PROTEIN PATTERNS FROM LENS OF BLUEBACK
HERRING (Alosa aestivalis) FROM
EASTERN NORTH CAROLINA
by
Jerome B. Leete
APPROVED BY:
SUPERVISOR OF THESIS
THESIS COMMITTEE
CHAIRMAN OF THE DEPARTMENT OF BIOLOGY
Charles E. Bland, Ph.D.
DEAN OF THE GRADUATE SCHOOL
Ï. Y. JOTiMEIÎ IJBBARY
Bast Carolina university;
^0 do-ir
Q.L
dm.
L Sy.
ABSTRACT
Jerome B. Leete. PROTEIN PATTERNS FROM LENS OF BLUEBACK HERRING
(Alosa aestivalis) FROM EASTERN NORTH CAROLINA. (Under the direction
of Charles W. O'Rear, Jr.) Department of Biology, August 1981.
Blueback herring were obtained from four river systems in eastern
North Carolina to determine if the populations were genetically diver-
gent. The proteins of eye lenses were studied using agarose gel
electrophoresis.
The protein patterns were subjected to chi-square analysis to
determine if blueback stocks could be identified by presence or absence
of specific banding patterns. The bands were described for each geo-
graphic location and tested for correlation with sex, year, and age.
Sokal's D values for geographic patterns indicated greatest
similarity among the closest river systems. The degree of divergence
did not allow sub-population identification from lenses proteins.
The banding patterns showed no significant influence when tested
for correlation with sex, year, and age. Length-frequency groupings
were used for age approximations. Further study was suggested using
a less conservative protein source or a combination of sources to gain
more insight than one source would allow.
PROTEIN PATTERNS FROM LENS OF BLUEBACK
HERRING (Alosa aestivalis) FROM
EASTERN NORTH CAROLINA
A Thesis
Presented to
the Faculty of the Department of Biology
East Carolina University
In Partial Fulfillment
of the Requirements of the
Master of Science Degree
in Biology
by
Jerome B. Leete
August 1981
ACKNOWLEDGEMENT
I wish to sincerely thank Dr. Charles O'Rear for his continuing
help as director of this thesis. I wish to thank my committee members,
Dr. Edward Ryan, Dr. James Smith, and Dr. Barney Kane, for their tech-
nical advice and assistance. I would like to thank Mrs. Martha Jones
for her technical assistance. I also wish to thank my wife, Suzanne,
for her continual editing and support.
TABLE OF CONTENTS
PAGE
LIST OF TABLES iv
LIST OF APPENDIX TABLES v
LIST OF FIGURES vi
INTRODUCTION 1
MATERIALS AND METHODS 13
RESULTS AND DISCUSSION 21
SUMMARY 40
REFERENCES 41
APPENDIX 44
LIST OF TABLES
PAGE
1. Percentage occurrence of bands by location, females 24
2. Percentage occurrence of bands by location, males 25
3. Percentage occurrence of bands by location, both sexes ... 26
4. Percentage occurrence of bands by length of the males.
N=74 32
5. Percentage occurrence of bands by length of the females.
N=140 33
6. Taxonomic distances calculated for different sample areas. . 37
LIST OF APPENDIX TABLES
PAGE
1. Pe8197 971991717797 1798 rcentage occurrence of bands by location, females, 452. Percentage occurrence of bands by location, females, 453. Percentage occurrence of bands by location, females, 464. Percentage occurrence of bands by location, males, 1979 .... 475. Percentage occurrence of bands by location, males, 1977 .... 486. Percentage occurrence of bands by location, males, 1978 .... 487. Percentage occurrence of bands by location, both sexes,1979 498. Percentage occurrence of bands by location, both sexes, 509. Percentage occurrence of bands bv location, both sexes, ... 50
10. Percentage occurrence of bands by length of the males,
1977. N=30 51
11. Percentage occurrence of bands by length of the males,
1978. N=27 52
12. Percentage occurrence of bands by length of the males,
1979. N=17 53
13. Percentage occurrence of bands by length of the females,
1977. N=54 54
14. Percentage occurrence of bands by length of the females,
1978. N=55 55
15. Percentage occurrence of bands by length of the feamles,
1979. N=31 56
LIST OF FIGURES
9.Len PAGE1. Blueback herring (Alosa aestivalis) 32. Alewife herring (Alosa pseudoharengus) 43. Study area in coastal North Carolina 144. Pegrittohn,eum color of Blueback herring 165. Peritoneum color of Alewife herring 176. Whole lens and nucleus of (left to right) Alewife herring,Blueback herring, and King mackerel (Scomberomoruscavalla) 187. Examples of lens patterns. Anode is to top of page for each.Application point is slot in film Chowan River, 1977, attop and Neuse River, 1977, at bottom 228. Examples of lens patterns. Anode is to top of page for each.Application point is slot in film. Neuse River, 1978, attop and Roanoke River, 1979, at bottom 23rreque^.cy for 19/.' .ample ¿./
10. Length-frequency for 1978 sample 28
11. Length-frequency for 1979 sample 29
12. Length-frequency for total sample 30
INTRODUCTION
The blueback herring, Alosa aestivalis (Mitchill), is an ana-
dromous fish that, when reaching sexual maturation, returns from the
open sea to spawn in natal streams. Many aspects of its life history
are unknown or sparsely studied. The migration from the sea begins
after four years of existence for most bluebacks (Marcy, 1969). The
spawning migration occurs primarily during the spring in the central
Atlantic area. The months of March and April have the highest activity
for North Carolina. The peak runs for this study area come in early
April with water temperatures between 50° and 60° F.
