Cellular & Molecular Imaging

Background Microsporidia sp. MB, a microsporidian symbiont found naturally in Anopheles mosquitoes, has potential as a novel malaria control tool since it can inhibit Plasmodium development and transmission. The most feasible MB-based Plasmodium control strategy would involve dissemination through live mosquito releases, or release of spores infective to mosquito larvae. To implement either strategy, establishment of stable mosquito colonies carrying MB at a high frequency is likely to be essential. The progeny of field caught An. gambiae s.l from Burkina Faso were isolated for individual egg laying and tested for MB. The progeny of the MB positive females were pooled and this process was repeated for multiple generations. The relative density of MB in different life stages and tissues of the An. coluzzii host was examined using a novel duplex qPCR assay. We also examined the impact of MB on fecundity through individualization for egg laying and counting of eggs. Finally, we examined laid eggs for presence of MB spores. Results Three An. coluzzii colonies and one An. gambiae s.l hybrid colony were established with high prevalence and density of MB and were maintained for more than two years with minimal intervention. MB prevalence and density was highest in eggs and adult females and lowest in L4 larvae; in adults density was highest in the gonads. Additionally, MB density increased in ovary samples following blood feeding which was likely due to the activation of sporogony. The production of spores is the reason why MB-carrying females lay more white non-hatching eggs and show a small reduction in fecundity. Conclusions Establishment of several stable MB carrying An. gambiae s.l colonies and understanding the impact of spores on fecundity are significant steps forward in developing MB as a malaria control tool.

SUMMARY. Here, we provide the image datasets supporting the development of CellCycleNet, a cell cycle stage classifier tool. We imaged thousands of fixed interphase mouse fibroblast cells containing a Fluorescent Ubiquitination-based Cell Cycle Indicator-2a (Fucci-2a) transgene, stained with DAPI, from two common fluorescent microscope modalities: widefield epifluorescence and spinning-disk confocal microscopy. ABSTRACT. The cell cycle governs proliferation of all eukaryotic cells. Profiling cell cycle dynamics is therefore central to basic and biomedical research. However, current approaches to cell cycle profiling involve complex interventions that may confound experimental interpretation. We developed CellCycleNet, a machine learning (ML) workflow, to simplify cell cycle staging from fluorescent microscopy data with minimal experimenter intervention and cost. CellCycleNet accurately predicts cell cycle phase using only a fluorescent nuclear stain (DAPI) in fixed interphase cells. Using the Fucci2a cell cycle reporter system as ground truth, we collected two benchmarking image datasets and trained 2D and 3D ML models—of support vector machine (SVM) and deep neural network architecture—to classify nuclei in the G1 or S/G2 phases. Our results show that 3D CellCycleNet outperforms SVM models on each dataset. When trained on two image datasets simultaneously, CellCycleNet achieves the highest classification accuracy (AUROC of 0.94-0.95). Overall, we found that using 3D features, rather than 2D features alone, significantly improves classification performance for all model architectures. We released our image data, models, and software as a community resource.

Background Microsporidia sp. MB, a microsporidian symbiont found naturally in Anopheles mosquitoes, has potential as a novel malaria control tool since it can inhibit Plasmodium development and transmission. The most feasible MB-based Plasmodium control strategy would involve dissemination through live mosquito releases, or release of spores infective to mosquito larvae. To implement either strategy, establishment of stable mosquito colonies carrying MB at a high frequency is likely to be essential. The progeny of field caught An. gambiae s.l from Burkina Faso were isolated for individual egg laying and tested for MB. The progeny of the MB positive females were pooled and this process was repeated for multiple generations. The relative density of MB in different life stages and tissues of the An. coluzzii host was examined using a novel duplex qPCR assay. We also examined the impact of MB on fecundity through individualization for egg laying and counting of eggs. Finally, we examined laid eggs for presence of MB spores. Results Three An. coluzzii colonies and one An. gambiae s.l hybrid colony were established with high prevalence and density of MB and were maintained for more than two years with minimal intervention. MB prevalence and density was highest in eggs and adult females and lowest in L4 larvae; in adults density was highest in the gonads. Additionally, MB density increased in ovary samples following blood feeding which was likely due to the activation of sporogony. The production of spores is the reason why MB-carrying females lay more white non-hatching eggs and show a small reduction in fecundity. Conclusions Establishment of several stable MB carrying An. gambiae s.l colonies and understanding the impact of spores on fecundity are significant steps forward in developing MB as a malaria control tool.

Fluorescent images of nuclear RelA and cRel dynamics upon CD40 and BCR activation. This is the data set behind figures 3L, 3M, 4F, and 4H.

