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Blood-feeding induces molecular signatures in I. scapularis hemocytes related to immunity, metabolism, and proliferation

Ticks rely solely on blood as a source of essential metabolites, ingesting ~100 times their body mass per meal19. During feeding, extensive modifications and tissue rearrangements are necessary to accommodate and digest such large volumes of blood19. Considering that hemocytes are the circulating cells in the hemolymph, we hypothesized that these immune cells sense and respond to physiological and microbial exposure during tick infestation on vertebrate hosts. We optimized a protocol to collect hemocytes from I. scapularis nymphs, a clinically relevant stage in the blacklegged tick, and identified three common hemocyte morphotypes reported in the literature (Fig.1a, Supplementary Fig.1)16,17,18. Prohemocytes, considered the stem cell-like hemocyte population, were the smallest cells, with a round or oval shape between 5.7 and 12.5m (average diameter of 8.5m). The cytoplasm was minute (high nuclear/cytoplasmic ratio) and homogeneous, with no apparent protrusions or granules (Supplementary Fig.1a, d). Granulocytes were round or oval shaped cells with diameters ranging from 10.4 to 22.8m (average of 16.7m). The position of the nucleus varied, appearing most often near the periphery of the cell. Their cytoplasm was filled with dark-blue or violet stained granules or vacuoles (Supplementary Fig.1b, d). Plasmatocytes varied in size (ranged from 14.8 to 31.2m, with an average of 21.3m) and shape (oval, ameboid-like, pyriform), and had cytoplasmic protrusions or pseudopodia-like structures. The cytoplasm was clear and had few dark-blue or violet stained granules or vacuoles. The nucleus was located either near the center or the periphery of the cell (Supplementary Fig.1c, d). We then investigated the impact of blood-feeding on hemocytes originating from I. scapularis nymphs. We observed an increased quantity of total hemocytes upon mammalian feeding (Fig.1b). The percentage of plasmatocytes increased in engorged ticks, whereas we noticed a decline in the proportion of prohemocytes and granulocytes in repleted nymphs compared to unfed (Fig.1b). Altogether, we demonstrate the impact of hematophagy on the distribution of tick hemocyte morphotypes.

a Schematic representation of the hemocyte-enriched collection procedure. b Total number of hemocytes (n=40, 25 and 19) and percentages of different morphotypes (prohemocytes, plasmatocytes and granulocytes; n=20, 13 and 17 for all cases) from unfed (ivory), partially fed (light blue) or engorged (dark blue) nymphs. c Functional enrichment analysis of the differentially expressed genes (DEGs) in hemocyte-enriched samples from engorged ticks (blue; Up) compared to unfed ticks (red; Down). Fold enrichment of significant categories (FDR<0.05) is depicted. The number of DEGs per category is shown in parentheses. d The expression of key genes upregulated during feeding in hemocyte-enriched samples from unfed (ivory), partially fed (light blue) and engorged (dark blue) ticks was evaluated by RT-qPCR (n=11, 5 and 7 for all cases; with 4080 pooled ticks per sample). b, d Results are represented as meanSD. At least three independent experiments were performed. Statistical significance was evaluated by Brown-Forsythe ANOVA test, and significant p-values (<0.05) are displayed in the figure. Source data are provided as a Source Data file. 4cl1=4-coumarate-CoA ligase 1; ahcy = adenosylhomocysteinase B; g6pc = glucose-6-phosphatase 2; impdh = inosine-5-monophosphate dehydrogenase 1.