Most of the spawning activity takes place in freshwater, but
brackish water is also utilized. Mortality of the spawned adults can
be high 'teaching above 50% in some case 'kissil, 1974). Duration on
the spawning grounds varie ,ii seve ys to several weeks for the
adults. After leaving the spawning areas, the adults begin to feed
again and return to the sea (Durbin, ^ 1979).
The blueback fry are highly salinity tolerant and can utilize
either freshwater or saltwater for nursery areas (Chittenden, 1972).
This is a significant advantage for the survival and growth of the
fish by increasing the potential food supply. The fry migrate down-
stream, remaining in the estuaries until fall before leaving for the
ocean (Burbridge, 1974). The adults range from St. John's River,
Florida, to Cape Breton, Nova Scotia (Loesch and Lund, 1977).
The herring fishery is an important source of revenue to commercial
fishermen in North Carolina. The fishery consists of the blueback and
2
the alewife. Alosa pseudoharengus (Wilson), which are not separated in
most catch statistics because of the virtually indistinguishable ex-
ternal morphology of the two fish. See Figures 1 and 2. They are
commonly referred to as river herring. Commercial uses of the river
herring range from human consumption to pet food (McKenzie and Martin,
1975). North Carolina and Virginia annually produce the highest
catches for the Atlantic coast and population parameters for these two
states reflect the condition of the fishery as a whole. In both states,
the fishery has been declining in recent years (Loesch and Lund, 1977).
The largest landings in North Carolina for river herring are from
the Albemarle Sound and its tributaties. Other productive waters
include the Pamlico and Neuse Rivers. North Carolina catches had a
recent peak in 1969 of 19,762,000 pounds with a dollar value of
$304,000. i ' decade of the 1970's proved to be disastrous for the
-erring i;i stry of North Carolina. The 1970 harvest recorded a 42%
drop in poundage and a 36% drop in dollar value from the previous year.
The yields dropped steadily and by 1975, the catch was down to
5,952,000 pounds. The only positive aspect for the fishermen was the
rise in price brought on by world demand for protein. The price in
1969 was 1.5 cents per pound, which increased to 3.9 cents per pound
in 1974. Slight yield increases for 1976-78 were followed by the
lowest catch on record in recent times with a poundage of 5,119,000
pounds for 1979. The increasing price, up to 6.1 cents per pound
brought $314,000 for the fishermen.
The central problem confronting the industry for the decade of
the 1970's was reduction of foreign catch in the Atlantic. The sharp
3
Figure 1. Blueback herring (Alosa aestivalis)
4
Figure 2. Alewife herring (Alosa pseudoharengus)
5
drop in inshore landings coincided with increased offshore harvest.
For relief, the United States negotiated bilateral treaties with
Russia, Poland, and Romania during 1975 and 1976. Catch limits were
set and certain North Carolina coastal waters were closed to these
countries in February and March to protect the herring spawning stock.
The Fishery Conservation and Management Act of 1976 extended the
United States' fishery jurisdiction out to 200 nautical miles. These
actions were successful in reducing the Atlantic catches of river
herring by foreign nations. The low catch in 1979 is thought to be
related to reduced water quality in the Chowan River and Albemarle
Sound. This problem is currently under investigation. These efforts
may help raise the yield and dollar value of the herring catch.
The dollar value does not reflect the value of the river herring
in their ecological role. During their spawning run. rivei" ring
provide nitrogen and phosphorous which increase microbial activity
thus releasing energy to the stream stored in leaf litter (Durbin,
1979). The river herring eggs hatch out at the same time that phyto-
plankton blooms are commonly occurring, leading to an increase in the
zooplankton. Burbridge, 1974, working with blueback fry in the James
River, Virginia, found that on a monthly average, seven zooplankton
species constituted not less than 96% of the plankton and not less
than 92% of the stomach contents. The river herring are an important
forage fish for predators in fresh water and marine environments
(Bigelow and Schroeder, 1953). These ecological roles are important
to community stability.
6
It would be helpful to the understanding and identification of
the herring's ecological benefits if the sub-populations spawning in a
particular stream could be identified. The impact of the spawning run
could be predicted if an estimate of sub-population spawners were
known. Also, fishery management scientists could manage the blueback
population better if sub-populations could be identified. Fishing
regulations could be adjusted to sub-population needs, allowing maximum
harvest without devastating the population.
Several methods are currently in use for sub-population identifi-
cation. Morphological characteristics have been used to identify sub-
populations, but problems are encountered in using this method. An
often found size gradient among fish occurs in which the more northern
member of a species is generally larger than its southern counterpart
(Barlow, 1961). This si variation is based on a tempe re dine
in which lower temperatures produce larger '/idual . the
Southern Hemisphere, the north-south variation in size reverses to
south-north. Also, within populations, there is variation in exprès-
si on of size. For example, similar fish of the same area will vary in
many comparative forms such as weight, size of head, or size of eye
orbit. There will be a high-low range common to the species in which
the measurements taken of the individuals will fall.
