Larval Cells Research Articles

Ferroptotic cyanobacteria as biocontrol agent of the southern house mosquito Culex quinquefasciatus

Culex quinquefasciatus is a hematophagous mosquito, widely distributed around the world, that plays a crucial role in public and veterinary health. As an efficient vector of etiological agents, it exhibits a marked preference for urban environments and human blood. Despite advances in mosquito-borne disease control, managing mosquito populations remains an economically efficient and safe strategy to reduce the impact of epidemic outbreaks. However, achieving this goal requires ecologically acceptable tools that ensure sustainability and minimize adverse environmental impacts. In the present work, we investigated the effect of a non-toxigenic model cyanobacterium on Cx. quinquefasciatus larvae through regulated cell death. We observed that heat stress treatment of Synechocystis PCC 6803 inducing ferroptosis, results in larval lipid oxidation, leading to their death. This effect can be mitigated by rearing larvae in an environment containing canonical inhibitors of ferroptosis, such as ferrostatin 1, or antioxidants, like glutathione and ascorbic acid. Furthermore, larval cell death induced by ferroptotic cyanobacteria is closely linked to oxidative dysregulation and lipid peroxidation, both hallmarks of ferroptosis. Moreover, while ferroptotic Synechocystis significantly affects larval development, it does not influence oviposition site selection by gravid females.

Characterization of cellular and molecular immune components of the painted white sea urchin Lytechinus pictus in response to bacterial infection.

Sea urchins are basal deuterostomes that share key molecular components of innate immunity with vertebrates. They are a powerful model for the study of innate immune system evolution and function, especially during early development. Here we characterize the morphology and associated molecular markers of larval immune cell types in a newly developed model sea urchin, Lytechinus pictus. We then challenge larvae through infection with an established pathogenic Vibrio and characterize phenotypic and molecular responses. We contrast these to the previously described immune responses of the purple sea urchin Strongylocentrotus purpuratus. The results revealed shared cellular morphologies and homologs of known pigment cell immunocyte markers (PKS, srcr142) but a striking absence of subsets of perforin-like macpf genes in blastocoelar cell immunocytes. We also identified novel patterning of cells expressing a scavenger receptor cysteine rich (SRCR) gene in the coelomic pouches of the larva (the embryonic stem cell niche). The SRCR signal becomes further enriched in both pouches in response to bacterial infection. Collectively, these results provide a foundation for the study of immune responses in L. pictus. The characterization of the larval immune system of this rapidly developing and genetically enabled sea urchin species will facilitate more sophisticated studies of innate immunity and the crosstalk between the immune system and development.

Contrasting the development of larval and adult body plans during the evolution of biphasic lifecycles in sea urchins.

Biphasic lifecycles are widespread among animals, but little is known about how the developmental transition between larvae and adults is regulated. Sea urchins are a unique system for studying this phenomenon because of the stark differences between their bilateral larval and pentaradial adult body plans. Here, we use single-cell RNA sequencing to analyze the development of Heliocidaris erythrogramma (He), a sea urchin species with an accelerated, non-feeding mode of larval development. The sequencing time course extends from embryogenesis to roughly a day before the onset of metamorphosis in He larvae, which is a period that has not been covered by previous datasets. We find that the non-feeding developmental strategy of He is associated with several changes in the specification of larval cell types compared to sea urchins with feeding larvae, such as the loss of a larva-specific skeletal cell population. Furthermore, the development of the larval and adult body plans in sea urchins may utilize largely different sets of regulatory genes. These findings lay the groundwork for extending existing developmental gene regulatory networks to cover additional stages of biphasic lifecycles.

Reduction of endocytosis and EGFR signaling is associated with the switch from isolated to clustered apoptosis during epithelial tissue remodeling in Drosophila.

Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Removal of cells is coordinated with the increase in number of newly dividing cells to maintain barrier function of the tissue. In Drosophila metamorphosis, larval epidermal cells (LECs) are replaced by adult precursor cells called histoblasts. Removal of LECs must counterbalance the exponentially increasing adult histoblasts. Previous work showed that the LEC removal accelerates as endocytic activity decreases throughout all LECs. Here, we show that the acceleration is accompanied by a mode switching from isolated single-cell apoptosis to clustered ones induced by the endocytic activity reduction. We identify the epidermal growth factor receptor (EGFR) pathway via extracellular-signal regulated kinase (ERK) activity as the main components downstream of endocytic activity in LECs. The reduced ERK activity, caused by the decrease in endocytic activity, is responsible for the apoptotic mode switching. Initially, ERK is transiently activated in normal LECs surrounding a single apoptotic LEC in a ligand-dependent manner, preventing clustered cell death. Following the reduction of endocytic activity, LEC apoptosis events do not provoke these transient ERK up-regulations, resulting in the acceleration of the cell elimination rate by frequent clustered apoptosis. These findings contrasted with the common perspective that clustered apoptosis is disadvantageous. Instead, switching to clustered apoptosis is required to accommodate the growth of neighboring tissues.

Haploidy-linked cell proliferation defects limit larval growth in zebrafish.

Haploid larvae in non-mammalian vertebrates are lethal, with characteristic organ growth retardation collectively called 'haploid syndrome'. In contrast to mammals, whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control larvae, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post-fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

Establishment and characterization of novel spontaneously immortalized larval cell lines from sablefish Anoplopoma fimbria.

Sablefish Anoplopoma fimbria is a groundfish of the North PacificOceantypically found in sea floor habitat at depths to 2700m. Prized as a food fish with exceptionally high market value, sablefish aquaculture has been sought to provide a sustainable source of this fish to meet market demands. While commercial culture has successfully produced market-sized fish in Pacific coastal environments, production has been hampered by disease and the overall lack of information on sablefish health and immunology. To begin to address these knowledge gaps, herein we describe the isolation and characterization of spontaneously immortalized sablefish larval cell lines (AFL). Six sublines were established from pools of early yolk-sac larvae, while attempts to develop tissue-specific-derived cell lines were unsuccessful. The six yolk-sac larval cell lines each display two morphologies in culture, an elongated fibroblast-like cell type, and a rounded squamous or epithelial-like cell type. Cytogenetic characterization suggests that both cell types are diploid (2n = 48) with 24 pairs of chromosomes, 23 pairs of autosomes, and 1 pair of sex chromosomes. A small proportion (11%) of AFL cells display tetraploidy. Incubation temperature and medium composition experiments revealed HEPES buffered L-15 media containing 10-20% FBS at temperatures between 15 and 18° C yielded optimal cell growth. These growth characteristics suggest that sablefish larval cells display a robustness for varying growth conditions. The establishment of AFL cell lines provides a foundational tool to study the physiology, health, immunology, and cell and molecular biology of sablefish.

In-vivo toxicological study of cysteamine modified carbon dots derived from Ruellia simplex on fruit fly for potential bioimaging

Modern medicine must shift its paradigm from conventional treatment strategies to theranostics to meet individuals' needs. Carbon dots, as a class of fluorescent materials, provide biocompatible and multifunctional solutions for a wide range of applications including clinical sensing, imaging, and drug delivery. Various studies have focused on their synthesis, photophysical properties, and innoxious nature but their potential biomedical applications for the in-vivo toxicity are yet limited. In this work, previously synthesized C-Dots (CDs) were utilized to determine their effect on the development of Drosophila melanogaster. Simultaneously, the genotoxic potential of CDs was evaluated on specific larval cell types that play important roles in immunological defence as well as growth and development. The gut organ toxicity of both CDs was studied using DAPI and DCFH-DA dyes wherein RS-CDs didn't show significant toxicity to the concentration 500 μg/mL whereas RS-Cys-CDs showed nuclear fragmentation and modest ROS (reactive oxygen species) production. Subsequently, trypan-blue assay, larvae crawling assay, touch sensitivity, adult phenotype, and survivability assay were performed. The trypan-blue assay shows the non-toxic nature of both CDs even at the concentration of 500 μg/mL. The high concentrations of RS-Cys-CD (500 μg/mL) were further associated with the alteration in touch behaviour and decrease in pupa hatching. In-vivo and ex-vivo fluorescence assessment of both the CDs exhibit bright fluorescence in green and red channels upon excitation at 485 and 577 nm respectively. The prominent imaging results from RS-Cys-CDs highlight the positive impact of surface modification.

