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Scanning electron microscopic studies on antenna of Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae)—A blow fly species of forensic importance
Accepted Manuscript Title: Scanning electron microscopic studies on antenna of Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae) − a blow fly species of forensic importance Authors: Garima Hore, Aniruddha Maity, Atanu Naskar, Waliza Ansar, Shyamasree Ghosh, Goutam Kumar Saha, Dhriti Banerjee PII: DOI: Reference:
S0001-706X(17)30104-3 http://dx.doi.org/doi:10.1016/j.actatropica.2017.04.005 ACTROP 4268
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
7-2-2017 11-4-2017 14-4-2017
Please cite this article as: Hore, Garima, Maity, Aniruddha, Naskar, Atanu, Ansar, Waliza, Ghosh, Shyamasree, Saha, Goutam Kumar, Banerjee, Dhriti, Scanning electron microscopic studies on antenna of Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae) − a blow fly species of forensic importance.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Scanning Electron Microscopic studies on antenna of Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae) – a blow fly species of forensic importance
Garima Horea, Aniruddha Maitya*, Atanu Naskara, Waliza Ansarb, Shyamasree Ghoshc, Goutam Kumar Sahad, Dhriti Banerjeea
a
Diptera Section, Zoological Survey of India, Ministry of Environment, Forest & Climate Change (Government of India), M-Block, New Alipore, Kolkata- 700 053. b
Department of Zoology, Behala College, Parnashree, Behala, Kolkata-700 060. School of Biological Sciences, National Institute of Science, Education and Research (NISER), Bhubaneswar- 751005, India. c
d
Department of Zoology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata- 700 019.
a*
Corresponding author: Aniruddha Maity, e-mail ID: [email protected]
Highlights Scanning electron microscopic studies on antenna of male and female Hemipyrellia ligurriens is carried out for the first time so as to generate a baseline data applicable in various fields of studies such as vector biology and forensic entomology. Five different types of sensilla and numerous microtrichiae were observed on the antennal surface. Chaetic sensilla were found on the scape and pedicel; styloconic sensilla found only on the pedicel whereas trichoid, basiconic and coeloconic sensilla were observed only on the flagellum. The fact that coeloconic sensilla, a type of multiporous sensilla was observed to be larger in females, can possibly add to the assumption that they serve the purpose of oviposition site detection in females.
Graphical abstract
Comparative analysis of size of flagellum between male and female of H. ligurriens 114.77
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Comparative analysis of size of Coeloconic sensilla between male and female of H. ligurriens 1.23 Coeloconic sensilla width (µm) 1.56
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Females had wider flagella than the males, females showed bigger coeloconic sensilla in length and width.
Play a pivotal role in locating successful sites for colonization and oviposition in corpses.
Abstract Blow flies (Diptera: Calliphoridae) are one of the foremost organisms amongst forensic insects to colonize corpses shortly after death, thus are of immense importance in the domain of forensic entomology. The blow fly Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae) is considered as a forensically important fly species globally and is also known for its medical and veterinary importance. In the present study, we report for the first time scanning electron microscopic studies on the morphology of sensilla of antenna of adult male and female of H. ligurriens is with profound importance in better understanding of the insect morphology from forensic entomological perspective, and also could aid in proper identification of the species from other calliphorid flies. The structural peculiarities observed in the (i) antenna of H. ligurriens with three segments- scape, pedicel and flagellum with dorsolaterally placed arista (ii) densely covered microtrichia and most abundant trichoid sensilla identified on the antenna (iii) observation of only one type of sensilla, chaetic sensilla (ChI) on the scape (iv) two types of chaetic sensilla (ChI and ChII) and styloconic sensilla on the pedicel (v) the flagellum with three types of sensilla- trichoid, basiconic and coeloconic sensilla (vi) Basiconic sensilla with multiporous surfaces with characteristic olfactory function. Moderate sexual dimorphism in the width of the flagellum, the females with wider flagella than the males, bear significance to the fact that they bear more multi-porous sensilla than the males, thus suffice their need to detect oviposition sites. Significant difference was observed in the length and width of coeloconic sensilla between the two sexes, the females showed bigger coeloconic sensilla, suggesting their function in oviposition site detection and successful colonization in corpses.
Keywords: Antenna; sensilla; Hemipyrellia ligurriens; scanning electron microscopy.
1. Introduction Hemipyrellia ligurriens (Wiedemann,1830), an important blow fly species with great medical and veterinary importance shows a fairly wide distribution comprising most of the Oriental region including India, Sri Lanka, China, Thailand, Malaysia, Indonesia, Java, Sumatra, Philippines and also Australia and New Guinea (Kurahashi et al., 1997; Kurahashi and
Chowanadisai, 2001; Senior-White et al., 1940; Tumrasvin et al., 1979). In the recent past, the species has been considered as a forensically important fly as their larvae have been collected from human corpses (Chen et al., 2004; Lee et al., 2004; Sukontason et al., 2007a). Moreover, the species can be a nuisance in market places and gardens, dry fish processing centers. The adults are the potential vectors of various enteric pathogens due to their attraction to human excreta near human-occupied environments (Sinha and Nandi, 2007). Larvae of H. ligurriens have also been recorded as parasites of living animals like earthworm, fish and toad in experimental conditions (Roy and Dasgupta, 1971). H. ligurriens also causes myiasis in domestic animals like goat and buffalo (Sinha, 2012). Olfaction, a sensory system which allows insects to detect chemicals in the environment is vital for their behaviour and ecology, as it helps them to locate food sources, mates and oviposition sites. The main olfactory organs of flies, like other insects are the paired antenna, which consists of numerous, morphologically distinct, microscopic sensilla each serving specific functions (Greenberg, 1970; Shanbhag et al., 1999). Thus in the case of forensically important insects, these sensillary structures present on the antenna are important in perception of chemical cues like ammonia and sulphur-rich volatiles, broadly classified as volatile organic compounds (VOCs) produced as a result of corpse decomposition and also used for Post mortem interval (PMI) estimation, thus having immense value in forensic investigations (Ishijima, 1967). In spite of its importance in medical, veterinary and forensic entomology, the morphology and fine structure of this species has received little attention in comparison to that of other blow fly genus like Chrysomya. In the initial phase, Ishijima (1967) provided some information on the adult and illustrated the third instar of H. ligurriens; whereas, some characters of the puparium have been recorded from light microscopy observations by Sukontason et al. (2007b). Life cycle studies of H. ligurriens in Sundarban Biosphere Reserve, West Bengal, India has been
done by Sinha and Nandi (2007). Light microscopic and scanning electron microscope studies on larval morphology has been done by Bunchu et al. (2012); Sukontason et al. (2008). However, no such efforts have been made to study the morphology of the adult H. ligurriens in detail, the olfactory organs like the antenna in particular. Moreover, forensic entomology requires accurate identification of insect specimens which can be provided by detailed morphological study of the species. So, the present study encompassing the ultrastructural characterization of the fly antenna with the help of Scanning Electron Microscopy has been carried out to fill in many of these lacunae.
