Individual Colonies Research Articles

Next-Generation Yeast Two-Hybrid Screening to Discover Protein-Protein Interactions.

Yeast two-hybrid is a powerful approach to discover new protein-protein interactions. Traditional methods involve screening a target protein against a cDNA expression library and assaying individual positive colonies to identify interacting partners. Here we describe a simple approach to perform yeast two-hybrid screens of a cDNA expression library in batch liquid culture. Positive yeast cell populations are enriched under selection and then harvested en masse. Prey cDNAs are amplified and used as input for next-generation sequencing libraries for identification, quantification, and ranking.

Foraging Behaviour and Population Dynamics of Asian Weaver Ants: Assessing Its Potential as Biological Control Agent of the Invasive Bagworms Metisa plana (Lepidoptera: Psychidae) in Oil Palm Plantations

The bagworm (Metisa plana) is a recurrent indigenous invasive defoliator in oil palm plantations. Moderate foliar injury can cost up to 40% yield loss and more for years. The main objective of this review is to disseminate published research demonstrating the versatile services that would benefit farmers by adopting the Asian weaver ant into their pest management agenda. Oecophylla smaragdina is a natural indigenous enemy applied as a successful biological control agent (BCA) and strong component of integrated pest management (IPM) against important damaging pest infestations of commercial crops in the Asia-Pacific region. Farmers facing invasion could benefit by introducing Oecophylla ants as a treatment. The foraging behavior and population dynamics of this species are poorly documented, and hence need further evaluation. Ants of the Oecophylla genus, while exhibiting an intrinsic obligate arboreal pattern, demonstrate additional lengthy diurnal ground activity. The absolute territorial characteristic via continuous surveillance is significantly valuable to maintain pest balance. The exploratory scheme of major workers over large territories is derived from their inner predation instinct. The insufficient understanding of the population dynamics of this weaver ant species diverges from the knowledge of underground species. However, population density estimations of weaver ants by direct nest visual recordings are practicable and viable. The abundance assessment of individual underground ant species colonies by excavation ends with their extinction, which is not a sustainable model for O. smaragdina. Mathematical model estimation by simulation could not resolve this issue, adding inaccuracy to the deficiency of experimental proof. Thus, long-term monitoring of the population dynamics in real time in the field is compulsory to obtain a valid dataset. Oecophylla colonies, with the criteria of population stability, individual profusion, and permanent daily patrol services, are eligible as a BCA and alternative IPM treatment. The last decades have witnessed the closing of the scientific applied research gap between Asian and African species in favor of O. longinoda with comprehensive novel findings. By introducing Oecophylla ants, two main goals are reached: easing the burden of management costs for injurious insects and ending the practice of applying highly toxic pesticides that are harmful to non-target taxa, thus promoting environmental restoration.

Decentralized Application Placement in Fog Computing

In recent years, cloud computing concepts have been extended towards the network edge, leading to paradigms like fog and edge computing. As a result, applications can be placed on a variety of resources, including fog nodes and cloud data centers. Application placement has significant impact on important metrics like latency. Finding an optimal application placement is computationally challenging, particularly because of the potentially huge number of infrastructure nodes and application components. To overcome the limited scalability of application placement algorithms, optimization can be decentralized, i.e., performed separately for different parts of the infrastructure. The infrastructure can be split into fog colonies, where a fog colony consists of the computational resources in a given geographical region. Application placement can then be performed for the individual fog colonies, thus mitigating the scalability problem. However, independent optimization of application placement in different fog colonies may lead to missed synergies and thus to sub-optimal overall results. Hence, some kind of coordination between fog colonies may be beneficial. In this article, we analyze the effects of decentralization and coordination on the optimization results. In particular, we compare empirically four different approaches: (i) centralized decision-making, where decisions are made in one go for the entire infrastructure, (ii) independent fog colonies, where optimization is carried out in each fog colony independently from each other, (iii) fog colonies with communication, where excess application components in one fog colony can be sent to a neighboring fog colony, and (iv) fog colonies with overlaps, where shared resources may be dynamically distributed between neighboring fog colonies. Our experiments show that, for large problem instances, decentralization combined with coordination leads to the best results.

Combination of Wild ginger, Honey, and Probiotics as Growth Prometer Candidates : An in Vitro Study

