The document discusses the application of DNA barcoding in conserving insect biodiversity, highlighting its strengths and weaknesses. It details the methodology of DNA barcoding, the significance of the CO1 gene, and the process of developing DNA barcode libraries for species identification. Additionally, it emphasizes the importance of integrating DNA barcoding with other conservation strategies for effective outcomes.

1. Vishnu G. M.
2014-09-111
B. Sc. – M. Sc.(Integrated) Biotechnology
College of Agriculture, Vellayani.
1
DNA barcodes in conservation of Insect
Biodiversity.

2. 2
Stork et al., 2018Total number of named species= 1M ; 80% to be
discovered

3. Why so slow?
3
• Phenotypic plasticity and genetic variability of characters used for identification
leads to misidentification
• Due to the presence of cryptic species.
• Morphological keys are only effective at some particular life stage
• Inadequate number of experts
Herbert et al., 2003

4. DNA Barcoding
• It is the use of universal polymerase chain reaction primers to amplify and
sequence traditionally the 600-800 segments of the mitochondrial cytochrome
oxidase subunit 1(CO1) to classify species (Rubinoff et al., 2005).
4

5. • The survey of just 15 of these nucleotide
positions creates the possibility of 4^15 (1
billion) codes, 100 times the number that would
be required to discriminate life if each taxon was
uniquely branded (Herbert et al., 2003).
5
Hollingsworth et al., 2008

6. An ideal DNA barcoding system must
have
• The gene region sequenced should be nearly identical among individuals of the
same species, but different between species.
• It should be standardized, with the same DNA region used for different taxonomic
groups.
• The target DNA region should contain enough phylogenetic information to easily
assign unknown or not yet ‘barcoded’ species to their taxonomic group (genus,
family, etc.).
• It should be extremely robust, with highly conserved priming sites and highly
reliable DNA amplifications and sequencing.
• The target DNA region should be short enough to allow amplification of degraded
DNA.
6Valentini et al., 2010

7. Why mitochondrial gene?
• Lack of introns
• Limited exposure to recombination
• Haploid mode of inheritance
• Availability of robust primers enables
recovery of specific segments of
mitochondrial genome (Herebert et
al., 2003)
7Rubinoff et al., 2006

8. Why CO1?
• Earlier mitochondrial genes encoding 16s,12s
ribosomes were used but it was found to disturb
the sequence alignment because of (indel)
insertion and deletions.
• In CO1 indels are very rare (Herbert et al.,
2003).
• Shows rapid evolution enough to allow
discrimination of speces.
8
G.-J. Brandon-Mong et al., 2015

9. Why CO1?
• Universal primers of the gene which enables recovery
of 5’ end (Folmer et al.,2008).
• Has high amount phylogenetic signal than any other
gene.
• Its third position nucleotide show high incidence of
substitution which results in a rate of molecular
evolution three times of 12s or 16s rDNA (Herbert et
al., 2003).
9
https://www.google.com/co1+gene+structure&gs

10. Problems with mtDNA
• Introgression due to hybridization creates confusion about species boundaries
between evolutionary lineages that would be normally distinct.
• Nuclear pseudogenes of mitochondrial orgin (numts).
• Because of maternal inheritance the rate of molecular divergence cant be accurately
predicted.
• Heteroplasmy; the presence of more than one type of organellar genome within a
cell or individual.
10Krishnamoorthy et al., 2015

11. Barcoding procedure:
From specimen to sequence to species
11Valentine et al., 2010

12. How barcoding works
• The CO1 gene region of organism amplified using the primers is sequenced.
• Sequence comparison using distance based algorithms with known sequences in
the database.
• Sample identification based on similarity of sequences.
12
If the similarity % is 3 then the sample is
identified if not, it needs further taxonomic
evaluation by a taxonomist.
Rubinoff et al., 2006

13. Building DNA barcode library
• It helps to identify the unknown
sample by matching query sequences
with known sequences(Kress et al.,
2015).
• It is a collection of DNA sequences
with a verified taxonomic
identification and ideally with voucher
specimens (Kress et al., 2015).
13Kress et al., 2015

14. Barcoding database
• The Barcode of Life is a project to
create a public collection of reference
sequences from vouchered specimens
of all species of life.
14www.ncbi.nlm.nih.gov/genbank/barcode/

15. 15

16. How are barcodes submitted?
• The NCBI provides a web-based Barcode
Submission Tool (BarSTool) that can be used
to submit sets of Barcode sequences to
GenBank .
16

17. Strengths of barcoding
• Offers alternative taxonomic identification tool for situations in which
morphology is inconclusive.
• Focus on one or a small number of genes provides greater efficiency of
effort.
• Cost of DNA sequencing is dropping rapidly due to technical advances.
• Potential capacity for high throughput and processing large numbers of
samples.
• Once reference database is established, can be applied by non-specialist.
17Rubinoff et al., 2006

18. Weakness of barcoding
• Assumes intraspecific variation is negligible, or at least lower than
interspecific values.
• No single gene will work for all taxa (e.g., COI is not appropriate for
vascular plants, or even for some animals).
• Single-gene approach is less precise than using multiple genes; may
introduce unacceptable error (Rubinoff et al.,2006).
18