2. Materials and methods 2.1. Collection and preservation of the fly specimens H. ligurriens male and female flies were collected from a dead Rohu fish (Labeo rohita), an Indian major carp, weighing about 1.5kg which was kept in natural environmental conditions in the garden area of the Zoological Survey of India (ZSI), Kolkata premises for observing and monitoring the successive colonisation, growth and development of flies on the dead organism. The study was conducted during the month of September, 2015 at ZSI New Alipore, Kolkata, West Bengal, India. A modified version of the Malaise trap was used for collection of adult specimens. Adult flies were killed using a killing-jar containing liquid benzene and they were preserved dry in insect-envelope. The specimens were sorted out by basic body plan assessment and relaxed in a relaxing-chamber for about 24 hours. The specimens were pinned and kept in a drying chamber for at least 4-5 days before being preserved in insect cabinet. 2.2. Processing of specimens for Scanning Electron Microscopic studies Initially, the male and female flies were sorted out under the microscope and then they were decapitated and only the head portion was processed for SEM studies. The procedure for
sample preparation for SEM studies were followed as mentioned by Sukontason et al. (2003a; 2003b). The head portion was rinsed briefly with normal saline solution several times to remove surface artifacts, then pre-fixed with a 2.5% glutaraldehyde mixture in phosphate buffer solution (PBS) (pH 7.4) at 4°C for 24 hours. The specimens were rinsed with PBS for two times at 10-min intervals, and then post fixed with 1% osmium tetroxide at room temperature for 3-4 days. Then the specimens were rinsed twice with PBS and dehydrated with ethyl alcohol, by sequentially subjecting the specimens to the following increased alcohol concentrations: 30, 50, 70, 80 and 90%. The specimens were kept in each concentration of alcohol for 12 h during each step of the dehydration process. After treatment, they were placed in absolute alcohol for 24 hours and lastly, treated with acetone for another 24 hours. Finally, the specimens were subjected to critical point drying, attached to double-stick tape on aluminium stubs and coated with platinum in a sputter coating apparatus in order to be viewed under a Zeiss EVO 18 Special Edition Scanning Electron Microscope (Zeiss, Germany). 2.3. Data analysis The terminology and nomenclature of antennae used to describe in this study follow Zacharuk (1985). Six adult H. ligurriens specimens of each sex were used in the study. Sensilla on the dorsal surfaces of the antennae of male and female H. ligurriens were identified and measured. Measurements (in μm) obtained from SEM micrographs of at least 15 individual sensilla of the same type were used to calculate the mean and standard error. Morphometric data obtained from the different antennal segments and sensilla of male and female flies were analysed by Student’s t-test using Microsoft Office Excel 2007, to determine any significant differences (P<0.05). 3. Results 3.1. Ultrastructural studies on the antennae of male and female H. ligurriens 3.1.1. Antenna
The aristate antenna of the calliphorid fly H. ligurriens consists of three segments: proximally located scape, distally located flagellum and pedicel located in between scape and flagellum. The arista is located dorso-laterally on the antenna (Fig. 1 b; 4b). Sensilla were observed on all antennal segments, though the most numerous sensilla were found located on the flagellum. The different types of sensilla identified on the antenna of H. ligurriens is morphologically similar to those described in many other fly species (Bisotto-De-Oliveira, 2011; Carrico, 2015; Setzu, 2011; Sukontason, 2004;). Scape The scape is the most proximal and shortest segment of the antenna which attaches it to the head capsule. The scape of H. ligurriens measured approximately 94.02±2.37 µm in length and 61.64±1.51 µm in width in female flies (n=6) and 102.02±2.04 µm in length and 81.4±1.22 µm in width in male flies (n=6). Significant difference was observed in the length of the scape between the two sexes (P < 0.05) as shown in Table 1. The surface of the scape was densely covered with microtrichia or spinules, which are small hair-like structures, lacking alveoli (sockets). Only one type of sensilla, the chaetic sensilla was found on the scape. The chaetic sensilla (ChI) are characterized by long, stout, sharp-tipped sensilla incised with longitudinal grooves. The base of the sensilla are fitted into smooth, round sockets. The chaetic sensilla were found arranged in a single curved row on the scape, interspersed by numerous microtrichiae (Fig.1d, 4e). The chaetic sensilla found on the scape measured approximately 34.49±2.82 µm in length and 5.45±0.35µm in width (basal region) in females and 56.82±8.15 µm in length and 6.77±0.64 µm in width (basal region) in males. Significant difference was observed in the length and width of the chaetic sensilla (ChI) found on the scape between the two sexes as depicted in Table 2. Pedicel
Pedicel, the second segment of the antenna measured approximately 153.51±4.32 µm in length and 129.52±1.30 µm in width in females and 167.11±1.81 µm in length and 151.6±1.73 µm in width in males of H. ligurriens. Table 1 shows significant difference in the length of the pedicel between the two sexes. The outer lateral side of the pedicel had a distinct longitudinal suture and the surface of the pedicel was also found densely covered with microtrichiae. Two types of sensilla were found on the pedicel as given below. 3.1.2. Chaetic sensilla Unlike the scape, two types of chaetic sensilla were observed on the pedicel. Small chaetic sensilla (ChI)- ChI found on the pedicel was structurally similar, though smaller to those found on the scape and was found in good numbers on both the halves of the pedicel (intersected by the longitudinal suture). The chaetic sensilla I (ChI), like those found on the scape are sharptipped, stout sensilla with longitudinal grooves, fitted into flexible sockets (Figs. 1e; 4c, d). ChI found on the pedicel measured approximately 29.60±7.17 µm in length and 5.56±0.72 µm in width (basal region) in females and 47.75±6.47 µm in length and 7.17±0.71 µm in width (basal region) in males. No significant difference was observed in ChI length and width between the two sexes (Table 2). Large chaetic sensilla (ChII)- ChII was the largest and broadest of all sensilla types found on the antenna. ChII was found to be few in number, characterized by usually one single, large, straight sensilla located dorso-centrally on the pedicel, emerging from sturdy, round sockets (Fig.1e; 4c). ChII measured approximately 345.37±2.38 µm in length in females and 324.87±14.62 µm in length in males. No significant difference was observed in ChII length between the two sexes as shown in Table 2. 3.1.3. Styloconic sensilla
Styloconic sensilla characterized by round sensillum with a seta which is bulbous at the base. A tuft of fine hairs was found situated near the setal socket in some sensilla (Fig. 1f; 4f). The styloconic sensilla were found located in groups in the medial region of the pedicel only in both the sexes and varied from 4-7 in number (Fig.1e, f; 4f). Styloconic sensilla measured approximately 12.01±0.12 µm in length and 11.76±0.11µm in width in females and 12.11±0.06 µm in length and 11.74±0.13 µm in width in males. No significant difference was observed in length and width of styloconic sensilla between the two sexes as shown in Table 2. Flagellum The flagellum, the longest and widest part of the antenna is the distal and most prominent segment of the antenna. Flagellum measured approximately 785.89±6.41 µm in length and 162.66±4.59 µm in width in females and 781.13±12.09 µm in length and 114.77±2.79 µm in width in males of H. ligurriens. As depicted in Table 1, significant difference was notably observed in the length of the flagellum between the two sexes. H. ligurriens, being a calliphorid fly, the antenna is of aristate type, that is a long, bristle-like appendage or arista is attached with the flagella and situated dorso-laterally along the flagellum (Fig.5a). The arista measured approximately 822.69±2.62 µm in length and 36.71±1.84 µm in width (basal region) in females and 911.64±4.67 µm in length and 49.61±2.24 µm in width (basal region) of males. Significant difference was observed in the width of the arista between the two sexes as mentioned in Table 1. The arista is plumose, that is covered with long hairs. The entire surface of the flagellum is covered with numerous microtrichiae (Fig.5f). The flagellum contains three different types of sensilla: trichoid sensilla, basiconic sensilla and coeloconic sensilla. 3.1.4. Trichoid sensilla
It is the longest, most conspicuous and abundant type of sensilla located on the flagellum. They are long, curved, hair-like structures with a swollen base and distally tapering ends. The surface are smooth and without any longitudinal grooves, unlike the chaetic sensilla (Fig.2; 5 b, c). The approximate length and width (basal region) of trichoid sensilla was found to be 16.94± 0.25 µm and 2.17±0.10 µm respectively in females and 17.25±0.16 µm and 2.21±0.16 µm respectively in males. As shown in Table 3, no significant difference was observed in the length and width of the flagellum between the two sexes. 3.1.5. Basiconic sensilla These are blunt-ended and digitiform (finger-like) processes with a smooth, multi-porous surface (Fig.2, 3; 5 b, d). Basiconic sensilla were found to be shorter in length and width than trichoid sensilla. These sensilla were found distributed throughout the flagellum in good numbers. The approximate length and width (apical region) of basiconic sensilla was 7.86±0.58 µm and 1.55±0.04 µm respectively in females and 8.01±0.63 µm and 1.62±0.11 µm respectively in males. No sex-biasness in length and width of basiconic sensilla were observed (Table 3). 3.1.6. Coeloconic sensilla These are short, cone-shaped peg-like structures with pointed tips, located centrally in a deep, round sacculus. The surface of coeloconic sensilla is incised with longitudinal grooves (Fig. 2, 3; 5 b, e). Coeloconic sensilla were found distributed throughout the flagellum but not as abundant as basiconic or trichoid sensilla. The approximate length and width (basal region) of coeloconic sensilla was 6.42±0.06 µm and 1.56±0.06 µm respectively in females and 5.93±0.08 µm and 1.23±0.04 µm respectively in males. Significant difference was observed in the length and width of the coeloconic sensilla between the two sexes (Table 3). 4. Discussion
