to 120 days post-hatching when too few larvae remained for sampling.
As temperature was not controlled the substage durations strictly apply only under the temperature
regime shown in Table 2. The mean sea-surface temperatures in the Firth of Clyde (Craig, 1958;
International Council for the Exploration of the Sea, 1962) during the period occupied by each
28-2
862 M. J. DOYLE
substage are also shown in Table 2. By applying a temperature correction, estimates of substage
durations under sea conditions were made. Battle (1930) reviewed the application of the van't
Hoff Law to fish development while Blaxter (1956) and Ryland (1966) subsequently applied the
law to herring and plaice {Pleuronectes platessa L). development respectively. The latter authors
found <2,0 to lie between 2-0 and 3-0 for the temperature range at whichfish development frequently
takes place in the laboratory. A Qlo value of 2-5 was assumed here and the formula.

to
was used where D2 is the corrected development time, Dt is the observed development time and
(ti — r2) is the temperature difference between laboratory water and sea-water temperatures.

1a

Fig. 2. Stage 1 herring larvae (substages l a , l b , and lc). Scale bar represents 2 mm.

RESULTS
Staging
The following stages are described and illustrated (Figs. 2-5).
Stage 1 (Fig. 2)
Post-hatching. Yolksac present.

Substage la
Depth of yolksac equal to, or exceeding, 2J times the depth of the myotomal
musculature which lies immediately adjacent and dorsal to the sac (Fig. 1 A).

Substage lb
Depth of yolksac about twice the depth of the myotomal musculature which lies
immediately adjacent and dorsal to the sac.
 LARVAL DEVELOPMENT OF HERRING 863

(Limits of substage lb - any specimen in which depth a in Fig. 1A is less than

2 | times but more than equal to depth b is taken as lb.)
Substage lc
Depth of yolksac equal to or less than depth of the myotomal musculature which
lies immediately adjacent and dorsal to the sac.
(Late limits of substage lc - any specimen which has a clear expansion of the
gut anterior to the ventral fin fold is taken as lc. Some larvae have tiny bumps of
yolk in this region. If radial elements are absent from the dorsal fin such larvae
are taken as lc; if radials are present they are taken as 2a.)

Fig. 3. Stage 2 herring larvae (substages 2a, 2b and 2c). Scale bar represents 2 mm.

Stage 2 (Fig. 3)
Yolksac absent. Dorsal fin differentiating from the dorsal primordial fin.

Substage 2a
Dorsal fin not yet separated posteriorly from primordial fin. Yolksac absent.
(Limits of substage 2a - separation from late substage lc is given above. Fin
separation is taken as complete when a continuous line is visible from the
dorsal margin of the dorsal fin to the body just posterior to the developing
radials. This is an arbitrary definition as it is often impossible to see whether
the fin is actually divided along the line without handling the delicate tissue.
In a few specimens a complete line did not form. These were taken as substage
2b when the fin was clearly divided for half its depth.)
864 M.J.DOYLE

Substage 2b
Division between the posterior edge of the dorsal fin and the posterior part of the
primordial fin complete. Lobe of dorsal fin not protruding markedly beyond
the posterior primordial fin nor growing along beside it.
(Late limit of substage 2b - 'to protrude markedly' is taken to mean that the
most posterior extremity of the dorsal fin is dorsal to the posterior part of the
primordial fin.)

3a

Fig. 4. Stage 3 herring larvae (substages 3 a, 3 b and 3 c). Scale bar represents 2 mm.

Substage 2c
Dorsal fin protruding markedly beyond or beside the primordial fin and extending
posteriorly above it. The dorsal fin is completely separate from the primordial fin
posteriorly. The posterior tip of the notochord is still more or less straight, i.e.
making an angle of less than about 10-150 dorsally with the remainder of the noto-
chord. (This is sometimes difficult to determine when the whole notochord is
contorted by fixation).

Stage 3 (Fig. 4)
Notochord turns dorsally at its posterior tip. The dorsal fin is clearly differentiated.
The pelvic fins are not yet visibly protruding ventrally below the gut when
larva is viewed from the side.
 LARVAL DEVELOPMENT OF HERRING 865

Substage 3a
Posterior tip of the notochord is turned dorsally less than 30-400 but more than
150 (angles estimated on the dorsal surface of the notochord). The anal fin is
not yet separated posteriorly by a line from the ventral primordial fin.
Substage 3b
Posterior tip of the notochord is turned dorsally 40-500. The anal fin is separated
posteriorly by a line from the ventral primordial fin.
Substage 3c
Posterior tip of the notochord is turned dorsally more than 50°.

4a

Fig. 5. Stage 4 herring larvae (substages 4a, 4b and 4c). Scale bar represents 2 mm.

Stage 4 (Fig. 5)
Pelvic fins are visible, protruding ventrally below the gut, when the larva is viewed
from the side. The gut is shortening relative to the body length.

Substage 4a
At least 24 myomeres are present between the posterior point of insertion of the
pelvic fin and the anus (Fig. 1 B).
Substage 4b
At least 21 such myomeres but less than 24.
866 M. J. DOYLE

Substage 4c (metamorphosis)
At least 18 such myomeres but less than 21.

Substage 4<1 (the beginning of post-metamorphic life)

At least 15 such myomeres but less than 18.

Substage durations
These are shown in Table 2. Durations could not be estimated after substage 4b
because of the paucity of larvae. The duration of 4b is the least certain as samples con-
taining 12 larvae were not taken after 114 days post-hatching. De Silva (1973) found
that Spring-spawned herring from either the Clyde ground or the Minch grounds
arrived in the Oban area by mid-June. They then had mean lengths of 40-45 mm with
a range of 30-57 mm. If spawning took place in early February, which is usual, the sub-
stage expected by mid-June would be 4b. The 9 substage 4b larvae measured in the pre-
sent work average 25 mm total length, but ranged up to 30 mm. The larger size of
the wild larvae could be explained by many factors including diet, sea temperatures
and genetics.

