Biology subjects

Shropshire, J. D.

Publications and source records attributed to Shropshire, J. D..

9 recordsLinked to original sources

bioRxiv2026

Insect COI barcoding data as an untapped resource for surveying Wolbachia symbioses

O_LIDNA barcoding of the mitochondrial cytochrome c oxidase I (COI) gene is widely used to characterise insect diversity and distributions; however, its potential to reveal information on species interactions, including host-symbiont associations, remains largely unexplored. Here, we assess whether COI amplicon data can be used to identify Wolbachia - one of the most widely distributed bacterial symbionts known to profoundly affect their hosts biology. C_LIO_LIWe demonstrate that several commonly used invertebrate COI primer sets perfectly match many reference Wolbachia genomes, leading to frequent co-amplification. C_LIO_LIBy screening 7,901 individual-insect COI amplicon libraries obtained with the popular BF3-BR2 primer set, we detected Wolbachia sequences in over 35% of samples, revealing that co-amplification is indeed widespread. After removing low-abundance reads, Wolbachia detection based on COI amplicons showed over 90% agreement with simultaneously generated 16S-V4 rRNA amplicon data from the same specimens. The degree of agreement, however, varied depending on the thresholds used, among datasets and insect clades. C_LIO_LIFurther, we show that Wolbachia abundance inferred from COI amplicons correlated with their abundance in metagenomic datasets for 152 specimens, supporting the quantitative relevance of the signal. C_LIO_LIFinally, we find that Wolbachia COI sequences provide greater phylogenetic resolution than 16S-V4 rRNA data (mean pairwise genetic distance of COI sequences - 9.6%, 16S-V4 rRNA - 2.8%), and the reconstructed Wolbachia COI-based genotype network largely agrees with genome-based phylogenies. C_LIO_LICollectively, our results demonstrate that off-target Wolbachia sequences recovered from standard insect COI barcoding data may reliably detect symbiont presence, provide phylogenetic insight, and guide sample selection for metagenomics. Given the rapid expansion of global insect barcoding initiatives, these findings highlight an opportunity for cost-effective monitoring of their most prevalent bacterial symbionts, offering new perspectives on how host-microbe interactions may shape insect communities. C_LI

ecology↗

bioRxiv2026

Temperature-sensitive cytoplasmic incompatibility across divergent Wolbachia partly reflects cifB transcription, not endosymbiont density

Maternally transmitted Wolbachia bacteria are common in insects, with many strains altering host reproduction through cytoplasmic incompatibility (CI). CI kills embryos fertilized by Wolbachia-bearing males unless those embryos also carry Wolbachia, which favors females with Wolbachia and drives the endosymbiont to higher frequencies in host populations. Strong CI now underpins successful applications that rely on maintaining pathogen-blocking Wolbachia transinfections in vector populations to reduce arboviral disease transmission. Temperature modulates CI strength (the proportion of embryos killed), with consequences for Wolbachia prevalence in natural and transinfected populations. Yet the mechanisms regulating temperature-sensitive CI-strength variation are poorly understood. We quantified CI strength across eight divergent Drosophila-associated Wolbachia strains at four temperatures (18{degrees}C-26{degrees}C), while characterizing development time, Wolbachia and Wovirus densities, and transcription of the CI-inducing gene cifB. Four of eight Wolbachia strains exhibited temperature-sensitive CI, three of which induced CI at multiple temperatures. Of these three, two expressed significantly more cifB at the temperature yielding stronger CI, whereas testes Wolbachia density did not predict CI strength. Notably, cifB-transcript levels were consistently decoupled from Wolbachia and Wovirus densities, suggesting that cifB transcription is not regulated solely by symbiont abundance. We also report temperature-sensitive rescue of CI, Wolbachia-associated developmental acceleration, and strain-specific Wovirus-Wolbachia covariance. Our findings reveal temperature as a pervasive modulator of Wolbachia-host interactions at multiple levels and extend evidence that cifB transcription partly predicts variable CI strength across strain identities, male ages, and now temperatures. CI variation unaccounted for by cifB transcription points toward additional regulatory or post-transcriptional mechanisms that we discuss.

molecular biology↗

bioRxiv2026

Calibrating and documenting host-switching and evolution of incompatibility loci for two closely related Wolbachia clades

