An attempt was made to detect spawning marks on the

otoliths (sagittae) of the northern anchovy, Engraulis

mordax, from waters of the Southern California Bight.

While no spawning marks were detected, a modification of

an existing technique for observing daily growth rings

was developed. A discussion is presented on detecting

spawning checks in the northern anchovy.  (15pp.

Coy, David G.

Gregory, Paul A.

English

Aquatic Commons

S t a t e  of C a l i f o r n i a  
The Resources A g e n c y  
DEPARTFENT O F  FISH AND GAPE 
RESULTS O F  A STUDY T O  D E T E C T  SPAWING PIARKS I N  OTOL'LTHS 
O F  NGRTIIERN ANCHOVY, EPIGFAULIS K0Ri)AX 
P a u l  A. Gregory 
and 
David G. C o y  
MARINE KESOURCES 
Administrative Report N o .  76-13 
December 1976 
RESULTS OF A STUDY TO DETECT SPAWNING MARKS I N  TOLITHS 
19 OF NORTHERN ANCHOVY, ENGRAULIS MORDAX- 
2/ Paul  A .  Gregory- 
and 
3  / David G. Coy- 
An a t tempt  was made t o  d e t e c t  spawning marks on t h e  
o t o l i t h s  ( s a g i t t a e )  of t h e  n o r t h e r n  anchovy, EngrauZis 
mordux, from wa te r s  of t h e  Southern C a l i f o r n i a  Bight.  
While no spawning marks were d e t e c t e d ,  a  modi f ica t ion  of 
. a n  e x i s t i n g  technique  f o r  observ ing  d a i l y  growth r i n g s  
was developed. A d i scuss ion  i s  presented  on d e t e c t i n g  
spawning checks i n  t h e  no r the rn  anchovy. 
11 
- 
Marine Resources Admin i s t r a t i ve  Report No. 76-13, 
December 1976 
- 
Operat ions Research Branch, 350 Golden Shore, Long Beach, 
C a l i f o r n i a  90802. 
3  / 
-
25574 - 1 8 t h  S t . ,  San Bernardino,  C a l i f o r n i a  92404 
INTRODUCTION 
Fecundity e s t ima te s  of t h e  no r the rn  anchovy, EngrauZia mordax, 
have been r epor t ed  by MacGregor (1968) a s  being 574 advanced eggs pe r  
gram of f i s h  410%. The spawning season of t h e  no r the rn  anchovy i s  
p r o t r a c t e d ;  some spawning occurs  i n  every  month, and f i s h  wi th  develop- 
i n g  ova m y  be found a t  any t i m e  of y e a r .  However, t h e r e  is  a d e f i n i t e  
annual  peak i n  spawning a s  shown by t h e  number of anchovy l a r v a e  taken 
i n  p lankton  tows o f f  t h e  C a l i f o r n i a  and Baja C a l i f o r n i a  c o a s t s  from 1951 
through 1957. Peak spawning occurred i n  January ( 1  y e a r ) ,  February 
(2  y e a r s ) ,  and March (4 yea r s )  w i t h  spawning during t h e  peak month r e -  
p re sen t ing  20 t o  33% of t h e  t o t a l  spawning f o r  each year .  This  pheno- 
menon toge the r  w i th  evidence of mu1tipl.e modes of ova l e n g t h  f r equenc ie s  
sugges ts  more than  one annual spawning f o r  t h e  ma jo r i t y  of female 
anchovies (MacGregor, 1968).  This  p o s s i b i l i t y  a f f e c t s  t h e  r e l i a b i l i t y  
of e s t ima te s  of spawning p o t e n t i a l  and e s t i m a t e s  of spawning biomass 
based on egg and l a r v a e  surveys.  
I n  a n  a t tempt  t o  determine t h e  number of t imes a female anchovy can 
spawn each yea r ,  a Department of F i sh  and Game s tudy  was funded by t h e  
Marine Research Committee (MRC) t o  examine t h e  o t o l i t h s  ( s a g i t t a e )  of 
a d u l t  female anchovies f o r  t h e  presence  of spawning checks o r  p a t t e r n s .  
Our examination of o t o l i t h s  was based on t h e  techniques  employed by 
Pannel la  (1971) who r epor t ed  observing d a i l y  growth l a y e r s ,  f o r t n i g h t l y  
and monthly p a t t e r n s ,  and spawning r i n g s  which were d i s t i n g u i s h a b l e  
from win te r  r i n g s  i n  o t o l i t h s  from v a r i o u s  western A t l a n t i c  marine 
f i s h e s .  The technique  has  been s u c c e s s f u l l y  used by v a r i o u s  r e s e a r c h e r s  
a t  t h e  Un ive r s i t y  of C a l i f o r n i a ,  San Diego, Scr ipps  I n s t i t u t e  of Oceano- 
graphy; Nat iona l  Marine F i s h e r i e s  Serv ice ,  La J o l l a  and Honolulu; and t h e  
Inter-American Tropical Tuna Commission, La Jolla. 
METHODS AND MATERIALS 
The otoliths used in this study were taken from two sources. The 
first was from fish routinely sampled for length, weight, reproductive 
