Wild life forensics

What is forensics???
Forensic science in the broadest sense applies the
knowledge and technology of science in the court of
law.
To understand the origin of wildlife forensics, it is
important to trace how the ﬁeld of forensic science in
general developed that would in turn form the
foundation of scientiﬁc protocols that are applied to
wildlife crime.

Wildlife forensics can be broadly deﬁned as the application of several
integrated aspects of natural and cultural sciences, e.g. biology,
chemistry, and anthropology in the court of law focused on the
regulation of wildlife protection and conservation .
crime against wildlife and their derivatives involves four major
categories:
(1) The illegal taking or poaching.
(2) possessing.
(3) trading, shipping or moving.
(4) inﬂicting cruelty or persecution of wildlife in breach of
these laws

The legality of wildlife killings depends not only on the species,
but also the circumstance of the killing as killing animals is not
always illegal.
In order to determine the legality of a case, wildlife
investigators first have to answer three questions:
What species is the victim??? Where does it come from???was
it captive bred???
Was the killing legal or illegal?
And if illegal, who committed the
crime?

Illegal wildlife trading nets approximately $20 billion a year
worldwide, with only illicit drugs and weapons sales earning a
higher profit Wildlife poaching, trafficking, and trading Are highly
lucrative businesses.
For instance, poachers can earn $1000 a gram for a rare animal
part, which is twenty times the profit of heroin.
In Brazil, recent estimates suggest that at least 40% of all illegal
drugs shipments are combined with wildlife trade is attractive to
criminals because weight-for-weight wildlife is equally or more
proﬁtable than drugs or arms and with less associated risk.

As a species becomes rarer from exploitation, its value on
the black market escalates making it even more desirable
despite the greater effort required to collect individuals from
declining populations

For example
Turtles worldwide are in peril with 3%
extinct or extinct in the wild, 9% critically endangered,
18% endangered, and 21% vulnerable
In Asia, the situation is even more dire
with 1% extinct or extinct in the wild, 20% critically
endangered, 31% endangered, and 25% vulnerable.
(IUCN) as the second greatest driver (only
by habitat destruction) of declines in endangered
animals,
impacting, and of threatened 33% mammals, 30% birds
and 6% amphibians, respectively.

Tigersare hunted illegally
for claws, bones, skins,
whiskers, and virtually every
part of their body which are
used in TCM.
♠ Lifting of ban on trade in tiger
parts would be deleterious to the
tiger conservation globally.
♠ CITES continues to keep the ban
trade in tiger parts.
According to a recent census by the
WWF only 3200 tigers exist in
the wild .This is a reduction of over
90% in the last century.

Leopards
are killed / trapped
for claws, bones,
skins, whiskers, any
many other body
parts used in the TCM

Rhino are poached for horns and skin. The population of black rhino (Diceros bicornis)
decreased by 96% between 1970 and 1992. In 1970, it was estimated that there were
approximately 65,000 black rhinos in Africa - but, by 1993, there were only 2300 surviving
in the wild, Intensive anti-poaching efforts there are now an estimated wild
population of 4420.

Elephants
continue to be killed
for their ivory.
Several techniques
including poisoned
arrows are used for
killing elephants

Wild animals as pets and for meat

♥ Otter skin trade is also prevalent in large volumes.
used as trimming for coats and other garments
♥ Musk Deer hunted for Musk pod
♥ Tibetan antelope hunted for wool for Shahtoosh
shawls
♥ Bear bile used in TCM
♥ Mongoose for hair for fine paint brushes
♥ Snakes skins for belts and leather purses
♥ Sea Turtles shells

Development of Wildlife Forensic Laboratories
Some of the basic tenets in forensic investigations involving human
suspects and victims include:
(1)processing and investigating crime scenes.
(2) determining cause and manner of death, and if a crime has occurred.
(3) identifying and comparing physical evidence in order to link a victim
to a crime scene.
(4) Testifying as an expert in the field.
regular crime labs for human crimes could not
justify working on animal cases above those involving
humans.