In the present study, the morphology and ultrastructure of the antennae of male and female H. ligurriens are studied for the first time with the help of scanning electron microscopy. SEM micrographs of the antennae of male and female H. ligurriens revealed different sensilla types which are similar in their morphological characters as seen in other dipteran species studied previously (Awad et al., 2014; Bisotto-De-Oliveira et al., 2011; Carrico et al., 2015; Setzu et al., 2011; Sukontason et al., 2004; Sukontason et al., 2005; Sukontason et al., 2007; Wang et al., 2014; Wasserman et al., 2003; Zhang et al., 2016). Out of the three antennal segments i.e. scape, pedicel and flagellum, moderate sexual dimorphism is evident in the fact that the length of the scape and pedicel was larger in males as shown in Table 1. Length of the flagellum is similar in both the sexes. However, flagellum is found to be significantly wider in females, suggesting that it contains multi-porous sensilla like basiconic sensilla more in number, which may allow females to locate oviposition sites (Keil et al., 2001; Mitchell et al., 1999). The aristae in both sexes are similar in length but in males it was found to be wider. Chaetic sensilla was found on both the scape and the pedicel of the antenna. The scape consists of only one type of chaetic sensilla (ChI) whereas, the pedicel shows two different types-ChI and ChII. The ChI sensilla in males are longer and wider than that of females. ChII sensilla on the pedicel is the largest sensilla of all types found on the antenna of the species. The probable function of the chaetic sensilla is either mechanotactile and/or chemosensitive as suggested in previous study (Sukontason et al., 2007). Another sensilla type, styloconic sensilla, is found only on the pedicel in H. ligurriens as reported in previous studies in other Calliphorids like Chrysomya megacephala, C. rufifacies, C. nigripes and Lucilia cuprina (Sukontason et al., 2004). The styloconic sensilla found in H. ligurriens is morphologically quite similar to that seen in P. dux (Sukontason et al., 2004). Studies show that styloconic sensilla are found more in calliphorids and sarcophagids than that of muscids as opined by Greenberg (1970). Regarding the functional aspect of styloconic sensilla, either mechano
and/or chemoreception has been suggested by previous authors (Sukontason et al., 2008; Zhang et al., 2016). Further research is to be carried out on this functional aspect as styloconic sensilla has so far been present in many forensically important calliphorids. The flagellum, the largest segment of the antenna, is covered with numerous microtrichiae, as well as three types of sensilla- trichoid, basiconic and coeloconic sensilla, which resemble those described in other calliphorid species by Sukontason et al. (2004) and in Protophormia terraenovae by Setzu et al. (2011). Trichoid sensilla is the most abundant type of sensilla found in both the sexes of H. ligurriens. No sexual dimorphism was seen in morphology of trichoid sensilla in the two sexes. It has multi-porous wall, indicating that these sensilla can accommodate olfactory receptor neurons (ORNs) which are bipolar receptor cells situated within porous-walled sensilla and helps in olfaction. Trichoid sensilla is suggested to serve a mechano-receptive function in most insects studied (De Freitas Fernandes et al., 2002; Merivee et al., 2002; ). In addition to this, as seen in previous studies, trichoid sensilla is presumed to be used for pheromone detection and well-developed in males, though in our study no such sex-related difference was observed (Sukontason et al., 2007). Basiconic sensilla were also found abundant on the flagellum in both the sexes. No sexual dimorphism was found in morphology of basiconic sensilla. Like the trichoid sensilla, the basiconic sensilla also consists of a multi-porous wall, thus functionally implies that, these too serve in olfaction as suggested in previous studies on Drosophila melanogaster (Shanbhag et al., 1995). Electron Microscopic studies on antenna of Hemipyrellia ligurriens has been done here. The findings are similar in case of other species also, for example, the observations by Keil et al., 2001; Mitchell et al., 1999 are that the wider flagellum in female suggesting it contains multi-porous sensilla like basiconic sensilla more in number and this helps females to locate oviposition sites.
Coeloconic sensilla was found to be larger in females in H. ligurriens, which is similar to that observed in Protophormia terraenovae (Setzu et al., 2011). Regarding the function of coeloconic sensilla, the probability of these sensilla types being involved with thermo or hygroreception is presumed (McIver, 1969; Zacharuk, 1985). Thus, as seen in our study, the larger size of the coeloconic sensilla in females, can possibly add to the fact that coeloconic sensilla is important in females for oviposition site detection. Antennae being the main olfactory organs, helps allow insects, like flies to evolve particular traits required to improve their fitness in environment (Ross, 1992; Sukontason et al., 2004; Zhang et al., 2012a; Zhang et al., 2012b). Taking this into consideration, phylogenetic relationships can be reconstructed in future based on antennal sensilla which serves as morphological characters for taxonomy and phylogeny. In context to forensically important dipteran families like Calliphoridae, Sarcophagidae and Muscidae, no such studies have been done, though ultrastructural studies on antenna have been carried out in a number of species. Ultrastructural studies on antenna of forensically important Calliphorids like Chrysomya megacephala, Chrysomya rufifacies, Chrysomya nigripes, Lucilia cuprina, Triceratopyga calliphoroides done previously (Sukontason et al., 2004; Zhang et al., 2014) shows both similarity and dissimilarity with respect to the sensilla types observed. Studies show that Sarcophagids (Sarcophaga dux) exhibit a high number of sensory pits as compared to Calliphorids and Muscids (Sukontason et al., 2004). Similar type of characterization studies of olfactory sensilla in S. calcitrans revealed an association of electrophysiological responses to odorant with host and oviposition media (Tangtrakulwanich et al., 2011). Flagellar sensilla observed in H.ligurriens in our study show similarity to that found in previous studies on C.megacephala, C.rufifacies, C.nigripes, L.cuprina (Sukontason et al.
2004). Coeloconic sensilla of Hemipyrellia ligurriens observed in our study shows structural similarity to that found in Lucilia cuprina, Triceratopyga calliphoroides and Protophormia terraenovae (Setzu et al. 2011; Sukontason et al. 2004; Zhang et al. 2014). Thus, phylogenetic studies based on antennal sensilla of forensically important dipteran families remains to be a crucial area of research and the data from the present study could aid in such research. 5. Conclusion The identification and characterization of the morphology and distribution of the five sensilla types found in Hemipyrellia ligurriens by using SEM is performed, so as to generate a baseline data of antennal ultrastructure of the species. Moreover, the probable function of each sensilla type is discussed with respect to the observations of investigations previously done on other species. Thus it can be concluded that the larger coeloconic sensilla in females of H. ligurriens in comparison to males play a pivotal role in locating successful sites for colonization and oviposition in corpses. The results have considerable significance in instigating further study on H. ligurriens not only in forensic entomology and entomo-toxicology but also in pest control as study of the sensilla chemoreceptors would allow use of specific insecticides that block the function of these sensilla, thus proving to be crucial from medical and veterinary perspective. The present study will provide the basis for future studies on antennal electrophysiology which would pave the path to better understanding of the functional significance of the sensilla types in the species. Another prospect of the study lies in the fact that the morphological features of the antenna examined can act as potential tools for reconstructing phylogenetic relationships among other forensic dipteran species of families Calliphoridae, Sarcophagidae and Muscidae. Competing interest Authors declare no conflict of interest.
Author contributions Conceived and designed the experiments: DB. Sample Collection and Identification: GH. Performed the experiment: GH, AM, AN. Analyzed the data: GH, AM, AN. Wrote the paper: GH, AM, AN. Critically revised the article: WA, SG, DB, GKS. Acknowledgements Present work is financially supported by Zoological Survey of India core funding. Facilities and support for the present study were provided by Dr. Kailash Chandra, Director, Zoological Survey of India, and Dr. K.C. Gopi, Additional Director and Divisional-in-charge, Entomology Division ‘A’ and ‘B’ are kindly acknowledged. We convey our sincere regards to Mr. Pachanan Parui, retired scientist, ZSI, for helping in taxonomical identification of the specimens. The authors express their gratitude to Dr. Vikas Kumar, Scientist D, Centre for DNA Taxonomy, Molecular Systematics Division, ZSI and Dr. Sagartirtha Sarkar, Associate Professor, Department of Zoology, University of Calcutta for providing the chemicals and consumables required for the SEM sample preparation. The authors are thankful to the operators of SEM, CRNN, University of Calcutta for their technical assistance in sputter-coating during Scanning Electron Microscopic study.
