Insect marking techniques and use of
mark-release-recapture studies to determine absolute population density
PDF of lab protocol
- Practice various marking techniques on different
kinds of insects and learn their usefulness and limitations.
- Use mark-release-recapture (MRR) technique to
determine absolute population size in a closed environment.
In preparation for
this laboratory exercise, read:
Hagler, J. R., and C. G. Jackson. 2001.
Methods for marking insects: current techniques and future prospects.
Annu. Rev. Entomol. 46: 511-543.
Henderson, P. A. 2003. Mark-recapture
methods for population size estimation, pp. 48-59. In Practical
Methods in Ecology, Blackwell Publishing, Oxford, UK.
If mark-recapture is likely to be an important area
of your research, you may want to get heavily into the math in the
Southwood, T. R. E., and P. A. Henderson. 2000.
Chapter 3 - Absolute population estimates using capture-recapture
experiments in Ecological Methods. Chapman and Hall, New
1. Marking techniques (summarized from Hagler
and Jackson 2001).
There are many reasons to
mark insects, whether individually or as a group, or to allow insects to
self-mark. You may wish to mark insects in laboratory experiments to
enable you to keep track of the behavior of individuals. In the
outdoor environment, marking is important to study insect movement
(foraging, migration and dispersal behaviors), to follow population
dynamics (birth, death and emigration rates), and to estimate population
size. Marking studies in the outdoor environment fall under two
broad categories: mark-release-recapture (MRR) and mark-capture.
In MRR studies, insects may be reared in the lab or collected from the
field. They are then marked, released into the field and recaptured
at a later date. The marked insects can be differentiated from
unmarked wild insects. Alternatively, in mark-capture experiments,
wild insects are marked in the field (without being individually trapped),
usually by mass spraying some component of their habitat or by being
attracted to marked bait that they incorporate into their bodies.
Insect populations are sampled at a later date and some of these
individuals will be marked. Some marking techniques amenable to MRR
studies cannot be used in mark-capture studies in the field because of the
difficulty of applying the mark.
When using any kind of
marking technique it is important that the mark be easy to use, durable,
inexpensive, nontoxic, and readily identifiable. The mark should
have no effects on the insect’s behavior or biology.
The type of marks that can
be used on insects include: tags, mutilation, paint and ink, dust, dye,
pollen, rare or trace elements, radioactive isotopes, genetic markers
(either natural or obtain via genetic engineering), and proteins.
- Tags - Paper, film, small plastic disks,
and wire have all been used to mark insects. Tags are good for
individually marking insects but are very tedious to apply and may not
stick well with any kind of adhesive. They can only be used on
larger insects that can handle the extra weight of a tag.
- Mutilation marking - Mutilation may involve
wing clipping, notching of the pronotum, removal of prolegs or
puncturing or branding of elytra. This technique is only useful on
large-winged or heavily sclerotized insects (beetles, Orthoptera,
butterflies, dragonflies, etc….). The mutilation must not affect
the insect’s normal behavior.
- Paint and ink marking - Paint and ink
materials that make good marks should be “durable, nontoxic, easy to
apply, quick drying, lightweight, available in several highly visible
colors and resistant to peeling and chipping” (Hagler and Jackson 2001).
- Dye marking - Oil-soluble dyes may be
retained in the body (especially fat body) after feeding. They are
used a lot in larval Lepidopterans, termites, adult fruit flies, ants,
etc… Some good dyes are Calco red N-1700, oil-soluble blue II,
rhodamine B, and Nile Blue. They are inexpensive and require
minimal handling of the insect; simply add it to the diet in oil.
Many dyes can be viewed from the outside but some require crushing the
insect and the use of a UV light source.
- Pollen marking - Pollen is a self-marking
substance for mark-capture studies. It is naturally adhesive to
insects and can be used to study migration in moths (remote pollen type)
and diet breadth in many insects. This techniques is relatively
little used because of difficulty in identifying pollen and pollen must
available (right time of year) and from a remote source.
- Genetic marking - Genetic marks can be due
to a visible, naturally occurring mutation (e.g., white-eye in
Drosophila) or an induced (radiation or mutagens) mutation.
Genetic marking could be useful in MRR studies using lab populations
that have many mutations, however it is relatively little used because
mutations may have invisible effects on insect fitness so that marked
insects don’t compete well with wild insects. Genetic marking
cannot be used in mark-capture studies. Genetic markers may be
biochemical such as differences in enzyme banding patterns in different
populations of insects (need polyacrylamide or starch gel
electrophoresis and lots of preliminary lab study to identify specific
marker enzymes that differentiate the populations.)
- Elemental marking (see Qureshi et al. 2004)
- Rare or trace element marking was developed in the 1970s to replace
marking with radioisotopes. The most common trace element mark is
rubidium chloride. Mark is applied by dipping or spraying insects
or by putting the element into artificial diet. The trace element
can also be used for self marking; inject a vertebrate host or spray or
irrigate a host plant with the natural trace element and attract wild
insects to the marked host. A limitation is the expense of some
natural elements and the expense for detection equipment and expertise.
