Sphingomyelinase activity in mother's milk is essential for juvenile development: a case from lactating tsetse flies

Sphingosine is a structural component of sphingolipids. The metabolism of phosphoethanolamine ceramide (sphingomyelin) by sphingomyelinase (SMase), followed by the breakdown of ceramide by ceramidase (CDase) yields sphingosine. Female tsetse fly is viviparous and generates a single progeny within her uterus during each gonotrophic cycle. The mother provides her offspring with nutrients required for development solely via intrauterine lactation. Quantitative PCR showed that acid smase1 (asmase1) increases in mother's milk gland during lactation. aSMase1 was detected in the milk gland and larval gut, indicating this protein is generated during lactation and consumed by the larva. The higher levels of SMase activity in larval gut contents indicate that this enzyme is activated by the low gut pH. In addition, cdase is expressed at high levels in the larval gut. Breakdown of the resulting ceramide is likely accomplished by the larval gut-secreted CDase, which allows absorption of sphingosine. We used the tsetse system to understand the critical role(s) of SMase and CDase during pregnancy and lactation and their downstream effects on adult progeny fitness. Reduction of asmase1 by short interfering RNA negatively impacted pregnancy and progeny performance, resulting in a 4-5-day extension in pregnancy, 10%-15% reduction in pupal mass, lower pupal hatch rates, impaired heat tolerance, reduced symbiont levels, and reduced fecundity of adult progeny. This study suggests that the SMase activity associated with tsetse lactation and larval digestion is similar in function to that of mammalian lactation and represents a critical process for juvenile development, with important effects on the health of progeny during their adulthood.     

The milk gland of the sheep ked, Melophagus ovinus: A comparison with Glossina

Structurally, the milk gland of the sheep ked, Melophagus ovinus, is quite similar to that of the tsetse fly, Glossina morsitans. In both insects the highly branched gland consists of two cell layers. An extracellular reservoir associated with each secretory cell initially receives the secreted milk. Milk then passes into the gland lumen through a dense cuticular rete. Gram-negative bacteria, presumably symbionts, are abundant in the lumen. Unlike tsetse, the secretory reservoir of the sheep ked is bi-lobed, and the secretory cell nucleus remains centrally located throughout the pregnancy cycle. Lipid droplets are much more abundant in the cytoplasm of the ked secretory cell, and analysis of larval milk shows 5–6% higher lipid content in the sheep ked. Results of histochemical analysis of ked milk are consistent with the analysis of tsetse milk. Four major milk proteins are detectable with polyacrylamide gel electrophoresis. Changes in abundance of ER and sizes of the secretory cell nucleus and secretory reservoir reflect a dramatic cycle of glandular activity during pregnancy. Unlike tsetse, the sheep ked remains constantly on its host and appears to take frequent, but small, blood meals. This strategy implies that the demand for nutrient storage is less than in tsetse.     

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

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.     

CHANGES IN THE RIBOSOME CONTENT, PRINCIPAL MICROSOMAL PROTEIN COMPOSITION, AND SECRETORY CHARACTER OF MAMMARY EPITHELIAL ROUGH ENDOPLASMIC RETICULUM DURING DIFFERENTIATION : Evidence that Messenger RNAs Specific for Milk Proteins are Incorporated into Rough Endoplasmic Reticulum Formed De Novo after Parturition

The equilibrium density distribution, protein composition, and secretory character of mouse mammary epithelial rough microsomes have been determined during differentiation. The density range exhibited by the rough microsomes broadens during mammary development; rough microsomes within the 1.25–1.29 g/ml density range appear soon after conception and then within the 1.30–1.34 range after the onset of lactation. The appearance of these denser microsomes represents the progressive increase of the average ribosome content of the rough endoplasmic reticulum (ER) during gestation and lactation. Fractionation of rough microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two proteins, having molecular weights of 57,000 and 76,000, occur to a significant extent only during lactation and are then most prominent in the very dense rough microsomes of the 1.30–1.34 range. Nascent polypeptide chains discharged (by incubation with puromycin) from 17-days lactation rough microsomes in either the 1.21–1.29 or 1.30–1.34 density range are distributed equally between the intra- and extravesicular compartments. Whereas 36% of the chains are discharged intravesicularly from 1-day lactation rough microsomes in the 1.30–1.34 range, only 25% are so discharged from those in the 1.21–1.29 range. The results indicate (a) that there is no correlation between the relative levels in lactation rough microsomes of the two microsomal proteins which become prominent during lactation and the extent of secretory activity and (b) that for a short period after parturition the rough ER elements bearing high surface densities of ribosomes have a greater proportion of ribosomes synthesizing milk proteins than the rough ER elements with moderate ribosome densities.     

Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with K_D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.     

Storage and mobilisation of nutriment for uterine milk synthesis by Glossina morsitans

Nitrogen content of eggs and larvae of Glossina morsitans was a constant proportion of dry weight and equivalent to ca. 55% protein assuming tsetse proteins contain 16% nitrogen. The larval gut content (uterine milk) contained 40% protein. Fatty acid composition of lipids in the milk and in the larval body was similar, with Palmitic (35–38%), Palmitoleic (31–35%) and Oleic acid (23–25%) predominating. Results support the hypothesis that uterine milk contains both protein and lipid and that its composition is relatively constant throughout the period of its synthesis and secretion.Patterns of incorporation of radioactivity by fertilized adult females from injected [¹⁴C]-leucine changed throughout a pregnancy cycle. High levels of incorporation into lipid (22–30%) during early pregnancy fell to around 10% during late pregnancy. Over the same period low levels of incorporation into protein (5%) increased to 15%. Results support the hypothesis that uterine milk is synthesized from a lipid store laid down in early pregnancy coupled with protein derived largely from blood meals ingested later. Such a system would not require the insect to store proteins for larval growth and is economical in terms of energy expenditure.     

Pten Regulates Development and Lactation in the Mammary Glands of Dairy Cows

Pten is a tumor suppressor gene regulating many cellular processes, including growth, adhesion, and apoptosis. In the aim of investigating the role of Pten during mammary gland development and lactation of dairy cows, we analyzed Pten expression levels in the mammary glands of dairy cows by using western blotting, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Dairy cow mammary epithelial cells (DCMECs) were used to study the function of Pten in vitro. We determined concentrations of β-casein, triglyceride, and lactose in the culture medium following Pten overexpression and siRNA inhibition. To determine whether Pten affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected. Pten expression was also assessed by adding prolactin and glucose to cell cultures. When Pten was overexpressed, proliferation of DCMECs and concentrations for β-casein, triglyceride, and lactose were significantly decreased. Overexpression of Pten down-regulated expression of MAPK, CYCLIN D1, AKT, MTOR, S6K1, STAT5, SREBP1, PPARγ, PRLR, and GLUT1, but up-regulated 4EBP1 in DCMECs. The Pten siRNA inhibition experiments revealed results that opposed those from the gene overexpression experiments. Introduction of prolactin (PRL) increased secretion of β-casein, triglyceride, and lactose, but decreased Pten expression levels. Introduction of glucose also increased β-casein and triglyceride concentrations, but did not significantly alter Pten expression levels. The Pten mRNA and protein expression levels were decreased 0.3- and 0.4-fold in mammary glands of lactating cows producing high quality milk (milk protein >3.0%, milk fat >3.5%), compared with those cows producing low quality milk (milk protein <3.0%, milk fat <3.5%). In conclusion, Pten functions as an inhibitor during mammary gland development and lactation in dairy cows. It can down-regulate DCMECs secretion of β-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways.     

Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10³ and 95.4×10³ CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10⁵ and 622.2×10⁵ cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.     

X-Ray Fluorescence Microscopy Reveals Accumulation and Secretion of Discrete Intracellular Zinc Pools in the Lactating Mouse Mammary Gland

Background

The mammary gland is responsible for the transfer of a tremendous amount of zinc (∼1–3 mg zinc/day) from maternal circulation into milk during lactation to support the growth and development of the offspring. When this process is compromised, severe zinc deficiency compromises neuronal development and immune function and increases infant morbidity and/or mortality. It remains unclear as to how the lactating mammary gland dynamically integrates zinc import from maternal circulation with the enormous amount of zinc that is secreted into milk.