Other factors such as sex can influence morphological character-
istics of fish. Pollard and Pichot (1971) in trying to classify fishes
of the genus Spi cara found their work difficult because of the color
variations caused by sexual dichromatism and state of sexual maturity.
These variations and changes make reliance on morphological
7
characteristics for sub-population markers insecure.
Another method employed in identifying fish sub-populations is
meristic counts of serial elements such as finray or vertebrae.
Barlow (1961) found that a slow developing rate in a fish produced
higher meristic counts in fish of the same age. Fish in lower tempera-
ture waters normally have a slower developing rate than warm water
counterparts resulting in meristic differences based upon temperature.
Other stress factors in the fish's environment can alter its rate of
development. An example would be a stream with high rates of sewage
discharge causing depressed values for dissolved oxygen. Stress
factors normally act by slowing development and increasing meristic
counts.
Messieh (1977), working with breeding stocks of alewives and blue-
backs from different areas of the St. John River, st d the groups
by comparison of eight meristic characu He was e to show signi-
ficant differences between the two species. He was not as successful
in differentiating between alewife breeding groups that were in the
same river.
A mean and range must be established for meristic counts and for
morphological studies. A disadvantage in establishing the mean is
that a relatively high sample number is required. This is a problem
in situations where only a small sample is available. Meristic counts
are a valuable research method, but more accurate methods are available
for identification of fish sub-populations.
Use of gene frequencies as a means of identifying populations has
been advanced in recent years by discoveries in molecular genetics
8
involving protein synthesis. One of the methods frequently used in
work involving sub-populations is a biochemical technique, electro-
phoresis. This technique is separation of the proteins in a support-
ing media while subjected to an electrical field. The rate of move-
ment depends primarily on the net electrical charge of the protein.
Also, size and shape of the protein alter its movement. The genetic
mechanisms for the use of electrophoresis in identifying fish popula-
tions centers around the "one gene-one protein" concept or more
accepted as "one gene-one polypeptide." Proteins can be studied for
variability reflecting the variance of the gene and how many forms it
may have, and the frequency of expression of each form.
Lane, £t ^ (1966) reported that the United States Department of
Agriculture was an early user of electrophoresis to identify species of
commercially orepared ;i’h fillets. Ground un muscle tissue was their
protein so "ce. Each _cies produced a unique, reproducible pattern.
Huntsman (1970) was successful in separating three species of Carpi odes
using electrophoretic techniques. Muscle tissue extracts were used
for the protein source. Morgan (1975) was able to distinguish larval
forms of white perch (Morone americana) from striped bass (Morone
saxatilis). He had not been able to do so using morphological or
meristic characters. McKenzie (1973) working with muscle tissue
protein established separate patterns for the blueback and the alewife
but was not able to determine any differences within each species, even
though the collection sites were geographically separated. He concluded
that muscle protein was not a suitable source of protein to study vari-
ation within a species.
9
Blood serum is frequently used in electrophoretic work. Morgan,
e^ ^ (1973) was able to detect intraspecific differences in serum
protein patterns of striped bass from several of the rivers sampled in
the Upper Chesapeake Bay. He felt this method was more accurate than
using meristic or morphological characters. McKenzie and Martin (1975)
worked with serum transferrins from blueback herring collected from
four areas of the St. John River, New Brunswick. The fish samples were
separated by time from geographically different collection points.
They concluded that the serum transferrins were of limited use in iden-
tifying sub-populations of blueback herring in the St. John River.
A problem in using serum proteins is the close relationship between
gene expression and the physiological condition of the fish. This
varies the quantity of an individual protein present in the blood
'
Ti. The concentrations of protein present may be altered by
vcr-.ous activities as feeding or spawning. Environmental factors may
stimulate quantitative changes in serum proteins. Booke (1964) found
eleven factors, physiological and environmental that effected the serum
proteins of fish. Dorfman (1973) reported that stressful methods of
capture could alter serum proteins. These possible influences make
precise interpretation of serum protein patterns difficult. Large
samples are needed because of these variables. Tissues containing
proteins that are less variable are desirable to use as sub-population
markers.
The eye lens of a fish provides such a tissue for use in sub-
population identification. The lens has no contact with the blood
10
circulatory system and is not as subject to alteration from physiologi-
cal or environmental influences. The lens is thirty-five percent
protein and ninety percent of these are water soluble (Bloemendal,
1977). The conservative trend of the lens is especially emphasized in
the lens' nucleus. Proteins found in the nucleus are present from the
time the fetus was formed (Bloemendal, 1977).
The nucleus is frequently isolated and used alone in searching
for genetic intraspecific variation in protein patterns. The cells of
the nucleus do not undergo mitotic activity after formation and are
buffered from contact with surrounding media, such as aqueous or
vitreous humors, by the lens cortex. This buffering from other
protein systems and lack of mitotic activity produce a source of
proteins that is not readily subject to qualitative or quantitative
change. These.oroteins are resistant to dénaturation (Smith ^od
Goldstein, 1967). Saunders and McKenzie (1971) used eye lens nuclei
in electrophoretic work with Arctic char. They found inter-population
variation in protein patterns and suggested that these variations
could identify sub-populations of char. Smith (1971) obtained best
results with eye lens nucleus while working with the rock fish family,
Scorpaenidae. He considered the nucleus to be of value in further
study of this family.