19. 19
Kress et al., 2014
Other gene used for barcoding

20. How DNA barcoding is useful in insect biodiversity
conservation?
• DNA barcoding, can contribute to conservation policy in two important
ways:
By speeding up local biodiversity assessments to prioritise conservation
areas or evaluate the success of conservation actions, and
By providing information about evolutionary histories and phylogenetic
diversity.
20

21. Rapid biodiversity assessments
• DNA barcoding has the potential of identifying species quickly and cheaply
(Leblois et al.,2008).
• After the initial investment is made to develop a reliable database, barcoding can be
used to quickly identify newly collected specimens.
• Quick and cheap biodiversity assessment is especially important because, majority
of undescribed species are in developing countries, where resources for
comprehensive biodiversity assessments are limiting (Krishnamurthy et al., 2012).
21

22. • Assessment of biodiversity is the first step to the successful design of any
conservation strategies
• Identification of the organism combined with detailed knowledge of their life
histories, species richness, endemism, rarity and the extent of morphological
and genetic data ae the essential components of biodiversity assessment
(Sabir et al., 2017).
22

23. Biodiversity assessments can be used for
• Identifying species where morphology is insufficient.
• Identifying species from small or incomplete samples.
• Cryptic species identification.
• Identification of new species.
• Identification of complex samples containing multiple organism (DNA
metabarcoding).
• Understanding ecological interactions in complex network of species.
23Kress et al., 2015

24. 24

25. 25Espiona et al., 2015
The forensic use of DNA barcodes for
identification of endangered species and
commercially useful plants and animals is
being expanded by local, state, and national
governments.

26. Identifying species where morphology is insufficient.
26
All three larval mtDNA COI sequences were identical.
Based on BLAST analysis, larval sequences showed
similarity to adult M. avidus sequences published in
GenBank within a range of 98.72–100%
Adric et al., 2014
Morphological keys are only applicable at particular
life stages

27. • From a tropical premontane forest in Costa
Rica, a comprehensive DNA barcode
library containing sequences of all species
from a community of Cephaloleia and
Chelobasis beetles was assembled from
tissue of adults. This reference library was
then used to quickly identify the species of
immature stages
27
Kress et al., 2015

28. Cryptic species identification.
28Kress et al., 2015

29. 29

30. Identification of complex samples containing multiple organism (DNA
metabarcoding).
• DNA sequences from complex
mixtures of organisms representing
different species are obtained through
NGS that localizes and simultaneously
recovers sequence data from all
individuals in a sample (Kress et a.,
2015).
30Kress et al., 2015

31. Understanding ecological interactions in complex
network of species.
• At La Selva Biological Station (Costa Rica),
interactions between plants in the order
Zingiberales and associated herbivores
(chrysomelid rolled-leaf beetles) were
determined by combining field records and
DNA bar code analysis (Kress et al., 2015).
31Kress et al., 2015

32. Assessment of phylogenetic diversity
• It is a species diversity indicator.
• It is measured as the sum of the lengths of all the branches that are members of
the corresponding minimum spanning path (Faith et al., 1992).
• Phylogenetic diversity can inform conservation strategies by ignoring tedious
species counts and using evolutionary lineages (phylogenies) to boost predictions
about biodiversity patterns (Mitchell et al., 2008).
32
Krishnamurthi et al.,2012

33. 33

34. NJ trees of 16 Formica species.
34

35. 35
Case study

36. Aim
• To reveal the little studied insects of Saharo-Arabian zoogeographic area by DNA
barcoding.
36

37. • Why Saharo-Arabian zoogeographic
area?
out of the 12
zoogeographic area of the world it is
the one with least amount of data about
insect diversity.
37
Ashraf et al., 2018

38. What if data is not known?
• Restricts the recognition of invasive
taxa
• Cant recognize the threatened species
• Lack of knowledge of community
structure and faunal evolution.
38Ashraf et al., 2018

39. Insect collection
• Sampling methods include
39
www.google.com/search?client=firefox

40. Traps were installed at
Pakistan: Islamabad
Saudi Arabia: Hada Al sham, near Makkha.
Egypt: Mosthafa kamal village and Antoniodes garden.
• Molecular analysis was done at,
CCDB(Canadian Center for DNA Barcoding)
40

41. 41Ashraf et al.,2018
• By considering sequence matches to records on
BOLD, the specimens with barcodes were
assigned to 254 families. Most of these families
(216/254) belonged to five orders:
Diptera (61)
Hymenoptera (44)
Coleoptera (42)
Lepidoptera (38)
Hemiptera (31)

42. Samples from
• Pakisthan had 214 families
• Soudhi arabia had 132 families.
• Egypt had 164 out of which site1 had 129 and site 2 had 137 families.
42

43. 43
• A similar study of analysis of 1.29 lakchs of insects from just .5 hectare took 8 years
and required contributions from 102 taxonomists.
• Here over a period of an year combining malaison trap method and DNA barcoding
they could study 50000 species and classicfy them.

44. Conclusion
• Evidence indicates that conservation planning informed by DNA barcoding
techniques is relatively easy to replicate and cost-efficient, with minimal field work.
• DNA barcoding can be used for the quick and cheap identification of species. But it
alone is not enough for strategizing conservation policies. It only adds genetic data
based on previous observations.
• DNA barcoding must be used in conjunction with other suitable methods for
successful conservation of insects.
44

45. 45