*2.7.4 Study of cross-sections*

The cross-sections of hair can be prepared by using a microtome. In case of nonavailability of microtome, a simple yet reliable method may be used [21]. This method requires a straw pipe, mounting wax and a razor blade for preparing cross-sections. Few hairs were inserted into a straw pipe, keeping them as straight as possible. Maintaining the vertical position of the hair, molten wax is slowly and carefully sucked into the straw. Once the molten wax rises past the hair samples, the straw pipe is constricted to prevent the molten wax from running down. The wax is allowed to solidify and the straw pipe is cut open to remove the wax stubs with embedded hair. These stubs with hair embedded in a vertical plane can be used for cutting fine cross-sections with a razor blade. The cut cross-sections are placed in a microscopic glass slide and a drop of xylene is added to remove the wax. These can be viewed under a light microscope at a magnification of 100× to 1000× depending upon the thickness of the hair. **Figure 3** depicts the cross-section of the hair takin (*Budorcas taxicolor*) under microscope 400× magnification.

The following parameters may be observed:


**39**

*Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis*

Examination of hair by considering these parameters usually helps to narrow down up to genus and species level. In some species like Tibetan antelope (*Pantholops hodgsonii*), mere examination of the cuticle can help in species characterization due to the unique scale pattern of the cuticle (**Figure 4**). The scales of the cuticle of the hair of Tibetan antelope (*Pantholops hodgsonii*) have a honeycomb-like shape that can be clearly appreciated in **Figure 4**. However, for most of the species,

*The cuticle of Tibetan antelope (Pantholops hodgsonii) under microscope 400× magnification. Note the* 

Sometimes, hair samples may not be in good shape, or may not be in an appropriate number, or may not be available at all. In such cases, it is worthwhile to use DNA-based techniques for species identification. The DNA-based methods are

In the past 2–3 decades, conservation genetics has evolved as an important tool to resolve problems faced in species conservation. It has wide applications in molecular ecology, population genetics, molecular phylogenetics, taxonomy and

A recognized field of conservation genetics, now drawing growing attention, is the advancement of analytical methods to offer strong DNA-based evidence to support conservation law enforcement, which is commonly known as "wildlife DNA forensics." Wildlife forensics is related to the identification of confiscated material to ascertain the species, individual identity or relationship, and source population of the sample. However, wildlife forensics has its challenges. Despite the implementation of national and international laws to protect degrading habitat, protect biological species diversity and secure long-term survival of species, DNA forensics

In the past decades, molecular techniques have evolved rapidly allowing forensic researchers to extract genomic DNA from small remains or quantity of biological samples left at the scene of a crime and to establish a connection with the wildlife species and the offender. Forensic scientists have utilized this methodology to monitor the illegal trade of ivory [31–33] and to detect the source population of whale meat confiscated from Japanese markets [34] and Bengal tiger body parts [35]. Wildlife DNA forensics has been proven to be powerful especially in remote

examination of all the parameters is necessary to reach a reliable result.

*DOI: http://dx.doi.org/10.5772/intechopen.98252*

discussed in the next section.

*honeycomb-like structure of the scales forensic.*

**Figure 4.**

phylogeography [29].

**3. DNA analysis in wildlife forensics**

has become a main probing tool to curb wildlife crime [30].

*Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis DOI: http://dx.doi.org/10.5772/intechopen.98252*

#### **Figure 4.**

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

time of keeping the hair cuttings under xylene. The following four observations can be made:

3.Medulla thickness in microns (using oculometer)

(*Budorcas taxicolor*) under microscope 400× magnification.

*Cross-section of hair of takin (Budorcas taxicolor) under microscope 400× magnification.*

The following parameters may be observed:

2.Medulla outline and configuration

3.Pigment distribution in the cortex

1.The medulla type

2.Medulla pattern

4.Medullary index

*2.7.4 Study of cross-sections*

1.Cross-section outline

in that part is clearly visible, whereas in the part where xylene has not infiltrated, the medulla shows dark globules. The dark globules can be cleared by increasing the

The cross-sections of hair can be prepared by using a microtome. In case of nonavailability of microtome, a simple yet reliable method may be used [21]. This method requires a straw pipe, mounting wax and a razor blade for preparing cross-sections. Few hairs were inserted into a straw pipe, keeping them as straight as possible. Maintaining the vertical position of the hair, molten wax is slowly and carefully sucked into the straw. Once the molten wax rises past the hair samples, the straw pipe is constricted to prevent the molten wax from running down. The wax is allowed to solidify and the straw pipe is cut open to remove the wax stubs with embedded hair. These stubs with hair embedded in a vertical plane can be used for cutting fine cross-sections with a razor blade. The cut cross-sections are placed in a microscopic glass slide and a drop of xylene is added to remove the wax. These can be viewed under a light microscope at a magnification of 100× to 1000× depending upon the thickness of the hair. **Figure 3** depicts the cross-section of the hair takin

**38**

**Figure 3.**

*The cuticle of Tibetan antelope (Pantholops hodgsonii) under microscope 400× magnification. Note the honeycomb-like structure of the scales forensic.*

Examination of hair by considering these parameters usually helps to narrow down up to genus and species level. In some species like Tibetan antelope (*Pantholops hodgsonii*), mere examination of the cuticle can help in species characterization due to the unique scale pattern of the cuticle (**Figure 4**). The scales of the cuticle of the hair of Tibetan antelope (*Pantholops hodgsonii*) have a honeycomb-like shape that can be clearly appreciated in **Figure 4**. However, for most of the species, examination of all the parameters is necessary to reach a reliable result.