Temperature influences immune cell development and body length in purple sea urchin larvae

Anthropogenic climate change has increased the frequency and intensity of marine heatwaves that may broadly impact the health of marine invertebrates. Rising ocean temperatures lead to increases in disease prevalence in marine organisms; it is therefore critical to understand how marine heatwaves impact immune system development. The purple sea urchin (Strongylocentrotus purpuratus) is an ecologically important, broadcast-spawning, omnivore that primarily inhabits kelp forests in the northeastern Pacific Ocean. The S. purpuratus life cycle includes a relatively long-lived (∼2 months) planktotrophic larval stage. Larvae have a well-characterized cellular immune system that is mediated, in part, by a subset of mesenchymal cells known as pigment cells. To assess the role of environmental temperature on the development of larval immune cells, embryos were generated from adult sea urchins conditioned at 14 °C. Embryos were then cultured in either ambient (14 °C) or elevated (18 °C) seawater. Results indicate that larvae raised in an elevated temperature were slightly larger and had more pigment cells than those raised at ambient temperature. Further, the larval phenotypes varied significantly among genetic crosses, which highlights the importance of genotype in structuring how the immune system develops in the context of the environment. Overall, these results indicate that larvae are phenotypically plastic in modulating their immune cells and body length in response to adverse developmental conditions.

Climate challenges for fish larvae: Interactive multi-stressor effects impair acclimation potential of Atlantic herring larvae

Fish early life stages are particularly vulnerable and heavily affected by changing environmental factors. The interactive effects of multiple climate change-related stressors on fish larvae remain, however, largely underexplored. As rising temperatures can increase the abundance and virulence of bacteria, we investigated the combination of a spring heat wave and bacterial exposure on the development of Atlantic herring larvae (Clupea harengus). Eggs and larvae of Western Baltic Spring-spawners were reared at a normal and high temperature ramp and exposed to Vibrio alginolyticus and V. anguillarum, respectively. Subsequently, mRNA and miRNA transcriptomes, microbiota composition, growth and survival were assessed. Both high temperature and V. alginolyticus exposure induced a major downregulation of gene expression likely impeding larval cell proliferation. In contrast, interactive effects of elevated temperature and V. alginolyticus resulted in minimal gene expression changes, indicating an impaired plastic response, which may cause cellular damage reducing survival in later larval stages. The heat wave alone or in combination with V. alginolyticus induced a notable shift in miRNA expression leading to the down- but also upregulation of predicted target genes. Moreover, both increased temperature and the Vibrio exposures significantly altered the larval microbiota composition, with warming reducing microbial richness and diversity. The outcomes of this study highlight the high sensitivity of herring early life stages towards multiple climate change-related stressors. Our results indicate that interactive effects of rapidly changing environmental factors may exceed the larval stress threshold impairing essential acclimation responses, which may contribute to the ongoing recruitment decline of Western Baltic Spring-Spawning herring.

The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera

The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.

Photocatalytic activity of TiO2-Carbon nanocomposite films against Culex pipiens mosquito larvae under sunlight irradiation

Recent research has focused on developing eco-friendly nanomaterials to combat mosquito infestations. This study demonstrates the enhancement of titanium dioxide (TiO2) nanoparticles through the addition of carbon nanoparticles (C-NPs), significantly boosting their photocatalytic efficiency. This enhancement allows TiO2 to purify water and act as an effective pesticide. Formulated TiO2-carbon (TiO2–C) nanocomposite films showed increased photocatalytic activity against third-instar larvae of Culex pipiens under natural sunlight. The addition of C-NPs improved sunlight absorption and reduced electron-hole recombination rates compared to pristine TiO2 (P–TiO2), making it more effective for mosquito control. The superior performance of the TiO2–C nanocomposite was confirmed through X-ray Photoelectron Spectroscopy (XPS), UV–vis diffuse reflectance spectra (UV–vis DRS), and Photoluminescence emission (PL). These analyses confirmed successful carbon incorporation, expanding the absorption spectrum and enhancing optical properties. The UV–vis DRS spectra showed a decrease in the bandgap energy (Eg) of P–TiO2 from 3.2 eV to 3.1 eV, improving its photocatalytic effectiveness under natural sunlight. Bioactivity tests, including catalase activity, reduced glutathione (GSH) colorimetric assay, and superoxide dismutase (SOD) assay, along with microscopic and histological examinations of treated larvae, indicated that the TiO2–C nanocomposite effectively reduces the mosquito population and causes significant physiological damage and abnormalities in larval structures and midgut cells. These findings highlight the enhanced photocatalytic capabilities of the TiO2–C nanocomposite, making it a novel and effective solution for mosquito control with significant public health and environmental benefits.

