Trophodynamics and Review of methods for Stomach

content analysis of fishes

P.U. Zacharia, Head, Demersal Fisheries Division

Central Marine Fisheries Research Institute, Kochi

Investigation of food and feeding of fishes has traditionally been an important

field of activity in fisheries biology, but it is one in which there are great
difficulties in correlating the results with the research made in the other fields
(FAO, 1974). Investigations of the food of the fish cannot be considered in
isolation but have to be discussed in relation to the whole marine environment, of
which the fish constitute single elements.

Food chains and trophic levels

The production of organic substances (food) by photosynthesis is a process

involving transformation of light energy into potential chemical energy. The
transfer of this food energy from the producers through a series of consumers is
called a food chain, each organism through which it is passed being a link in the
chain.

Three different food chains may be recognized

1. The carnivore chain, where the energy is passed from smaller to larger
organisms.
2. The parasite chain, where the energy is passed from larger to smaller
organisms.
3. The saprophyte chain, where the energy is passed from dead organic
matter to micro-organism in most cases.

In reality food may be passed through parts of all three chains before it is finally
decomposed into inorganic nutrients by the bacteria and fungi found at the end of
every food chain. In other words, the species population within a community or
ecosystem form many food chains which interconnect, anastomose or cross each
other in a complex pattern, which is usually referred to as the food web.

Organisms which belong to the same link of the food chain as counted from the
producer level are said to belong to the same trophic level. Thus the plants
constitute the first trophic level, the herbivores the second, and the carnivores
feeding on herbivores the third trophic level. Secondary carnivores feeding on
third level carnivores belong to the fourth trophic level and so forth. However,
there is a very definite limit to the number of possible links in a food chain, and
consequently also to the number of trophic levels in any ecosystem. The reason
for this is that only about 10 percent of the available energy is assimilated in
passing from one trophic level to the next. At the top of the food chain there are
usually only one or two major predators. The number of species in each trophic

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layer increases with approach to the first layer, giving rise to what is called a
pyramid of numbers. For the major predators introduction of small amounts of
pollutants into the first trophic layer can have fatal consequences because it is
eventually concentrated in them.

Gross production and net production

Only a very small portion of the light energy absorbed by green plants that is
transformed into food energy (gross production) because most of it is dispersed as
heat. Furthermore, some of the synthesized gross production is used by the plants
in their own respiratory processes, leaving a still smaller amount of potential
energy (the net production) available for transfer to the next trophic level.

True production of organic matter takes place only in the chlorophyll-possessing

plants and certain synthetic bacteria, and this has been referred to as the primary
production. Copepods and euphausids, convert plant material into protein that can
be assimilated by the animals which eat them but which themselves could not
exist on plant material. In reality, of course, they only assimilate and store energy
derived from the primary producers. They are called secondary producers, a term
which of course fits animals at higher trophic levels just as well because they too -
although indirectly - utilize the primary production of the plants. The loss of
energy is generally referred to as the respiratory loss because the organisms utilize
the food energy by oxidizing it. Because of the respiratory losses the food chains
cannot be very long and the number of trophic levels in natural communities is
therefore seldom more than four or five and often only three. It also means that
the total amount of food available decreases with increasing trophic level. For this
reason, the largest animals are found feeding on either plants or other animals
which are in a low trophic level as, for example, whales on krill and elephants on
plants.