Growth Promoter Antibiotics are used to prevent disease and promote growth and production in poultry. Repeated admnistration of feed can have a microorganic resistance effect, accumulation of antibiotic residues in animal and environmental products and imbalance of normal microflora in the intestine. The antibacterial and carbohydrate content of some natural ingredients can be potential as a replacement candidate for AGP. This study aims to determine the role of a combination of wild ginger, honey, and probiotics (Bacillus subtilis and Lactobacillus acidophilus) as AGP candiate in Vitro. The antibacterial activity of the combination of wild ginger and honey against pathogens (E. coli) and their use against probiotics was tested by disk diffusion method, while the calculation of optical density values to determine the minimum inhibitory concentration and minimum bactericidal concentration was carried out on E. coli. The ability of inhibition of probiotics against pathogens is also done by the disk diffusion method. The disk diffusion test results showed the best combination was extract of 25% wild ginger aquades + 100% Lombok honey with inhibition zone diameter (8,53 ±, 03). Optical density values indicate this combination is able to inhibit and kill E. coli (DO 0,00 ±, 002) and support B. subtilis (DO 0,18 ±, 002) and L. acidophilus (DO 0.25 ±, 005) significantly better than positive control. MIC value of wild ginger aquades extract and honey combination against E. coli is wild ginger aquades extract 3.13% + Lombok honey 25%, while MBC value is wild ginger aquades extract 6,25% + Lombok honey 25%. The combination of B. subtilis and L. acidophilus showed the largest inhibitory zone diameter against E. coli (7,30 ±, 02 mm) compared to individual colonies. The combination of wild ginger and honey, in addition to inhibiting also able to kill pathogens and support the growth of probiotics, so this formula can be used as one of the replacement candidates for AGP.

Restoration of Dystrophin Expression in Mdx-Derived Muscle Progenitor Cells Using CRISPR/Cas9 System and Homology-Directed Repair Technology.

Duchenne muscular dystrophy (DMD) is a progressive myopathy caused by mutations in genes encoding dystrophin proteins that ultimately lead to depletion of myogenic progenitor cells (MPCs). Several approaches have been used to correctly express the dystrophin gene in induced pluripotent stem cells (iPSCs), including deletion of mutated exon 23 (ΔEx23) by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated gene 9 (Cas9)-mediated gene editing technology. However, this approach is labor-intensive due to individual colony picking and genotyping to verify allelic modification. Here, we present a protocol to restore the function of the dystrophin gene by using homology-directed repair (HDR)-based CRISPR/Cas9 and inducing myogenic program of reprogrammed iPSCs from Mdx mice by inducible muscle-specific transcription factor MyoD.

Clonal Analysis Reveals Functional and Transcriptional Heterogeneity of Self-Renewing Fetal Liver Hematopoietic Stem Cells

The fetal liver (FL) is a key organ supporting the expansion of hematopoietic stem cells (HSCs) during development. The ability to expand transplantable HSCs in vitro is a long-standing challenging; thus, decoding the gene pathways that regulate proliferation and self-renewal during FL-HSC development is highly relevant to engineering HSCs for therapeutic applications. Moreover, while heterogeneity in self-renewal and engraftment properties of HSCs in adult bone marrow has been extensively studied, these properties remain largely uncharacterized in FL-HSCs. To explore FL-HSC properties at the single cell level, we engineered a serum-free culture platform designed to recapitulate the murine FL endothelial niche (FL-EC) (Figure1), and sorted single FL-HSC (CD45+GR1-F4/80-SCA1highEPCRhigh) into individual wells containing FL-EC. Following co-culture, we observed emergence of immunophenotypically distinct types of hematopoietic colonies as determined by flow cytometry. Only colonies containing cells predominantly expressing high levels of HSC markers SCA1 and EPCR (Figure1, HSC colonies) provided multilineage engraftment as determined by transplantation assays, whereas colonies containing cells expressing HSC markers and more differentiated progeny (mixed colonies) were unable to provide serial multilineage engraftment. Furthermore, cellular progeny from a single HSC colony transplanted into multiple recipients demonstrated serial engraftment, indicating the FL-EC niche could amplify single FL-HSC with long-term reconstitution capacity. These studies suggest that transplantable FL-HSCs are uniquely distinguished by their propensity for symmetric self-renewal in this assay. However, not all HSC colonies provided engraftment in secondary recipients, suggesting heterogeneity in self-renewal properties of immunophenotypically defined FL-HSCs that required deeper analysis. Thus, to further assess transcriptional heterogeneity in HSC colonies, we next integrated single-cell RNA sequencing (scRNAseq) into the clonal FL-HSC assay (Figure 2). Residual cells from six HSC colonies were divided for simultaneous scRNAseq and transplantation. Consistent with experiments above, we observed heterogeneity in long-term serial engraftment from individual colonies. Remarkably, cells from the colony capable of serial engraftment were distinctly enriched in genes associated with dormant HSCs in adult marrow (Cabezas-Wallscheid et.al., Cell 2017). Conversely, cells from HSC colonies that lacked serial engraftment capacity exhibited increased expression of Myc target genes by Gene Set Enrichment Analysis (GSEA), and ribosome biogenesis and mitochondrial translation by Gene Ontogeny (GO) analysis, which are hallmarks of biosynthetically activated HSCs in adult marrow that are primed for differentiation to progenitor states. To further assess the transcriptional heterogeneity of primary FL-HSC, we next performed scRNAseq with freshly isolated FL-HSC. Unsupervised clustering and co-regulated modules of differentially expressed genes identified a rare subset of cells highly enriched for genes associated with HSC dormancy, while the other cells featured higher expression of genes involved in biosynthetic processes via GO analysis (e.g. cytoplasmic translation, rRNA processing). This data further supports transcriptional heterogeneity within the FL-HSC compartment, which includes a unique biosynthetically dormant subset. Collectively, our studies elucidate previously unrecognized heterogeneity in FL-HSCs, highlighting a state of relative biosynthetic dormancy as a key to symmetric self-renewal of serially engraftable FL-HSCs. These findings complement recent studies demonstrating distinct developmental origins for multipotent progenitors and functional engraftable HSCs in definitive hematopoiesis (Dignum et.al., Cell Reports 2021; Patel et.al., Nature 2022), suggesting functional heterogeneity in immunophenotypically defined FL-HSCs in our study may in part reflect these distinct ontogenies. Building on these studies, we are currently using CRISPR-Cas9 gene knockout to define the roles of candidate genes identified by our scRNAseq analysis that are implicated in regulating dormancy and symmetric self-renewal of FL-HSCs, which would provide essential insight for future strategies for ex vivo amplification of HSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CBFB-MYH11 Is Required for the Maintenance of Inv(16) AML

