Comparative sequence studies of the repeat elements in diverse insect species can provide useful information on how to make use of them for developing abundant markers that can be used in those species; $ At the moment, a total of 8 species are in genome assembly stages and another 35 are in progress for genome sequencing; $ Different molecular marker systems in the field of entomology are expected to provide new directions to study insect genomes in an unprecedented way in the years to come

1. Molecular Markers: MajorMolecular Markers: Major
applications in insectsapplications in insects
SARAMITA DE (CHAKRAVARTI)SARAMITA DE (CHAKRAVARTI)
M. Phil (Zoology), 2M. Phil (Zoology), 2ndnd
SemesterSemester
Roll: BUR MP ZOO No.: 2008 / 9Roll: BUR MP ZOO No.: 2008 / 9
Registration No.: 2546 of 2008 – 2009Registration No.: 2546 of 2008 – 2009
The University of BurdwanThe University of Burdwan
Burdwan – 713 104Burdwan – 713 104
West Bengal, IndiaWest Bengal, India

2. Concept of molecular markers inConcept of molecular markers in
insect ecology- prologueinsect ecology- prologue
What makes insects so diverged and how the genes and genetic
make – up of insects contribute to their adaptable life – forms
and most importantly how they may affect human life either
directly or indirectly? (Speight et. al. 2005)
Common visible markers including body colour, body spots,
bands, hairs, spines were used as phenotypic markers – in
studying of pattern of dispersal, mating behaviour and
inheritance of genetic traits in insects.

3. Drawbacks of visible markers ----
 visible phenotypes are relatively infrequent;
 hard to score;
 difficult and time – consuming to induce genetic mutations in laboratory
populations;
 interfere with the overall fitness of the organism;
 they are rare;
So use of them in a trait is problematic.

4. Protein Markers:Protein Markers:
Electrophoretic pattern of allozymes were used to identify different alleles of a given
gene; e.g. insecticide resistance, pathogen identification, chromosome mapping and
even in detection of prey in insect predators.
DNA markers:DNA markers:
Greater level of polymorphism could be obtained by using (Molecular markers)
thereafter. Because mutations in introns or even in the codons of a gene can
potentially provide variation at the DNA level. DNA markers contributed- genetic
relatedness, phylogeny, population dynamics or gene and genome mapping in
insects.

5. Molecular marker is any kind of molecule indicating the
existence of a chemical or physical process.
In biology and medicine, a molecular marker (biomarker)
can be a substance native to the organism whose
detection indicates a particular disease state.
In genetics, a molecular marker (genetic marker) is a
fragment of DNA sequence that is associated to a part
of the genome.

6.  DNA sequences are very highly specific;DNA sequences are very highly specific;
they can be identified with the help of thethey can be identified with the help of the
known molecular markers which can find outknown molecular markers which can find out
a particular sequence of DNA from a groupa particular sequence of DNA from a group
of unknown;of unknown;
molecular biology;molecular biology;
biotechnology ;biotechnology ;
other scientific experiments to identify aother scientific experiments to identify a
particular sequence of DNA.particular sequence of DNA.

7.  Elucidation of ecological interactions ;Elucidation of ecological interactions ;
 Possess taxonomic importance;Possess taxonomic importance;
 Discrimination of recently diverged taxa e.g. biotypes, races,Discrimination of recently diverged taxa e.g. biotypes, races,
species,species,
subspecies, cryptic species, sibling species and immature lifesubspecies, cryptic species, sibling species and immature life
stagesstages
that show morphological differences.that show morphological differences.
Importance of MolecularImportance of Molecular
MarkersMarkers

8. Molecular Markers in Insects
Insects comprise the largest species composition;
900,000 insect species approx. in the entire animal;
nearly 75% of all the recorded animal species;
possess a vast undiscovered genetic diversity and gene
pool that can be better explored using molecular
marker techniques.

9. Conventional
Marker System
Novel
Marker System

10. Conventional Marker
System
 mt DNA (Mitochondrial DNA)
 Microsatellites
 RAPD - PCR (random amplified polymorphic DNA - PCR)
 ESTs (expressed sequence tags)
 AFLP (amplified fragment length polymorphism)
They have contributed significantly for progresses towards
understanding genetic basis of insect diversity and for
mapping medically and agriculturally important genes and
quantitative trait loci (QTL) in insect pests.

11. Novel Marker System
 Transposon display
 Sequence – specific amplification polymorphism (s – SAP)
 Repeat – associated PCR (RA – PCR)
But use of some methodologies like whole – genome
microarray and single nucleotide polymorphism (SNP)
assays has not gained widespread popularity in
entomological studies due to time consuming and cost –
effective reasons.

