This document discusses various methods for marking insects that are currently used or have potential future applications. It describes 12 main marking techniques: tags and related markers, mutilation marking, paint and ink marking, dust marking, dye marking, pollen marking, biochemical genetic markers, radioactive isotope marking, elemental marking, protein marking, and genetically engineered marking. Each technique is explained in terms of how it is applied to insects, advantages, and disadvantages. The document provides many examples of specific studies that have used each marking method.

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2. METHODS FOR MARKING
INSECTS: CURRENT TECHNIQUES AND
FUTURE PROSPECTS
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Welcome
To
Seminar -iii
Ravi Biradar
PhD 2nd
year

3. Introduction
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4. Characteristics of Effective
Markers
Economic basis
 Durable
 Inexpensive
 Nontoxic
 Easily applied
 Clearly identifiable.
Biological base
 Hinder
 Irritate the insect
 Affect its normal behavior
 Growth, reproduction or life span.
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5. HOWTO CHOOSE
Insect being marked
Habitat of the insects
Nature of the experiment
Individually or in large groups
Durability of the study
Purpose of the experiment
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6. Types of Studies
Marking procedure for two broad categories of research
 Mark-Release-Recapture
 Mark-Capture
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7. Mark-Release-Recapture (MRR)
For studies using MRR techniques
 The researchers collects insects either from laboratory
colonies or from the field.
 The collected insects are then marked, released into field
and recapture at given time and distance interval after
their release.
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(Reynold, 1990)

8. Mark-Recapture (MR)
For studies using MR techniques
 The researchers applies the marker to the
insects directly in the field.
 The most useful markers for MR studies are
those material that are inexpensive and can be
easily applied.
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(Reynold, 1990)

9. • Tags and Related Markers
• Mutilation Marking
• Paint and Ink Marking
• Dust Marking
• Dye marking
• Pollen Marking
• Biochemical Genetic Markers
• Radioactive-Isotope Marking
• Elemental Marking
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What are the option are there…..?

10. Recent Development in Insect
Marking
 Protein Marking
 Genetically Engineered marking
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11. Tags and Related
markers
 Early researchers glued numbered pieces or
cellophane on insects.
 Tags were made by photographically reducing a series
of two alphanumeric codes, printed in a continues,
nonrepeating series onto a film.
 Small plastic tags made from photographic film have
been used to mark screw worms (Co chlio m yia
ho m inivo rax).
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(Rubink, 1988)

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13.  Honey bees (Apis m e llife ra) are frequently marked with tags,
this can simply be glued onto thorax of bees.
 This metal –tagging procedure was useful for examining the
flight range and forage activity of individual bees.
 Individual member of an ant colony have been tagged by using
an elaborate wire-banding system.
 Various combinations of wire diameter, placement and knots
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(Gray, 1991)

14. Cont…
Advantages of Tags and Related markers
• Inexpensive and they can be used to identify
insects in an individual basis for MRR studies.
• Tags are most useful for long term studies
Disadvantages
• Application of individual tags is tedious and time
consuming.
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15. Mutilation Marking
This techniques involves clipping, punching, or etching a
distinctive mark.
Lepidoptera are sometimes tagged by clipping the wings
of adults or the prolegs of larvae (eg. Gypsy moth
larvae).
A notching technique : orthopteran species.
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(Querci, 1936 and Weseloh, 1985)
(Gangwere and chavin, 1994)

16.  Distinctive marks have been etched or punctured onto
the elytra of adult beetle using insect pins.
 Some beetles produce a melanized dark spot when they
are punctured.
 This puncturing (Colorado potato beetle) and etching do
not produce any noticeable side effects on beetle life
span.
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(Best et al., 1981)

17. Advantages of Mutilation Marking
 The marks usually persisted and that they can
readily and accurately recognized in the field
without the aid of any specialized equipment.
Disadvantages
 It can be tedious and time consuming.
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18. Paints and InkMarking
These were among the first materials used to
mark insects and they are still among the most
commonly used material for marking individual
and groups of insects
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20. Marking individual insects
with Paints and Inks
 Paints and inks are applied to individual insects with toothpicks,
insects pins, fine-tipped pens, or fine-haired brushes.
 Red fire ants (Solenopsis invicta) were marked with ballpoint paint
pens.
 Wineriter and Walker (1984) tested 26 paints and ink on two
cricket and one beetle species and found that water insoluble paints
had the greatest durability on their surfaces.
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23.  Ideal paint or ink marker: durable, nontoxic, easy to apply, quick drying,
lightweight.
 White & Singer (1995) assigned numeric code to each larval segment,
enabling each larvae to be coded with any 1 to 50 different combination.
 Ex: Late instar larvae of nymphaid Euphydryas editha bayensis were
marked by strategically placing three dots of paints on each side of the
larvae.
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25. Marking Group of insects with Paints
and Inks
 A variety of paints and inks can be applied topically to large
batches of insects for MRR studies using various spraying
devices including hand atomizers and spray guns.
 Mass marking with paints and inks is easy, rapid and inexpensive
 These marker are usually recognizable on recaptured insects
without microscope examination.
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(Davey, 1956)