Sometimes, hair samples may not be in good shape, or may not be in an appropriate number, or may not be available at all. In such cases, it is worthwhile to use DNA-based techniques for species identification. The DNA-based methods are discussed in the next section.

#### **3. DNA analysis in wildlife forensics**

In the past 2–3 decades, conservation genetics has evolved as an important tool to resolve problems faced in species conservation. It has wide applications in molecular ecology, population genetics, molecular phylogenetics, taxonomy and phylogeography [29].

A recognized field of conservation genetics, now drawing growing attention, is the advancement of analytical methods to offer strong DNA-based evidence to support conservation law enforcement, which is commonly known as "wildlife DNA forensics." Wildlife forensics is related to the identification of confiscated material to ascertain the species, individual identity or relationship, and source population of the sample. However, wildlife forensics has its challenges. Despite the implementation of national and international laws to protect degrading habitat, protect biological species diversity and secure long-term survival of species, DNA forensics has become a main probing tool to curb wildlife crime [30].

In the past decades, molecular techniques have evolved rapidly allowing forensic researchers to extract genomic DNA from small remains or quantity of biological samples left at the scene of a crime and to establish a connection with the wildlife species and the offender. Forensic scientists have utilized this methodology to monitor the illegal trade of ivory [31–33] and to detect the source population of whale meat confiscated from Japanese markets [34] and Bengal tiger body parts [35]. Wildlife DNA forensics has been proven to be powerful especially in remote

wild areas and the marine environment where poaching of protected or threatened species is tough to detect [36]. This portion of the chapter introduces different methods used in wildlife DNA forensics.

### **3.1 Methods used in forensic genetic identification**

#### *3.1.1 Species identification*

The genetic-based analysis is commonly used in wildlife forensics to identify the species from a confiscated item. Species identification is useful in illegal poaching cases to examine the trace amount of evidence from the possession of a suspect or scene of crime [37]. It has also been proven useful in detecting species from shark fins [38, 39], products generally used in wildlife trades such as traditional Chinese medicines (TCMs [40–42], hair [43] decorative items such as ivory idol [44] and burnt samples [45] where morphological identification is not possible or reliable.

Species identification is based on genetic markers that exhibit variation in DNA sequence among species, but are highly conserved or similar within a species [30]. Mitochondrial DNA (mtDNA) is generally preferred as a genetic marker over nuclear DNA (nDNA) for species identification as it is easier to extract from highly degraded and processed tissues or samples. This is because of the presence of multiple copies of mtDNA per cell compared to a single copy of nuclear DNA [46, 47]. Besides, universal mtDNA primers can be utilized to amplify the informative sequence of mtDNA across taxa that are less time-consuming in method development [48]. Polymerase chain reaction (PCR) is especially used for gene amplification [49].

In animals, mitochondrial cytochrome b (Cyt b) and cytochrome oxidase 1 (CO1) genes are commonly used as universal mtDNA markers for species identification [50–58]. The Cyt b gene is a useful mtDNA marker for the identification of several vertebrate species from illegal trade items including seals [59], snakes [60], tigers [32, 56, 61–64], sharks [52], turtles [64] and birds [65].

Sequencing of a fragment (600 bp) of the CO1 gene is highly informative and has been recommended as the inexpensive, fast and efficient approach to characterize species. Researchers around the world are making efforts to utilize the CO1 gene to catalog the entire vertebrate biodiversity on earth (www.barcodinglife.org) [47]. Furthermore, pyrosequencing is another method of DNA sequencing based on "sequencing by synthesis" that facilitates further rapid screening of DNA samples compared to methods used in conventional DNA sequencing [61]. Pyrosequencing can sequence only short fragments (50–500 bp) of DNA, which can restrict its use in DNA forensics unless we target very informative and highly variable regions of a gene [61]. Pyrosequencing has been used to identify twenty-eight European mammal species using very short fragments of 12S rRNA (17–18 bases) and 16S rRNA (15–25 bases) gene regions of mtDNA [66].

However, DNA nucleotide sequencing is a key method followed by comparing sequenced DNA fragments with reference DNA sequences of different species. The similarity or sequence homology between the unknown and reference sequences facilitates to ascertain the species of origin. Moreover, the International Society for Forensic Genetics (ISFG) has approved and validated the use of the DNA sequencing method [67, 68] and validated this method as a method for application in the detection of forensic casework [69]. Furthermore, the important advantage of DNA sequencing is that universal PCR primers can be used to amplify the DNA from unknown or random forensic case samples [56].

Species of unknown samples is assigned by analyzing and calculating the sequence homology with the reference DNA sequences [50, 70] available on DDBJ/EMBL-EBI/NCBI database collaboration (The International Nucleotide Sequence Database

**41**

*Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis*

the CBOL (Consortium for the Barcoding of Life, www.barcodinglife.com).

