Analysis of milk gland structure and function in Glossina morsitans: Milk protein production, symbiont populations and fecundity

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Abstract

A key process in the tsetse reproductive cycle is the transfer of essential nutrients and bacterial symbionts from mother to intrauterine offspring. The tissue mediating this transfer is the milk gland. This work focuses upon the localization and function of two milk proteins (milk gland protein (GmmMGP) and transferrin (GmmTsf)) and the tsetse endosymbionts (Sodalis and Wigglesworthia), in the context of milk gland physiology. Fluorescent in situ hybridization (FISH) and immunohistochemical analysis confirm that the milk gland secretory cells synthesize and secrete milk gland protein and transferrin. Knockdown of gmmmgp by double stranded RNA (dsRNA) mediated RNA interference results in reduction of tsetse fecundity, demonstrating its functional importance in larval nutrition and development. Bacterial species-specific in situ hybridizations of milk gland sections reveal large numbers of Sodalis and Wigglesworthia within the lumen of the milk gland. Sodalis is also localized within the cytoplasm of the secretory cells. Within the lumen, Wigglesworthia localize close to the channels leading to the milk storage reservoir of the milk gland secretory cells. We discuss the significance of the milk gland in larval nutrition and in transmission of symbiotic bacteria to developing offspring.

Introduction

Tsetse (Diptera: Glossinidae) are important vectors of African trypanosomes which cause disease of both medical and agricultural importance. Trypanosomes are the causative agents of sleeping sickness (Human African Trypanosomiasis—HAT) and nagana in sub-Saharan Africa. There are limited tools for control of these diseases in the mammalian host but control has been realized via tsetse population reduction methods in previous efforts. Given the low reproductive capacity of tsetse, additional knowledge on its reproductive physiology can provide new avenues for control.

Flies have developed various forms of viviparous reproduction. However, most of these undergo facultative viviparity, resulting in the deposition of developing embryos or larvae. Tsetse reproductive physiology is unique as the female carries and nourishes their offspring for their entire larval developmental cycle. Females develop a single oocyte at a time. The oocyte is ovulated, fertilized and undergoes embryonic development in the uterus. The resulting larva hatches and is carried and nourished in the intrauterine environment for the duration of its development. Within an hour of parturition, the larva burrows into the earth and pupates. This viviparous strategy is termed pseudo-placental unilarviparity and has been observed in three other families of flies, Hippoboscidae, Nycteribiidae and Streblidae (Meier et al., 1999). All of these families are close relatives of tsetse and are haematophagous (blood feeding). Viviparity and blood feeding may be associated due to the nutritional demands of the intrauterine larva. Blood is one of the few sources of nutrition rich enough support this reproductive strategy.

Tsetse reproductive physiology differs from other Diptera in significant ways. These differences accommodate the requirements of the viviparous reproductive strategy. The uterus is a modified vaginal canal that is covered with highly tracheated muscle tissue. The uterus has the capacity to hold a mature third instar larva equivalent in weight to the mother. Larval nutrition is provided via a modified accessory gland (milk gland) that empties into the uterus. The milk gland is connected to the dorsal side of the uterus and expands throughout the abdominal cavity of the fly as bifurcating tubules intertwining with fat body tissue (Tobe and Langley, 1978). The lumen of the milk gland is surrounded by secretory and epithelial cells. The epithelial cells secrete and maintain the chitinous lining of the lumen. The secretory cells contain large nuclei and surround an extracellular secretory reservoir which changes size dynamically as it fills with and empties milk secretions over the course of pregnancy. The opening of the reservoir into the lumen is covered by a fibrous plug which is confluent with the lining of the lumen (Ma et al., 1975, Tobe et al., 1973).

Tsetse has a biological association with three bacterial species, Sodalis glossinidius, Wigglesworthia glossinidia and Wolbachia pipientis (Aksoy, 2000). Wolbachia is predominantly localized in the ovaries and is passed from generation to generation by transovarial transmission. Sodalis and Wigglesworthia are both hypothesized to be transmitted to the larva via milk gland secretions (Denlinger and Ma, 1975). Sodalis is detectable in multiple tissues within the fly, including the milk gland, as evidenced by a symbiont specific PCR amplification assay (Cheng and Aksoy, 1999). Wigglesworthia resides in specialized cells (bacteriocytes) that form the bacteriome in the midgut. The transmission route of Wigglesworthia from mother to the developing progeny was also assumed to be via the milk. Electron microscopy analysis showed bacteria in the lumen of the milk gland and based upon their large size, these bacteria were thought to be Wigglesworthia (Denlinger and Ma, 1975). However, the identity of these bacteria remains unconfirmed.