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22. Sinha, S.K., Nandi, B.C., 2007. Studies on life history of Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) in Sundarbans Biosphere Reserve, West Bengal, India. Rec. zool. Surv. India, 107(1), 63-70. 23. Sinha, S. K., 2012. Myiasis in domestic animals: new records of calyptrate Diptera. J. Parasit. Dis. 36(2), 277-279. 24. Sukontason, K., Sukontason, K.L., Piangjai, S., Boonchu, N., Chaiwong, T., Vogtsberger, R.C., 2003a. Mouthparts of Megaselia scalaris (Loew) (Diptera: Phoridae). Micron, 34, 345-350. 25. Sukontason, K., Sukontason, K.L., Piangjai, S., Chaiwong, T., Boonchu,N., Kurahashi, H., Vogtsberger, R.C., 2003b. Larval ultrastructure of Parasarcophaga dux (Thomson) (Diptera: Sarcophagidae). Micron 34, 359-364. 26. Sukontason, K., Sukontason, K.L., Piangjai, S., Boonchu, N., Chaiwong, T., Ngern-klun, R., Sripakdee, D., Vogtsberger, R.C., Olson, J.K., 2004. Antennal sensilla of some forensically important flies in families Calliphoridae, Sarcophagidae and Muscidae. Micron, 35, 671-679. 27. Sukontason, K., Sukontason, K.L., Vogtsberger, R.C., Boonchu, N., Chaiwong, T., Piangjai, S., Disney, H., 2005. Ultrastructure of coeloconic sensilla on postpedicel and maxillary palp of Megaselia scalaris (Diptera: Phoridae). Ann. Entomol. Soc. Am. 98, 113118. 28. Sukontason, K., Narongchai, P., Kanchai, C., Vichairat, K., Sribanditmongkol, P., Bhoopat, T., Kurahashi, H., Chockjamsai, M., Piangjai, S., Bunchu, N., Vongvivach, S., Samai, W., Chaiwong, T., Methanitikorn, R., Ngern-klun, R., Sripakdee, D., Boonsriwong, W., Siriwattanarungsee, S., Srimuangwong, C., Hanterdsith, B., Chaiwan, K., Srisuwan, C., Upakut, S., Moopayak, K., Vogtsberger, R.C., Olson, J.K., Sukontason, K.L., 2007a.
Forensic entomology cases in Thailand: a review of cases from 2000 to 2006. Parasitol Res 101:1417–1423. 29. Sukontason, K.L., Ngern-klun, R., Sripakdee, D., Sukontason, K., 2007b. Identifying fly puparia by clearing technique: application to forensic entomology. Parasitol. Res. 101, 1407–1416. 30. Sukontason, K., Methanitikorn, R., Chaiwong, T., Kurahashi, H., Vogtsberger, R.C., Sukontason, K.L., 2007c. Sensilla of the antenna and palp of Hydrotaea chalcogaster (Diptera: Muscidae). Micron, 38, 218-223., 31. Sukontason, K.L., Sribanditmongkol, P., Chaiwong, T., Vogtsberger, R.C., Piangjai, S., Sukontason, K., 2008. Morphology of immature stages of Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) for use in forensic entomology applications. Parasitol. Res., 103(4), 877-887. 32. Tangtrakulwanich, K., Chen, H., Baxendale, F., Brewer, G., Zhu, J.J., 2011. Characterization of olfactory sensilla of Stomoxys calcitrans and elctrophysiological responses to odorants compounds associated with hosts and oviposition media. Med. Vet. Entomol., 25, 327-336. 33. Tumrasvin, W., Kurahashi, H., Kano, R., 1979. Studies on medically important flies in Thailand. VII. Report on 42 species of calliphorid flies, including the taxonomic keys (Diptera: Calliphoridae). Bull. Tokyo Med. Dent. Univ., 26(4), 243–272. 34. Wang, Q. K., Liu, X. H., Lu, P. F., Zhang, D., 2014. Ultrastructure of antennal sensilla in Hydrotaea armipes (Fallén) (Diptera: Muscidae): New evidence for taxonomy of the genus Hydrotaea. Zootaxa, 3790(4), 577-586. 35. Wasserman, S.L., Itagaki, H., 2003. The olfactory responses of the antenna and maxillary palp of the fleshfly, Neobellieria bullata (Diptera: Sarcophagidae), and their sensitivity to blockage of nitric oxide synthase. J. Insect Physiol. 49(3), 271-280.33
36. Zacharuk, R.Y., 1985. Antennal sensilla. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comparative Insect Physiology, Biochemistry and Pharmacology. Volume 6. Pergamon Press, Oxford, U.K. pp. 1-69. 37. Zhang, D., Wang, Q.K., Hu, D.F., Li, K., 2012a. Sensilla on the antennal funiculus of the horse stomach bot fly, Gasterophilus nigricornis. Med. Vet. Entomol., 26, 314–322. 38. Zhang, D., Wang, Q.K., Hu, D.F., Li, K., 2012b. Cuticular structures on antennae of the bot fly, Portschinskia magnifica (Diptera: Oestridae). Parasitol. Res., 111, 1651–1659. 39. Zhang, D., Liu, X.H., Wang, Q.K., Li, K., 2014. Sensilla on the antenna of blow fly, Triceratopyga calliphoroides Rohdendorf (Diptera: Calliphoridae). Parasitol. Res., 113, 2577–2586.
40. Zhang, Z., Li, X., Chen, L., Wang, L., Lei, C., 2016. Morphology, distribution and abundance of antennal sensilla of the oyster mushroom fly, Coboldia fuscipes (Meigen) (Diptera: Scatopsidae). Rev. Bras. entomol., 60(1), 8-14.
Figure legends Figure 1: SEM micrographs of features on antenna of female Hemipyrellia ligurriens. a. Frontal view of head showing compound eyes, antennae and mouthparts. b. Antenna in magnified view comprised of the three antennal segments: scape (I), pedicel (II) and flagella (IV) bearing a dorso-laterally located arista (III). c. Magnified view of the scape, pedicel and anterior part of the flagella. d. Scape in magnified view showing chaetic sensilla (ChI). e. Pedicel in magnified view showing two types of chaetic sensilla-small chaetic sensilla (ChI) and large chaetic sensilla (ChII) and styloconic sensilla (St). f. Four styloconic sensilla on the pedicel in magnified view (arrow); a tuft of fine hairs located above the seta is shown by white arrow.
Figure 2: SEM micrograph of features of antennal flagella of female Hemipyrellia ligurriens. Four types of structures are observed on the cuticular surface of flagella- covered with numerous thin microtrichia or spinules (Mc); trichoid sensilla (Tr) characterized by somewhat curved setae with thicker bases; basiconic sensilla (black arrows) are shorter, with blunt ends and coeloconic sensilla (Cs) which bears a cone-shaped projection centrally located within a sacculus. Bar = 2µm. Figure 3: Basiconic and coeloconic sensilla of female Hemipyrellia ligurriens at higher magnification. Basiconic sensillum (Bs) bears a multi-porous surface; coeloconic sensillum (Cs) has longitudinal grooves along its surface. Bar = 200 nm. Figure 4: SEM micrographs of features on antenna of male Hemipyrellia ligurriens. a. Frontal view of head showing compound eyes, antennae and mouthparts. b. Antenna in magnified view comprised of the three antennal segments: scape (I), pedicel (II) and flagella (III) bearing a dorso-laterally located arista (IV). c. Magnified view of the scape and pedicel showing two types of chaetic sensilla- chaetic sensilla (ChI) and large chaetic sensilla (ChII).d. Two chaetic sensilla (ChI) located on the pedicel in magnified view-note that they emerge from flexible sockets and have a longitudinally grooved cuticular surface. e. Scape in magnified view showing chaetic sensilla (ChI) and microtrichia (Mc) or spinules. f. Pedicel in magnified view showing seven styloconic sensilla (St) and microtrichia (Mc); a tuft of fine hairs located above the styloconic sensilla is shown by arrow. Figure 5: SEM micrographs of antenna of male Hemipyrellia ligurriens. a. Arista (a) of flagella, (b) Bar = 20µm. b. Sensilla types observed on antennal flagella- Four types of structures are observed on the surface of flagella- covered with numerous thin microtrichia or spinules (Mc); trichoid sensilla (Tr) characterized by somewhat curved setae with thicker bases; basiconic sensilla, which are shorter than the trichoid sensilla are shown by white arrows and coeloconic sensilla (Cs) which bears a cone-shaped projection centrally located within a
sacculus. Bar = 1µm. c. Trichoid sensillum magnified (shown by white arrow). d. Basiconic sensillum (shown by white arrow) - its multi-porous surface can be seen. e. A coeloconic sensillum among trichoid sensilla and microtrichia is magnified (shown by white arrow) – showing the longitudinal grooves along the surface of the sensilla. f. Microtrichiae- slender, curved processes shown by black arrows.