In contrast to isolating the nucleus, many researchers prefer to
use the entire lens. The outermost layer of the lens is the only site
of mitotic activity. This activity decreases as the fish ages, except
when the lens is injured. The outer section of the lens, or cortex,
does not contain proteins from blood or muscle systems. The proteins
of the cortex are more subject to environmental influences than the
nucleus but are more conservative than either blood or muscle protein
systems.
Cobb, et ^ (1968) used the entire eye lens and worked with
thirteen different species of saltwater fish ranging from Atlantic
croaker (Micropogon undulatus) to bonnethead shark (Sphyrna tiburo).
The analyzed pattern of their eye lens proteins showed each species
identifiable from the results. Pollard and Pichot (1971) selected the
eye lens for its protein constancy to identify three forms of the
genus Spiraca. Eckroat (1971) separated brook trout (Salvelinus
fontinalis) that came from different sample sites. He employed the
eye lens as a protein source. Brassington and Fergusson (1975) iden-
tified hybrids of roach, rudd, and bream on the basis of their protein
from the entire eye lens.
A statistical method usable to analyze presence or absence fre-
quencies in protein banding patterns was developed by Sokal (1961).
Sokal developed a distance coefficient computation for measurement of
similarities between pairs of taxa. Equally weighted characters of
observed values must be used in his formula to compare pairs of taxa.
Weinstein and Yerger (1976) employed Sokal's taxonomic distance
measure to compare protein pattern results among four sub-populations
of seatrout. Values for the calculations were obtained from the per-
centage occurrence of the observed protein bands. As taxonomic dis-
tance between compared pairs increases, the calculated values increase.
The whole lens provides more protein bands for study than the
12
isolated nucleus. It is relatively constant and less variable than
protein systems from either the blood or muscle systems. The lens is
easy to remove from fish and requires little more equipment than a
scalpel and containers. Its compact size makes possible storage of
samples in a small area.
The objective to be accomplished is the identification of sub-
populations of blueback stocks. The specific objectives are to:
identify stocks by presence or absence of specific banding patterns
using eye lens as the protein source; describe banding patterns from
various sample areas; correlate sex, year, and size with any bands
they influenced.
MATERIALS AND METHODS
Collections of spawning blueback herring were made in the spring
of 1977, 1978, and 1979. The herring were sampled for these three
years from the Roanoke River, Meherrin River, Chowan River, and the
Neuse River. Single samples were taken in 1977 from the Albemarle
Sound and Blounts Creek in Camden County (see Figure 3). Data derived
from samples for these last two collection sites was determined to be
of less significance than data from the other sample locations. This
was done because of the small sample size from the same year. The
Meherrin, Chowan, and Roanoke Rivers are all tributaries of the Albe-
marie Sound. Blounts Creek is a tributary of the Pasquotank River
which also flows into the Albemarle Sound. The Neuse River is a
tributary of the Pamlico Sound and is geroraphically separately from
the other sources. Tables '' through 3 give locations and s of
fish sampled by sex and year. The herring were purchased from net
fishermen who caught the fish in gill nets. Samples were also obtained
for me and kept frozen by employees of the North Carolina Division of
Marine Fisheries at Elizabeth City.
The non-herring species sampled consisted of gray trout, croaker,
and bluefish. Single samples were taken for the study in the summer of
1978. These fish were caught by hook and line in the Pamlico Sound
near the vicinity of the Neuse River entrance buoy.
With fresh fish, preparation was done primarily in the field. The
fork length of the fish was measured on a board with a built-in metric
ruler. The fish were sexed by applying palm pressure to the fish's
14
2. Chowan River
3. Neuse River
4. Meherrin River
15
abdomen and observing the vent for release of milt or eggs.
The bluebacks were identified from the alewives by opening the
body cavity and checking the abdominal peritoneum for color, noting
that bluebacks have a bluish-black peritoneum, and alewives have a
pink peritoneum (see Figures 4 and 5).
The entire eyeball was then removed and dissected for the lens
(see Figure 6). Both lenses were placed in a glass vial, then on ice,
and transported to the lab. In the lab, the lenses were processed
immediately or frozen for later use. Freezing the lenses did not
alter their protein patterns which was in agreement with published
literature (Brassington and Freguson, 1976), (Utter and Folmar, 1978),
Weinstein and Yerger, 1976).
The Marine Fisheries employees froze the fish they collected.
Th'. frozen fish were pirked up and transported to thp lab or ice í'nd
ixópt frozen until processed. When required for use, these samples
were thawed at room temperature and immediately processed. There was
no refreezing of the lens. Once defrosted, the fish were subjected to
the same procedure as the non-frozen samples, except that all the work
was done in the lab.
The first step in lens preparation was a wash in distilled water,
repeated three times, to remove any protein sources attached to the
outside. Next, I added one milliliter of 0.018 percent saline solu-
tion to a test tube and placed the lens from one fish in the solution.