26. Cont…
Advantages of Marking with Paints and Inks
• Inexpensive , durable and easy to apply.
• Specimens can be nondestructively sampled and resample over the
course of long-term study.
Disadvantages
• Process is tedious and time consuming.
• The marker or solvent is often toxic to insects.
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27. Dust Marking (known as powders)
 They are probably the most commonly used materials for externally
marking a variety of insects.
 Dusts are most useful for marking large insects or insects with hairy
surfaces.
 An invisible green fluorescent dust was among the first dusts used
to mark insects (it is easily detectable under the UV-light).
 The most commercially dust used to mark insects is Day-Glo.
(Beier and Craig,
1982)
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28. Cont
 Tumbling devices have been used to mark adult boll weevils and
fruit fly pupae.
 Mosquitoes may be marked with dusts by “puffing” them, using
insufflators.
(Stern and Mueller, 1988)
 Schroeder & Mitchell (1992) used dusts to mass-mark millions of
sterilized tephritid fruit flies. Fruit fly pupae were “tumbled” with dust
just prior to adult emergence. The dust was contacted by the
expanded ptilinum at emergence and retained in the face of the
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29.  Self-marking techniques were developed for mark-capture studies
in which dusts were placed strategically near insect nest and hive
entrances, insect floral-visitation sites, insect bait stations and
insect traps (ex: Honeybees).
 Price & Slosser (1995) placed fluorescent dust in pheromone-baited
traps modified to allow for the escape of trapped boll weevils
exposed to the dust. Adult bark beetles (Dendroctonus frontalis
and Ips grandicollis) were self-marked as they emerged from logs
that were treated with dust.
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Self-marking technique

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 21% of foraging bees and 32% of nurse bees had their gut contents
contaminated with the fluorescent powder.

31. Dye Marking
 Internally marking of the insects.
 Certain oil soluble dyes accumulate in insects body fluids or tissue
 Gast&Landin (1996) examined the feasibility of marking the adult stage of
Anthonomus grandis by adding 60 oil-soluble dyes to their larval diet.
Ex: deep black BB, calco red N-1700, oil-soluble blue II, rhodamine B
 Calco red N-1700 produced a highly visible and long lasting color that did
not yield any adverse side effects on insects.
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32. Cont…
 Calco red N-1700 accumulates in the fat body of the larvae,
pupae, and newly emerged adults (Antho no m o us g randis ).
 Many lepidopteron pests were marked with various dyes by
adding dyes directly into larval diets (He lio this vire sce ns).
(Graham, 1991)
 Sudan deep-black BB turns the haemolymph of the melon fly
(Dacus cucurbitae ) black without affecting its growth and
development.
(Schroeder, 1974)
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35.  Internal dyes are used in MRR studies to investigate
various aspects of termite ecology.
 Field collected termites are placed on filter paper or
paper toweling that has been stained with on oil
soluble dye.
 Marked individual can then be released and
recaptured for studies of dispersal, nest mate behavior
and territoriality between nests.
35
Hagler et al., 2010

36. Cont…
Advantages of Dye Marking
 Most oil soluble dyes are inexpensive required minimal additional labor for
marking.
 It is a self marking procedure that avoids extraneous handling of insects.
Disadvantages
 Only minimum number of dyes are proven to be effective.
 Most dyes have too short retention or are harmful to insects.
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37. Pollen Marking
 It can be used as a self-marking material for mark–capture
studies.
 Pollen is an outstanding natural marking material because
 Pollen that naturally adheres to insects in pollination process
 Pollenium
 Pollen grains are distinctive
 Distribution and periods of flowering
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38. Cont…
 Mikkola(1971) suggested that pollen attached to insect’s
surface could be used to provide circumstantial evidence
of migratory pattern and feeding activities.
 In study conducted in Arkansas, 68% of the He lico ve rpa
z e a moths collected in pheromone traps possessed
pollen from false mesquite (Calliandra spp. ).
 Closest location of these plants to trapping site was in
Texas which suggest that these moths must have
migrated > 750km.