CED-5/CED-12 (DOCK/ELMO) can promote and inhibit F-actin formation via distinct motifs that may target different GTPases.

Coordinated activation and inhibition of F-actin supports the movements of morphogenesis. Understanding the proteins that regulate F-actin is important, since these proteins are mis-regulated in diseases like cancer. Our studies of C. elegans embryonic epidermal morphogenesis identified the GTPase CED-10/Rac1 as an essential activator of F-actin. However, we need to identify the GEF, or Guanine-nucleotide Exchange Factor, that activates CED-10/Rac1 during embryonic cell migrations. The two-component GEF, CED-5/CED-12, is known to activate CED-10/Rac1 to promote cell movements that result in the engulfment of dying cells during embryogenesis, and a later cell migration of the larval Distal Tip Cell. It is believed that CED-5/CED-12 powers cellular movements of corpse engulfment and DTC migration by promoting F-actin formation. Therefore, we tested if CED-5/CED-12 was involved in embryonic migrations, and got a contradictory result. CED-5/CED-12 definitely support embryonic migrations, since their loss led to embryos that died due to failed epidermal cell migrations. However, CED-5/CED-12 inhibited F-actin in the migrating epidermis, the opposite of what was expected for a CED-10 GEF. To address how CED-12/CED-5 could have two opposing effects on F-actin, during corpse engulfment and cell migration, we investigated if CED-12 harbors GAP (GTPase Activating Protein) functions. A candidate GAP region in CED-12 faces away from the CED-5 GEF catalytic region. Mutating a candidate catalytic Arginine in the CED-12 GAP region (R537A) altered the epidermal cell migration function, and not the corpse engulfment function. We interfered with GEF function by interfering with CED-5's ability to bind Rac1/CED-10. Mutating Serine-Arginine in CED-5/DOCK predicted to bind and stabilize Rac1 for catalysis, resulted in loss of both ventral enclosure and corpse engulfment. Genetic and expression studies strongly support that the GAP function likely acts on different GTPases. Thus, we propose CED-5/CED-12 support the cycling of multiple GTPases, by using distinct domains, to both promote and inhibit F-actin nucleation.

A mechanical transition from tension to buckling underlies the jigsaw puzzle shape morphogenesis of histoblasts in the Drosophila epidermis.

The polygonal shape of cells in proliferating epithelia is a result of the tensile forces of the cytoskeletal cortex and packing geometry set by the cell cycle. In the larval Drosophila epidermis, two cell populations, histoblasts and larval epithelial cells, compete for space as they grow on a limited body surface. They do so in the absence of cell divisions. We report a striking morphological transition of histoblasts during larval development, where they change from a tensed network configuration with straight cell outlines at the level of adherens junctions to a highly folded morphology. The apical surface of histoblasts shrinks while their growing adherens junctions fold, forming deep lobules. Volume increase of growing histoblasts is accommodated basally, compensating for the shrinking apical area. The folded geometry of apical junctions resembles elastic buckling, and we show that the imbalance between the shrinkage of the apical domain of histoblasts and the continuous growth of junctions triggers buckling. Our model is supported by laser dissections and optical tweezer experiments together with computer simulations. Our analysis pinpoints the ability of histoblasts to store mechanical energy to a much greater extent than most other epithelial cell types investigated so far, while retaining the ability to dissipate stress on the hours time scale. Finally, we propose a possible mechanism for size regulation of histoblast apical size through the lateral pressure of the epidermis, driven by the growth of cells on a limited surface. Buckling effectively compacts histoblasts at their apical plane and may serve to avoid physical harm to these adult epidermis precursors during larval life. Our work indicates that in growing nondividing cells, compressive forces, instead of tension, may drive cell morphology.

Gene Protein Sequence Evolution Can Predict the Rapid Divergence of Ovariole Numbers in the Drosophila melanogaster Subgroup.

