Tsetse fly (Diptera: Glossinidae) viviparous reproductive physiology remains to be explored at the molecular level. Adult females carry their young in utero for the duration of embryonic and larval development, all the while supplying their offspring with nutrients in the form of a "milk" substance secreted from a modified accessory gland. Flies give birth to fully developed third instar larvae that pupariate shortly after birth. Here, we describe the spatial and temporal expression dynamics of two reproduction-associated genes and their products synthesized during the first and second gonotrophic cycles. The proteins studied include a putative yolk protein, Glossina morsitans morsitans yolk protein 1 (GmmYP1) and the major protein found in tsetse "milk" secretions (Glossina morsitans morsitans milk gland protein, GmmMGP). Developmental stage and tissue-specific expression of GmmYP1 show its presence exclusively in the reproductive tract of the fly during oogenesis, suggesting that GmmYP1 acts as a vitellogenic protein. Transcripts for GmmMGP are present only in the milk gland tissue and increase in coordination with the process of larvigenesis. Similarly, GmmMGP can be detected at the onset of larvigenesis in the milk gland, and is present during the full duration of pregnancy. Expression of GmmMGP is restricted to the adult stage and is not detected in the immature developmental stages. These phenomena indicate that the protein is transferred from mother to larvae as nourishment during its development. These results demonstrate that both GmmYP1 and GmmMGP are involved in tsetse reproductive biology, the former associated with the process of oogenesis and the latter with larvigenesis.

Iron is an essential element for metabolic processes intrinsic to life, and yet the properties that make iron a necessity also make it potentially deleterious. To avoid harm, iron homeostasis is achieved via proteins involved in transport and storage of iron, one of which is transferrin. We describe the temporal and spatial aspects of transferrin (GmmTsf) expression and its transcriptional regulation in tsetse where both the male and female are strictly hematophagous. Using Northern, Western and immunohistochemical analysis, we show that GmmTsf is abundant in the hemolymph and is expressed in the adult developmental stages of male and female insects. It is preferentially expressed in the female milk gland tubules and its expression appears to be cyclical and possibly regulated in synchrony with the oogenic and/or larvigenic cycle. Although no mRNA is detected, GmmTsf protein is present in the immature stages of development, apparently being transported into the intrauterine larva from the mother via the milk gland ducts. Transferrin is also detected in the vitellogenic ovary and the adult male testes, further supporting its classification as a vitellogenic protein. Similar to reports in other insects, transferrin mRNA levels increase upon bacterial challenge in tsetse suggesting that transferrin may play an additional role in immunity. Although transferrin expression is induced following bacterial challenge, it is significantly reduced in tsetse carrying midgut trypanosome infections. Analysis of tsetse that have cured the parasite challenge shows normal levels of GmmTsf. This observation suggests that the parasite in competing for the availability of limited dietary iron may manipulate host gene expression.

Beetles (Coleoptera) are the most diverse animal group on earth and interact with numerous symbiotic or pathogenic microbes in their environments. The red flour beetle Tribolium castaneum is a genetically tractable model beetle species and its whole genome sequence has recently been determined. To advance our understanding of the molecular basis of beetle immunity here we analyzed the whole transcriptome of T. castaneum by high-throughput next generation sequencing technology. Here, we demonstrate that the Illumina/Solexa sequencing approach of cDNA samples from T. castaneum including over 9.7 million reads with 72 base pairs (bp) length (approximately 700 million bp sequence information with about 30× transcriptome coverage) confirms the expression of most predicted genes and enabled subsequent qualitative and quantitative transcriptome analysis. This approach recapitulates our recent quantitative real-time PCR studies of immune-challenged and naïve T. castaneum beetles, validating our approach. Furthermore, this sequencing analysis resulted in the identification of 73 differentially expressed genes upon immune-challenge with statistical significance by comparing expression data to calculated values derived by fitting to generalized linear models. We identified up regulation of diverse immune-related genes (e.g. Toll receptor, serine proteinases, DOPA decarboxylase and thaumatin) and of numerous genes encoding proteins with yet unknown functions. Of note, septic-injury resulted also in the elevated expression of genes encoding heat-shock proteins or cytochrome P450s supporting the view that there is crosstalk between immune and stress responses in T. castaneum. The present study provides a first comprehensive overview of septic-injury responsive genes in T. castaneum beetles. Identified genes advance our understanding of T. castaneum specific gene expression alteration upon immune-challenge in particular and may help to understand beetle immunity in general.

The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6–8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite–host coevolution, and disease prevalence are discussed.

The regulation of iron is critical for maintaining homeostasis in the tsetse fly (Diptera:Glossinidae), in which both adult sexes are strict blood feeders. We have characterized the cDNAs for two putative iron-binding proteins (IBP) involved in transport and storage; transferrin (GmmTsf1) and ferritin from Glossina morsitans morsitans. GmmTsf1 transcripts are detected in the female fat body and in adult reproductive tissues, and only in the adult developmental stage in a bloodmeal independent manner. In contrast, the ferritin heavy chain (GmmFer1HCH) and light chain (GmmFer2LCH) transcripts are expressed ubiquitously, suggesting a more general role for these proteins in iron transport and storage. Protein domain predictions for each IBP suggest both the conservation and loss of several motifs present in their vertebrate homologues. In concert with many other described insect transferrins, putative secreted GmmTsf1 maintains 3 of the 5 residues necessary for ironbinding in the N-terminal lobe, but exhibits a loss of this iron-binding ability in the C-terminal role as well as a loss of large sequence blocks. Both putative GmmFer1HCH and GmmFer2LCH proteins have signal peptides, similar to other insect ferritins. GmmFer2LCH has lost the 5'UTR ironresponsive element (IRE) and, thus, translation is no longer regulated by cellular iron levels. On the other hand, GmmFer1HCH maintains both the conserved ferroxidase center and the 5'UTR IRE; however, transcript variants suggest a more extensive regulatory mechanism for this subunit.