This saline solution helped extract water soluble proteins and caused
no interference in their patterns (Smith and Clemens, 1973). This was
16
Figure 4 Peritoneum color of Blueback
herring
17
Figure 5. Peritoneum color of Alewife
herring
18
Figure 6. Whole lens and nucleus of
(left to right) Alewife herring,
Blueback herring, and King
mackerel (Scomberomorus cavalla)
19
followed by cell destruction with a manual tissue grinder. After
grinding, the mixture was sealed and placed in a cold room on a shaker
for twenty-four hours to enhance extraction of water soluble proteins.
When removed from the shaker, the solution was centrifuged for five
minutes at twelve hundred revolutions per minute and the supernatant
was used for the sample.
The system used to run the samples was the Corning-ACI Agarose
Film/Cassette System using Universal Electrophoresis Film Agarose.
This film utilizes Agarose as the media through which the proteins
migrate and is imprinted with eight slots for sample application.
One microliter of sample was applied to each slot using the Corning
ACI microliter sample dispenser and disposable sample tips. This
system was calibrated to deliver one microliter of sample. The power
source was designed to oroduce a constant ninety volts. The samples
were run for forty minutes. Longer running times expanded and
lightened the bands making them difficult to read. Shorter running
times kept the bands close together and caused severe masking of the
proteins. The buffer used was Barbitol Buffer with EDTA and a pH of
8.6. Amido Black lOB stain was used to mark the location of the
proteins on the film after running, and excess stain was removed with
a five percent acetic acid wash. The film was then allowed to dry.
Proteins were studied on the basis of electrophoretic migration.
The migration distance of the protein bands were read visually by
using the film as an overlay on one millimeter graph paper. Distances
were recorded in millimeters. Protein appearance frequencies have
been treated statistically to determine degrees of similarity between
20
populations of a species by the author.
A zone electrophoretic method utilizing an agarose gel media was
chosen by me for separating the lens proteins. This method allowed a
wide range of application with basic apparatus that could be operated
by technicians without long periods of training. There should be less
technician error and less technician bias introduced in a method of
this sort. Expenses from supplies were lower than for many electro-
phoretic systems. The speed of this method was high allowing greater
sample numbers to be run and less technician time devoted to the
mechanics of the system.
Computer key punch cards were prepared for each fish recording all
data for statistical analysis by computer. Statistical Analysis
System (SAS 76, 1976), and the Statistical Package for the Social
Sciences (SPS'' 1976) were used for analysis. Sokal's method was used
L.. "jmpare ta/. ..omic distance between sample sites.
RESULTS AND DISCUSSION
The lens protein patterns for the blueback herring produced a
maximum number of eight bands and a minimum number of seven. All bands
were not distinct in each fish. The bands migrating the least stained
darkest, and those migrating farthest were the lightest. The bands
with highest consistency in staining intensity and distance traveled
were three bands migrating toward the negative pole. These proteins
possessed a net positive charge. They were present in 100 percent of
the samples run and displayed little variation in band characteristics.
The other bands migrated toward the positive pole, representing a net
negative charge for the protein. Of these five bands, the band
closest to the point of sample application showed highest consistency
in staining. Bands occurring in nearly ‘'00 percent of the samples for
all locations were of leas, interpretive value in statis analy
See Figures 7 and 8 for typical patterns.
The protein patterns were compared by collection location and sex.
Data are presented in Tables 1 through 3. Summaries are given by each
sex and for both sexes combined. Frequency of occurrence for bands
one through four were consistent for all locations and for both sexes.
Bands firve through eight displayed more variation based on presence or
absence.
For age analysis of protein patterns, data was grouped by sex and
by size. Figures 9 through 12 are the length frequency histograms.
The bluebacks were separated by age and by sex, followed by a summary
for the three years. Herring were sorted by size to determine
22
Figure 1. Zxaaples of lens patterns. fjiode is to top
of po-ge for each. Application point is slot
in film. Chowan River, 1977, o-t top and
ITeuse River, 1977, at bottom.
23
8
7
D
5
4
1
2
3
Figure 8, Examples of lens patterns. -'node is to top
of page for each. Application point is slot
in film. House River, lt78, at top and
Ro-uioke River, 1979» at bottom.
Table 1. Percentage occurrence of bands by location, females.
Location Band Band Band Band Band Band Band Band N
1 2 3 4 5 6 7 8
Roanoke River 100 100 100 98 64 88 69 64 42
Chowan River 100 100 100 100 33 74 67 85 17
Neuse River 100 100 100 100 52 100 83 93 27
Meherrin River 100 100 100 100 91 36 91 36 22
Albemarle Sound 100 100 100 100 100 100 0 88 8
Blounts Creek 100 100 100 100 67 67 17 42 12
Table 2. Percentage occurrence of bands by location, males.
Location Band Band Band Band Band Band Band Band N
1 2 3 4 5 6 7 8
Roanoke River 100 100 100 100 53 90 58 47 19
Chowan River 100 100 100 100 13 47 87 67 15
Neuse River 100 100 100 100 61 83 100 100 18
Meherrin River 100 100 100 100 100 50 93 29 14
Albemarle Sound 100 100 00 100 100 100 0 75 4
Blounts Creek 100 100 100 100 0 100 50 0 4
ro
cn
Table 3. Percentage occ.'?"''ence of bands by location, both sexes.