Ovaries play key roles in fitness and evolution: they are essential female reproductive structures that develop and house the eggs in sexually reproducing animals. In Drosophila, the mature ovary contains multiple tubular egg-producing structures known as ovarioles. Ovarioles arise from somatic cellular structures in the larval ovary called terminal filaments (TFs), formed by TF cells and subsequently enclosed by sheath (SH) cells. As in many other insects, ovariole number per female varies extensively in Drosophila. At present, however, there is a striking gap of information on genetic mechanisms and evolutionary forces that shape the well-documented rapid interspecies divergence of ovariole numbers. To address this gap, here we studied genes associated with Drosophila melanogaster ovariole number or functions based on recent experimental and transcriptional datasets from larval ovaries, including TFs and SH cells, and assessed their rates and patterns of molecular evolution in five closely related species of the melanogaster subgroup that exhibit species-specific differences in ovariole numbers. From comprehensive analyses of protein sequence evolution (dN/dS), branch-site positive selection, expression specificity (tau), and phylogenetic regressions (phylogenetic generalized least squares), we report evidence of 42 genes that showed signs of playing roles in the genetic basis of interspecies evolutionary change of Drosophila ovariole number. These included the signaling genes upd2 and Ilp5 and extracellular matrix genes vkg and Col4a1, whose dN/dS predicted ovariole numbers among species. Together, we propose a model whereby a set of ovariole-involved gene proteins have an enhanced evolvability, including adaptive evolution, facilitating rapid shifts in ovariole number among Drosophila species.

Fruit ripening retardant Daminozide induces cognitive impairment, cell specific neurotoxicity, and genotoxicity in Drosophila melanogaster

BackgroundWe explored neurotoxic and genotoxic effects of Daminozide, a fruit ripening retardant, on the brain of Drosophila melanogaster, based on our previous finding of DNA fragmentation in larval brain cell in the flies experimentally exposed to this chemicals. MethodsAdult flies were subjected to two distinct concentrations of daminozide (200 mg/L and 400 mg/L) mixed in culture medium, followed by an examination of specific behaviors such as courtship conditioning and aversive phototaxis, which serve as indicators of cognitive functions. We investigated brain histology and histochemistry to assess the overall toxicity of daminozide, focusing on neuron type-specific effects. Additionally, we conducted studies on gene expression specific to neuronal function. Statistical comparisons were then made between the exposed and control flies across all tested attributes. ResultsThe outcome of behavioral assays suggested deleterious effects of Daminozide on learning, short term and long term memory function. Histological examination of brain sections revealed cellular degeneration, within Kenyon cell neuropiles in Daminozide-exposed flies. Neurone specific Immuno-histochemistry study revealed significant reduction of dopaminergic and glutaminergic neurones with discernible reduction in cellular counts, alteration in cell and nuclear morphology among daminozide exposed flies. Gene expression analyses demonstrated upregulation of rutabaga (rut), hb9 and down regulation of PKa- C1, CrebB, Ace and nAchRbeta-1 in exposed flies which suggest dysregulation of gene functions involved in motor neuron activity, learning, and memory. ConclusionTaken together, our findings suggests that Daminozide induces multifaceted harmful impacts on the neural terrain of Drosophila melanogaster, posing a threat to its cognitive abilities.

CAMP/PKA signaling regulates TDP-43 aggregation and mislocalization

Cytoplasmic mislocalization and aggregation of TDP-43 protein are hallmarks of amyotrophic lateral sclerosis (ALS) and are observed in the vast majority of both familial and sporadic cases. How these two interconnected processes are regulated on a molecular level, however, remains enigmatic. Genome-wide screens for modifiers of the ALS-associated genes TDP-43 and FUS have identified the phospholipase D (Pld) pathway as a key regulator of ALS-related phenotypes in the fruit fly Drosophila melanogaster [M. W. Kankel et al., Genetics 215, 747-766 (2020)]. Here, we report the results of our search for downstream targets of the enzymatic product of Pld, phosphatidic acid. We identify two conserved negative regulators of the cAMP/PKA signaling pathway, the phosphodiesterase dunce and the inhibitory subunit PKA-R2, as modifiers of pathogenic phenotypes resulting from overexpression of the Drosophila TDP-43 ortholog TBPH. We show that knockdown of either of these genes results in a mitigation of both TBPH aggregation and mislocalization in larval motor neuron cell bodies, as well as an amelioration of adult-onset motor defects and shortened lifespan induced by TBPH. We determine that PKA kinase activity is downstream of both TBPH and Pld and that overexpression of the PKA target CrebA can rescue TBPH mislocalization. These findings suggest a model whereby increasing cAMP/PKA signaling can ameliorate the molecular and functional effects of pathological TDP-43.