Studying food and feeding of fishes

The study of the feeding habits of fish and other animals based upon analysis of
stomach content has become a standard practice (Hyslop 1980). Stomach content
analysis provides important insight into fish feeding patterns and quantitative
assessment of food habits is an important aspect of fisheries management. Lagler
(1949) pointed out that the gut contents only indicate what the fish would feed on.
Accurate description of fish diets and feeding habits also provides the basis for
understanding trophic interactions in aquatic food webs. Diets of fishes represent
an integration of many important ecological components that included behavior,
condition, habitat use, energy intake and inter/intra specific interactions. A food
habit study might be conducted to determine the most frequently consumed prey
or to determine the relative importance of different food types to fish nutrition and
to quantify the consumption rate of individual prey types. Each of these questions
requires information on fish diets and necessitates different approaches in how
one collects and analyzes data. Here, we outline qualitative and quantitative
techniques used to describe food habits and feeding patterns of fishes. For a better

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understanding of diet data and for accurate interpretation of fish feeding patterns,
time of day, sampling location, prey availability and even the type of collecting
gear used need to be considered before initiating a diet study or analyzing existing
diet data.

Stomach contents can be collected either from the live or fresh died fish.
Regardless of the method, investigators should ensure that the removal technique
effectively samples all items in the gut. Other wise data will be skewed toward
items that are more easily displaced from the stomach. Alternatively, live fish can
be sacrificed and stomach contents removed for analysis. If fish are to be
sacrificed, they should be preserved immediately either by freezing or by fixing in
formalin. Stomach contents will continue to digest, rendering rapid preservation
of the fish or removed contents necessary to prevent loss of resolution. As in
most fish groups feeding behavior of juveniles and adults vary distinctly attention
should be taken to encounter more samples which will include all size groups of
the particular fish. The specimens either from live or preserved should be
measured to its total length to the nearest 1mm and weight to the nearest 0.1 g.
Cut open the fish and record the sex and maturity stage of the fish. Remove the
stomach and preserve them in 5% neutralized formalin for further analysis. For
the analysis, a longitudinal cut must be made across the stomach and the contents
are transferred into a petri dish. The contents then keep for five minutes to
remove excess formalin and then examine under binocular microscope. Identify
the gut content up to the genus and if possible up to species level depending up on
the state of digestion. Various taxa digest at different rates. As such, all recently
consumed taxa may be present in the foregut but only resistant items remain in the
hindgut. To avoid bias when both easily digested prey and resistant prey are
present, only the immediate foregut (e.g., stomach) should be sampled.

Prey items in fish stomachs are often not intact. Hard parts such as otoliths,
scales, cleithra or backbones have diagnostic, species specific characteristics
useful for identifying prey. Alternatively, partially digested prey may be identified
using unique biochemical methods such as allozyme electrophoresis, or
immunoassays. An important fact assessed by the examination of the stomach is
the state or the intensity of feeding. This is judged by the degree of distension of
the stomach or by the quantity of food that is contained in it. The distension of the
stomach is judged and classified as ‘gorged or distended’, ‘full’, ‘3/4full’,
‘1/2full’ etc by eye estimation.

Fish diets can be measured in a variety of ways. Methods of gut contents analysis
are broadly divisible into two, viz., qualitative and quantitative. The qualitative
analysis consists of a complete identification of the organisms in the gut contents.
Only with extensive experience and with the aid of good references it is possible
to identify them from digested, broken and finely comminuted materials.
Quantitative methods of analysis are three types, viz., numerical, gravimetric and
volumetric. All these types of analysis are widely employed by different workers.
The following outline of methods is based mainly on the reviews by Hynes
(1950), Pillay (1952), Windell (1968), Hyslop (1980) and Chipps et al (2002).

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Quanttatve methods

1) Numerical methods
The numerical methods are based on the counts of constituent items in the gut
contents. The numerical methods have been adapted in different ways to assess
the relative importance of food items and these can be classified under four
distinct heads, viz., a) Occurrence, b) Dominance, c) Number and d) Point
(Numerical) methods.

a) Frequency of Occurrence. Stomach contents are examined and the individual

food organisms sorted and identified. The number of stomachs in which each item
occurs is recorded and expressed as a percentage of the total number of stomachs
examined.
J
Frequency of Occurrence, Oi = i
P

Where, J i is number of fish containing prey i and P is the number of fish with
food in their stomach.

