New Record: Study on Morphometric and Molecular Taxonomy of Vespidae Species (Hymenoptera: Vespidae: Polistinae) in Different Districts of Tamil Nadu, India

New Record: Study on Morphometric and Molecular Taxonomy of Vespidae Species (Hymenoptera: Vespidae: Polistinae) in Different Districts of Tamil Nadu, India | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article New Record: Study on Morphometric and Molecular Taxonomy of Vespidae Species (Hymenoptera: Vespidae: Polistinae) in Different Districts of Tamil Nadu, India BALACHANDAR M, RAJA MALAIYAPAN, R. AZHAGU RAJ This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4507993/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hymenoptera is one of the largest orders of insects. It consists many species of bees, wasps, hornets, sawflies, and ants. Wasps are important to natural and biological pest due to their predaceous parasitoid nature. Stinging behavior is one of the salient aspects of social hymenopterans. The mortality and morbidity from these bites reported in many districts of Tamil Nadu and around the world. The current study investigates the molecular phylogeny, histology of venom gland and kairomones profile of chosen Vespidae species from the northwest districts (1.Villupuram-Thadagam, 2.Nagapattinam - Mudhalaimedu, 3. Dharmapuri- Lourdhupuram and 4. Chennai – Kilpauk cemetery) of Tamil Nadu. The hornets nests were carefully observed in day time and collected at night time. The phylogenetic relationship analysis was carried out using cytochrome oxidase subunit I (COI) and PCR was performed to amplify COI fragment. The amplified COI regions (the range from 577 base pairs to 601 base pairs nucleotides in length) were sequenced, analysed and DNA barcoded. The four novel nucleotide sequences of COI gene were deposited into the GenBank database and obtained accession numbers for Vespa tropica haematodes ( MN893829.1), Vespa affinis (MN 853754.1), Polistes olivaceus (MT133311.1), and Ropalidia marginata (MT178807.1). Multiple sequence alignment was performed using clustal W in MEGA 6.0. and Phylogenetic analysis, Maximum likelihood, and Neighbour joining tree was constructed. The results revealed that the V. tropica , and V. affinis are sister group and share a common ancestor and V. tropica, V. affinis were found to be more close relatives than P. olivaceous and R. marginata. This study provides more information on Vespa species for researchers, agriculturalists and others in the prevention of the casualties by Vespa bites and the annoyance inflicted on the people. Morphology Molecular Phylogeny Vespa tropica haematodes Vespa affinis continentalis Polistes olivaceus and Ropalidia marginata Vespidae Tamil Nadu Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Insects have extraordinary species richness: over a million species have been identified, and even more await discovery and classification. Given their abundance and diversity, insects are excellent teaching tools for science classrooms. However, accurate insect identification can be especially challenging for beginning students. There are millions of insect species that belong to at least 30 different orders (i.e., the rank below class and above family), making them the most abundant and species-rich animal group. The most successful animals on our planet are the phylum Arthropods. It consists of more individuals than all other groups of animals on earth. It is estimated that around 85 percent of all known animal species belong to arthropods. Insects are the largest group of arthropods, whether measured in terms of numbers of species or number of individuals (Johnson, 2003 ). Insects are reported to be divided into nine 9 major orders represented as Coleoptera 350,000, Diptera 120,000, Lepidoptera 120,000, Hymenoptera 100,000, Orthoptera 20,000, Odonata 5,000, Isoptera 2,000, Siphanoptera 1,200, Hemiptera and Homoptera 60,000 which were the approximate no of named species (Raven et al., 2011 ). Insects play important roles in biological research. Insects are widely used as experimental animals in various fields of biology such as genetics, toxicology, and neurobiology (Pedigo and Rice, 2009 ). The Vespidae are a morphologically and ecologically similar group of wasps all of whom are eusocial. They are found in both temperate and tropical climates over most of the northern hemisphere – and they can also now be recorded in New Zealand and Southern Australia, where they have been introduced. They usually feed their larvae on insects, with the larger Hornets such as the European Vespa crabro , specialising in preying on the rest of the Vespidae – though some species are partial to Honey bees (Apidae). Vespidae queens overwinter or diapause during temperate climate, in the cracks in trees and other suitable places, emerging in the spring to commence nest building. The nest site is chosen by the queen, with utmost care taking into consideration criteria – such as temperature, humidity, protection from other aspects of the weather and expansion space. Different species use different sites for the nests. The Hymenoptera is an order of insects with more than 155,000 well described species (Aguiar et al., 2013 ). Hymenoptera derived from two words i.e. HYMEN means membranous and PETRON means wings (naked membranous wings). This is the most beneficial order in the class Insecta comprising of parasites, predators, and bees involved in pollination and honey production. Hymenoptera is a large component, especially in the parasitic wasp groups. The commonly found hymenoptera can be recognized by the narrow "waist." During flight, the wings form two membranous pairs that can be hooked together. Ovipositors of hymenoptera are usually well developed and are the modified form of stinger in females. The male wasps usually do not sting. Majority of hymenoptera are extremely small and are thus difficult to identify even to family. This group of hymenoptera consists of some familiar types, such as Hornets, Spider Wasps, and Hunting Wasps. Sawflies are also a group of wasps, having of several families, and noteworthy because they have no "waist" that is present in all other Hymenoptera. Archer ( 1991 ) and Nguyen et al. ( 2006 ) reported that the genus Vespa is one of the four genera of the subfamily Vespinae; it is composed of 22 extant species of hornets. Most species have a distribution confined to Asia, with the highest diversity found in northern Indo Malaya (Matsuura and Yamane, 1990 : Archer, M.E. 1991b, reported that V. tropica is associated with terrain forest in the lowlands and up to 2100m altitude. It nests in hollow trees usually within 3m above the ground or in underground cavities usually to a depth of 20cm including inside a dead log partly buried in the soil. Near human habitations it nests under roofs and in attics and sheds. c Hymenopteran insects are famous for their sting and their ability to cause severe anaphylaxis and even in some cases death. This allergic reaction is due to allergens present in the venom. Hence, the present study was carry out the identification of medically important Vespa species using molecular tools and analysing the important information on wasp and hornet workers in different districts of Tamil Nadu. Materials and Methods Collection Sites The hornets’ nests were carefully observed in day time and collected at night time (around 9pm) from the northwest districts of Tamil Nadu during the month of June 2018 to November 2019 (Fig. 1). 1. Thadagam Village is located in Gingee Taluk, Villupuram District of Tamil Nadu State, India (Longitude 79.25935and Altitude 12.20247). It is located 39 KM towards North from District headquarters Viluppuram.149 KM from State capital Chennai, Thadagam, Pin code is 604152. Thadagam is surrounded by Vallam Taluk towards East, Melmalayanur Taluk towards North, Keelpennathur Taluk towards west, Thellar Taluk towards North. 2. Mudhalaimedu village is located in Sirkali Tehsil of Nagapattinam district in Tamil Nadu, India (latitude 11.3498974 and longitude 79.770492). It is situated 21km away from sub-district headquarter Sirkali and 80km away from district headquarter Nagapattinam. 3. Lourdhupuram is a small Village/hamlet in Pappireddipatti Block in Dharmapuri District of Tamil Nadu State, India (latitude 11.9476° N, and longitude 78.2798° E). It comes under B Pallipatty Panchayath. It is located 28 KM towards South from District headquarters Dharmapuri. 9 KM from Pappampadi. 288 KM from State capital Chennai. 4. Kilpauk cemetery, Kilpauk Garden Road, Kilpauk is in Chennai City in Tamil Nadu State, India. Latitude (Width) 13°05'08.0"N (13.0855600°) and longitude (Length) 80°14'16.0"E (80.2377800°). Insect Collection and Identification The Wasp Vespa species were collected from the northwest districts of Tamil Nadu by using sweep net near the wasp nest. All the collected wasp specimens were fixed in 10% v/v formalin in the field as a voucher (Fig. 2). For molecular studies the Vespa sp. leg or muscle samples were preserved in 95% v/v ethanol in a glass vial for further studies. The collected wasp species brought to the laboratory and stored in vials for identification. The preserved specimen wasps were deposited to Dr. Grishkumar, Scientist, Zoological Survey of India, Kozhikode, Kerala for identification. After being fixed, the specimens were counted and separated by morphospecies. Each specimen was identified using the dichotomic keys proposed by Richards (1978) and Carpenter, J.M. & Kojima, J.,(1997). Keys for identification of Vespidae species Carina : a slightly raised ridge on the integument (referred to as the "keel"in some Polistes Keys). Corona : a more or less rectangular yellow area just above and between the antennal bases. Gaster : the main portion of the abdomen posterior to the pedicel. Genal band : a narrow area immediately posterior to each compound eye. Jugal lobe : the basal, posterior lobe of the hind wing of Polistes (often referred to as the anal lobe). Maculation : a pattern of colors; in Vespula, the pattern of yellow or white against a black background on the gaster or clypeus. Ocular sinus : a small yellow area lying within the medial identification of each compound eye. Oculomalar space : a narrow area immediately beneath each compound eye and just above each mandible base. Pedicel : the"waist"between the gaster and the propodeum; also, the second antennal segment. Propodeum : the posterior plate of the thorax continuous with the pedicel (actually, the propodeum is the first true abdominal segment united with the last thoracic segment). Key to the Subfamilies of Eusocial Vespidae Gastral tergum I narrowed anteriorly and convex in lateral view: c1ypeusrounded or narrowly truncate ventrally; hindwing with a jugal lobe: slender wasps; nest a single, exposed comb......................................................................................................... Polistinae Gastral tergum I strongly truncate anteriorly and arising nearly vertically from the pedicel in lateral view; clypeus broadly truncate and concave ventrally; hindwing lacking a jugal lobe; robust wasps; nest consists of multiple combs enclosed in a paper envelope................................................................................................................. Vespinae Morphology The wasp Vespa species morphometric measurements (head, mouthparts, legs and wings) of the wasp sp. and nest will be studied by standard methodology. The microphotograph was taken with Canon camera (EOS 77D), Sensor: APS-C CMOS senor with 24.2 MP, image processor: DIGIC 7with 45 autofocus points. Lens mount: EF/EF-s, Focal length equivalent to 1.6x the focal length of the lens. Image stabilisation: optical image stabilizer on compatible lens, image processing: digital photo professional 4 for RAW image processing. Measurements and indices TL Total length - The added lengths of head (includ­ing mandibles), mesosoma, petiole, and gaster; HW Head width - Maximum width of head in full-face view, excluding eyes if laterally protruding; HL Head length - Maximum length of head in full-face view, excluding mandibles, measured par­allel to midline from anterior-most point of clypeus to midpoint of occipital margin (in minor workers) or to midpoint of an imaginary line connecting the apices of posterior projections (major workers and some gynes); HS Head size - (HW+HL) / 2. ; EL Eye length- Maximum diameter of compound eye; EI (eye index): EL/HW x 100 ; REL (relative eye length)- EL/HW x 100 ; MandI - MandL/HL x 100 ; SI (scape index) - SL/HW x 100 ; SL Scape length - Maximum length of antennal scape in dorsal view excluding basal neck and condyle. Genomic DNA Isolation Genomic DNA was isolated from the tissues of female wasp workers using NucleoSpin® Tissue Kit (Macherey-Nagel) following manufacturer’s instructions. Tissues were placed in a 1.5 ml microcentrifuge tube. 180µl of T1 buffer and 25µl of proteinase K was added and incubated at 56 o C in a water bath until the tissue was completely lysed. After lysis, 5µl of RNase A (100mg/ml) was added and incubated at room temperature for 5 minutes. 200µl of B3 buffer was added and incubated at 70 o C for 10 minutes. 210 µl of 100% ethanol was added and mixed thoroughly by vortexing. The mixture was pipetted into NucleoSpin® Tissue column placed in a 2 ml collection tube and centrifuged at 11000 x g for 1 minute. The NucleoSpin® Tissue column was transferred to a new 2 ml tube and washed with 500 µl of BW buffer. Wash step was repeated using 600 µl of B5 buffer. After washing the NucleoSpin® Tissue column was placed in a clean 1.5 ml tube and DNA was eluted out using 50 µl of BE buffer. The quality of the DNA isolated was checked using agarose gel electrophoresis. 1µl of 6X gel-loading buffer (0.25% bromophenol blue, 30% sucrose in TE buffer pH-8.0) was added to 5µl of DNA. The samples were loaded to 0.8% agarose gel prepared in 0.5X TBE (Tris-Borate-EDTA) buffer containing 0.5 µg/ml ethidium bromide. Electrophoresis was performed with 0.5X TBE as electrophoresis buffer at 75 V until bromophenol dye front has migrated to the bottom of the gel. The gels were visualized in a UV trans illuminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad). PCR Analysis The PCR amplification was carried out in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems). COX1 gene using the forward Primer LCO Sequence (5’ à 3’) GGTCAACAAATCATAAAGATATTGG and reverse primer HCO Sequence (5’ à 3’) TAAACTTCAGGGTGACCAAAAAATCA Agarose Gel electrophoresis of PCR products The PCR products were checked in 1.2% agarose gels prepared in 0.5X TBE buffer containing 0.5 µg/ml Ethidium Bromide. 