Scanning electron microscopy of the mouthparts of autumnalis, Musca domestica and Musca osiris (Diptera: )

Dr. Ferenc K0VÁCS-SZ. Veterinary Medical Research Institnte, Hungarian Academy of Sciences, Budapest, Hungary

"Scanning electron microscopy of the mouthparts of Musca autumnalis, Musca domestica and Musca osiris (Diptera, Muscidae." - Kovács-Sz., F. - Parasit. hung., 20: 91-98. 1987.

ABSTRACT. Scanning electron microscopic (SEM) observations on the mouth­ parts of three species of muscoid (Musca autumnalis, M. domestica, M. osiris) are described in order to draw attention to those structures which are able to injure the surfaces they sucked from, particularly the non-hornified epi- thelia of the host . The morphological data (toughness of chitinization, shape, length, position and sharpness of labrum and hypopharynx and those of the prestomal teeth) show that all these structures would be able to cut through almost all the layers of the different epithelia. However, a functional study of these structures (on living tissues) has proved that the microlesions were caus­ ed by the prestomal teeth and the pseudotracheal rows only; no harmful effect of the labrum and the hypopharynx could be detected. On the basis of earlier SEM morphological studies pseudotracheal rows had not been considered to have the ability to cause mechanical lesions; because of their small size, but it was found that their considerable rubbing effect cause harm to the epithelia. The features of the prestomal teeth of the three Musca species are also described from a taxonomic point of view.

KEY WORDS: Musca autumnalis, M. domestica, M. osiris, mouthparts, SEM studies, transmission of diseases.

All these three muscoid species are secretophagous as regards their feeding strategy, which is also reflected in the similar structures of their mouthparts. Mouthpart structures and their life-habits closely connected with animal husbandry, determine by and large their eco­ nomic importance.

This is a morphological study to describe those features of the mouthparts of the three spe­ cies of Musca, which enable those flies to injure the surfaces of animal epithelia, resulting in opening gates for pathogens. A mere list of the names of pathogenic organisms spread by the Musca species covers 67 printed pages in GREENBERG's (1973) review. The morphology of the proboscis of the secretophagous flies was analysed by GREENBERG (1973) and McAL- PINE (1981), a more functional version is involved in WEST'S (1951) book; these books and other works not mentioned here concluded from the morphological observations to the details of the movement of proboscis but without a functional verification of the movements of the mouthparts. GRAHAM-SMITH's (1930) light microscopic observations on the elements of the mouthparts of Calliphora erythrocephala and on their print on agar plates were a ba­ sis for all the later morphological descriptions. Scanning electron microscopy has opened new prospects for researchers to discover unknown morphological details (PAYSINGER et al., 1983; BROCE and ELZINGA, 1984; IWASA, 1984). The new SEM observations indeed, improved our knowledge of morphology, however the func­ tion of the mouthparts of the secretophagous flies and their harmful effect to the host animals were not much dealt with in those works. MAHANKO (1973) described the morphology of the prestomal teeth of 13 species of the genera Hydrotaea and Musca, and he classified those muscoid species in three categories of "parasitism" based on the size, measurements of the prestomal teeth and the life-habits of the given species (where e. g. the place of Musca do­ mestica was rather questionable.

Below not only those parts of the proboscis are described which have the ability to cause harm to the epithelia (see e.g. MEDVECZKY et al., 1988) but evidence was also sought for the details of the effects of the mouthparts causing microlesions. Of course, several pic­ tures are also included here which show hitherto known features and do not provide new find­ ings but which promote understanding a morphological-functional treatment, or, which show simply the usefulness of the SEM studies in this field.

MATERIALS AND METHODS

The specimens of a WHO strain Musca domestica and those of Musca autumnalis were ob­ tained alive from the insectarium of the Parasitological Department, University of Veteri­ nary Science, Budapest; the specimens (dry museum specimens) of Musca osiris were ob­ tained from the collection of the Zoological Department, Hungarian Natural History Museum, Budapest.