This method demonstrates what organisms are being fed upon, but it gives no
information on quantities or numbers and doest not take in to consideration the
accumulation of food organisms resistant to digestion. For instance, three
organisms in a stomach, say, prawn, rotifers and diatoms, present in the ratio of
1:200:2000 would all be treated by this method as 1:1:1 with reference to the
stomach in question. This method holds good even when there is differential
distribution of various food organisms in the water for the same reason that it is
not biased by size or numbers of organism comprising the food. Many have used
this method as an indicator of inter-specific competition while some utilized this
method to illustrate the seasonal changes in diet composition.

b) Number method. The number of individual of each food type in each stomach
is counted and expressed as a percentage of the total number of food items in the
sample studied, or as a percentage of the gut contents of each specimen examined,
from which the total percentage composition is estimated.

Ni
Percent by number, N i = Q

∑N
i1
i

Where, N i is the number of food category i

This method has been employed successfully by several workers in studies on the
food of plankton feeding fishes where the items can be counted with ease. In the
basic number method, no allowance is made for the differences in size of food
items. So in the studies on the food of fishes other than plankton feeders, the

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number method has very limited use. The counting of comminuted plant matter in
the stomach of fish is impracticable and will not yield correct evaluations. So also
in the analysis of the gut contents of a carnivore which may consist of only one
large sized fish and a couple of small larvae, the counting are of little value
computations. These are summed to give totals for each kind of food item in the
whole sample, and then a grand total of all items. The quotient of these gives the
percentage representation, by number, of each type of food item.

c) Dominance method. Essentially the dominance method is a partial

improvement of the occurrence method, viz., the lack of consideration of the
quantities of the food items present in the stomach, sought to be remedied. The
stomach contents comprising the main bulk of the food materials present, is
determined and the number of fish in which each such dominant food material is
present is expressed as a percentage of the total number of fishes examined. The
percentage composition of the dominant food materials can also be expressed by
this method as in the occurrence method.

Though in an analysis of dominance the bulk of the food material is taken in to

account, it can yield only a very rough picture of the dietary of a fish. More over,
items which are less dominant due to environmental reasons may escape notice.
Though this defect can also be remedied to a certain extent by the examination of
large samples spread over a long period of time, a system of assay that takes in to
account the relative importance of food constituents will obviously be more
suitable in gut content analysis.

d) Points (Numerical) Method. The points method is an improvement on the

numerical method where consideration is given to the bulk of the food items. The
simple form of points method is the one in which the counts are computed falling
a certain organisms as the unit. In a more modified form, the food items are
classified as ‘very common’, ‘common’, ‘frequent’, ‘rare’, etc., based on rough
counts and judgments by the eye. In this arbitrary classification the size of the
individual organisms is also given due consideration. The contents of all stomachs
are then tabulated and as a further approximation, different categories are allotted
a certain number of points and the summations of the points for each food item
are reduced to percentages to show the percentage composition of the diet. This
method is essentially a numerical one; the volume being only a secondary
consideration and it is only in the counts that a certain amount of accuracy can be
claimed.

2) Volumetric methods
Many workers consider the volume as a more satisfactory method for quantitative
analysis of gut contents. As Hynes (1950) pointed out, volume forms a very
suitable means of assessment, this is especially so in the case of herbivorous and
mud feeding fishes where the numerical methods “become meaningless as well as
inaccurate”. Even in cases where the numerical methods are suitable, volume has
been considered as an essential factor to be reckoned with, and in all improved

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numerical methods the volume of the food items is taken in to consideration in
some way or other. The chief methods that are employed in assessing the volume
of food items in the gut contents of fishes are:

a) Eye estimation method: - This is probably the simples and easiest means of
determining the volume of food constituents. In this method the contents of each
sample is considered as unity, the various items being expressed in terms of
percentage by volume as estimated by inspection. This method of analysis is
subjective in nature and the investigators personal bias is likely to influence the
results very greatly. This defect can be minimized to a great extent by the
examination of large samples conducted over a long period.