\
Table 1: Mean ± SE of the length and width (µm), at the larger diameter, in the antennae segments of females and males of Hemipyrellia ligurriens. Means followed by the same lowercase letter on the lines are not statistically significantly different (Student’s t-test, P < 0.05, n = 6). Sensilla Sex of flies
Length (µm) Females
Width (µm) Males
Females
Males
Scape
94.02 ± 2.37 a
102.02 ± 2.04 b
61.64 ± 1.51 a
81.4 ± 1.22 a
Pedicel
153.51 ± 4.32 a
167.11 ± 1.81 b
129.52 ± 1.30 a
151.6 ± 1.73 a
Flagellum
785.89 ± 6.41 a
781.13 ± 12.09 a 162.66 ± 4.59 a
114.77 ± 2.79 b
Arista
822.69 ± 2.62 a
911.64 ± 4.67 a
49.61 ± 2.24 b
36.71 ± 1.84 a
Table 2: Mean ± SE of the length and width (µm) of sensilla on the antennal scape and pedicel of females and males of Hemipyrellia ligurriens. Means followed by the same lower-case letter on the lines are not statistically significantly different (Student’s t-test, P< 0.05, n=6). - = undetermined.
Scape Sensilla
Pedicel Length (µm)
Sex of Females flies 34.49 ± Chaetic 2.82 a sensilla (ChI) Chaetic sensilla (ChII) Styloconic sensilla -
Width (µm)
Length (µm)
Width (µm)
Males
Females
Males
Females
Males
Females
Males
56.82 ± 8.15 b
5.45 ± 0.35 a
6.77 ± 0.64 b
29.60 ± 7.17 a
47.75 ± 6.47 a
5.56 ± 0.72 a
7.17 ± 0.71 a
345.37 ± 2.38 a
324.87 ± 14.62 a
12.01 ± 0.12 a
12.11 ± 0.06 a
-
-
-
-
-
-
11.76 ± 0.11 a
11.74 ± 0.13 a
Table 3: Mean ± S of the length and width (µm) of sensilla on the antennal flagellum of females and males of Hemipyrellia ligurriens. Means followed by the same lower-case letter on the lines are not statistically significantly different (Student’s t-test, P< 0.05, n=6).
Sensilla
Length (µm)
Width (µm)
Sex of flies
Female
Male
Female
Male
Trichoid
16.94 ± 0.25 a
17.25 ± 0.16 a
2.17 ± 0.10 a
2.21 ± 0.16 a
Basiconic
7.86 ± 0.58 a
8.01 ± 0.63 a
1.55 ± 0.04 a
1.62 ± 0.11 a
Coeloconic
6.42 ± 0.06 a
5.93 ± 0.08 b
1.56 ± 0.06 a
1.23 ± 0.04 b
S0001-706X(17)30104-3 http://dx.doi.org/doi:10.1016/j.actatropica.2017.04.005 ACTROP 4268
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
7-2-2017 11-4-2017 14-4-2017
Please cite this article as: Hore, Garima, Maity, Aniruddha, Naskar, Atanu, Ansar, Waliza, Ghosh, Shyamasree, Saha, Goutam Kumar, Banerjee, Dhriti, Scanning electron microscopic studies on antenna of Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae) − a blow fly species of forensic importance.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Scanning Electron Microscopic studies on antenna of Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae) – a blow fly species of forensic importance
Garima Horea, Aniruddha Maitya*, Atanu Naskara, Waliza Ansarb, Shyamasree Ghoshc, Goutam Kumar Sahad, Dhriti Banerjeea
a
Diptera Section, Zoological Survey of India, Ministry of Environment, Forest & Climate Change (Government of India), M-Block, New Alipore, Kolkata- 700 053. b
Department of Zoology, Behala College, Parnashree, Behala, Kolkata-700 060. School of Biological Sciences, National Institute of Science, Education and Research (NISER), Bhubaneswar- 751005, India. c
d
Department of Zoology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata- 700 019.
a*
Corresponding author: Aniruddha Maity, e-mail ID: [email protected]
Highlights Scanning electron microscopic studies on antenna of male and female Hemipyrellia ligurriens is carried out for the first time so as to generate a baseline data applicable in various fields of studies such as vector biology and forensic entomology. Five different types of sensilla and numerous microtrichiae were observed on the antennal surface. Chaetic sensilla were found on the scape and pedicel; styloconic sensilla found only on the pedicel whereas trichoid, basiconic and coeloconic sensilla were observed only on the flagellum. The fact that coeloconic sensilla, a type of multiporous sensilla was observed to be larger in females, can possibly add to the assumption that they serve the purpose of oviposition site detection in females.
Graphical abstract
Comparative analysis of size of flagellum between male and female of H. ligurriens 114.77
Flagellum width (µm)
162.66
781.13
Flagellum length (µm)
785.89
0
100
200
300 Male
400
500
600
700
800
900
Female
Comparative analysis of size of Coeloconic sensilla between male and female of H. ligurriens 1.23 Coeloconic sensilla width (µm) 1.56
5.93 Coeloconic sensilla length (µm) 6.42
0
1
2
Male
Female
3
4
5
6
7
Females had wider flagella than the males, females showed bigger coeloconic sensilla in length and width.
Play a pivotal role in locating successful sites for colonization and oviposition in corpses.
Abstract Blow flies (Diptera: Calliphoridae) are one of the foremost organisms amongst forensic insects to colonize corpses shortly after death, thus are of immense importance in the domain of forensic entomology. The blow fly Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae) is considered as a forensically important fly species globally and is also known for its medical and veterinary importance. In the present study, we report for the first time scanning electron microscopic studies on the morphology of sensilla of antenna of adult male and female of H. ligurriens is with profound importance in better understanding of the insect morphology from forensic entomological perspective, and also could aid in proper identification of the species from other calliphorid flies. The structural peculiarities observed in the (i) antenna of H. ligurriens with three segments- scape, pedicel and flagellum with dorsolaterally placed arista (ii) densely covered microtrichia and most abundant trichoid sensilla identified on the antenna (iii) observation of only one type of sensilla, chaetic sensilla (ChI) on the scape (iv) two types of chaetic sensilla (ChI and ChII) and styloconic sensilla on the pedicel (v) the flagellum with three types of sensilla- trichoid, basiconic and coeloconic sensilla (vi) Basiconic sensilla with multiporous surfaces with characteristic olfactory function. Moderate sexual dimorphism in the width of the flagellum, the females with wider flagella than the males, bear significance to the fact that they bear more multi-porous sensilla than the males, thus suffice their need to detect oviposition sites. Significant difference was observed in the length and width of coeloconic sensilla between the two sexes, the females showed bigger coeloconic sensilla, suggesting their function in oviposition site detection and successful colonization in corpses.
Keywords: Antenna; sensilla; Hemipyrellia ligurriens; scanning electron microscopy.
1. Introduction Hemipyrellia ligurriens (Wiedemann,1830), an important blow fly species with great medical and veterinary importance shows a fairly wide distribution comprising most of the Oriental region including India, Sri Lanka, China, Thailand, Malaysia, Indonesia, Java, Sumatra, Philippines and also Australia and New Guinea (Kurahashi et al., 1997; Kurahashi and
Chowanadisai, 2001; Senior-White et al., 1940; Tumrasvin et al., 1979). In the recent past, the species has been considered as a forensically important fly as their larvae have been collected from human corpses (Chen et al., 2004; Lee et al., 2004; Sukontason et al., 2007a). Moreover, the species can be a nuisance in market places and gardens, dry fish processing centers. The adults are the potential vectors of various enteric pathogens due to their attraction to human excreta near human-occupied environments (Sinha and Nandi, 2007). Larvae of H. ligurriens have also been recorded as parasites of living animals like earthworm, fish and toad in experimental conditions (Roy and Dasgupta, 1971). H. ligurriens also causes myiasis in domestic animals like goat and buffalo (Sinha, 2012). Olfaction, a sensory system which allows insects to detect chemicals in the environment is vital for their behaviour and ecology, as it helps them to locate food sources, mates and oviposition sites. The main olfactory organs of flies, like other insects are the paired antenna, which consists of numerous, morphologically distinct, microscopic sensilla each serving specific functions (Greenberg, 1970; Shanbhag et al., 1999). Thus in the case of forensically important insects, these sensillary structures present on the antenna are important in perception of chemical cues like ammonia and sulphur-rich volatiles, broadly classified as volatile organic compounds (VOCs) produced as a result of corpse decomposition and also used for Post mortem interval (PMI) estimation, thus having immense value in forensic investigations (Ishijima, 1967). In spite of its importance in medical, veterinary and forensic entomology, the morphology and fine structure of this species has received little attention in comparison to that of other blow fly genus like Chrysomya. In the initial phase, Ishijima (1967) provided some information on the adult and illustrated the third instar of H. ligurriens; whereas, some characters of the puparium have been recorded from light microscopy observations by Sukontason et al. (2007b). Life cycle studies of H. ligurriens in Sundarban Biosphere Reserve, West Bengal, India has been
done by Sinha and Nandi (2007). Light microscopic and scanning electron microscope studies on larval morphology has been done by Bunchu et al. (2012); Sukontason et al. (2008). However, no such efforts have been made to study the morphology of the adult H. ligurriens in detail, the olfactory organs like the antenna in particular. Moreover, forensic entomology requires accurate identification of insect specimens which can be provided by detailed morphological study of the species. So, the present study encompassing the ultrastructural characterization of the fly antenna with the help of Scanning Electron Microscopy has been carried out to fill in many of these lacunae.