Methodology/Principal Findings

Herein we utilized X-ray fluorescence microscopy (XFM) which allowed for the visualization and quantification of the process of zinc transfer through the mammary gland of the lactating mouse. Our data illustrate that a large amount of zinc first accumulates in the mammary gland during lactation. Interestingly, this zinc is not cytosolic, but accumulated in large, discrete sub-cellular compartments. These zinc pools were then redistributed to small intracellular vesicles destined for secretion in a prolactin-responsive manner. Confocal microscopy identified mitochondria and the Golgi apparatus as the sub-cellular compartments which accumulate zinc; however, zinc pools in the Golgi apparatus, but not mitochondria are redistributed to vesicles destined for secretion during lactation.

Conclusions/Significance

Our data directly implicate the Golgi apparatus in providing a large, mobilizable zinc storage pool to assist in providing for the tremendous amount of zinc that is secreted into milk. Interestingly, our study also provides compelling evidence that mitochondrial zinc pools expand in the mammary gland during lactation which we speculate may play a role in regulating mammary gland function.     

Determinants of Human African Trypanosomiasis Elimination via Paratransgenesis

Human African trypanosomiasis (HAT), transmitted by tsetse flies, has historically infected hundreds of thousands of individuals annually in sub-Saharan Africa. Over the last decade, concerted control efforts have reduced reported cases to below 10,000 annually, bringing complete elimination within reach. A potential technology to eliminate HAT involves rendering the flies resistant to trypanosome infection. This approach can be achieved through the introduction of transgenic Sodalis symbiotic bacteria that have been modified to produce a trypanocide, and propagated via Wolbachia symbionts, which confer a reproductive advantage to the paratransgenic tsetse. However, the population dynamics of these symbionts within tsetse flies have not yet been evaluated. Specifically, the key factors that determine the effectiveness of paratransgenesis have yet to be quantified. To identify the impact of these determinants on T.b. gambiense and T.b. rhodesiense transmission, we developed a mathematical model of trypanosome transmission that incorporates tsetse and symbiont population dynamics. We found that fecundity and mortality penalties associated with Wolbachia or recombinant Sodalis colonization, probabilities of vertical transmission, and tsetse migration rates are fundamental to the feasibility of HAT elimination. For example, we determined that HAT elimination could be sustained over 25 years when Wolbachia colonization minimally impacted fecundity or mortality, and when the probability of recombinant Sodalis vertical transmission exceeded 99.9%. We also found that for a narrow range of recombinant Sodalis vertical transmission probability (99.9–90.6% for T.b. gambiense and 99.9–85.8% for T.b. rhodesiense), cumulative HAT incidence was reduced between 30% and 1% for T.b. gambiense and between 21% and 3% for T.b. rhodesiense, although elimination was not predicted. Our findings indicate that fitness and mortality penalties associated with paratransgenic symbionts, as well as tsetse migration rates, are instrumental to HAT elimination, and should be a key focus in the development of paratransgenic symbionts.     

Use of vector control to protect people from sleeping sickness in the focus of Bonon (Côte d’Ivoire)