state, and age during the 1970-71 commercial fishing season. We 
selected otoliths from 165 female fish based on normal appearance of 
the bones and from the oldest fish available. Older (ergo larger) bones 
provided more possibility for repeated spawning marks and were easier 
to handle. The second source was the bones from five running ripe fish 
taken in a midwater trawl during sea survey cruise 76-A-3. It was hoped 
that the margins of these otoliths would reveal some pattern indicative 
of prespawning physiological conditions. All fish were taken in the 
Southern California Bight area. 
Essentially, pannellals technique entails grinding the inner surface 
of the otolith, etching the surface with 1% hydrochloric acid, making 
an acetate peel of the etched surface, and examining the acetate peel 
under oil immersion with a compound microscope. Unfortunately, anchovy 
otoliths do not seem to be suitable for acetate peels. We tried grinding 
with assorted grits and etching with various concentrations of hydro- 
chloric acid for differing periods of time with poor results. On the 
advice of David Kramer (National Marine Fisheries Service), Edward 
Brothers, (Cornell University, Ithaca, N.Y.), and Terrence Foreman 
(Inter-American Tropical Tuna Commission), we tried cleaning the surface 
with enzymes (the laundry product "Biz" and Adolph's Meat Tenderizer), 
household bleach (5.25%,sodium hypochlorite), and etching the surface 
with other reagents (phosphoric acid and sodium hydroxide). The results 
were still unsatisfactory. The peels revealed an irregular scaly surface 
w i t h  incomplete pa tches  of d a i l y  growth r i n g s .  During t h e  course  of 
experimentat ion,  i t  was discovered t h a t  when t h e  o t o l i t h  was examined 
under t h e  microscope whi le  t h e  a c e t a t e  f i l m  was s t i l l  i n  p l ace ,  a n e a r l y  
complete p rog res s ion  of s h o r t  pe r iod  growth r i n g s  became v i s a b l e .  The 
a c e t a t e  f i l m  had reso lved  t h e  s u r f a c e  i r r e g u l a r i t i e s  and allowed f o r  
microscopic examination of t h e  f i n e s t  increments  of l a y e r i n g  w i t h i n  t h e  
bone. This  f i n d i n g  l e d  t o  t h e  fo l lowing  product ion technique which was 
used t o  s ea rch  f o r  spawning checks. 
The o u t e r  ( d i s t a l )  s u r f a c e  of t h e  o t o l i t h  was l i g h t l y  ground under 
f i n g e r  p re s su re  on 400 g r i t  l a p i d a r y  paper .  This  was done t o  reduce t h e  
th i ckness  of t h e  c e n t r a l  p o r t i o n  t o  a i d  i n  l i g h t  t r ansmis s ion ,  b u t  c a r e  
was taken  t o  no t  make t h e  bone too  f r a g i l e .  The bone was then  mounted, 
ground s i d e  down, onto a drop of epoxy cement on a microscope s l i d e .  An 
# 
a t tempt  was made t o  s i n k  t h e  bone evenly i n t o  t h e  cement f o r  maximum 
suppor t  without  enveloping t h e  bone. A f t e r  cu r ing  overn ight ,  t h e  ex- 
posed s u r f a c e  (concave s i d e )  was ground on 400 g r i t  l a p i d a r y  paper  
whi le  occas iona l ly  checking t h e  p rog res s  under a compound scope a t  80 
d iameters .  A t  a p o i n t  s u b j e c t i v e l y  determined t o  b e  optimum, t h e  bone 
was g e n t l y  po l i shed  wi th  600 g r i t  l a p i d a r y  paper and then  cleaned i n  
f u l l  s t r e n g t h  househqld b leach  f o r  1-2 minutes .  The bone and s l i d e  were 
then  wiped o f f  and allowed t o  a i r  dry.  An a p p r o p r i a t e  s i z e d  s l i p  of 
.00088 inch  a c e t a t e  was quick ly  immersed i n  ace tone  and g e n t l y  l a i d  
a c r o s s  t h e  bone and mounting cement. The o t o l i t h  was then  ready f o r  
examination (Figure 1 )  . 
The r i n g s  were counted and measured wh i l e  examining t h e  o t o l i t h  
under o i l  immersion a t  1250 d iameters  wi th  t r ansmi t t ed  l i g h t .  An 
o p t i c a l  micrometer was used t o  measure t h e  d i s t a n c e  between unique r i n g s ,  
FIGURE 1. Diagram of comp1,eted otolith preparation. 
c racks ,  o r  o t h e r  landmarks and then  t h e  inc luded  r i n g s  were counted. Af t e r  