Acute myeloid leukemia (AML) is a heterogenous disease arising from the immature blood cells in the bone marrow. One of the most common chromosomal aberrations, inversion of chromosome 16 [inv(16)], generates the fusion gene Cbfb-MYH11 (CM) which is known to initiate leukemogenesis. Because the fusion protein is invariably found in patients at relapse, it is assumed that CM is also required after leukemic transformation. However, knockdown of CM in the inv(16) immortalized cell line ME-1, caused relatively subtle effects on differentiation but did not affect viability. This raises the possibility that CM may be dispensable after leukemic transformation. To address this possibility in primary leukemia cells, we generated a new knock-in mouse model with the CM fusion gene flanked by loxP sites (CbfbflMYH11). This allows for the fusion gene to be deleted by Cre Recombinase (Cre) after leukemia development. To test the effect of loss of CM in vitro, we transduced Cbfb+/flMYH11 leukemia cells from three independent CbfbflMYH11 mice with a lentivirus expressing Cre. We confirmed the deletion of CM by qPCR and western blot. In Cbfb+/flMYH11, Cre+ cells, we observed a statistically significant increase in Annexin V staining and a decrease in colony forming ability as compared to control Cbfb+/flMYH11 cells. To determine if the colonies that did grow from Cre-infected cells had successfully deleted CM, individual colonies were picked. We found that majority of the colonies retained the unexcised CM allele, but 5-20% of colonies had successfully deleted CM, indicating that a rare population of leukemia cells can survive without CMin vitro. To test the requirement for CM in vivo, we used an inducible shRNA against CM rather than Cre+CbfbflMYH11 leukemia cells, as the inducible knockdown model will allow us to avoid potential defects in engraftment. We transduced CM expressing leukemic cells with a lentiviral construct with a doxycycline (Dox) inducible shRNA against MYH11 (shMYH11) that also expresses GFP. To test the feasibility of this approach in vitro, the sorted shCM+ leukemia cells were incubated with Dox for 48 hours. We found that Cbfb-MYH11 mRNA and protein were decreased in Dox treated leukemia cells, as compared to control cells. In addition, Dox treated shCM+ cells showed increased apoptosis and decreased colony forming ability, similar to Cbfb+/flMYH11Cre+ cells. To test the effect of CM knockdown in vivo, shCM transduced leukemia cells were transplanted into sub-lethally irradiated mice and monitored for leukemic engraftment. Once significant leukemic burden in the peripheral blood was detected, mice were given Dox or vehicle in their drinking water. Starting on day 7, we observed a statistically significant decrease in leukemia burden in Dox treated mice in the peripheral blood and spleen, with the percent of leukemia cells below 5% starting on day 14. In the bone marrow, there was a statistically significant decrease in leukemic burden starting on day 14, but the leukemia cells remained at 30-40% through day 28. Our initial tests indicate that the remaining GFP+ leukemia cells in the bone marrow express reduced levels of CM, implying that these cells may not strictly require the fusion protein or may be able to survive with only low levels of Cbfb-MYH11 activity. In a separate, small cohort of shCM+ transplanted mice, we treated the mice with Dox or control water and monitored for leukemia development. All the control treated mice developed fatal leukemia by day 21, whereas the majority of the Dox treated mice survived until day 42 of treatment. Interestingly, among the 6 Dox treated mice, 2 showed re-emergence of GFP+ leukemia cells in the peripheral blood on day 21 and 28, respectively. We are currently testing whether these relapsed leukemia cells re-express CM. Taken together, our results indicate that Cbfb-MYH11 is largely required for leukemia maintenance, although a subset of cells may be able to survive without the fusion gene. These results imply that drugs targeting the Cbfb-MYH11 have the potential to be effective treatments for inv(16) AML, although substantial inhibition of the fusion protein will likely be needed for effective treatment of the disease.

