Prevalence of enlarged salivary glands in wild populations of Glossina pallidipes in Kenya, with a note on the ultrastructure of the affected organ

JOURNAL

OF

INVERTEBRATE

36, 113- 118 (1980)

PATHOLOGY

Prevalence of Enlarged Salivary Glands in Wild Populations Glossina pallidipes in Kenya, with a Note on the Ultrastructure of the Affected Organ L. H. OTIENO, The International

Centre

E. D. KOKWARO, of Insect

Physiology

M. CHIMTAWI, and Ecology

(ICIPE).

of

AND P. ONYANGO P.O.

Bor

30772,

Nairobi,

Kenya

Received July 3, 1979 Enlarged salivary gland was found to be widespread among wild populations of GIossina palin Kenya. The incidence of this abnormality varied from 0.9% in Meru National Park in Central Kenya to 5.4% in the Shimba hills area on the Kenya coast. Ultrastructurally, the enlarged glands were multinucleated with lumen reduced substantially in size. A large number of viruses filled both the lumen and the broken pieces of epitheliaf cytoplasm. In some cases Tr.~panosoma bracci trypanosomes were seen in the lumen of the enlarged glands. The epithelial cytoplasm was heavily vacuolated. Comment is made on the suitability of the diseased flies as transmitters of T.

lidipes

brctcei. KEY WORDS: pathology oj:

Glossina

pallidipes;

Trypanosoma

INTRODUCTION

While investigating Glossina pallidipes in the Umfolosi game reserve in Zululand, Whitnall, (1934) found that a number of flies had hypertrophied salivary glands. He observed this abnormality in both sexes of adult and young flies. Burtt (1945) later noticed that the hypertrophied salivary glands were peculiar to G. pallidipes. The affected glands were about four times the normal width and had a chalky appearance. Jaenson (1978) observed that in G. pallidipes with enlarged salivary glands, the males were often completely sterile and the young females had an abnormal growth of the ovarioles. He found the abnormalities in both wild and laboratory-bred flies. In this communication, it is shown that hypertrophy of G. pallidipes salivary glands may be a common feature among wild populations of this group of tsetse flies in Kenya. An ultrastructural account of the affected gland is also given. MATERIALS

AND METHODS

Study Areas

The tsetse flies used for this work were from different lo-

G. pallidipes collected

brucei;

salivary

gland,

insect;

salivary

gland.

calities in Kenya, namely: (1) Kibwezi Forest (2” 25’S, 37” 55’E) some 200 km southeast of Nairobi; (2) Lambwe Valley Game Reserve (0” 40’S, 34” 10’E) situated in South Nyanza district, Western Kenya; (3) Meru National Park (0’ lO’N, 38” 19’E) on the slopes of Mt. Kenya, Central Kenya; and (4) Shimba Hills National Park Reserve (4” 30’S, 38” 45’E) on the Kenya Coast near Mombasa. Trapping Methods

See Swynnerton (1936) for details of various trapping methods. In Lambwe Valley where the fly population was high, Landgride and Moloo traps were used. The flies collected were examined at the ICIPE field station, Mbita Point, on the shores of Lake Victoria. The fly population at Kibwezi Forest was very low and consequently the “fly-round” hand net method was used to collect the flies. The flies collected were examined at a nearby Range Research Station, Kiboko. Challier traps were used both in Shimba Hills and Meru National Park. The flies caught along the Kenya coast were examined at the ICIPE coastal research station, Mombasa, 113

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0 1980 by Academic Press. Inc. of reproduction in any form reserved.

114

OTIENO

while flies caught from the Met-u National Park were examined at Leopard Rock Banda Camp. Detection

of Enlarged

Salivary

Glands

The flies caught were kept in individual plastic tubes (4.2 x 3.2 cm) with nylon gauze at both ends. In this way, it was easy to collect salivary secretions from each fly using the technique described by Youdeowei (1975). After saliva collection, the flies were killed, dissected, and salivary glands pulled out and examined under low power (250x) microscopy. Possible trypanosome infections in the hypertrophied salivary glands were noted. Histology

of Affected

Salivary

Glands

Tissues for light microscope studies were prepared by standard techniques for electron microscopy (Hayat, 1970). Before dissection, the flies were perfused with 2.5% glutaraldehyde buffered to pH 7.4 with 0.05 M sodium cacodylate containing 5% sucrose. They were then dissected out in the same glutaraldehyde solution as used for perfusion, and left to fix at room temperature for 2-3 hr. The tissues were subsequently washed several times in 0.05 M sodium cacodylate and postfixed for 1 hr in 1% osmium tetroxide buffered at pH 7.4 with 0.05 M sodium cacodylate. Dehydration was carried out at room temperature through an ethanol series, 10 min each and 30 min in 100% ethanol. This was followed by two changes in propylene oxide, 10 min in all. Embedding was done in araldite prepared by the method of Glauert and Glauert (1958). Sections 1 pm thick were cut with an LKB ultramicrotome and stained for general histology in 3% toluidine blue made up in 3% borax. Enlarged salivary glands. These were ground and blended in a microhomogenizor with phosphate buffer, pH 7.4., for 2 min and then sonicated. To reveal virus particles, the mixture was negatively stained with 2% phosphotungstic acid (PTA, pH 6.5) for l-2 sec. These were examined and

ET

AL.

