biodiversity genomics and future of insect systematics

BIODIVERSITY GENOMICS &

FUTURE OF INSECT SYSTEMATICS

Presented By:Varun Saini

Ph.D ENTOMOLOGY 1st Year

PAU, LUDHIANA

CREDIT SEMINAR- IENT-691

INTRODUCTION

‘Genome’ + ‘Omics’ = GENOMICS• ‘Genome’- set of all genetic information of an

organism encoded in the DNA of the nucleus and organelles.

• ‘Omics’ means High thoughtput.

GENOMICS refers to the study of structure and function of entire genome of a living organism.

Term GENOMICS was first used by Thomas Roderick in 1986.

GENETICS vs GENOMICS vs PROTEOMICS

•Genetics looks at single genes, one at a time, like a picture or snapshot.

• Genomics looks at the big picture and examines all the genes as an entire system.

• It involves the systemic analysis, identification and characterization of all of the proteins in an organism

(Palli et al., 2012)

TYPES OF GENOMICSSTRUCTURAL FUNCTIONAL COMPARATIVE

• Sequence organization.

• It determines size of the genome of a species in Megabases (Mb) and also the number of genes present in the entire genome of a species

• Ambitious high-throughput approach to understanding the genome

• Studies how genes and other sequences of DNA act in relation to the entire organism.

•Analyzing DNA sequence patterns of different organisms side by side to identify genes and determine functions.• Looking for similar genes in different species to determine possible relationships and genomic variations.


TOOLS USED IN GENOMICS

Molecular Marker Technology

Microarray Chip Technology

Comparative Genomics

Molecular Marker Technology

1. Conventional Marker System

• RFLP• Microsatellites• RAPD- PCR• ESTs (expressed sequence tags)• AFLP (amplified fragment

length polymorphism)Mainly used for understanding the genetic basis of insect biodiversity and agriculturally important genes and Quantitative Traits Loci (QTL) in insect pests.

(Agarwal et al., 2008)

2. Novel Marker System

1. Transposon Display2. s- SAP (sequence- specific

amplification polymorphism)3. SNP

Major Applications of Marker System

in Insect Science1. Mating, Parentage and Kinship

2. Insect- Plant interactions

3. Insect- Pathogen interactions

4. Insecticide Resistance

5. Prey, Predator and Parasites

6. QTL mapping

7. Behavioural studies

8. Comparative genomics and Cytogenetics

9. Ecology, Phylogeny and Evolutionary trajectories(Speight et al., 2005)

Microarray Chip Technology

• Microarrays are a way to study the expression of many genes or even the whole genome at once. Analyzing the results of a microarray experiment, however, can by quite complex.

• Mainly used for comparison of gene expression between two species. A single microarray experiment can

generate tens of thousands of data points.

(Zhu et al., 2010).

http://www.surrey.ac.uk/SBMS/Fgenomics/Microarrays/index.html

http://www.surrey.ac.uk/SBMS/Fgenomics/Metabolic_flux/index.html

Applications of Microarray Technology in Insects

Applications DescriptionGene Expression Measuring global gene expression

pattern under various biological conditions

miRNA profiling Genome-wide detection of the expression of miRNAs (small non-coding RNAs)

SNP detection Detecting polymorphisms within a population

Pathogen and virus detection Low-density DNA microarray for the identification of viruses and pathogens




Comparative Genomics• It is the process of comparing newly sequenced

genomes to more well-cured reference genomes.• The Contigs or scaffolds from a newly assembled

genome can be mapped to the reference, or the shorter reads can be mapped and assembled in a hybrid approach.

• It will also provide a much better understanding of practical matters such as insecticide resistance


BIODIVERSITY GENOMICS

Biodiversity means all Living Things on Earth• First Prokaryote: Haemophilus influenzae• First Eukaryote: Saccharomyces cerevicae (Yeast)• First Plant: Arabidopsis thaliana (Mouse Ear Cress)• First Animal: Caenorhabditis elegans (Nematode)

• Most Complex and Largest Genome sequence project started in 1986 and completed in 2003.

Opening of door of New Science

Further Science is moving to understand more……• Genome sequencing of Mouse, Mus musculus- For comparing and understanding the contents of the

human genome.• Also moving on CEREAL GENOMICS.- To breakthrough in Breeding programs of cereals.• Genome sequencing of Most diverse animals on earth

Breakthrough in INSECT GENOMICS

Genome sequencing of Drosophila melanogaster

Why Sequence Insect GENOMES ?