Maternally inherited Wolbachia alphaproteobacteria are the most common arthropod endosymbionts. Often Wolbachia spread to high frequencies through cytoplasmic incompatibility, in which cif loci act through sperm to kill embryos lacking Wolbachia. Closely related Wolbachia with diverse cif loci often associate with anciently diverged hosts, but the timescale of associations remains uncertain. We produce new calibrations based on filarial nematodes with vertically inherited Wolbachia that codiverge with their hosts. Applying these calibrations to Wolbachia variants closely related to pathogen-blocking wMel from Drosophila melanogaster, we demonstrate that over a timescale of 1-2 million years, a core set of single-copy Wolbachia loci evolve largely through bifurcation rather than by gene exchange with distant Wolbachia. Dating bifurcating core genomes, we show that "wMel-like" Wolbachia diverged 2.1x105-2.4x106 years inhabit dipteran and hymenopteran hosts diverged more than 108 years. Previous published analysis of variants related to wRi from D. simulans, the first Wolbachia found in a drosophilid, concluded that "wRi-like" Wolbachia spread among different Drosophila in tens of thousands of years. However, our new calibrations suggest these estimates from a mutation-based calibration underestimated wRi-like spread by about a factor of seven. In addition, cif exchanges between wMel-like and wRi-like Wolbachia genomes have occurred over [~]104-106 years. Comparing intact cif loci found in various Wolbachia, we find function-preserving selection in their evolution. We discuss these results in light of theoretical predictions concerning selection on cytoplasmic incompatibility phenotypes within and among host lineages. The wMel variants analyzed may offer new options for Wolbachia-based biocontrol efforts. Article SummaryMaternally transmitted Wolbachia endosymbionts associate with many arthropods. Closely related Wolbachia are known to occupy anciently diverged hosts, but the timescale of associations remains uncertain. Using genomic data from obligate Wolbachia codiverging with filarial nematodes, we calibrate Wolbachia molecular evolution. wMel-like variants that diverged about 104-106 years ago occur in hosts that diverged more than 108 years ago. Incompatibility loci move even more rapidly among these Wolbachia genomes. As these variants diverge, selection occurred on loci involved in both inducing and protecting against cytoplasmic incompatibility. These results elucidate Wolbachia host-range and genome dynamics and provide potential material for applications.

microbiology↗

bioRxiv2025

Counting cytoplasmic incompatibility factor mRNA using digital droplet PCR

Wolbachia bacteria inhabit over half of all insect species and often spread through host populations via efficient maternal transmission and cytoplasmic incompatibility (CI), killing aposymbiotic embryos when fertilized by symbiotic males. Wolbachias cifB gene triggers CI in males, while cifA, expressed in females, rescues embryos from CI-induced lethality. In some systems, cifA also contributes to CI induction. CI strength--the percentage of embryos that die from CI--is a key determinant of Wolbachias prevalence in host populations, and cifB mRNA levels in testes generally correlate with CI strength. Yet, cifBs rarity can hamper precise quantification, necessitating tissue pooling for reverse transcription quantitative PCR (RT-qPCR) to achieve reliable measurements, obscuring variation at the level of individual insect tissues. Here, we present four RT digital droplet PCR (RT-ddPCR) assays to count rare cifA and cifB mRNA from wMel Wolbachia in Drosophila melanogaster. These assays count cif transcripts alongside a synthetic spike-in RNA or a D. melanogaster housekeeping gene to normalize for technical or biological variation. These assays have a limit of detection of about 1 cifA and 3 cifB copies per reaction. We expect these methods to be useful for mosquito-control programs that use wMel to block the spread of pathogens from Aedes aegypti to humans. Moreover, the oligos were designed with homology to cifA and cifB sequences from at least 33 Wolbachia strains, suggesting utility beyond wMel. These methods will allow researchers to measure cif mRNA levels from individual insect tissues, enabling efforts to pair molecular and phenotypic data at unprecedented resolutions. ImportanceWolbachia, a maternally transmitted bacterium, is found in over half of all insect species. Its ability to induce cytoplasmic incompatibility (CI), which prevents Wolbachia-free eggs from hatching, significantly contributes to its high prevalence in host populations. Public health experts use CI to spread pathogen-blocking Wolbachia through mosquito populations, thereby controlling pathogen spread. CI is often weak, resulting in few egg deaths and consequently slowing Wolbachias spread. We recently discovered that weak CI often correlates with low CI factor B (cifB) mRNA levels. However, our understanding of CI-strength variation remains limited because cifB is transcribed at low levels, making it challenging to measure in individual insects. Here, we report four RT-ddPCR assays to overcome this challenge. These assays offer high sensitivity for rare targets and maintain accuracy and precision across a wide dynamic range. We expect these tools will enhance efforts to understand CI-strength variation in both natural and applied populations.