2. Materials and methods 2.1. Collection and preservation of the fly specimens H. ligurriens male and female flies were collected from a dead Rohu fish (Labeo rohita), an Indian major carp, weighing about 1.5kg which was kept in natural environmental conditions in the garden area of the Zoological Survey of India (ZSI), Kolkata premises for observing and monitoring the successive colonisation, growth and development of flies on the dead organism. The study was conducted during the month of September, 2015 at ZSI New Alipore, Kolkata, West Bengal, India. A modified version of the Malaise trap was used for collection of adult specimens. Adult flies were killed using a killing-jar containing liquid benzene and they were preserved dry in insect-envelope. The specimens were sorted out by basic body plan assessment and relaxed in a relaxing-chamber for about 24 hours. The specimens were pinned and kept in a drying chamber for at least 4-5 days before being preserved in insect cabinet. 2.2. Processing of specimens for Scanning Electron Microscopic studies Initially, the male and female flies were sorted out under the microscope and then they were decapitated and only the head portion was processed for SEM studies. The procedure for
sample preparation for SEM studies were followed as mentioned by Sukontason et al. (2003a; 2003b). The head portion was rinsed briefly with normal saline solution several times to remove surface artifacts, then pre-fixed with a 2.5% glutaraldehyde mixture in phosphate buffer solution (PBS) (pH 7.4) at 4°C for 24 hours. The specimens were rinsed with PBS for two times at 10-min intervals, and then post fixed with 1% osmium tetroxide at room temperature for 3-4 days. Then the specimens were rinsed twice with PBS and dehydrated with ethyl alcohol, by sequentially subjecting the specimens to the following increased alcohol concentrations: 30, 50, 70, 80 and 90%. The specimens were kept in each concentration of alcohol for 12 h during each step of the dehydration process. After treatment, they were placed in absolute alcohol for 24 hours and lastly, treated with acetone for another 24 hours. Finally, the specimens were subjected to critical point drying, attached to double-stick tape on aluminium stubs and coated with platinum in a sputter coating apparatus in order to be viewed under a Zeiss EVO 18 Special Edition Scanning Electron Microscope (Zeiss, Germany). 2.3. Data analysis The terminology and nomenclature of antennae used to describe in this study follow Zacharuk (1985). Six adult H. ligurriens specimens of each sex were used in the study. Sensilla on the dorsal surfaces of the antennae of male and female H. ligurriens were identified and measured. Measurements (in μm) obtained from SEM micrographs of at least 15 individual sensilla of the same type were used to calculate the mean and standard error. Morphometric data obtained from the different antennal segments and sensilla of male and female flies were analysed by Student’s t-test using Microsoft Office Excel 2007, to determine any significant differences (P<0.05). 3. Results 3.1. Ultrastructural studies on the antennae of male and female H. ligurriens 3.1.1. Antenna
The aristate antenna of the calliphorid fly H. ligurriens consists of three segments: proximally located scape, distally located flagellum and pedicel located in between scape and flagellum. The arista is located dorso-laterally on the antenna (Fig. 1 b; 4b). Sensilla were observed on all antennal segments, though the most numerous sensilla were found located on the flagellum. The different types of sensilla identified on the antenna of H. ligurriens is morphologically similar to those described in many other fly species (Bisotto-De-Oliveira, 2011; Carrico, 2015; Setzu, 2011; Sukontason, 2004;). Scape The scape is the most proximal and shortest segment of the antenna which attaches it to the head capsule. The scape of H. ligurriens measured approximately 94.02±2.37 µm in length and 61.64±1.51 µm in width in female flies (n=6) and 102.02±2.04 µm in length and 81.4±1.22 µm in width in male flies (n=6). Significant difference was observed in the length of the scape between the two sexes (P < 0.05) as shown in Table 1. The surface of the scape was densely covered with microtrichia or spinules, which are small hair-like structures, lacking alveoli (sockets). Only one type of sensilla, the chaetic sensilla was found on the scape. The chaetic sensilla (ChI) are characterized by long, stout, sharp-tipped sensilla incised with longitudinal grooves. The base of the sensilla are fitted into smooth, round sockets. The chaetic sensilla were found arranged in a single curved row on the scape, interspersed by numerous microtrichiae (Fig.1d, 4e). The chaetic sensilla found on the scape measured approximately 34.49±2.82 µm in length and 5.45±0.35µm in width (basal region) in females and 56.82±8.15 µm in length and 6.77±0.64 µm in width (basal region) in males. Significant difference was observed in the length and width of the chaetic sensilla (ChI) found on the scape between the two sexes as depicted in Table 2. Pedicel
Pedicel, the second segment of the antenna measured approximately 153.51±4.32 µm in length and 129.52±1.30 µm in width in females and 167.11±1.81 µm in length and 151.6±1.73 µm in width in males of H. ligurriens. Table 1 shows significant difference in the length of the pedicel between the two sexes. The outer lateral side of the pedicel had a distinct longitudinal suture and the surface of the pedicel was also found densely covered with microtrichiae. Two types of sensilla were found on the pedicel as given below. 3.1.2. Chaetic sensilla Unlike the scape, two types of chaetic sensilla were observed on the pedicel. Small chaetic sensilla (ChI)- ChI found on the pedicel was structurally similar, though smaller to those found on the scape and was found in good numbers on both the halves of the pedicel (intersected by the longitudinal suture). The chaetic sensilla I (ChI), like those found on the scape are sharptipped, stout sensilla with longitudinal grooves, fitted into flexible sockets (Figs. 1e; 4c, d). ChI found on the pedicel measured approximately 29.60±7.17 µm in length and 5.56±0.72 µm in width (basal region) in females and 47.75±6.47 µm in length and 7.17±0.71 µm in width (basal region) in males. No significant difference was observed in ChI length and width between the two sexes (Table 2). Large chaetic sensilla (ChII)- ChII was the largest and broadest of all sensilla types found on the antenna. ChII was found to be few in number, characterized by usually one single, large, straight sensilla located dorso-centrally on the pedicel, emerging from sturdy, round sockets (Fig.1e; 4c). ChII measured approximately 345.37±2.38 µm in length in females and 324.87±14.62 µm in length in males. No significant difference was observed in ChII length between the two sexes as shown in Table 2. 3.1.3. Styloconic sensilla
Styloconic sensilla characterized by round sensillum with a seta which is bulbous at the base. A tuft of fine hairs was found situated near the setal socket in some sensilla (Fig. 1f; 4f). The styloconic sensilla were found located in groups in the medial region of the pedicel only in both the sexes and varied from 4-7 in number (Fig.1e, f; 4f). Styloconic sensilla measured approximately 12.01±0.12 µm in length and 11.76±0.11µm in width in females and 12.11±0.06 µm in length and 11.74±0.13 µm in width in males. No significant difference was observed in length and width of styloconic sensilla between the two sexes as shown in Table 2. Flagellum The flagellum, the longest and widest part of the antenna is the distal and most prominent segment of the antenna. Flagellum measured approximately 785.89±6.41 µm in length and 162.66±4.59 µm in width in females and 781.13±12.09 µm in length and 114.77±2.79 µm in width in males of H. ligurriens. As depicted in Table 1, significant difference was notably observed in the length of the flagellum between the two sexes. H. ligurriens, being a calliphorid fly, the antenna is of aristate type, that is a long, bristle-like appendage or arista is attached with the flagella and situated dorso-laterally along the flagellum (Fig.5a). The arista measured approximately 822.69±2.62 µm in length and 36.71±1.84 µm in width (basal region) in females and 911.64±4.67 µm in length and 49.61±2.24 µm in width (basal region) of males. Significant difference was observed in the width of the arista between the two sexes as mentioned in Table 1. The arista is plumose, that is covered with long hairs. The entire surface of the flagellum is covered with numerous microtrichiae (Fig.5f). The flagellum contains three different types of sensilla: trichoid sensilla, basiconic sensilla and coeloconic sensilla. 3.1.4. Trichoid sensilla
