Paratransgenic manipulation of a tsetse microRNA alters the physiological homeostasis of the fly's midgut environment.

Tsetse flies are vectors of parasitic African trypanosomes, the etiological agents of human and animal African trypanosomoses. Current disease control methods include fly-repelling pesticides, fly trapping, and chemotherapeutic treatment of infected people and animals. Inhibiting tsetse's ability to transmit trypanosomes by strengthening the fly's natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines the insect midgut and serves as a protective barrier that inhibits infection with pathogens. African trypanosomes must cross tsetse's PM in order to establish an infection in the fly, and PM structural integrity negatively correlates with trypanosome infection outcomes. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse's gut lumen early during the infection establishment, and free VSG molecules are internalized by the fly's PM-producing cardia. This process results in a reduction in the expression of a tsetse microRNA (miR275) and a sequential molecular cascade that compromises PM integrity. miRNAs are small non-coding RNAs that are critical in regulating many physiological processes. In the present study, we investigated the role(s) of tsetse miR275 by developing a paratransgenic expression system that employs tsetse's facultative bacterial endosymbiont, Sodalis glossinidius, to express tandem antagomir-275 repeats (or miR275 sponges). This system induces a constitutive, 40% reduction in miR275 transcript abundance in the fly's midgut and results in obstructed blood digestion (gut weights increased by 52%), a significant increase (p-value < 0.0001) in fly survival following infection with an entomopathogenic bacteria, and a 78% increase in trypanosome infection prevalence. RNA sequencing of cardia and midgut tissues from paratransgenic tsetse confirmed that miR275 regulates processes related to the expression of PM-associated proteins and digestive enzymes as well as genes that encode abundant secretory proteins. Our study demonstrates that paratransgenesis can be employed to study microRNA regulated pathways in arthropods that house symbiotic bacteria.

Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis.

Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their host's metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the fly's resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetse's viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetse's reproductive fitness. Using a Gff lab line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.

Gene-Level, but Not Chromosome-Wide, Divergence between a Very Young House Fly Proto-Y Chromosome and Its Homologous Proto-X Chromosome.

X and Y chromosomes are usually derived from a pair of homologous autosomes, which then diverge from each other over time. Although Y-specific features have been characterized in sex chromosomes of various ages, the earliest stages of Y chromosome evolution remain elusive. In particular, we do not know whether early stages of Y chromosome evolution consist of changes to individual genes or happen via chromosome-scale divergence from the X. To address this question, we quantified divergence between young proto-X and proto-Y chromosomes in the house fly, Musca domestica. We compared proto-sex chromosome sequence and gene expression between genotypic (XY) and sex-reversed (XX) males. We find evidence for sequence divergence between genes on the proto-X and proto-Y, including five genes with mitochondrial functions. There is also an excess of genes with divergent expression between the proto-X and proto-Y, but the number of genes is small. This suggests that individual proto-Y genes, but not the entire proto-Y chromosome, have diverged from the proto-X. We identified one gene, encoding an axonemal dynein assembly factor (which functions in sperm motility), that has higher expression in XY males than XX males because of a disproportionate contribution of the proto-Y allele to gene expression. The upregulation of the proto-Y allele may be favored in males because of this gene's function in spermatogenesis. The evolutionary divergence between proto-X and proto-Y copies of this gene, as well as the mitochondrial genes, is consistent with selection in males affecting the evolution of individual genes during early Y chromosome evolution.

Temperature-dependent effects of house fly proto-Y chromosomes on gene expression could be responsible for fitness differences that maintain polygenic sex determination.