12. Conventional MarkerConventional Marker
SystemSystem

13.  Maternal inheritance haploid status and highMaternal inheritance haploid status and high
rate of evolutionrate of evolution
 Loci can be readily amplified by usingLoci can be readily amplified by using
universal primers designed from highlyuniversal primers designed from highly
conserved mt genesconserved mt genes (Lanave(Lanave et. al.et. al. 2002)2002);;
 Upon amplification these loci can be used forUpon amplification these loci can be used for
genotyping by RFLP by easy and simplegenotyping by RFLP by easy and simple
restriction digestions and gel electrophoresisrestriction digestions and gel electrophoresis
(Behura(Behura et. al.et. al. 2001)2001);;
 mt DNA undergoes selective sweep, paternalmt DNA undergoes selective sweep, paternal
linkage and even nuclear organizations, so inlinkage and even nuclear organizations, so in
insects these markers are difficult to use ininsects these markers are difficult to use in
evolutionary and phylogenetic studies;evolutionary and phylogenetic studies;
Fig. Structure of mt DNA

14.  Easy – to – perform and easy – to –Easy – to – perform and easy – to –
score procedurescore procedure (Black 1993)(Black 1993)..
 Reliability and reproducibilityReliability and reproducibility
poor;poor;
 unsuitable for population studies.unsuitable for population studies.
[E[Example inxample in Gypsy mothGypsy moth
((Lymantria disparLymantria dispar Linnaeus),Linnaeus), it wasit was
found that a fragment was present infound that a fragment was present in
the RAPD amplification products ofthe RAPD amplification products of
F1 progeny although it was notF1 progeny although it was not
amplifiable from either parent].amplifiable from either parent].
Fig. Standard PCRFig. Standard PCR

15. RAPD Reaction : 1RAPD Reaction : 1 RAPD Reaction : 2RAPD Reaction : 2

16.  It combines the easiness of RAPD and the reliability of RFLP.It combines the easiness of RAPD and the reliability of RFLP.
 It is relatively time – consuming and often utilizes radiolabeled primers.It is relatively time – consuming and often utilizes radiolabeled primers.
 AFLP loci may contain repeats such as microsatellites in their sequences and mayAFLP loci may contain repeats such as microsatellites in their sequences and may
pose difficulty in scoring the allelespose difficulty in scoring the alleles (Wong(Wong et. al.et. al. 2000)2000)..
 Advantage of AFLP system is that the selective PCR generates more numbers ofAdvantage of AFLP system is that the selective PCR generates more numbers of
marker loci, on average 50 – 100 bands per primer pairs per sample.marker loci, on average 50 – 100 bands per primer pairs per sample.
 AFLP loci are highly reproducible and codominant in nature.AFLP loci are highly reproducible and codominant in nature.
 AFLP markers suitable for mapping of genes and QTL and to generate the linkageAFLP markers suitable for mapping of genes and QTL and to generate the linkage
maps of genesmaps of genes (Behura(Behura et. al.et. al. 2004; Ruppell2004; Ruppell et. al.et. al. 2004)2004) and can also be applied toand can also be applied to
cDNA and the results can be used to detect differentially expresses genes incDNA and the results can be used to detect differentially expresses genes in
insectsinsects (Reineke(Reineke et. al.et. al. 2003)2003)..

17.  Highly polymorphic in nature and are also abundant to generate large numbers of markersHighly polymorphic in nature and are also abundant to generate large numbers of markers
compared to AFLP system.compared to AFLP system.
 Large numbers of microsatellites can be isolated by generating a genomic library of smallLarge numbers of microsatellites can be isolated by generating a genomic library of small
fragments either amplified by PCR or generated by partial digestion of genomic DNA andfragments either amplified by PCR or generated by partial digestion of genomic DNA and
enriched with simple sequences repeats.enriched with simple sequences repeats.
 Amenable for high throughput genotyping by non – radioactive labeling and scoring byAmenable for high throughput genotyping by non – radioactive labeling and scoring by
automated sequencing machines.automated sequencing machines.
 Initial set up a cost – effective than that of the AFLP, but this is often compromised inInitial set up a cost – effective than that of the AFLP, but this is often compromised in
population and ecological studies where more numbers of genotypes are required forpopulation and ecological studies where more numbers of genotypes are required for
meaningful statistical analyses.meaningful statistical analyses.
 In some cases they are not neutral markers as they are involved in gene regulation, geneticIn some cases they are not neutral markers as they are involved in gene regulation, genetic
hitchhiking and sex – specific differential selection and hence, those loci may not be useful forhitchhiking and sex – specific differential selection and hence, those loci may not be useful for
evolutionary and phylogenetic studies.evolutionary and phylogenetic studies.