b) Points (Volumetric) method: - This method is a variation of the eye estimation

method. Here instead of directly assessing the volume by sight as in the previous
method, each food item in the stomach is allotted a certain number of points based
on its volume. Certain workers have taken into account both the size of the fish
and the fullness of the stomach in the allotment of points. The diet component
with highest volume was given 16 points. Every other component was awarded
16, 8, 4, 2, 1 and 0 points depending on the volume relative to the component with
the highest volume. Percentage volumes within each subsample were calculated
as:
Number of points allocated to component α
α = X100
Total points allocated to sub sample

Where,
α is the percentage volume of the prey component α

This method is quite useful for analyzing omnivorous and herbivores where
measuring volumes of microscopic organisms such as diatoms and filamentous
algae are very difficult.

c) Displacement method: - The displacement method is probably the most

accurate one for assessing the volume. The volume of each food item is measured
by displacement in a graduated container such as a cylinder with the smallest
possible diameter for accuracy. This method is eminently suited in the estimation
of the food of carnivorous fishes. But the differential rate of digestion of the food
items may sometimes affect he accuracy of the observations. However, if the
collections are made when the fish are on feed, this defect can be easily
overcome. A knowledge of the volumes of the different size groups of the food
items ay be of great help in estimating the volume of the whole item form the
semi digested fragments

3. Gravimetric method

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 The gravimetric method consists of the estimation of the weight of each of the
food items, which is usually expressed as percentages of the weight of the total
gut contents as in other quantitative methods.

Wi
Percent by weight, Wi = Q

∑W
i1
i

Where, Wi is the weight of the prey i

Generally the wet weigh of the food after removing superfluous water buy
pressing it dry between filter papers is taken for this purpose. Dry weight
estimation is more time consuming and is usually employed where accurate
determinations of calorific intake is required. The limitation of weight as a
criterion of analysis has already been referred in the consideration of the method
of assessing the condition of feed. Besides these, the accurate weighing of small
quantities of food matter is extremely difficult and impracticable in studies of
large collections. This method is, therefore generally employed only in
conjunction with other methods to demonstrate seasonal variations in the intensity
of feeding.

Table: Example of results obtained using different methods of estimation of stomach

contents for two Lactarius lactarius

L. lactarius 1 (LL1). 1. Stolephorus bataviensis, 9 cm long, weight 5 g, volume 7

ml, 6 Acetes each 3.0cm long, weight 300mg vol. 2ml, 1 Bregmaceros ,4cm, 1 g,
vol. 1 ml.
L. lactarius 2 (LL2). 1. Stolephours bataviensis, 7 cm long, weight 3 g, volume 4
ml, 4 Acetes 2.5 cm long, weight 250 mg, vol.1 ml.

L. lactarius 1 (LL1). 1. Stolephorus bataviensis, 9 cm long, weight 5 g, volume 7 ml, 6

Acetes each 3.0cm long, weight 300mg vol. 2ml, 1 Bregmaceros ,4cm, 1 g, vol. 1 ml.
L. lactarius 2 (LL2). 1. Stolephours bataviensis, 7 cm long, weight 3 g, volume 4 ml,
4 Acetes 2.5 cm long, weight 250 mg, vol.1 ml.