1 µl of 6X loading dye was mixed with 4 µl of PCR products and was loaded and electrophoresis was performed at 75V power supply with 0.5X TBE as electrophoresis buffer for about 1-2 hours, until the bromophenol blue front had migrated to almost the bottom of the gel. The molecular standard used was a 2-log DNA ladder (NEB). The gels were visualized in a UV trans illuminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad). Sequencing Sequencing reaction was done in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) using the BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, USA). The sequence quality was checked using Sequence Scanner Software v1 (Applied Biosystems). Sequence alignment and required editing of the obtained sequences were carried out using Geneious Pro v5.1 (Drummond et al., 2010). Sequenced nucleotides for four species of Vespidae were submitted to NCBI gene bank and obtained accession number. Phylogenetic analysis The DNA sequences were subjected to pairwise distance analysis and the following phylogenetic trees were constructed: Maximum Parsimony, Maximum Likelihood and Neighbor-Joining by using MEGA 6.0 software (Tamura et al., 2011). Pairwise distance analysis was carried out with gap opening penalty 15 and gap extension penalty 6.66(Clustalw) (Thompson et al., 1994). Maximum parsimony: The maximum parsimony analyses were carried out using MEGA 5 (Tamura et al., 2011). Bootstrap method was used with 100 replications and gap/missing data treatment by complete selection and the search method was Subtree-Pruning –Regrafting (SPR) and substitution based on nucleotide sequences. The percentage of replicate trees in which the associated taxa clustered together in the boostrap test (100 replicates) was used (Felsenstein,1985).The Maximum parsimony tree was obtained using the Subtree-Pruning- Regrafting (SPR) algorithm (Nei and Kumar,2000),using search level 1. The substitution type based nucleotide sequences and the codon positions included were first +second+third+noncoding and all the positions containing gaps and missing data were eliminated. Maximum Likelihood: Maximum Likelihood analyses were run in MEGA 5 (Tamura et al., 2011). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei 1993). Initial tree for the heuristic search was obtained automatically by applying Neighbor- Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The substitution type based nucleotide sequences and the codon positions included were first+second+third+noncoding and all the positions containing gaps and missing data were eliminated. Neighbor-Joining: Neighbor-Joining analyses were determined using MEGA 5 (Tamura et al., 2011). The evolutionary history inferred using the Neighbor-Joining method (Saitou and Nei,1987) .The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test(100replicates) was used (Felsenstein,1985). The evolutionary distances were computed using the Tajima-Nei method (Tajima and Nei, 1984). Codon positions included were first+second+third+noncoding and all positions containing gaps and missing data were eliminated. Statistical analysis The collected data from the morphometric studies of four Vespidae species were analysed with statistical tools (SPSS version 14.0). The mean and standard deviation was calculated for the morphometric data and presented in the table. Results Identification of specimens The research specimen wasp species was collected from different districts namely Villupuram (Thadagam), Nagapattianam (Muthalaimedu), Dharmapuri (Lourdhupuram) and Chennai (Kilpauk cemetery) of Tamil Nadu during 2018 to 2019. Collected specimens was identified and confirmed by using standard taxonomy keys and Dr. P. Girish Kumar, Scientist-C, Zoological Survey of India, Kerala for morphologically similar species namely Vespa tropica haematodes, Vespa affinis, Polistes olivaceus and Ropalidia marginata ( Fig. 3-6). The distribution, habitat and nesting characteristics were identified and presented in table 1. Table 1: Distribution, habitat and nesting characteristics of the Vespidae species Species D i stribution H abitat N esting Vespa tropica Widely distributed from Afghanistan and Pakistan to the Philippines, including the Indochinese peninsula and Peninsular Malaysia, it extends south to Indonesia, including Timor, the Moluccas and Papua New Guinea, but it is absent from Taiwan, And it has been introduced into Guam (USA), Korea and Southern Manchuria. Nocturnal, probably a forest species Probably aerial nesting Vespa affinis Widely distributed from India to the Ryukyu Islands, including Taiwan, the Philippines, the Indochinese and Malay Peninsula and Indonesia to New Guinea. Introduced in California (USA), Queensland (Australia) and New Zealand. Nocturnal, lowland habitats Usually aerial nesting Polisters olivaceus India: Andhra Pradesh, Arunachal Pradesh, Assam, Gujarat, Himachal Pradesh, Jammu & Kashmir, Jharkhand, Karnataka, Madhya Pradesh, Manipur, Meghalaya, Nagaland, Odisha, Rajasthan, Sikkim, Tripura, Uttarakhand, Uttar Pradesh, West Bengal. Elsewhere: Afghanistan, Amirantes, Australia, Bangladesh, Cambodia, Chagos Archipelago, Chile (introduced), China, Egypt, Fiji, Indonesia, Iran, Laos, Madagascar, Malaysia, Marianas, Marquesas, Mauritius, Myanmar, Nepal, New Caledonia, New Zealand, Oman, Pakistan, Philippines, Reunion, Samoa, Seychelles, Singapore, Society Is., Sri Lanka, Tanzania, Thailand, Tongas, Tuamotu Archipelago, U.S.A., Vietnam. Nocturnal, lowland habitats Usually aerial nesting Ropalidia marginata India: Andhra Pradesh, Gujarat, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Odisha, Punjab, Rajasthan, Tamil Nadu, Uttarakhand, West Bengal. Elsewhere: Australia, Bali, Bangka, Borneo, Java, Kariman Djawa, Lombok, Malay Peninsula, Mariana Islands, Myanmar, New Britain, New Guinea, Palau Islands, Pakistan, Philippine Islands, Sri Lanka, Sulawesi, Sumatra, Sumba, Sumbawa, Talud Islands, Tukang Besi Islands, Vietnam, Volcano Islands. Various habitats Usually subterranean nesting 1. Vespa tropica haematodes (Bequaert) (Fig.3) Scientific classification Kingdom : Animalia Phylum : Arthropoda Class : Insecta Order : Hymenoptera Super family : Vespoidea Family : Vespidae Genus : Vespa Species : tropica haematodes (Bequaert) 2. Vespa affinis continentalis haematodes (Bequaert) (Fig.4) Scientific classification Kingdom : Animalia Phylum : Arthropoda Class : Insecta Order : Hymenoptera Super family : Vespoidea Family : Vespidae Genus : Vespa Species : affinis continentalis 3. Polistes (Gyrostoma) olivaceus (De Geer) (Fig.5) Scientific classification Kingdom : Animalia Phylum : Arthropoda Class : Insecta Order : Hymenoptera Super family : Vespoidea Family : Vespidae Genus : Polistes Species : olivaceus 4. Ropalidia marginata (Lepeletier) (Fig.6) Scientific classification Phylum : Arthropoda Class : Insecta Super family : Vespoidea Family : Vespidae Order : Hymenoptera Genus : Ropalidia Species : marginata Morphometric studies on Vespa species Morphometry studies was conducted on four species of Vespidae using standard procedure mainly based on the work of Richard (1956, 1973), Spradbery (1973) and van der Vecht (1975). The morphological terms used for describing different parts of the body namely width and length of head, mandible, antennae, eyes, legs, tergum, fore wing, hind wing and total length was measured (Table 2). Body length was measured From the anterior margin of head to the end of second metasomal tergite along the mid dorsal line with a graduated eye piece and graticule, thus comprising the length of head, mesosoma and 1 st + 2 nd metasomal segments. Fore wing length was measured from base to apex in millimeter (mm). Photographs were taken with a Veho USB microscope (Nikon SMZ800 streozoom microscope) and labelled in Adobe Photoshop (Fig. 7-10). V. tropica haematodes : Head length (0.5 ± 0.23) and width (0.56 ±0.04); Mandible length (0.3 ± 0.0) and width (0.2 ± 0.0); Antennae length (0.9 ± .006) and width (0.27 ± 0.41); Eye length (0.33 ± 0.05) and width (0.2 ± 0.0); Leg Meta femur length (0.49 ± 0.03), Meta tibia length (0.47 ± 0.04), Hind femur length (0.52 ± 0.078), Hind tibia length (0.51 ± 0.087); Tergum length (0.49 ± 0.03) and width (0.95 ± 0.05); Total length (2.4 ± 0.231); Fore wing length (2.01 ± 0.07) and width (0.81 ± 0.12); Hind wing (1.21 ± 0.057) and width (0.35 ± 0.052) (Table 2, Fig. 7). V. affinis continentalis haematodes: Head length (0.5 ± 0.23) and width (0.56 ±0.04); Mandible length (0.3 ± 0.0) ad width (0.2 ± 0.0); Antennae length (0.9 ± .006) and width (0.27 ± 0.41); Eye length (0.33 ± 0.05) and width (0.2 ± 0.0); Leg Meta femur length (0.49 ± 0.03), Meta tibia length (0.47 ± 0.04), Hind femur length (0.52 ± 0.078), Hind tibia length (0.51 ± 0.087); Tergum length (0.49 ± 0.03) and width (0.95 ± 0.05); Total length (2.4 ± 0.231); Fore wing length (2.01 ± 0.07) and width (0.81 ± 0.12); Hind wing (1.21 ± 0.057) and width (0.35 ± 0.052) (Table 2, Fig. 8). P. olivaceus : Head length (0.545 ± 0.049) and width (0.521 ±0.027); Mandible length (0.3 ± 0.041) and width (0.275 ± 0.048); Antennae length (0.745 ± .049) and width (0.362 ± 0.039); Eye length (0.32 ± 0.078) and width (0.22 ± 0.078); Leg Meta femur length (0.48 ± 0.042), Meta tibia length (0.5 ± 0.067), Hind femur length (0.51 ± 0.0875), Hind tibia length (0.51 ± 0.087); Tergum length (0.6 ± 0.094) and width (0.54 ± 0.080); Total length (2.13 ± 0.094); Fore wing length (1.74 ± 0.117) and width (0.375 ± 0.135); Hind wing (1.25 ± 0.057) and width (0.235 ± 0.041) (Table 2, Fig. 9). R. marginata : Head length (0.39 ± 0.021) and width (0.34 ±0.021); Antennae length (0.31 ± 0.061); Eye length (0.295 ± 0.028) and width (0.255 ± .036); Leg Meta femur length (0.245 ± 0.043), Meta tibia length (0.15 ± 0.040), Hind femur length (0.32 ± 0.105), Hind tibia length (0.275 ± 0.0485); Tergum length (0.3 ± 0.033) and width (0.28 ± 0.034); Total length (1.26 ± 0.051); Fore wing length (0.565 ± 0.052) and width (0.145 ± 0.036); Hind wing (0.835 ± 0.052) and width (0.78 ± 1.13) (Table 2, Fig. 10). Molecular Taxonomy Genomic DNA Isolation The mitochondrial gene sequence of COI gene data of wasp species from Tamil Nadu, India are presented and analyzed. The collection sites were Thadagam, Sirkali, Dharmapuri and Chennai districts. The barcode results indicated that the PCR products with COI (Forward/reverse) primer were 596, 586, 601,577 bp. Base pair length in V. tropica, V. affinis, P. olivaceus and R. marginata respectively (Figure 11). Nucleotide composition of Vespidae species DNA always contains equal amounts of adenine and thymine, guanine and cytosine, in pairs usually denoted A=T and G=C. These two regularities give rise to a third one. The nucleotide compositions of five Vespidae COX gene have been identified in the family Vespidae. The overall A+T content of Polistes olivaceus and Vespa tropica is 72.55% and 71.60% respectively. The highest total content of nucleotides composition of P. olivaceus is 601.0 and least Vespa tropica 567.0 was observed (Table 3). In this study the wasp COI gene, 5 ’ → 3 ’ standard forward primer (LCO) - GGTCAACAAATCATAAAGATATTGG and reverse primer (HCO) TAAACTTCAGGGTGACCAAAAAATCA with amplified PCR products were the range from 577 base pairs to 601 base pairs nucleotides in length. The amplified PCR products were visualized on 1.27 agarose gel electrophoresis. The amplified PCR products was cleaned up and sequenced in ABI 3500. DNA analyzer (Applied Biosystems). DNA Barcoding The mitochondrial gene sequences of COI the wasp’s species V. tropica, V. affinis, P. olivaceus and R. marginata in this study were submitted in NCBI gene bank and accession numbers were assigned for all the four species. The sequence quality was checked in sequence scanner software V1.0. The aligned sequences were identified as cytochrome C oxidase with BLAST procedure and the COI gene sequence was manually edited in Finch T.V. All sequences were deposited in NCBI gene bank. Multiple sequence alignment was performed using Clustal W MEGA 6.0. NCBI gene bank accession number for cytochrome c oxidase COI gene sequences for each wasp species were presented in the table 4. The mitochondrial cytochrome c oxidase gene sequences of four wasp species NCBI gene bank accession numbers MN893829.1, MN 853754.1, MT133311.1 and MT178807.1 for V. tropica, V. affinis, P. olivaceus and R. marginata respectively. From the gene sequences and phylogenetic tree to topology structure results was confirmed that the wasp species Vespa tropica and Vespa affinis very strong relationship between this species. Table 4: Mitochondrial DNA sequences of Cytochrome C oxidase in wasp species subjected to phylogenetic analysis. S.No Species name NCBI Gen bank no /Accession No Base-pairs (bp) 1 Vespa affinis MN853754.1 596 2 Vespa tropica haematodes MN893829.1 586 3 Polistes olivaceus MT133311.1 601 4 Polistes olivaceus MT136727.1 601 5 Ropalidia marginata MT178807.1 577 Phylogenetic analysis The Cytochrome C Oxidase (Co-I) sequences of wasp species were subjected to pairwise distance analysis and the following phylogenetic trees were constructed: Maximum Parsimony, Maximum Likelihood and Neighbor-Joining by using MEGA 6 software (Tamura et al., 2011). Pairwise distance analysis was carried out with gap opening penalty 15 and gap extension penalty 6.66 (Clustalw) (Thompson et al., 1994). Maximum Likelihood: Maximum Likelihood analyses were run in MEGA 6 (Tamura et al., 2011). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei 1993). Initial tree for the heuristic search was obtained automatically by applying Neighbor- Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The results of Maximum Likelihood tree constructed based on Cytochrome c oxidase (Fig.12) with two parallel clusters; Ropalida marginata and Polistes olivaceus evolved as separate lineage, and the second cluster with Vespa affinis and Vespa tropica haematodes shows their inter and intra generic closeness. But they share a common ancestral node suggesting an affinity between these four wasp species. Among them, the two morphologically related Vespa affinis and Vespa tropica haematodes share a common node exhibiting their affinity. Ropalida marginata and Polistes olivaceus are morphologically different; they share a common node which shows their closeness. Maximum Likelihood tree based on CO I variations showing the relationships of wasp species. The genetic similarity between the Vespa affinis and Vespa tropica haematodes as observed in the Maximum Likelihood tree. Neighbor-Joining tree: Neighbor-Joining analyses were determined using MEGA 6 (Tamura et al., 2011). The evolutionary history inferred using the Neighbor-Joining method (Saitou and Nei,1987) .The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test(100replicates) was used (Felsenstein,1985). The evolutionary distances were computed using the Tajima-Nei method (Tajima and Nei 1984). The results of Neighbor-Joining tree and UPGMA constructed based on Cytochrome c oxidase (Fig. 13 and Fig. 14) with two parallel clusters; Ropalida marginata and Polistes olivaceus evolved as separate lineage, and the second cluster with Vespa affinis and Vespa tropica haematodes shows their inter and intra generic closeness. But they share a common ancestral node suggesting an affinity between these four wasp species. Among them, the two morphologically related Vespa affinis and Vespa tropica haematodes share a common node exhibiting their affinity. Ropalida marginata and Polistes olivaceus are morphologically different; they share a common node which shows their closeness. The affinity between any two species is similar in phylograms revealing uniform phylogenetic relationship and suggesting monophyly. Maximum parsimony: The maximum parsimony analyses were carried out using MEGA 6 (Tamura et al., 2011). Bootstrap method was used with 100 replications and gap/missing data treatment by complete selection and the search method was Subtree-Pruning –Regrafting (SPR) and substitution based on nucleotide sequences. The percentage of replicate trees in which the associated taxa clustered together in the boostrap test (100 replicates) was used (Felsenstein,1985).The Maximum parsimony tree was obtained using the Subtree-Pruning- Regrafting (SPR) algorithm (Nei and Kumar, 2000),using search level 1. The results of Maximum Parsimony tree constructed based on Cytochrome c oxidase (Fig.15) with two parallel clusters; Vespa affinis and Vespa tropica haematodes, forms a cluster. Ropalida marginata evolved as a separate lineage and Polistes olivaceus evolved as single separate lineage. Maximum parsimony shows inter and intra generic closeness between species and they share a common ancestral node suggesting an affinity between these four wasp species. Discussion Wasps belong to the Vespidae family, which is a worldwide order of Hymenoptera (Class: Insecta). The members of this family are mostly found in tropical areas of the world, and there are about 5000 species in total. Hornet wasps are a species of wasps belonging to the genus Vespa Linnaeus. They are social wasps with an excessive degree of evolution. Hornet wasps may be observed mainly within side the Oriental and Palaearctic areas of the Globe. There are a complete of 23 recognized species within side the world, with sixteen species from the Indian subcontinent and 15 species from India (Carpenter and Kojima, 1997 ). The nests are made of paper and are connected to wood, metal, or rock (Cranshaw et al., 2011 ). Vespa affinis , a species of hornet found largely in Asia, develops vast aerial nests with up to tens of thousands of workers (Archer, 1997 ). Other insects, particularly honey bees near their hives, are preyed upon by these wasps. Their nests are made of a combination of plant fibres and salivary secretions, and they can range in size from a baseball-sized nest with a few thousand cells to a nest with hundreds of thousands of cells. Nests are typically annual, but in warmer areas, a few species will establish huge perennial nests. They are dangerous because they sting people and domesticated animals. In all social wasps and their close solitary relatives, the females have the apparatus and ability needed to inflict painful stings. They are honey bees' natural enemy, and they harm the fruit eating with them right before harvest (Kimsey and Carpenter, 2012 ). Ceylonese specimens differ from those from the rest of India in that their thorax is heavily reddened. Since specimens from Tenasserim, part of the Indo-Malayan mountain region in Southeast Asia, have similar colour, it appears difficult to distinguish them as a unique variety. Mangalore, Shembaganur near Madura, and Nagapattinam are all in India (Bridwell and Bequaert, 1918 ; Balachandar et al., 2019 ). The maximum not unusual place Hornets of the Oriental and Indo-Malayan areas are the large black or fusoous wasps with the second one or first and 2nd stomach segments in part or thoroughly orange or orange-yellow. In maximum collections those wasps stand as Vespa cincta and Vespa affinis , the latter being frequently seemed as a number of the former. A take a look at of structure, however, discloses that pretty wonderful species are generally pressured beneath those names. This fact, unfortunately, changed into now no longer identified via way of means of Dubuysson, while he wrote his monograph of the Vespinae (1905). At the Paris Museum, he located specimens of each species beneath V. cincta var. affinis , in addition to beneathneath V. cincia . Koeniger, N., et al. ,1994 first observed the confusion, while he defined his V. indosinensis as wonderful from V. cincta . The present study to carry out to identify the species from different study area of district of Tamil Nadu. The specimens were collected, preserved, identified and confirmed with suitable taxonomy key and the wasp expert Dr. P. Girish Kumar, Zoological survey of India, Kerala. The identified specimens namely, Vespa tropica haematodes (Thadagam - Villupuram district), Vespa affinis (Sirkali - Nagapattianam District), Polistes olivaceus (Lourdhupuram – Dharmapuri district) and Ropalidia marginata (Kilpauk Garden cemetry- Chennai district). Bingham (1897) and Das & Gupta ( 1989 ) made significant advances to Vespidae taxonomy in India and its neighbouring nations. The understanding of Rajasthan's vespid fauna is limited and fragmented. In scattered literatures, 14 genera and 20 species have been recorded from Rajasthan till date (Nurse, 1903 ; van der Vecht, 1941 ; Chhotani & Ray, 1975 ; Giordani Soika, 1982 ; Gusenleitner, 2006 ; Girish Kumar, 2010 , 2011, 2012 a & b; Girish Kumar & Lambert Kishore, 2012; Girish Kumar & Carpenter, 2013 ; Girish Kumar et al. , 2013). The goal of this paper is to present a comprehensive analysis of the Vespidae family's fauna in the state of Rajasthan. In 2014, Girish Kumar and Gaurav Sharma reported that, in Rajasthan, 15 genera and 26 species and from this 1 genus and 6 species are new records for Rajasthan. The Indian species of the genus Eumenes Latreille with a key to species is provided by Girish kumar et al. , 2017. The species Eumenes pomiformis (Fabricius, 1781) is recorded at Kozhikode, Kerala for the first time from India, while E. affinissimus de Saussure, 1852, E. pomiformis (Fabricius, 1781) and E. persimilis kojimardani Soika, 1960 are recorded from Pakistan. Based on colour variation, Kimsey and Carpenter ( 2012 ) proposed that Vespula intermedia (du Buysson, 1905 ) be considered a genuine species rather than a synonym of Vespula rufa (Linnaeus, 1758 ). This idea is not supported by our Bayesian inference data. Depending on locus, the divergence is minor or non-existent, implying that V. rufa and V. intermedia were separated for just a brief time, if at all. We believe that V. intermedia should be considered a synonym of V. rufa because the species delimitation is solely based on colour variation. The multi-species coalescent tree and the species delimitation analyser also corroborate this. Morphometric studies on Vespidae species The morphometric studies was conducted for all the four species V. tropica haematodes, V. affinis , P. olivaceus and R. marginata collected from different districts of Tamil Nadu based on the work of Richard (1971, 1977), Philip Spradbery J ( 1973 ) and van der Vecht ( 1975 ). The Vespa species morphometric measurements like length and width of head, mouthparts, legs and wings, antenna, eye and tergum was recorded and tabulated. Body length was measured From the anterior margin of head to the end of second metasomal tergite along the mid dorsal line with a graduated eye piece and graticule, thus comprising the length of head, mesosoma and 1st + 2nd metasomal segments. Fore wing length was measured from base to apex in millimeter (mm). Photographs were taken with a Veho USB microscope and labelled in Adobe Photoshop and presented. The similar study was conducted by Roohollah Abbasi ( 2012 smith) on sexual dimorphism in the wing morphology of social vespid wasps Polistes laterille using geographic morphometries. Size differentiation is considered as an initial step for the origin of morphological castes in the four main groups of social hymenoptera. In social wasps, the differences are more conspicuous in Vespinae and more complex in Polistinae. All the four species are different in morphological characters, size, colour and length and width of the body. Vespa tropica and Vespa affinis both the species are morphologically similar but genetically dissimilar. The length and width of the species vary from species to species Molecular taxonomy Barcoding will provide vital new tools for appreciating and managing Earth’s immense and changing biodiversity. Traditional taxonomy has been used in Systematics for over 250 years with the identification of more than 1.75 million species. However, in many countries as in India, traditional taxonomy is yet to adopt the latest molecular techniques available for a better and accurate identification of taxa. Enhanced taxonomic infrastructure with the help of DNA information can certainly speed up the process of species exploration, description and classification of biological diversity. The nucleotide sequence of this particular 640 nucleotide region (Folmer et al., 1994 ) shall be the unique identification code for all species to facilitate the correct determination of species and the discovery of new species (Moritz and Cicero, 2004). Many studies have shown that taxon identification based on DNA sequences can facilitate the recognition of known species and the discovery of new species (Blaxter et al., 2005 ; Hebert et al., 2003a , b ). DNA sequences of the mitochondrial COX-1 gene can serve as a molecular genetics for identifying all kinds of animals (Hebert et al., 2004a ), especially cryptic species in tropical regions (Berkov, 2002; Monaghan et al, 2005 ; Hajibabaei et al., 2006 ) and insect at different growth stages (Janzen et al ., 2004). Our Vespa bicolor (Fabricius, 1787 ) specimens' results are perplexing and difficult. We have numerous individuals who morphologically match each other and one individual who differs. The specimen that diverges (U3481) matches Archer's (1989) description of a V. bicolor . On BOLD or BLAST, however, this specimen does not match any other V. bicolor . The additional morphologically similar individuals (U3480, U3656, U3657, U3673, U3674, and U3675) do not meet Archer's (1989) description for a V. bicolor or anything else, but the closest match is for a V. bicolor . However, using both BOLD and BLAST systems, they identified a 99 percent identification match to other V. bicolor . As per the report of Edward Laidlaw Smith 1970 The Hymenoptera has the least sexually dimorphic combined with the least specialized genitalia among the Pterygota. Based on physical features, two specimens identified as Paravespula flaviceps , one from China and the other from Japan, appear to represent two different evolutionary lineages. The species delimitation analyser, the Bayesian inference species tree, the COI SLA, the multi-species coalescent tree, and the Bayesian inference species tree all support this. The morphology of the Japanese specimen U3936 matches that of a P. flaviceps described by Archer ( 1989 ). The sample had no matches in BOLD systems but 99 percent identity to P. flaviceps in BLAST, including the Federico Lopez-Osorio et al., ( 2014 ) specimen (also from Japan). The Chinese specimen U3678 has a somewhat dimpled surface. According to Carpenter et al. (2011) indicates that there is a divergence between the specimens from Japan and China, and since a species has been identified from that region, it 28 could be the same as our specimen. However, before we can make any recommendations, we need to compare their samples to ours. Carpenter & Glare ( 2010 ) investigated whether the North American species Vespula alascensis (Packard, 1870 ) was a synonym of the European Paravespula vulgaris (Linnaeus, 1758 ) or a species sui generis using morphological and genetic data. They used specimens from North America and Europe and found that they were two distinct species based on male genetalia and molecular divergence. This theory is supported by our results. On the basis of morphological features, specimens of Dolichovespula pacifica were identified as D. pacifica ; nonetheless, they appear to belong to three different evolutionary lineages. All of our Swedish specimens form a sister group with a subset of our Japanese specimens in two of these lineages. The third lineage, which includes a subset of our Japanese samples, is related to our D. saxonica samples from Sweden. The species delimitation analyser, the Bayesian inference species tree, the COI SLA, the multispecies coalescent tree, and the Bayesian inference species tree all support this. In BOLD systems, the specimens U1742 and U1922 from Sweden were confirmed to be 100% identical to Dolichovespula norvegicoides (early release, no information for country of collection). The closest match after that was 96 percent identity to D. albida and D. norwegica . The closest match in BLAST was 96 percent identity to D. albida . The Japanese specimens U3926 and U3937 belong to the same family as the Swedish specimens. In BOLD systems, the specimens were found to be 97.8% identical to D. norvegicoides . After that, D. norwegica was the closest match with 96.9% identity. In BLAST, the specimens had a 97 percent identity to D. albida , which was the closest match. The Japanese specimens U3924 and U3928 are sister species to D. saxonica . In BOLD systems, the specimens had 100 percent identity to KMP268, and 98 percent identity to D. saxonica . In BLAST, the outcomes are the same. The results of the BLAST and BOLD systems are intriguing since the specimens are more closely related to D. saxonica and D. albida than to other D. pacifica specimens. One probable explanation is that Dolichovespula is a very new and unresolved group containing closely related species. The species' divergence occurred recently and over a brief period of time. Based on morphological features, Pekkarinen ( 1995 ) determined that Dolichovespula pacifica was a synonym of Dolichovespula norvegicoides . Based on physical characteristics, Archer ( 1989 ) classified them as two distinct species. Sladen ( 1918 ) used a type specimen found in Canada to describe D. norvegicoides . Birula ( 1930 ) used a type specimen taken in Eastern Siberia to describe D. pacifica . Our findings support the idea that they are two distinct species, with D. pacifica (Birula, 1930 ) as a legitimate designation. Many species of social wasps are also important in the biological control of pests, mainly lepidoptera of crops such as eucalyptus, vegetables, coffee, corn and tomatoes (Bach, 1951 ; Marques and Carvalho, 1993 ; Prezoto and Machado, 1999 ; Richter, 2000 ). Six subfamilies are recognized under Vespidae , and their relationships have been established with greater confidence than for most of the other families of Vespoidea. They are ( Euparagiinae + ( Masarinae + (Eumeninae + (Stenogastrinae + (Vespinae + Polistinae ). Since good estimates of phylogeny are required for the proper investigation of many other topics of particular interest in the context of the aculeates, notably behavioural and physiological adaptations such as sociality, nesting behaviour, host switching, etc., and the possible times of their origin, it is important that further work in this area be pursued. Conclusion The present study concludes and gives more information on morphology, molecular phylogeny, histology and cuticular hydrocarbons (kairomones) of the four species namely, Vespa affnis, Vespa tropica , Polistes olivaceus and Ropalidia marginata and its very useful for researcher, farmers, agriculturalist and others for prevention of the casualties and annoyances inflicted on the people of the state of Tamil Nadu, India as well as those in other countries through appropriate cognizance on Vespidae (wasp) species. Since this is the first research report of this kind in the state of Tamil Nadu. 