Sex determination, the developmental process by which sexually dimorphic phenotypes are established, evolves fast. Evolutionary turnover in a sex determination pathway may occur via selection on alleles that are genetically linked to a new master sex determining locus on a newly formed proto-sex chromosome. Species with polygenic sex determination, in which master regulatory genes are found on multiple different proto-sex chromosomes, are informative models to study the evolution of sex determination and sex chromosomes. House flies are such a model system, with male determining loci possible on all six chromosomes and a female-determiner on one of the chromosomes as well. The two most common male-determining proto-Y chromosomes form latitudinal clines on multiple continents, suggesting that temperature variation is an important selection pressure responsible for maintaining polygenic sex determination in this species. Temperature-dependent fitness effects could be manifested through temperature-dependent gene expression differences across proto-Y chromosome genotypes. These gene expression differences may be the result of cis regulatory variants that affect the expression of genes on the proto-sex chromosomes, or trans effects of the proto-Y chromosomes on genes elswhere in the genome. We used RNA-seq to identify genes whose expression depends on proto-Y chromosome genotype and temperature in adult male house flies. We found no evidence for ecologically meaningful temperature-dependent expression differences of sex determining genes between male genotypes, but we were probably not sampling an appropriate developmental time-point to identify such effects. In contrast, we identified many other genes whose expression depends on the interaction between proto-Y chromosome genotype and temperature, including genes that encode proteins involved in reproduction, metabolism, lifespan, stress response, and immunity. Notably, genes with genotype-by-temperature interactions on expression were not enriched on the proto-sex chromosomes. Moreover, there was no evidence that temperature-dependent expression is driven by chromosome-wide cis-regulatory divergence between the proto-Y and proto-X alleles. Therefore, if temperature-dependent gene expression is responsible for differences in phenotypes and fitness of proto-Y genotypes across house fly populations, these effects are driven by a small number of temperature-dependent alleles on the proto-Y chromosomes that may have trans effects on the expression of genes on other chromosomes.

Is Multifactorial Sex Determination in the House Fly, Musca domestica (L.), Stable Over Time?

Sex determination pathways evolve rapidly, usually because of turnover of master regulatory genes at the top of the developmental pathway. Polygenic sex determination is expected to be a transient state between ancestral and derived conditions. However, polygenic sex determination has been observed in numerous animal species, including the house fly, Musca domestica House fly males carry a male-determining factor (M) that can be located on any chromosome, and an individual male may have multiple M factors. Females lack M and/or have a dominant allele of the Md-tra gene (Md-tra D ) that acts as a female-determining locus even in the presence of multiple copies of M. We found the frequency and linkage of M in house flies collected in Chino, CA (USA) was relatively unchanged between 1982 and 2014. The frequency of females with Md-tra D in the 2014 collection was 33.6% (n = 140). Analysis of these results, plus previously published data, revealed a strong correlation between the frequencies of Md-tra D and multiple M males, and we find that these populations are expected to have balanced sex ratios. We also find that fitness values that allow for the invasion and maintenance of multiple sex determining loci suggest that sexually antagonistic selection could be responsible for maintaining polygenic sex determination in house fly populations. The stability over time and equilibrium frequencies within populations suggest the house fly polygenic sex determination system is not in transition, and provide guidance for future investigations on the factors responsible for the polymorphism.

Genomic evidence of sex chromosome aneuploidy and infection-associated genotypes in the tsetse fly Glossina fuscipes, the major vector of African trypanosomiasis in Uganda.

The primary vector of the trypanosome parasite causing human and animal African trypanosomiasis in Uganda is the riverine tsetse fly Glossina fuscipes fuscipes (Gff). Our study improved the Gff genome assembly with whole genome 10× Chromium sequencing of a lab reared pupae, identified autosomal versus sex-chromosomal regions of the genome with ddRAD-seq data from 627 field caught Gff, and identified SNPs associated with trypanosome infection with genome-wide association (GWA) analysis in a subset of 351 flies. Results from 10× Chromium sequencing greatly improved Gff genome assembly metrics and assigned a full third of the genome to the sex chromosome. Results from ddRAD-seq suggested possible sex-chromosome aneuploidy in Gff and identified a single autosomal SNP to be highly associated with trypanosome infection. The top associated SNP was ∼1100 bp upstream of the gene lecithin cholesterol acyltransferase (LCAT), an important component of the molecular pathway that initiates trypanosome lysis and protection in mammals. Results suggest that there may be naturally occurring genetic variation in Gff in genomic regions in linkage disequilibrium with LCAT that can protect against trypanosome infection, thereby paving the way for targeted research into novel vector control strategies that can promote parasite resistance in natural populations.

