# Studies on Molecular Taxonomy and Morphological Characteristics of *Haematobia irritans exigua* (de Meijere, 1903); A New Range Extension in the Bargur Hills of Erode District in Tamil Nadu, India

Yaswanthkumar.S<sup>a</sup>, Ganapathi. P<sup>b</sup>, Velusamy R<sup>c</sup>
and Venkitachalam. R<sup>a*</sup>

## Authors' contributions

This work was carried out in collaboration among all authors. Author YS did the insect collection, morphological and molecular analysis and written original manuscripts. Authors VR and GP as the research supervisors and review and edit the manuscript. Author VR does review and edits the manuscript. All authors read and approved the final manuscript.

## Article Information

DOI: https://doi.org/10.56557/upjoz/2024/v45i154256

**Open Peer Review History:**

This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://prh mbimph com/review-history/3738

Received: 15/05/2024

Accepted: 11/07/2024

Published: 16/07/2024

**Original Research Article**

<footnote><sup>a</sup> Department of Zoology, Kongunadu Arts and Science College, Coimbatore-29, Tamil Nadu, India.</footnote>

<footnote><sup>b</sup> Bargur Cattle Research Station (TANUVAS), Bargur, Anthiyur Taluk, Erode-638 501, Tamil Nadu, India.</footnote>

<footnote><sup>c</sup> Department of Veterinary Parasitology, Veterinary College and Research Institute, Orathanadu-25, Tanjavur, Tamil Nadu, India.</footnote>

<footnote>*Corresponding author: Email: venkitachalam zo@kongunaducollege.ac.in;</footnote>

<footnote>**Cite as:** S, Yaswanthkumar, Ganapathi. P, Velusamy R, and Venkitachalam. R. 2024. "Studies on Molecular Taxonomy and Morphological Characteristics of Haematobia Irritans Exigua (de Meijere, 1903); A New Range Extension in the Bargur Hills of Erode District in Tamil Nadu, India". UTTAR PRADESH JOURNAL OF ZOOLOGY 45 (15):394-405. https://doi.org/10.56557/upjoz/2024/v45i154256.</footnote>
ABSTRACT

The flies of the genus *Haematobia* (Diptera: Muscidae) are hematophagous ectoparasites of medical and veterinary importance. The morphological identification of these flies is often complicated due to their similarities. This was the first documentation of *Haematobia irritans exigua* in Tamil Nadu which affected free-ranging Bargur Cattle (*Bos indicus*) from Bargur Hills of Tamil Nadu. The aim of this research was to provide the details on the molecular characterization and molecular divergence of the cytochrome c oxidase subunit 1 gene (COI) and 28S Ribosomal RNA of *Haematobia irritans exigua* and to compare them with the morphological features. The results of this study showed that the nucleotide composition of the COI gene of *Haematobia irritans exigua* had a higher AT (69.5%) and a lower GC (30.4%). The rate of transition was higher than that of transversion. The intra-specific distance based on COI gene analysis did not exceed 1.1%, while the inter-specific distance between different species ranged from 0.9% to 14.1%. A neighbor-joining tree and an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree were constructed using 1000 bootstrapped samples to analyze the phylogenetic relationship between different muscidae species based on the COI gene as an identification marker. The analysis of 28S Ribosomal RNA showed an intra-specific distance of 1.6% and an inter-specific distance ranging from 1.49% to 8.56%. The 28S rRNA revealed the presence of intra-specific variation among the species. Additionally, this study found notable color variations on the thorax of the flies in the same population, along with a minute phenotypical variation with minimum intraspecific distance among the flies in the same population. This taxonomic data of the hematophagous fly *Haematobia irritans* *exigua* would be well-intentioned for identification.

Keywords: *Bos indicus*; DNA barcoding; COI; 28S Ribosomal RNA; *Haematobia irritans* exigua (de Meijere, 1903).