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Mol Biol Evol 28:2731–2739. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10(3):512–526. Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speciÞc gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680. Van Der Vecht J (1975) A review of the genus Stenogaster Guerin (Hymenoptera: Vespoidea). Journal of the Australian Entomological Society 14: 283–308. Vecht J, Van Der (1941) The Indo-Australian species of the genus Ropalidia (= Icaria ) (Hym., Vespidae) (first part). Treubia18: 103–190. Table Table 2 is available in the Supplementary Files section. Supplementary Files TableMorphometry2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4507993","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371942397,"identity":"09c69a33-d15e-48d2-b5eb-e5bdb51de72b","order_by":0,"name":"BALACHANDAR M","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9838-2489","institution":"Loyola College","correspondingAuthor":true,"prefix":"","firstName":"BALACHANDAR","middleName":"","lastName":"M","suffix":""},{"id":371942398,"identity":"0615c923-7a6f-4e0a-b9ae-6625695085ec","order_by":1,"name":"RAJA MALAIYAPAN","email":"","orcid":"","institution":"Loyola College","correspondingAuthor":false,"prefix":"","firstName":"RAJA","middleName":"","lastName":"MALAIYAPAN","suffix":""},{"id":371942399,"identity":"59f8ee57-a4e2-4981-993c-00f9899bcdc4","order_by":2,"name":"R. AZHAGU RAJ","email":"","orcid":"","institution":"St Xavier's College","correspondingAuthor":false,"prefix":"","firstName":"R.","middleName":"AZHAGU","lastName":"RAJ","suffix":""}],"badges":[],"createdAt":"2024-05-31 09:48:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4507993/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4507993/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68531857,"identity":"27d2d790-2402-4a4b-8a7e-7f231a9b4781","added_by":"auto","created_at":"2024-11-08 09:19:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1305908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVespidae species nest built on different 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11","display":"","copyAsset":false,"role":"figure","size":304233,"visible":true,"origin":"","legend":"\u003cp\u003eThe barcode results indicated that the PCR products with COI gene\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4507993/v1/601e15e4d60d115a8893350e.png"},{"id":68531855,"identity":"a3fb47dc-b0c7-4942-b644-8b2b7bc3a2de","added_by":"auto","created_at":"2024-11-08 09:19:38","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":118705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum Likelihood tree based on Cytochrome c oxidase subunit gene variations showing the relationships of wasp species.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4507993/v1/c2f05e036386a5ee049d0c50.png"},{"id":68531858,"identity":"1d7eceb7-9354-41b3-82da-1d2a9082aea8","added_by":"auto","created_at":"2024-11-08 09:19:40","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":121567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeighbor-Joining tree based on Cytochrome c oxidase subunit gene variations showing the relationships of wasp species.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-4507993/v1/388c7ad4eda2a7a0f7f1092a.png"},{"id":68533050,"identity":"9dffa570-a421-491b-941f-2e4e7ac4fee9","added_by":"auto","created_at":"2024-11-08 09:27:38","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":121751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUPGMA tree based on Cytochrome c oxidase subunit gene variations showing the relationships of wasp species.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-4507993/v1/9ec49e46018be22d3684847e.png"},{"id":68531297,"identity":"1401b2cb-063f-4e80-a63d-559a4116fae3","added_by":"auto","created_at":"2024-11-08 09:11:38","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":140125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum parsimonybased on Cytochrome c oxidase subunit gene variations showing the relationships of wasp species.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-4507993/v1/599769ca918eb67756b90feb.png"},{"id":78599700,"identity":"505df355-3e32-45c3-8086-bdb243e330cc","added_by":"auto","created_at":"2025-03-16 07:46:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7326438,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4507993/v1/3f97c167-f21c-4b32-b15f-ceceadfe1a6f.pdf"},{"id":68529728,"identity":"fd0f5747-8a55-4caa-a36c-8935c0d050ff","added_by":"auto","created_at":"2024-11-08 09:03:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16508,"visible":true,"origin":"","legend":"","description":"","filename":"TableMorphometry2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4507993/v1/c29d2235d2c327c7303ee722.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eNew Record: Study on Morphometric and Molecular Taxonomy of Vespidae Species (Hymenoptera: Vespidae: Polistinae) in Different Districts of Tamil Nadu, India\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInsects have extraordinary species richness: over a million species have been identified, and even more await discovery and classification. Given their abundance and diversity, insects are excellent teaching tools for science classrooms. However, accurate insect identification can be especially challenging for beginning students. There are millions of insect species that belong to at least 30 different orders (i.e., the rank below class and above family), making them the most abundant and species-rich animal group. The most successful animals on our planet are the phylum Arthropods. It consists of more individuals than all other groups of animals on earth. It is estimated that around 85 percent of all known animal species belong to arthropods. Insects are the largest group of arthropods, whether measured in terms of numbers of species or number of individuals (Johnson, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Insects are reported to be divided into nine 9 major orders represented as Coleoptera 350,000, Diptera 120,000, Lepidoptera 120,000, Hymenoptera 100,000, Orthoptera 20,000, Odonata 5,000, Isoptera 2,000, Siphanoptera 1,200, Hemiptera and Homoptera 60,000 which were the approximate no of named species (Raven et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Insects play important roles in biological research. Insects are widely used as experimental animals in various fields of biology such as genetics, toxicology, and neurobiology (Pedigo and Rice, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Vespidae are a morphologically and ecologically similar group of wasps all of whom are eusocial. They are found in both temperate and tropical climates over most of the northern hemisphere \u0026ndash; and they can also now be recorded in New Zealand and Southern Australia, where they have been introduced. They usually feed their larvae on insects, with the larger Hornets such as the European \u003cem\u003eVespa crabro\u003c/em\u003e, specialising in preying on the rest of the \u003cem\u003eVespidae\u003c/em\u003e \u0026ndash; though some species are partial to Honey bees (Apidae). \u003cem\u003eVespidae\u003c/em\u003e queens overwinter or diapause during temperate climate, in the cracks in trees and other suitable places, emerging in the spring to commence nest building. The nest site is chosen by the queen, with utmost care taking into consideration criteria \u0026ndash; such as temperature, humidity, protection from other aspects of the weather and expansion space. Different species use different sites for the nests.\u003c/p\u003e \u003cp\u003eThe Hymenoptera is an order of insects with more than 155,000 well described species (Aguiar et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Hymenoptera derived from two words i.e. HYMEN means membranous and PETRON means wings (naked membranous wings). This is the most beneficial order in the class Insecta comprising of parasites, predators, and bees involved in pollination and honey production. Hymenoptera is a large component, especially in the parasitic wasp groups. The commonly found hymenoptera can be recognized by the narrow \"waist.\" During flight, the wings form two membranous pairs that can be hooked together. Ovipositors of hymenoptera are usually well developed and are the modified form of stinger in females. The male wasps usually do not sting. Majority of hymenoptera are extremely small and are thus difficult to identify even to family.\u003c/p\u003e \u003cp\u003eThis group of hymenoptera consists of some familiar types, such as Hornets, Spider Wasps, and Hunting Wasps. Sawflies are also a group of wasps, having of several families, and noteworthy because they have no \"waist\" that is present in all other Hymenoptera. Archer (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) and Nguyen et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) reported that the genus Vespa is one of the four genera of the subfamily Vespinae; it is composed of 22 extant species of hornets. Most species have a distribution confined to Asia, with the highest diversity found in northern Indo Malaya (Matsuura and Yamane, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1990\u003c/span\u003e: Archer, M.E. 1991b, reported that \u003cem\u003eV. tropica\u003c/em\u003e is associated with terrain forest in the lowlands and up to 2100m altitude. It nests in hollow trees usually within 3m above the ground or in underground cavities usually to a depth of 20cm including inside a dead log partly buried in the soil. Near human habitations it nests under roofs and in attics and sheds.\u003c/p\u003e \u003cp\u003ec Hymenopteran insects are famous for their sting and their ability to cause severe anaphylaxis and even in some cases death. This allergic reaction is due to allergens present in the venom. Hence, the present study was carry out the identification of medically important \u003cem\u003eVespa\u003c/em\u003e species using molecular tools and analysing the important information on wasp and hornet workers in different districts of Tamil Nadu.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eCollection Sites\u003c/p\u003e\n\u003cp\u003eThe hornets\u0026rsquo; nests were carefully observed in day time and collected at night time (around 9pm) from the northwest districts of Tamil Nadu during the month of June 2018 to November 2019 (Fig. 1).\u003c/p\u003e\n\u003cp\u003e1. Thadagam Village is located in Gingee Taluk, Villupuram District of Tamil Nadu State, India (Longitude 79.25935and Altitude 12.20247). It is located 39 KM towards North from District headquarters Viluppuram.149 KM from State capital Chennai, Thadagam, Pin code is 604152. Thadagam is surrounded by Vallam Taluk towards East, Melmalayanur Taluk towards North, Keelpennathur Taluk towards west, Thellar Taluk towards North.\u003c/p\u003e\n\u003cp\u003e2. Mudhalaimedu village is located in Sirkali Tehsil of Nagapattinam district in Tamil Nadu, India (latitude 11.3498974 and longitude 79.770492). It is situated 21km away from sub-district headquarter Sirkali and 80km away from district headquarter Nagapattinam.\u003c/p\u003e\n\u003cp\u003e3. Lourdhupuram is a small Village/hamlet in Pappireddipatti Block in Dharmapuri District of Tamil Nadu State, India (latitude 11.9476\u0026deg; N, and longitude 78.2798\u0026deg; E). It comes under B Pallipatty Panchayath. It is located 28 KM towards South from District headquarters Dharmapuri. 9 KM from Pappampadi. 288 KM from State capital Chennai.\u003c/p\u003e\n\u003cp\u003e4. Kilpauk cemetery, Kilpauk Garden Road, Kilpauk is in Chennai City in Tamil Nadu State, India. Latitude (Width) 13\u0026deg;05\u0026apos;08.0\u0026quot;N (13.0855600\u0026deg;) and longitude (Length) 80\u0026deg;14\u0026apos;16.0\u0026quot;E (80.2377800\u0026deg;).\u003c/p\u003e\n\u003cp\u003eInsect Collection and Identification\u003c/p\u003e\n\u003cp\u003eThe Wasp\u003cem\u003e\u0026nbsp;Vespa\u003c/em\u003e species were collected from the northwest districts of Tamil Nadu by using sweep net near the wasp nest. All the collected wasp specimens were fixed in 10% v/v formalin in the field as a voucher (Fig. 2). For molecular studies the \u003cem\u003eVespa\u0026nbsp;\u003c/em\u003esp. leg or muscle samples were preserved in 95% v/v ethanol in a glass vial for further studies. The collected wasp species brought to the laboratory and stored in vials for identification. The preserved specimen wasps were deposited to Dr. Grishkumar, Scientist, Zoological Survey of India, Kozhikode, Kerala for identification. After being fixed, the specimens were counted and separated by morphospecies. Each specimen was identified using the dichotomic keys proposed by Richards (1978) and Carpenter, J.M. \u0026amp; Kojima, J.,(1997).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eKeys for identification of Vespidae species\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCarina\u003c/em\u003e : a slightly raised ridge on the integument (referred to as the \u0026quot;keel\u0026quot;in some Polistes\u003c/p\u003e\n\u003cp\u003eKeys).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCorona\u003c/em\u003e: a more or less rectangular yellow area just above and between the antennal bases.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGaster\u003c/em\u003e : the main portion of the abdomen posterior to the pedicel.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGenal band\u003c/em\u003e: a narrow area immediately posterior to each compound eye.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJugal lobe\u003c/em\u003e: the basal, posterior lobe of the hind wing of Polistes (often referred to as the anal lobe).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMaculation\u003c/em\u003e: a pattern of colors; in Vespula, the pattern of yellow or white against a black background on the gaster or clypeus.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOcular sinus\u003c/em\u003e: a small yellow area lying within the medial identification of each compound eye.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOculomalar space\u003c/em\u003e: a narrow area immediately beneath each compound eye and just above each mandible base.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePedicel\u003c/em\u003e: the\u0026quot;waist\u0026quot;between the gaster and the propodeum; also, the second antennal segment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePropodeum\u003c/em\u003e: the posterior plate of the thorax continuous with the pedicel (actually, the propodeum is the first true abdominal segment united with the last thoracic segment).