Genetic and environmental components of sperm function in Drosophila melanogaster

Sperm function has been studied in multiple research fields as it is essential to male fertility. In previous studies a variety of sperm traits have been examined as an assessment of sperm function. Among those traits, sperm viability, sperm motility and sperm metabolism are often commonly examined. However, sperm function can be influenced by both environmental and genetic factors. Specifically, nuclear genome has been demonstrated to play a role in sperm function, especially in sperm competitive capacity. There are increasing evidence for effects of mitochondrial genome on sperm function. Mitochondrial genetic variance has been suggested to affect sperm length and sperm viability in seed beetle and sperm metabolism in rodent. Given the coordinated collaborations between nuclear and mitochondrial genomes in OXPHOS, replication and transcription of mitochondrial genome as well as intergenomic signalling, potential mitonuclear effects on sperm function are expected even though empirical evidence so far remains less. A recent review summarised all the previous work on environmental effects on sperm and found that various factors affects sperm function but largely neglected in ecology and evolution. In the study, we used D. melanogaster as a model to disentangle both genetic and environmental components of sperm function at sperm cell, ejaculate and offspring levels. We found environmental effects on sperm function in D. melanogaster. Specifically, sperm incubation buffers affect sperm viability in chapter 2 and dietary PUFAs influence sperm volume and metabolism in chapter 4. Nuclear effects were found on sperm viability, sperm quality and male fertility in chapter 3. Mitochondrial genome was found to have an effect on sperm function, i.e. sperm viability and sperm quality differed among mitochondrial haplotypes examined. In addition, sperm function was further modified by the interaction of nuclear and mitochondrial genomes in ageing male. Sperm quality and fertilization success were suggested to be dependent on age-related mitonuclear interaction in chapter 3. Moreover, we examined the mitonuclear coadaptation hypothesis in the function of D. melanogaster sperm. No evidence for mitonuclear coadaptation hypothesis was found for sperm function in D. melanogaster as there were no difference between coadapted and non-coadapted lines in sperm traits examined. Lastly, we found that sperm viability, sperm quality and sperm metabolic rate cannot predict male fertility in D. melanogaster as correlation analysis revealed no relationship between them. Our experiment explored and disentangled the genetic and environmental components of sperm function at multiple levels in D.melanogaster systematically. Our results suggested that both mitochondrial and nuclear genome as well as the interaction between them play a role in sperm function in D. melanogaster. In addition to genetic components, our findings revealed environmental components of Drosophila sperm and suggested that it was phenotypic plastic

Antioxidative Defense and Fertility Rate in the Assessment of Reprotoxicity Risk Posed by Global Warming

The objective of this review is to briefly summarize the recent progress in studies done on the assessment of reprotoxicity risk posed by global warming for the foundation of strategic tool in ecosystem-based adaptation. The selected animal data analysis that was used in this paper focuses on antioxidative markers and fertility rate estimated over the period 2000–2019. We followed a phylogenetic methodology in order to report data on a panel of selected organisms that show dangerous eects. The oxidative damage studies related to temperature fluctuation occurring in biosentinels of dierent invertebrate and vertebrate classes show a consistently maintained physiological defense. Furthermore, the results from homeothermic and poikilothermic species in our study highlight the influence of temperature rise on reprotoxicity

Adaptive gene regulatory polymorphisms in natural populations of Drosophila melanogaster