Carbon tetrachloride (CCI4) was used for killing the flies and assuring the reproduction of the physiological positions of the labella (for details see KOVÁCS-SZ. and GONDÁR, 1986). After a few minutes of air-drying - which removes CCI4 residues - a cleaning procedure was applied by a bio-active washing powder. Then the heads of flies were cut off and after trans- versally halving and air-drying they were mounted on SEM stubs. Specimens were vacuum- coated with a thin layer of coal and gold prior to examining them on a Jeol JSM-35 scanning electron microscope. For examination of the living tissues with SEM - which served as tests showing the harmful effect of the mouthparts - the method of SULOCHANA and DERBYSHIRE (1977) was adopted with the alterations described in the paper of MEDVECZKY et al. (1988). For testing the harmful effect of the different parts of the proboscis, flies were immobilized on the same way as it has been previously described (MEDVECZKY et al., 1988).

RESULTS

Based on the hitherto known references, first of all as regards the functions of proboscis, our examinations were concentrated to the haustellum and to the labella (Fig. 1), considering also the aims of our trials with living flies. Prior to an actual functioning, the proboscis of the muscoid flies emerges from the subcranial cavity through the movement of the muscles inserted to the inner chitinous skeleton of the proboscis and through the extra amount of hae- molympha which is pressed into the volumen of proboscis. The contraction of the proboscis is also a result of active contractures (WEST, 1951; GREENBERG, 1973). However, this stretchy organ contains rather numerous stiffening elements, particularly so for the haustel­ lum. For causing microlesions, the labrum and the hypopharynx seem the most dangerous elements at first glance, since both these structures are borne on a firm base on the rostrum, both are well-sclerotized and extremely sharp (WEST, 1951); the tips of both these struc­ tures terminate between the lamellae of the labella. The labrum, which covers the dorsal surface of the proboscis, is comparatively easy to examine (Fig. 2). Contrarily, the other "dagger", namely the hypopharynx which forms the ventral wall of the food-channel in the haustellum, is hardly accessible and in addition, is easily breakable while preparing off the plates of the mentum (Figs 3-6). The complex of the labrum and the hypopharynx form the food-channel, and they are extremely stiff when forming that tube. Our Fig. 7 shows clearly the position of the labrum-hypopharynx inside the haustellum, where we managed to bend the labrum-hypopharynx out of the axis of the proboscis, i. e. out of the plates of the mentum (like a blade of a jack-knife). Nevertheless, this picture represents an artificial position in order to prove that the labrum and the hypopharynx together constitute a functional unit. If the labrum-hypopharynx complex is bent back to their original position, their apices termi­ nate exactly between the lamellae of the labella (Figs 8-10). It can be supposed referring to these data that in the sucking position of the labella (see Fig. 31) the apex of the labrum and the hypopharynx (above and below the entrance of the food-channel) may touch and injure the surface where the is sucking from. However, we regard this as a morphological possibil­ ity only, which we did not manage to prove in direct trials.

As for the labella (Fig. 10), the morphology and the function of the prestomal teeth and of the pseudotracheae were studied. The name "prestomal tooth" itself refers to some cutting and grinding function; indeed, they have a role in cutting up of food into proper pieces. This function can be deducted from the morphology and position of the prestomal teeth but mor­ phology alone would leave questions open as for the way and expressivity of this function. In haematophagous species the labella is much reduced but prestomal teeth function as sharp cutting devices (McALPINE, 1981). The prestomal teeth of the three Musca species involved in our studies are rather different (Fig. 32). These teeth can precisely be characterized one by one but some common features are observable through the SEM micrographs. Schematic drawings of the prestomal teeth in lateral view were made on such a general impression.

The prestomal tooth of the house fly (M. domestica) (Figs 11-12) has a thick base, narrow­ ing in its "neck" and the crown of the tooth consists of a strong high, slightly indented main branch and of two weaker laterally directed side branches. Mean length of the teeth (based on SEM micrographs) is 65 to 70 jum, they are of 11- 15 urn at widest and 7. 5-8. 5 jum at their "neck".

The prestomal tooth of the face fly (M. autumnalis) (Figs 13-15) is longer, 75 to 85jum, slimmer than that of M. domestica. That tooth is 11-13 jum at widest and 3.7 to 5. 5 jum at its "neck". The base of the tooth is narrower than that of M. domestica, the two lateral branches of its crown are longer than the main branch and they are not indented. The main branch is indented and emerges from a concave emairgination between the lateral branches.