UBASH3B knockout Downregulates STAT1 Mediated Interferon Signalling and Megakaryocyte Related Genes in a JAK2 V617F Positive MPN Model

Interferon (IFN) signalling via STAT1 plays an important role in the pathogenesis and treatment of myeloproliferative neoplasms (MPN). Single cell sequencing approaches have implicated megakaryocyte (MKC) biased stem cells with enhanced intrinsic activation of IFN signalling pathways in MPN patients as critical mediators of disease initiation and maintenance. UBASH3B is a histidine phosphatase and has been identified as a potential regulator of malignant haematopoiesis and as a mediator of JAK/STAT1 IFN signalling in lymphoid cells. RNA sequencing (RNA-seq) data in JAK2 V617F positive UKE-1, HEL (both own lab) and SET2 (GSE91062) cells demonstrate that UBASH3B is significantly downregulated following administration of the JAK inhibitor ruxolitinib (RUX). We observed that UBASH3B expression in CD34+ cells in patients with primary myelofibrosis (GSE53482) is significantly enhanced but reduced in patients with essential thrombocythaemia(ET) and polycythaemia (GSE103237) and hypothesized that UBASH3B may contribute to MPN pathogenesis. UKE-1 is a JAK2 V617F positive primitive haematopoietic cell line derived from an ET patient. It shows features of intrinsic IFN signalling and expresses genes related to MKC development and differentiation providing a useful model of MPN stem cell behaviour. CRISPR-Cas9 mediated KO of UBASH3B was performed in UKE-1 cells. A ribonucleoprotein complex of guide RNA and Cas9 protein was nucleofected into cells. Individual colonies were assessed for UBASH3B status using western blotting and Sanger sequencing. Four distinct, healthy KO clones were established for the UKE-1 cell line and compared to the original wildtype cell population. RNA-seq and phospho-proteomic analysis were undertaken in two KO clones. Extracted RNA was sequenced on Illumina platforms with a minimum of 45 million individual paired reads per sample. Differential gene expression was analysed using DESEQ2. Whilst, matched cell lysates underwent tandem mass tagging, high pH fractionation, and phospho-peptide enrichment was undertaken prior to liquid chromatography-mass spectrometry for total and phospho-proteomic analysis. RNA-seq and total proteomic analysis were consistent in demonstrating a significant downregulation of STAT1 (Log2FC-0.79, FDR 9.03E-49) and STAT2 (Log2FC -0.70, FDR 9.05E-20) with over-enrichment analysis identifying downregulation of type 1 IFN signalling pathways and pathways related to MKC and platelet function as shown in Figure 1. The significant downregulation of genes associated with platelet function, MKC development and differentiation included GATA1, GP1BA, ITGA2B, SELP, VWF and genes identified as mediators of fibrosis in MPN as demonstrated in Figure 2. Targeted real time PCR for STAT1, GP1BA and LTPB1 confirmed the same transcriptional patterns were present in both additional knockout clones. Knockout clones show a diminished response to exogenous IFN alpha administration consistent with downregulation of these pathways. Consistently reduced proliferation is observed in the KO clones by a variety of methodologies. Comparison to the transcriptional response in RUX treated UKE-1 cells demonstrates that at 24 hours post RUX treatment significant STAT1 downregulation is observed but not accompanied by the same pattern of down-regulation of MKC and fibrosis associated genes. The same was seen in cells persistently treated with high dose RUX for greater than 5 weeks. These results show a significant downregulation of IFN signalling in UBASH3B KO cells linked to a significant downregulation of MKC associated genes further highlighting the link observed in MPN stem cells. They implicate UBASH3B as a potential mediator of IFN signalling in a JAK2 V617F context and support a hypothesis that downregulating IFN signalling in MPN stem cells may have disease modifying potential by altering differentiation and pro-fibrotic potential. RUX treatment downregulates STAT1 without activating the same transcriptional programme associated with MKC gene downregulation and this may help explain the limited disease modifying effect of RUX observed in patients. This work has generated an important KO cell line model which will permit the molecular characterisation of differential STAT1/IFN pathway activation and allow further study to understand the regulation of MKC differentiation and pro-fibrotic gene transcription observed in MPN stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Germline Loss-of-Function Mutations in MDM4 Cause p53-Dependent Hematopoietic Cell Death in Patients with Variable Bone Marrow Failure Phenotypes