It has long been recognized that changes in gene regulation, specifically mutations in cis-regulatory elements that tend to be stable and additive, are important to adaptive processes and phenotypic evolution. Since cis-regulatory elements are found in the vicinity of the genes they regulate, the direct effect of changes in these sequences is typically limited to a particular gene that allows for refined, situation-specific control of gene expression but are not exclusive of downstream or trans-acting elements. This dissertation focuses on examining mechanisms responsible for maintaining adaptive cis-regulatory polymorphisms in two Drosophila melanogaster genes: fezzik (fiz) and Metallothionein A (MtnA) and their associated effect on gene expression and organismal phenotype. Previous experiments show that the 3’ untranslated region of MtnA contains an insertion/deletion (indel) polymorphism, wherein the deletion is rare or absent in the ancestral African range but its deletion frequency appears to follow a latitudinal cline in derived populations worldwide. By genotyping biannual collections of wild caught D. melanogaster across a 5-year period, I show that the deletion is maintained at a high frequency (~90%) in a single German population with no evidence for overdominant, seasonally fluctuating or sexually antagonistic selection. Expression analysis on pairs of nearly-isogenic lines and on data from a North American population indicated significant differences in expression associated to the indel. Furthermore, the data from this North American population showed that expression variation was only partially explained by the deletion and the effect on oxidative stress tolerance was significantly associated with menadione sodium bisulfite and not paraquat. Altogether these findings suggested a scenario in which MtnA expression and consequently oxidative stress tolerance is likely a polygenic adaptation that varies with genomic background. Indeed, the effect of the deletion allele on oxidative stress tolerance was dependent on the genomic background with some indication of sign epistasis. Transcriptomic analysis revealed that MtnA expression was induced by oxidative stress independent of the indel status, indicating a general role of this gene in stress tolerance as well as suggesting additional levels of context-dependent expression regulation. The transcriptional response to oxidative stress between lines with and without the deletion was mostly similar but interestingly, there were consistently larger numbers of differentially expressed genes associated with the deletion which is possibly related to regulatory cascades resulting from aberrant microRNA epigenetic regulation due to the loss of microRNA binding sites in the deleted region. In general, the response to MSB indicated the significance of functional categories such as general stress response, oxidative stress response, metabolism, apoptosis and autophagy. In particular among the differentially expressed genes with the largest fold-change in response to MSB-induced oxidative stress were several genes related to glutathione metabolism and biosynthesis, suggesting a strong association between this pathway and oxidative stress tolerance. Another instance of expression divergence between ancestral and cosmopolitan populations being associated with a regulatory polymorphism is represented by a single nucleotide polymorphism (SNP) located 67 base pairs upstream of the start codon, in the enhancer region of the gene fezzik, referred to here as “SNP67”. SNP67 has two variants segregating in natural populations of D. melanogaster: the ancestral “C” variant, and the derived “G” variant that is found outside of the ancestral range at intermediate frequencies and is associated with increased fiz expression. Previous studies suggest that this SNP was a recent target of balancing selection; therefore to determine the forces of selection maintaining this SNP in cosmopolitan populations we genotyped biannually collected wild-caught D. melanogaster from a single European (Munich, Germany) population. A model-based approach using allele and genotype frequency data of the SNP67 variants across seasons and sexes was employed. The model indicated that sexually antagonistic and temporally fluctuating selection may help maintain variation at this site, with the derived variant likely being female-beneficial but there was some uncertainty of dominance estimates in the model. Gene expression and body-size phenotypes that were dependent on genomic background and developmental stage indicated that variable dominance may play a role in the maintenance of this polymorphism. Lastly, we identified a novel sex-dependent association between fiz expression and starvation resistance that may suggest that this trait is a potential phenotypic target of selection. Interestingly our findings for the MtnA and fiz regulatory polymorphisms both indicated that the relationship between gene expression divergence and population-level genetic mechanisms underlying phenotypic evolution is potentially complicated by context-dependent factors such as genomic background or spatial and temporal differences. By integrating extensive experimental work to identify the mechanisms of selection in natural populations along with functional characterizations, a refined understanding of these adaptive regulatory polymorphisms was achieved

Wolbachia colonization in drosophila midguts and its effects on intestinal stem cells