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKey to the Subfamilies of Eusocial Vespidae\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eGastral tergum I narrowed anteriorly and convex in lateral view: c1ypeusrounded or narrowly truncate ventrally; hindwing with a jugal lobe: slender wasps; nest a single, exposed comb.........................................................................................................\u003cstrong\u003ePolistinae\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003eGastral tergum I strongly truncate anteriorly and arising nearly vertically from the pedicel in lateral view; clypeus broadly truncate and concave ventrally; hindwing lacking a jugal lobe; robust wasps; nest consists of multiple combs enclosed in a paper envelope.................................................................................................................\u003cstrong\u003eVespinae\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eMorphology\u003c/p\u003e\n\u003cp\u003eThe wasp\u003cem\u003e\u0026nbsp;Vespa\u003c/em\u003e species morphometric measurements (head, mouthparts, legs and wings) of the wasp sp. and nest will be studied by standard methodology. The microphotograph was taken with Canon camera (EOS 77D), Sensor: APS-C CMOS senor with 24.2 MP, image processor: DIGIC 7with 45 autofocus points. Lens mount: EF/EF-s, Focal length equivalent to 1.6x the focal length of the lens. Image stabilisation: optical image stabilizer on compatible lens, image processing: digital photo professional 4 for RAW image processing.\u003c/p\u003e\n\u003cp\u003eMeasurements and indices\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTL Total length -\u003c/strong\u003e The added lengths of head (includ\u0026shy;ing mandibles), mesosoma, petiole, and gaster; \u003cstrong\u003eHW Head width -\u003c/strong\u003e Maximum width of head in full-face view, excluding eyes if laterally protruding; \u003cstrong\u003eHL Head length -\u003c/strong\u003e Maximum length of head in full-face view, excluding mandibles, measured par\u0026shy;allel to midline from anterior-most point of clypeus to midpoint of occipital margin (in minor workers) or to midpoint of an imaginary line connecting the apices of posterior projections (major workers and some gynes); \u003cstrong\u003eHS Head size\u003c/strong\u003e - (HW+HL) / 2. ; \u003cstrong\u003eEL Eye length-\u003c/strong\u003e Maximum diameter of compound eye; \u003cstrong\u003eEI\u0026nbsp;\u003c/strong\u003e(eye index): EL/HW x 100 ; \u003cstrong\u003eREL\u0026nbsp;\u003c/strong\u003e(relative eye length)- EL/HW x 100 ; \u003cstrong\u003eMandI -\u003c/strong\u003e MandL/HL x 100 ; \u003cstrong\u003eSI\u0026nbsp;\u003c/strong\u003e(scape index) - SL/HW x 100 ; \u003cstrong\u003eSL Scape length -\u003c/strong\u003e Maximum length of antennal scape in dorsal view excluding basal neck and condyle.\u003c/p\u003e\n\u003cp\u003eGenomic DNA Isolation\u003c/p\u003e\n\u003cp\u003eGenomic DNA was isolated from the tissues of female wasp workers using NucleoSpin\u0026reg; Tissue Kit (Macherey-Nagel) following manufacturer\u0026rsquo;s instructions. Tissues were placed in a 1.5 ml microcentrifuge tube. 180\u0026micro;l of T1 buffer and 25\u0026micro;l of proteinase K was added and incubated at 56\u003csup\u003eo\u003c/sup\u003eC in a water bath until the tissue was completely lysed. After lysis, 5\u0026micro;l of RNase A (100mg/ml) was added and incubated at room temperature for 5 minutes. 200\u0026micro;l of B3 buffer was added and incubated at 70\u003csup\u003eo\u003c/sup\u003eC for 10 minutes. 210 \u0026micro;l of 100% ethanol was added and mixed thoroughly by vortexing. The mixture was pipetted into NucleoSpin\u0026reg; Tissue column placed in a 2 ml collection tube and centrifuged at 11000 x g for 1 minute. The NucleoSpin\u0026reg; Tissue column was transferred to a new 2 ml tube and washed with 500 \u0026micro;l of BW buffer. Wash step was repeated using 600 \u0026micro;l of B5 buffer. After washing the NucleoSpin\u0026reg; Tissue column was placed in a clean 1.5 ml tube and DNA was eluted out using 50 \u0026micro;l of BE buffer.\u003c/p\u003e\n\u003cp\u003eThe quality of the DNA isolated was checked using agarose gel electrophoresis. 1\u0026micro;l of 6X gel-loading buffer (0.25% bromophenol blue, 30% sucrose in TE buffer pH-8.0) was added to 5\u0026micro;l of DNA. The samples were loaded to 0.8% agarose gel prepared in 0.5X TBE (Tris-Borate-EDTA) buffer containing 0.5 \u0026micro;g/ml ethidium bromide. Electrophoresis was performed with 0.5X TBE as electrophoresis buffer at 75 V until bromophenol dye front has migrated to the bottom of the gel. The gels were visualized in a UV trans illuminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PCR amplification was carried out in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems). COX1 gene using the forward \u003cstrong\u003ePrimer LCO Sequence (5\u0026rsquo;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026agrave;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;3\u0026rsquo;)\u0026nbsp;\u003c/strong\u003eGGTCAACAAATCATAAAGATATTGG and reverse primer HCO \u003cstrong\u003eSequence (5\u0026rsquo;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026agrave;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;3\u0026rsquo;)\u0026nbsp;\u003c/strong\u003eTAAACTTCAGGGTGACCAAAAAATCA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAgarose Gel electrophoresis of PCR products\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PCR products were checked in 1.2% agarose gels prepared in 0.5X TBE buffer containing 0.5 \u0026micro;g/ml Ethidium Bromide. 1 \u0026micro;l of 6X loading dye was mixed with 4 \u0026micro;l of PCR products and was loaded and electrophoresis was performed at 75V power supply with 0.5X TBE as electrophoresis buffer for about 1-2 hours, until the bromophenol blue front had migrated to almost the bottom of the gel. The molecular standard used was a 2-log DNA ladder (NEB). The gels were visualized in a UV trans illuminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequencing reaction was done in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) using the BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, USA). The sequence quality was checked using Sequence Scanner Software v1 (Applied Biosystems). Sequence alignment and required editing of the obtained sequences were carried out using Geneious Pro v5.1 (Drummond \u003cem\u003eet al.,\u003c/em\u003e 2010). Sequenced nucleotides for four species of Vespidae were submitted to NCBI gene bank and obtained accession number.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA sequences were subjected to pairwise distance analysis and the following phylogenetic trees were constructed: Maximum Parsimony, Maximum Likelihood and Neighbor-Joining by using MEGA 6.0 software (Tamura \u003cem\u003eet al.,\u003c/em\u003e 2011). Pairwise distance analysis was carried out with gap opening penalty 15 and gap extension penalty 6.66(Clustalw) (Thompson \u003cem\u003eet al.,\u003c/em\u003e 1994).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMaximum parsimony:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eThe maximum parsimony analyses were carried out using MEGA 5 (Tamura \u003cem\u003eet al.,\u003c/em\u003e2011). Bootstrap method was used with 100 replications and gap/missing data treatment by complete selection and the search method was Subtree-Pruning \u0026ndash;Regrafting (SPR) and substitution based on nucleotide sequences. The percentage of replicate trees in which the associated taxa clustered together in the boostrap test (100 replicates) was used (Felsenstein,1985).The Maximum parsimony tree was obtained using the Subtree-Pruning- Regrafting (SPR) algorithm (Nei and Kumar,2000),using search level 1. The substitution type based nucleotide sequences and the codon positions included were first +second+third+noncoding and all the positions containing gaps and missing data were eliminated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMaximum Likelihood:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eMaximum Likelihood analyses were run in MEGA 5 (Tamura \u003cem\u003eet al.,\u003c/em\u003e 2011). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei 1993). Initial tree for the heuristic search was obtained automatically by applying Neighbor- Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. The substitution type based nucleotide sequences and the codon positions included were first+second+third+noncoding and all the positions containing gaps and missing data were eliminated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNeighbor-Joining:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eNeighbor-Joining analyses were determined using MEGA 5 (Tamura et al., 2011). The evolutionary history inferred using the Neighbor-Joining method (Saitou and Nei,1987) .The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test(100replicates) was used (Felsenstein,1985). The evolutionary distances were computed using the Tajima-Nei method (Tajima and Nei, 1984). Codon positions included were first+second+third+noncoding and all positions containing gaps and missing data were eliminated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected data from the morphometric studies of four Vespidae species were analysed with statistical tools (SPSS version 14.0). The mean and standard deviation was calculated for the morphometric data and presented in the table.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIdentification of specimens\u003c/p\u003e\n\u003cp\u003eThe research specimen wasp species was collected from different districts namely Villupuram (Thadagam), Nagapattianam (Muthalaimedu), Dharmapuri (Lourdhupuram) and Chennai (Kilpauk cemetery) of Tamil Nadu during 2018 to 2019. Collected specimens was identified \u0026nbsp;and confirmed by using standard taxonomy keys and Dr. P. Girish Kumar, Scientist-C, Zoological Survey of India, Kerala for morphologically similar species namely Vespa tropica haematodes, Vespa affinis, Polistes olivaceus and Ropalidia marginata ( Fig. 3-6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe distribution, habitat and nesting characteristics were identified and presented in table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003eDistribution,\u0026nbsp;habitat\u0026nbsp;and\u0026nbsp;nesting\u0026nbsp;characteristics\u0026nbsp;of\u0026nbsp;the\u0026nbsp;Vespidae species\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"658\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.9574%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38.4498%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003cstrong\u003estribution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.7964%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003eabitat\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.7964%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003cstrong\u003eesting\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.9574%;\"\u003e\n \u003cp\u003eVespa tropica\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.4498%;\"\u003e\n \u003cp\u003eWidely distributed from Afghanistan and Pakistan to the Philippines, including the Indochinese peninsula and Peninsular Malaysia, it extends south to Indonesia,\u003c/p\u003e\n \u003cp\u003eincluding Timor, the Moluccas and Papua\u003c/p\u003e\n \u003cp\u003eNew Guinea, but it is absent from Taiwan,\u003c/p\u003e\n \u003cp\u003eAnd it has been introduced into Guam (USA), Korea and Southern Manchuria.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.7964%;\"\u003e\n \u003cp\u003eNocturnal, probably\u0026nbsp;a\u003c/p\u003e\n \u003cp\u003eforest\u0026nbsp;species\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.7964%;\"\u003e\n \u003cp\u003eProbably\u0026nbsp;aerial\u0026nbsp;nesting\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.9574%;\"\u003e\n \u003cp\u003eVespa affinis\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.4498%;\"\u003e\n \u003cp\u003eWidely distributed from India to the Ryukyu Islands, including Taiwan, the Philippines, the Indochinese and Malay Peninsula and Indonesia to New Guinea.\u003c/p\u003e\n \u003cp\u003eIntroduced in California (USA), Queensland (Australia) and New Zealand.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.7964%;\"\u003e\n \u003cp\u003eNocturnal, lowland\u003c/p\u003e\n \u003cp\u003ehabitats\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.7964%;\"\u003e\n \u003cp\u003eUsually\u0026nbsp;aerial\u0026nbsp;nesting\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.9574%;\"\u003e\n \u003cp\u003ePolisters olivaceus\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.4498%;\"\u003e\n \u003cp\u003eIndia: Andhra Pradesh, Arunachal Pradesh, Assam, Gujarat, Himachal Pradesh, Jammu \u0026amp; Kashmir, Jharkhand, Karnataka, Madhya Pradesh, Manipur, Meghalaya, Nagaland, Odisha, Rajasthan, Sikkim, Tripura, Uttarakhand, Uttar Pradesh, West Bengal. Elsewhere: Afghanistan, Amirantes, Australia, Bangladesh, Cambodia, Chagos Archipelago, Chile (introduced), China, Egypt, Fiji, Indonesia, Iran, Laos, Madagascar, Malaysia, Marianas, Marquesas, Mauritius, Myanmar, Nepal, New Caledonia, New Zealand, Oman, Pakistan, Philippines, Reunion, Samoa, Seychelles, Singapore, Society Is., Sri Lanka, Tanzania, Thailand, Tongas, Tuamotu Archipelago, U.S.A., Vietnam.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.7964%;\"\u003e\n \u003cp\u003eNocturnal, lowland\u003c/p\u003e\n \u003cp\u003ehabitats\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.7964%;\"\u003e\n \u003cp\u003eUsually\u0026nbsp;aerial\u0026nbsp;nesting\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.9574%;\"\u003e\n \u003cp\u003eRopalidia marginata\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.4498%;\"\u003e\n \u003cp\u003eIndia: Andhra Pradesh, Gujarat, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Odisha, Punjab, Rajasthan, Tamil Nadu, Uttarakhand, West Bengal. Elsewhere: Australia, Bali, Bangka, Borneo, Java, Kariman Djawa, Lombok, Malay Peninsula, Mariana Islands, Myanmar, New Britain, New Guinea, Palau Islands, Pakistan, Philippine Islands, Sri Lanka, Sulawesi, Sumatra, Sumba, Sumbawa, Talud Islands, Tukang Besi Islands, Vietnam, Volcano Islands.