Collaboration, www.insdc.org) and the Barcode of Life Data system (BOLD, boldsystems.org), which is the cloud-based data storage and analysis platform and the part of

Another method for identifying species is the construction of a phylogenetic tree. Such tree analysis helps understand the evolutionary relationship between unknown and the reference DNA sequences [56, 71, 72]. Phylogenetic trees allow identifying the reference species as likely source species if it is located closest to the unknown sample. Trees can be constructed using different methods such as neighbor-joining, maximum likelihood, Bayesian and maximum parsimony [73] and in wildlife forensics, there is no consensus over which method to use [74, 75]. Although methods that target single nucleotide polymorphisms (SNPs) other than whole DNA sequences confine their capacity to detect species, it enables researchers to analyze samples that contain multiple species, opposite to DNA sequencing using universal primers [30]. Identification of endangered species from traditional Chinese medicines that may contain plant and animal products has been successfully performed using allele-specific PCR primers and probes [63, 76, 77]. DNA sequencing–based species identification and SNP typing–based ability to examine mixed DNA of multiple species can be mixed [30]. Species-specific primers are used to sequence target species from mixed-species samples like TCMs. This tool has been used to ascertain the body parts of shark [39] and bear bile in TCMs [42].

The great concern to wildlife conservation is to ascertain the geographic origin of confiscated items to curtail illegal poaching within the country's boundary and cross-border trafficking of wildlife derivates. In addition to species identification, it is necessary to trace the source population of individual forensic samples to implement wildlife protection laws and CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) regulations. Given this, genetic studies have been widely conducted to infer the source of origin of the poached items and can be used to identify marine stocks harvested illegally. However, few

Ascertaining geographic origin or source population is based on the ability to assign an unknown or a confiscated sample to its population of origin, counting on the availability of population genetic data from several areas and requiring an adequate genetic differentiation of the source population from other populations. Despite these restrictions, a large number of recent conservation researchers are now emphasizing the urgent need for enforcement methods to efficiently ascertain

In divergent populations, phylogeographic analyses can determine the geographic distribution of the genealogical lineages in which unique haplotypes of mtDNA are correlated with large geographic areas [30, 71]. Generally, the D-loop or hypervariable control region of mtDNA is applied as a genetic marker in ascertaining geographic origin based on haplotypes (individual sequence types of the control region) related to specific populations [30]. This method has been successfully applied to identify the large geographic origins of the Chinese sika deer (*Cervus nippon*) [78] and four species of seahorse (*Hippocampus ingens, H. trimaculatus, H. barbouri* and *H. spinosissimus*)

Furthermore, populations with a subtle genetic variation or with insufficient variation in mtDNA can be identified using population assignment methods by employing nuclear genetic markers that exhibit differences among regions more efficiently than phylogeographic analyses. Population assignment methods are useful in assigning individuals to their source population after testing with all

published studies are using these methods in forensic investigations.

*DOI: http://dx.doi.org/10.5772/intechopen.98252*

*3.1.2 Identification of the geographic origin*

the geographic origin of samples [30].

from forensic samples [79].

#### *Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis DOI: http://dx.doi.org/10.5772/intechopen.98252*

Collaboration, www.insdc.org) and the Barcode of Life Data system (BOLD, boldsystems.org), which is the cloud-based data storage and analysis platform and the part of the CBOL (Consortium for the Barcoding of Life, www.barcodinglife.com).

Another method for identifying species is the construction of a phylogenetic tree. Such tree analysis helps understand the evolutionary relationship between unknown and the reference DNA sequences [56, 71, 72]. Phylogenetic trees allow identifying the reference species as likely source species if it is located closest to the unknown sample. Trees can be constructed using different methods such as neighbor-joining, maximum likelihood, Bayesian and maximum parsimony [73] and in wildlife forensics, there is no consensus over which method to use [74, 75].

Although methods that target single nucleotide polymorphisms (SNPs) other than whole DNA sequences confine their capacity to detect species, it enables researchers to analyze samples that contain multiple species, opposite to DNA sequencing using universal primers [30]. Identification of endangered species from traditional Chinese medicines that may contain plant and animal products has been successfully performed using allele-specific PCR primers and probes [63, 76, 77]. DNA sequencing–based species identification and SNP typing–based ability to examine mixed DNA of multiple species can be mixed [30]. Species-specific primers are used to sequence target species from mixed-species samples like TCMs. This tool has been used to ascertain the body parts of shark [39] and bear bile in TCMs [42].

#### *3.1.2 Identification of the geographic origin*

The great concern to wildlife conservation is to ascertain the geographic origin of confiscated items to curtail illegal poaching within the country's boundary and cross-border trafficking of wildlife derivates. In addition to species identification, it is necessary to trace the source population of individual forensic samples to implement wildlife protection laws and CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) regulations. Given this, genetic studies have been widely conducted to infer the source of origin of the poached items and can be used to identify marine stocks harvested illegally. However, few published studies are using these methods in forensic investigations.

Ascertaining geographic origin or source population is based on the ability to assign an unknown or a confiscated sample to its population of origin, counting on the availability of population genetic data from several areas and requiring an adequate genetic differentiation of the source population from other populations. Despite these restrictions, a large number of recent conservation researchers are now emphasizing the urgent need for enforcement methods to efficiently ascertain the geographic origin of samples [30].