DISCUSSION
There is no accepted framework for the description of vertebrate larval stages. Witschi
(1956) proposed a standard set of criteria for use in vertebrate embryology, but the whole
of larval development was classified merely as ' Stage VII - Metamorphosis'.
For simplicity a single character has been stressed here in each main stage. The
presence of the yolk sac had previously been used in herring by Blaxter & Hempel (1961)
and Blaxter (1962). The depth of the sac was chosen by Ryland (1966) as a parameter in
larval plaice development. It is the most obviously changing feature in living early post-
hatching herring. The differentiation of the dorsal fin is conspicuous after the yolk sac
stage, followed by dorsal upturning of the posterior notochord tip. Late premetamorphic
development is difficult to categorize without resource to myomere counts as a measure
of relative gut shortening. Lebour (1921) has used myomere counts and the position of
the pelvic fins relative to the pylorus when grouping herring larvae.
Metamorphosis is taken to include any post-embryonic developmental changes in non-
reproductive structures which are completed within a time period short by comparison
with that of the total developmental period, and by which a larva adapted to one mode
of life is transformed into a juvenile with a different mode of life (Etkin, 1964; Dent,
1968). This process is not easy to pinpoint in the herring. Substages 4a and 4b are con-
sidered larviform and in 4d the fish are clearly adult-like in appearance. Thus substage
4c was considered to end the larval period.
The larval stages described here were used in studies of variation in the rate of growth
and development in aquarium populations (Doyle 1975, M.Sc. Thesis, University of
Stirling). Over 500 living larvae were examined in addition to three hundred fixed speci-
mens in the present paper. The desiderata set out above were fully met except that a
 LARVAL DEVELOPMENT OF HERRING 867
simple myomere count was necessary in stage 4. This was readily performed however,
on lightly anaethetized larvae which subsequently recovered fully.
Duplicate staging of samples showed the results to be repeatable providing the limits
described here were adhered to in borderline cases.
I am grateful to Dr J. H. S. Blaxter and to Professor F. G. T. Holliday for their advice and
interest. Dr Blaxter also supplied the preserved larvae used in the preliminary study. The
Director and staff of the Dunstaffnage Marine Research Laboratory provided facilities. The
financial support given by the Natural Environment Research Council is gratefully acknowledged.

REFERENCES
BATTLE, H. I., 1930. Effects of extreme temperatures and salinities on the development of
Enchelyopus cimbrius (L.). Contributions to Canadian Biology and Fisheries, 5, 107-192.
BLAXTER, J. H. S., 1956. Herring rearing. II. The effect of temperature and other factors on
development. Marine Research, 1956, No. 5, 1-19.
BLAXTER, J. H. S.J 1962. Herring rearing IV. Rearing beyond the yolk-sac stage. Marine Research,
1962, No. 1, 1-18.
BLAXTER, J. H. S., 1968. Rearing herring larvae to metamorphosis and beyond. Journal of the
Marine Biological Association of the United Kingdom, 48, 17-28.
BLAXTER, J. H. S. & HEMPEL, G., 1961. Biologische Beobachtungen bei der Aufzucht von Herings-
brut. Helgoldnder wissenschaftliche Meeresuntersuchungen, 7, 260-282.
CRAIG, R. E., 1958. Hydrography of Scottish coastal waters. Marine Research, 1958, No. 2, 1-30.
CRISP, D. J., 1954. The breeding of Balanus porcatus (da Costa) in the Irish Sea. Journal of the
Marine Biological Association of the United Kingdom, 33, 473-496.
DENT, J. N., 1968. Survey of amphibian metamorphosis. In Metamorphosis (ed. W. Etkin and
L. I. Gilbert), pp. 271-312. New York: Appleton-Century-Crofts.
DE SILVA, S. S., 1973. Abundance, structure, growth and origin of inshore Clupeid populations of
the West Coast ofScotland. Journal of Experimental Marine Biology and Ecology, 12,119-144.
EHRENBAUM, E., 1909. Eier und Larven von Fischen des Nordisches Plankton. Nordisches Plankton,
Zool. Tiel, 1 (1), 1-413.
ETKIN, W., 1964. Metamorphosis. In The Physiology of Amphibia (ed. J. Moore), pp. 427-468.
New York and London: Academic Press.
FAGE, L., 1920. Engraulidae, Clupeidae. Report on the Danish Oceanographical Expeditions,
1908-10, to the Mediterranean, 2, Biol. A 9, 1-140.
INTERNATIONAL COUNCIL FOR THE EXPLORATION OF THE SEA, 1962. Mean monthly temperature and
salinity of the surface layer of the North Sea and adjacent waters from 1905-1954. In
Bulletin Hydrographique, 318 pp. Charlottenlund, Denmark: International Council for the
Exploration of the Sea.
LEBOUR, M. V., 1921. The larval and post-larval stages of the pilchard, sprat and herring from
Plymouth Sound. Journal of the Marine Biological Association of the United Kingdom, 12,
427-457.
MASTERMAN, A. T., 1896. On the rate of growth of the food fishes. Scientific Investigations. Fishery
Board of Scotland, 14th Annual Report, pp. 294-302.
RYLAND, J. S., 1966. Observations on the development of larvae of the plaice, Pleuronectes platessa
L., in aquaria. Journal du Conseil, 30, 177-195.
WITSCHI, E., 1953. Proposals for an international agreement on normal stages in vertebrate
embryology. In Proceedings of the 14th Congress of Zoology, Copenhagen, pp. 260-262.