Amelioration of Reproduction-Associated Oxidative Stress in a Viviparous Insect Is Critical to Prevent Reproductive Senescence

<div><p>Impact of reproductive processes upon female health has yielded conflicting results; particularly in relation to the role of reproduction-associated stress. We used the viviparous tsetse fly to determine if lactation, birth and involution lead to damage from oxidative stress (OS) that impairs subsequent reproductive cycles. Tsetse females carry an intrauterine larva to full term at each pregnancy cycle, and lactate to nourish them with milk secretions produced by the accessory gland ( = milk gland) organ. Unlike most K-strategists, tsetse females lack an apparent period of reproductive senescence allowing the production of 8–10 progeny over their entire life span. In a lactating female, over 47% of the maternal transcriptome is associated with the generation of milk proteins. The resulting single larval offspring weighs as much as the mother at birth. In studying this process we noted an increase in specific antioxidant enzyme (AOE) transcripts and enzymatic activity at critical times during lactation, birth and involution in the milk gland/fat body organ and the uterus. Suppression of <i>superoxide dismutase</i> (<i>sod</i>) decreased fecundity in subsequent reproductive cycles in young mothers and nearly abolished fecundity in geriatric females. Loss of fecundity was in part due to the inability of the mother to produce adequate milk to support larval growth. Longevity was also impaired after <i>sod</i> knockdown. Generation of OS in virgin females through exogenous treatment with hydrogen peroxide at times corresponding to pregnancy intervals reduced survival, which was exacerbated by <i>sod</i> knockdown. AOE expression may prevent oxidative damage associated with the generation of nutrients by the milk gland, parturition and milk gland breakdown. Our results indicate that prevention of OS is essential for females to meet the growing nutritional demands of juveniles during pregnancy and to repair the damage that occurs at birth. This process is particularly important for females to remain fecund during the latter portion of their lifetime.</p></div

Oxidative stress markers recovered from females during their third pregnancy following <i>sod</i> knockdown during the first two reproductive cycles.

<p>A. Protein oxidation by measurement of protein carbonyl levels. Mean ± SE of five groups of 3 flies. B. Lipid oxidation levels measured by lipid peroxidation. Samples were collected from mothers carrying a 3^rd instar larvae in their uterus. Mean ± SE of five groups of 3 flies.</p

Population modeling following <i>sod</i> knockdown.

<p>A. Frequency of growth rate. B. Reduction in growth rate between <i>sod</i> knockdown and control (<i>siGFP</i>). Results represent 10,000 simulated replicates.</p

Survival of pregnant and virgin females following <i>sod</i> knockdown and exogenous treatment with H₂O₂.

<p>A. Longevity following <i>sod</i> knockdown in mated and unmated females. Mean ± SE of 15 flies. B. Survival of groups of virgin flies to mimic subjected to one of four treatments: H₂O or H₂O₂ injections at intervals during the peak of lactation that matched those of tsetse fly pregnancy, <i>Mn/Fe</i> and <i>Cu/Zn sod</i> during the first three pregnancy cycles and <i>Mn/Fe</i> and <i>Cu/Zn sod</i> during the first three pregnancy cycles along with H₂O₂ at intervals that match those of pregnancy. Survival data was measured using a Kaplan-Meier plot along with a log rank test. Arrows indicate treatment with H₂O or H₂O₂.</p

Effect of RNA interference of <i>Mn/Fe sod</i> and <i>Cu/Zn sod</i> on tsetse fecundity.

<p>A. Average number of pupae produced per female during generation of the 1^st (L1), 2^nd (L2) and 3^rd (L3) pregnancy cycle after <i>sod</i> knockdown in the 14^th day of the fly development and 5^th day of subsequent pregnancy cycles. Mean ± SE of three groups of 15 flies. B. Length of the gonotrophic cycles analyzed under similar treatment as A. Mean ± SE of three groups of 15 flies are shown. Expression of <i>milk gland proteins</i> (<i>mgp1</i>, <i>mgp7</i> and <i>asmase1</i>) during the early (C, 1^st instar larva present in uterus) and late stages (D, 3^rd instar larva present in uterus) of lactation after <i>sod</i> knockdown during the first two pregnancy cycles.</p

Summary for the role of oxidative stress and antioxidant enzyme expression during tsetse fly reproduction.

<p>Developmental images adapted from Benoit et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Benoit3" target="_blank">[104]</a>. Cross sections adapted from Ma and Denlinger <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Ma1" target="_blank">[28]</a>, Hecker and Moloo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Hecker1" target="_blank">[105]</a> and Yang et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Yang1" target="_blank">[41]</a>.</p

Tsetse fly investment in their progeny during lactation.

<p>A. Changes in dry mass of single intrauterine larva throughout development. B. Predicted read abundance for the 12 major milk protein genes (<i>milk gland protein 1–10</i>, <i>transferrin</i> and <i>acid sphingomyelinase 1</i>) throughout lactation based on fold changes in milk proteins in relation to transcriptome analysis measured at the peak of lactation (17–18 d) and 24–48 h after parturition according to Benoit et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Benoit2" target="_blank">[40]</a>. C. Total lipid content in females through pregnancy.</p

Age-related fecundity patterns in various species in comparison to tsetse flies.

<p>A. Medfly, (eggs/day) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Carey1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Carey2" target="_blank">[47]</a>, human (traditional Ache population, progeny/year) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Hill1" target="_blank">[101]</a>, lions (progeny/year) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Packer1" target="_blank">[102]</a>, <i>Drosophila melanogaster</i> (eggs/day) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Novoseltsev1" target="_blank">[103]</a> and tsetse fly (this study, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087554#pone.0087554-Langley3" target="_blank">[33]</a>). B. Age-related fecundity patterns in tsetse fly after knockdown <i>Mn/Fe sod</i> and <i>Cu/Zn sod</i>. Mean ± SE for three groups of 15 flies.</p

The Homeodomain Protein Ladybird Late Regulates Synthesis of Milk Proteins during Pregnancy in the Tsetse Fly (<i>Glossina morsitans</i>)

<div><p>Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (<i>Glossina</i>), 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 (<i>milk gland protein</i> 1:<i>mgp1</i>) by utilizing a combination of molecular techniques in tsetse, <i>Drosophila</i> transgenics, transcriptomics and <i>in silico</i> sequence analyses. The function of this promoter is conserved between tsetse and <i>Drosophila</i>. In transgenic <i>Drosophila</i> the <i>mgp1</i> promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of <i>Glossina</i>. Analysis of the minimal required regulatory region of <i>mgp1</i>, and the regulatory regions of other <i>Glossina</i> milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of <i>Glossina</i> homeodomain proteins identified <i>ladybird late</i> (<i>lbl</i>) as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of <i>lbl</i> 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.</p></div

RNA-seq identification of lactation associated homeodomain genes.

<p>RNA–seq statistics comparing the differential expression of putative tsetse homeodomain proteins between transcript datasets from lactating and non-lactating flies. The fold change between samples was tested for significance by Kal's Z-test analysis.</p