Also there are some adverse biological effects on the insect when
applied at high dosages.
- Protein marking - Insects are marked with
vertebrate-specific proteins. The presence of the mark is
confirmed using sandwich enzyme-linked immunosorbent assay (ELISA) using
vertebrate specific antibodies. Proteins can be applied with a
perfume atomizer or a nebulizer (medical instrument) to the outside of
the insect or given to them in artificial diet. This technique has
only been used for MRR studies so far. The marker proteins and
immunolabeling reagents are relatively inexpensive. Proteins are
persistent in the field and also resistant to light, heat and water.
Protein markers have been used for trophic studies, flow of nectar in
honey bee colonies, and migration of parasitoids.
- Genetically engineered marking -
Transposable elements (P, hobo, Hermes, piggyBac, etc…) are used to
genetically and stably transform insects with visible mutations (such as
white eye or something like that) or to express a fluorescent coloration
such as the green fluorescent protein (GFP) from jellyfish.
Potential use for permanent marking of mass-reared SIT insects.
Advantages of GFP transformation are that it is permanent (in the
genome) and potentially could be present during all lifestages.
2. Use of marking for estimation of absolute
population size (Henderson 2003).
Basic premise - “If a sample from a population is marked, returned to the
original population, and then, after complete remixing, resampled, the
number of marked individuals in the second sample will have the same ratio
to the total numbers in the second sample as the total of marked
individuals originally released have to the total population”.
- Marking has no effect on animal’s behavior or
- Marked insects are completely mixed in the
- Same probability of capturing a marked animal as
an unmarked animal.
- Sampling must be a small proportion of the total
time of the study.
Populations to be estimated
may be open or closed. A closed population does not change during
the duration of the study (no migration, death or natality) so study must
be of short duration. An open population may increase or decrease
during the time interval between release and population estimation due to
a combination of natality, mortality, and migration. Different
methods are used for estimating the absolute density of the two types of
Closed population - most simple way to
estimate a closed population is to use the Petersen-Lincoln index where
all four assumptions are met, population is closed and there is constant
probability of capture.
= estimate of the number of individuals in the
a = total number marked in the first capture
n = total number of individuals in the
recapture (second sample),
r = total recapture of marked individuals
When n is predetermined (e.g., sample until 5
individuals are caught) and approximately equal to a, variance of
The above equations are good for large samples where
r is fairly large (> 20). With small samples, a different
estimate may be better.
with a variance of:
Other simple single recapture methods and those that
involve more than one recapture can be used on closed populations.
Open populations - the population must be
marked on at least two occasions. On the second and subsequent
occasions the recaptured insects are remarked and released again. An
addition assumption necessary for multiple markings is that being captured
one or more times does not affect an insect’s subsequent chance of capture
(i.e., they do not become harder or easier to catch after being captured
methods and equations have been suggested for estimating the absolute
density of open populations (see Southward and Henderson 2000 for
larval density using mark-release-recapture techniques.
We will determine the effect of sample size (n, in
Peterson-Lincoln Index equations) and number of insects marked (a, in
Peterson-Lincoln Index equations), in two different experiments, on our
estimates of the absolute population density of bean bugs in a plastic
box. In a third experiment we will determine how accurate the population
estimation equations are when the absolute population density of insects
A) No. marked (a) varied; No. capture (n) constant
– Casey and Lucy set up four clear plastic boxes filled with wheat bran
with 200 bean bugs each. A different percentage of the population was
marked in each box (5, 10, 20 or 40%); white "bean bugs" are unmarked
whereas red "bean bugs" are marked. Each pair of students will “sample” the wheat bran
and pull out 20 bean bugs (10% of the population) (with eyes closed and in a
haphazard manner) from each of the four densities. Run your fingers
through the flour and pick out a bean bug when you feel one. Some of your
bean bugs will be marked (red beans) and some will not (white beans). Record the number of marked
individuals (red beans) (out of the 20 bean bugs sampled) on the board.
B) No. marked (a) constant; No. captured (n)
varied - Casey and Lucy set up one clear plastic box filled with
wheat bran and 200 bean bugs. This time 20% of the population was marked
(40 individuals) and you will sample 5, 10, 20 or 40% of the population
(10, 20, 40 or 80 individuals). Resample the box in the same manner as
before, first taking 10 bean bugs and recording the number marked (red
taking another 10 (to get 20 total) and recording the number marked, then
taking another 20 and finally another 40 bean bugs. Record the number of
marked bean bugs on the board at each sampling intensity (5, 10, 20 or 40%
of the population sampled).
C) Population size varied; No. marked (a) and No.
sampled (n) constant – Casey and Lucy again set up four clear
plastic boxes filled with wheat bran but this time the boxes held
different populations of bean bugs (50, 100, 200 or 400). The same
number of individuals is marked in each box (20) but the proportion of the
population marked declines with increasing population density (40, 20, 10
or 5%). You will sample 20 bean bug from each box and write the number of
marked bean bugs (red) that you collected on the board.
ease of use, durability and any mortality effects of several
Pair up and choose 3
different insects on which to try at least 3 different marking techniques.
