Ultrastructure of Adult Gasterophilus Intestinalis (Diptera: Gasterophilidae) and Its Puparium
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International Journal of Tropical Insect Science https://doi.org/10.1007/s42690-019-00084-9 ORIGINAL RESEARCH ARTICLE Ultrastructure of adult Gasterophilus intestinalis (Diptera: Gasterophilidae) and its puparium Marwa M. Attia1 & Nagla M.K. Salaeh2 Received: 12 June 2019 /Accepted: 2 December 2019 # African Association of Insect Scientists 2019 Abstract This research was conducted to identify the most common equine stomach bot fly by using light and scanning electron microscopy. Third instar larvae (n = 200) of Gasterophilus intestinalis were collected from stomach of slaughtered donkeys (Equus asinus) at Giza Zoo abattoir, Egypt, in July 2017 (the donkeys chosen were from Giza; Egypt). One hundred only were full mature 3rd instar larvae (the mature 3rd instar was active and has brown bands on its dorsal surface) which were incubated at 32 °C and 80–85% Relative Humidity (RH), for the development of adult. The following pupal parameters were recorded: prepupal period, number of pupated larvae, pupal period, and number of emerged adult and determination of adult sex ratio. Morphological description of pupae, puparium and adults were provided using light and scanning electron microscopy (SEM). The length of prepupal duration was five days and about 90% of the collected G. intestinalis larvae successfully pupated with pupal duration lasting for 21–25 days prior to emergence of the adult stage. Adults sorted according to sex show a female to male ratio of 8:1 (i.e. 80 females to 10 males). The pupae of G. intestinalis were brown to black in color. The adult head, thorax, abdomen, legs and wings of male and female were morphologically described using light and SEM. This research fully describes the adult G. intestinalis male; female and its puparium to distinguish it from other specific myiasis producing flies within its family. Keywords Adult fy . Equine bot fly . Gasterophilus intestinalis . Light and scanning electron microscope (SEM) . Pupa and Puparium . Specific myiasis Introduction Gasterophilus intestinalis eggs do not hatch prior to its ingestion by host (family: equidae). Depending on the en- Gasterophilus intestinalis (Oestridae: Gasterophilinae) is a vironmental temperature and relative humidity, the first Palaearctic species. It is now the most common and widely stage larvae take about 5–10 days to emerge from eggs distributed equine bot fly worldwide (Huang et al. 2016; (Kettle 1995;Piloetal.2015). Zhang et al. 2016; Attia et al. 2018). In the tropics and The adult flies are diurnal (in early afternoon) with short subtropics areas, there could be continuous or overlapping life span that could last only for a day (Catts 1979; Kettle generation of Gasterophilus intestinalis (G. intestinalis) 1995). An adult G. intestinalis is bee like with hairy head every year (more than one generation in one year). The life and thorax, where the female had numerous abdominal seg- cycle of G. intestinalis begins with a female laying eggs in ments than other cyclorrapha flies and the abdomen is ventral- apreferablesiteinthehairsoffetlockarea(Zumpt1965). ly recurved. The wings have a broad median band and dark areas at the end of median vein and around the wing apex (Kettle 1995). * Marwa M. Attia [email protected] Almost all equines and donkeys were infested with these larvae all the years (Huang et al. 2016; Attia et al. 2018). In 1 Egypt, Hilali et al. (1987) recorded the prevalence rate of Parasitology Department, Faculty of Veterinary Medicine, Cairo G. intestinalis larvae as 98.3% infestation in the donkeys University, Giza 12211, Egypt 2 while Attia et al. (2018) recorded prevalence rate of 97.2% Zoology department, Faculty of Science, Aswan University, in donkeys with G. intestinalis. Aswan, Egypt Int J Trop Insect Sci Infestation of equine and donkeys with G. intestinalis lar- Table 1 Mean length of male and female and their abdomen vae leads to severe pathological lesions due to attachment of Total length Length of abdomen larval mouth hooks which leads to chronic gastritis, ulcera- tion, anemia, loss of condition, diarrhea (Yang et al. 2013). On Male 14.60 ± 0.40 b 7.93 ± 0.12 b the other hand, peritonitis may occur due to intestinal rupture Female 20.90 ± 0.46 a 8.82 ± 0.15 a (Otranto et al. 2005). In addition, there are many reports of P- value 0.0001* 0.0001* human myiasis, such as ophthalmo-myiasis among human, which are caused by Gasterophilus spp. larvae (Yang et al. Means of dimensions of males and females were significantly different (P = 0.0001) 2013). Limited information is available about the adult SE Standard error G. intestinalis male and female and its pupae. Only, Zayed a,b Different superscripts indicate significant difference at P <0.05 (1988) and Zhang et al. (2016) were partially described the morphology of this adult