The Biting Mouthparts of a Black Fly (Simulium) and an Attached Mite (Arrenurus), an Sem and Edxa (X-Ray Scan) Study Using a Dual Beam Scanning Electron Microscope
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Pakistan J. Parasitol., 68; December 2019: 1-9 THE BITING MOUTHPARTS OF A BLACK FLY (SIMULIUM) AND AN ATTACHED MITE (ARRENURUS), AN SEM AND EDXA (X-RAY SCAN) STUDY USING A DUAL BEAM SCANNING ELECTRON MICROSCOPE Richard Heckmann Dept. of Biology 1114 MLBM, Brigham Young University, Provo, Utah 84602 USA Abstract The mouthparts of a black fly (Simulium) and its ectoparasitic mite (Arrenurus) are evaluated using SEM and EDXA. Parts of the mouthparts of both were cut with a gallium beam (LMIS: Liquid Metal Ion Source) using a dual beam SEM and analyzed for chemical elements. One element, chlorine was present in the chelicerae of both the black fly and mite with the highest quantity in the mite. Possible functions of the chemical elements are listed. The mite drains hemolymph from the black fly which is depicted by the close association of the mite with the host (SEM figures). Keywords: EDXA, Simulium, Arrenurus, Chelicerae, Host-Parasite Interaction. INTRODUCTION Our lab published a paper on mites infesting black flies along a major drainage in the USA (Heckmann et al., 2011). This is an extension of that paper stressing the biting mouthparts of both the host (Black fly) and its ectoparasite (Mite). This paper will also include the elemental scan (X-ray) of the biting mouthparts of both organisms and the gallium beam cuts of these critical parts of chelicerae and mandibles. Dual beam scanning electron microscopy has become a critical part of our studies at Brigham Young University, Provo Utah. The technique allows us to study not only the fine structure but also to analyze chemical elements playing a major role in the daily activity of the parasite (Heckmann et al., 2012; Amin et al., 2016, 2018). Black flies of the family Simulidae are common around the world and have other common names (buffalo gnat, white socks, etc). The species of blackfly are above 2000 amongst them fifteen are extinct. Most black flies feed on the blood of mammals as well as humans. They are generally small, black or grey, with antennae and short legs. They are a common nuisance for humans. They distribute several diseases, including river blindness in Africa (Simulium damnosum and S. neavei) and the Americas (S. callidum and S. metallicum in Central America, S. ochraceum in Central and South America) (Crosskey, 1990; Hahn, 2015; Weiser and Undeen, 1981). The simulids have a wide array of parasites and symbiotic relationships (Ezenwa, 1974; Harkrider, 1988; Ledin, 1994; Molloy, 1981; McCreadie and Adler, 1999). Mites like ticks have no prominent head, the head, thorax and abdomen are fused into one part called the cephalothorax. With its two pair of mouthparts it becomes a biting and feeding machine. Eight legs divided into 6-7 segments with the terminal part containing modifications for attachment such as claws. The main mouthpart for feeding is the reed-like chelicerae (Heckmann, 2018a; Heckmann et al., 2011). Mites, along with ticks, are not large members to the subclass Acari (also known as Acarina) and the class Arachnida. Mites favor a moist conditions with high organic matter (Heckmann, 2016). Mites are successful and most diverse of all the invertebrate groups. They have exploited all of the notable natural environments due to their small size, they are not noticeable. Many live freely in the soil or water but there are also a large number of species that live as parasites on plants, animals and some that feed on mold. They are approximately 48,200 species of mites have been recorded (Iverson et al., 1996; Crosskey, 1990). Mites reside in a wide range of ecological niches. For example, Oribatid mites occur in a number of habitats and good decomposers. They feed upon diverse material which include living and dead plant and fungal material, carrion and lichens; some are even predatory, though no species of Oribatid mite are parasites (Mullin, 1977; Heckmann, 2016). 