• Phylogeny and Evolution(Hymenoptera considered as most evolved Insect order. Why ?)• Insecticide Resistance Mechanism in insects• Various Gene Expression Patterns• Detecting Polymorphism within population• Host Plant relationship• Tritrophic interaction studies• Symbiotic association of insects with microorganisms• Taxonomy/species/strain and clone identification• Identifying novel insecticide targets

(Agarwal et al., 2008)

INSECT GENOMICSIntroduction

• Genomic sequencing has become a routinely used molecular biology tool in many insect science laboratories.

• Completion of the sequencing of the first insect genome, the fruit fly Drosophila melanogaster, in 2000 was followed by a outbreak of activities aimed at sequencing the genomes of several additional insect species.

• Research on insects, especially in the areas of physiology, biochemistry, and molecular biology, has undergone notable transformations during the past two decades.


Milestones of INSECT GENOMICS

COLEOPTERA

Dendroctonus ponderosae, Pine Beetle (2013)Tribolium castaneum (2008)

DIPTERA

Drosophila spp. (2000-2014)Anopheles spp. (2002-2015)Aedes spp. (2007)Culex quinquefasciatus (2010)Clogmia albipunctata, Moth midge (2013)Episyrphus balteatus, Hoverfly (2011) Megaselia abdita, Scuttle fly (2013)

HEMIPTERA

Acyrthosiphon pisum, Pea Aphid (2010)PHTHIRAPTERA

Pediculus humanus, Sucking Louse (2010)

LEPIDOPTERA

Bombyx mori, Silk worm (2004) Danaus plexippus, Monarch Butterfly (2011) Heliconius melpomene, Butterfly (2012) Plutella xylostella, Diamondback Moth (2013)

HYMENOPTERA Acromyrmex echinatior, Leafcutter Ant (2011) Apis mellifera, Honey bee (2006) Atta cephalotes, Leaf-cutter Ant (2011) Camponotus floridanus, Ant (2010) Cerapachys biroi, Clonal Raider Ant (2014) Harpegnathos saltator, Ant (2010) Linepithema humile, Argentine Ant (2011) Nasonia spp. Parasitoid Wasp (2010) Pogonomyrmex barbatus, Harvester Ant (2011) Solenopsis invicta, Fire Ant (2011)

As around 28 insect species genome sequences has been completed till now.

(Chilana et al., 2012)(http://www.wikipedia.org (Access on 25th March, 2016)

http://www.wikipedia.org/

Genome Sequencing in Insects

Isolation of high molecular weight genomic DNA from nuclei isolated

from isogenic lines of insects.

Genomic DNA is sheared with Bal31 nuclease/ T4 DNA polymerase primers.

Genomic fragments are then inserted into restriction enzyme- linearised plasmid vectors.

The plasmid DNA is purified, isolated, sequenced, and assembled using bioinformatics tools.

1

3

2

4

ASSEMBLING

Genomes assemble from Short reads

Contigs created and lumped together by a assemblers

Assemble contigs in to scafolds

Map scafolds to chromosomes

Genome Map(Staden, 1979)

Whole-genome Shotgun

Sequencing (WGS) method

(Staden, 1979)

HOW MUCH SCIENCE REACHED

?

(Korb et al ., 2015)

A genomic comparison of two termites species with different social complexity

They found that protein coding genes less in Z. nevadensis (15,876 vs. 16,310 in M. natalensis).

M. natalensis genome contained a much higher proportion of repeat sequences (67.1 vs. 26.0% in Z. nevadensis).

Sequence Divergence Rates of Transposable Elements (TEs)

- LINE (Long Interspersed Nuclear Elements)- SINE (Short Interspersed Nuclear Elements)- LTR (Long Terminal Repeat)


They also found that TE sequence divergence (i.e., percentage of different base pairs) expansion more in M. natalensis than Z. nevadensis because of multiplied TE copies

hence, M. natalensis genome is much larger in size than Z. nevadensis genome.


Other findings………..• Z. nevadensis had significantly

more ORs (Odorant genes) related to chemical communication than M. natalensis.

GNBPs more important in species with closed nests, whereas Termicins seem to be in foraging termites with subterranean nests.

The major differences between the two termite genomes are related to genome architecture and the presence of transposons that can explain the much larger genome size of M. natalensis.

O- OdourOBP- Odour Binding Proteins• Receptor on nerve

(Kapheim et al., 2015)

The evolution of simple eusociality involves increased regulatory flexibility of ancestral gene networks to create specialized reproductive and non-reproductive individuals, and the evolution of complex eusociality requires genetic novelty to

coordinate emergent properties of group dynamics.