Antibacterial effect of singlet oxygen depending on bacteria surface charge.

Here, we investigated the bactericidal effects of two types of photoinduced reactive oxygen species (ROS), superoxide anion and singlet oxygen, on bacteria with distinct surface charges. We fabricated photofunctional polymer films (PFPFs) capable of generating both types of ROS, and they were subjected to photodynamic inactivation tests for 12 various strains of Acinetobacter baumannii. The results showed that the type I ROS (superoxide anion) was significantly dependent on the surface charge of the bacteria owing to charge-charge repulsion, while the type II ROS (singlet oxygen) was independent of the surface charge of the bacteria. These results could be significant in enhancing treatment efficiency in the clinical field.

Minimal Effects of Proto-Y Chromosomes on House Fly Gene Expression in Spite of Evidence that Selection Maintains Stable Polygenic Sex Determination.

Sex determination, the developmental process by which organismal sex is established, evolves fast, often due to changes in the master regulators at the top of the pathway. Additionally, in species with polygenic sex determination, multiple different master regulators segregate as polymorphisms. Understanding the forces that maintain polygenic sex determination can be informative of the factors that drive the evolution of sex determination. The house fly, Musca domestica, is a well-suited model to those ends because natural populations harbor male-determining loci on each of the six chromosomes and a biallelic female determiner. To investigate how natural selection maintains polygenic sex determination in the house fly, we assayed the phenotypic effects of proto-Y chromosomes by performing mRNA-sequencing experiments to measure gene expression in house fly males carrying different proto-Y chromosomes. We find that the proto-Y chromosomes have similar effects as a nonsex-determining autosome. In addition, we created sex-reversed males without any proto-Y chromosomes and they had nearly identical gene expression profiles as genotypic males. Therefore, the proto-Y chromosomes have a minor effect on male gene expression, consistent with previously described minimal X-Y sequence differences. Despite these minimal differences, we find evidence for a disproportionate effect of one proto-Y chromosome on male-biased expression, which could be partially responsible for fitness differences between males with different proto-Y chromosome genotypes. Therefore our results suggest that, if natural selection maintains polygenic sex determination in house fly via gene expression differences, the phenotypes under selection likely depend on a small number of genetic targets.

Water striders adjust leg movement speed to optimize takeoff velocity for their morphology.

Water striders are water-walking insects that can jump upwards from the water surface. Quick jumps allow striders to avoid sudden dangers such as predators' attacks, and therefore their jumping is expected to be shaped by natural selection for optimal performance. Related species with different morphological constraints could require different jumping mechanics to successfully avoid predation. Here we show that jumping striders tune their leg rotation speed to reach the maximum jumping speed that water surface allows. We find that the leg stroke speeds of water strider species with different leg morphologies correspond to mathematically calculated morphology-specific optima that maximize vertical takeoff velocity by fully exploiting the capillary force of water. These results improve the understanding of correlated evolution between morphology and leg movements in small jumping insects, and provide a theoretical basis to develop biomimetic technology in semi-aquatic environments.

BIOMECHANICS. Jumping on water: Surface tension-dominated jumping of water striders and robotic insects.

Jumping on water is a unique locomotion mode found in semi-aquatic arthropods, such as water striders. To reproduce this feat in a surface tension-dominant jumping robot, we elucidated the hydrodynamics involved and applied them to develop a bio-inspired impulsive mechanism that maximizes momentum transfer to water. We found that water striders rotate the curved tips of their legs inward at a relatively low descending velocity with a force just below that required to break the water surface (144 millinewtons/meter). We built a 68-milligram at-scale jumping robotic insect and verified that it jumps on water with maximum momentum transfer. The results suggest an understanding of the hydrodynamic phenomena used by semi-aquatic arthropods during water jumping and prescribe a method for reproducing these capabilities in artificial systems.