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 Food Method Fish % Total of which
LL1 LL2 % expressed
Occurrence
S. bataviensis 1 1 2 40
Acetes 1 1 2 40 All food
Bregmaceros 1 0 1 20 occurrences
Numerical
S. bataviensis 1 1 2 15.4 All food
Acetes 6 4 10 76.9 organisms
Bregmaceros 1 0 1 7.7
Dominance
S. bataviensis 1 1 2 100 All fish
Acetes 1 1 2 100
Bregmaceros 1 0 1 50
Total Volume
S. bataviensis 7 4 11 73.3 Total food
Acetes 2 1 3 20 volume
Bregmaceros 1 0 1 6.7
% volume
S. bataviensis 70 80 75 75 Food volume
Acetes 20 20 20 20
Bregmaceros 10 0 5 5
Gravimetric
S. bataviensis 5 3 8 67.8 Total weight of
Acetes 1.8 1 2.8 23.7 food
Bregmaceros 1 0 1 8.5

Food analysis indices

A. Simple indices
1) Index of fullness. This is measured as the ratio of food weight to body weight
as an index of fullness, which is very widely employed. (The ratio of
corresponding volume can also be used.) This index can be applied to the food in
the stomach, or to that in the whole digestive tract. It is usually expressed as parts
per 10,000 (%00, or parts per decimile); that is:

weight of the stomch contents x 10,000

Fullness index =
weight of fish

2) Index of selection or forage ratio. Most fishes have a scale of preference for
the organisms in their environment, so that some are consumed in large numbers,
others moderately, some not al all. A quantitative index of such differences called
as the forage ratio. A study of the quantities of different organisms available to
the fish is made, and also of the various items in their stomachs; then;

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 s
Selection index = forage ratio =
b
Where, s = percentage representation by weight, of a food organism in the
stomach and b = percentage representation of the same organism in the
environment. The lower limit for this index is 0; its upper limit is indefinitely
large.

3) Index of electivity, Ivlev (1961) proposed a somewhat different quantitative

measure of selection which has been widely used as mean of comparing the
feeding habits of fishes and other aquatic organisms with the availability of
potential food resources in natural habitats. The relationship is defined as
s-b
Electivity index = E =
s+b

The index has a possible range of -1 to +1, with negative values indicating
avoidance or inaccessibility of the prey item, zero indicating random selection
form the environment, and positive values indicating active selection.

B. Compound indices

In an attempt to consolidate the desirable properties of individual diet measures

(e.g., Ni, Wi. Foi), compound indices were developed that combine two or more
measures into a single index. The belief is that compound indices capture more
information than do single component measures (Chipps et al 2002).

1) Index of Preponderance: - (Natarajan and Jhingran, 1961)

This index gives a summary picture of frequency of occurrence as well as bulk of

various food items. It provides a definite and measurable basis of grading the
various food elements. The bulk of food items can be evaluated by 1) Numerical
2) volumetric and 3) Gravimetric methods. As the numerical method is not suited
to the index with the frequency of occurrence it magnifies the importance of
smaller organisms which may appear in enormous numbers. Therefore either
volumetric or gravimetric are best to assess the food items quantitatively. If we Vi
and Oi are the volume and occurrence index of food item i. then,
Vi Oi
Index of preponderance I i = Χ100
∑Vi Oi
Example: The ‘Index of Preponderance’ of food items of Catla catla (Ham.) is
given in the table 2 with rankings in brackets.

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Table 2 : Index of Preponderance (Natarajan and Jhingran, 1961) of adult Catla

Food items Percentage of Percentage of Vi Oi Vi Oi

Χ100
occurrence
( Oi )
volume ( Vi )
∑Vi Oi

Crustaceans 24.5 57.1 1398.95 64.50 (1)

Algae 27.3 24.0 655.20 30.06 (2)
Plants 6.4 8.2 52.48 2.41 (3)
Rotifers 10.8 2.4 25.92 1.19 (4)
Insects 3.6 6.0 21.60 0.99 (5)
Protozoa 0.6 0.3 0.18 0.01 (8)
Molluscs …. …. …… …...
Polyzoa …. …. …… . .…
Detritus 10.0 1.3 13.00 0.60 (6)
Sand and mud 16.8 0.7 11.76 0.54 (7)

∑ 100 100 2179.09 100

According to the index crustacea and algae constitute 1 and 2 ranks in Catla catla.
While third, fourth and fifth places are held by plants, rotifers and insects. In
grading the food elements accidental and incidental inclusions like sand, mud,
etc., may be left out of consideration.