- Simple Eusociality from solitary ancestor

- Complex Eusociality


Increasing social complexity is associated with increasing presence of cis-regulatory TFBS in promoter regions. Each bar represents a TFBS for which presence correlates significantly

with social complexity (blue: positive; red: negative).

Increase number of methylated genes

More will be Complex Eusociality

TFBS - Transcription Factor Binding Sites


Social complexity among genes evolving faster (solid) or slower (hatched) in lineages with different levels of

social complexity

Complex eusocial species have a reduced proportion of repetitive

DNA compared to other bees

(Puinean et al., 2010)

This is the first report of a P450 gene amplification event associated with insecticide

resistance in an agriculturally important insect pest.

M. persicae Clone (5191A):

Over expresses a single P450, CYP6CY3 has increased metabolism of the insecticide.

Over expresses genes encoding cuticular proteins and has reduced penetrationof the insecticide

(Erdmanis et al., 2012)

Neonicotinoids kill insects but not ticks

This study aimed to identify the molecular basis for Imidacloprid insensitivity to ticks

In this studies, A homology model of the nAChR binding domain was generated on the basis of the crystal structure of an acetylcholine-binding protein with the insecticide

Imidacloprid bound.

(Erdmanis et al., 2012)

Residues same in all species (selected Blue

region)

Red region: Glutamine in Arachnids Green region: Arginine in insects

Models of nAChRs withGlu - neonicotinoid doesn’t bindArg - neonicotinoid does bind

Why Nasonia ?Nasonia is the second genus of Hymenoptera to have whole-genome sequencing, after

Apis mellifera

Nasonia are now emerging as genetic model organisms, particularly for complex trait analysis, developmental genetics, and evolutionary genetics

TE diversity in Nasonia is 30% higher (2.9 TE types/Mb) than the next most diverse insect (Bombyx mori, 2.1 TE types/Mb)

(Werren et al., 2010)

Functional and Evolutionary insights from the genome of parasitoid Nasonia species

(Pteromalidae: Hymenoptera)


The Nasonia genome encodes a full DNA methylation tool kit, including all three DNA cytosine-5-methyltransferase (Dnmt) types viz., Dnmt 1, Dnmt 2 and Dnmt 3 contrast with D. melanogaster, which has only Dnmt2.DNA methylation is important in Apis caste development and is suggested for Nasonia sex determination

A. Mellifera shows an expansion of the yellow/major royal jelly (yellow/MRJP) genes that are linked to caste formation and sociality. Nasonia has the largest number of yellow/MRJP

genes so far found in any insect, including an independent amplification of MRJP-like proteins


This finding is consistent with the rapid evolutionary rate of Nasonia mitochondria and studies implicating nuclear-

mitochondrial incompatibilities in F2 hybrid breakdown

Nasonia also has the largest number of ankyrin (ANK) repeat containing proteins (over 200) so far found in any insect, mainly

for protein-protein interactions


Recent lateral gene transfers from the bacterial endosymbiont Wolbachia into

the genomes of Nasonia has been identified

Both Wolbachia and Nasonia have ANK-PRANC genes

The Nasonia PRNAC are integrated in genome and are expressed in different life

stages

PRNAC (Pox proteins repeats of ankyrin- C terminal)

Phylogenetic analysis of PRNAC domain sequences suggests that Nasonia acquired one or more of these proteins from Wolbachia

Such lateral gene transfer could be an important source of evolutionary innovation

Future of insect systematics

Insect systematics: past and present

(Narendran, 2001)

Foreign workers mainly studied the taxonomy of Indian insects.

Almost forgotten Science now a days

Hardly 100 insect taxonomists in india

Only few scientists like Ramakrishna Ayyar, Narayanan, Pruthi, Mani made taxonomic studies.

At present, ZSI, IARI, FRI and a few universities and institutions carry out taxonomic research on Indian insects to a

limited extent.

Why it’s happening to Taxonomy ?• Shortage of man power• Young scientists rarely recruited• Lack of funding• Unavailability of old literature• Prejudiced and biased approach• Lack of confidence • Lack of will and commitment• Lack of consistency• Lack of vision• Lack of credibility

(Tahseen, 2014)

Future of insect systematics• Less than 100,000 species are already discovered from India and

many more yet to be discovered, because of presence of two major biodiversity hot spots.

• Accelerating taxonomic discovery through automated character extraction.