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39. Genetic
Marking
Visible genetic Marking
• Laboratory reared insects have been used to identify insects
for MRR studies.
• In some instances mutation are induced by exposing insects
to ionizing radiation or mutagenic chemicals.
• Part of the insect
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40. Cont…
 Body and Eye colors are the most common and conspicuous visible
markers (Geocoris punctipes).
 Mosquito visible genetic markers: silver mesonotum, spotted
abdomen, bronzed tarsi and black palpi.
 An ebony body color mutation was discovered in Anthonomus
grandis, this codominant phenotype which persisted over 9
generations in the laboratory and in field cages.
(Bartlett, 1967)
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41. Biochemical Genetic Marker
 Genetic variation between insect races and biotypes
 Insect-specific enzymes can identify distinct insect populations. These
enzyme “fingerprints” are invisible to the naked eye, so their banding
patterns must be detected by various techniques, including
Polyacrylamide and Starch Gel Electrophoresis.
 Use of biochemical markers include studying insect dispersal, mating
immigrant origin, population relationships, and insecticides resistance.
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(Pashley and Bush, 1979)

42. Radioactive-Isotope Marking
 This technique was a popular insects marking method
from the 1950s to 1970s.
 Stricter environmental protection laws coupled with
development of simpler, less expensive and more
reliable methods have reduced the usefulness of these
techniques.
(Service, 1993)
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Mastrangelo - 2011

43. Elemental Marking
 Element were used successfully to mark at least 8 orders and 30
families of insects, ticks and spiders.
 Elements used to mark are rubidium, strontium, cesium,
manganese, hafnium, and iridium.
 An easy and effective method to externally mark insects for
MRR studies is to dip the pupae or topically spray the adults.
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44.  Mosquito larvae marked by adding RbCl to water in which they were
reared.
 Tsetse fly were labeled by adding europium and dysprosium to their
blood meal.
 Fire ants were marked feeding on baits containing a trace element.
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(Showers and Knaus, 1990)

45. Factors affecting the elemental
marking
 Trace element are distributed in small quantities in earth’s crust.
 The uptake of element varies among species of plants and insects.
 Large insects contain more of the marker than small insects.
 Concentration of element applied to an insect has a direct linear
relationship with the amount of the element retained by the
insect.
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46. Advantages of elemental marking
 These markers are not radioactive.
 These are retained during non feeding, over winter stages (Pink bollworm).
 Multistage, multigenerational and multitrophic marking.
 Mating studies (tobacco budworm and beet armyworm).
Disadvantages
 Detection of element can be difficult, expensive and time consuming and requires a
technical expertise.
 Increased mortality in Rb - marked H. zea
 Increaesd larval development time in T. ni
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Nitrogen-15
marking
Nitrogen-15
marking

48. Protein marking
 Insects were marked with vertebrate specific protein and then
examined for presence of protein by sandwich ELISA using
vertebrate-specific antibodies.
 L. hesperus : vertibrate proetin, rabbit immunoglobulin G (IgG)
 The adult stage of Trichogrammatoidea bactrae, Chelonus
curvimaculatus, Encarsia formosa, and Eretmocerus emiratus were
marked with protein externally by contact exposure or topically mist
and internally by feeding by feeding them a honey solution containing
rabbit IgG.
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(Hagler, 2001)

49.  11.2% T. bactrae emerging from marked pink bollworm eggs and 50
% of E. emiratus emerging from whitefly nymphs contained
detectable traces.
 Protein marking was used to investigate the flow of incoming
nectars in A.mellifera by feeding a sucrose solution labeled with
rabbit IgG to foraging bees.
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(Hagler, 2001)

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51. Genetic Engineered
Marking
 Gene transfer systems that use transposable elements (hobo, Hermes, mariner,
Minos etc).
 Transposable elements may be used to permanently mark mass-reared insects
forSIRstudies programmes.
 PiggyBac transposable element containing the White-eyed mutant gene of the
Mediterranean fruit fly, C.capitata was used to transform C.Capitata and
D.melanogaster.
 The resulting transformations yielded a certain percentage of flies possessing
highly visible “white eyes” that remained stable for15 generations.
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Handleret al., 1999

52.  Transposable elements carrying green fluorescent protein
 GFB used to study : sperm competition, egg development and neurobiology
 Pink bollworm was transformed with piggyBac transposable element
carrying enhanced GFP and have planned to introduce this marker gene
into the pink bollworm colony used for the SIR programme.
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(Peloquin and miller, 2000)

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54.  GMOs containing GFP are permanently marked.
 Expression of GFP can potentially be expressed during
one or all life stages.
 GFP can detected using a fluorescence microscope,
spectrofluorometer.
 Alternative approach for detecting GFP was developed
using PCR
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Advantages

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