The mouse double minute 4 (MDM4) negatively regulates p53 through the p53/MDM2/MDM4 regulatory network and is commonly overexpressed in several cancers. Here, we describe two unrelated pedigrees with germline MDM4 mutations with clinical features of bone marrow (BM) failure. Patient 1 (P1) presented at the age of 11 months with chronic pure red cell aplasia requiring chronic red blood cell transfusions and BM findings with absent erythroid precursors and dysplastic megakaryocytes. Patient 2 (P2) presented at age of 4 weeks with neutropenia with hypogranulation of neutrophils and a normocellular bone marrow with hypoplastic myelopoiesis and dysplasia of all three lineages. Both patients had normal chromosomal breakage and normal for age telomere length in peripheral blood. Whole genome and panel sequencing identified germline heterozygous variants in MDM4: p.Leu31Thrfs*15 in P1 and p.T454M in P2 as the only relevant candidate variants. Both mutations are absent in >140 thousand individuals in gnomAD database, and the constraint analysis indicates that MDM4 gene is intolerant to missense variation (observed to expected (O/E) ratio of 181/258, Z-score 1.71) and highly intolerant to protein truncating variation (O/E: 0/27 with a pLI score of 1). MDM4 protein consists of four key domains: p53 binding N-terminus, an acidic region, a zinc finger domain, and a C-terminal RING domain, which heterodimerizes with MDM2 to ubiquitinate and promote p53 degradation. MDM4 p.T454M missense mutation found in P2 was previously reported by Toufektchan et al. in 2020 in one patient with BM failure phenotype with short telomeres who carried additional TERT variant. The MDM4 p.T454M mutation was shown to result in lower protein levels (consistent with a loss-of-function effect) and had altered capacity to dimerize with the MDM2 RING domain, causing p53 hyperactivity. MDM4 p.Leu31Thrfs*15 mutation described in P1 is localized in the N-terminal p53 binding domain and results in RNA transcript loss, as confirmed by us in patient cells using RNA sequencing showing a two-fold reduction of the dominant transcript [ENST00000367182.8]. This was corroborated by Western blot demonstrating decreased levels of MDM4 protein. We sought to elucidate the impact of p.Leu31Thrfs*15 and p.T454M mutations on hematopoiesis and regulation of p53 expression. In two human induced pluripotent stem cell (iPSC) lines derived from male and female healthy donors, we knocked-in the MDM4 p.T454M and p.Leu31Thrfs*15 mutations by CRISPR/Cas9 engineering. Of note, MDM4 p.Leu31Thrfs*15 homozygous knock-in line could not be established despite multiple attempts. We discovered statistically significant decreased erythroid and myeloid differentiation capacity from MDM4-mutant iPSCs compared to isogenic control iPSCs (p <0.05). There were statistically significantly more AnnexinV+/DAPI+ mutant iPSC-derived myeloid and erythroid progenitors compared to isogenic control-derived progenitors (p <0.05), pointing to a higher rate of cell death in MDM4-mutant cells. We further demonstrated increased expression of p53 and p21 proteins in MDM4 p.Leu31Thrfs*15 iPSC clones. To better understand the loss-of-function effect of MDM4, we knocked out MDM4 in healthy CD34+ cells using CRISPR/Cas9 editing. Knock out of MDM4 with an efficiency of >80% was achieved in multiple biological replicates, resulting in increased p53 activity and fewer blast-forming erythroid (BFU-E) and myeloid colonies compared to control cells that were Cas9-edited at the AAVS1 safe harbor locus. This impairment was rescued by disruption of TP53 gene. Sequencing of individual BFU-E colonies did not reveal any cells with biallelic loss of MDM4, while similar percentages of unedited colonies and heterozygous disrupted MDM4 colonies were detected. Xenotransplantation studies using immunodeficient NBSGW mice are currently underway to explore the impact of MDM4-edited CD34+ cells in vivo. In summary, we describe two patients with germline loss-of-function MDM4 mutations that cause p53 hyperactivity which causes the unifying feature of hematopoietic defect across a BMF disorder spectrum without telomere shortening. Sharma and Bhoopalan are equal contributors

Water provisioning increases caged worker bee lifespan and caged worker bees are living half as long as observed 50 years ago

The high loss rates of honey bee colonies drive research for solutions aimed to mitigate these losses. While honey bee colonies are superorganisms, experiments that measure the response to stressors often use caged individuals to allow for inference in a controlled setting. In an initial experiment, we showed that caged honey bees provisioned with various types of water (deionized, 1%NaCl in deionized, or tap) have greater median lifespans than those that did not. While researching the history of water provisioning in cage studies, we observed that the median lifespan of caged honey bees has been declining in the US since the 1970’s, from an average of 34.3 days to 17.7 days. In response to this, we again turned to historical record and found a relationship between this trend and a decline in the average amount of honey produced per colony per year in the US over the last 5 decades. To understand the relationship between individual bee lifespan and colony success we used an established honey bee population model (BEEHAVE) to simulate the predicted effects of decreased worker lifespans. Declines in downstream measures of colony population, overall honey production, and colony lifespan resulted from reduced worker bee lifespans. Modeled colony lifespans allowed us to estimate colony loss rates in a beekeeping operation where lost colonies are replaced annually. Resulting loss rates were reflective of what beekeepers’ experience today, which suggests the average lifespan of individual bees plays an important role in colony success.

Coral bleaching resistance variation is linked to differential mortality and skeletal growth during recovery.

