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
is predominantly localized in the ovaries and is passed from generation to generation by transovarial transmission. Sodalis
are both hypothesized to be transmitted to the larva via milk gland secretions (Denlinger and Ma 1975
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
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 6–8 post-eclosion and begins embryonic development. At days 10–13 the embryo hatches and larval development begins. Development continues to between days 19–21 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.