fly. Therefore, the objective of our study was to fully describe the pupae; puparium and adult Pupation of mature 3rd stage larvae stages of the commonly distributed bot fly in donkeys. The 3rd instar larvae were incubated at 32 °C and 80–85% Relative Humidity (RH), where each 10 larvae were separate- Materials and methods ly placed in a beaker of 250 CC, containing sterile sand, cov- ered with gauze and fixed with rubber band. The beakers were Collection of larvae incubated in a large dissector containing suitable amount of saturated salt solution to adjust the relative humidity Gasterophilus intestinalis 3rd instar larvae (n = 200) were col- (Sukhapesna et al. 1975;Zayed1988). The larvae were exam- lected from stomachs of donkeys, usually the glandular part of ined daily, and to determine the prepupal period, number of the stomach. These donkeys were slaughtered at the Giza Zoo pupated larvae, pupal period and number of emerged adults abattoir, Egypt, in July 2017. The collected larvae examined, with determination of sex ratio. and separation of the fully mature larvae from the less mature 3rd instar of G. intestinalis (the full mature larva (n =100)was Morphological studies active; dark in color and had brown bands on its dorsal sur- face). The full mature larva was identified according to the Macroscopic identification published morphological keys (Zumpt 1965; Colwell et al. 2007). All Institutional and National Guidelines for the care All specimens (pupae; puparium and adult) were preserved in and use of animals were followed with number 70% ethanol and examined under stereoscopic microscope VetCu10102019096. (X100). Fig. 1 Puparium of G. intestinalis. a:dorsalviewof puparium showing persistent spines in its segments and triangular shape openings. b, c and d Scanning electron micrograph of puparium showing; b: the shape and length (2.54 mm) of triangular aperture in which adult emerge also the arrow refer to the remnant of cephalopharyngeal skeleton, c and d: showing persistent structure of 3rd instar larvae persistent spines and diameter of puparium, 6.2 mm Int J Trop Insect Sci Microscopic studies by light microscope Results The preserved adults were placed into 10% Caustic Soda Pupation parameters (10% NaOH) and left at room temperature for up to 24 h. The adults (both male and female) were dissected to isolate The parameters of pupation of G. intestinalis 3rd instar larvae head, thorax, abdomen, wings and legs. Then thorax and ab- incubated under controlled condition (32 °C and 80–85% domen were evacuated from their contents. All parts were Relative Humidity) were recorded as follows: the prepupal washed several times with water to remove the contents and period took 5–7 days, the number of pupated larvae was 90 residual of NaOH. The adult parts were dehydrated through out of 100 larvae (90%), and the pupal period took 21–25 days. passing in ascending serial degrees of ethyl alcohol (30%, The sex ratio recorded as 80 females to 10 males. 50%, 70%, 90 and absolute) for 1 h each. The specimens were cleared using clove oil, then put in xylene for 5 min, finally mounted by Canada balsam and then incubated at 40 °C to dry The pupae and puparium (Soulsby 1986). Morphological identification of specimens was carried out using published keys (Zumpt 1965). The mor- The pupae of G. intestinalis were brown and black in color phological parameters were recorded in 10 specimens from ranging in length from 15 to 18 mm (16.8 mm ± 1.0) in length each stage of pupae; puparia and adult male and female. All and from 6 to 9 mm (7.9 mm ± 0.5) in width. At the start of dimensions (length; width of each part of adult and pupa) were pupal period, the morphological characters resemble the 3rd recorded after examination with light microscope according to instar larvae with persistent spines on their dorsal and ventral (Zhang et al. 2016). segments but with darkened and hardened tegument. With the release of adult from the triangular opening at the anterior end dorsally, this opening was 2.54 mm long and 2.8–3.3 mm Scanning electron microscopy (SEM) wide and showed inside it the remnant of cephalopharyngeal skeleton (Fig. 1). For SEM, puparium as well as adult male and female (their body parts were dissected for observation into heads, thorax, abdomen, legs and antenna) were washed Adult stage of G. intestinalis several times using phosphate buffered saline (PH 7.2). The samples were prepared by serial washing in saline It is a large sized hairy bee like fly, 12–16 mm (15 mm ± 1.0) solution and fixed in 2.5% glutaraldehyde at 4 °C for in male while 19–23 mm (20 mm ± 1.3) long in female; 24 h as described by Zhang et al. 2016 for adults and Table 1. Adult males and females were yellowish brown in Cepeda-Palacios et al. 2015 for puparium. Specimens colour, with large pairs of coloured wings, eyes broadly sep- were dehydrated through passes in serial ascending etha- arated from each other in both sexes.