2 Heckmann The larvae of water mite are parasites, and they require a host to live on, such as black flies and mosquitos (Iverson et al., 1996; Heckmann, 2016). Other insects selected to be hosts are dragonfly naiads, fly larvae, damselfly naiads, true bugs and stone fly nymphs. Twenty mites may be carried by a single insect (Heckmann, 2016). The larvae feed with the aid of Chelicerae and other mouthparts (Heckmann, 2016). When a mite larva has grown substantially, its exoskeleton is shed and develops into a bag-like. The larvae remains inside the bag and becomes a nymph. As it changes, the “bag” stays fastened to the host (Public Health Control, 2010). When the nymph is ready, it leaves the host and has 8 legs and resembles like an adult water mite (Heckmann, 2016). MATERIALS AND METHODS For SEM studies: specimens of infested black flies were earlier fixed in 70% ethanol and then placed in critical-point drying baskets and dehydrated using an ethanol series of 95% and 100% for at least 10 min per soak followed by critical-point drying (Lee, 1992). On SEM sample mounts (stubs) samples were mounted, gold coated and observed with a dual beam scanning electron microscope (FEI Helios Dual Beam Scanning Electron Microscope, Hillsboro, OR, USA). Digital images of the structures were prepared using digital imaging software connected to a computer. Figures were stored on a USB. For X-ray microanalysis (EDXA), standard methods for SEM preparation (Lee, 1992) were employed. Specimens coated were examined with an FEI Helios Dual Beam Scanning Electron Microscope equipped with an Apollo 40 Silicon Drift Detector (SDD) X-ray detector (FEI, Hillsboro, OR, USA). X-ray spot analysis and line scan analysis were carried out at 15 kV and results were represented in charts and recorded on digital imaging software attached to a computer. Data were then stored on a USB. Weight percent and atom percent for the chemical elements were recorded as results. The cutting of each chelicerae of the mite and the mouthparts of the black fly were done with a gallium beam using the FEI Helios Dual Beam Electron Microscope. The mouthpart was arranged at the eccentric position of the stage and cut longitudinally using a 30 kV gallium ion gun (LMIS) functioning at 2.8 nA. A cross-sectional pattern was used followed by a cleaning cross-section to make available a clean cut surface to image. Images were obtained of the cut surface using a 5 kV electron beam at 0.17 nA followed by X-ray spectrum analysis using a 15 kV electron beam. The Energy Disruptive X-Ray Analysis (EDXA) Genesis System was carried out utilizing the Apollo 40 SDD (Silicon Drift Detector) X-ray detector (FEI, Hillsboro, OR, USA) with results stored on a USB. This procedure is similar to the sequence followed in; Heckmann et al. (2007; 2012), Amin et al. (2018). RESULTS Tables 1 and 2 represent the results of the chemical analysis (EDXA) of the black fly and attached mite including the gallium cuts with the latter compared to a similar mouthpart of the honey bee mite ( Varroa destructor). Note the chlorine content of the chelicerae for the mites. Calcium is more prominent for the mouth region of the black fly in relation to the mite. The SEM and X-ray spectrum figures are represented by Figs. 1 to 18 and Figs. 19 to 20. The black fly (Simulium) has well defined mouthparts as well as the parasitic mite (Figs. 1, 2 and 16). Note the close association of the mite with the back fly (Figs. 1, 2 and 4). Note the chelicerae (Figs. 11, 14 and 15) which aids in penetrating the soft parts of the host which is usually at the joint area. Mites are found among many different hosts including humans (Crosskey 1990; Hahn 2015). The Simulid has mouthparts whereby it can obtain food from hosts which are in turn a host for other parasites such as mites. The SEM figures on the plates represent a visual picture of the host-parasite association. Gallium cut areas (Figs. 19 and 20 insets) show the internal structure of the biting apparatus of each and the chemical mineralization of these structures (Tables 1 and 2). There is a close similarity for the chelicerae of the bee mite (Varroa) and the black fly mite (Arrenurus). The trace amounts of chlorine in the chelicerae helps in disinfecting the host area being penetrated. The dual-beam electron microscope is a major tool in understanding host-parasite relationships especially the means of parasite attachment. The structure of the biting mouthparts of the fly (Figs. 5, 6, 7, 9 and 10) and the mite (Figs. 4, 11, 12, 13 and 14) are demonstrated. The chelicerae of the mite are shown (Figs. 13, 14 and 15). THE BITING MOUTHPARTS OF A BLACK FLY 3 Fig. 1: Host, black fly covered with mites (M) which have attached at joint location of host. Fig. 2: Mite (M) attached to the joints of the host appendage. Fig. 3: Head region of black fly (BF). Antenae and compound eyes are visible. Fig. 4: Hi magnification of Fig. 2, (M) mite at host appendage. Fig. 5: Mouth (M) region of black fly (Simulium). Note host piercing structures. Fig. 6: Hi magnification of mandibles (M) used for piercing host tissue. 4 Heckmann Fig. 7: Chelicerae (C) of black fly, within mouth of host. Fig. 8: Hi magnification of Chelicerae (C) for black fly host. Fig. 9: Enface view of simulid host with prominent mouth parts. Fig. 10: Lateral view of the mouth region of the black fly showing piercing mouth parts. Fig. 11: Mite (M) attached to eye region of the black fly host. Fig.