t h i s  in format ion  was recorded on a d a t a  s h e e t ,  t h e  d i s t a n c e  between each 
r i n g  was computed (F igure  2).  
Due t o  g r ind ing  d e f e c t s ,  some a r e a s  lacked  observable  r i n g s .  An 
e s t ima te  of t h e  number of r i n g s  no t  s een  was obta ined  by d i v i d i n g  t h e  
measured d i s t a n c e  i n  ques t ion  by t h e  q u o t i e n t  of t h e  sum of t h e  p r i o r  
and subsequent r i n g  counts  d iv ided  by t h e  sum of t h e i r  r e s p e c t i v e  
d i s t a n c e s .  For example, us ing  t h e  fo l lowing  d a t a  (from Figure  2) an 
unknown count can be  es t imated:  unknown count and 75 u n i t s ,  p r i o r  count = 
13 r i n g s  and 50 u n i t s ,  and subsequent count = 10 r i n g s  and 60 u n i t s .  
50 + 60 - 75 
unknown count = 75 t 13 + - - = 16 r i n g s  4.8 u n i t s l r i n g  
These e s t ima te s  were recorded i n  p a r e n t h e s i s  on t h e  d a t a  shee t .  This  i s  
t h e  p o i n t  when t h e  l a r g e s t  source  of e r r o r  i s  introduced.  I n  a d d i t i o n  
t o  t h e  aforementioned equipment, a necessary  i tem i n  ma.ny o t o l i t h  
s t u d i e s  i s  imaginat ion.  This  i s  c e r t a i n l y  t r u e  i n  t h i s  s tudy  because 
of t h e  n e c e s s i t y  t o  focus  i n  and out  wh i l e  counting v a r i o u s l y  d i s c e r n i b l e  
r i n g s  which makes them appear  and d i sappea r  i n  t h e  process .  
RESULTS 
Both s a g i t t a e  from each s u b j e c t  f i s h  were ground i n  p r e p a r a t i o n  t o  
be mounted b u t  on ly  67 of t h e  330 bones s e l e c t e d  from t h e  r educ t ion  
f i s h e r y  samples surv ived  long enough t o  be mounted i n  cement. Of those  
67 mounted 22 were considered r eadab le ,  10 were r eadab le  i n  t h e  nuc leus  
a r ea ,  25 had zones of readable  s e r i e s ,  f i v e  were considered anomalous, 
and f i v e  were t o t a l l y  unreadable.  Of t h e  22 which were r eadab le ,  t h e  
r i n g  measurement d a t a  f o r  f i v e  t y p i c a l  bones were s e l e c t e d  t o  be  graphed 
f o r  t h i s  r e p o r t  (F igure  3 ) .  Estimated chronology was back-calculated 
FIGURE 2. Example of data record. Encircled portion representi an area 
of adjustment . 
from the capture data, assuming that each ring counted was a daily incre- 
ment and that none was missed (a poor but necessary assumption). Growth, 
as revealed by the data, is accelerated in two steps for the first year 
or so, which can be expected (Sakagawa and Kimura, 1976), and then assumes 
a variable but moderate rate thereafter, The mean growth rate after 
stabilization was computed and included in the graph along with plus 
and minus one standard deviation (Figure 3). The growth rate for any 
individual bone shows periodic variations beyond one standard devia- 
tion and indeed suggests a cyclic pattern. Some graphs demonstrate an 
acceleration during August each year, which can be expected, but only 
August 1968 and April 1970 are associated with accelerated growth on 
the majority of bones. At any rate it is our conclusion that these bones 
have too much variation and lack enough clearly defined structures to 
say that any spawning marks are present. Another illustration of the 
high variability is the fact that graphs of bones from the same fish are 
not similar in appearance (Figure 3, fish B and C). 
The growth rates of bones from three of the running ripe fish taken 
in 1976 also show inconsistent variations and lack indications of depressed 
growth rates prior to spawning (Figure 4). Aside from this attempt to 
document growth rate patterns, some of the patterns described by Pannella 
(1971, 1974) were evident. Evidence of weekly, fortnightly, and lunar 
patterns can be demonstrated by a frequency distribution of the number 
of rings in each measured group (Figures 2 and 5). While the limits of 
each group to be measured were not chosen towards this end, the pro- 
nounced peaks at seven and 14 rings per group do suggest weekly and 
fortnightly periodicity. Other peaks are probably artifacts of using 
cracks and other irregularities as limits for measurement. 