PREVALENCE

photographed microscope.

with a Philips

OF

ENLARGED

201 electron

Trypanosome-infected hypertrophied and normal salivary glands. Thin sections

were cut from the same epon blocks which provided semithin sections for histological studies. Thin sections were picked up on uncoated 200- to 300-mesh copper grids and stained with uranyl acetate and lead citrate according to Reynolds (1963). Observations were made with a Philips 201 electron microscope. RESULTS

Milky, viscous salivary secretions were obtained only from flies with enlarged salivary glands. However, since many flies with hypertrophied salivary glands secreted normal (clear, colorless, and slightly viscous) saliva, the total number of flies found with enlarged glands were based only on dissections. Table 1 shows the number of flies examined from the various localities, the number with the enlarged salivary glands, and sex distribution among flies found with this abnormality. It may be seen from the table that the enlarged salivary glands were found in G. pallidipes from the four localities examined. The incidence of this abnomality varied from 0.9% in Meru National Park to 5.4% in the Shimba Hills area. The abnormality was nonetheless absent in 9 G. brevipalpis from Shimba Hills and 96 G. longipennis from Meru National Park. Histology Glands

and Ultrastructure

of Salivary

The morphology of normal and enlarged salivary glands was compared (Figs. 1, 2). The epithelium of the normal gland is made up of a single layer of cells (Fig. 1) enveloped by a thick basal lamina and a muscular coat. The epithelium includes much rough endoplasmic reticulum throughout the cytoplasm (Fig. 3) and Golgi complexes. The surfaces of adjacent cells are closely apposed to each other at the intercellular membranes (Fig. 3). The gland has

TSETSE

SALIVARY

GLANDS

115

a wide lumen (Fig. 1) tilled with loosely arranged matrix containing numerous electron-opaque filaments (Fig. 4). Cells from a hypertrophied gland were multinucleated and the gland had a reduced lumen (Fig. 2). In heavily infected glands the lumen was tilled with viruses and pieces of broken down epithelial cytoplasm and in some cases trypanosomes (Fig. 5). Cell membranes separating adjacent epithelial cells were indistinct, giving an impression of an amorphous tissue partitioned with virus-filled intercellular spaces (Figs. 2, 6). The nucleoli were circular to oval and the chromatin strands were condensed and were electron opaque (Fig. 7). The epithelial cytoplasm was heavily vacuolated (Fig. 8). The negatively stained salivary gland suspension contained rod-shaped virions similar to those found in tissue sections. The rods had a flattened profile (Fig. 9) where the ends were not obviously broken. The following tissues from infected flies contained no viruses: male accessory glands, testes, flight muscle, fat body, and spermathecae. Similar organisms were found only in the gut. DISCUSSION

The presence of viruslike particles in G. has been reported by Jenni (1973) and Jenni and Steiger (1974a). They also noted similar organisms in the nuclei of the midgut epithelial cells of G. fuscipes (see Jenni and Steiger, 1974b). In these reports, no evidence was shown of any cytopathic effects of the viruslike particles on the testes, although they were led to believe that the organism caused high mortality of young G. fuscipes and to the low hatching rate. Whitnall (1934), on the other hand, observed a peculiar abnormality in the structure of G. pallidipes salivary glands and hypopharynx. The 2.8% infected of the 1129 flies that he examined had distinct thickening of the salivary glands. The abnormality was seen in males, females, and young flies. Jaenson (1978) morsitans

FIG. 1. Photomicrograph of l-pm-thick transverse section through a normal salivary gland stained by toluidine blue-borax. The monolayered epithelium (Ep) is enveloped by a muscular coat (MC). The epithelium surrounds a darkly staining secretory material in the lumen (Lu) of the gland. x3375. FIG. 2. Photomicrograph showing salivary gland infected with viruses. Long arrow, nuclei; Lu, lumen: V. vacuole; short arrow, viruses; MC, muscle. x845. FIG. 3. Transmission electron micrograph of a portion of Figure 1 showing the relationship between the lumen (Lu) and the epithelium (Ep). The cytoplasm is composed of mitochondria (mi), ribosomes (R), rough endoplasmic reticulum (RER), and Golgi complexes (G). BL. Basal lamina. x9190. FIG. 4. Electron micrograph showing the lumen of a healthy gland. Filaments (F) are embedded in matrix (Mx). ~24,940. 116