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.7964%;\"\u003e\n \u003cp\u003eVarious habitats\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.7964%;\"\u003e\n \u003cp\u003eUsually\u0026nbsp;subterranean\u003c/p\u003e\n \u003cp\u003enesting\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e1. Vespa tropica haematodes (Bequaert) (Fig.3)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScientific classification\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKingdom \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Animalia\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhylum \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Arthropoda\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClass \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Insecta\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOrder \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Hymenoptera\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSuper family \u0026nbsp; : Vespoidea\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFamily \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Vespidae\u003c/p\u003e\n\u003cp\u003eGenus \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Vespa\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSpecies \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: tropica haematodes (Bequaert)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Vespa affinis continentalis\u0026nbsp;haematodes (Bequaert) (Fig.4)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScientific classification\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKingdom \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Animalia\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhylum \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Arthropoda\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClass \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Insecta\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOrder \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Hymenoptera\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSuper family \u0026nbsp; : Vespoidea\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFamily \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Vespidae\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGenus \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Vespa\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSpecies \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: affinis continentalis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Polistes\u0026nbsp;(Gyrostoma) olivaceus (De Geer) (Fig.5)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScientific classification\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKingdom \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Animalia\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhylum \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Arthropoda\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClass \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Insecta\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOrder \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Hymenoptera\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSuper family \u0026nbsp; : Vespoidea\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFamily \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Vespidae\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGenus \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Polistes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSpecies \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: olivaceus\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Ropalidia marginata\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(Lepeletier)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(Fig.6)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScientific classification\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhylum \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Arthropoda\u003c/p\u003e\n\u003cp\u003eClass \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Insecta\u003c/p\u003e\n\u003cp\u003eSuper family \u0026nbsp; \u0026nbsp;: Vespoidea\u003c/p\u003e\n\u003cp\u003eFamily \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Vespidae\u003c/p\u003e\n\u003cp\u003eOrder \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; : Hymenoptera\u003c/p\u003e\n\u003cp\u003eGenus \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Ropalidia\u003c/p\u003e\n\u003cp\u003eSpecies \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: marginata\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphometric studies on Vespa species\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphometry studies was conducted on four species of Vespidae using standard procedure mainly based on the work of Richard (1956, 1973), Spradbery (1973) and van der Vecht (1975). The morphological terms used for describing different parts of the body namely width and length of head, mandible, antennae, eyes, legs, tergum, fore wing, hind wing and total length was measured (Table 2). \u0026nbsp;Body length was measured From the anterior margin of head to the end of second metasomal tergite along the mid dorsal line with a graduated eye piece and graticule, thus comprising the length of head, mesosoma and 1\u003csup\u003est\u003c/sup\u003e + 2\u003csup\u003end\u003c/sup\u003e metasomal segments. Fore wing length was measured from base to apex in millimeter (mm). Photographs were taken with a Veho USB microscope (Nikon SMZ800 streozoom microscope) and labelled in Adobe Photoshop (Fig. 7-10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eV. tropica haematodes\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eHead length (0.5 \u0026plusmn; 0.23) and width (0.56 \u0026plusmn;0.04); Mandible length (0.3 \u0026plusmn; 0.0) and width (0.2 \u0026plusmn; 0.0); Antennae length (0.9 \u0026plusmn; .006) and width (0.27 \u0026plusmn; 0.41); Eye length (0.33 \u0026plusmn; 0.05) and width (0.2 \u0026plusmn; 0.0); \u0026nbsp;Leg Meta femur length (0.49 \u0026plusmn; 0.03), \u0026nbsp;Meta tibia length (0.47 \u0026plusmn; 0.04), Hind femur length (0.52 \u0026plusmn; 0.078), Hind tibia length \u0026nbsp;(0.51 \u0026plusmn; 0.087); Tergum length (0.49 \u0026plusmn; 0.03) and width (0.95 \u0026plusmn; 0.05); Total length (2.4 \u0026plusmn; 0.231); Fore wing length (2.01 \u0026plusmn; 0.07) and width (0.81 \u0026plusmn; 0.12); Hind wing (1.21 \u0026plusmn; 0.057) and width (0.35 \u0026plusmn; 0.052) (Table 2, Fig. 7). \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eV. affinis continentalis\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ehaematodes:\u0026nbsp;\u003c/strong\u003eHead length (0.5 \u0026plusmn; 0.23) and width (0.56 \u0026plusmn;0.04); Mandible length (0.3 \u0026plusmn; 0.0) ad width (0.2 \u0026plusmn; 0.0); Antennae length (0.9 \u0026plusmn; .006) and width (0.27 \u0026plusmn; 0.41); Eye length (0.33 \u0026plusmn; 0.05) and width (0.2 \u0026plusmn; 0.0); \u0026nbsp;Leg Meta femur length (0.49 \u0026plusmn; 0.03), \u0026nbsp;Meta tibia length (0.47 \u0026plusmn; 0.04), Hind femur length (0.52 \u0026plusmn; 0.078), Hind tibia length \u0026nbsp;(0.51 \u0026plusmn; 0.087); Tergum length (0.49 \u0026plusmn; 0.03) and width (0.95 \u0026plusmn; 0.05); Total length (2.4 \u0026plusmn; 0.231); Fore wing length (2.01 \u0026plusmn; 0.07) and width (0.81 \u0026plusmn; 0.12); Hind wing (1.21 \u0026plusmn; 0.057) and width (0.35 \u0026plusmn; 0.052) (Table 2, Fig. 8). \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP. olivaceus\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eHead length (0.545 \u0026plusmn; 0.049) and width (0.521 \u0026plusmn;0.027); Mandible length (0.3 \u0026plusmn; 0.041) and width (0.275 \u0026plusmn; 0.048); Antennae length (0.745 \u0026plusmn; .049) and width (0.362 \u0026plusmn; 0.039); Eye length (0.32 \u0026plusmn; 0.078) and width (0.22 \u0026plusmn; 0.078); \u0026nbsp;Leg Meta femur length (0.48 \u0026plusmn; 0.042), \u0026nbsp;Meta tibia length (0.5 \u0026plusmn; 0.067), Hind femur length (0.51 \u0026plusmn; 0.0875), Hind tibia length \u0026nbsp;(0.51 \u0026plusmn; 0.087); Tergum length (0.6 \u0026plusmn; 0.094) and width (0.54 \u0026plusmn; 0.080); Total length (2.13 \u0026plusmn; 0.094); Fore wing length (1.74 \u0026plusmn; 0.117) and width (0.375 \u0026plusmn; 0.135); Hind wing (1.25 \u0026plusmn; 0.057) and width (0.235 \u0026plusmn; 0.041) (Table 2, Fig. 9). \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eR. marginata\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eHead length (0.39 \u0026plusmn; 0.021) and width (0.34 \u0026plusmn;0.021); Antennae length (0.31 \u0026plusmn; 0.061); \u0026nbsp;Eye length (0.295 \u0026plusmn; 0.028) and width (0.255 \u0026plusmn; .036); \u0026nbsp;Leg Meta femur length (0.245 \u0026plusmn; 0.043), \u0026nbsp;Meta tibia length (0.15 \u0026plusmn; 0.040), Hind femur length (0.32 \u0026plusmn; 0.105), Hind tibia length \u0026nbsp; (0.275 \u0026plusmn; 0.0485); Tergum length (0.3 \u0026plusmn; 0.033) and width (0.28 \u0026plusmn; 0.034); Total length (1.26 \u0026plusmn; 0.051); Fore wing length (0.565 \u0026plusmn; 0.052) and width (0.145 \u0026plusmn; 0.036); Hind wing (0.835 \u0026plusmn; 0.052) and width (0.78 \u0026plusmn; 1.13) (Table 2, Fig. 10). \u0026nbsp;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Taxonomy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA Isolation\u003c/p\u003e\n\u003cp\u003eThe mitochondrial gene sequence of COI gene data of wasp species from Tamil Nadu, India are presented and analyzed. The collection sites were Thadagam, Sirkali, Dharmapuri and Chennai districts. The barcode results indicated that the PCR products with COI (Forward/reverse) primer were 596, 586, 601,577 bp. Base pair length in V. tropica, V. affinis, P. olivaceus and R. marginata respectively (Figure 11).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNucleotide composition of Vespidae species\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA always contains equal amounts of adenine and thymine, guanine and cytosine, in pairs usually denoted A=T and G=C. These two regularities give rise to a third one. The nucleotide compositions of five Vespidae COX gene have been identified in the family Vespidae. The overall A+T content of Polistes olivaceus and Vespa tropica is 72.55% and 71.60% respectively. The highest total content of nucleotides composition of P. olivaceus is 601.0 and least Vespa tropica 567.0 was observed (Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cimg 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\" width=\"818\" height=\"607\"\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cbr\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn this study the wasp COI gene, 5\u003csup\u003e\u0026rsquo;\u0026nbsp;\u003c/sup\u003e\u003csub\u003e\u0026rarr;\u0026nbsp;\u003c/sub\u003e3\u003csup\u003e\u0026rsquo;\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003estandard forward primer (LCO) - GGTCAACAAATCATAAAGATATTGG and reverse primer (HCO) TAAACTTCAGGGTGACCAAAAAATCA with amplified PCR products were the range from 577 base pairs to 601 base pairs nucleotides in length. The amplified PCR products were visualized on 1.27 agarose gel electrophoresis. The amplified PCR products was cleaned up and sequenced in ABI 3500. DNA \u0026nbsp;analyzer (Applied Biosystems).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDNA Barcoding \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe mitochondrial gene sequences of COI the wasp\u0026rsquo;s species V. tropica,\u0026nbsp;\u003cbr\u003e\u0026nbsp;V. affinis, P. olivaceus and R. marginata in this study were submitted in NCBI gene bank and accession numbers were assigned for all the four species. The sequence quality was checked in sequence scanner software V1.0. The aligned sequences were identified as cytochrome C oxidase with BLAST procedure and the COI gene sequence was manually edited in Finch T.V. All sequences were deposited in NCBI gene bank. Multiple sequence alignment was performed using Clustal W MEGA 6.0. NCBI gene bank accession number for cytochrome c oxidase COI gene sequences for each wasp species were presented in the table 4.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe mitochondrial cytochrome c oxidase gene sequences of four wasp species NCBI gene bank accession numbers MN893829.1, MN 853754.1, MT133311.1 and MT178807.1 for V. tropica, V. affinis, P. olivaceus and R. marginata respectively. From the gene sequences and phylogenetic tree to topology structure results was confirmed that the wasp species Vespa tropica and Vespa affinis very strong relationship between this species.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTable 4: Mitochondrial DNA sequences of Cytochrome C oxidase in wasp species subjected to phylogenetic analysis.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"573\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10.4712%;\"\u003e\n \u003cp\u003eS.No\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32.6353%;\"\u003e\n \u003cp\u003eSpecies name\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.6021%;\"\u003e\n \u003cp\u003eNCBI Gen bank no /Accession No\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.2914%;\"\u003e\n \u003cp\u003eBase-pairs (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10.4712%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32.6353%;\"\u003e\n \u003cp\u003eVespa affinis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.6021%;\"\u003e\n \u003cp\u003eMN853754.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.2914%;\"\u003e\n \u003cp\u003e596\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10.4712%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32.6353%;\"\u003e\n \u003cp\u003eVespa tropica haematodes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.6021%;\"\u003e\n \u003cp\u003eMN893829.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.2914%;\"\u003e\n \u003cp\u003e586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10.4712%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32.6353%;\"\u003e\n \u003cp\u003ePolistes olivaceus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.6021%;\"\u003e\n \u003cp\u003eMT133311.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.2914%;\"\u003e\n \u003cp\u003e601\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10.4712%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32.6353%;\"\u003e\n \u003cp\u003ePolistes olivaceus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.6021%;\"\u003e\n \u003cp\u003eMT136727.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.2914%;\"\u003e\n \u003cp\u003e601\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 10.4712%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32.6353%;\"\u003e\n \u003cp\u003eRopalidia marginata\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.6021%;\"\u003e\n \u003cp\u003eMT178807.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.2914%;\"\u003e\n \u003cp\u003e577\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe Cytochrome C Oxidase (Co-I) sequences of wasp species were subjected to pairwise distance analysis and the following phylogenetic trees were constructed: Maximum Parsimony, Maximum Likelihood and Neighbor-Joining by using MEGA 6 software (Tamura et al., 2011). Pairwise distance analysis was carried out with gap opening penalty 15 and gap extension penalty 6.66 (Clustalw) (Thompson et al., 1994).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMaximum Likelihood:\u0026nbsp;\u003c/strong\u003eMaximum Likelihood analyses were run in MEGA 6 (Tamura et al., 2011). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei 1993). Initial tree for the heuristic search was obtained automatically by applying Neighbor- Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe results of Maximum Likelihood tree constructed based on Cytochrome c oxidase (Fig.12) with two parallel clusters; Ropalida marginata and Polistes olivaceus \u0026nbsp;evolved as \u0026nbsp; separate lineage, and the second cluster with Vespa affinis and Vespa tropica haematodes \u0026nbsp;shows their inter and intra generic closeness. But they share a common ancestral node suggesting an affinity between these four wasp species. \u0026nbsp;Among them, the two morphologically related Vespa affinis and Vespa tropica haematodes share a common node exhibiting their affinity. Ropalida marginata and Polistes olivaceus are morphologically different; they share a common node which shows their closeness. Maximum Likelihood tree based on CO I variations showing the relationships of wasp species. \u0026nbsp;The genetic similarity between the Vespa affinis and Vespa tropica haematodes as observed in the Maximum Likelihood tree.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eNeighbor-Joining tree:\u0026nbsp;\u003c/strong\u003eNeighbor-Joining analyses were determined using MEGA 6 (Tamura et al., 2011). The evolutionary history \u0026nbsp;inferred using the Neighbor-Joining method (Saitou and Nei,1987) .The percentage of replicate trees in which the associated taxa clustered \u0026nbsp; together in the bootstrap test(100replicates) was used (Felsenstein,1985). The evolutionary distances were computed using the Tajima-Nei method (Tajima and Nei 1984).\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe results of Neighbor-Joining tree and UPGMA constructed based on Cytochrome c oxidase (Fig. 13 and Fig. 14) with two parallel clusters; Ropalida marginata and Polistes olivaceus \u0026nbsp;evolved as \u0026nbsp; separate lineage, and the second cluster with Vespa affinis and Vespa tropica haematodes \u0026nbsp;shows their inter and intra generic closeness. But they share a common ancestral node suggesting an affinity between these four wasp species. \u0026nbsp;Among them, the two morphologically related Vespa affinis and Vespa tropica haematodes share a common node exhibiting their affinity. Ropalida marginata and Polistes olivaceus are morphologically different; they share a common node which shows their closeness. The affinity between any two species is similar in phylograms revealing uniform phylogenetic relationship and suggesting monophyly.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMaximum parsimony:\u0026nbsp;\u003c/strong\u003eThe maximum parsimony analyses were carried out using MEGA 6 (Tamura et al., 2011). Bootstrap method was used with 100 replications and gap/missing data treatment by complete selection and the search method was Subtree-Pruning \u0026ndash;Regrafting (SPR) and substitution based on nucleotide sequences. The percentage of replicate trees in which the associated taxa clustered together in the boostrap test (100 replicates) was used (Felsenstein,1985).The Maximum parsimony tree was obtained using the Subtree-Pruning- Regrafting (SPR) algorithm (Nei and Kumar, 2000),using search level 1.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe results of Maximum Parsimony tree constructed based on Cytochrome c oxidase (Fig.15) with two parallel clusters; Vespa affinis and Vespa tropica haematodes, forms a cluster. Ropalida marginata \u0026nbsp;evolved as a separate lineage and Polistes olivaceus \u0026nbsp;evolved as single \u0026nbsp;separate lineage. Maximum parsimony shows inter and intra generic closeness between species and they share a common ancestral node suggesting an affinity between these four wasp species. \u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWasps belong to the Vespidae family, which is a worldwide order of Hymenoptera (Class: Insecta). The members of this family are mostly found in tropical areas of the world, and there are about 5000 species in total. Hornet wasps are a species of wasps belonging to the genus \u003cem\u003eVespa Linnaeus.\u003c/em\u003e They are social wasps with an excessive degree of evolution. Hornet wasps may be observed mainly within side the Oriental and Palaearctic areas of the Globe. There are a complete of 23 recognized species within side the world, with sixteen species from the Indian subcontinent and 15 species from India (Carpenter and Kojima, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The nests are made of paper and are connected to wood, metal, or rock (Cranshaw et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eVespa affinis\u003c/em\u003e, a species of hornet found largely in Asia, develops vast aerial nests with up to tens of thousands of workers (Archer, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Other insects, particularly honey bees near their hives, are preyed upon by these wasps. Their nests are made of a combination of plant fibres and salivary secretions, and they can range in size from a baseball-sized nest with a few thousand cells to a nest with hundreds of thousands of cells. Nests are typically annual, but in warmer areas, a few species will establish huge perennial nests. They are dangerous because they sting people and domesticated animals. In all social wasps and their close solitary relatives, the females have the apparatus and ability needed to inflict painful stings.\u003c/p\u003e \u003cp\u003eThey are honey bees' natural enemy, and they harm the fruit eating with them right before harvest (Kimsey and Carpenter, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Ceylonese specimens differ from those from the rest of India in that their thorax is heavily reddened. Since specimens from Tenasserim, part of the Indo-Malayan mountain region in Southeast Asia, have similar colour, it appears difficult to distinguish them as a unique variety. Mangalore, Shembaganur near Madura, and Nagapattinam are all in India (Bridwell and Bequaert, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1918\u003c/span\u003e; Balachandar et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe maximum not unusual place Hornets of the Oriental and Indo-Malayan areas are the large black or fusoous wasps with the second one or first and 2nd stomach segments in part or thoroughly orange or orange-yellow. In maximum collections those wasps stand as \u003cem\u003eVespa cincta\u003c/em\u003e and \u003cem\u003eVespa affinis\u003c/em\u003e, the latter being frequently seemed as a number of the former. A take a look at of structure, however, discloses that pretty wonderful species are generally pressured beneath those names. This fact, unfortunately, changed into now no longer identified via way of means of Dubuysson, while he wrote his monograph of the \u003cem\u003eVespinae\u003c/em\u003e (1905). At the Paris Museum, he located specimens of each species beneath \u003cem\u003eV. cincta\u003c/em\u003e var. \u003cem\u003eaffinis\u003c/em\u003e, in addition to beneathneath \u003cem\u003eV. cincia\u003c/em\u003e. Koeniger, N., \u003cem\u003eet al.\u003c/em\u003e,1994 first observed the confusion, while he defined his \u003cem\u003eV. indosinensis\u003c/em\u003e as wonderful from \u003cem\u003eV. cincta\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe present study to carry out to identify the species from different study area of district of Tamil Nadu. The specimens were collected, preserved, identified and confirmed with suitable taxonomy key and the wasp expert Dr. P. Girish Kumar, Zoological survey of India, Kerala. The identified specimens namely, \u003cem\u003eVespa tropica haematodes\u003c/em\u003e (Thadagam\u003cem\u003e-\u003c/em\u003eVillupuram district), \u003cem\u003eVespa affinis\u003c/em\u003e (Sirkali \u003cem\u003e-\u003c/em\u003e Nagapattianam District), \u003cem\u003ePolistes olivaceus\u003c/em\u003e (Lourdhupuram \u003cem\u003e\u0026ndash;\u003c/em\u003e Dharmapuri district) and \u003cem\u003eRopalidia marginata\u003c/em\u003e (Kilpauk Garden cemetry- Chennai district).\u003c/p\u003e \u003cp\u003eBingham (1897) and Das \u0026amp; Gupta (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) made significant advances to Vespidae taxonomy in India and its neighbouring nations. The understanding of Rajasthan's vespid fauna is limited and fragmented. In scattered literatures, 14 genera and 20 species have been recorded from Rajasthan till date (Nurse, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1903\u003c/span\u003e; van der Vecht, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1941\u003c/span\u003e; Chhotani \u0026amp; Ray, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Giordani Soika, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Gusenleitner, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Girish Kumar, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, 2011, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e a \u0026amp; b; Girish Kumar \u0026amp; Lambert Kishore, 2012; Girish Kumar \u0026amp; Carpenter, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Girish Kumar \u003cem\u003eet al.\u003c/em\u003e, 2013). The goal of this paper is to present a comprehensive analysis of the Vespidae family's fauna in the state of Rajasthan. In 2014, Girish Kumar and Gaurav Sharma reported that, in Rajasthan, 15 genera and 26 species and from this 1 genus and 6 species are new records for Rajasthan. The Indian species of the genus \u003cem\u003eEumenes Latreille\u003c/em\u003e with a key to species is provided by Girish kumar \u003cem\u003eet al.\u003c/em\u003e, 2017. The species \u003cem\u003eEumenes pomiformis\u003c/em\u003e (Fabricius, 1781) is recorded at Kozhikode, Kerala for the first time from India, while \u003cem\u003eE. affinissimus\u003c/em\u003e de Saussure, 1852, \u003cem\u003eE. pomiformis\u003c/em\u003e (Fabricius, 1781) and \u003cem\u003eE. persimilis\u003c/em\u003e kojimardani Soika, 1960 are recorded from Pakistan.\u003c/p\u003e \u003cp\u003eBased on colour variation, Kimsey and Carpenter (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) proposed that\u003c/p\u003e \u003cp\u003e \u003cem\u003eVespula intermedia\u003c/em\u003e (du Buysson, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1905\u003c/span\u003e) be considered a genuine species rather than a synonym of \u003cem\u003eVespula rufa\u003c/em\u003e (Linnaeus, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1758\u003c/span\u003e). This idea is not supported by our Bayesian inference data. Depending on locus, the divergence is minor or non-existent, implying that\u003c/p\u003e \u003cp\u003e \u003cem\u003eV. rufa\u003c/em\u003e and \u003cem\u003eV. intermedia\u003c/em\u003e were separated for just a brief time, if at all. We believe that\u003c/p\u003e \u003cp\u003e \u003cem\u003eV. intermedia\u003c/em\u003e should be considered a synonym of \u003cem\u003eV. rufa\u003c/em\u003e because the species delimitation is solely based on colour variation. The multi-species coalescent tree and the species delimitation analyser also corroborate this.\u003c/p\u003e \u003cp\u003eMorphometric studies on Vespidae species\u003c/p\u003e \u003cp\u003eThe morphometric studies was conducted for all the four species \u003cem\u003eV. tropica haematodes, V. affinis\u003c/em\u003e, \u003cem\u003eP. olivaceus\u003c/em\u003e and \u003cem\u003eR. marginata\u003c/em\u003e collected from different districts of Tamil Nadu based on the work of Richard (1971, 1977), Philip Spradbery J (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1973\u003c/span\u003e) and van der Vecht (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). The \u003cem\u003eVespa\u003c/em\u003e species morphometric measurements like length and width of head, mouthparts, legs and wings, antenna, eye and tergum was recorded and tabulated.\u003c/p\u003e \u003cp\u003eBody length was measured From the anterior margin of head to the end of second metasomal tergite along the mid dorsal line with a graduated eye piece and graticule, thus comprising the length of head, mesosoma and 1st\u0026thinsp;+\u0026thinsp;2nd metasomal segments. Fore wing length was measured from base to apex in millimeter (mm). Photographs were taken with a Veho USB microscope and labelled in Adobe Photoshop and presented. The similar study was conducted by Roohollah Abbasi (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2012\u003c/span\u003esmith) on sexual dimorphism in the wing morphology of social vespid wasps \u003cem\u003ePolistes laterille\u003c/em\u003e using geographic morphometries.\u003c/p\u003e \u003cp\u003eSize differentiation is considered as an initial step for the origin of morphological castes in the four main groups of social hymenoptera. In social wasps, the differences are more conspicuous in \u003cem\u003eVespinae\u003c/em\u003e and more complex in \u003cem\u003ePolistinae.\u003c/em\u003e All the four species are different in morphological characters, size, colour and length and width of the body. \u003cem\u003eVespa tropica\u003c/em\u003e and \u003cem\u003eVespa affinis\u003c/em\u003e both the species are morphologically similar but genetically dissimilar. The length and width of the species vary from species to species\u003c/p\u003e \u003cp\u003eMolecular taxonomy\u003c/p\u003e \u003cp\u003eBarcoding will provide vital new tools for appreciating and managing Earth\u0026rsquo;s immense and changing biodiversity. Traditional taxonomy has been used in Systematics for over 250 years with the identification of more than 1.75\u0026nbsp;million species. However, in many countries as in India, traditional taxonomy is yet to adopt the latest molecular techniques available for a better and accurate identification of taxa. Enhanced taxonomic infrastructure with the help of DNA information can certainly speed up the process of species exploration, description and classification of biological diversity. The nucleotide sequence of this particular 640 nucleotide region (Folmer et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) shall be the unique identification code for all species to facilitate the correct determination of species and the discovery of new species (Moritz and Cicero, 2004). Many studies have shown that taxon identification based on DNA sequences can facilitate the recognition of known species and the discovery of new species (Blaxter et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hebert et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2003a\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003eb\u003c/span\u003e). DNA sequences of the mitochondrial COX-1 gene can serve as a molecular genetics for identifying all kinds of animals (Hebert et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e), especially cryptic species in tropical regions (Berkov, 2002; Monaghan et al, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hajibabaei et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and insect at different growth stages (Janzen \u003cem\u003eet al\u003c/em\u003e., 2004).