This distinct disadvantage when enforcing
the law on violators could only be
remedied through the establishment of a
wildlife crime laboratory. In 1989, federal
funds were allocated to construct
National Fish and
Wildlife Forensics
(NF&WS)
Laboratory in Ashland,
Oregon, U.S.A.
to provide services
(analytical techniques and
expert witness )

lab processes approximately 700–800 cases annually
and utilizes the assembly of six team sections: criminalistics,
pathology, morphology, genetics(molecular biology),
analytical chemistry, and digital analysis.
Agents on these teams evaluate wildlife evidence with the central
purpose of:
(1) identifying the species or animal or plant from the
evidence collected whether is the entire organism, parts or
pieces or even products such as clothing, jewellery,
or processed meats; and
(2) determining the cause-of-death (COD)

Functions of different units
Criminalistics Unit is to evaluate the physical
evidence collected from crime scenes such as
ballistic evidence, tool markings, soil,
questioned documents (such as falsiﬁed permits,
licenses and other documents), and animal/human
prints.
Sometimes, both the Biology and Chemistry Units
are integrated into trace evidence assessments.

♣ The Pathology Unit functions much like the medical
examiner’s office that deals with human cases, in that the
primary objective for this section is to conduct necropsies
(animal autopsies) to determine the COD, mechanism of death
(due to injury or disease) as well as the manner of death
While the Pathology Unit generally works on intact carcasses,
♣ The Morphology Unit typically uses zoological and
botanical form and structure from items such as fur/fibers,
feathers, teeth/bones, claws, seeds, flowers, etc to ascertain the
species in question.
This work is often facilitated with optical and scanning electron
microscopy of this and other evidentiary items.

Majority times when morphological characters (such
as feathers or fur) are not available or collected as
evidence, therefore, it is the lab’s
♣ Genetic (Molecular Biology) section that utilizes
serological proteins and DNA analytical methods,
mitochondrial (mtDNA) and nuclear DNA for animal
and plant species identiﬁcation

When the cause of death or species identiﬁcation
cannot be determined using biological analytical
approaches, the
♣ Chemistry Unit of the NF&WS forensic lab by using
blood or tissue evidence, or any derivative product to
examine the chemical and molecular structures for
species identiﬁcation and COD. In addition, this
section can provide toxicological methodologies to
identify toxins or poisons useful in determining the
manner of death

♣ Digital Analysis Unit that not only utilizes state-of-
the-art computer technology, but also audio and
visual evidence collected to facilitate jury
comprehension of the interpretations presented in
court.

Monitoring trade in wildlife
It requires firstly the identification
of the species traded, then assessment of whether
they are derived from legal or illegal trade.
♠ Morphological traits have traditionally been used as
markers, but they are not suitable when traded products are
degraded or highly processed.
♠ Molecular markers are ideal for species identification
because unlike morphological markers they do not require intact
specimens.

Forensic science relating to wildlife crime
♫ The first being the ability to identify a particular species.
♫ The second is the ability to determine whether the biological material
can be assigned with confidence to a particular individual member of
that species.

DNA can be readily extracted from highly processed
and degraded products commonly encountered in
wildlife trade markets such as:
cooked and dried meats , claws left on tanned
hides , dried shark ﬁns , egg shells , animal hairs ,
bone , ivory , rhinoceros horns ,turtle shell ,
feathers and ﬁsh scales.

Molecular technologies have great utility for wildlife
forensics. Assigning geographic origins of trade products
can also be achieved using molecular methods, a task
that is often impossible using morphological traits alone.
Knowledge of geographic origin can be used to:
♫ to distinguish between legal and illegal products,
♫ to assist in the repatriation of seized animals back to
their
source population, and
♫ to identify which populations are most intensively
harvested for trade

Morphological traits
(1) Animal Tracks
To identify animal
behavior, movement,
Activity.

WHAT CAN YOU TELL BY LOOKING AT A TRACK???
Type of Animal
Species
– Plantigrade: “big butt” animal, rear foot bigger
• Ex. Bear, raccoon
– Digitigrade: “big chest” animal, front foot bigger
• Ex. Elk, deer
Dog v. cat family
– Dogs have claws that show in tracks
– Cats don’t have claws that show in tracks
(retractable)
Digger v. climber
– Skunk v. squirrel
– Climbers will have longer, widespread digits

Size of Animal
• Measuring feet will give an idea of the overall size of the animal
• Adults v. children (babies)
Number of feet
• 2 feet, bipedal– birds, humans
• 4 feet, quadrapedal—dogs, cats, rodents, ruminants
Speed
• Stride/Gait
– Tracks farther apart indicate running
• Direction of animal movement
• Actions/activity of animal
– Chasing, other animal encounters, jumping, finding holes/streams/etc.