The prevalence of global coral bleaching has focused much attention on the possibility of interventions to increase heat resistance. However, if high heat resistance is linked to fitness tradeoffs that may disadvantage corals in other areas, then a more holistic view of heat resilience may be beneficial. In particular, overall resilience of a species to heat stress is likely to be the product of both resistance to heat and recovery from heat stress. Here, we investigate heat resistance and recovery among individual Acropora hyacinthus colonies in Palau. We divided corals into low, moderate, and high heat resistance categories based on the number of days (4-9) needed to reach significant pigmentation loss due to experimental heat stress. Afterward, we deployed corals back onto a reef in a common garden 6-month recovery experiment that monitored chlorophyll a, mortality, and skeletal growth. Heat resistance was negatively correlated with mortality during early recovery (0-1 month) but not late recovery (4-6 months), and chlorophyll a concentration recovered in heat-stressed corals by 1-month postbleaching. However, moderate-resistance corals had significantly greater skeletal growth than high-resistance corals by 4 months of recovery. High- and low-resistance corals on average did not exhibit skeletal growth within the observed recovery period. These data suggest complex tradeoffs may exist between coral heat resistance and recovery and highlight the importance of incorporating multiple aspects of resilience into future reef management programs.

First Report of Bacterial Leaf Scald of Plum Caused by Xylella fastidiosa in Texas.

Xylella fastidiosa is the etiological agent of Plum Leaf Scald (Greco et al. 2021). The disease was first reported in Argentina (Fernandez-Valiela et al. 1954) and then Brazil and Paraguay (French et al. 1978). In the USA, Plum Leaf Scald has been reported in the Southeastern United States (Wells et al. 1981a) and California (Hernandez-Martinez et al. 2009). In August 2021, during the Stone Fruit Survey of FY2020, plum trees (Mexican variety, Prunus mexicana) with symptoms of leaf scald, were observed in a Central Texas orchard with approximately 7% of trees exhibiting symptoms. Leaf margins were asymmetrically scorched, with necrotic areas that transitioned into chlorotic and healthy green tissues. To detect the presence of the pathogen, leaf sample petioles were tested using a double-antibody sandwich (DAS) ELISA® with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) according to manufacturer's guidelines. X. fastidiosa was detected in 20 of the 35 symptomatic samples. To confirm ELISA results, total DNA was extracted from the plant samples using the Plant DNeasy® kit (Qiagen Co. Hilden, Germany) following the manufacturer's protocol. All 20 ELISA-positive samples tested positive in a X. fastidiosa-specific real time PCR assay, using the primers XF1F and XF1R and probe XF1p (Schaad et al. 2002). Moreover, the ELISA-negative samples were also negative for PCR assay. Symptomatic samples were used to isolate the pathogen. Samples were debarked, surface-sterilized and xylem fluid collected. The fluid was gently imprinted on buffered charcoal yeast extract (BCYE) (Wells et al. 1981b) or periwinkle wilt modified (PWM) agar plates (Summer et al. 2010). After 10 days of incubation, individual colonies were observed. The colonies were slightly convex, white, opalescent, mucoid, circular with entire margins and with smooth surfaces on both media plates. Isolated colonies were triple-streak single colony purified and archived. Genomic DNA was extracted from four purified isolates using the DNeasy Blood and Tissue Qiagen® Kit, to conduct conventional PCR using HL5/HL6 (Francis et al. 2006), which identified the isolates as X. fastidiosa. Using the 16S rRNA primer pair U3/U4 (James 2010), amplicons were sequenced and compared against the NCBI database using the BLASTn algorithm. Comparative sequence analysis of amplicons from the four isolates were identical and indicated that the isolates were 100% identical to X. fastidiosa subsp. multiplex RIV5 (CP064326.1) from cherry plum, and IVIA5901 (CP047134.1) from almond. The sequences of all four isolates were deposited into NCBI GenBank, with the accession numbers OM617940 (467), OM617941 (470), OM617942 (471) and OM617943 (468). To our knowledge, this is the first report of X. fastidiosa associated with plum leaf scald in Texas, extending the geographical range of this important bacterial disease, in the Southern United States. This study highlights the importance of routine scouting of agricultural settings with a view to assessing and detecting early threats from either pests or disease and implementing relevant management strategies.

Characterizing in vivo loss of virulence of an HN878 Mycobacterium tuberculosis isolate from a genetic duplication event

The increase of global cases of drug resistant (DR) Mycobacterium tuberculosis (M.tb) is a serious problem for the tuberculosis research community and the goals to END TB by 2030. Due to the need for advancing and screening next generation therapeutics and vaccines, we aimed to design preclinical DR models of Beijing lineage M.tb HN878 strain in different mouse backgrounds. We found escalating sensitivities of morbidity due to low dose aerosol challenge (50–100 bacilli) in CB6F1, C57BL/6 and SWR mice, respectively. We also observed that pulmonary bacterial burden at morbidity endpoints correlated inversely with survival over time between mouse strains. Interestingly, with in vitro passaging and in the process of selecting individual DR mutant colonies, we observed a significant decrease in in vivo HN878 strain virulence, which correlated with the acquisition of a large genetic duplication. We confirmed that low passage infection stocks with no or low prevalence of the duplication, including stocks directly acquired from the BEI resources biorepository, retained virulence, measured by morbidity over time. These data help confirm previous reports and emphasize the importance of monitoring virulence and stock fidelity.