\u003c/p\u003e \u003cp\u003eOur \u003cem\u003eVespa bicolor\u003c/em\u003e (Fabricius, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1787\u003c/span\u003e) specimens' results are perplexing and difficult. We have numerous individuals who morphologically match each other and one individual who differs. The specimen that diverges (U3481) matches Archer's (1989) description of a\u003c/p\u003e \u003cp\u003e \u003cem\u003eV. bicolor\u003c/em\u003e. On BOLD or BLAST, however, this specimen does not match any other \u003cem\u003eV. bicolor\u003c/em\u003e. The additional morphologically similar individuals (U3480, U3656, U3657, U3673, U3674, and U3675) do not meet Archer's (1989) description for a \u003cem\u003eV. bicolor\u003c/em\u003e or anything else, but the closest match is for a \u003cem\u003eV. bicolor\u003c/em\u003e. However, using both BOLD and BLAST systems, they identified a 99 percent identification match to other \u003cem\u003eV. bicolor\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAs per the report of Edward Laidlaw Smith \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1970\u003c/span\u003e The Hymenoptera has the least sexually dimorphic combined with the least specialized genitalia among the Pterygota. Based on physical features, two specimens identified as \u003cem\u003eParavespula flaviceps\u003c/em\u003e, one from China and the other from Japan, appear to represent two different evolutionary lineages. The species delimitation analyser, the Bayesian inference species tree, the COI SLA, the multi-species coalescent tree, and the Bayesian inference species tree all support this. The morphology of the Japanese specimen U3936 matches that of a \u003cem\u003eP. flaviceps\u003c/em\u003e described by Archer (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). The sample had no matches in BOLD systems but 99 percent identity to \u003cem\u003eP. flaviceps\u003c/em\u003e in BLAST, including the Federico Lopez-Osorio et al., (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) specimen (also from Japan). The Chinese specimen U3678 has a somewhat dimpled surface. According to Carpenter \u003cem\u003eet al.\u003c/em\u003e (2011) indicates that there is a divergence between the specimens from Japan and China, and since a species has been identified from that region, it 28 could be the same as our specimen. However, before we can make any recommendations, we need to compare their samples to ours. Carpenter \u0026amp; Glare (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) investigated whether the North American species \u003cem\u003eVespula alascensis\u003c/em\u003e (Packard, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1870\u003c/span\u003e) was a synonym of the European \u003cem\u003eParavespula vulgaris\u003c/em\u003e (Linnaeus, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1758\u003c/span\u003e) or a species sui generis using morphological and genetic data.\u003c/p\u003e \u003cp\u003eThey used specimens from North America and Europe and found that they were two distinct species based on male genetalia and molecular divergence. This theory is supported by our results. On the basis of morphological features, specimens of \u003cem\u003eDolichovespula pacifica\u003c/em\u003e were identified as \u003cem\u003eD. pacifica\u003c/em\u003e; nonetheless, they appear to belong to three different evolutionary lineages. All of our Swedish specimens form a sister group with a subset of our Japanese specimens in two of these lineages. The third lineage, which includes a subset of our Japanese samples, is related to our \u003cem\u003eD. saxonica\u003c/em\u003e samples from Sweden. The species delimitation analyser, the Bayesian inference species tree, the COI SLA, the multispecies coalescent tree, and the Bayesian inference species tree all support this.\u003c/p\u003e \u003cp\u003eIn BOLD systems, the specimens U1742 and U1922 from Sweden were confirmed to be 100% identical to \u003cem\u003eDolichovespula norvegicoides\u003c/em\u003e (early release, no information for country of collection). The closest match after that was 96 percent identity to \u003cem\u003eD. albida\u003c/em\u003e and \u003cem\u003eD. norwegica\u003c/em\u003e. The closest match in BLAST was 96 percent identity to \u003cem\u003eD. albida\u003c/em\u003e. The Japanese specimens U3926 and U3937 belong to the same family as the Swedish specimens. In BOLD systems, the specimens were found to be 97.8% identical to \u003cem\u003eD. norvegicoides\u003c/em\u003e. After that, \u003cem\u003eD. norwegica\u003c/em\u003e was the closest match with 96.9% identity.\u003c/p\u003e \u003cp\u003eIn BLAST, the specimens had a 97 percent identity to \u003cem\u003eD. albida\u003c/em\u003e, which was the closest match. The Japanese specimens U3924 and U3928 are sister species to \u003cem\u003eD. saxonica\u003c/em\u003e. In BOLD systems, the specimens had 100 percent identity to KMP268, and 98 percent identity to\u003c/p\u003e \u003cp\u003e \u003cem\u003eD. saxonica\u003c/em\u003e. In BLAST, the outcomes are the same. The results of the BLAST and BOLD systems are intriguing since the specimens are more closely related to \u003cem\u003eD. saxonica\u003c/em\u003e and\u003c/p\u003e \u003cp\u003e \u003cem\u003eD. albida\u003c/em\u003e than to other \u003cem\u003eD. pacifica\u003c/em\u003e specimens. One probable explanation is that \u003cem\u003eDolichovespula\u003c/em\u003e is a very new and unresolved group containing closely related species. The species' divergence occurred recently and over a brief period of time.\u003c/p\u003e \u003cp\u003eBased on morphological features, Pekkarinen (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) determined that \u003cem\u003eDolichovespula pacifica\u003c/em\u003e was a synonym of \u003cem\u003eDolichovespula norvegicoides\u003c/em\u003e. Based on physical characteristics, Archer (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) classified them as two distinct species. Sladen (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1918\u003c/span\u003e) used a type specimen found in Canada to describe \u003cem\u003eD. norvegicoides\u003c/em\u003e. Birula (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1930\u003c/span\u003e) used a type specimen taken in Eastern Siberia to describe \u003cem\u003eD. pacifica\u003c/em\u003e. Our findings support the idea that they are two distinct species, with \u003cem\u003eD. pacifica\u003c/em\u003e (Birula, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1930\u003c/span\u003e) as a legitimate designation.\u003c/p\u003e \u003cp\u003eMany species of social wasps are also important in the biological control of pests, mainly lepidoptera of crops such as eucalyptus, vegetables, coffee, corn and tomatoes (Bach, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1951\u003c/span\u003e; Marques and Carvalho, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Prezoto and Machado, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Richter, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Six subfamilies are recognized under \u003cem\u003eVespidae\u003c/em\u003e, and their relationships have been established with greater confidence than for most of the other families of Vespoidea. They are (\u003cem\u003eEuparagiinae\u003c/em\u003e + (\u003cem\u003eMasarinae + (Eumeninae + (Stenogastrinae + (Vespinae\u0026thinsp;+\u0026thinsp;Polistinae\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eSince good estimates of phylogeny are required for the proper investigation of many other topics of particular interest in the context of the aculeates, notably behavioural and physiological adaptations such as sociality, nesting behaviour, host switching, etc., and the possible times of their origin, it is important that further work in this area be pursued.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study concludes and gives more information on morphology, molecular phylogeny, histology and cuticular hydrocarbons (kairomones) of the four species namely, \u003cem\u003eVespa affnis, Vespa tropica\u003c/em\u003e, \u003cem\u003ePolistes olivaceus\u003c/em\u003e and \u003cem\u003eRopalidia marginata\u003c/em\u003e and its very useful for researcher, farmers, agriculturalist and others for prevention of the casualties and annoyances inflicted on the people of the state of Tamil Nadu, India as well as those in other countries through appropriate cognizance on \u003cem\u003eVespidae\u003c/em\u003e (wasp) species. Since this is the first research report of this kind in the state of Tamil Nadu.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe authors thank the Loyola College management and Fire and Rescue departments (Sirkahzi and Vettavalam), Tamil Nadu for their support to carry out this research work. Our sincere gratitude to Dr. Grish Kumar P, Scientist, Zoological Survey of India, Kozhicode, Kerala for his taxonomical keys and help in photographing the specimens inclusion described here.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbdalla FC, Cruz-Landim C. (2001). Dufour glands in the hymenopterans (Apidae, Formicidae, Vespidae): a review. Braz. J. Biol. 61:95\u0026ndash;106.\u003c/li\u003e\n \u003cli\u003eAguiar AP, Deans AR., Engel MS (2013) Order Hymenoptera. 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London: British Museum of Natural History.\u003c/li\u003e\n \u003cli\u003eRichards OW (1978) \u003cem\u003eThe social wasps of the Americas, excluding the Vespinae.\u003c/em\u003e London:\u0026nbsp;Schneider D (1963) Electrophysiological investigation of insect olfaction. In: Olfaction and Taste, I. Zotterman Y (ed), Pergamon Press, Oxford.\u003c/li\u003e\n \u003cli\u003eRichards\u0026nbsp;OW (1971) The biology of the social wasps. Biol. Rev.\u003cem\u003e,\u003c/em\u003e 46: 483\u0026ndash;528.\u003c/li\u003e\n \u003cli\u003eRichter MR (2000) Social wasp (Hymenoptera, Vespidae) foraging behavior. Annual Review of Entomology 45(1):121\u0026ndash;150.\u003c/li\u003e\n \u003cli\u003eRoohollah Abbasi (2012) On the distribution and taxonomy of vespine wasps of Iran (Hymenoptera: Vespidae: Vespinae): ACTA Mus Morav Sci Biol\u003cem\u003e\u0026nbsp;\u003c/em\u003e97: 69\u0026ndash;86.\u003c/li\u003e\n \u003cli\u003eRoohollah Abbasi\u0026nbsp;(2009) Sexual dimorphism in the wing morphology of social vespid wasps \u0026ndash; a case study on the genus \u003cem\u003ePolistes\u003c/em\u003e Latreille using geometric morphometrics, Zoology in the Middle East 47(1): 83\u0026ndash;92.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSaitou N, Nei\u0026nbsp;M\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003e1987)\u003c/em\u003e The neighbor-joining method: a new method for reconstructing phylogenetic trees.\u0026nbsp;\u003cem\u003eMol Biol Evol\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003e4 (4): 406\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003e425.\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003eSladen\u0026nbsp;FW L\u0026nbsp;(1918) The genus\u0026nbsp;\u003cem\u003eVespa\u003c/em\u003e in Canada. Ottawa Nat. 32: 71\u0026ndash;72.\u003c/li\u003e\n \u003cli\u003eSmith EL (1970) Evolutionary Morphology of the External Insect Genitalia. 2. Hymenoptera. Annals of the Entomological Society of America,63: 1 \u0026ndash;27.\u003c/li\u003e\n \u003cli\u003eTajima\u0026nbsp;F, Nei M (1984) Estimation of Evolutionary Distance between Nucleotide-Sequences.\u0026nbsp;Molecular biology and evolution 1\u0026nbsp;(3):269\u0026ndash;285\u003c/li\u003e\n \u003cli\u003eTamura K, et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol\u003cem\u003e\u0026nbsp;\u003c/em\u003e 28:2731\u0026ndash;2739.\u003c/li\u003e\n \u003cli\u003eTamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10(3):512\u0026ndash;526.\u003c/li\u003e\n \u003cli\u003eThompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speci\u0026THORN;c gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673\u0026ndash;4680.\u003c/li\u003e\n \u003cli\u003eVan Der Vecht J (1975) A review of the genus Stenogaster Guerin (Hymenoptera: Vespoidea). Journal of the Australian Entomological Society 14: 283\u0026ndash;308.\u003c/li\u003e\n \u003cli\u003eVecht J, \u0026nbsp;Van Der (1941)\u0026nbsp;The Indo-Australian species of the genus\u003cem\u003e\u0026nbsp;Ropalidia\u0026nbsp;\u003c/em\u003e(=\u003cem\u003eIcaria\u003c/em\u003e) (Hym., Vespidae) (first part). Treubia18: 103\u0026ndash;190.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 2 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Morphology, Molecular Phylogeny, Vespa tropica haematodes, Vespa affinis continentalis, Polistes olivaceus and Ropalidia marginata, Vespidae, Tamil Nadu","lastPublishedDoi":"10.21203/rs.3.rs-4507993/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4507993/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHymenoptera is one of the largest orders of insects. It consists many species of bees, wasps, hornets, sawflies, and ants. Wasps are important to natural and biological pest due to their predaceous parasitoid nature. Stinging behavior is one of the salient aspects of social hymenopterans. The mortality and morbidity from these bites reported in many districts of Tamil Nadu and around the world. The current study investigates the molecular phylogeny, histology of venom gland and kairomones profile of chosen \u003cem\u003eVespidae\u003c/em\u003e species from the northwest districts (1.Villupuram-Thadagam, 2.Nagapattinam - Mudhalaimedu, 3. Dharmapuri- Lourdhupuram and 4. Chennai \u0026ndash; Kilpauk cemetery) of Tamil Nadu. The hornets nests were carefully observed in day time and collected at night time. The phylogenetic relationship analysis was carried out using cytochrome oxidase subunit I (COI) and PCR was performed to amplify COI fragment. The amplified COI regions (the range from 577 base pairs to 601 base pairs nucleotides in length) were sequenced, analysed and DNA barcoded. The four novel nucleotide sequences of COI gene were deposited into the GenBank database and obtained accession numbers for \u003cem\u003eVespa tropica haematodes (\u003c/em\u003eMN893829.1), \u003cem\u003eVespa affinis\u003c/em\u003e (MN 853754.1), \u003cem\u003ePolistes olivaceus\u003c/em\u003e (MT133311.1), and \u003cem\u003eRopalidia marginata\u003c/em\u003e (MT178807.1). Multiple sequence alignment was performed using clustal W in MEGA 6.0. and Phylogenetic analysis, Maximum likelihood, and Neighbour joining tree was constructed. The results revealed that the \u003cem\u003eV. tropica\u003c/em\u003e, and \u003cem\u003eV. affinis are\u003c/em\u003e sister group and share a common ancestor and \u003cem\u003eV. tropica, V. affinis\u003c/em\u003e were found to be more close relatives than \u003cem\u003eP. olivaceous and R. marginata.\u003c/em\u003e This study provides more information on Vespa species for researchers, agriculturalists and others in the prevention of the casualties by Vespa bites and the annoyance inflicted on the people.\u003c/p\u003e","manuscriptTitle":"New Record: Study on Morphometric and Molecular Taxonomy of Vespidae Species (Hymenoptera: Vespidae: Polistinae) in Different Districts of Tamil Nadu, India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-08 09:03:33","doi":"10.21203/rs.3.rs-4507993/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b28393b1-46e9-430f-955b-0a626192502d","owner":[],"postedDate":"November 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-16T07:38:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-08 09:03:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4507993","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4507993","identity":"rs-4507993","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}