Injuries/Mutations
• Limb loss
• Dragging legs
– Some animals drag legs normally (crows, otters)
Fur/Webbing on Feet
• Fur shows up as lines in the track
– Ex. jackrabbits
• Webbing appears near/above toes
– Ex. beavers

(2) SKULLS
What can we learn from
studying skulls?

• Diet of animal
– Herbivore, omnivore, carnivore
– Based on teeth
• Relative age (using teeth)
– Baby vs adult
• Facial structure
– Cat vs dog family
• Brain Size
• Predator vs prey
• Strength of bite/jaw
• What species it might be!!!
– Size, specialized traits
We can tell…

Teeth only 1 pair of canines (if any); all teeth behind canines are molars–
some are tiny!
diastema

Herbivores
– Flat molars for grinding
– No canines (or very small)
– Incisors are flat
• Herbivore gnawers
• 2 curving incisors in front
• They must chew all their lives to
keep those teeth short
• Ex: beavers, squirrels, chipmunks,
hamsters, rats
Elephants –
6 sets of teeth

herbivore gnawer omnivore
beaver
opossum

Facial Structure
• Cats
– Shorter, rounder
faces
• Dogs
– Long snouts,
– sharper faces
bobcat vs coyote skulls

Predator vs Prey
• Predators have eyes in
front/forward
• Prey have eyes on sides of
head
"Eyes in the front,the animal hunts.Eyes on the side,the animal hides."

Omnivores/Carnivores
• Molars are more
pointed
– Tearing ability for
meat
• Canine teeth usually
present
• Some have a fused
molar (carnassial tooth)
– Combination of flat
and sharp molars
• Dogs, wolves

(3) Horns vs Antlers
Horns
Animals: goats, sheep, bison,
cattle
Gender: both – commonly
Traits:
- unbranched
- grow throughout life
- bony core
- hollow keratin sheath
covers bony core
Antlers
Animals: deer, elk,moose,
caribouGender: male
Traits:
- branched
- shed each year
- bone
- skin over bone; skin dries up
and peels

Horns
Pronghorn Antelope
only animal in the world
with horns that are
pronged and are shed
(like antlers)

Whitetail deer vs. Mule Deer
Whitetail Deer
antlers curved
tines extend from main
beam like teeth on a comb
Mule deer
Antlers branch off of main
beam. Not curved

Bighorn sheep
Horns curving with
spiral shape
Mountain
Goat
Horns gently curving
black in color
no forking

Cuticle – outer covering of hair – look at
edges of hair – you can’t see scales easily
coronal – crown-like scales - rodents
spinous – petal-like scales - cats
imbricate –flattened scales
– humans, many other animals

Medulla Type – focus on this trait
lattice – deer family
uniserial or multiserial - rabbits
vacuolated – dog,fox family
amorphous - humans

Medullary Index
0 = no medulla 1= medulla is thickness of whole hair
estimate: ¼, ½, ¾

Medulla Pattern
fragmented
intermittent
continuous
absent

HAIR OF DIFFERENT ANIMALS
RACOON BEAR SKUNK OPOSSUM BOB CAT

CARNIVORES RED FOX BEAVER DEER RABBIT

Molecular markers
Several approaches have been adopted for identiﬁcation
of wildlife species distinguished by the DNA target
(mitochondrial or nuclear) and the technique applied to
develop the genetic marker.
Some techniques, such as sequencing, can be applied to
investigate both types of DNA, while other techniques
are speciﬁc to nuclear DNA (nDNA).