Next, we aimed to examine global transcriptional changes induced by hematophagy through bulk RNA-seq. We collected hemocyte-enriched samples from unfed and engorged nymphs and observed drastic changes in gene expression, with a total of 6134 differentially expressed genes (DEGs) (Fig.1c; Supplementary Data1). Samples from unfed I. scapularis nymphs were enriched for housekeeping genes, including those involved in mRNA transcription (e.g., RNA splicing, mRNA processing, histone modification), protein synthesis (e.g., peptide biosynthetic process, cellular protein modification process, ribosome biogenesis) and membrane receptor signaling pathways (e.g., transmembrane signaling receptor activity, G protein-coupled receptor signaling pathway, protein kinase activity) (Fig.1c; Supplementary Data2). In contrast, hemocyte-enriched samples from engorged ticks exhibited an overrepresentation of gene signatures related to immunity, metabolic pathways, cell proliferation/growth, and arthropod molting/development (Fig.1c; Supplementary Fig.2; Supplementary Data2). Key genes modulated during feeding, including 4-coumarate-CoA ligase 1 (4cl1), adenosylhomocysteinase B (ahcy), glucose-6-phosphatase 2 (g6pc), inosine-5-monophosphate dehydrogenase 1 (impdh), Brahma chromatin remodeling complex subunit osa (osa), runt-related transcription factor 1 (runx) and frizzled-5 (frizzled), were independently validated using RT-qPCR (Fig.1d; Supplementary Fig.3). Notably, the overall expression levels of these genes in hemocyte-enriched samples from partially fed ticks were intermediate compared to those in unfed and engorged ticks, suggesting a transitional phenotype. Collectively, this dataset indicated that I. scapularis hemocytes exhibit a dynamic genetic program during hematophagy.

To uncover whether the transcriptional changes observed through bulk RNA-seq are accompanied with heterogeneity among hemocytes, we performed scRNA-seq. We collected hemocyte-enriched samples from (1) unfed nymphs, (2) engorged nymphs fed on uninfected mice, and engorged nymphs fed on mice infected with either (3) the rickettsial pathogen A. phagocytophilum or (4) the Lyme disease spirochete B. burgdorferi. After stringent quality controls, we profiled a total of 20,432 cells (unfed = 4630; engorged uninfected = 6000; engorged A. phagocytophilum-infected = 6287; engorged B. burgdorferi-infected = 3515), with a median of 744 unique molecular identifiers (UMIs), 261 genes and 6.2% of mitochondrial transcripts per cell across conditions (Supplementary Fig.4). Consistent with the bulk RNA-seq results, the principal component analysis (PCA) identified distinct distributions between unfed and fed conditions, reinforcing the notion that significant cellular and/or transcriptional changes occur following a blood meal (Supplementary Fig.5).

Following unsupervised clustering, we identified seven clusters in unfed ticks and thirten clusters in engorged nymphs (Supplementary Fig.6). Based on similarities in marker gene profiles, two clusters in the unfed and two clusters in the engorged datasets were merged (Supplementary Data3, 4). Thus, six and twelve clusters remained, respectively, with each cluster expressing a unique set of cell type-defining genes (Fig.2ac, Supplementary Data57). One cluster in each dataset showed high expression of gut-associated genes cathepsinB, cathepsinD and boophilinH2 (Fig.2ac, Supplementary Data57) and two additional clusters in the engorged dataset had gene expression profiles indicative of cuticle and salivary glands tissues (Fig.2ac, Supplementary Data5 and 7). Therefore, these clusters were excluded from subsequent analysis. We assigned putative functions to the top 50 DEGs per cluster using information for tick genes in VectorBase, the sequence homology to D. melanogaster in FlyBase and the presence of functionally annotated protein domains in InterPro (Fig.2ce, Supplementary Data6, 7). We also performed functional enrichment analysis based on gene ontology (GO) using the entire list of DEGs for each cluster (Supplementary Data8, 9). Altogether, we defined five hemocyte clusters from unfed and nine clusters in engorged I. scapularis nymphs.

Hemocyte-enriched samples pooled from individual unfed (n=90) or engorged uninfected, A. phagocytophilum- or B. burgdorferi-infected I. scapularis ticks (n=50 for each) were collected immediately post-detachment from the host. t-Distributed Stochastic Neighbor Embedding (t-SNE) plot clustering of samples collected from (a) unfed (4,630 cells) and (b) engorged (15,802 cells) nymphs. The engorged t-SNE includes cells from uninfected (6000 cells), A. phagocytophilum-infected (6287 cells) and B. burgdorferi-infected (3515 cells) ticks. c Dot plot of the top 5 marker genes present in clusters from engorged ticks based on average gene expression. Color intensity demarks gene expression level, while the size of the dot indicates the percentage of cells within individual clusters expressing the corresponding gene. d Heatmap depicting the expression of marker genes for hemocyte subtypes from engorged ticks. The top 20 marker genes per cluster based on gene expression are included, with representative genes highlighted. e The top 50 marker genes from each hemocyte cluster were manually annotated using the VectorBase, FlyBase, and UniProt publicly available databases. The percentage of predicted functional categories, such as ncRNA/pseudogenes (yellow), protein synthesis (black), secreted/extracellular matrix (blue), unknown (orange), actin/cell rearrangement (brown), detoxification (white), cell proliferation/differentiation (grey), metabolism (green), hormone-related (purple), and immunity (red) are shown. f Pseudotime analysis using slingshot defined six hemocyte lineages (indicated by arrows) in engorged ticks. Tick images in (a, b) were created with BioRender.com.