1. INTRODUCTION

The flies belong to the order Diptera of the cosmopolitan distributed genus *Musca* (Linnaeus, 1758) with evolved mouthparts required for blood feeding causing blood loss for the host and acting as the vector for the transmission of pathogens [1,2]. Insect identification is important to understand the interaction among the host [3] and to report the essential queries in evolution, economic and ecological threats, and conservation biology [4]. The morpho-taxonomy has some limitations such as a lack of experts, resolving cryptic species, sexual dimorphism, adaptations, etc. [5,6]. The misidentification of veterinary and medicinal important flies will lead to ineffective control and management [7,8,9]. The proper identification of flies is essential for both epidemiological studies and to formulate control strategies [10]. Molecular identification is a potential tool to identify the species by matching unknown genes with known genes of a species [11]. Molecular gene markers are the more sensitive and accurate method for the identification of parasitic flies affecting livestock farms [12]. DNA barcoding is a complementary tool in the identification process by using some conserved and standardized regions of DNA [13]. The mitochondrial DNA, mitochondrial cytochrome c

oxidase subunit I gene (COI gene) is one of the
most useful gene markers for the identification of
insects and analyses of the phylogenetic
relationship [14]. COI marker helps in the
identification of cryptic species and provides
taxonomic-based diversity estimation [13]. In
addition to the Mitochondrial gene, 18S and 28S
Ribosomal RNA were used for the molecular
identification of the lower taxonomy up to the
species level [15]. The taxonomic status of
*Haematobia* taxa is still misperception because
of the similarity in morphological characteristics
[16]. There are variations between the taxa are
minor. The Buffalo flies (*Haematobia irritans
exigua* de Meijere) and horn flies (*Haematobia
irritans* Linnaeus) are the major flies with a minor
morphological variation. The *Haematobia irritans
exigua* (Buffalo fly) is a dipteran hematophagous
fly affecting cattle and buffalo in the oriental
region [17]. Both male and female flies feed
solely on blood, piercing the animal's skin
through their sharp mouthparts only ovipositing
on the cattle dung. The heavy infestations of
these flies may include several thousand flies per
animal, with each feeding up to 40 times daily,
irritating cattle and causing economic losses and
welfare issues, reducing hide value and making
cattle less appealing to consumers [18]. These
lesions can range from dry and alopecic or scab-
encrusted to severe open ulceration which lead
to secondary bacterial infections [19]. They are most commonly found beneath the cattle's eyes, but can also be found on the neck, dewlap, belly, and flanks [20], and their development and persistence have been linked to a currently unidentified variety of filarial nematode (*Stephanofilaria sp*.), which is transmitted by buffalo flies [21]. Bargur cattle are the indigenous free-ranging cattle endemic to the Bargur hills of Anthiyur taluk of Erode district in Tamil Nadu [22]. The free-ranging Bargur cattle were affected by *Haematobia irritans exigua*. The study aims to identify the molecular and morphological taxonomic status of *Haematobia irritans exigua* affecting the bargur cattle. The present study on identification and characterization of flies by using molecular markers COI and 28S rRNA and compares with the morphological character to provide clear data on the taxonomical identification of *Haematobia irritans exigua*. This was the first report on Bargur hills of Tamil Nadu.

## 2. MATERIALS AND METHODOLOGY

### 2.1 Study Area

The Bargur hills is located between latitude of 11.805° and longitude of 77.534° in the Anthiyur

taluk of Erode district in the Eastern Ghats region with elevation of about 1000 m above the mean sea level (MSL). The average temperature ranging from 15°C to 32°C and rainfall about 350 cm per annum. There are 34 hamlets are located in the Bargur hills namely Bargur, Thamarakarai, Thalakarai, Thurusanampalayam, Bejilpalayam, Oosimalai, Eeretti, Devarmalai, Bejilitti and Ondanai. The highest point is at Thamarakarai with 1078 m above MSL and lowest at Bejiletti with 988m above MSL. These areas receive rainfall during both the south-west and north-east monsoon. The Ragi, Maize, Dry paddy, Tapiocca and other rainfed crops are majorly cultivated agricultural crops. There numerous cattle are being reared in and around the Bargur areas but indigenous Bargur cattle are maintained under zero input with unique 'Patti' system. These cattle are coexisting with the wild animals especially with elephants which huge existing in this forest so called as semi wild types of cattle. The total area of the Bargur cattle breeding tract is about 34,043 ha and most of which comes under reserve forest category and now Bargur forest is announced a Periyar Wildlife Sanctuary by Tamil Nadu Forest Department.

<figure><img src="image_1.png" /><figcaption>Map. 1. Study Area Bargur Hills in Tamil Nadu, India</figcaption></figure>
## 2.2 Sample Collection and Identification

The ectoparasites were collected from all over the body of the Bargur cattle by using the handmade insect collection net. The collected insect was sprayed with 99% ethanol and then transferred into the glass bottles. The insect samples were taken to the laboratory for further morphological analysis. The parasites were identified with the help of a standard identification key.