In divergent populations, phylogeographic analyses can determine the geographic distribution of the genealogical lineages in which unique haplotypes of mtDNA are correlated with large geographic areas [30, 71]. Generally, the D-loop or hypervariable control region of mtDNA is applied as a genetic marker in ascertaining geographic origin based on haplotypes (individual sequence types of the control region) related to specific populations [30]. This method has been successfully applied to identify the large geographic origins of the Chinese sika deer (*Cervus nippon*) [78] and four species of seahorse (*Hippocampus ingens, H. trimaculatus, H. barbouri* and *H. spinosissimus*) from forensic samples [79].

Furthermore, populations with a subtle genetic variation or with insufficient variation in mtDNA can be identified using population assignment methods by employing nuclear genetic markers that exhibit differences among regions more efficiently than phylogeographic analyses. Population assignment methods are useful in assigning individuals to their source population after testing with all

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

methods used in wildlife DNA forensics.

*3.1.1 Species identification*

**3.1 Methods used in forensic genetic identification**

chain reaction (PCR) is especially used for gene amplification [49].

tigers [32, 56, 61–64], sharks [52], turtles [64] and birds [65].

(15–25 bases) gene regions of mtDNA [66].

unknown or random forensic case samples [56].

In animals, mitochondrial cytochrome b (Cyt b) and cytochrome oxidase 1 (CO1) genes are commonly used as universal mtDNA markers for species identification [50–58]. The Cyt b gene is a useful mtDNA marker for the identification of several vertebrate species from illegal trade items including seals [59], snakes [60],

Sequencing of a fragment (600 bp) of the CO1 gene is highly informative and has been recommended as the inexpensive, fast and efficient approach to characterize species. Researchers around the world are making efforts to utilize the CO1 gene to catalog the entire vertebrate biodiversity on earth (www.barcodinglife.org) [47]. Furthermore, pyrosequencing is another method of DNA sequencing based on "sequencing by synthesis" that facilitates further rapid screening of DNA samples compared to methods used in conventional DNA sequencing [61]. Pyrosequencing can sequence only short fragments (50–500 bp) of DNA, which can restrict its use in DNA forensics unless we target very informative and highly variable regions of a gene [61]. Pyrosequencing has been used to identify twenty-eight European mammal species using very short fragments of 12S rRNA (17–18 bases) and 16S rRNA

However, DNA nucleotide sequencing is a key method followed by comparing sequenced DNA fragments with reference DNA sequences of different species. The similarity or sequence homology between the unknown and reference sequences facilitates to ascertain the species of origin. Moreover, the International Society for Forensic Genetics (ISFG) has approved and validated the use of the DNA sequencing method [67, 68] and validated this method as a method for application in the detection of forensic casework [69]. Furthermore, the important advantage of DNA sequencing is that universal PCR primers can be used to amplify the DNA from

Species of unknown samples is assigned by analyzing and calculating the sequence

homology with the reference DNA sequences [50, 70] available on DDBJ/EMBL-EBI/NCBI database collaboration (The International Nucleotide Sequence Database

wild areas and the marine environment where poaching of protected or threatened species is tough to detect [36]. This portion of the chapter introduces different

The genetic-based analysis is commonly used in wildlife forensics to identify the species from a confiscated item. Species identification is useful in illegal poaching cases to examine the trace amount of evidence from the possession of a suspect or scene of crime [37]. It has also been proven useful in detecting species from shark fins [38, 39], products generally used in wildlife trades such as traditional Chinese medicines (TCMs [40–42], hair [43] decorative items such as ivory idol [44] and burnt samples [45] where morphological identification is not possible or reliable. Species identification is based on genetic markers that exhibit variation in DNA sequence among species, but are highly conserved or similar within a species [30]. Mitochondrial DNA (mtDNA) is generally preferred as a genetic marker over nuclear DNA (nDNA) for species identification as it is easier to extract from highly degraded and processed tissues or samples. This is because of the presence of multiple copies of mtDNA per cell compared to a single copy of nuclear DNA [46, 47]. Besides, universal mtDNA primers can be utilized to amplify the informative sequence of mtDNA across taxa that are less time-consuming in method development [48]. Polymerase

**40**

#### *Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

populations within a large geographic area or landscape complex [35]. Therefore, a strategy to first test individuals with mtDNA haplotypes to identify the wide geographic origin and to second detect a particular source population in its large geographic area where other populations exist can be followed [35].

The frequency of the alleles at hypervariable nuclear DNA (nDNA) genetic marker observed in a natural population can be utilized to characterize population genetic structure and to estimate the probability of an individual or a sample belonging to its putative population of origin. Similarly, a forensic sample is assigned to its probable source population or geographic area [30, 35, 80–83]. The most commonly used hypervariable nDNA genetic markers for population assignment are microsatellites (**Box 1**) and AFLPs (**Box 1**) [47].

#### 1. DNA sequencing

DNA sequencing detects each nucleotide base within a target region of a specific genetic DNA marker. For species identification, DNA sequencing of a fragment (nearly 500 bases) is most commonly utilized to offer species-specific DNA sequence. DNA sequencing facilitates the development of single nucleotide polymorphisms (SNPs), Indels and microsatellites with specific regions of DNA sequence variation.