The molecular features and differentiation process of I. scapularis hemocytes is currently unknown. Thus, based on GO enrichment and marker gene profiles, we were able to characterize clusters of hemocytes shared by both unfed and engorged ticks (Supplementary Figs.7, 8, Supplementary Data57). The Immune 1 cluster showed high expression of genes related to phagocytosis or cytoskeleton organization; coagulation and agglutination functions, such as lectins (hemocytin, techylectin-5A), chitin-binding and clotting proteins; and secreted proteins related to immunity, such as astakine, microplusin, mucins and cystatin domain peptides (Fig.2ce, Supplementary Data57). The Immune 2 cluster displayed genes encoding secreted proteins involved in immunity, such as antimicrobial peptides (AMPs) and clotting related peptides (Fig.2ce, Supplementary Data57). The Proliferative 1 cluster was enriched with mitochondrial genes, characteristic of stem cells in ancient arthropods, such as crayfish20. This cell cluster also had high expression of genes related to actin polymerization, cell proliferation and differentiation (Fig.2ce, Supplementary Data57). The Proliferative 2 cluster displayed a high percentage of transcription factors, RNA binding proteins and genes related to actin dynamics. Marker genes for this cluster included several genes involved in hormone-related responses, suggesting they may be responsive to ecdysteroids synthesized after a blood meal (Fig.2ce, Supplementary Data57). Lastly, both datasets had a Transitional cluster indicative of intermediate subtypes (Fig.2ce, Supplementary Data57).

Four hemocyte clusters were only observed in engorged I. scapularis ticks. The Immune 3 cluster displayed an enrichment in secreted proteins and genes related to immune functions. This cluster was enriched for chitinases, matrix and zinc metalloproteinases, peptidases, and actin binding proteins, which have roles related to wound healing or tissue rearrangement. Several glycine-rich proteins (GRPs), commonly associated with antimicrobial properties or structural proteins, were also present (Fig.2ce, Supplementary Data5, 7). The Immune 4 cluster showed an overrepresentation of genes related to protein degradation, immune function, and cell proliferation (Fig.2ce, Supplementary Data5, 7). Thus, we posit that the Immune 4 cluster represents an intermediate state between the Immune 2 and the Proliferative 2 clusters. Two clusters displayed an enrichment for genes related to metabolic functions. The Metabolism 1 cluster represented genes involved in sulfonation of proteins, lipids and glycosaminoglycans, transmembrane solute transporter, nucleotide, and protein metabolism (Fig.2ce, Supplementary Data5, 7). The Metabolism 2 cluster displayed genes related to detoxification, histamine binding, lipid metabolism, methionine and juvenile hormone metabolism (Fig.2ce, Supplementary Data5, 7).

Based on these findings, we predicted hemocyte ontogeny using pseudotime analysis, which orders hemocyte clusters based on their gene expression profiles, enabling us to infer developmental lineages (Fig.2f)21. We found six trajectories considering the cluster Proliferative 1 as a stem cell-like subpopulation. Lineage 1 and 2 ended with the Immune 2 and Proliferative 2 clusters, respectively, with the Immune 4 cluster serving as an intermediate state. Lineages 3 and 4 gave rise to the Immune 3 and Immune 1 clusters, respectively. Finally, two lineage trajectories ended with the metabolic clusters, Metabolism 1 and Metabolism 2. Overall, our findings suggest the presence of an oligopotent subpopulation that differentiates into more specialized subtypes involved in immune and metabolic functions, a process evoked by hematophagy.