SOYBEAN AS AN ALTERNATIVE NUTRIENT MEDIUM FOR Bacillus subtilis GROWTH

Nutrient agar is a commonly used medium for the isolation and growth of a broad range of microorganisms. The feasibility of using soybean as a base medium in the development of alternative growth media was assessed in this study. Nutrient agar was used as a standard guide to evaluating the performance of the formulated soybean agar. Bacillus subtilis was inoculated and allowed to grow on nutrient agar and soybean agar. Their growth was compared within 24 h after inoculation based on the morphology of individual colonies formed on both media and the pattern of bacterial growth. Our results showed that soybean agar had comparable performance to nutrient agar as the morphological characteristics of B. subtilis colonies formed on both media are generally identical in terms of texture, margin, optical properties, colour, elevation, and shape. However, due to the similar appearance of the bacterial colonies and the soybean agar, the colonies formed on the soybean agar were slightly larger than those formed on nutrient agar. In addition, our findings also revealed that agar strips formed the best soybean agar compared to gelatin and agar powder. Ultimately, this study has shown that locally available soybeans and agar strips can be easily formulated as an alternative to commercial nutrient agar and have great potential for bacteriological research.

The effects of marine heatwaves on acute heat tolerance in corals.

Scleractinian coral populations are increasingly exposed to conditions above their upper thermal limits due to marine heatwaves, contributing to global declines of coral reef ecosystem health. However, historic mass bleaching events indicate there is considerable inter- and intra-specific variation in thermal tolerance whereby species, individual coral colonies and populations show differential susceptibility to exposure to elevated temperatures. Despite this, we lack a clear understanding of how heat tolerance varies across large contemporary and historical environmental gradients, or the selective pressures that underpin this variation. Here we conducted standardised acute heat stress experiments to identify variation in heat tolerance among species and isolated reefs spanning a large environmental gradient across the Coral Sea Marine Park. We quantified the photochemical yield (Fv /Fm ) of coral samples in three coral species, Acropora cf humilis, Pocillopora meandrina, and Pocillopora verrucosa, following exposure to four temperature treatments (local ambient temperatures, and + 3°C, +6°C and + 9°C above local maximum monthly mean). We quantified the temperature at which Fv /Fm decreased by 50% (termed ED50) and used derived values to directly compare acute heat tolerance across reefs and species. The ED50 for Acropora was 0.4-0.7°C lower than either Pocillopora species, with a 0.3°C difference between the two Pocillopora species. We also recorded 0.9°C to 1.9°C phenotypic variation in heat tolerance among reefs within species, indicating spatial heterogeneity in heat tolerance across broad environmental gradients. Acute heat tolerance had a strong positive relationship to mild heatwave exposure over the past 35 years (since 1986) but was negatively related to recent severe heatwaves (2016-2020). Phenotypic variation associated with mild thermal history in local environments provides supportive evidence that marine heatwaves are selecting for tolerant individuals and populations; however, this adaptive potential may be compromised by the exposure to recent severe heatwaves.

Emergent multilevel selection in a simple spatial model of the evolution of altruism.

Theories on the evolutionary origins of altruistic behavior have a long history and have become a canonical part of the theory of evolution. Nevertheless, the mechanisms that allow altruism to appear and persist are still incompletely understood. It is well known, however, that the spatial structure of populations is an important determinant. In both theoretical and experimental studies, much attention has been devoted to populations that are subdivided into discrete groups. Such studies typically imposed the structure and dynamics of the groups by hand. Here, we instead present a simple individual-based model in which altruistic organisms spontaneously self-organize into spatially separated colonies that themselves reproduce by binary fission and hence behave as Darwinian entities in their own right. Using software to automatically track the rise and fall of colonies, we are able to apply formal theory on multilevel selection and thus quantify the within- and among-group dynamics. This reveals that individual colonies inevitably succumb to defectors in a within-colony “tragedy of the commons”. Even so, altruism persists in the population because more altruistic colonies reproduce more frequently and drive less altruistic ones to extinction. Evidently, the colonies promote the selection of altruism but in turn depend on altruism for their existence; the selection of altruism hence involves a kind of evolutionary bootstrapping. The emergence of the colonies also depends crucially on the length scales of motility, altruism, and competition. This reconfirms the general relevance of these scales for social evolution, but also stresses that their impact can only be understood fully in the light of the emergent eco-evolutionary spatial patterns. The results also suggest that emergent spatial population patterns can function as a starting point for transitions of individuality.

