DNA Barcoding of Pest Rodents: An Approach in Integrated Rodent Pest Management

The most significant vertebrate pests in agriculture are rodents and their habitation, dispersal, mass, and productive importance differs depending upon the intake of crops, climatic seasons, and terrestrial areas across India. Molecular identification using DNA barcoding with the COI gene provides a high level of taxonomic differentiation (>95%) at the species level. An attempt was made to identify certain pest species of rodent namely Bandicota indica, Rattus rattus, Millardia meltada, and Tatera indica , from six different places through the COI gene in the context of rodent pest management program (RPM). The present study also covered the population survey, seasonal variation, and species of rodent pest morphology. The study revealed that the number of rodents seems to be equal in pre-monsoon and post-monsoon and there is a seasonal variation between the numbers of species in different localities. However, there were no morphological changes within the species. The present investigation showed that there were no intra-specific nucleotide alterations within M. meltada and T. indica and low intraspecific variation within B. indica and R. rattus at a greatest 0.3% and 0.5%, respectively. T. indica and R. rattus had the most elevated interspecific divergence, around 22%, and M. meltada and B. indica had the lowest, around 4%. The current results revealed that the COI gene’s 650 bp is accurate for the finding of major species of rodents from other species. Consequently, molecular identification of individual rodent pest species would generally assist to develop rodent pest programs by using species-specific (i


INTRODUCTION
R odents are the most abundant and the most prosperous class of mammals on the planet.Most predominant rodent pests such as Bandicota indica, Bandicota bengalensis, Mus booduga, Rattus rattus, Millardia meltada, and Tatera indica are found in south India and affects agriculture and warehouse, etc.Some of them are widely distributed while others are restricted in their dispersal.Attempts have been made to develop methods for the rodent pest management (RPM) using different techniques but none was successful in management (Archunan and Achiraman, 2006;Capizzi et al., 2014).Pheromones involved in chemical communication may have a major part in RPM (Robert, 2003;Archunan, 2009).Earlier on urine and scent glands of rodents used as lone sources with bait showed partial success in RPM (Selvaraj and Archunan, 2002).Since pheromones are speciesspecific their influence makes an impact on reproduction and social behaviour.It is necessary to identify the different rodents in the field and then only the pheromone cues can be used to develop a technique for the RPM program.Conventional approaches for the identification of rodent pests have traditionally been based on external morphological features and morphometric analysis such as colour, fur, teeth, skull, etc.Unfortunately, due to the lack

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of taxonomic experts, the identification of these rodents is often difficult (Bacher, 2012).For implementing integrated pest management (IPM), accurate species identification is important to understand the distribution and life history of a particular species.For this reason, the identification of rodents based on molecular methods such as DNA barcoding is imperative (Dobigny et al., 2011).DNA barcoding plays a key role in the anticipation of well-known pests to the database from all over the world.In DNA barcoding accurate identification is made at the species level based on the sequence similarity of Cytochrome C oxidase 1 (COI) gene rather than on morphological characters of (Hebert et al., 2003).The combined use of both DNA barcoding and traditional taxonomy species identification can be put together as an effective tool for identifying species identification across a wide variety of taxa.The main objective of this study is to identify the selected four rodent pest species by morphometric analysis and DNA barcoding.In addition, population status and seasonal variation of selected four rodent pest species in and around the selected six study areas were done.The identified four rodent pest species were compared with the other species for similarities and divergence by phylogenetic analysis.This attempt has been made as support of developing a pheromone trap controlling for rodent pests as part of IPM.

Study animals
Bandicota indica (Bechstein, 1800), commonly known as greater bandicoot rat, is found throughout most parts of India, Sri Lanka, Bangladesh, the lowlands of Nepal, Myanmar, southern China, Taiwan, Thailand, Cambodia, Vietnam and the island of Java.Millardia meltada (Gray, 1837) also called a South Asian field rat is commonly known as a metad and soft-furred field rat.M. meltada is.It is widely distributed from India, Nepal, Pakistan and Sri Lanka.Rattus rattus (Linnaeus, 1758) is commonly known as house rat, black rat, roof rat and ship rat.It is originally an Indohimalayan species.A native of the Indian sub-continent, this rat has now spread throughout the world as a result of human activities.Tatera indica (Hardwicke, 1807), commonly called Indian Gerbil has been recorded from southeastern Turkey, eastern Syria, Kuwait and Iraq, ranging through much of central and southern Iran to Pakistan and Afghanistan, Furthermore, it ranges throughout India and Sri Lanka.