## 2.3 Insect Measurements

The male and female insects were identified based on their morphological characteristic features. The following measurements such as total body length, wing length, and wing breath were taken by using a vernier caliper.

## 2.4 COI Gene Isolation and Amplification

DNA was extracted from the whole insect sample using a DNeasy Blood and Tissue kit (Qiagen; Cat # 69504) Kit following the manufacturer's instructions. The DNA of each individual was eluted in 50µl and stored at -20°C until further use of DNA amplification. The COI gene was amplified using the primer LCO1490 (5'-CACGACGTTGTAAAACGACGTCAACAAATC ATAAAGATATTGG-3') and HCO2198 (5'GGATAACAATTTCACACAGGTAAACTTCAG GGTGACCAAAAAATCA-3') [23]. The COI genes of insect DNA were amplified in a 20 µl reaction volume containing 1 µl DNA (10-50 ng), 1 µl each of Forward and Reverse primers (10 picomoles µl-1), and 10 µl Emerald Amp GT PCR master mix. The cycling conditions used were as follows: initial denaturation at 95°C for 2 min followed by cycle denaturation at 95°C for 40 sec, annealing at 45°C for 40 sec., extension at 72°C for 1.5 min for a total of 30 cycles, and a final extension for 10 min at 72°C. The success of the PCR reaction was confirmed by running 5µl PCR product on 1 % agarose gel with ethidium bromide at 120 V; for ~45min in 1X TAE Buffer. The PCR products were further treated with ExoSAP-I. PCR Product Clean-up Reagents were used as a template for sequencing PCR product. Sequencing PCR product was done with ABI PRISM Big Dye terminator ready reaction mix (Life Technologies, USA) available in Enfys Lifesciences Pvt Ltd, Cochin, Kerala.

## 2.5 28s Ribosomal RNA Isolation and Amplification

The whole sample was picked up and placed in a mortar and homogenized with 1 ml of extraction

buffer and the homogenate was transferred to a 2 ml-microfuge tube. The DNA isolation was done by phenol chloroform method and to remove RNA 5 µl of DNAse free RNAse (10 mg/ml) was added to the DNA. 177 ng of extracted DNA is used for amplification along with 10 pM of each primer. The PCR product size is ~0.8 kb using the forward primer sequence (5'-ACTACCCCCTGAATTTAACAT-3') and the reverse primer (5' GACTCCTTGG TCCGTGTTTGAAG-3'). Each reaction mixture for the amplification of the 28S rRNA gene by PCR consists of 1 ul DNA, 2µl 28s Forward Primer, 2µl Reverse Primer, 4 ul dNTPs (2.5mM each), 10 ul 10X Taq DNA polymerase Assay Buffer, 1 ul Taq DNA Polymerase Enzyme (3U/ml), 30 ul Water. The PCR cycle conditions were as follows: 94°C for 3 minutes; followed by 94°C for 3 minutes, 50°C for 1 minute and 72°C for 2 minutes repeats for 30 cycles, and a final extension of 72°C for 7 minutes. The PCR product was sequenced Bi-directionally. Sequencing was done by using the ABI 3130xl platform in Biokart genomic lab, Bangalore.

## 2.6 Bioinformatics Analysis

The sequences were analysed, trimmed, and edited by using Bio edit software. The sequences obtained from the present study were compared with already submitted sequences with NCBI to analyse the nucleotide composition, Homogeneity of Substitution, Genetic Distance, and Phylogenetic analysis were done by using Mega 11 software.

# 3. RESULTS

## 3.1 Morphological Measurements of Male and Female *Haematobia irritans* exigua

The average body length of the male flies was 4.55 (0.48 ± 0.09) mm, wing length was 2.32 (0.50 ± 0.10) mm and wing breadth was 1.04 (0.25 ± 0.05) mm. The average body length of female flies was 4.09 (0.54 ± 0.10) mm, wing length was 1.95 (0.44 ± 0.08) mm and wing breadth was 1.14(0.3 ± 0.06) mm. The body length and wing length (r=0.8) and the wing length and wing breadth (r=0.7) show a positive correlation.