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NUMBER OF RINGS YER GROUP MEASUKEMENT 
FIGURE 5. Histogram of relative frequencies of number of rings per 
group measurement. Frequencies are totaled from the data 
records from the eight otoliths in Figures 3 and 4. 
DISCUSSION 
One observa t ion  of n o t e  i s  t h a t  o f t e n  t h e  t o t a l  number of r i n g s  
counted d id  n o t  correspond wi th  t h e  age  of t h e  f i s h  ass igned  by t h e  
o t o l i t h  r eade r  i n  1971. This  i s  n o t  s u r p r i s i n g  i n  t h a t  agreement 
between anchovy o t o l i t h  r eade r s  i s  about  68% and s e l f  agreement is  
about 75% (John Sunada, C a l i f o r n i a  Department of F i s h  and Game, pe r s .  
commun. ) . 
I n  t h e  course  of t h e  s tudy  some hypotheses about o t o l i t h  growth and 
spawning r i n g  s t r u c t u r e  were formulated.  The fo l lowing  thoughts  a r e  
supported by t h e  exper iences  of o t h e r  s c i e n t i s t s  (John Hunter,  Gary 
Sakagawa, Nat ional  b r i n e  F i s h e r i e s  Se rv i ce ,  p e r s .  commun.). During a 
f i s h ' s  immature l i f e  s t a g e ,  food which i s  inges t ed  i s  used f o r  mainte- 
nance metabolism and growth. A t  onse t  of ma tu r i t y ,  growth i s  de-emphasized 
and a l a r g e  p o r t i o n  of ex t rametabol ic  energy goes toward development of 
r ep roduc t ive  products .  F luc tua t ions  i n  energy consumed by gonad develop- 
ment i s  probably n o t  r e f l e c t e d  by o t o l i t h  growth a s  much a s  a r e  s easona l  
v a r i a t i o n s  i n  t h e  f i s h ' s  environment. Therefore ,  w e  concluded t h a t  i f  i t  
w e r e  p o s s i b l e  t o  document a spawning check dur ing  a f i s h ' s  l i f e ,  i t  would 
be a t  t h e  onse t  of ma tu r i t y .  This  indeed may account f o r  t h e  dramatic  
drop i n  t h e  growth r a t e s  observed f o r  c e r t a i n  f i s h  dur ing  1967 (F igure  3 ,  
f i s h  A, B y  C ,  and E). However, t h e  p r o b a b i l i t y  of m u l t i p l e  spawnings i n  
t h e  f i r s t  year  of ma tu r i t y  i s  no t  expected t o  be h igh .  
ACKNOWLEDGMENTS 
W e  would l i k e  t o  express  our  thanks t o  t h e  fo l lowing  s c i e n t i s t s  who 
shared wi th  us  t h e i r  knowledge and e x p e r t i s e :  David Kramer, John Hunter,  
and Gary Sakagawa, a l l  w i t h  t h e  Nat iona l  Marine F i s h e r i e s  Se rv i ce  a t  La 
J o l l a .  We a r e  e s p e c i a l l y  g r a t e f u l  t o  Micaela Wolfe f o r  typ ing  t h i s  
r e p o r t  and t o  Herbert  Frey f o r  c r i t i c a l l y  reviewing t h e  manuscript ,  
REFERENCES 
MacGregor, John S. 1968. Fecundity of t h e  no r the rn  anchovy, E"nyrauZis 
rnordax Girard.  C a l i f .  F i sh  and Game, 54(4):281-288. 
Messersmith, James D.  1969. The n o r t h e r n  anchovy (Engrauzis Mordcz) 
and i ts  f i s h e r y  1965-1968. C a l i f .  Dept. F ish  and Game, F ish  Bul l . ,  
(147) : 1-102. 
Pannel la ,  Giorgio.  1971. F i s h  o t o l i t h s :  Dai ly growth l a y e r s  and 
p e r i o d i c a l  p a t t e r n s .  Science,  (173):1124-1127. 
. 1974. O t o l i t h  growth p a t t e r n s :  An a i d  i n  age de termina t ion  i n  
temperate and t r o p i c a l  f i s h e s . :  28-39. I n  Bagenal, T.  D.  ( E d ) .  
The ageing of f i s h .  Unwin Eros.  L td . ,  Surrey,  England. 234 p .  
Sakagawa, Gary T. and Makoto Kimura. 1976. Growth of labora tory- reared  
no r the rn  anchovy, EngrauZis mordax, from southern  C a l i f o r n i a .  F i sh .  
Bu l l .  74 (2)  : 271-279. 
Smith, Paul  E. 1972. The i n c r e a s e  i n  spawning biomass of no r the rn  
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Results of a study to detect spawning marks in otoliths of Northern anchovy, Engraulis mordax

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Results of a study to detect spawning marks in otoliths of Northern anchovy, Engraulis mordax

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