The prestomal tooth of M. osiris (Figs 16-18) is 70 to 80 jum long, it is 15 to 20 jum at wid­ est and ca. 5 jam wide at its "neck". The base of the tooth is narrow (like in M. autumnalis), the three branches on its crown are nearly evenly high. The lateral branches are without in­ dentation, the main branch is strongly indented. The main branch - since it is as strong as the lateral branches - does not emerge from an emargination between the lateral branches (in contrast to that of M. autumnalis).

Comparing these sizes of teeth to the mean thickness of animal epithelia (i. e. 30-40 urn) we may conclude that such a tooth is capable of cutting through the whole epithelium. There are weighty arguments to support this morphological possibility when studying the fixation of these teeth. The teeth sit on a firm base on the discal sclerite. Their bases are connected to one another up to their neck by a chitinous plate and this way a one-sided row of teeth form a coherently moving -system. In addition, the teeth are connected one by one to the lamellae of labella through a chitinous plate (Figs 11-14, 16-18, 19-21). This latter chitinous plate is movable in harmony with the discal sclerite and these two connecting systems allow the ver­ tical and also the horizontal movements of the prestomal teeth between the different positions (Fig. 31) and also those in a given position. All these movements are results of the changing pressure of the haemolympha and of the work of strings and muscles (WEST, 1951). Our trials with flies sucking ("biting") on living tissues furnish documentary proofs for these movements (Figs 22, 23). The mouthparts of the house fly are capable of biting out three epi­ thelial cell-layers during the short period of a "bite". The heaps on the micrograph indicate the entrance of the food-channel, the breaks beside them are results of tearing of the pre­ stomal teeth. An exact print of a prestomal tooth is well discernible on the surface of the epithelium (Fig. 23). These results are of another nature, and thus they are published else­ where (MEDVECZKY et al., 1988, KOVÁCS-SZ. et al., in print). Fig. 1. Proboscis of Musca osiris, emerged from the subcranial cavity (R: rostrum, H: haustellum, L: labella) (60 x).

Fig. 2. Proboscis of Musca domestica in dorsal view (Lb: labrum) (86 x).

Fig. 3. Labrum (Lb) and hypopharynx (Hp) of Musca domestica (mentum removed) (78 x).

Fig. 4. Labrum (Lb) and hypopharynx (Hp) of Musca domestica in ventral view (mentum removed) (130 x)

Fig. 5. Labrum (Lb) and food channel (F) of Musca osiris in ventral view (mentum and hy­ popharynx removed) (130 x).

Fig. 6. Labrum (Lb) and food (F) channel of Musca osiris, seen from the entrance of the food channel. The base of the removed hypopharynx is indicated by the salivary duct (Sd) (360 x).

Fig. 7. Labrum (Lb) and hypopharynx (Hp) of Musca autumnalis, bent out from the axis of proboscis (86 x).

Fig. 8. Proboscis of Musca autumnalis in dorsal view (the tip of labrum /Lb/ is immediate­ ly below the lamellae of the labella, latter in a scraping position) (100 x).

Fig. 9. Labella of Musca osiris, in anterior view. The lamellae of labella are open above the food channel: this gap allows the labrum to reach the surface of feeding (600 x).

Fig. 10. Labella of Musca domestica, in anterior view. The gap between the lamellae of la­ bella is discernible above the entrance of the food channel (Ef: entrance of food channel, P: prestomal teeth, Ps: pseudotracheal rows) (150 x).

Fig. 11. Prestomal teeth of Musca domestica; a chitinous membrane (Cm) chains them to each other (lettering as in Fig. 10) (660 x).

Fig. 12. Prestomal teeth and the chitinous laths (C) connecting them with the labella in Mus­ ca domestica (1300 x).

Fig. 13. Labella (L), prestomal teeth (P) and pseudotracheal rows (Ps) in Musca autumnalis (200 x). Fig. 14. Prestomal teeth and chitinous laths (C) connecting them with the labella in Musca autumnalis (540 x).

Fig. 15. Prestomal teeth of Musca autumnalis (480 x).

Fig. 16. Prestomal teeth (P) and chitinous membrane (Cm) connecting their bases in Musca osiris (Ef: entrance of the food channel, Ps: pseudotracheal rows) (440 x).

Fig. 17. Prestomal teeth (P) and chitinous laths (C) connecting them with the labella in Mus­ ca osiris (720 x).