The successful recovery of DNA from biological evidence is the most
important stage in any forensic genetic investigation. The diversity of DNA
sources available to human forensic scientists has been well publicized and
includes soft body tissues, bones, teeth, hair, saliva, sweat, urine and faeces.
The methods used to extract DNA from these sample types can often be
transferred to other species;
However, wildlife forensic geneticists may be faced with quite different
sample types such as fish scales, feathers, fruits or processed timber.
Conservation geneticists have developed techniques For Recovering DNA
From A Remarkable Array Of Sample types (e.g. snake venom, Pook &
McEwin 2005; moulted feathers, Horvath et al. 2005; fish scales, Kumar et al.
2007; porcupine quills, Oliveira et al. 2007; historic eggs, Lee & Prys-Jones
2008), enabling genetic information to be recovered from almost any
biological material.

Sample types can be characterized in terms of the quantity of DNA initially
present, its protection from environmental degradation and the ease with
which purified DNA can be recovered.
For example, hard materials such as bone, tooth, horn and ivory may contain
Relatively Little DNA, Which Is difficult to extract, but which is preserved in
the sample for many years (Yang et al. 1998).
In contrast, soft tissues tend to contain more DNA which is simple to recover,
but which is prone to rapid decomposition.
Plant tissues also vary widely in composition, and different techniques need to
be used when dealing with root fibres, leaves, fruit and seeds or solid timber.

Problems associated with DNA recovery from
wildlife samples:
environmental degradation due to bacterial
breakdown, physical destruction,
damage from natural UV light (lindahl 1993).
Another complication to DNA recovery is that
crimes against wildlife often involve the illegal
trade in processed parts and derivatives, such as
in TMs, and the investigator is often faced with
needing to identify heavily treated sample types
containing multiple individuals or species’ DNA
(gill et al. 2006, tobe & linacre 2008).

Very low quantities of cellular material may limit analysis to
mitochondrial or chloroplast DNA.
Degraded DNA will fragment, restricting analysis to small target
sequences.
Although processes such as ‘whole genome amplification’ Are
Now Being Applied To Increase The Success Rate Of non-human
Genetic analysis,
The quality and quantity of DNA is critical to downstream
applications.
The implications for wildlife crime investigations are that there is
a higher success rate for techniques that rely on mitochondrial
markers, such as species identification, and techniques that target
short fragments of DNA, such as SNP genotyping.

The DNA-based methods used in wildlife crime
investigations were adapted from those used in human
identification and
In the case of species identification, from taxonomic and
phylogenetic studies.The DNA loci used in species testing
are located on the mitochondrial genome rather than
being nuclear DNA based.
Mitochondrial DNA typing has become a standard
process in species testing, allowing inter-laboratory
comparison and permitting a means to standardize
methodologies.

Mitochondrial genome in eukaryotes encodes a total of 37 genes, 22
genes encode transfer RNA (tRNA) , 2 genes encode ribosomal (rRNA)
molecules and the other 13 encode proteins involved primarily with the process of
oxidative respiration .
The number of genes on the mt genome is largely invariant for all vertebrates but the
order may alter. The order of the loci on the mitochondrial genome is the same within
mammalian species
The order is different between avian and mammalian mitochondrial genomes.
Vertebrate mitochondrial DNA: has 2 strands .The heavy or H-strand and the light or L-
strand.
H-strand is the sense strand for one
protein-coding gene (ND6) and 8tRNA genes.
L-strand is the sense strand for 12 protein-coding genes,
2rRNA and 14 tRNA genes.

A major reason for using mitochondrial DNA (mtDNA)
(1) There is no recombination of mtDNA.
(2) All maternal descendants will have the same mitochondrial DNA
sequence,
(3) With the exception of mutations, and all loci are linked.
(4) There is little DNA on the mtDNA that is non-coding, no
introns or pseudogenes within the mammalian mtDNA.
(5) All mitochondrial genes coding for proteins or RNA molecules involved
in respiration.
(6) There would be conservation of sequence as any change in the
proteins or RNA molecules could adversely affect the organism.
(7) Unlike the nucleus, no error reading enzyme exists in the mitochondria
to correct DNA bases added incorrectly during DNA replication.
Therefore, the accumulation of single base changes is up to 5 times higher
in mtDNA compared to errors due to DNA replication in nuclear DNA.