Insights on the bacterial composition of Parmigiano Reggiano Natural Whey Starter by a culture-dependent and 16S rRNA metabarcoding portrait

Natural whey starters (NWS) are undefined bacterial communities produced daily from whey of the previous cheese-making round, by application of high temperature. As a result, in any dairy plant, NWS are continuously evolving, undefined mixtures of several strains and/or species of lactic acid bacteria, whose composition and performance strongly depend on the selective pressure acting during incubation. While NWS is critical to assure consistency to cheese-making process, little is known about the composition, functional features, and plant-to-plant fluctuations. Here, we integrated 16S rRNA metabarcoding and culture-dependent methods to profile bacterial communities of 10 NWS sampled in the production area of Parmigiano Reggiano cheese. 16S rRNA metabarcoding analysis revealed two main NWS community types, namely NWS type-H and NWS type-D. Lactobacillus helveticus was more abundant in NWS type-H, whilst Lactobacillus delbrueckii/St. thermophilus in NWS type-D, respectively. Based on the prediction of metagenome functions, NWS type-H samples were enriched in functional pathways related to galactose catabolism and purine metabolism, while NWS type-D in pathways related to aromatic and branched chain amino acid biosynthesis, which are flavor compound precursors. Culture-dependent approaches revealed low cultivability of individual colonies as axenic cultures and high genetic diversity in the pool of cultivable survivors. Co-culturing experiments showed that fermentative performance decreases by reducing the bacterial complexity of inoculum, suggesting that biotic interactions and cross-feeding relationships could take place in NWS communities, assuring phenotypic robustness. Even though our data cannot directly predict these ecological interactions, this study provides the basis for experiments targeted at understanding how selective regime affects composition, bacterial interaction, and fermentative performance in NWS.

Coral community data Heron Island Great Barrier Reef 1962–2016

Here we describe benthic composition data derived from benthic photoquadrats collected over 41 surveys between 1962 and 2016 at four sites on Heron reef, at the southern end of Australia’s Great Barrier Reef, to assess change in coral composition over time. Surveys have often been annual, in a few years sub-annual, and the longest gap is six years. A subset of the data from two sites with the most complete records has been fully processed to allow the size of all individual colonies, and changes in species composition and cover, to be tracked over time. The taxonomy in these quadrats has been carefully checked for internal consistency, and is generally at the species level. A second subset has been processed, but has not been through full quality control, while a third subset exists as images only. This is the longest, 56 years, regular photographic record of coral cover in existence, and provides a valuable temporal contrast dating back in time to more recent studies of greater geographic extent and/or resolution.

DIVERSITY AND DISTRIBUTION OF CORALS AT POSHITRA, GULF OF KACHCHH, GUJARAT

Corals are the tiny invertebrates and are keystone species. The individual colony are called “polyps” which secrete calcium carbonate. Such colony forms a coral reef which play a major role in providing shelter to marine animals. Amongst four major reefs in India one is Gulf of Kachchh. Gujarat has a longest coastline of about 1650 km among all the marine states in India and the Gulf of Kachchh (22⁰15’ - 23⁰40’N; 68⁰20’ - 70⁰40’E) is the biggest gulf along the west coast of India. It is endowed with 42 islands with fringing reef of corals and mangroves which provided congenial habitat to many marine animals (Roy et al., 2015). In the present study, the diversity and distribution of corals were enumerated for three (03) different sites at Poshitra in the Gulf of Kachchh. The study was carried out during December 2019 to March 2020. The method used was random quadrant method. Total 18 hard corals and 01 soft coral were recorded. Further, diversity indices of corals were carried in which site 2 showed highest species richness which is also called alpha diversity, for beta diversity number of species variety were more in site 1 and site 2 and for evenness, the species were more evenly distributed in site 2.

On the fate of dead coral colonies

Abstract Carbonate budgets dynamically balance production and loss of calcium carbonate (CaCO3) on coral reefs. To sustain or expand the coral reef framework, CaCO3 production by calcifying organisms must be higher than erosion. However, global climate change has been negatively impacting carbonate production, with bleaching events causing widespread coral mortality. Although bleaching and coral mortality are well documented, the fate of coral colonies after their death, including their erosion rates, are still poorly known. We followed the fate of 143 recently dead individual coral colonies with complex growth forms (arborescent, caespitose, corymbose, digitate and tabular), whose mortality was triggered by two consecutive bleaching events. These colonies, spread over 16 km2 of the Lizard Island reef complex, were tracked for up to 5 years, allowing detailed examination of erosion rates and post‐mortality structural persistence. We also tested how variables that are commonly used in coral reef erosion studies relate to spatial and temporal variability in the erosion rates of dead coral colonies. We revealed rapid erosion of dead coral colonies, with an average of 79.7% of dead colonies completely disintegrating within 60 months. The predicted half‐life of a dead coral colony was 40 months, with limited variation among wave exposure levels. Remarkably, we found no effect of estimated parrotfish bioerosion, wave exposure, nor coral growth form, on observed erosion rates. Our results suggest that our understanding of the erosion of dead corals may be more limited than previously thought. The rapid loss of coral colonies on our study sites calls for a re‐evaluation of the role of corals with complex growth forms in reef growth and of parrotfishes in reef erosion. Read the free Plain Language Summary for this article on the Journal blog.