It is the longest, most conspicuous and abundant type of sensilla located on the flagellum. They are long, curved, hair-like structures with a swollen base and distally tapering ends. The surface are smooth and without any longitudinal grooves, unlike the chaetic sensilla (Fig.2; 5 b, c). The approximate length and width (basal region) of trichoid sensilla was found to be 16.94± 0.25 µm and 2.17±0.10 µm respectively in females and 17.25±0.16 µm and 2.21±0.16 µm respectively in males. As shown in Table 3, no significant difference was observed in the length and width of the flagellum between the two sexes. 3.1.5. Basiconic sensilla These are blunt-ended and digitiform (finger-like) processes with a smooth, multi-porous surface (Fig.2, 3; 5 b, d). Basiconic sensilla were found to be shorter in length and width than trichoid sensilla. These sensilla were found distributed throughout the flagellum in good numbers. The approximate length and width (apical region) of basiconic sensilla was 7.86±0.58 µm and 1.55±0.04 µm respectively in females and 8.01±0.63 µm and 1.62±0.11 µm respectively in males. No sex-biasness in length and width of basiconic sensilla were observed (Table 3). 3.1.6. Coeloconic sensilla These are short, cone-shaped peg-like structures with pointed tips, located centrally in a deep, round sacculus. The surface of coeloconic sensilla is incised with longitudinal grooves (Fig. 2, 3; 5 b, e). Coeloconic sensilla were found distributed throughout the flagellum but not as abundant as basiconic or trichoid sensilla. The approximate length and width (basal region) of coeloconic sensilla was 6.42±0.06 µm and 1.56±0.06 µm respectively in females and 5.93±0.08 µm and 1.23±0.04 µm respectively in males. Significant difference was observed in the length and width of the coeloconic sensilla between the two sexes (Table 3). 4. Discussion
In the present study, the morphology and ultrastructure of the antennae of male and female H. ligurriens are studied for the first time with the help of scanning electron microscopy. SEM micrographs of the antennae of male and female H. ligurriens revealed different sensilla types which are similar in their morphological characters as seen in other dipteran species studied previously (Awad et al., 2014; Bisotto-De-Oliveira et al., 2011; Carrico et al., 2015; Setzu et al., 2011; Sukontason et al., 2004; Sukontason et al., 2005; Sukontason et al., 2007; Wang et al., 2014; Wasserman et al., 2003; Zhang et al., 2016). Out of the three antennal segments i.e. scape, pedicel and flagellum, moderate sexual dimorphism is evident in the fact that the length of the scape and pedicel was larger in males as shown in Table 1. Length of the flagellum is similar in both the sexes. However, flagellum is found to be significantly wider in females, suggesting that it contains multi-porous sensilla like basiconic sensilla more in number, which may allow females to locate oviposition sites (Keil et al., 2001; Mitchell et al., 1999). The aristae in both sexes are similar in length but in males it was found to be wider. Chaetic sensilla was found on both the scape and the pedicel of the antenna. The scape consists of only one type of chaetic sensilla (ChI) whereas, the pedicel shows two different types-ChI and ChII. The ChI sensilla in males are longer and wider than that of females. ChII sensilla on the pedicel is the largest sensilla of all types found on the antenna of the species. The probable function of the chaetic sensilla is either mechanotactile and/or chemosensitive as suggested in previous study (Sukontason et al., 2007). Another sensilla type, styloconic sensilla, is found only on the pedicel in H. ligurriens as reported in previous studies in other Calliphorids like Chrysomya megacephala, C. rufifacies, C. nigripes and Lucilia cuprina (Sukontason et al., 2004). The styloconic sensilla found in H. ligurriens is morphologically quite similar to that seen in P. dux (Sukontason et al., 2004). Studies show that styloconic sensilla are found more in calliphorids and sarcophagids than that of muscids as opined by Greenberg (1970). Regarding the functional aspect of styloconic sensilla, either mechano
and/or chemoreception has been suggested by previous authors (Sukontason et al., 2008; Zhang et al., 2016). Further research is to be carried out on this functional aspect as styloconic sensilla has so far been present in many forensically important calliphorids. The flagellum, the largest segment of the antenna, is covered with numerous microtrichiae, as well as three types of sensilla- trichoid, basiconic and coeloconic sensilla, which resemble those described in other calliphorid species by Sukontason et al. (2004) and in Protophormia terraenovae by Setzu et al. (2011). Trichoid sensilla is the most abundant type of sensilla found in both the sexes of H. ligurriens. No sexual dimorphism was seen in morphology of trichoid sensilla in the two sexes. It has multi-porous wall, indicating that these sensilla can accommodate olfactory receptor neurons (ORNs) which are bipolar receptor cells situated within porous-walled sensilla and helps in olfaction. Trichoid sensilla is suggested to serve a mechano-receptive function in most insects studied (De Freitas Fernandes et al., 2002; Merivee et al., 2002; ). In addition to this, as seen in previous studies, trichoid sensilla is presumed to be used for pheromone detection and well-developed in males, though in our study no such sex-related difference was observed (Sukontason et al., 2007). Basiconic sensilla were also found abundant on the flagellum in both the sexes. No sexual dimorphism was found in morphology of basiconic sensilla. Like the trichoid sensilla, the basiconic sensilla also consists of a multi-porous wall, thus functionally implies that, these too serve in olfaction as suggested in previous studies on Drosophila melanogaster (Shanbhag et al., 1995). Electron Microscopic studies on antenna of Hemipyrellia ligurriens has been done here. The findings are similar in case of other species also, for example, the observations by Keil et al., 2001; Mitchell et al., 1999 are that the wider flagellum in female suggesting it contains multi-porous sensilla like basiconic sensilla more in number and this helps females to locate oviposition sites.
Coeloconic sensilla was found to be larger in females in H. ligurriens, which is similar to that observed in Protophormia terraenovae (Setzu et al., 2011). Regarding the function of coeloconic sensilla, the probability of these sensilla types being involved with thermo or hygroreception is presumed (McIver, 1969; Zacharuk, 1985). Thus, as seen in our study, the larger size of the coeloconic sensilla in females, can possibly add to the fact that coeloconic sensilla is important in females for oviposition site detection. Antennae being the main olfactory organs, helps allow insects, like flies to evolve particular traits required to improve their fitness in environment (Ross, 1992; Sukontason et al., 2004; Zhang et al., 2012a; Zhang et al., 2012b). Taking this into consideration, phylogenetic relationships can be reconstructed in future based on antennal sensilla which serves as morphological characters for taxonomy and phylogeny. In context to forensically important dipteran families like Calliphoridae, Sarcophagidae and Muscidae, no such studies have been done, though ultrastructural studies on antenna have been carried out in a number of species. Ultrastructural studies on antenna of forensically important Calliphorids like Chrysomya megacephala, Chrysomya rufifacies, Chrysomya nigripes, Lucilia cuprina, Triceratopyga calliphoroides done previously (Sukontason et al., 2004; Zhang et al., 2014) shows both similarity and dissimilarity with respect to the sensilla types observed. Studies show that Sarcophagids (Sarcophaga dux) exhibit a high number of sensory pits as compared to Calliphorids and Muscids (Sukontason et al., 2004). Similar type of characterization studies of olfactory sensilla in S. calcitrans revealed an association of electrophysiological responses to odorant with host and oviposition media (Tangtrakulwanich et al., 2011). Flagellar sensilla observed in H.ligurriens in our study show similarity to that found in previous studies on C.megacephala, C.rufifacies, C.nigripes, L.cuprina (Sukontason et al.