18.  Use of commercial kits for preparation of cDNA libraries andUse of commercial kits for preparation of cDNA libraries and
automated sequencing methods has made it possible toautomated sequencing methods has made it possible to
generate large sets of ESTs in a relatively fast and efficientgenerate large sets of ESTs in a relatively fast and efficient
manner.manner.
 Mainly used for transcriptosomes analysesMainly used for transcriptosomes analyses (Nakabachi(Nakabachi et. al.et. al.
2005)2005), integrated linkage mapping of insect genomes, integrated linkage mapping of insect genomes
(Graham(Graham et. al.et. al. 2004)2004)..

19. Major Applications of ConventionalMajor Applications of Conventional
Marker Systems in studying insectMarker Systems in studying insect
ecologyecology
DNA markers are used ----
 to provide raw information based on which an ecologist makes
estimates or predicts migration and colonization history (Bosio et. al.
2005);
 to infer phylogeny and biogeography of insect populations;
 to provide the means to differentiate sympatric species from allopatric
species and parapatric species (Margonari et. al. 2004);
 to understand modes of evolution and evolutionary trajectories (Prasad
et. al. 2005);
 for diagnostic purposes of the individual traits (Ullman et. al. 2003).

20. Major ApplicationsMajor Applications
 Mating, parentage and kinship;Mating, parentage and kinship;
 Insect – plant interaction;Insect – plant interaction;
 Insect – pathogen interaction;Insect – pathogen interaction;
 Insecticide resistance;Insecticide resistance;
 Prey, predator and parasites;Prey, predator and parasites;
 Gene, genome and QTL mapping;Gene, genome and QTL mapping;
 Behavioural studies;Behavioural studies;
 Comparative genomics and cytogeneticsComparative genomics and cytogenetics..

21. Mating, parentage and kinship -Mating, parentage and kinship -
 DNA markers can unravel information to determine parentage and kinshipDNA markers can unravel information to determine parentage and kinship
relations in insects;relations in insects;
 RAPD markers:RAPD markers: determined paternity in two odonate species of anisopterandetermined paternity in two odonate species of anisopteran
dragonflies (dragonflies (Anax parthenopeAnax parthenope Julius Braner) and keeled skimmersJulius Braner) and keeled skimmers
((Orthetrum coerulescensOrthetrum coerulescens)) [Hadreys[Hadreys et. al.et. al. 1993]1993];;
 RAPD markers:RAPD markers: showed how the females in some non – parthenogeneticshowed how the females in some non – parthenogenetic
insect species such as white – pin weevil (an important forest prey), carryinsect species such as white – pin weevil (an important forest prey), carry
sperms of more than one male from one season to the nextsperms of more than one male from one season to the next (Lewis(Lewis et. al.et. al.
2002)2002);;

22.  Microsatellite markers:Microsatellite markers: used in bush cricketused in bush cricket Requena verticalisRequena verticalis Walker toWalker to
find out that the ejaculated sperm quantity between males is the determiningfind out that the ejaculated sperm quantity between males is the determining
factor in establishing paternity identity of the offsprings;factor in establishing paternity identity of the offsprings;
 Microsatellite markers:Microsatellite markers: in identification of egg paternity to validatein identification of egg paternity to validate
theoretical models that predict the influence on parental care ontheoretical models that predict the influence on parental care on
reproductive success in some insects like golden bugs (reproductive success in some insects like golden bugs (PhyllomorphaPhyllomorpha
laciniatalaciniata Villers)Villers) [Garcia – Gonzalenz[Garcia – Gonzalenz et. al.et. al. 2003]2003];;
 Microsatellite markers:Microsatellite markers: used to test the polyandry as a mating strategy inused to test the polyandry as a mating strategy in
cricket (cricket (Gryllus bimaculatususeGryllus bimaculatususe de Geer) – to minimize genetic inbreeding ofde Geer) – to minimize genetic inbreeding of
their brood;their brood;

23.  Microsatellite markers:Microsatellite markers: measured genetic diversitymeasured genetic diversity
between sexual and asexual aphid populations;between sexual and asexual aphid populations;
 RAPD markers:RAPD markers: associated with life cycle variation andassociated with life cycle variation and
breeding traits in some insects.breeding traits in some insects.