## 3.2 Morphological Characteristics of *Haematobia irritans* exigua

**Head:** The both male and female adult flies had piercing and sucking mouth part. The maxillary palp was shorter than the proboscis and held
apart. The antenna has six to seven aristae. The
eyes were dark red in fresh samples (Fig. 1.).
The space between the eyes was the main
distinguishable character between males and
females and male. The eyes are close together
in the male (Holoptic) and wider spaced in
female (Dicoptic). A single row of bristles was
present on each side of the midline between the
eyes (Fig. 7.).

**Thorax:** Thorax has two dark inconspicuous stripes on the mesothorax region (Scutum) (Fig. 5.). A pair of presultural supra-alar seta and a pair of postsultural supra-alar seta was present. A pair of postalar seta and a pair of intra-alar seta were present at the lateral proximal end of the scutum region. A pair of dorsocentral seta and a pair of acrostichal seta were present in the middle above the scutoscutellar suture. The scutellum region was dark with a pair of basal scutellar seta and a pair of apical scutellar seta (Fig. 6.).

**Abdomen:** The abdomen was oval, dark without any markings, attached anteriorly to the thorax. In females, the distal segments were modified to form an ovipositor (Figs. 3 and 4).

**Legs:** The presence of two long hairs at the distal tibia followed by two long hairs with curled tip on the first segment and 4 to 8 long hairs with curled tip on the second segment of the hind tarsus of male flies while this character was absent in female flies (Fig. 2).

**Wings:** Wing vein M1 is gently curved forward. The wings had a dark hint due to minuscule pubescence (Fig. 8).

**3.3 Molecular analysis of *Haematobia irritans* exigua**

The NCBI (GenBank) sequences accession
numbers are PP335804, PP336418 for the COI
gene, PP335205, PP335233 for 28S ribosomal
RNA. Also, sequences were recorded in Barcode
Index Number Registry BOLD:AAC7987 in the
Barcode of Life Data Systems (BOLD).

**Molecular Analysis Based on Mitochondrial Cytochrome c Oxidase Subunit I Gene**

**Nucleotide composition of specimen:** The nucleotide sequence and particular nucleotide percent have been evaluated due to their importance in the study of variations among the different species. The nucleotide composition of the sample was analyzed to know the percentage of base content by using Mega 11 software. The average percentage of T (40.5%), A (29.0%), C (14.2%), and G (16.3%). The

higher AT percentage (69.5%) and the lower GC percentage (30.4) were found in *Haematobia irritans exigua*. The variations in the A+T content was higher than G+C. The higher A+T-rich region is the distinguishing makeup of insect mtDNA [24].

**Maximum composite likelihood estimate of the pattern of nucleotide substitution:** The entry shows that probability of substitution (r) from one base (row) to another base (column). The sum of r values was made equal to 100. The nucleotide frequencies are (28.90%) A, (40.14%) T/U, (15.49%) C, and (15.46%) G. The transition/transversion rate ratios are k₁ = 0.741 (purines) and k₂ = 4.515 (pyrimidines). The overall transition/transversion bias was R = 1.272, where R = [A*G*k₁ + T*C*k₂]/[(A+G) *(T+C)]. The most frequent transitions were T to C type. There were 24 nucleotide sequences involved for the analysis. The codon positions included were 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were total of 356 positions in the final dataset.

**Test of the homogeneity of substitution patterns between sequences:** The sequences have evolved with the same pattern of substitutions and the probability of rejecting the null hypothesis that, as judged from the extent of differences in base composition biases between sequences (Disparity Index test). A Monte Carlo test (500 replicates) was used to estimate the P-values, which are shown below the diagonal. The P-values smaller than 0.05 are considered significant. The estimates of the disparity index per site are shown for each sequence pair above the diagonal. Codon positions include 1st+2nd+3rd+Noncoding for 24 nucleotide sequences.