microbiology↗

bioRxiv2024

Counting rare Wolbachia endosymbionts using digital droplet PCR

Wolbachia is the most widespread animal-associated intracellular microbe, living within the cells of over half of insect species. Since they can suppress pathogen replication and spread rapidly through insect populations, Wolbachia is at the vanguard of public health initiatives to control mosquito-borne diseases. Wolbachias abilities to block pathogens and spread quickly are closely linked to their abundance in host tissues. The most common method for counting Wolbachia is quantitative polymerase chain reaction (qPCR), yet qPCR can be insufficient to count rare Wolbachia, necessitating tissue pooling and consequently compromising individual-level resolution of Wolbachia dynamics. Digital droplet PCR (ddPCR) offers superior sensitivity, enabling the detection of rare targets and eliminating the need for sample pooling. Here, we report three ddPCR assays to measure total Wolbachia abundance, Wolbachia abundance adjusted for DNA extraction efficiency, and Wolbachia density relative to host genome copies. Using Drosophila melanogaster with wMel Wolbachia as a model, we show these ddPCR assays can reliably detect as few as 7 to 12 Wolbachia gene copies in a 20 L reaction. The designed oligos are homologous to sequences from at least 106 Wolbachia strains across Supergroup A and 53 host species from the Drosophila, Scaptomyza, and Zaprionus genera, suggesting broad utility. These highly sensitive ddPCR assays are expected to significantly advance Wolbachia-host interactions research by enabling the collection of molecular data from individual insect tissues. Their ability to detect rare Wolbachia will be especially valuable in applied and natural field settings where pooling samples could obscure important variation.

microbiology↗

bioRxiv2022

Nuclease proteins CifA and CifB promote spermatid DNA damage associated with symbiont-induced cytoplasmic incompatibility

The worldwide endosymbiosis between arthropods and Wolbachia bacteria is an archetype for reproductive parasitism. This parasitic strategy rapidly increases the proportion of symbiont-transmitting mothers, and the most common form, cytoplasmic incompatibility (CI), impacts insect evolution and arboviral control strategies. During CI, sperms from symbiotic males kill embryos of aposymbiotic females via two nuclear-targeting proteins, CifA and CifB, that alter sperm chromatin organization in Drosophila melanogaster. Here we hypothesize that Cif proteins metabolize nucleic acids of developing sperm to initiate genome integrity changes. Using in vitro and in situ transgenic, mutant, enzymatic, and cytochemical assays, we show that CifA is a previously-unrecognized DNase and RNase, and CifB is a DNase. Notably, in vitro nuclease activity translates to in situ spermatid DNA damage at the canoe stage of spermiogenesis. Evolution-guided mutations ablate Cif enzymatic activity. Nucleic acid metabolism by Cif enzymes expands a fundamental understanding of the mechanism of symbiont-mediated reproductive parasitism.

biochemistry↗

bioRxiv2021

Male age and Wolbachia dynamics: Determining how fast and why bacterial densities and cytoplasmic incompatibility strengths vary

Endosymbionts can influence host reproduction and fitness to favor their maternal transmission. For example, endosymbiotic Wolbachia bacteria often cause cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-modified sperm. Infected females can rescue CI, providing them a relative fitness advantage. Wolbachia-induced CI strength varies widely and tends to decrease as host males age. Since strong CI drives Wolbachia to high equilibrium frequencies, understanding how fast and why CI strength declines with male age is crucial to explaining age-dependent CIs influence on Wolbachia prevalence. Here, we investigate if Wolbachia densities and/or CI gene (cif) expression covary with CI-strength variation and explore covariates of age-dependent Wolbachia-density variation in two classic CI systems. wRi CI strength decreases slowly with Drosophila simulans male age (6%/ day), but wMel CI strength decreases very rapidly (19%/ day), yielding statistically insignificant CI after only three days of D. melanogaster emergence. Wolbachia densities and cif expression in testes decrease as wRi-infected males age, but both surprisingly increase as wMel-infected males age, and CI strength declines. We then tested if phage lysis, Octomom copy number (which impacts wMel density), or host immune expression covary with age-dependent wMel densities--only host immune expression correlated with density. Together, our results identify how fast CI strength declines with male age in two model systems and reveal unique relationships between male age, Wolbachia densities, cif expression, and host immunity. We discuss new hypotheses about the basis of age-dependent CI strength and its contributions to Wolbachia prevalence. ImportanceWolbachia are the most common animal-associated endosymbionts due in large part to their manipulation of host reproduction. Many Wolbachia cause cytoplasmic incompatibility (CI) that kills uninfected host eggs. Infected eggs are protected from CI, favoring Wolbachia spread in natural systems and in transinfected mosquito populations where vector-control groups use strong CI to maintain pathogen-blocking Wolbachia at high frequencies for biocontrol of arboviruses. CI strength varies considerably in nature and declines as males age for unknown reasons. Here, we determine that CI strength weakens at different rates with age in two model symbioses. Wolbachia density and CI gene expression covary with wRi-induced CI strength in Drosophila simulans, but neither explain rapidly declining wMel-induced CI in aging D. melanogaster males. Patterns of host immune gene expression suggest a candidate mechanism behind age-dependent wMel densities. These findings inform how age-dependent CI may contribute to Wolbachia prevalence in natural systems and potentially in transinfected systems.