2. SNP typing

Generally, single bp variations in the DNA sequence at a genetic marker causes differences among species, termed as single nucleotide polymorphisms (SNPs). SNP typing, also known as genotyping, investigates the specific regions with variation in the DNA sequence. SNP typing facilitates cheaper and faster tests that do not need long segments of high- or good-quality DNA but provides less information compared to conventional DNA sequencing. Three most commonly used SNP typing methods in forensics are given below:

2.1 PCR-RFLP

PCR-RFLP (Restriction Fragment Length Polymorphism) utilizes restriction endonuclease enzymes that recognize specific cleavage sites to cut DNA. The resulting nicked fragments are analyzed using agarose gel electrophoresis.

2.2 Allele-specific PCR

PCR primers, employed in the amplification of genetic markers, can be designed for highly conserved DNA regions or fragments (universal primers) or areas where highly variable DNA sequences occur between any species or populations (allele-specific primer).

2.3 Allele-specific probes

In this approach, a combination of universal primers and a specific probe that attaches to a specific variant of DNA sequence is used. Such probes allow detecting the base situated at SNP site.

3. Microsatellite DNA genotyping

Microsatellites or short tandem repeats (STRs) or simple sequence repeats (SSRs) are tandem stretches of 1-6 bp long-short nucleotide sequence motifs (e.g., ATATATAT) that occur randomly and are widely distributed in all eukaryotic genomes [84–86]. Variations in the number of repeat units lead to the difference in the size of both DNA fragments (alleles) that can be resolved and visualized on gel electrophoresis [86, 87]. These polymorphic loci are generally used in genetics and forensics studies. These are codominant markers.

4. AFLP (Amplified Fragment Length Polymorphism)

AFLPs are dominant DNA markers in which an allele is present or absent in an individual. AFLP locus cannot determine the heterozygosity of any individual. Therefore, in contrast to the codominant microsatellite DNA markers, AFLPs have less resolving power to assign an individual to its population of origin [88]; generally, at least 50 AFLP loci and 8 microsatellite loci are recommended to conduct population assignment tests [89]. Furthermore, in comparison to microsatellites, high-quality DNA requirement and greater genotyping errors of AFLPs have proven them to be less versatile [88, 90].

#### **Box 1.**

*Commonly used techniques in wildlife DNA forensic* [30]*.*

Generally, a panel of highly polymorphic microsatellite loci is first selected and used to generate the genetic profile of a test sample or forensic specimen [91, 92]. This profile is assigned to a particular population by matching and comparing its observed alleles with the observed allele frequency in the population. There are several analytical methods available to perform assignment tests [83] and freely available statistical software packages [93]. Population assignment tests are highly

**43**

*Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis*

meticulous when the genetic database of all candidate populations has been developed, population or species boundaries are distinct, sampling is random and all population represents Hardy–Weinberg equilibrium (random mating, no inbreeding, the balance between mutation and genetic drift). Conversely, these assumptions are not feasible for several populations, for instance, when population boundaries are not evident or the genetic variations between populations are minimal or low, and populations are small [94]. Where populations are genetically widely distributed, stable isotopes (nongenetic substitutes) may be more appropriate to ascertain

For the last three decades, individual identification of forensic specimens based

Highly polymorphic SNP or microsatellite markers are used to generate a DNA profile with a series of gene variants or alleles (**Box 1**). The inclusion of more number of loci or markers reduces the chance that two different individuals will have the same DNA profile. Samples are identified to be from the same or different individuals based on the same or different DNA profiles, respectively. It is important to calculate the probability of identity that two individuals may share the same DNA profile [30]. In Canada, a database of DNA profiles has been established and is commonly used to support forensic investigations of the poaching cases of black bear (*Ursus americanus*), caribou (*Rangifer tarandus*), moose (*Alces alces*), elk (*Cervus canadensis*), white-tailed deer (*Odocoileus virginianus*) and mule deer (*Odocoileus hemionus*) (www.forensicdna.ca/dnadatabases.html). Similarly, DNA profiling has been used to generate strong evidence by matching the blood left on the suspect's knife to the

Furthermore, the ability to validate familial relatedness is also a major application of wildlife DNA forensics. In a forensic investigation, the focus of establishing levels of relatedness lies predominantly on the discrimination of wild-caught animals from captive-bred [30]. Genetic or DNA markers are inherited from both parents from one generation to the next that allows using DNA profiles to validate parent–offspring relationships. Microsatellite loci–based DNA profile database is used in Australia and Europe to authenticate captive bird breeding, whereas parentage DNA analysis is applied to verify caviar of captive sturgeon (*S. Rastorguev pers.* comm.) [30].

Apart from species identification from unknown wildlife sample, quite often it also becomes imperative to identify the geographic origin of the sample. In addition to the DNA-based methods for ascertaining the possible geographical origin of samples, the study of the elemental profiles of samples is also a reliable means for predicting the geographic origin of the samples. This also becomes important when a particular species is protected in one area and not in another, and further when animals from the wild are captured and traded as captive-bred [98]. Among the elemental analysis techniques, a comparison of the ratios of different isotopes using methods such as inductively coupled plasma mass spectrometry (ICP-MS) and

on a unique DNA profile has revolutionized human forensic studies [30]. This technique can be employed to detect the number of individuals used in the commercial market or trade, even from highly processed or powdered products [47]. Baker et al. [96] used a partial fragment of the mtDNA control region (464 bp) and 8 microsatellite loci to identify the minimum number of individual North Pacific

minke whales (*Balaenoptera acutorostrata* spp.) from 12 markets.