• Collaboration of digital technology, information science and computer engineering.

• Decreasing cost of imaging.• Use of RAPID (Robotic Automated Pest ID).• CyberTaxonomy• Preparation of ‘Character Matrix’• Digitalization of Taxonomic Databases• DNA barcoding• Comparative genomics

Grieneisen et al., 2014

• Zoobank- launched in Jan, 2008

• LepIndex- developed by NHM, London for updating data of lepidopterans

• Online database, “GLOBIZ”, at the State Museum of Zoology, Dresden,

Germany for Pyraloidea

• DNA sequencing in taxonomy- Genomics

Future of insect systematics

Grieneisen et al., 2014

Steps For saving taxonomy in future

The students encouraged to take up taxonomy as their career. Employment opportunities for taxonomists in museum,

universities and other research institutions without bias. Proper funding to taxonomists or concerned organizations

for work. The work of taxonomists particularly original authors should

be cited along with taxon’s name. Training in taxonomy Publication of research papers Library facilities Role of repositories Identification service

Narendran, 2001

APPLICATION OF INSECT GENOMICS IN PEST MANAGEMENT

Insect Transgenesis• Transgenic strains is created to improve

existing biocontrol programmes such as sterile insect management technique.

• The improved understanding of genome sequencing of the pest insect will stimulate the design of new classes of transgene microorganisms to be used in pest control. Ex. Baculoviruses and Bacillus thuringiensis

Insect Transgenesis• Transgenic strains is created to improve

existing biocontrol programmes such as sterile insect management technique.

• The improved understanding of genome sequencing of the pest insect will stimulate the design of new classes of transgene microorganisms to be used in pest control. Ex. Baculoviruses and Bacillus thuringiensis

(Tahseen, 2001)

Pest Genomics and Bio-rational Target Sites

• Developing novel pest-control products like biorational insecticides. Genomics is applied to identify the target sites (proteins) that can be exploited for the developing these biorationals.

Comparative genomics approaches• Comparative genomics approaches have identified genes that encode

proteins unique to insects or to specific insect taxa.• It will also provide a much better understanding of practical matters

such as insecticide resistance.

Insect genomics, biotechnology and insect cell lines have begun to provide powerful

tools for the identification of new lead compounds. Insect cell lines together with

HTS procedures (High Thoughtput Screening), can enable the discovery of new modes of action for insecticide candidates.(Tahseen, 2001)

Future Perspective of insect genomics

• Drosophila genomic sequence is a major milestone for genomics and it proved as new strategy for sequencing large eukaryotic genomes and as a model system to understand biological functions.

• At present, only three agricultural pest insect genomes, that of the red flour beetle, pea aphid and DBM have been fully sequenced, but within the next few years several pest sequences will be available.

• Genomic information increase our knowledge for understanding the biology of insects and insecticide resistance.

• In future, comparative genomics will be an increasingly useful approach for pinpointing common and different genes across species.

(Garcia, 2008)

Future Perspective………..• Genomic sequence and organization will be useful to explore

gene functions.• Functional genomics is being applied more and more in every

aspect of life sciences research, including ecology and evolution.

• In future, there is a growing tendency for insect molecular scientists.

(Garcia, 2008)

“Manhattan Project of

Entomology”

March 2011

Creating a Buzz About Insect Genomes

The National Agricultural Library (NAL), Beltsville, USA has implemented the i5kWorkspace@NAL to help meet the i5k initiative’s genome hosting needs.

Source :http://i5k.nal.usda.gov

Started in 1st April, 2014 five-year National Science Foundation Research Coordination Network focusing on Insect Genetic Technologies

David O’ BrochtaUniversity of Maryland,

College ParkIGTRCN DirectorSource: http://www.igtrcn.org

$ The insect genomic databases are goldmines with information on all the proteins, biochemical and physiological process of an insect.

$ Insect pest control will soon enter the genomic era with all its surprises and discoveries, as pest and parasitoids genomes are now available.

$ Different molecular marker systems in the field of entomology are expected to provide new directions to study of insect genomes in an unprecedented way in years to come.

$ The emergence of a large number of insect genome projects during the last 10 years has revolutionize insect research.

$ Comparative sequence studies in diverse insect species can provide useful information on how to make use of them for developing markers.

$ High quality taxonomic research is vital for sustainable agriculture, forestry, fisheries, combating insect pests and human diseases and for sustainable national and international trade in biological products without endangering indigenous plant and animal species.

Concluding Remarks

Databases available on Insect Genome sequences

Thank you for kind attention