Location Band Band S' - .. •’ Band Band Band Band Band N
1 2 4 5 6 7 8
Roanoke River 100 100 100 98 61 89 66 59 61
Chowan River 100 100 ICO 100 26 64 74 79 32
Reuse River 100 100 ICO 100 55 94 89 96 45
Meherrin River 100 100 100 100 94 42 92 33 36
Albemarle Sound 100 100 ICO 100 100 100 0 83 12
Blounts Creek 100 100 ICO 100 50 75 25 31 16
ro
CTi
Length-frenuency for 1977 sample.
40
35
30
25
20
15
10
5
55
Male
35
N=30
30
25
20
L5
10
5
t t
21 22 23 24 25 26 27 28
Fork length (cm)
Figure 10, Length-frequency for 1978 sample.
(F%requen)cy
Fork length (cm)
re 11. Length-freauency for 1979 sample.
40
Female
35 N=31
30
25
20
15
10
5
Male
50
N=17
45
25
20
15
10
5
1 1 [
23 24 25 26 27 28
Fork length (cm)
30
re Length-frequency for total sample.
40 Male
N=74
35
30
25
20
15
10
5
40 Female i
N=140
35
30
25
20
15
10
5
?+-
21 29
Fork Length (cm)
31
approximate age according to the data of Holland and Yelverton (1973).
The major portion of the herring were estimated to be four to six years
old.
Tables 4 and 5 present the percentage occurrence of bands by
length of fish. The appendix tables separate the fish by sex and by
year of capture. Variance in terms of present or absent began with
band five. The female values for percent present were not as variable
as the male. The males' presence values ranged from 40 to 100 percent.
The band showed no significant relationship to age.
The range of frequency of occurrence of band six did not differ
greatly by sex. It was present in all samples for males either twenty-
two or twenty-six and above centimeters in length. Band six showed a
nonsignificant relationship with age, as did bands seven and eight.
Band seven . especially consistent for f.vslis ranging frnm a per-
centage present of 61 to 71 to,’ ¡1 size c.asses.
Protein patterns for non-blueback species in this study produced
interspecific variation that permitted species identification for fish
utilized. Cobb ^ al_ (1968) used lens protein patterns to identify
thirteen saltwater fish. Croaker and trout were included in their
study. The soluble protein distributions for each fish were character-
istic for the species. The limited croaker, bluefish, and trout samples
in my work were used primarily as a check on technique, procedure, and
sensitivity of the method employed.
The literature on the homing ability of the blueback and alewife
herring shows that these fish return to their natal streams for spawning.
32
Table 4. Percentage occurrence of bands by length of the males. N=74
Length (cm)
22 23 24 25 26
Band No. N=4 N=30 N=30 N=9 N=1
1 100 100 100 100 100
2 100 100 100 100 100
3 100 100 100 100 100
4 100 100 100 100 100
5 100 50 43 89 100
6 100 73 67 78 100
7 75 67 93 89 100
8 25 37 77 89 100
33
Table 5. Percentage occurrence of bands by length of the females.
N=140
Length (cm)
Band 22 23 24 25 26No. N=21 N=37 N=34 N=48
1 100 100 100 100
2 100 100 100 100
3 100 100 100 100
4 100 97 100 100
5 IP. 62 n 67
6 81 70 74 88
7 29 49 82 85
8 43 46 88 85
34
Bigelow & Schroeder (1953) reported that alewife runs could be intro-
duced into suitable unpopulated rivers by allowing gravid adults to
spawn upstream. Their work showed that the offspring of the adults
returned 3 to 4 years later upon reaching sexual maturity. This would
support a home stream capability for the herring. Durbin, Nixon, and
Oviatt (1979) in a paper on the impact of the spawning alewives on
nutrient cycles found the homing ability of the alewife to be similar
to the salmon in returning to natal waters. Thunberg (1971) tested
alewives for their ability to select home waters from other waters
that may or may not have supported an alewife run. The other waters
were selected from streams in the same geographic area as the home
stream. In his results, the alewives were able to detect and select
water from their natal streams. These results support the concept of
hrming abil" for the alewives (197' ' ( ! a twc year study of
alewife spawning movement at Lake .lattamuskeeu. North Carolina. The
lake is connected to the Pamlico Sound by four separate canals. For
the two year study, 97.5% of the fish taken for samples were from the
same canal. All four canals were set up in a similar manner for
sampling. The canal being almost exclusively used by the spawning
herring for access to Lake Mattamuskeet is also the oldest of the four
canals. This is consistent with a homing theory as a possible explana-
ti on.
Messieh (1977) used meristic counts as a basis for a study includ-
ing the homing ability of the alewives. Eight meristic characters were
examined and analyzed. More separation was found between groups of
35
fish during spawning than before spawning. Messieh felt that with the
amount of overlap he found that the alewife was not as specific in
homing to a smaller brook or stream as the shad or salmon, after enter-
ing the parent stream.
As a summary of the works presented, the evidence strongly sug-
gests that the blueback and alewife do return to home streams for
spawning. These fish have some mechanism for imprinting the home
stream waters because a minimum of three to four years pass before the
fish ever return to spawn.
Much of the home stream theory work has been done with alewives,
but with the similarities of the alewives and bluebacks, I believe that
the homing ability of the blueback would be equivalent to the alewife.