2) Index of Relative Importance (IRI):- Leo Pinkas et al (1971)

This index is an integration of measurement of number, volume and frequency of

occurrence to assist in evaluating the relationship of the various food items found
in the stomach. It is calculated by summing the numerical and volumetric
percentages values and multiplying with frequency of occurrence percentage
value.;

Index of relative importance, IRIi = (% N i +% Vi ) % O i ,

Where, N i , Vi and O i represent percentages of number, volume and frequency of

occurrence prey i respectively.

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Example:. Index of Relative Importance of pelagic preflexion summer flounder,
Paralichthys dentatus larvae (Grover, 1998).

Prey % Ni % Vi % Oi (% N i +% Vi ) % O i %IRI
Tintinnids 28.7 3.3 37.6 1203.2 19.3
Copepod nauplii 20.0 10.2 41.2 1244.24 20.0
Copepodites 16.0 61.4 30.0 2322 37.3
Calanoids 0.6 4.9 2.0 11 0.2
Cyclopoids 0.6 2.0 2.4 6.24 0.1
Copepod eggs 16.0 1.2 34.8 598.56 9.6
Bivalve larvae 12.1 14.8 28.0 753.2 12.1
Invertebrate eggs 3.7 0.9 11.6 53.36 0.9
Other 2.3 1.3 9.2 33.12 0.5

In pelagic preflexion summer (Paralichthy dentatus) larvae, copepodites

composed the bulk of the diet (61.4% Vol, 37.3 % IRI) and formed the most
important prey. Copepod nauplii, the second most important prey, composed
20.0% (N and IRI). Tintinnids, despite being the most abundantly ingested prey
(28.7% N); ranked third in importance at 19.3% (IRI). Bivalve larvae and
copepod eggs were the only other prey that accounted for >1% of the diet, and
together they composed 21.7% (IRI).

References

Chipps S.R and E.J. Garvey 2002. Assessment of Food Habits and Feeding
Patterns, USGS South Dakota Cooperative Fish and Wildlife Research Unit,
Department of Wildlife and Fisheries Sciences, South Dakota State
University, Brookings, SD 57007.
FAO, 1974. Manual of Fisheries Science Part 2 - Methods of Resource
Investigation and their Application. Fish.Tech paper 115.p.255.
Grover, J.J. 1998. Feeding habits of pelagic summer flounder, Paralichthys
dentatus, larvae in oceanic and estuarine habitats. Fish.Bull, 90 (2): 248-257.
Hynes, H.B. N. 1950. The food of the freshwater sticklebacks (Gastrosteus
aculeatus) and Pygosteus pungitius) with a review of methods used in studies
of the food of fishes. J. Anim. Ecol., 19: 36-58.
Hyslop, E. J. 1980. Stomach contents analysis: a review of methods and their
application. J.Fish. Biol, 17:411-429.
Ivlev, V.S. 1961. Experimental ecology of the feeding of fishes. Yale University
Press, New Haven, Conn.
Lagler, K.F. 1949. Studies in freshwater biology, Ann Arbor, Michigan.
Natarajan, A.V and A. C. Jhingran. 1961. ‘Index of preponderance’-a
method of grading the food elements in the stomach analysis of
fishes. Indian J. Fish, 8: 54-59.

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Pillay, TV.R. 1952. A critique of the methods of study of food of fishes. J. zool.
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Pinkas, L., M.S.Olipahnt, and I.L.K.Iverson 1971. Food habits of albacore,
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Seaburg, K.G. 1957. A stomach sampler for live fish. Progre. Fish. Cult. 19: 137-
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Shchoener, T. W. 1970. Non synchronous spatial over lapof lizards in patchy
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Strauss, R. E. 1979. Reliability estimates for Ivlev’s electivity index, the forage
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