Wolbachia is a vertically transmitted, obligate intracellular bacterium infecting ~40% of all known species of arthropods, as well as filarial nematodes. The nature of Wolbachia-host interactions ranges from reproductive parasitism to increased fecundity and pathogen protection. Wolbachia reduces the ability of mosquitoes to transmit human pathogens, which is being explored as a novel method for the control of vector-borne diseases like Dengue and Zika. The mechanisms of Wolbachia blocking the transmission of these diseases are not fully understood. There are studies indicating that Wolbachia-induced changes in the insect immunity could block the virus, however there is no consensus in the literature. A necessary step in the transmission of these diseases is the viral entry into the insect vector. This occurs trough the gut epithelium, highlighting the importance of understanding the interaction of this tissue with microorganisms. We have recently shown that Wolbachia colonizes the Drosophila gut epithelium and affects the gut microbiome composition. Wolbachia’s presence did not affect the gene expression of immune effector molecules from the main regulators of gut immunity, Imd and ROS pathways. Our understanding of the mechanisms of Wolbachia’s colonization of the gut epithelium and modulation of gut microbiome are still very limited. This work characterizes Wolbachia’s kinetics of colonization in Drosophila midguts. Imaging analysis revealed that Wolbachia colonizes adult and larval midguts in different patterns. We have also characterized a preferential colonization in specific adult midgut sub-regions. We observed that Wolbachia patches are confined to specific midgut subregions, in a pattern similar to the arrangement of intestinal stem cell (ISC) clones. These results led us to hypothesize that Wolbachia colonizes Drosophila midguts by infecting intestinal progenitor cells and spreading vertically to their progeny with limited lateral transmission between neighboring cells. We provide evidence to support this hypothesis by showing that Wolbachia is present in intestinal progenitor cells in all stages of the fly’s life cycle as well as by analyzing the infection status of ISC clones and differentiated cells surrounding ISCs. Finally, we found that ISC proliferation is reduced by the intracellular presence of Wolbachia, which also decreases ISC tumor incidence triggered by the downregulation of Notch signaling specifically in ISCs. These findings will aid in our understanding of Wolbachia tropisms and its phenotypic consequences. It has been shown that in the Wolbachia wMelPop strain excessive growth of intracellular bacteria leads to damage to the host cell, suggesting a mechanism of controlling intracellular growth in other strains. To better understand the molecular mechanisms behind Wolbachia-Drosophila interactions, we turned to the gonads, as Wolbachia colonization of these tissues has been well characterized. We chose to investigate the interplay between Reactive Oxygen Species (ROS) and Wolbachia, as intracellular ROS could regulate bacterial density but also be affected by Wolbachia and play a role in symbiont-related phenotypes. Using direct and indirect measurements of ROS, we show that the pathogenic strain wMelPop increases ROS in the germarium, while the symbiotic strains wMel and wMelCS reduce ROS in the terminal filaments. None of the Wolbachia strains tested affected ROS levels in the testes. In addition, genetically altering ROS levels in the germline or systemically in the fly did not affect Wolbachia levels in the ovaries. We conclude that ROS does not significantly affect Wolbachia in the fruit fly gonads

Conditional Handicaps in Exuberant Lizards: Bright Color in Aggressive Males Is Correlated with High Levels of Free Radicals

abstract: The maintenance of genetic variation and signal honesty may be explained, in part, through the genic capture hypothesis in sexual selection biology. Polygenic traits, like body condition, could help maintain variation in signaling traits under strong, directional sexual selection while maintaining signal honesty. Here we consider the genic capture hypothesis in a study of morph-specific condition and free radical effects on signaling traits (head coloration) in males of a polymorphic lizard, the Australian painted dragon (Ctenophorus pictus). Males differ in head color (red, orange, yellow, and a “blue” morph that has no yellow or red pigments). The red and yellow morphs were the first described and we have previously demonstrated that red morphs are aggressive and dominant over yellow morphs that are better at sperm completion than the red morphs. Body condition varied significantly in its relationship with superoxide among the four morphs, with males in better condition showing higher superoxide levels in “yellow-orange-reds” morphs (least so in red morphs). Blue morphs contrasted markedly by showing lower superoxide levels in males in better condition, perhaps facilitated by no (or reduced) dermal deposition of pigmentation on the head. Color degradation with loss in condition from yellow to red morphs, suggesting that red morphs are better able to maintain color with superoxide acting as a potential handicap. This result is consistent with condition-dependent signal expression and the genic capture hypothesis; males with the more pronounced signal carry a higher potential cost (higher superoxide levels) when being in better body condition, while maintaining more vivid coloration (the condition-dependent trait).View the article as published at https://www.frontiersin.org/articles/10.3389/fevo.2017.00001/ful

Immunostaining for allatotropin and allatostatin-A and -C in the mosquitoes Aedes aegypti and Anopheles albimanus

