Insect Ecology

ENY 6203 and ENY 6203L



Insect marking techniques and use of mark-release-recapture studies to determine absolute population density

PDF of lab protocol

2014 Data


  1. Practice various marking techniques on different kinds of insects and learn their usefulness and limitations.
  2. 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. PDF

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 following paper:

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 York.  

Background information

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”. 

Basic assumptions

  1. Marking has no effect on animal’s behavior or survival.
  2. Marked insects are completely mixed in the population.
  3. Same probability of capturing a marked animal as an unmarked animal.
  4. 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 populations. 

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 population,

a = total number marked in the first capture (sample),

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 estimate is: 

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 once). 

        Various methods and equations have been suggested for estimating the absolute density of open populations (see Southward and Henderson 2000 for details):

  • Fisher-Ford method

  • Bailey’s triple-catch method

  • Jolly-Seber stochastic method

Laboratory exercises 

1.  Estimate "bean bug" 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 varies.

A) No. marked (a) varied; No. capture (n) constant – 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 - 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 ones), then 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 – 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.

2.  Evaluate ease of use, durability and any mortality effects of several insect-marking techniques. 

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 insects. 

Insects available:

  • American cockroaches
  • termite workers
  • housefly adults
  • mosquitoes
  • Chilagnathus sp. soldier beetles
  • Tenebrio molitor beetles
  • mealworms (T. molitor larvae) 
  • mole crickets
  • slugs
  • pepper weevils
  • big-eyed bugs
  • aphids
  • butterflies

Marking materials available:

Paints (oil base)

  • UniPaint markers (orange, blue, red)

Paints (water base)

  • Fabric paint (Polymark - blue, green, yellow, orange, red)

  • 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 buff, blue)

  • Liquid Paper correction fluid (white - multi fluid, bond white, pen & ink)


  • Stamp pad (black) and stamps

  • Waterproof drawing ink (orange)


  • Rhodamine B (purple)

  • Nile Blue (blue)

Dry pigments (to be used as dusts)

  • Day-glo daylight fluorescent (Yellow, orange, red, pink, purple, blue, green)

  • USD UV fluorescent (yellow-orange, green, blue, yellow, TV red)

  • Rich gold dust

  • Chalk (white, yellow, blue)


  • Sharpie permanent ultra-fine markers (black, red, yellow, blue, orange, purple, green, brown)

  • Colored thread

  • Circular plastic disks (red)

  • Pressure sensitive labels (red, green, yellow, blue)

  • Fine-tipped scissors (for mutilation)

Glues or solvents

  • Rubber cement (Craft Bond)

  • Elmer’s Glue-All white glue

  • Clear nail polish (Artmatic)

  • Super Glue (Loctite)

  • Instant Crazy Glue

  • Patch Attach

  • Quick dry tacky glue (Aleene’s)

  • Glue stick (Duck)

  • Mineral spirits (to thin paints if necessary)


  • Hamilton paint pots

  • Artists’ brushes

  • Swab sticks

  • Insect pins

  • Straightened paper clips

  • Nasal suction bulbs (to be used a dust puffers)

  • Syringes and needles


  • Plastic gloves

  • Vacuum holder

  • Ice chest with ice

  • Mini refrigerator

  • Petri dishes with cork and fine netting

  • Netting of various mesh size

  • Soft forceps

  • Fine-tipped forceps

  • Filter paper (9 cm)

  • Vials, test tubes, cages, etc… for holding 

Lab assignment 

A lab assignment is due October 29th.  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 Friday, October 10th (2014 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.

1.  Estimate bean bug 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 techniques. 

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?

Other references on marking:

Hagler, J. R. 1997. Field retention of a novel mark-release-recapture method.  Environ. Entomol. 26: 1079-1088. PDF

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. PDF

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. PDF

Walker, T. J., and S. A. Wineriter.  1981.  Marking techniques for recognizing individual insects. Fla. Entomol. 64: 18-29. PDF

Wineriter, S. A., and T. J. Walker.  1984. Insect marking techniques: durability of markers.  Entomol. News 95: 117-123. PDF

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. PDF 



                                            Web page last updated 10/8/2014 by Heather J. McAuslane