**Genetic distance:** The intra-specific genetic distance range of flies varies between 0.3% to 1.1%. Similarly, the interspecific genetic distance between the *Muscidae flies* was 1.1% to 14.1%. The maximum genetic distance was observed with *Musca domestica* 13.0% to 14.1% followed by *Haematobosca sanguinolenta* with 9.4% to 13.8% and *Stomoxys indicus* with 6.3% to 12.9%. The least inter-specific distance was found with *Haematobia irritans* 0.3% to 1.7%. The minimum genetic distance was noted because the *Haematobia irritans exigua* was a subspecies of *Haematobia irritans*. The *Tabanus biannularis* was taken as the out-group and shows the maximum genetic distance of 20.6% to 22.7%.
<figure><img src="image_2.png" /><figcaption>**Fig. 1** Adult *Haematobia irritans exigua*
(female)</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Fig. 3.** Female *Haematobia irritans exigua*</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 5. Dorsal view of Thorax</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 7. Head variation</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 2. Hind Leg of Male *Haematobia irritans*
*exigua*</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 4. Male *Haematobia irritans* exigua</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 6. Scutellum region</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 8. Wing Venation</figcaption></figure>

**Phylogenetic analysis:** The evolutionary
relationship of the COI gene obtained during the
study together with the sequences available
from NCBI by using the Neighbor-Joining method

(Fig. 9.) and Unweighted Pair Group Method with
Arithmetic Mean (UPGMA) method (Fig. 10.).
The percentage of replicate trees in which the
associated taxa clustered together in the
bootstrap test with 1000 replicates. The
Phytogenic tree is drawn to scale, with branch
lengths in the same units as those of the
evolutionary distances used to infer the
phylogenetic tree. The evolutionary distances
were computed using the Maximum Composite
Likelihood method and units of the numbers of
base substitutions per site. The proportion of
sites where at least one unambiguous base was
present in at least one sequence for each
descendent clade was shown next to each
internal nodes of the tree. The phylogenetic
analysis revealed that species belonging to the
same genus because the same node formed with
two branches. The first branch had two clades of
*Haematobia irritans exigua*. The first clade was
occupied by *Haematobia irritans exigua* from
India and Japan and the second clade with
Thailand flies. The *Haematobia irritans* was listed
in the second branch and formed three clades.
The specimen shows 100% similarity with the
*Haematobia irritans exigua*. The *Tabanus
biannularis* was taken as the outgroup for the
phylogenetic analysis.

**3.4 Molecular Analysis Based on 28S Ribosomal RNA**

**Genetic distance:** The intra-specific variations showed between the sample was 1.16%. The variation between the same genus was 1.49%, and the results *Haematobia irritans* may be a sub species varies from *Haematobia irritans* because inter-specific variation ranges from 3.52% to 8.56%.

**Phylogenetic analysis:** The neighbor-joining analysis of 28S rRNA with 1000 bootstraps by the Kimura 2-parameter model showed similar topologies. The node-linking sequences of the same *Haematobia* species had high bootstrap values. The tree clustered into four major clades, the first clade consists of *Haematobia irritans exigua*, the second clade consists of *Stomoxys calcitrans*, and the third clade consists of Musca domestica (Fig. 11). The phylogenetic analysis shows that the sample was more similarity with the *Haematobia irritans exigua* with minimum variations.

<figure><img src="image_10.png" /><figcaption>Fig. 9. Neighbour-Joining Tree of COI gene</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Fig. 10. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) Tree of COI gene</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>Fig. 11. Neighbour-joining tree of 28S Ribosomal RNA</figcaption></figure>
4. DISCUSSION

The Stomoxyini is the bloodsucking insects
under the tribe of Muscinae subfamily of
Muscadiae comprising about 50 species in the
world [1]. In Stomoxyini, the horn flies
*Haematobia irritans* with two closely related
subspecies of *Haematobia*, the horn fly, *H.
irritans irritans* and the buffalo fly, *H. irritans
exigua* (De Meijere), have morphological and
genetic differences [25,16]. *H. irritans* is mostly
distributed in the Polarctic Region and
*Haematobia exigua* is distributed throughout the
Oriental and Australasian Regions.
Morphological differences between these two
species are very minor. *Haematobia Lepeletier &
Serville, Haematobia minuta* (Bezzi,1872),
*Haematobia sanguisugens* (Austen,1909),
*Haematobia titillans* (Bezzi,1907), *Haematobia
exigua* were found in India [26]. The body length
and number of frontal setulae between the
populations of *Haematobia irritans* and
*Haematobia exigua* collected from various
localities were reported along with the
morphological variation based on the latitudinal
gradient [27]. The genetic variability in the
Brazilian population of *H.irritans* which dispersed
throughout the country through cattle trade [28].
In *H. exigua*, variations in male and female body
length and number of frontal setulae in males
between Taiwan and Vietnam may be due to
minor genetic variations (0.3–0.4%) and more
variable from Vietnam (0.4%) suggested as the
presence of more intraspecies gene diversity in
the tropical region [14]. *H.exigua* prefers water
buffalo than cattle in the Oriental region [29]. The
*H.irritans* and *H.exigua* had adapted based on
the different geographical areas [14]. The
distinguishable character of *Haematobia exigua*
from other *Haematobia* species is the presence
of 4 to 6 long hairs with curled tips on the second
segments of the male’s hind tarsus [27]. The
*Haematobia irritans* and *Haematobia exigua*
have often been regarded as different species
[30,31], or as subspecies [25,31]. There are only
a few studies that have looked at the molecular
characterization and variability of *Haematobia*
species in terms of genetic surveys [32]. The
genetic and morphological differences between
*Haematobia irritans* and *H. exigua* and molecular
phylogeny of Japanese stomoxyini flies were
reported [14]. The two species are genetically
distinct, as shown by recent studies on molecular
differentiation of the two species using the
mtDNA COI, cytochrome B (Cytb), NADH
dehydrogenase subunit 5 (ND5), nuclear, and
18S and 28S ribosomal RNA regions [16,17].