*DOI: http://dx.doi.org/10.5772/intechopen.98252*

the origin of the samples [95].

*3.1.3 Individual identification and parentage*

carcass of a wild boar in northern Italy [97].

**4. Study of isotopes in wildlife forensics**

#### *Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis DOI: http://dx.doi.org/10.5772/intechopen.98252*

meticulous when the genetic database of all candidate populations has been developed, population or species boundaries are distinct, sampling is random and all population represents Hardy–Weinberg equilibrium (random mating, no inbreeding, the balance between mutation and genetic drift). Conversely, these assumptions are not feasible for several populations, for instance, when population boundaries are not evident or the genetic variations between populations are minimal or low, and populations are small [94]. Where populations are genetically widely distributed, stable isotopes (nongenetic substitutes) may be more appropriate to ascertain the origin of the samples [95].

#### *3.1.3 Individual identification and parentage*

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

geographic area where other populations exist can be followed [35].

ment are microsatellites (**Box 1**) and AFLPs (**Box 1**) [47].

1. DNA sequencing

2. SNP typing

are given below: 2.1 PCR-RFLP

gel electrophoresis.

2.2 Allele-specific PCR

2.3 Allele-specific probes

3. Microsatellite DNA genotyping

between any species or populations (allele-specific primer).

4. AFLP (Amplified Fragment Length Polymorphism)

*Commonly used techniques in wildlife DNA forensic* [30]*.*

genotyping errors of AFLPs have proven them to be less versatile [88, 90].

populations within a large geographic area or landscape complex [35]. Therefore, a strategy to first test individuals with mtDNA haplotypes to identify the wide geographic origin and to second detect a particular source population in its large

The frequency of the alleles at hypervariable nuclear DNA (nDNA) genetic marker observed in a natural population can be utilized to characterize population genetic structure and to estimate the probability of an individual or a sample belonging to its putative population of origin. Similarly, a forensic sample is assigned to its probable source population or geographic area [30, 35, 80–83]. The most commonly used hypervariable nDNA genetic markers for population assign-

DNA sequencing detects each nucleotide base within a target region of a specific genetic DNA marker. For species identification, DNA sequencing of a fragment (nearly 500 bases) is most commonly utilized to offer species-specific DNA sequence. DNA sequencing facilitates the development of single nucleotide polymorphisms (SNPs), Indels and microsatellites with specific regions of DNA sequence variation.

Generally, single bp variations in the DNA sequence at a genetic marker causes differences among species, termed as single nucleotide polymorphisms (SNPs). SNP typing, also known as genotyping, investigates the specific regions with variation in the DNA sequence. SNP typing facilitates cheaper and faster tests that do not need long segments of high- or good-quality DNA but provides less information compared to conventional DNA sequencing. Three most commonly used SNP typing methods in forensics

PCR-RFLP (Restriction Fragment Length Polymorphism) utilizes restriction endonuclease enzymes that recognize specific cleavage sites to cut DNA. The resulting nicked fragments are analyzed using agarose

PCR primers, employed in the amplification of genetic markers, can be designed for highly conserved

In this approach, a combination of universal primers and a specific probe that attaches to a specific

Microsatellites or short tandem repeats (STRs) or simple sequence repeats (SSRs) are tandem stretches of 1-6 bp long-short nucleotide sequence motifs (e.g., ATATATAT) that occur randomly and are widely distributed in all eukaryotic genomes [84–86]. Variations in the number of repeat units lead to the difference in the size of both DNA fragments (alleles) that can be resolved and visualized on gel electrophoresis [86, 87]. These polymorphic loci are generally used in genetics and forensics studies. These are codominant markers.

AFLPs are dominant DNA markers in which an allele is present or absent in an individual. AFLP locus cannot determine the heterozygosity of any individual. Therefore, in contrast to the codominant microsatellite DNA markers, AFLPs have less resolving power to assign an individual to its population of origin [88]; generally, at least 50 AFLP loci and 8 microsatellite loci are recommended to conduct population assignment tests [89]. Furthermore, in comparison to microsatellites, high-quality DNA requirement and greater

DNA regions or fragments (universal primers) or areas where highly variable DNA sequences occur

variant of DNA sequence is used. Such probes allow detecting the base situated at SNP site.

Generally, a panel of highly polymorphic microsatellite loci is first selected and used to generate the genetic profile of a test sample or forensic specimen [91, 92]. This profile is assigned to a particular population by matching and comparing its observed alleles with the observed allele frequency in the population. There are several analytical methods available to perform assignment tests [83] and freely available statistical software packages [93]. Population assignment tests are highly

**42**

**Box 1.**

For the last three decades, individual identification of forensic specimens based on a unique DNA profile has revolutionized human forensic studies [30]. This technique can be employed to detect the number of individuals used in the commercial market or trade, even from highly processed or powdered products [47]. Baker et al. [96] used a partial fragment of the mtDNA control region (464 bp) and 8 microsatellite loci to identify the minimum number of individual North Pacific minke whales (*Balaenoptera acutorostrata* spp.) from 12 markets.