Mark at least 5 individuals of each species with one of the techniques
(you can try more than one technique on different individuals of one
species). Handle 5 control insects in the same manner but do not mark
them. Examine them to make sure that they have been marked.
Put the marked and unmarked insects in different containers with the
appropriate food and moisture source so that they can survive 2 days.
Two days after marking determine the mortality of your control and marked
insects and determine whether the mark is still visible on your marked
adult Blatella germanica cockroaches
banded cucumber beetle
sp. soldier beetles
mealworms (T. molitor larvae)
American bird grasshoppers
Marking materials available:
Paints (oil base)
Paints (water base)
Fabric paint (Polymark - blue, green, yellow, orange,
Acrylic paint (Delta Ceramcoat - green, blue, orange, red, yellow)
Nail polish and correction fluid
Nail polish (color)
Liquid Paper correction
fluid (Paper Mate ledger green, green, pink, ivory, canary yellow, ledger
Liquid Paper correction
fluid (white - multi fluid, bond white, pen & ink)
Rhodamine B (purple)
Nile Blue (blue)
Dry pigments (to be used as dusts)
Day-glo daylight fluorescent (Yellow, orange, red, pink, purple, blue,
USD UV fluorescent (yellow-orange, green, blue, yellow, TV red)
Rich gold dust
Chalk (white, yellow, blue)
ultra-fine markers (black, red, yellow, blue, orange, purple, green,
Circular plastic disks
Pressure sensitive labels
(red, green, yellow, blue)
Fine-tipped scissors (for
Glues or solvents
Rubber cement (Craft Bond)
Elmer’s Glue-All white glue
Clear nail polish (Artmatic)
Super Glue (Loctite)
Instant Crazy Glue
Quick dry tacky glue (Aleene’s)
Glue stick (Duck)
Mineral spirits (to thin paints if necessary)
Ice chest with ice
Petri dishes with cork and fine netting
Netting of various mesh size
Filter paper (9 cm)
Vials, test tubes, cages, etc… for holding
A lab assignment is due October 23rd.
This will be worth 10% of your grade for the lab course. I will
upload the "bean bug" mark-release-recapture data to the web by
October 4th (2013 data at the top of this web page). After I have uploaded the class data, summarize and analyze the data as
outlined below. Also address the questions about the marking techniques
that you chose for the other insects.
larval density using mark-release-recapture techniques.
Using the average number of marked insects captured
in each experiment (average over all replicates), estimate the
absolute population density of each of the plastic boxes using the
Petersen-Lincoln index equations. Use both equations (one for large
sample sizes and one for small sample sizes) and both variance terms.
Give a table of estimated densities (± standard deviation = square root of the
variance) using the two
equations versus actual densities. Which estimates are closer to the
actual densities, the ones from the large sample equations or the ones
from the small sample equations? Which estimates have the
smallest variance? What can you conclude about the effects
of the proportion of the population that you mark and the proportion of
the population that you sample on the accuracy (i.e., its closeness to the
“true” mean) and precision (i.e., repeatability) of your estimate ?
2. Evaluate ease of
use, durability and any mortality effects of several insect-marking
What 3 marking techniques did you choose? What
affected your choice of mark for each particular insect species? How did
you apply the marks to the insects that you chose? What were the
difficulties? Did you learn any tricks on how to handle them? Was there
any mortality associated with your marking technique (either during
marking or in the 2-day holding period afterwards)? Was the mark still
visible 2 days after marking? What insect do you work on? How would you
mark it and why?
Hagler, J. R. 1997. Field retention of a novel
mark-release-recapture method. Environ. Entomol. 26: 1079-1088.
Hagler, J. R., C. G. Jackson, T. J. Henneberry, and
J. R. Gould. 2002. Parasitoid mark-release-recapture
techniques -- II. Development and application of a protein marking
technique for Eretmocerus spp., parasitoids of Bemisia
argentifolii. Biocon. Sci. Technol. 12: 661-675.
Qureshi, J. A., L. L. Buschman, S. B. Ramaswamy, J.
E. Throne, and P. M. Whaley. 2004. Evaluation of rubidium chloride
and cesium chloride incorporated in a meridic diet to mark Diatraea
grandiosella (Lepidoptera: Crambidae) for dispersal studies.
Environ. Entomol. 33: 487-498.
Walker, T. J., and S. A. Wineriter. 1981.
Marking techniques for recognizing individual insects. Fla. Entomol. 64:
Wineriter, S. A., and T. J. Walker. 1984.
Insect marking techniques: durability of markers. Entomol. News 95:
Wojcik, D. A., R. J. Burges, C. M. Blanton, and D. A. Focks. 2000. An improved and quantified method for marking
individual fire ants. Fla. Entomol. 83: 74-78.