Honey bee (Apis mellifera) nurse bee visitation of worker and drone larvae increases Varroa destructor mite cell invasion.

The life cycle of Varroa destructor, the ectoparasitic mite of honey bees (Apis mellifera), includes a dispersal phase, in which mites attach to adult bees for transport and feeding, and a reproductive phase, in which mites invade worker and drone brood cells just prior to pupation to reproduce while their bee hosts complete development. In this study, we wanted to determine whether increased nurse bee visitations of adjacent drone and worker brood cells would increase the likelihood of Varroa mites invading those cells. We also explored whether temporarily restricting the nurses' access to sections of worker brood for 2 or 4h would subsequently cause higher nurse visitations, and thus, higher Varroa cell invasions. Temporarily precluding larvae from being fed by nurses subsequently led to higher Varroa infestation of those sections in some colonies, but this pattern was not consistent across colonies. Therefore, removing highly infested sections of capped worker brood could be further explored as a potential mechanical/cultural method for mite control. Our results provide more information on how nurse visitations affect the patterns of larval cell invasion by Varroa. Given that the mite's successful reproduction depends on the nurses' ability to visit and feed developing brood, more studies are needed to understand the patterns of Varroa mite invasion of drone and worker cells to better combat this pervasive honey bee parasite.

Increasing temperature and time in glasshouses increases honey bee activity and affects internal brood conditions

Honey bees are globally important pollinators, key to many aspects of ecosystem function and agricultural production. However they are facing an increasing array of stress factors. These stressors include exposure to pathogens and pesticides, agricultural intensification, and changes in climate, and likely contribute to colony dysfunction and colony losses. Here we use temperature-controlled glasshouse experiments to investigate the impact of a field-realistic temperature-range on honey bee colonies, including temperatures based on projections for near-future local conditions. We show that increased temperatures have a significant impact on honey bee worker activity, with increased worker movement in and out of colonies, particularly over 30 °C. In addition, increased glasshouse temperatures led to significantly higher brood (egg, larval and pupal cells) humidity. Finally, temperature had a more severe impact at the later end of the experiment than at the start (on worker movement and brood conditions), suggesting that colonies under stress (either due to exposure to thermal stress or glasshouse confinement) have more difficulty in manging thermoregulation. These results indicate the potential impact of higher temperatures on the healthy functioning of these important pollinators.

Determination of the larval precursor configuration of the Drosophila adult hindgut by G-TRACE analysis

The Drosophila hindgut is a classical model to study organogenesis. The adult hindgut originates from the precursor cells in the larval hindgut. However, the territory of these cells has still not been well determined. A ring of wingless (wg)-expressing cells lies at the anterior zone of both the larval and adult hindgut. The larval Wg ring was thought as a portion of precursor of the adult hindgut. By applying a cell lineage tracing tool (G-TRACE), we demonstrate that larval wg-expressing cells have no cell lineage contribution to the adult hindgut. Additionally, adult Wg ring cells do not divide and move posteriorly to replenish the hindgut tissue. Instead, we determine that the precursors of the adult pylorus and ileum are situated in the cubitus interruptus (ci)-expressing cells in the anterior zone, and deduce that the precursor stem cells of the adult rectum locate in the trunk region of the larval pylorus including hedgehog (hh)-expressing cells. Together, this research advances our understanding of cell lineage origins and the development of the Drosophila hindgut.

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction.

Although it is particularly valuable in revealing membrane potential changes, intracellular recording has a number of limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of miniature synaptic potentials (minis) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only under low-release conditions when nonlinear summation is not a major issue. As the EJP amplitude increases, it creates a "ceiling effect," because the same amount of transmitter will be less effective in depolarizing the membrane when the potential is approaching the reversal potential of glutamate receptors/channels. To overcome these limitations, the voltage-clamp technique can be used, which uses negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In voltage-clamp mode, the amplitude and kinetics of membrane currents can be determined. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval neuromuscular junction preparation.