Highly polymorphic SNP or microsatellite markers are used to generate a DNA profile with a series of gene variants or alleles (**Box 1**). The inclusion of more number of loci or markers reduces the chance that two different individuals will have the same DNA profile. Samples are identified to be from the same or different individuals based on the same or different DNA profiles, respectively. It is important to calculate the probability of identity that two individuals may share the same DNA profile [30].

In Canada, a database of DNA profiles has been established and is commonly used to support forensic investigations of the poaching cases of black bear (*Ursus americanus*), caribou (*Rangifer tarandus*), moose (*Alces alces*), elk (*Cervus canadensis*), white-tailed deer (*Odocoileus virginianus*) and mule deer (*Odocoileus hemionus*) (www.forensicdna.ca/dnadatabases.html). Similarly, DNA profiling has been used to generate strong evidence by matching the blood left on the suspect's knife to the carcass of a wild boar in northern Italy [97].

Furthermore, the ability to validate familial relatedness is also a major application of wildlife DNA forensics. In a forensic investigation, the focus of establishing levels of relatedness lies predominantly on the discrimination of wild-caught animals from captive-bred [30]. Genetic or DNA markers are inherited from both parents from one generation to the next that allows using DNA profiles to validate parent–offspring relationships. Microsatellite loci–based DNA profile database is used in Australia and Europe to authenticate captive bird breeding, whereas parentage DNA analysis is applied to verify caviar of captive sturgeon (*S. Rastorguev pers.* comm.) [30].

## **4. Study of isotopes in wildlife forensics**

Apart from species identification from unknown wildlife sample, quite often it also becomes imperative to identify the geographic origin of the sample. In addition to the DNA-based methods for ascertaining the possible geographical origin of samples, the study of the elemental profiles of samples is also a reliable means for predicting the geographic origin of the samples. This also becomes important when a particular species is protected in one area and not in another, and further when animals from the wild are captured and traded as captive-bred [98]. Among the elemental analysis techniques, a comparison of the ratios of different isotopes using methods such as inductively coupled plasma mass spectrometry (ICP-MS) and

isotope ratio mass spectrometry (IRMS) is an established method for predicting the geographic origin of wildlife samples. Variations in the concentration of elements and ratios of the isotopes have been used widely to ascertain the geographical origins of the African Rhinoceros horn [99–101]. Further, Amin et al. used mass spectrometry to study carbon and nitrogen isotopes and laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) to measure the relative abundance of isotopes of various elements to ascertain the geographic origin of African Rhinoceros horns [102]. Recently, Alexander et al. used stable isotope analyses to monitor illegally traded African gray parrots [103].

#### **4.1 What are isotopes?**

Isotopes are atoms of the same element that have a different number of neutrons in their nucleus; hence, they have the same atomic number but their atomic mass is different. Isotopes can be two types, that is, radioactive isotopes or stable isotopes.

Radioactive isotopes have an unstable nucleus that tends to attain a stable form by emitting radiation. Hence, these isotopes are called radioactive isotopes. The process is also radioactive decay, and during the process, particles and photons are emitted. Carbon-14 (14c), which is widely used in the dating of archeological samples and is a good example of a radioactive isotope. It has a nucleus with six protons and eight neutrons. With time, it decays into the nonradioactive nitrogen-14.

Stable isotopes have stable nuclei and hence do not exhibit radioactivity; that is, they do not undergo radioactive decay. Any element that has isotopes will have a lighter isotope (with a lesser number of neutrons) and a heavier isotope (with a higher number of neutrons). Further, the relative abundance of these isotopes with respect to each other varies significantly with geographical location. Hence, for an unknown sample, if the relative abundance of isotopes of a particular element is determined, it becomes possible to predict the geographical origin of a sample, based on the relative abundance of the isotopes. The elements and isotopes thereof enter the food chain and hence get incorporated into the tissues of living organisms. Further, the elements along with their isotopes remain in the remains of the organisms. As species are distributed according to ecozones and geography, their elemental profile including the relative abundance of stable isotopes is bound to vary with the geographical origins. This is used in wildlife forensics for predicting the geographical origins of wildlife samples with analysis of stable isotopes and elemental profiles. The isotopes most commonly used in forensic science for this purpose are generally H, C, N and O [98, 104].

#### **4.2 Techniques used for isotope studies in wildlife forensics**

#### *4.2.1 ICP-MS*

Inductively coupled plasma mass spectrometry (ICP-MS) is a highly sensitive technique of elemental analysis with a capability to detect metals and nonmetals and very low concentrations. Further, it has the capability of detecting isotopes of an element in a given sample.

#### *4.2.2 IRMS*

Isotope-ratio mass spectrometry (IRMS) is a specialization form of mass spectrometry, in which mass spectrometric methods are used to determine the relative abundance of isotopes in a sample.

**45**

India

**Author details**

Vivek Sahajpal1

\*, Sudhanshu Mishra2

2 Sichuan Agricultural University, Chengdu, China

provided the original work is properly cited.