FIG. 5. Lumen of virus-infected salivary gland studded with trypanosomes (T) and viruses (arrows). x6100. FIG. 6. Between the adjacent cells there are rather broad intercellular spaces (Is), and they contain viruses (arrow). Note viruses (arrow) in the nucleus (N). x6100. FIG. 7. Epithelial cells from salivary gland of infected fly. Several vacuoles (V), nuclei (N). and virus-containing nucleoli (NC) are shown. x2760. FIG. 8. Epithelium filled with large vacuoles (V) and vesicles (Ve) of various sizes containing viruses (arrow). x 13,125. FIG. 9. Electron micrograph of a negatively stained virus (arrow). ~57,000. 117

118

OTIENO

has recently given an account of enlarged salivary glands in both laboratory and field collected G. pallidipes. In this account he describes the presence of many long, rodlike, or slightly curved viruslike particles in the nuclei, cytoplasm, intercellular spaces, and in the lumina of enlarged salivary glands of G. pallidipes collected from Kibwezi Forest in Kenya. We have observed similar organisms. Our observations based on specimens obtained from G. palfidipes collected from Lambwe Valley, Western Kenya, some 700 km from the Kiboko Forest, showed that the cells from hypertrophied salivary glands were multinucleated thus reducing considerably the gland lumen. Cell membranes separating adjacent epithelial cells were partitioned with virus filled intercellular spaces. Negatively stained virus suspension contained rod-shaped virus similar to those described by Jaenson (1978). If these organisms are indeed similar, then it may be safe to assume that the enlarged salivary glands are caused by the virus. Attempts are currently being made to isolate the virus particles from the infected flies and to test their pathogenicity to tsetse flies. If found to be pathogenic, the virus could be considered as a biological control candidate. There are no records of virus causing epizootics among tsetse populations apart from the above reports. In this communication attention is drawn to the widespread incidence of the enlarged salivary glands (and by inference, virus infection) among wild populations of G. pallidipes in Kenya. The presence of a large number of trypanosomes in the enlarged salivary gland suggest that the degenerating tissue does not present an adverse environment to T. brucei, neither do the virions present any danger to the trypanosomes. However, the reduced lumen of the hypertrophied glands, and presumably the small quantities of viscous saliva secreted by such flies, could be

ET AL.

assumed to work against the efficient transmission of T. brucei. Whether these trypanosomes are transmitted with the same efficiency as those contained in normal healthy glands is worth examining. ACKNOWLEDGMENTS We are grateful to Professor M. Locke of the University of Western Ontario, Canada, and also Visiting Director of Research in the ICIPE Insect Histology and Fine Structure Unit for his useful comments on the micrographs. This work was done under the guidance of Professor Thomas R. Odhiambo and received financial support from the Rockefeller Foundation, New York, and the International Atomic Energy Agency (IAEA), Vienna, to whom we are greatly indebted.

REFERENCES BURTT, E. 1945. Hypertrophied salivary glands in Glossina: Evidence that G. pallidipes with this abnormality is peculiarly suited to trypanosomes infection. Ann. Trop. Med. Parasitol.. 39, ll- 13. GLAUERT, A. M., AND GLAUERT, R. H. 1958. Araldite as an embedding medium for electron microscopy. J. Biophys.

Biochem.

Cytol.,

4, 191-194.

HAYAT, M. A. 1970. “Principles and Techniques of Electron Microscopy: Biological Applications.” Vol. I. Van Nostrand Reinhold, New York. JAENSON, T. G. T. 1978. Virus-like rods associated with salivary gland hyperplasia in tsetse GIossina pallidipes. 234-238.

Trans.

Roy.

Sot.

Trop.

Med.

Hyg.,

72,

JENNI, L. 1973. Virus-like particles in a strain of G. morsitans centralis Machado 1970. Trans. Roy. Sot. Trop.

Med.

Hyg.,

67, 295.

JENNI, L., AND STEIGER, R. 1974a. Virus-like particles of Glossina fuscipes fuscipes Newst. 1910. Acta. Trop.,

31, 177-180.

JENNI, L., AND STEIGER, R. F. 1974b. Virus-like particles in the tsetse fly, Glossina morsitans sspp., preliminary results. Rev. Suisse Zool., 81,663-666. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17, 208-212. SWYNNERTON. C. F. M. 1936. The tsetse flies of East Africa. Trans. Rev. Entomol. Sot. Londun, 84, 33-36.

WHITNALL, A. B. M. 1934. The trypanosome infections of Gloss&a pallidipes in the Umfolosi Game Reserve, Zululand. Onderstepoort J. Vet. Anim. Ind., 11, 7-21. YOUDEOWEI, A. 1975. A simple technique for observing and collecting the saliva of tsetse flies (Diptera, Glossinidae). Bull. Entomol. Res., 65, 65-67.