(8) There are multiple copies of mitochondrial DNA per cell compared to only two copies of
nuclear DNA .
(9) Within each cell there are multiple mitochondria within which there are multiple copies of
mtDNA.
(10) The result is that there can be many thousands of mtDNA copies in each cell .
(11) Mitochondria have a protein coat that helps protect the mtDNA from degradation.
Highly degraded biological material is therefore
more likely to be used for mtDNA typing
compared to a nuclear DNA.

Species identification methods
Now universal mtDNA markers are available which makes
amplification(PCR) and typing of specific region of DNA easy
and are successfully applied in identification of wild life
forensics.
The commonly used universal markers for species
identification are mitochondrial
cytochrome b (Cyt b) and cytochrome c oxidase 1 (CO1)
genes.
Species discrimination using cyt b or CO1 can be used directly
on DNA sequence differences between species.

The (Cyt b) gene is an informative marker used in
the identiﬁcation of many vertebrate species from
trade products including sharks, snakes, marine
turtles, seals, birds, and tigers.
Sequencing of a 600 base pair (bp) portion of the
(CO1) gene has been proposed to be an efﬁcient,
fast, and inexpensive way to characterise species.

cytochrome b (Cyt b)
The main locus used in taxonomic and phylogenetic
studies until recently was cytochrome b (cyt b) which
occurs between bases 14,747-15,887 (1140 bp) in
mtDNA and encodes a protein 380 amino acids in
length.
The cyt b locus has been used extensively in
taxonomic and forensic studies, including tiger body
parts, turtle eggs and shells, crocodile skins, rhino
horn, elephant ivory, peafowl and bear bile.

cytochrome c oxidase 1 (CO1)
recently, cytochrome c oxidase I (COI) has increased owing
primarily to its adoption by the Barcode for Life Consortium
http://www.boldsystems.org
COI is found between bases 5904-7445 (1541 bp) in mtDNA, and
used initially to identify invertebrate species.
It soon became the locus of choice in forensic entomology to
identify the beetle larvae on a corpse.
As this one locus could identify these species, it was used more
widely as the locus of choice for identification of all animal
species.
Now the COI locus as a Barcode has been proposed for many
types of organisms.

Other gene loci on the mtDNA used in species
identification.
These include the 12S rRNA and 16S rRNA loci
and the NDH family of genes.
The D-loop (displacement loop)
used less in species identification
but more in intra-species identification.
Due to the greater sequence variation
at this non-coding locus, it is now being
used as a tool for identifying the
presence of
particular species within mixture of many species

Process of species identification
species identification in forensic science is
becoming routine but has not been standardized to
one
single locus.
Regardless of the locus used, sample is analysed by
amplifying a section of the gene (PCR),
predominantly of the cyt b gene or the COI gene.

This polymerase chain reaction (PCR) fragment is then sequenced
directly and the DNA sequence is compared to those registered on an
open DNA databank such as GenBank
http://www.ncbi.nlm.nih.gov/genbank/
GenBank currently has over
108 million sequences (as
of August 2009)
there is a high chance that
the unknown sample will
match a DNA sequence
from a reference sample
deposited on the database

Figure shows an example of the match between a sample taken from a shatoosh shawl,
woven from the fine under hairs of the Tibetan antelope (P. hodgsonii), to the DNA sequence
held on the GenBank database.

Interpretation
The availability of open access DNA sequence database, such as GenBank, has
undoubtedly facilitated much scientific research and, in this regard, forensic science and
species identification has also benefited.
If the DNA sequence from the unknown sample shows a 100% match to the reference
sequence for (P. hodgsonii) and an 84% match to the next closest species, there is
confidence that the unknown sample is that of the Tibetan antelope. So that the
questioned sample came from the Tibetan antelope and not any other species assumes:
(1) all species are held on the database and there is not another species of the
same DNA sequence yet to be analysed;
(2) all members of the Tibetan antelope have the same DNA sequence as
that registered on GenBank (there is no intra species variation);
(3) the sequence data for the next closest match (in Figure this is the goat) is also
representative for this species and no members of this species have, by chance, the same
sequence as that of the questioned sample.