The most informative genes for differentiating the
two species were the COI and Cytb genes. All
evidence points to a very close relationship
between buffalo and horn flies, regardless of
whether they can be classified as distinct species
[33]. The transitional substitution frequency is
known to be higher than the transversional
substitutions in a genome [34]. The high level of
sequence divergence with transition saturation
indicates saturation in data. Our study shows a
higher rate of transversions than the transitions
lead to considerable saturation of the sequences.
By analyzing these substitutions, the
transversion contains a stronger phylogenetic
signal. The invertebrate DNA barcoding shows 3
to 3.9% of intra-specific variation [35]. According
to Iwasa and Ishiguro (2010), the mt-DNA in the
COI to COII genes of buffalo flies from Taiwan
and Vietnam and horn flies collected from Japan
showed sequence divergence of 1.8% to 1.9%
between the two species. Based on the COI
gene, the least inter-specific divergence was
found with *Haematobia irritans* 0.3 % to 1.7%.
The intra-specific genetic divergence range of
flies varies between 0.3% to 1.1%. The genetic
distance between the haplogroups of *H.exigua*
was about 0.12-0.95% was lower than the
distances between the *H.irritans* and *H.exigua*
[17]. Based on the analysis of 28S rRNA the
intra-specific variation between the sample flies
was 1.16%. and the variation between the same
genus is 1.49%, The inter-specific variation
ranges from 3.52% to 8.56%. According to the
10X rule, the percentage of nucleotide
divergence between intraspecies should be less
than 3%, while interspecies divergence should
exceed 3%. The sequence analysed in this study
showed significant inter-species variability based
on nucleotide sequences and the intraspecific
divergence was high enough to distinguish
between individuals. The mitochondrial DNA
variations provide the phylogenetic relationship
of various insect groups at a generic level
[36,37].

5. CONCLUSION

This was the first report of *Haematobia irritans*
*exigua* with morphological and molecular
identification on Bargur Cattle of Tamil Nadu,
India. The molecular analysis shows that
*Haematobia irritans exigua* is a subspecies of
*Haematobia irritans* with morphological
characteristics of *Haematobia exigua*. This study
can provide clear morphological and molecular
data on the *Haematobia irritans exigua* for
identification.
## 6. SIGNIFICANCE OF THIS WORK

This is the first record of the *Haematobia irritans exigua* infested on Bargur Cattle which is one of the indigenous and endemic cattle found in Bargur Hills of Eastern Ghats of Tamil Nadu, India. This paper can provide clear data on the Morphological and Molecular identification of *Haematobia irritans exigua*.

## DISCLAIMER (ARTIFICIAL INTELLIGENCE)

Author(s) hereby declares that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc.) and text-to-image generators have been used during writing or editing of manuscripts.

## ACKNOWLEDGEMENT

The authors are thankful to Tamil Nadu Veterinary and Animal Science University, Chennai, and Kongunadu Arts and Science College, Coimbatore, Tamil Nadu, India for providing us with the opportunity and Support to carry out this work. The authors would like to thank the farmers of Bargur Hills of Erode District, Tamil Nadu, India for their cooperation.

## COMPETING INTERESTS

Authors have declared that no competing interests exist.

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