In this work, we used an in silico approach to search for unique milk proteins in a tsetse cDNA database and describe functional aspects of two novel milk genes resulting from that search (gmmmgp2 and gmmmgp3). The predicted open reading frames of both genes contain a signal peptide at the N-terminus suggesting that these proteins are secretory products. The expression profiles for both of these genes is female and milk gland tubule specific and expression levels correlate with intrauterine embryonic and larval development events during the gonotrophic cycle. Knockdown of gmmmgp2 by RNAi results in reduced fecundity suggesting that this protein component is required for ovulation and intrauterine larval development. Flies in the gmmmgp2 knockdown group also appeared to have higher mortality levels than the controls. Knockdown of gmmmgp3 does not result in an effect upon fecundity suggesting that this protein may be non-essential or redundant.
Previous work on the tsetse milk proteins identified a number of unknown proteins produced by the milk gland tubules over the course of a pregnancy (Riddiford and Dhadialla 1990
). Data mining and analysis of the fat body/milk gland specific cDNA library facilitated the analysis of these two novel proteins that appear to function either as nutrient proteins, regulatory/signaling proteins or both. The phenotype associated with the gmmmgp2
knockdown suggests that this protein may have regulatory properties. Female gmmmgp2
knockdown flies undergo oogenesis, however the fully developed oocytes are never ovulated into the uterus and appear to be broken down and absorbed back into the ovary. These flies also appear to have a higher mortality rate. The cause of this increase is not apparent and further research will be required to understand the physiological consequences of losing the function of this protein. The mode and site of action for this protein remains unknown and bears further analysis to understand its role in the reproductive cycle and tsetse physiology in general. It is possible that GmmMGP2 is provided to the larva as a component of the milk. However, another possibility is that it functions as a regulatory protein that is secreted from the milk gland tubule into the hemolymph rather than the milk gland lumen where it effects ovulation by acting on the uterus/ovaries. Alternatively, the protein could be a milk component that acts as a signal for ovulation from within the uterus. In contrast, knockdown of the second protein, GmmMGP3, resulted in no apparent negative effects upon larval development and deposition. This suggests that this protein may be a non-essential or redundant component of the milk the loss of which is compensated for by other milk proteins.
The development of milk production by females to nourish offspring has evolved multiple times. Mammals, marsupials and a small number of insect species have developed lactation in a convergent manner. While lactation has developed in independent systems, there are interesting parallels in the type of proteins expressed in the milk secretions produced by these dramatically different organisms. The major milk protein in tsetse GmmMGP is in the lipocalin family of proteins (Attardo et al. 2006a
). Lipocalins bind small hydrophobic molecules and are thought to act as transporters for insoluble moieties (Flower et al. 2000
). Lipocalins are an important component in the milk secretions of viviparous cockroaches (Williford et al. 2004
), marsupials (Piotte et al. 1998
; Trott et al. 2002
) and mammals (Kontopidis et al. 2004
). Proteins with iron binding capability such as transferrin and lactoferrin are another common component of milk secretions and are associated with immune function through the sequestration of iron from pathogenic bacteria in the digestive tract of the offspring (Guz et al. 2007
; Raiha 1985
). The novel proteins identified here may not have structural orthologs in other lactation systems, but they may be representative of orthologous functions in other species. Further functional characterization of these proteins may reveal a unique solution to a common need in lactation or they may represent a specific function required by tsetse’s physiology.
Understanding of the transcriptional regulation of these proteins is important for the identification of the signals and factors regulating lactation in tsetse. The reduced (gmmmgp2) or absent (gmmmgp3) expression of these genes in virgin flies suggests that their regulation is associated with a mating stimulus (such as the transfer of male accessory gland proteins to the female) or a developmental stimulus resulting from larvigenesis.
The role of mating stimuli on female Diptera mating behavior and reproductive physiology is well documented. After mating Drosophila
enter a phase called the “Long-Term Post Mating Response” (LTR). During this period female flies are non-receptive to mating advances from males and the rate of egg production and deposition is increased (Manning 1967
). These changes in behavior and physiology are activated by the transfer of accessory gland proteins and sperm from the male to the female during copulation. One of the accessory proteins, “Sex Peptide or Acp70A”, stimulates the LTR response temporarily (1–2 days) if injected alone (Chen et al. 1988
). However, accessory gland proteins appear to require sperm for the effect to last as females mating with males that secrete accessory gland proteins but not sperm also show a temporary LTR response (Xue and Noll 2000
Recent work, demonstrates that accessory gland protein function requires a complex network of interactions amongst the proteins as well as physical association with sperm for mobilization to the spermathica which is required for longevity of the LTR response (Ram and Wolfner 2009
). This system appears to be conserved as orthologus proteins are present in Aedes aegypti (Sirot et al. 2008
) which undergo behavioral and physiological changes including suppression of host seeking behavior, stimulation of vitellogenesis, oviposition and decreased mating receptivity (Klowden 1999
). While we do see transcription of some milk proteins in unmated tsetse (gmmmgp1
, and gmmmgp2
) the level of transcription appears to be significantly lower than mated and pregnant females. Also gmmmgp3
does not appear to be transcribed at all in unmated females suggesting that mating status could play a larger role in its expression.
Another possible mechanism of milk protein activation is via stimuli related to larvigenesis. As is seen in , gmmmgp transcription increases over time in correlation with the size and demand for nutrients by the larvae. Transcriptional activation of milk-protein genes could be associated with feedback from stimulation associated with larval development (such as uterine stretch receptors) and/or the volume of milk in the storage reservoirs of the secretory cells lining the milk gland tubules. Transcriptional behavior of the GmmMGP2 and GmmMPG3 proteins can be studied in association with GmmMGP and GmmTsf to build a consensus of genes regulated by the pregnancy cycle in tsetse to understand the signals and factors regulating milk gland protein transcription and translation processes.
Comparative analysis of the regulatory regions of these and other genes will be important for identifying key transcription factors responsible for milk gland tissue specificity and for regulating expression levels during larval development to compensate for larval growth and demand for nutrients. Identification of the regulatory mechanisms controlling milk production in tsetse is essential to development of novel tsetse specific population control strategies.