Pyrosequenscing
Pyrosequencing is an alternative method for direct sequencing of
DNA
templates that uses a series of enzymatic reactions to detect visible light emitted
during the synthesis of DNA and enables more rapid screening of samples
compared to conventional sequencing methods.
Only short fragments of 10–500 bp of DNA can be sequenced with
pyrosequencing methods, which can limit its application in forensics unless highly
variable and informative regions are targeted.
Karlsson and Holmlund (2007) used pyrosequencing to develop a highly sensitive
assay to identify 28 species of European mammals based on short fragments of
the mitochondrial 12S rRNA (17–18 bp)and 16S rRNA (15–25 bp) regions.

PCR-restriction fragment length polymorphism
(PCR-RFLP)
targets specific areas of genetic variation among samples.
Initially, the DNA segment of interest is amplified using PCR to generate
billions of copies of the gene, and then subjected to digestion by
restriction enzymes.
These enzymes recognise specific base pair sequence motifs
(that are often mirror images such as ATTA, GATTAG, etc.) and cuts
amplified fragment at these sites generates DNA fragments of differing
lengths (i.e. polymorphic fragments) in which the number and size of
the fragments depends on the number of cutting sites in the DNA
fragment.
Electrophoresis of samples through an agarose or
polyacrylamide gel separates fragments based on size and the different
taxa will have characteristic banding patterns.

1 ← DNA LADDER AT LANES → 12 ← DNA LADDER AT LANES → 22

PCR-Single Strand Confirmation Polymorphism
(SSCP)
SSCP is a method based on the electrophoresis of single-stranded
(ss) DNA fragments of suitable size through a polyacrylamide gel,
followed by visualization
This method has been used for identification based on a region of
the mitochondrial cytochrome b gene
this method is only valuable for short PCR fragments (normally <250
bp)
it easily distinguishes single nucleotide substitutions
SSCP could be a valuable tool for the differentiation of (sub)
populations
SSCP requires reference data from wild populations which are
sometimes available from public gene banks

Discrimination of sturgeon species based on Single Strand Confirmation
Polymorphism of cytb-fragments. This figure was presented by Rehbein (pers.
comm., 2006).

Species-specific primers-PCR
PCR using species-specific
primers presented by N.
Mugue during the 2
Status Workshop in Berlin
at the IZW from 29 25 Th
Sep. to 1St Oct. 2006
(Mugue et al., 2006).
Lane 1/12 are length
standards, lane 2/3 are A.
gueldenstaedtii, lane 4 is
A. baerii, lane 5 is A.
ruthenus, lane 6 is A.
schrenckii, lane 7 is A.
stellatus, lane 8 is A.
nudiventris, lane 9 is H.
huso and lane 10 is H.
dauricus.

Nuclear DNA techniques
Random Amplified Polymorphic DNA (RAPD)
In RAPD analysis, the target sequences to be amplified are unknown. Random
primers (only one short primer is used in each PCR reaction) are used for a
nonspecific, PCR reaction using nuclear DNA as template.
Run on agarose gels or sometimes on polyacrylamide gels, are Scored as
presence/absence for each generated fragment in the different individuals.
The Many Amplification products are simultaneously synthesised from Different
Genomic regions, with different degrees of variability: Some fragments ( = bands)
are very well conserved amongst different individuals while some others show
lower frequencies.
Accordingly, species-specific, population-specific, and individual specific Bands
can be identified.

RAPD band pattern for H. huso, A. stellatus and A. gueldenstaedtii
[modified after Barmintsev et al., (2001)]

Amplified Fragment Length Polymorphism
(AFLP)
AFLP is a combination of RAPD and RFLP
This method is very valuable for the identification
of hybrids
Microsatellites
Tre tandemly repeated motifs of 1-6 nucleotides found in all
eukarotes and many prokaryotic genomes. These usually non-coding
motifs are inherited in a Mendelian fashion.
They are also called simple sequence repeats (SSR), short
tandemrepeats (STR),variable number tandem repeats (VNTR)

Future developments – DNA barcoding
DNA barcode: a short
standardized sequence from a genome used
to identify species

http://wccb.gov.in/ http://www.wwfindia.org/ http://www.fws.gov/lab/
http://www.bear-tracker.com/

Submitted by,
Dr k.santosh kumar,
M.V.Sc Veterinary Biotechnology,
santoshkmr787@gmail.com

Wild life forensics

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