The homeodomain protein ladybird late regulates synthesis of milk proteins during pregnancy in the tsetse fly (Glossina morsitans)

Abstract

Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (Glossina), the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. Here we identify and delineate the regulatory sequence of a major milk protein gene (milk gland protein 1:mgp1) by utilizing a combination of molecular techniques in tsetse, Drosophila transgenics, transcriptomics and in silico sequence analyses. The function of this promoter is conserved between tsetse and Drosophila. In transgenic Drosophila the mgp1 promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of Glossina. Analysis of the minimal required regulatory region of mgp1, and the regulatory regions of other Glossina milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of Glossina homeodomain proteins identified ladybird late (lbl) as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of lbl in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This system is critical for tsetse fecundity and provides a potential target for development of a reproductive inhibitor.

Figure 1. Pre and post parturition timecourse of mgp1 transcript and protein levels in Glossina.

A. Quantitative PCR analysis of mgp1 transcript levels just prior to parturition and in 24 hour periods after parturition. Pregnant Glossina flies carrying 3^rd instar larvae were collected and staged by time of parturition within 24 hour windows. The values represent the mean mgp1 transcript level from 3 individual flies at each point. Error bars represent standard error. All qPCR data was normalized to tubulin. Statistical notation: a = not significantly different than pregnant; b = significantly different from pregnant (P-value <0.05 by students t-test); c = significantly different from pregnant (P-value <0.01 by students t-test). B. Western blot analysis of MGP1 levels in flies from the same samples as described in A. Values represent quantification of the mean signal intensity from 3 individuals for each point and are normalized to tubulin levels. Statistical notation is as described above.

Figure 2. mgp1 driven reporter expression in transgenic Drosophila.

Tissue and stage specific expression of mgp1 driven ß-gal and egfp reporter genes. All qPCR analyses normalized to Drosophila tub. Error bars represent standard error. Letter groups (a,b,c) represent significant statistical differences of P-value <0.05 by students t-test. A. Accessory gland specific staining of transgenic Drosophila reproductive tissue (mgp1- β-gal-2.0). B. Florescent microscopy of accessory gland specific egfp expression in transgenic Drosophila (mgp1-egfp-509). C. qPCR analysis of sex/stage specific egfp levels in transgenic line mgp1-egfp-509. Samples represent the average of three groups of 20 flies. D. qPCR analysis of egfp levels in 3–5 day old mated adult females from the mgp1-egfp-509, mgp1-egfp-236, mgp1-egfp-112 or mgp1-egfp-13 lines. Samples represent three groups of 20 flies. E. 3–5 day old transgenic Drosophila (509 bp-mgp1/egfp) were reared on control or nutrient deficient media. Data represent the mean eggs/day from five groups of 60 flies. Statistical notation: a = statistical difference between minimal and control media (P-value <0.05 by students t-test). F. qPCR analysis of egfp levels in mgp1-egfp-509 flies reared on control or minimal media. Data represents the mean egfp transcript level from three groups of 20 flies. Statistical notation: as in E.

Figure 3. Schematic of the 124mgp1 regulatory sequence required for tissue and stage specific transgene expression.

This schematic represents a scale model of in silico predicted homeodomain (DHOM) binding sites. The associated table lists the number of DHOM binding sites predicted within the 500 bp upstream sequence from the 11 other milk protein genes and the associated p-value of these findings.

Figure 4. Tissue specificity and siRNA analysis of the lbl gene in Glossina.

All qPCR analyses normalized to Tsetse tub. Error bars represent standard error. A. qPCR analysis of lbl tissue specificity. Samples represent 3 replicates of tissues isolated from 5 individuals. Statistical notation: a = significant difference between groups (P-value <0.05 by students t-test). B. qPCR analysis of tsetse lbl abundance 4 days post sigfp (control) or silbl injection. Statistical notation: a = significant difference between groups (P-value <0.05 by students t-test). C. Relative levels of asmase, mgp1 and mgp2 following injection of sigfp (control) or silbl. Statistical notation: a = statistical difference from control (P-value <0.01 by students t-test). All flies were injected 5–6 days after adult emergence and tested at 11–13 days for lbl suppression and 16–17 days for asmase, mgp1 and mgp2 interference.

Figure 5. Fecundity effects of lbl knockdown.

2 groups of 50 flies were injected with either sigfp or silbl at 7 days post eclosion and monitored daily for mortality and pupal deposition. A. Rate of larval deposition per fly per day after injection of either sigfp or silbl. Error bars represent standard error. Statistical notation: a = statistical difference between groups (P-value <0.05 by students t-test). B. Cumulative count of larval deposition post injection over the course of the first gonotrophic cycle.