Animal collection
Animals were collected from the residential houses, agriculture fields, and storage godowns with intensive trapping procedures for twelve months.Live trapping methods in which single and multiple traps were used to capture R. rattus and B. indica (Flowerdew et al., 2004).The traps were placed in places where the signs of regular rodent activity showed.Net trapping and burrow identification method were used for capturing T. indica and M. meltada (Neelanarayanan et al., 1994;Palanivelu, 2004).The seasonal variation of these four rodent species

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was calculated.To determine the trap success for B. indica and R. rattus, trapping was carried out bimonthly with 10 and 20 traps, respectively.Trap success (abundance) was calculated for R rattus and B. indica by using the formula described by (Lathiya et al., 2003).The animals that have been captured were incarcerated in cages made of steel and polypropylene, before being transported to the animal house of Bharathidasan University.The collected and cage animals were identified to the species class using morphological features (Aplin et al., 2003).

Population survey and morphometric analysis
As a preliminary study, we analyzed a population study of four species of rodents in diverse localities to know the variations among the species.The number of animals captured was recorded and surveys of rodent species were done according to the procedure of Aplin et al. (2003).As many as 586 adult rodent pests of four different species from six different localities were taken for morphometric analysis.The external morphometric characters were measured and analyzed by one-way ANOVA using SPSS (Ver.16.0) Software.The p-value <0.01 is considered significant in the present investigation.

Sequence and phylogenetic analysis
DNA was extracted from liver samples, amplified, and sequenced (Lakshminarayanan et al., 2015) which was checked, aligned, and manually edited by using CLUSTAL W (Thompson et al., 1994).The COI sequences were blasted in the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) for similarity searches.All the appropriate sequence information was documented in the barcode of life database (BOLD, www.barcodinglife.org)(Ratnasingham and Hebert, 2007).The exact specimen record and sequence data for each rodent pest species are available on the BOLD in the project file (Rodents in India).Every sequence has been presented to Genbank and accession numbers were obtained.Thirteen rodent species available in the Genbank database along with the four study species were used for phylogenetic analysis.All the seventeen species included in the study belonged to Murinae, Arvicolinae, Cricetomyinae, Sigmodontinae, Gerbillinae subfamilies.Interspecific and intraspecific nucleotide sequence divergences were calculated for the rodent species founded on the Kimura-2 parameter (Kimura, 1980) for distinctive COI sequences.The neighbour-joining (NJ) tree was also created by utilizing the MEGA X version software (Kumar et al., 2018) with a bootstrap value of 1500 replicates.All the sequences were aligned and trimmed evenly for balanced weight, for the appropriate species assessment phylogenetic tree analysis.

Population survey and seasonal variation
Among the rodents collected, R. rattus was highest in number and B. indica was found to be lowest in number.By considering the predominance of individual species populations and the twelve-month field survey among the six sites Bhudhalur showed the highest population of B. indica (n=46), M. meltada (n=45), and R. rattus (n=63).Similarly, Keeranur had the highest population of T. indica (n=61) and the lowest number of B. indica (n=23) was recorded in both Lalgudi and Nidur, however, the number of species is equal in both study areas.Astonishingly, the lower population of M. meltada (n=29) and R. rattus (n=34) was found only in Lalgudi.Similarly, the lowest population of T. indica (n=47) was identified in Ootangadu when compared to that of other respective localities.Invariably in all three seasons R. rattus and T. indica were found in maximum number when compared with other species of B. indica and M. meltada irrespective of localities.However, R. rattus and T. indica were more predominant in the premonsoon than in the monsoon and post-monsoon periods.Among the subordinate groups, the B. indica and M. meltada were less abundant in pre-monsoon and monsoon respectively when compared to post-monsoon season.

Morphometric analysis and species abundance
Table I shows the comparative analysis of eight external morphometric cheracteristics, which differed statistically (P<0.01)among the four rodent pest species.Present results revealed that there is no significant statistical difference in morphological observation within the species of the different localities.The current investigation showed that an increased abundance of B. indica was recorded in Nidur, while Ootangadu had a lower abundance.The percentage of dissimilarity in the mass of B. indica was statistically significant (P<0.01).Likewise, a high abundance of R. rattus was observed in Nidur whereas its abundance was low in Keeranur.The percentage of difference in abundance of R. rattus was statistically significant (P<0.01).

Genetic identification
In the present study, the COI proteins were found to be conserved in which no insertion, deletion, or stop codons were observed in the sequence.The BLAST result of the COI sequence for all four species showed more than 97% identity with the same species found in the Genbank.The intraspecific nucleotide divergences observed within B. indica and R. rattus were 0.3% and 0.5%, respectively.By distinction, there were no interspecific nucleotide variations within the species of M. meltada and T. indica (Table II).The interspecific nucleotide divergence among rodents is as follows: B. indica and R. rattus were 15%, B. indica and T. indica were 19%, M. meltada, T. indica were 19%, R. rattus and M. meltada were 16%.The most elevated nucleotide variation was found between T. indica and R.