The milk secreted from the milk gland into the uterus consists primarily of protein and lipids (Cmelik et al., 1969). Two milk proteins were characterized, the milk gland protein (GmmMGP) and transferrin (GmmTsf) (Attardo et al., 2006, Guz et al., 2007). Other milk proteins have been detected and remain uncharacterized (Riddiford and Dhadialla, 1990). GmmMGP and GmmTsf are synthesized by the adult female and are transferred to the developing larva. During the first gonotrophic cycle, gmmmgp expression correlates with larval development. The first oocyte is ovulated into the uterus between days 6 and 8 post-eclosion and begins embryonic development. At days 10–13 the embryo hatches and larval development begins. Development continues to between days 19 and 21 when the mother undergoes parturition. Over the course of larval development, gmmmgp transcript levels make a dramatic increase in abundance beginning around day 8 post-eclosion through partuition. After the first gonotrophic cycle expression of gmmmgp remains constitutive. However, GmmMGP protein is almost undetectable in the mother upon larval deposition illustrating its transfer from mother to larva (Attardo et al., 2006).

Expression of gmmtsf differs somewhat from that of gmmmgp. In females transcript abundance of gmmtsf is cyclic and correlates with oogenesis and larvigenesis. Expression of gmmtsf also differs from gmmmgp as it is expressed in both males and females. GmmTsf protein levels are constitutive and can be found in the milk gland, hemolymph, reproductive tract and developing larva. An increase in GmmTsf is observed during larval development, however, it is not equivalent to observed levels of GmmMGP (Guz et al., 2007).

The primary goals of this research are: (1) to investigate the physiological and functional characteristics of the major milk proteins, GmmMGP and GmmTsf, in the specific context of the milk gland and (2) to identify and localize the symbionts residing in the milk gland. The role of milk proteins in fecundity and vertical transmission biology of tsetse's symbionts are discussed.

Section snippets

Fly rearing

The Glossina morsitans morsitans colony maintained in the insectary at Yale University was originally established from puparia from fly populations in Zimbabwe. Newly emerged flies are separated by sex and mated at 3–4 days post-eclosion. Flies are maintained at 24 ± 1 °C with 50–55% relative humidity, and receive defibrinated bovine blood every 48 h using an artificial membrane system (Moloo, 1971).

Tissue dissection and sample preparation

Fat body and milk gland were collected from pregnant females. Fat body and milk gland were detached

Milk gland protein and mRNA localization within the milk gland

Florescent light microscopy of sectioned milk gland and fat body tissue was performed to expand upon the previous whole mount immunohistochemical analysis of the milk gland (Attardo et al., 2006). Fat body and milk gland tubules were analyzed by in situ hybridization and immunohistochemical analysis to identify cellular and subcellular expression and localization of milk gland protein transcript and protein.

DAPI staining reveals the secretory cell nuclei (Fig. 1A and E). In situ hybridization

Discussion

Data presented confirm the synthesis and secretion of GmmMGP and GmmTsf by the milk gland secretory cells for use in larval nutrition. Functional evidence of GmmMGP's role in larvigenesis is demonstrated by the negative impact on fecundity resulting from its knockdown. Bacterium specific FISH analysis indicates the presence of both tsetse symbionts, Sodalis and Wigglesworthia, in the milk gland. Sodalis resides both in the milk gland lumen and within the milk gland secretory cells, while

Acknowledgements

We would like to acknowledge Severine Balmand (UMR203 Biologie Fonctionnelle Insectes et Interactions, IFR41, INRA, INSA-Lyon, F-69621 Villeurbanne, France) for her technical assistance and training in FISH techniques. This work was generously funded by grants to S.A. from NIHGM (069449), NIAID (051584), Li Foundation and Ambrose Monell Foundation. G.M.A was funded by the NIH Ruth Kirshstein Postdoctoral Training Award F32 GM077964.

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