BackgroundGambian human African trypanosomiasis (gHAT) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by tsetse flies (Glossina). In Côte d’Ivoire, Bonon is the most important focus of gHAT, with 325 cases diagnosed from 2000 to 2015 and efforts against gHAT have relied largely on mass screening and treatment of human cases. We assessed whether the addition of tsetse control by deploying Tiny Targets offers benefit to sole reliance on the screen-and-treat strategy.Methodology and principal findingsIn 2015, we performed a census of the human population of the Bonon focus, followed by an exhaustive entomological survey at 278 sites. After a public sensitization campaign, ~2000 Tiny Targets were deployed across an area of 130 km² in February of 2016, deployment was repeated annually in the same month of 2017 and 2018. The intervention’s impact on tsetse was evaluated using a network of 30 traps which were operated for 48 hours at three-month intervals from March 2016 to December 2018. A second comprehensive entomological survey was performed in December 2018 with traps deployed at 274 of the sites used in 2015. Sub-samples of tsetse were dissected and examined microscopically for presence of trypanosomes. The census recorded 26,697 inhabitants residing in 331 settlements. Prior to the deployment of targets, the mean catch of tsetse from the 30 monitoring traps was 12.75 tsetse/trap (5.047–32.203, 95%CI), i.e. 6.4 tsetse/trap/day. Following the deployment of Tiny Targets, mean catches ranged between 0.06 (0.016–0.260, 95%CI) and 0.55 (0.166–1.794, 95%CI) tsetse/trap, i.e. 0.03–0.28 tsetse/trap/day. During the final extensive survey performed in December 2018, 52 tsetse were caught compared to 1,909 in 2015, with 11.6% (5/43) and 23.1% (101/437) infected with Trypanosoma respectively.ConclusionsThe annual deployment of Tiny Targets in the gHAT focus of Bonon reduced the density of Glossina palpalis palpalis by >95%. Tiny Targets offer a powerful addition to current strategies towards eliminating gHAT from Côte d’Ivoire.     

In Silico Identification of a Candidate Synthetic Peptide (Tsgf118–43) to Monitor Human Exposure to Tsetse Flies in West Africa

Background

The analysis of humoral responses directed against the saliva of blood-sucking arthropods was shown to provide epidemiological biomarkers of human exposure to vector-borne diseases. However, the use of whole saliva as antigen presents several limitations such as problems of mass production, reproducibility and specificity. The aim of this study was to design a specific biomarker of exposure to tsetse flies based on the in silico analysis of three Glossina salivary proteins (Ada, Ag5 and Tsgf1) previously shown to be specifically recognized by plasma from exposed individuals.

Methodology/Principal Findings

Synthetic peptides were designed by combining several linear epitope prediction methods and Blast analysis. The most specific peptides were then tested by indirect ELISA on a bank of 160 plasma samples from tsetse infested areas and tsetse free areas. Anti-Tsgf1_18–43 specific IgG levels were low in all three control populations (from rural Africa, urban Africa and Europe) and were significantly higher (p<0.0001) in the two populations exposed to tsetse flies (Guinean HAT foci, and South West Burkina Faso). A positive correlation was also found between Anti-Tsgf1_18–43 IgG levels and the risk of being infected by Trypanosoma brucei gambiense in the sleeping sickness foci of Guinea.

Conclusion/Significance

The Tsgf1_18–43 peptide is a suitable and promising candidate to develop a standardize immunoassay allowing large scale monitoring of human exposure to tsetse flies in West Africa. This could provide a new surveillance indicator for tsetse control interventions by HAT control programs.     

Protein synthesis by the milk gland and fat body of the tsetse fly,Glossina pallidipes

The patterns of protein synthesis by the milk gland and the fat body of female Glossinapallidipes during the pregnancy cycle were studied by incubation with [³⁵S]methionine both in vivo and in vitro. The pattern of protein synthesis by the milk gland changed with the stage of the larva in the uterus. Very little synthesis occurred in the milk gland until the first instar larva hatched. Then four proteins (13, 16, 24 and 72 kDa) were prominently synthesized. As the larva matured, the synthesis of 19, 38, 40 and 72 kDa proteins increased, whereas that of the 13 and 24 kDa proteins decreased. Just before larviposition, only the 16 and 72 kDa proteins were still being synthesized. The milk gland secreted into the medium primarily the 13, 16, 19 and 72 kDa proteins, all of which were found in the larval gut after a 5 hr pulse of labeled methionine in vivo. During most of the pregnancy cycle protein synthesis in the fat body was low compared to that of the milk gland and only small amounts of several low molecular weight proteins (less than or equal to 16 kDa) were released into the medium. But when a large third instar larva was present in the uterus, the fat body synthesized and secreted a 72 kDa and a 15–17 kDa complex of proteins.