Fig. 18. Prestomal teeth (P) and chitinous membrane (Cm) connecting their bases in Musca osiris (Ps: pseudotracheal rows) (1000 x).

Fig. 19. Prestomal teeth (P) and chitinous membrane (Cm) connecting their bases in Musca osiris (400 x).

Fig. 20. Prestomal teeth (P) and chitinous laths (C) connecting them with the labella and with the pseudotracheal rows (Ps) (1000 x).

Fig. 21. Prestomal teeth (P) and chitinous laths (C) connecting them with the labella in Mus­ ca autumnalis (540 x). Fig. 22. A lesion on the surface of living tissue (cornea) caused by sucking of a house fly. A small heap indicates the entrance of the food channel (ef) (p: the place of the tear­ ing of prestomal teeth, 1,2,3: layers of epithelial cells) (600 x).

Fig. 23: A tearing line left the exact print of a prestomal tooth on the surface of the epithe­ lium (1 500 x). Fig. 24: The labella of Musca domestica in a filtering position in anterior view (Ps: pseu­ dotracheal rows) (150 x). Fig. 25: Pseudotracheal rows have an annular structure (2 600 x).

Fig. 26: Pseudotracheal rows (Ps) in dorsal view. One end of an incomplete ring has. the shape of a fish-tail (H), the other end forms a wish-bone (V) (K: chitinous plates leaning to the surface). The pseudotracheal rows are as far open as possible, which permits filtering of low efficacy only (2 000 x).

Fig. 27: Pseudotracheal rows (Ps) in dorsal view (V: wish-bone end, K: chitinous plates leaning to the surface) (2 000 x).

Fig. 28: Chitinous plates (K) leaning out of the pseudotracheal rows, and their articular con­ nection (Kc). The plates leave open small spaces for filtering (10 000 x).

Fig. 29: Pseudotracheal rows with filtering fissures reduced to a minimum (1 100 x).

Fig. 30: The surface of a tissue-culture is abraded by a sucking of a house fly. The remains of cells and nuclei are still discernible, the distinct contour of cells is blurred (1 000 x).

Fig. 31: A diagramm of longitudinal sections of the labella (1= resting position; II-VI = feeding positions; 11= filtering position, 111= cupping position, IV= intermediate position, V= scraping position, VI= sucking position). From Graham-Smith, 1930 and Green- berg, 1973.

Fig. 32: A schematic lateral view (contour) of the prestomal teeth of: a) Musca autumnalis, b) Musca domestica, c) Musca osiris (drawn from SEM pictures).

Fig. 33: Filtering fissures and structure of the chitinous skeleton of the pseudotracheae based on light-microscopic studies: a) dorsal view; b) alternating relationships of the extremities of the pseudotracheal rings; c)interbifid space of a pseudotracheal ring in a lateral view. From McAlpine, 1981.

Fig. 34: A schematic view of: a) the cross-section of pseudotracheae; b) the structures of pseudotracheae seen from the external surface, based on SEM pictures (H = fish­ tail end, V= wish-bone end, K= chitinous plate, Kc= articular connection of chitin­ ous plates). The other structures of labella, the pseudotracheae (Fig, 10) were also studied. The pseudo­ tracheae touch the surface where the fly is sucking from, from the filtering to the intermedi­ ate positions of the labella (see Fig. 31). The pseudotracheae of the three Musca species are not much different in their structure and since they are rather small (ca. 10 jam), they are insignificant in differentiation of these species. The pseudotracheae are open (or closed) channels running from the periphery to the entrance of the food-channel and they play a role both in filtering (differentiating) the food particles and in the transport of food (Fig. 25). The fine structure of these channels was described by GRAHAM-SMITH (1930) based on studies by traditional light microscopy. His observations can be complemented by SEM micrographs in some details. Our Figs 33-34 compare their schematic structure based on light micro­ scopy and on SEM micrographs. Their annular structure is also discernible on SEM pictures (Fig. 25) but fish-tail ends and wish-bone ends are hardly distinguishable (Figs 33, 34). In fact, one fish-tail end and laterally directed twig of its neighbouring two wish-bone ends are joint into such a chitinous plate which turns laterally and slightly ventrally (Figs 26-271 These latter ventrally directed chitinous plates have one process each towards their two neighbour­ ing plates and they are articularly connected (Fig. 28). These same chitinous plates are con­ tinued into the membrane covering the haemocoel of the labella. This fact becomes impor­ tant when the haemocoel is filled up with haemolympha: the membrane is raised and these chitinous plates tighten the fissures of the pseudotracheal rows (Figs 33, 34, 28, 29). An ef­ flux of the haemocoel results in the opening of the pseudotracheal rows through a reverse mechanism (Fig. 26). This latter process has been surveyed by SEM micrographs only and no more documentative data were searched for. For the harmful effect on the surface (epi­ thelium) caused by the secretophagous flies, nothing but any movement of the pseudotracheal rows is to be considered. The significance of these movements is proved by those minor in­ juries (abrasions) which are seen on projecting points of a tissue-culture caused by a suck­ ing of a house fly (Fig. 30) (for details see MEDVECZKY et al., 198 8, KOVÁCS SZ. et al. , in print).