2004). Coeloconic sensilla of Hemipyrellia ligurriens observed in our study shows structural similarity to that found in Lucilia cuprina, Triceratopyga calliphoroides and Protophormia terraenovae (Setzu et al. 2011; Sukontason et al. 2004; Zhang et al. 2014). Thus, phylogenetic studies based on antennal sensilla of forensically important dipteran families remains to be a crucial area of research and the data from the present study could aid in such research. 5. Conclusion The identification and characterization of the morphology and distribution of the five sensilla types found in Hemipyrellia ligurriens by using SEM is performed, so as to generate a baseline data of antennal ultrastructure of the species. Moreover, the probable function of each sensilla type is discussed with respect to the observations of investigations previously done on other species. Thus it can be concluded that the larger coeloconic sensilla in females of H. ligurriens in comparison to males play a pivotal role in locating successful sites for colonization and oviposition in corpses. The results have considerable significance in instigating further study on H. ligurriens not only in forensic entomology and entomo-toxicology but also in pest control as study of the sensilla chemoreceptors would allow use of specific insecticides that block the function of these sensilla, thus proving to be crucial from medical and veterinary perspective. The present study will provide the basis for future studies on antennal electrophysiology which would pave the path to better understanding of the functional significance of the sensilla types in the species. Another prospect of the study lies in the fact that the morphological features of the antenna examined can act as potential tools for reconstructing phylogenetic relationships among other forensic dipteran species of families Calliphoridae, Sarcophagidae and Muscidae. Competing interest Authors declare no conflict of interest.
Author contributions Conceived and designed the experiments: DB. Sample Collection and Identification: GH. Performed the experiment: GH, AM, AN. Analyzed the data: GH, AM, AN. Wrote the paper: GH, AM, AN. Critically revised the article: WA, SG, DB, GKS. Acknowledgements Present work is financially supported by Zoological Survey of India core funding. Facilities and support for the present study were provided by Dr. Kailash Chandra, Director, Zoological Survey of India, and Dr. K.C. Gopi, Additional Director and Divisional-in-charge, Entomology Division ‘A’ and ‘B’ are kindly acknowledged. We convey our sincere regards to Mr. Pachanan Parui, retired scientist, ZSI, for helping in taxonomical identification of the specimens. The authors express their gratitude to Dr. Vikas Kumar, Scientist D, Centre for DNA Taxonomy, Molecular Systematics Division, ZSI and Dr. Sagartirtha Sarkar, Associate Professor, Department of Zoology, University of Calcutta for providing the chemicals and consumables required for the SEM sample preparation. The authors are thankful to the operators of SEM, CRNN, University of Calcutta for their technical assistance in sputter-coating during Scanning Electron Microscopic study.
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Figure legends Figure 1: SEM micrographs of features on antenna of female Hemipyrellia ligurriens. a. Frontal view of head showing compound eyes, antennae and mouthparts. b. Antenna in magnified view comprised of the three antennal segments: scape (I), pedicel (II) and flagella (IV) bearing a dorso-laterally located arista (III). c. Magnified view of the scape, pedicel and anterior part of the flagella. d. Scape in magnified view showing chaetic sensilla (ChI). e. Pedicel in magnified view showing two types of chaetic sensilla-small chaetic sensilla (ChI) and large chaetic sensilla (ChII) and styloconic sensilla (St). f. Four styloconic sensilla on the pedicel in magnified view (arrow); a tuft of fine hairs located above the seta is shown by white arrow.
Figure 2: SEM micrograph of features of antennal flagella of female Hemipyrellia ligurriens. Four types of structures are observed on the cuticular surface of flagella- covered with numerous thin microtrichia or spinules (Mc); trichoid sensilla (Tr) characterized by somewhat curved setae with thicker bases; basiconic sensilla (black arrows) are shorter, with blunt ends and coeloconic sensilla (Cs) which bears a cone-shaped projection centrally located within a sacculus. Bar = 2µm. Figure 3: Basiconic and coeloconic sensilla of female Hemipyrellia ligurriens at higher magnification. Basiconic sensillum (Bs) bears a multi-porous surface; coeloconic sensillum (Cs) has longitudinal grooves along its surface. Bar = 200 nm. Figure 4: SEM micrographs of features on antenna of male Hemipyrellia ligurriens. a. Frontal view of head showing compound eyes, antennae and mouthparts. b. Antenna in magnified view comprised of the three antennal segments: scape (I), pedicel (II) and flagella (III) bearing a dorso-laterally located arista (IV). c. Magnified view of the scape and pedicel showing two types of chaetic sensilla- chaetic sensilla (ChI) and large chaetic sensilla (ChII).d. Two chaetic sensilla (ChI) located on the pedicel in magnified view-note that they emerge from flexible sockets and have a longitudinally grooved cuticular surface. e. Scape in magnified view showing chaetic sensilla (ChI) and microtrichia (Mc) or spinules. f. Pedicel in magnified view showing seven styloconic sensilla (St) and microtrichia (Mc); a tuft of fine hairs located above the styloconic sensilla is shown by arrow. Figure 5: SEM micrographs of antenna of male Hemipyrellia ligurriens. a. Arista (a) of flagella, (b) Bar = 20µm. b. Sensilla types observed on antennal flagella- Four types of structures are observed on the surface of flagella- covered with numerous thin microtrichia or spinules (Mc); trichoid sensilla (Tr) characterized by somewhat curved setae with thicker bases; basiconic sensilla, which are shorter than the trichoid sensilla are shown by white arrows and coeloconic sensilla (Cs) which bears a cone-shaped projection centrally located within a
sacculus. Bar = 1µm. c. Trichoid sensillum magnified (shown by white arrow). d. Basiconic sensillum (shown by white arrow) - its multi-porous surface can be seen. e. A coeloconic sensillum among trichoid sensilla and microtrichia is magnified (shown by white arrow) – showing the longitudinal grooves along the surface of the sensilla. f. Microtrichiae- slender, curved processes shown by black arrows.
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Table 1: Mean ± SE of the length and width (µm), at the larger diameter, in the antennae segments of females and males of Hemipyrellia ligurriens. Means followed by the same lowercase letter on the lines are not statistically significantly different (Student’s t-test, P < 0.05, n = 6). Sensilla Sex of flies
Length (µm) Females
Width (µm) Males
Females
Males
Scape
94.02 ± 2.37 a
102.02 ± 2.04 b
61.64 ± 1.51 a
81.4 ± 1.22 a
Pedicel
153.51 ± 4.32 a
167.11 ± 1.81 b
129.52 ± 1.30 a
151.6 ± 1.73 a
Flagellum
785.89 ± 6.41 a
781.13 ± 12.09 a 162.66 ± 4.59 a
114.77 ± 2.79 b
Arista
822.69 ± 2.62 a
911.64 ± 4.67 a
49.61 ± 2.24 b
36.71 ± 1.84 a
Table 2: Mean ± SE of the length and width (µm) of sensilla on the antennal scape and pedicel of females and males of Hemipyrellia ligurriens. Means followed by the same lower-case letter on the lines are not statistically significantly different (Student’s t-test, P< 0.05, n=6). - = undetermined.
Scape Sensilla
Pedicel Length (µm)
Sex of Females flies 34.49 ± Chaetic 2.82 a sensilla (ChI) Chaetic sensilla (ChII) Styloconic sensilla -
Width (µm)
Length (µm)
Width (µm)
Males
Females
Males
Females
Males
Females
Males
56.82 ± 8.15 b
5.45 ± 0.35 a
6.77 ± 0.64 b
29.60 ± 7.17 a
47.75 ± 6.47 a
5.56 ± 0.72 a
7.17 ± 0.71 a
345.37 ± 2.38 a
324.87 ± 14.62 a
12.01 ± 0.12 a
12.11 ± 0.06 a
-
-
-
-
-
-
11.76 ± 0.11 a
11.74 ± 0.13 a
Table 3: Mean ± S of the length and width (µm) of sensilla on the antennal flagellum of females and males of Hemipyrellia ligurriens. Means followed by the same lower-case letter on the lines are not statistically significantly different (Student’s t-test, P< 0.05, n=6).
Sensilla
Length (µm)
Width (µm)
Sex of flies
Female
Male
Female
Male
Trichoid
16.94 ± 0.25 a
17.25 ± 0.16 a
2.17 ± 0.10 a
2.21 ± 0.16 a
Basiconic
7.86 ± 0.58 a
8.01 ± 0.63 a
1.55 ± 0.04 a
1.62 ± 0.11 a
Coeloconic
6.42 ± 0.06 a
5.93 ± 0.08 b
1.56 ± 0.06 a
1.23 ± 0.04 b