The blueback herring spawning runs, in returning to natal waters,
provide» a genetic basis for the possible development of discrete local
popui. ?> ).is of herring that are discernible from other blueback popu-
lations. Any spawning isolation would be a result of natal waters
serving as a boundary. Sharper boundaries would tend to heighten dif-
ferences in local populations while crossover would tend to diminish
differences.
As stated earlier, proteins selected for study in this paper came
from the lens. The conservative nature of the lens proteins lends
credence to any differences found in the protein patterns being genetic
with little environmental influence. Environmentally influenced
proteins could change rapidly and be affected by short periods of high
stress such as capture methods or low amounts of dissolved oxygen in
the water.
36
An application of Sokal's taxonomic distances was used to compare
herring from the Chowan, Neuse, Meherrin, and Roanoke Rivers. Values
utilized were percent occurrence of lens proteins for fish from each
location. This data appears in Table 3 (bands five through eight),
and the results of the analysis are in Table 6. Based on the distance
values in Table 6, the protein patterns in fish from the Chowan and
Meherrin Rivers were most similar. This could possibly be explained
by the closeness of the two rivers as the Meherrin is a tributary of
the Chowan. A chance for overlap and intermingling would exist between
the populations of these two rivers as the fish spawning in the Meherrin
must first traverse the greater part of the Chowan. The Neuse is the
most geographically isolated of the river systems utilized. It is a
tributary of the Pamlico Sound. The other three rivers fees into the
Albemarle Sound. This isolation provides for less po? ility of
intermingling between fish in the Neuse and those of the Roanoke,
Chowan, or Meherrin.
The calculated distance values correlate with geographic distance,
increasing in value as the compared taxa grow farther apart. Geographic
distance can be a genetically isolating mechanism resulting in less gene
exchange between populations which allows for greater variance in gene
frequencies between populations of a species. Isolation and genetic
drift could account for the Neuse samples being the most divergent.
Although Sokal's test indicated possible divergent trends in the popu-
lations, no protein bands were present or absent in a pattern that
allowed positive population identification from the sample areas.
37
Table 6. Taxonomie distances calculated for different sample areas
Roanoke Chowan Meherrin Neuse
River Ri ver River River
Roanoke
River ... 2.0040 2.543 3.8800
Chowan
River 1.2099 3.1323
Meherrin
River 3.2308
38
Another possible interpretation for the found degree of divergency
in the patterns is that they resulted from likeness of environmental
factors in the streams. Weinstein and Yerger (1976) suggested this as
a possibility in their sea trout comparisons using serum proteins.
This explanation is less plausible for the herring because of the con-
servative nature of the lens proteins.
The protein bands were also subjected to analysis in length-
frequency classes. Length-frequency grouping is often used as an age
approximation for fish. Eckroat (1971) worked with lenses of 9
natural populations of brook trout (Salvelinus fortinalis). The
protein patterns were found to be independent of age using length-
frequency groupings.
Eckroat and Wright (1969) worked with brook trout proteins and
found the results not to be influenced by age of Mi fish. Hasler
and Wright (1967) studied the serum proteins of white bass (Roccus
chrysops), and the results were examined for age-dependency relation-
ships. The serum proteins, less conservative than lens proteins, were
found not to be influenced by age.
Using chi square analysis and length-frequency groupings for the
blueback, there was no significant correlation between age and presence
or absence relationships of the protein patterns.
The blueback lens patterns were tested for any significant in-
fluence of sex on the bands. No significant differences appeared in
the protein bands that were attributed to sex.
Hasler and Wright (1967), in the study referred to earlier, tested
39
for sexual influences on serum protein bands in the white bass.
Presence or distance of migration of the bands was not affected. One
band present in males and females, stained darker in spawning females.
Huntsman (1970) compared results for sex differences in protein
patterns of the northern hogsucker (Hypentelium nigricans) and the
river carpsucker (Carpiodes carpió). No observable differences were
found.
The results of the blueback study have indicated that lens pro-
teins are too conservative for utilization as a population marker with
this system of electrophoresis. The protein patterns do not allow
natal stream identification of individual fish but would identify
groups of fish. The group samples of bluebacks would have to spawn in
one of the streams sampled and their protein pattern percentages
bracketed by data of this study. The differences in the protein
patterns may be slight because of some degree ov inter-mingling of
spawning sub-populations. This would reduce genetic variation by
increasing the gene pool available to the spawning herring. Geogra-
phic analysis by Sokal's test has indicated that some degree of popu-
lation divergence does exist.
Further study is needed before the extent of this divergence can
be determined. Protein sources more influenced by factors such as
environment may be desirable for study. Examples are muscle tissue
or serum. The use of more than one protein source from each fish may
be beneficial. The combination of results could be compared by
statistical analysis for ascertaining the degree of divergence among
the blueback populations.
SUMMARY
The geographically distributed protein patterns, tested by Sokal's
test for taxonomic distance, showed some degree of divergence for the
sample areas. D values for the closest river systems were lowest and
d values for the farthest separated river systems were highest.
Length-frequency groupings, representing age brackets, had no
significant correlation with the protein patterns. None of the bands
were correlated with the sex of the fish. Year and location of capture
had no significant correlation with the protein patterns.
The croaker, trout, and bluefish patterns were species specific
allowing identification by the patterns. The lens protein system is
too conservative to allow determination of the extent of some divergence
in the sample areas.