microbiology↗

bioRxiv2020

Evolution-guided mutagenesis of the cytoplasmic incompatibility proteins: Identifying CifAs complex functional repertoire and new essential regions in CifB

Wolbachia are the worlds most common, maternally-inherited, arthropod endosymbionts. Their worldwide distribution is due in part to a selfish drive system termed cytoplasmic incompatibility (CI) that confers a relative fitness advantage to females that transmit Wolbachia to their offspring. CI results in embryonic death when infected males mate with uninfected females but not infected females. Under the Two-by-One genetic model of CI, males expressing the two phage WO proteins CifA and CifB cause CI, and females expressing CifA rescue CI. While each protein is predicted to harbor three functional domains, there is no knowledge on how sites across these Cif domains, rather than in any one particular domain, contribute to CI and rescue. Here, we use evolution-guided, substitution mutagenesis of conserved amino acids across the Cif proteins, coupled with transgenic expression in uninfected Drosophila melanogaster, to determine the functional impacts of conserved residues evolving mostly under purifying selection. We report that amino acids in CifAs N-terminal unannotated region and annotated catalase-related domain are important for both complete CI and rescue, whereas C-terminal residues in CifAs putative domain of unknown function are solely important for CI. Moreover, conserved CifB amino acids in the predicted nucleases, peptidase, and unannotated regions are essential for CI. Taken together, these findings indicate that (i) all CifA amino acids determined to be involved in rescue are correspondingly involved in CI, (ii) an additional set of CifA amino acids are uniquely important in CI, and (iii) CifB amino acids across the protein, rather than in one particular domain, are all essential for CI. We discuss how these findings advance an expanded view of Cif protein evolution and function, inform the mechanistic and biochemical bases of Cif-induced CI/rescue, and continue to substantiate the Two-by-One genetic model of CI. Article summaryWolbachia are maternally-transmitted, intracellular bacteria that occur in approximately half of arthropod species worldwide. They can spread rapidly though host populations via the cytoplasmic incompatibility (CI) drive system. CI causes embryonic death when infected males mate with infected females, but offspring of infected females are rescued. Two proteins, CifA and CifB, underlie the genetic basis of CI and rescue, but how amino acid sites across these proteins contribute to CI and/or rescue remain unknown. Here, we employed evolution-guided, combinatorial mutagenesis on conserved amino acids to understand their relative contributions to CI and rescue. The results of this study reveal a phenotypic complexity underlying the expression of these proteins and provide relevance to the biochemical and mechanistic bases of CI and rescue.

microbiology↗

bioRxiv2018

A single prophage WO gene rescues cytoplasmic incompatibility in Drosophila melanogaster

Wolbachia are maternally-inherited, intracellular bacteria at the forefront of vector control efforts to curb arbovirus transmission. In international field trials, the cytoplasmic incompatibility (CI) drive system of wMel Wolbachia is deployed to replace target vector populations, whereby a Wolbachia- induced modification of the sperm genome kills embryos. However, Wolbachia in the embryo rescue the sperm genome impairment, and therefore CI results in a strong fitness advantage for infected females that transmit the bacteria to offspring. The two genes responsible for the wMel-induced sperm modification of CI, cifA and cifB, were recently identified in the eukaryotic association module of prophage WO, but the genetic basis of rescue is unresolved. Here we use transgenic and cytological approaches to demonstrate that cifA independently rescues CI and nullifies embryonic death caused by wMel Wolbachia in Drosophila melanogaster. Discovery of cifA as the rescue gene and previously one of two CI induction genes establishes a new Two-by-One model that underpins the genetic basis of CI. Results highlight the central role of prophage WO in shaping Wolbachia phenotypes that are significant to arthropod evolution and vector control.\n\nSignificance StatementThe World Health Organization recommended pilot deployment of Wolbachia-infected mosquitoes to curb viral transmission to humans. Releases of mosquitoes are underway worldwide because Wolbachia can block replication of these pathogenic viruses and deterministically spread by a drive system termed cytoplasmic incompatibility (CI). Despite extensive research, the underlying genetic basis of CI remains only half-solved. We recently reported that two prophage WO genes recapitulate the modification component of CI in a released strain for vector control. Here we show that one of these genes underpins rescue of CI. Together, our results reveal the complete genetic basis of this selfish trait and pave the way for future studies exploring WO prophage genes as adjuncts or alternatives to current control efforts.

genetics↗