DISCUSSION

The morphology of the mouthparts of three species of Musca was studied from the point of view of a better knowledge of those structures which enable the flies to injure the animal epi­ thelia while sucking. The importance of this point of view is underlined by the fact that flies frequently vomit. This way the microbial pathogens sucked in other places (a diseased ani­ mal, etc. ) will be scarified into the host animal through epithelial microlesions caused by the sucking and biting of the flies (e. g. MEDVECZKY et al., 1988). The secretophagous flies which have traditionally been regarded as passive carriers, this way are active causative agents ensuring the establishment of infections through their "bites". Our present work is aimed at surveying the morphological aspects of this intricate process. Originally, the hypo­ pharynx and the labrum of the haustellum and the chitinous structures of the labella seem to be the objectives to be considered.

In the haustellum, the labrum and the hypopharynx are acute structures, borne on a firm base and forming the food-channel. Their apices (below and above the food-channel) reach the level of the lamellae of the labella. In the sucking position when the lamellae of the label­ la are reclinate (and the prestomal teeth are directed laterally), the apex of the labrum-hy- popharynx can reach the surface where the fly is sucking from, and may cause injuries on the animal epithelia. We cannot exclude this as a real possibility, though our trials to detect the nature of the microlesions did not prove any lesion as a result of this kind of function of the labrum-hypopharynx. Possibly, studies of other kind can exclude the importance of this function.

The prestomal teeth are the sharpest chitinous structures on the labella even at first glance (Fig. 9-10). The main features of the prestomal teeth of the different species of Musca have been described by light microscopic studies. In the present study schematic drawings of these teeth were made based on SEM micrographs, emphasizing their main contours in or­ der to present a basis for differentiating these species in the proper position of the mouth- parts also for light microscopic studies. This study was supplemented by a functional test of the prestomal teeth. By this latter test, the morphological possibility that the prestomal teeth are also capable of biting out small pieces of epithelia has been proved.

In the course of this study the abrasing effect caused by the movements of pseudotracheae has attracted attention. These latter structures rise 5 to 10 um over their bases. In the filtering, cupping and intermediate positions of the labella the pseudotracheae touch the surface of the object where the fly is feeding from. In the meantime a strong suction pressure is generated and together with the movements of the pseudotracheae this caused a strong abrasing effect. Our SEM studies on the pseudotracheae have revealed some morphological details which slightly modify and supplement the model of the structure suggested by GRAHAM-SMITH (1930), and thus they are contributions to a better understanding of the function, movements and driving of the pseudotracheae.

The above morphological observations and functional studies are recommended to the atten­ tion of applied parasitologists and epizootiologists. They are aimed at a survey of those in­ terrelations which emerge with the transmission of animal diseases by secretophagous flies. After completing our studies, we believe, these three secretophagous Musca species (M. au- tumnalis, M. domestica, M. osiris) are shown to have a more significant and active role in spreading several diseases than thought before and expressed in terms like "passive trans­ port".

Acknowledgement. I am greatly indebted to Mrs. Erzsébet GONDÁR (ENNO VA TEXT, Buda­ pest) for preparing the SEM micrographs.