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42
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APPENDIX
45
Appendix Table 1. Percentage occurrence of bands by location.
females, 1977
Location Band Band Band Band N
5 6 7 8
1-Roanoke River 21 79 14 0 14
2-Chowan River 100 100 0 89 9
3-Neuse River 0 100 0 60 3
4-Meherrin River 100 100 100 0 6
5-Albemarle Sound 100 100 0 88 8
6-Blounts Creek 67 67 17 42 12
/ opendix Table 2. Percentage : 'rrence of bands by location,
’ 'f .les , "'
Location Band Band Band Band N
5 6 7 8
1-Roanoke River 83 91 96 96 23
2-Chowan River 0 0 100 86 7
3-Neuse River 100 100 100 100 15
4-Meherrin River 80 10 80 80 10
46
Appendix Table 3. Percentage occurrence of bands by location,
females, 1979
Location Band Band Band Band
5 6 7 8
1-Roanoke River 100 100 100 100
2-Chowan River 0 100 100 82
3-Neuse River 0 100 100 100
4_^ i^-^rrin River 100 100 0 6
47
Appendix Table 4. Percentage occurrence of bands by location, males,
1979
Location Band Band Band Band
5 6 7 8
1-Roanoke River 100 100 100 100
2-Chowan River 0 100 100 60
3-Neuse River 0 100 100 100
4-Meherrin River 100 50 1 Tí 0 2
48
Appendix Table 5. Percentage occurrence of bands by location. males,
1977
Location Band Band Band Band N
5 6 7 8
1-Roanoke River 0 90 20 0 10
2-Chowan River 100 100 0 0 2
3-Neuse River 100 0 100 100 3
4-Meherrin River 100 86 86 14 7
5-Albemarle Sound 100 100 0 75 4
6-Blounts Creek 0 100 50 0 4
Appendix Table 5. Percentage occurrence cf bands by locatio;'1, nidljS,
1978
Location Band Band Band Band N
5 6 7 8
1-Roanoke River 100 100 100 100 6
2-Chowan River 0 0 100 88 8
3-Neuse River 100 100 100 100 8
4-Meherrin River 100 0 100 60 5
49
Appendix Table 7. Percentage occurrence of bands by loation, both
sexes, 1979
Location Band Band Band Band
5 6 7 8
1-Roanoke River lOO 100 100 100
2-Chowan River 0 100 100 64
3-Neuse River 0 100 100 100
^^-Meherrin River 25 100 8
50
Appendix Table 8. Percentage occurrence of bands by location, both
sexes, 1977
Location Band Band Band Band N
5 6 7 8
1-Roanoke River 12 84 17 0 24
2-Chowan River 100 100 0 73 11
3-Neuse River 50 50 50 80 6
4-Meherrin River 100 93 93 8 13
Appendix Table 9. Percentage ioccurrence of bands by location. both
sexes, 1978
Location Band Band Band Band N
5 6 7 8
1-Roanoke River 87 93 97 97 29
2-Chowan River 0 0 100 87 15
3-Neuse River 100 100 100 100 23
4-Meherrin River 87 7 87 73 15
51
Appendix Table 10. Percentage occurrence of bands by length of the
males, 1977. N=30
Length (cm)
22 23 24 25
Band No. N=4 N=17 N=7 N=2
1 100 100 100 100
2 100 100 100 100
3 100 100 100 100
4 100 100 100 100
5 100 53 29 100
"1ÜU 82 /I 50
7 75 24 71 50
8 25 18 29 50
52
Appendix Table 11. Percentage occurrence of bands by length of the
males. 1978. N=27
Length (cm)
23 24 25 26
Band No. N=5 N=14 N=7 N=1
1 100 100 100 100
2 100 100 100 100
3 100 100 100 100
4 100 100 100 100
5 80 57 86 K
6 0 50 U
7 100 100 100 100
8 60 93 100 100
53
Appendix Table 12, Percentage occurrence of bands by length of the
males, 1979. N=17
Length (cm)
24 25
Band No. N=8 N=9
1 100 100
2 100 100
3 100 100
4 100 100
5 25 33
6 100 89
7 100 100
8 63 89
54
Appendix Table 13. Percentage occurrence of bands by length of the
females, 1977. N=54
Length (cm)
23 24 25 26
Band No. N=19 N=22 N=7 N=6
1 100 100 100 100
2 100 100 100 100
3 100 100 100 100
4 100 96 100 100
5 79 64 43
6 84 86 86 100
7 21 23 14 0
8 42 32 57 67
55
Appendix Table 14. Percentage occurrence of bands by length of the
females, 1978. N=55
Length (cm)
24 25 26
Band No. N=6 N=18 N=31
100 100 100
100 100 100
100 100 100
100 100 100
83 67 81
17 o n
* J
67 lOO 97
8 67 94 97
56
Appendix Table 15. Percentage occurrence of bands by length of the
females, 1979. N=31
Length (cm)
23 24 25 26
Band No. N=2 N-9 N=9 N=ll
1 100 100 100 100
2 100 100 100 100
3 100 100 100 100
4 100 100 100 100
5 50 44 1'. 46
6 50 67 100 91
7 100 100 100 100
8 50 67 100 64