Our results on the characterization of transferrin in tsetse suggest that it plays multiple physiological roles in immunity, iron metabolism and reproduction similar to that observed in other insects and vertebrates. However, the extreme physiological adaptations of the tsetse fly required for its blood specific diet and its viviparous reproductive strategy reveal further insights to the function of transferrin in insects in general as well as raise new questions.
expression in tsetse has similarities and differences with other insects in which it has been characterized. Northern analysis of tissue specificity shows that transferrin transcript is abundant in the fat body/milk gland fraction with lower levels of expression in the reproductive tract. Immunohistochemical experimental results indicate that this protein appears to be predominantly synthesized in the milk gland tissue of tsetse and not the fat body. This is different to what is seen in other insects where transferrin is expressed primarily in the fat body (Harizanova et al. 2005
; Hirai et al. 2000
; Yoshiga et al. 1999
). This suggests that the accessory (milk) gland in tsetse may have taken up the role of the fat body in the synthesis of this protein. Its abundant presence in the milk ducts indicates that it is transferred into the intrauterine larvae in the mother’s “milk” to nurture its development. The presence of transferrin protein in the hemolymph and oocytes, suggests that the protein synthesized in the milk gland is also secreted into the hemolymph. This would explain the areas of intense staining that are observed at the tips of the milk gland tubules in the immunohistochemical analysis. The presence of GmmTsf in the oocytes suggests that it is being taken up as a vitellogenic protein. The predominant yolk protein, GmmYP1 is expressed exclusively by the ovaries (Attardo et al., 2006
). The GmmTsf
gene differs in that it is expressed predominantly in the fat body/milk gland tissues with significantly lower levels expressed in the ovaries ( Lanes 2+3). One possible way that transferrin is entering the reproductive tract is via milk secretions. If milk spills into the ovaries from the uterus, this would put GmmTsf in proximity with the oocytes and allow the protein to be taken up by receptor mediated endocytosis. Another possibility is that the low levels of GmmTsf
transcript generated in the ovary are enough to supply adequate GmmTsf protein to the developing oocytes.
In tsetse, transferrin is expressed in both sexes although its spatial and temporal pattern differs. In females the majority of expression is in the fat body/milk gland tissues whereas in males the gene is expressed in the reproductive tract (Strickler-Dinglasan et al. 2006
). GmmTsf protein can be detected in the hemolymph and reproductive tracts of both males and females. The fact that the protein is found in the reproductive tract of both sexes suggests that this protein plays an important role in reproduction and/or development.
The mechanism of how GmmTsf gets into the male hemolymph from the reproductive tract is not yet known; however microscopic examination of the male reproductive tract has shown that the male has two large glandular organs attached to the reproductive tract that appear physiologically similar to the milk gland in the female (data not shown). These glands may be male analogues of the milk gland that are used for protein production and secretion. Further research will be needed to confirm these hypotheses.
The temporal expression of transferrin in tsetse also differs from that seen in other insects. In tsetse GmmTsf
is only significantly expressed in the adult stages of development. This differs from Drosophila
and Aedes aegypti
where transferrin is expressed in the late larval, pupal and adult stages (Harizanova et al. 2005
; Yoshiga et al. 1999
). The differences in the expression pattern of transferrin between tsetse and other dipterans may be a result of the fact that the immature stages in tsetse (larvae and pupae) rely on their mother’s nutritional resources for their development. The presence of GmmTsf in the ovaries and in the milk provided to the larvae suggests that it is playing an important role in the development of these stages. Interestingly, lactoferrin (a member of the transferrin family) is commonly found in high concentrations in mammalian milk secretions with human colostrum containing up to 7 grams per liter (Farnaud and Evans 2003
). The presence of transferrin in milk secretions suggests functional conservation for this aspect of reproductive biology.
Multiple functions have been attributed to transferrin, such as iron binding/transport, immune peptide and vitellogenic protein (Nichol et al. 2002
). In vertebrates transferrin has also been associated with induction of neutrophilic end-stage maturation, activation of enzymes regulating cell growth in conjunction with an insulin like growth factor and upregulation of chemokine synthesis by human proximal tubular epithelial cells (Evans et al. 1989
; Tang et al. 2002
; Wang et al. 1995
). If these types of functions are also conserved in insects then they have the potential to be important for oogenesis, larvigenesis and metamorphosis. Further analysis of these functions will be important to determine the developmental role of GmmTsf in tsetse.
High levels of GmmTsf in the hemolymph of males and females suggest that the protein is also playing an important role in adults. Transferrins are know to function as iron transporters in blood feeding insects, moving iron from the digested blood meal through the hemolymph to target tissues for storage in ferritin protein complexes (Huebers et al.
, 1988). In a recent microarray based global expression analysis study in tsetse, we have observed significantly higher levels of transferrin expression in females in agreement with our analysis here (Attardo and Aksoy, unpublished observations). In these same experiments, the Drosophila
NRAMP2 (DMT1) homolog is also detected as a highly expressed female specific gene. NRAMP2 in vertebrates has been shown to transport iron with high affinity in the intestines (Mackenzie and Hediger 2004
). The potential interaction of transferrin with NRAMP2 and the role of this interaction in iron metabolism will require further investigation.
Transferrin has also been shown to be an important part of the immune system in insects and vertebrates. Upregulation of its transcription following immune challenge has been observed in a number of insects including Ae. aegypti
, Bombyx mori
(Yoshiga et al. 1999
; Yoshiga et al. 1997
; Yun et al. 1999
) and also in the studies reported here in tsetse. Recent work on transferrin expression in goldfish (Carassius auratus
) has shown that transferrin acts to activate the macrophage antimicrobial response (Stafford and Belosevic 2003
). Specifically it results in the generation of the free radical nitrous oxide, which in turn is used to kill invading microbes. It is possible that in insects, transferrin is similarly acting upon the blood cells, hemocytes similar to vertebrate macrophages, in order to induce host immune responses. It is interesting that in the goldfish system transferrin was only active after proteolytic cleavage at a specific site. Proteolytic cleavage of transferrins has also been observed in D, melanogaster
and Ae. aegypti
(Harizanova et. al., 2005
). Alignment of multiple insect transferrins (including tsetse) reveals conservation of the proposed cleavage site that was identified in the goldfish transferrin gene. Cleavage of transferrin appears to occur in Glossina
as well, as multiple transferrin bands can be detected in the larval and pupal developmental stages. Whether transferrin is being cleaved to regulate development, immunity or both in the immature stages is not known and will require further study. Transferrin cleavage in immune challenged flies also has not yet been examined and requires further study to see if this mechanism may play a role in activating the host’s antimicrobial defenses.
Another point of interest is that tsetse flies with a stable midgut trypanosome infection express lower levels of transferrin transcripts than uninfected flies. Furthermore, flies that were challenged but resisted trypanosome infection have close to normal levels of transferrin transcripts. This suggests the possibility that the trypanosomes in the infected flies might be influencing host gene expression in order to provide more hospitable living conditions, such as greater iron availability and lower levels of free radicals. Such a strategy would provide additional nutritional resources and security to the replicating parasites, but as a result undermine the host’s reproductive fitness.
In conclusion, transferrin apparently plays a complex role in the life history of the tsetse fly. It appears to be involved in reproduction, development, homeostasis and immunity. Further analysis of this factor will yield important insights into the biology of the tsetse fly and the role of transferrin in all animals.