\*Address all correspondence to: viveksahajpal@gmail.com

1 Directorate of Forensics Services, Junga, Shimla, Himachal Pradesh, India

3 Institute of Forensics Science, University of Mumbai, Mumbai, Maharashtra,

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Deepika Bhandari1,3

*Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis*

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a highly sensitive elemental and isotopic analysis method in which analysis can be directly performed on solid samples. It used a process called laser ablation in which a laser beam is focused on the surface to generate fine particles. These particles are then transported to an ICP-MS for digestion and ionization and subsequent detec-

Elaboration of these techniques shall be beyond the scope of this chapter. The usefulness of the technique has been demonstrated by several workers in this field and the technology has great potential in identifying the geographic origin of

As discussed earlier, the spectrum of wildlife forensics is very wide, and to address different queries of investigation, the use of different techniques may be required. Some of these techniques, even though readily available, still may require standardization with respect to wildlife crime samples. The matters get further complicated when the evidence material is very limited; hence, it becomes imperative to have a precise idea of what technique(s) should be employed for the purpose. In this chapter, we have covered some important techniques that may be useful for dealing with wildlife crime cases. However, to get a more refined and working

knowledge of the techniques, referring to detailed texts is advised.

*DOI: http://dx.doi.org/10.5772/intechopen.98252*

*4.2.3 LA-ICP-MS*

wildlife samples.

tion of elements and isotopes.

*Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis DOI: http://dx.doi.org/10.5772/intechopen.98252*

#### *4.2.3 LA-ICP-MS*

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

monitor illegally traded African gray parrots [103].

purpose are generally H, C, N and O [98, 104].

**4.2 Techniques used for isotope studies in wildlife forensics**

**4.1 What are isotopes?**

nitrogen-14.

*4.2.1 ICP-MS*

*4.2.2 IRMS*

an element in a given sample.

abundance of isotopes in a sample.

isotope ratio mass spectrometry (IRMS) is an established method for predicting the geographic origin of wildlife samples. Variations in the concentration of elements and ratios of the isotopes have been used widely to ascertain the geographical origins of the African Rhinoceros horn [99–101]. Further, Amin et al. used mass spectrometry to study carbon and nitrogen isotopes and laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) to measure the relative abundance of isotopes of various elements to ascertain the geographic origin of African Rhinoceros horns [102]. Recently, Alexander et al. used stable isotope analyses to

Isotopes are atoms of the same element that have a different number of neutrons in their nucleus; hence, they have the same atomic number but their atomic mass is different. Isotopes can be two types, that is, radioactive isotopes or stable isotopes. Radioactive isotopes have an unstable nucleus that tends to attain a stable form by emitting radiation. Hence, these isotopes are called radioactive isotopes. The process is also radioactive decay, and during the process, particles and photons are emitted. Carbon-14 (14c), which is widely used in the dating of archeological samples and is a good example of a radioactive isotope. It has a nucleus with six protons and eight neutrons. With time, it decays into the nonradioactive

Stable isotopes have stable nuclei and hence do not exhibit radioactivity; that is, they do not undergo radioactive decay. Any element that has isotopes will have a lighter isotope (with a lesser number of neutrons) and a heavier isotope (with a higher number of neutrons). Further, the relative abundance of these isotopes with respect to each other varies significantly with geographical location. Hence, for an unknown sample, if the relative abundance of isotopes of a particular element is determined, it becomes possible to predict the geographical origin of a sample, based on the relative abundance of the isotopes. The elements and isotopes thereof enter the food chain and hence get incorporated into the tissues of living organisms. Further, the elements along with their isotopes remain in the remains of the organisms. As species are distributed according to ecozones and geography, their elemental profile including the relative abundance of stable isotopes is bound to vary with the geographical origins. This is used in wildlife forensics for predicting the geographical origins of wildlife samples with analysis of stable isotopes and elemental profiles. The isotopes most commonly used in forensic science for this

Inductively coupled plasma mass spectrometry (ICP-MS) is a highly sensitive technique of elemental analysis with a capability to detect metals and nonmetals and very low concentrations. Further, it has the capability of detecting isotopes of

Isotope-ratio mass spectrometry (IRMS) is a specialization form of mass spectrometry, in which mass spectrometric methods are used to determine the relative

**44**

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a highly sensitive elemental and isotopic analysis method in which analysis can be directly performed on solid samples. It used a process called laser ablation in which a laser beam is focused on the surface to generate fine particles. These particles are then transported to an ICP-MS for digestion and ionization and subsequent detection of elements and isotopes.

Elaboration of these techniques shall be beyond the scope of this chapter. The usefulness of the technique has been demonstrated by several workers in this field and the technology has great potential in identifying the geographic origin of wildlife samples.

As discussed earlier, the spectrum of wildlife forensics is very wide, and to address different queries of investigation, the use of different techniques may be required. Some of these techniques, even though readily available, still may require standardization with respect to wildlife crime samples. The matters get further complicated when the evidence material is very limited; hence, it becomes imperative to have a precise idea of what technique(s) should be employed for the purpose. In this chapter, we have covered some important techniques that may be useful for dealing with wildlife crime cases. However, to get a more refined and working knowledge of the techniques, referring to detailed texts is advised.