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rattus at 22% and the lower between M. meltada and B. indica at 4 % (Table III).The phylogenetic tree analysis using COI fragments clearly showed that T. indica was grouped under the Gerbillinae subfamily whereas the other three species (i.e.,) B. indica, M. meltada, and R. rattus were grouped under murinae subfamily (Fig. 2).Furthermore, all four pest species of rodents (i.e.) B. indica, M. meltada, R. rattus, and T. indica are grouped under a single separate clade individually.All the species described additionally than one sample formed 99% -100% supported clades.The phylogenetic tree further indicated a vital species distinct from other species.The subfamily and genera relationship was also extremely endorsed with 100% bootstrap values within 1500 replicates.All the COI sequences from the four rodent species were further analyzed in BOLD.Among the four different species, three species, (B.indica, M. meltada,

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Tactic of Rodent Pest Management and R. rattus) belonged to the murinae subfamily and one, (T.indica) belonged to the Gerbillinae subfamily.The BOLD database showed 100% sequence similarity within the species for all four rodent species.The NJ phylogenetic tree revealed a straightforward identification and showed a single cluster of individuals for the four rodent pest species (Fig. 3).Further, an illustrative barcode was generated by BOLD based on the COI sequence obtained from the four rodent species.

DISCUSSION
It is critical for efficient biosecurity and biosurveillance programmes to intercept potential invasive species at ports of entry.However, taxonomic assessment of immature stages of most arthropods is difficult; identifying features are frequently dependent on adult morphology and reproductive systems (Madden et al., 2019).The fundamental purpose of DNA barcoding is to distinguish all species from one another and to build a reference library for all organisms.There have been numerous studies in different animals using DNA barcoding and a portion of the COI gene (Waugh, 2007).There are recent reports that genome skimming can be used to widen the process of DNA barcoding to develop the standard barcode (Eric et al., 2016) species through the DNA barcode method.The genetic distance between these two species T. indica and R. rattus is found to be high (22%) and low between M. meltada and B. indicia (4%), while the genetic distance between B. indica and R. rattus is low (0.3% and 0.5%, respectively).The present study clearly showed that the phylogenetic analysis of all four different species comes under the same cluster.Sequences from identical species were grouped with lower divergences and exhibited 100% bootstrap supports in phylogenetic analysis.It is noted that near-interconnected species from the identical genus and subfamilies were also found separated in the analysis.This NJ phylogenetic tree analysis further shows a strong species distinct from other species.
Based on the results obtained in the population survey, it is found the fluctuations in the population size of rodents related to the sample sites, showed that there is a variation in population size from place to place.Further, the species abundance is varying due to their habitat in different geographical regions.The size of a population is likely to be based on the seasonal model of reproduction, like other Didelphis marsupialis (Cerqueira, 1988).In the present study, a total of eight morphologic characteristics are considered and found that all the characteristics do not vary significantly (P<0.01)within the rodent species in different localities, by contrast, it differs statistically, among the four different pest species of rodents irrespective of the study area.
The outcomes of the current analysis inferred that though the population density of rodent species was altered by the geographical regions, the COI gene provides strong help to the downward taxonomy up to the level of subfamily than to that of upward taxonomy.Our findings showed that 650 bp of COI gene sequence has been generally accurate and can be used as a novel technique for the identification of rodent pest species.

CONCLUSION
The identification of species among rodents through the COI gene provides strong support to choose a particular species in a particular region.Therefore, it could be a possible way to identify the species and control the use of pheromone compounds against the conspecific rodent.The present investigation does not cover all the species available in the study area and the same approach may be adapted and implemented for identifying other pests in future studies.

Fig. 1 .
Fig. 1.The geographical location of the study areas in Tamilnadu, India.

Fig. 2 .
Fig. 2. NJ evolutionary phylogenetic tree constructed based on (K2P) inferred from 604 bp sequence of COI sequence.The analysis involved 18 nucleotide sequences.Philarctus bergrothi were utilized as an outgroup to implant the tree.NJ, Neighbor Joining

Fig. 3 .
Fig. 3. NJ phylogenetic tree constructed based on (K2P) inferred from COI of B. indica, M. meltada, R. rattus, and T. indica.The bootstrap values are displayed adjacent to the branches.The study included 51 nucleotide sequences.All positions with gaps and missing data were removed.The final dataset contained 614 positions in total.NJ, Neighbor Joining.

Table I . Morphometric characteristics of B. indica, M. meltada, R. rattus, T. indica collected from six different localities.
Values are expressed as mean ± SD for eight morphometric parameters for four rodent pest species.Statistical comparisons were made with one-way analysis of variance (ANOVA).No statisticaldifferences (p < 0.01) were observed within the each morphometric characters.