24. Insect – plantInsect – plant
interaction -interaction -
• DNA markers provide utility in tagging and mapping genes in
important crop plants that provide resistance to damaging insect
pests;
• These are also useful in characterizing avirulence genes in the
insects interacting with the host plants (Harris et. al. 2003);
• RAPD – PCR: identified distinct loci specific to individual strains
(or biotypes) of Asian Rice gall midges (Orseolia oryzae Wood –
Mason), were identified (Behura et. al. 2000);

25. • RAPD and AFLP markers: employed to identify major
avirulence genes in Hessian fly (Behura et. al. 2004);
• RAPD – PCR: established a major difference existed between
winged phenotypes and the wingless phenotypes of the
asexual adult aphids;

26. • RAPD banding pattern: showed the differences between the
feeding habits on different hosts of grasses and cereals of the
grain aphids (Sitobion avenae Fabricus);
• Microsatellite markers: studied genetic basis of host plant
association in lettuce root aphid (Pemphigus bursarius
Linnaeus) [Miller et. al. 2005];
• RAPD markers: evaluated degree of virulence
(aggressiveness) of individual clones in pea aphids in response
to natural resistance in alfa alfa (Bournoville et. al. 2000).

27. Insecticide Resistance -Insecticide Resistance -
 Molecular markers are used for identification and
mapping of resistance genes in insects against
insecticides;
 Microsatellite markers: in mapping experiments
identifies QTL in Anopheles gambiae (a major malaria
vector in Africa) that determines the DDT resistance
phenotypes (Ranson et. al. 2000);

28.  RFLP markers: discovery of the Kdr trait (associated
with DDT resistance in houseflies Musca domestica);
 PCR markers: to examine expression pattern of the
cytochrome P450 genes near a DDT resistance gene in
Drosophila (Brandt et. al. 2002);

29.  RAPD markers: mapping of genetic loci in lesser grain
borer (Rhyzopertha dominica Fabricus) – that
determines high level resistance to phosphine
(Schlipalius et. al. 2002);
 AFLP markers: in the identification of resistance loci in
Colorado potato beetle to pyrethroid (Hawthorne 2001)
and in diamond back moth to Bt toxins.

30. Gene, genome and QTLGene, genome and QTL
mapping -mapping -
Genetic linkage mapping based on recombination
frequency uses molecular markers for tagging and
mapping of specific genes and QTL in insects;
QTLs – stretches of DNA – closely linked to the genes
that underlie the trait in question.
When more than one gene govern a trait (polygenic
trait), markers identify those links to the chromosomal
region containing the genes.

31. QTLs are molecularly identified (by PCR or AFLP) to
help map regions of the genome that contain genes
involved in specifying a quantitative trait;
AFLP markers: used to generate genetic maps of
silkworms, Colorado potato beetles, red flour beetles,
European corn borers, Hessian flies, butterflies;
RAPD markers: for genomes of honeybees, silkworms,
beetles, sawflies;

32. Microsatellite markers: for mapping of genomes of Drosophila mojavensis and in honeybees
and also in bumblebees and ants to identify genes responsible for diversity in foraging range
and mating behaviour (Knight et. al. 2005), host – parasitization (Solignac et. al. 2005),
colonization (Jensen et. al. 2005) and kinship relation (Troutti et. al. 2005);
AFLP markers: used to map QTL governing aging in Drosophila melanogaster (Luckinbill and
Goldenberg 2002), foraging behaviour in honeybees (Ruppell et. al. 2004), susceptibility in
beetles to tapeworm parasites (Zhong et. al. 2005), variation in pheromone composition in
Heliothis sp (Groot et. al. 2004) and Bt resistance in diamond back moths (Heckel et. al. 1999);
AFLP markers: employed in pea aphids (Acyrthosiphon pisum) [Braendle et. al. 2005] to trace
the phenotypic traits of wing size , in moth species (Thaumetopoea pityocampa and T.
wilkinsoni) to study genetics of male dispersal (Salvato et. al. 2002) and in Venturia canesceus
wasps to investigate the genetic basis of haplodiploidy (Reineke and Lobmann 2005);

33. Behavioural studies -
ESTs: used as expression markers in microarray
formats to predict the nursing and foraging behaviour in
the individual honeybees (Apis mellifera) [Whitfield et.
al. 2003];
RAPD markers: determined two QTLs responsible for
the foraging behaviour in honeybees in backcross
populations between bees collecting nectar and those
collecting pollen;

34. mt DNA: traced the movement of females between colonies
in Australian meat ants (Iridomyrmex purpureus Smith);
AFLP markers: employed in bumble bees to study genetic
basis of ecological implications of foraging range and nest
density behaviours (Knight et. al. 2005);
Microsatellite markers: used in dissecting the guarding and
stinging behaviours in honeybees (Arechavaleta – Velasco
et. al. 2003).

35. Concluding RemarksConcluding Remarks
$ Comparative sequence studies of the repeat elements in diverse
insect species can provide useful information on how to make use
of them for developing abundant markers that can be used in
those species;
$ At the moment, a total of 8 species are in genome assembly stages
and another 35 are in progress for genome sequencing;
$ Different molecular marker systems in the field of entomology are
expected to provide new directions to study insect genomes in an
unprecedented way in the years to come.