A szerző három szekretofág Musca faj szájszerveit vizsgálta pásztázó elektronmikroszkópos felvételek alapján. Célja az volt, hogy felhívja a figyelmet azokra a szájszervi struktúrákra, amelyek segítségével ezek a legyek apró sérüléseket képesek létrehozni azokon a helyeken, ahol élő állaton táplálkoznak. Különösen fontosak ezek az ismeretek a gazdaállatok nem sza- rusodó hámfelületein (nyálkahártyák, a szemek szaruhártyája) okozott mikroléziókkal kapcso­ latosan A morfológiai adatok (a kitinizáció keménysége, a szervek alakja, hossza, elhe­ lyezkedése és élessége) alapján a labrum, a hypopharynx és a prestomalis fogak mind képe­ sek nemcsak mikrosérülések létrehozására, hanem akár az egész nyálkahártyát átmetszhe­ tik. Ezen struktúrák funkcionális vizsgálata (élő szöveteken) azonban megmutatta, hogy a va­ lóságos mikrosérüléseket a prestomalis fogak és a pszeudotrachea sorok okozzák, a labrum és a hypopharynx káros hatását nem sikerült bizonyítani. A szerzőt megelőző SEM morfoló­ giai vizsgálatok alapján a pszeudotracheák sorait nem tekintették olyan szervnek, amely ké­ pes mechanikai sérüléseket okozni; a szerző azonban bebizonyította, hogy azok dörzsölő mozgása felsérti a nyálkahártyát. A három Musca faj prestomalis fogainak jellegzetességeit taxonómiai megkülönböztetésükre is fel lehet használni.

;j7 REFERENCES

BROCE, A.B. - ELZINGA, R.J. (1984): Comparison of prestomal teeth in the face fly (Mus­ ca autumnalis) and the house fly (Musca domestica) (Diptera: Muscidae). - J. Med. En­ tomol., 21(1): 82-85.

GRAHAM-SMITH, G.S. (1930): Further observations on the anatomy and function of the pro­ boscis of the blow-fly, Calliphora erythrocephala L. - Parasitology, 22: 47-115.

GREENBERG, B. (1973): Flies and disease. Volume II. Biology and disease transmission. - Princeton University Press, Princeton, pp. 447.

IWASA, M. (1983): A comparative study on the mouth-parts of medically and veterinarily im­ portant flies, with special reference to the development and origin of the prestomal teeth in cyclorrhaphous Diptera. - Jap. J. Sanit. Zool., 34: 177-206.

KOVÁCS-SZ., F. (1988): A simple scanning electron microscope preparatory method for the mouthparts of muscoid flies (Diptera). - Acta biol. hung., in press.

KOVÁCS-SZ., F. - G ON DAR, E. (1986): SEM studies on the mouthparts of Musca autumna­ lis, M. domestica and M. osiris. - In: DARVAS, B. - PAPP, L. (eds): Abstracts of the First International Congress of Dipterology, Budapest, p. 131.

MAHANKO, E.V. (1973): Parasitism rate and structure of oral teeth in synantropic flies of the genera Hydrotaea R. -D. and Musca L. (Diptera, Muscidae). - Ent. Obozr., 52(4): 768-781 (in Russian). McALPINE, J. F. (1981): Morphology and terminology - adults. In: Manual of Nearctic Di­ ptera, Volume 1. - Research Branch, Agriculture Canada, Monograph No. 2J: 9-63.

MEDVECZKY, I. - KOVÁCS, L. - KOVÁCS-SZ., F. - PAPP, L. (1988): The role of the house fly, Musca domestica, in the spread of Aujeszky's disease (pseudorabies). - Med. Vet. Entomol., 2(1): 81-86.

PAYSINGER, H.T. - NOBLET, R. - ADKINS, T.R. - VAUGLER, E.T. (1978): Scanning electron microscopy of the adult mouthparts of the ham fly and the face fly. - J. Geor­ gia Ent. Soc, 13: 28-39. SULOCHANA, S. - DERBYSHIRE, J.B. (1977): Scanning electron microscopical observa­ tions on the cytopathology of porcine enteroviruses on PK-15 cells. - J. Gen. Virol., 37: 415-418.

WEST, L. S. (1951): The housefly, its natural history, medical importance, and control. - Comstock Publ. Co., Ithaca, New York, pp. 584.

Received: 1 August, 1987 Dr. KOVÁCS-SZ., F. Veterinary Medical Research Institute Hungarian Academy of Sciences H-1581 Budapest P.O.B. 18 HUNGARY