Polyphasic studies on cyanobacterial biodiversity are scarce in the Caribbean and have never been conducted in the Dominican Republic. In this study we analysed sixteen cyanobacterial isolates sampled from biofilms growing on rocks and related to freshwater, saline and thermal environments across the country. A polyphasic approach was employed, incorporating 16S rRNA gene phylogenetic analysis, molecular identity assessments (p-distance), 16S–23S ITS sequence comparison and secondary structure analysis, alongside morphological characterization and habitat comparison. The strains were distributed across the orders Nodosilineales, Oculatellales, Oscillatoriales and Nostocales. Eleven strains were identified as belonging to Almyronema, Euryhalinema, Salileptolyngbya, Nodosilinea, Reticulonema, Vacuolonema, Capilliphycus, Purpureonostoc, Violetonostoc, Desmonostoc and Hapalosiphon. Additionally, four strains formed three distinct clades and presented low 16S rRNA gene identities with their phylogenetically closest genera, suggesting the existence of three undescribed lineages pending comprehensive polyphasic study. At the species level, four strains were considered undescribed lineages pending comprehensive polyphasic study and one strain (LEGE 171504) was described as a new species of Salileptolyngbya. This study represents the first investigation into the cyanobacterial diversity of Dominican Republic expanding the distribution of the identified taxa. Moreover, this is the first report of Salileptolyngbya in the Caribbean region and in brackish waters, as well as the first newly described cyanobacterial species from the Dominican Republic.

Bacterial endosymbionts inhabit diverse niches within eukaryotic cells, yet their direct associations with mitochondria have remained enigmatic. Here we introduce mitobiosis, the phenomenon of bacterial residency inside mitochondria or tight association with them. Using a multimodal volumetric electron microscopy (vEM) framework spanning whole-cell reconstructions to subnanometer-scale electron tomography, we investigated two ecologically important eukaryotes: the tick Ixodes ricinus and the marine protist Diplonema japonicum. We show that Midichloria mitochondrii, the maternally transmitted symbiont of I. ricinus, penetrates into the intracristal space of host mitochondria, inducing ≥60-fold cristal expansion exceeding 1 μm in width. In diplonemids, we observed staged endosymbiotic interaction with host mitochondrion, including cases of complete engulfment by both mitochondrial membranes. These findings establish mitochondria as dynamic symbiotic niches and highlight vEM as a powerful tool for uncovering hidden organelle-microbe interactions. Mitobiosis extends the spectrum of organelle-associated symbioses and represents a conceptual advance in cell biology.

Tara Expeditions are global scientific voyages that probe morphological and molecular diversity, evolution and ecology of marine plankton to explore how they are impacted by changes in the Earth's climate. The first expeditions collected samples of marine plankton containing viruses, bacteria, archaea, protists and planktonic metazoans living in the photic layer of the world's oceans. These expeditions, the first taking place between 2009 and 2013, include Tara Oceans: a global view, and Tara Oceans Polar Circle, both of which followed the same sampling protocol. This dataset includes 1074 pictures of 284 planktonic taxa (mainly microalgae and other Ciliate and Radiolarian protists) collected from the vicinity of the Marquesas Islands in the Southern Pacific Ocean during the Tara Oceans expedition. Multiple samples particularly of the size fractions 5-20 and 20 180 um from four sites and two depths were processed with different methods and studied in detail using scanning electron microscopy.

The Darwin Tree of Life project has the goal to sequence the genomes of 70,000 species of eukaryotic organisms in Britain and Ireland. This is a collection of photographs of the samples included in the study, provided by the National History Museum (NHM).

Confocal sample dataset for the "Step by step guide to post-acquisition correlation of confocal and FIB/SEM volumes using Amira software" chapter in the MCB: Correlative Light and Electron Microscopy IV Volume 162 book, by Allon Weiner

Bacterial endosymbionts inhabit diverse niches within eukaryotic cells, yet their direct associations with mitochondria have remained enigmatic. Here we introduce mitobiosis, the phenomenon of bacterial residency inside mitochondria or tight association with them. Using a multimodal volumetric electron microscopy (vEM) framework spanning whole-cell reconstructions to subnanometer-scale electron tomography, we investigated two ecologically important eukaryotes: the tick Ixodes ricinus and the marine protist Diplonema japonicum. We show that Midichloria mitochondrii, the maternally transmitted symbiont of I. ricinus, penetrates into the intracristal space of host mitochondria, inducing ≥60-fold cristal expansion exceeding 1 μm in width. In diplonemids, we observed staged endosymbiotic interaction with host mitochondrion, including cases of complete engulfment by both mitochondrial membranes. These findings establish mitochondria as dynamic symbiotic niches and highlight vEM as a powerful tool for uncovering hidden organelle-microbe interactions. Mitobiosis extends the spectrum of organelle-associated symbioses and represents a conceptual advance in cell biology.