Confocal laser-scanning microscopy was used to carry out a comparative study of the immunostaining for three families of neuropeptides, viz., allatostatin-A (AS-A), allatostatin-C (AS-C) and allatotropin (AT), in adult female mosquitoes of Aedes aegypti and Anopheles albimanus. The specific patterns of immunostaining for each of the three peptides were similar in both species. The antisera raised against AT, AS-A, and AS-C revealed intense immunoreactivity in the cells of each protocerebral lobe of the brain and stained cells in each of the ventral ganglia and neuronal projections innervating various thoracic and abdominal tissues. Only the AS-A antiserum labeled immunoreactive endocrine cells in the midgut. The distribution of the peptides supports the concept that they play multiple regulatory roles in both species

Identification of phenotypic variation across levels of cSOD activity in Drosophila melanogaster reveals that phenotypes are most influenced by second chromosome genetic background and sex at high cSOD activity

This thesis examined how variable the effects of changes in cSOD activity were on phenotypes across genetic backgrounds and between sexes. Analysis of variance (ANOVA), and the effect size partial eta squared (η2p) were used to partition the amount of variation attributable to sex, cSOD activity, and genetic background across the distal and proximal phenotypes assayed. The absence of cSOD activity results in pervasive changes in phenotypic expression, and these changes are only slightly modified by sex or genetic background. Higher levels of cSOD activity generally result in phenotypic expression closer to wild-type levels, though phenotypes were more susceptible to modification depending on sex and genetic background when some cSOD activity was present. Results here indicate that the cSOD-null syndrome is pervasive, and the significant influence of sex and genetic background across phenotypes supports the utilization of both sexes and multiple genetic backgrounds in genetic analyses.Master of Science (MSc) in Biolog

NEUROTOXICOLOGICAL EFFECTS OF CHLORPYRIFOS AND DELTAMETHRIN ON DOPAMINE AND ACETYLCHOLINE SYSTEMS IN DROSOPHILA MELANOGASTER

Emerging evidence suggests a positive association between pesticide exposure and sporadic Parkinson\u27s disease (PD) development. The molecular mechanisms of PD and other neurodegenerative diseases are not fully understood, which hinders the development of therapeutic agents to cure or prevent the development of such diseases. Drosophila has been widely used as a model organism to study various neurodegenerative diseases and to screen for promising therapeutic agents. The aims of this study were: (i) investigating the toxic effect of 24 hours exposure to chlorpyrifos (CPF) and deltamethrin (DLM) on the dopaminergic system and AChE activity in Drosophila melanogaster; (ii) comparing different methodologies to test negative geotaxis behavior in D. melanogaster; and (iii) investigating the combined neuroprotective effects of ferulic acid (FA), and Thymoquinone (TQ) natural compounds on DLM induced neurotoxicity. Adult male wild-type flies were exposed to either CPF, DLM, for 24 hours, or concomitantly exposed to DLM and individual neuroprotective agents, or their mix for 72 hours in 10% sucrose on a cotton swab. CPF/DLM-treated flies climbed shorter distances in the negative geotaxis assay as well as had a higher incidence of mortality when compared to the control group. Evidently, CPF/DLM exposure caused a disturbance in the expression of DA-related genes. The DLM exposure for 72 hours caused a higher incidence of mortality and severe locomotor defects. Co-treatment with neuroprotective agents offered protection against these toxic effects of DLM after 72 hours. DLM caused significant inhibition of AChE which was ameliorated with the concomitant exposure with FA. Our results suggest that FA and TQ were effective in reducing the toxicity induced by DLM in D. melanogaster as well as confirm the utility of this model to investigate potential therapeutic strategies on movement disorders, including PD. The present study indicates that a single molecule can interact and affect multiple systems that are not related to their main mechanism of action. Data gathered in the present study may be important for the assessment of the safety of insecticides that humans are at risk of daily exposure to them. Moreover, this study presents a potential natural compound that can ameliorate and protect against the neurotoxicity that is caused by these insecticides

Free Radical Medicine and Biology

Although free radicals perform some useful immune functions, they can also damage healthy cells through a process called oxidation. Antioxidants reduce the effect of free radicals by binding together with these harmful molecules, decreasing their destructive power. This book highlights various issues of free radical biology from the perspective of antioxidant defense mechanisms. It also provides useful information on gene modulation, radiation-generated reactive oxygen species-induced apoptosis in cancer, and environmental aspects associated with free radicals’ exposure on living systems