The impact of bacterial infection on subtypes of tick hemocytes remains elusive. Thus, we collected hemocytes from I. scapularis nymphs fed on uninfected, A. phagocytophilum- or B. burgdorferi-infected mice and determined morphotype percentages. During infection with the rickettsial agent A. phagocytophilum, a relative decrease in prohemocytes and increase in plasmatocytes was noted (Fig.3a). Conversely, only a slight decrease in the proportion of prohemocytes was observed during infection with the Lyme disease spirochete B. burgdorferi (Fig.3a). No difference in total hemocyte numbers was observed across infection conditions (Supplementary Fig.9). However, partitioning the engorged scRNA-seq datasets by treatments revealed a reduction in the Transitional cluster with an expansion in the Metabolism 1 cluster during B. burgdorferi infection (Fig.3b, c). We next analyzed transcriptional changes at the cellular level in engorged uninfected nymphs compared to engorged infected ticks. Hemocyte clusters were grouped according to three molecular programs: Immune (Immune 1-4), Proliferative (Proliferative 1-2 and Transitional) and Metabolism (Metabolism 1-2). During A. phagocytophilum infection, we identified 177 DEGs within the Proliferative clusters, 53 DEGs in the Metabolism clusters, and 5 DEGs among the Immune clusters (Fig.3d, Supplementary Data10). Conversely, during B. burgdorferi infection, we detected 244 DEGs within the Proliferative clusters, 81 DEGs among the Metabolism clusters, and 12 DEGs associated with the Immune clusters (Fig.3d, Supplementary Data10). In total, we identified 188 and 257 unique DEGs across all hemocyte clusters during A. phagocytophilum and B. burgdorferi infection, respectively. Notably, 11 genes were differentially expressed during both bacterial infections and shared amongst all cluster groupings, in which 36.4% (4 out of 11) were marker genes of the Immune 1 cluster (Fig.3e). Collectively, our results defined specific hemocyte subpopulations and genes that are differentially expressed in I. scapularis in response to A. phagocytophilum or B. burgdorferi infection.

a Hemocyte morphotypes (prohemocytes, plasmatocytes and granulocytes) in I. scapularis nymphs fed on A. phagocytophilum- (Ap, pink) or B. burgdorferi- (Bb, green) infected mice compared to uninfected [(-), dark blue] (n=16 and 12; n=14 and 14, respectively). Results are presented as meanSD. A minimum of two independent experiments were conducted. Statistical significance was determined using an unpaired two-tailed t test with Welchs correction, and significant p-values (<0.05) are displayed in the figure. b t-Distributed Stochastic Neighbor Embedding (t-SNE) plot clustering of cells collected from the hemolymph of uninfected (6000 cells), A. phagocytophilum- (6287 cells) or B. burgdorferi-infected (3515 cells) I. scapularis nymphs. c Percentage of hemocyte clusters in uninfected, A. phagocytophilum- or B. burgdorferi-infected ticks. d Venn diagram illustrating the number of differentially expressed genes (DEGs) between groups of hemocyte clusters during A. phagocytophilum (top) or B. burgdorferi (bottom) infection compared to uninfected ticks. Hemocyte clusters were categorized into three molecular programs: Immune (Immune 1-4), Proliferative (Proliferative 1-2 and Transitional) and Metabolism (Metabolism 1-2). DEGs were determined using pairwise comparisons against uninfected. e Heatmap representing the average expression patterns of DEGs altered during infection and shared between all 3 cluster groups. For each DEG, the mean average across all experimental conditions was centered at zero for each hemocyte group. The # symbol indicates Immune 1 marker genes. Source data are provided as a Source Data file. Tick images in (b) were created with BioRender.com. (-)=Uninfected. Anaplasma=A. phagocytophilum. Borrelia=B. burgdorferi.

The preceding findings suggested that the Immune 1 hemocyte cluster represented a subpopulation of cells that responded to bacterial infections in ticks (Fig.3d). Therefore, our focus shifted to two marker genes from the Immune 1 hemocyte cluster: hemocytin and astakine (Fig.4a, Supplementary Data6, 7). Hemocytin is homologous to hemolectin in D. melanogaster and von Willebrand factors of mammals22,23. Hemocytin encodes for a large multidomain adhesive protein involved in clotting, microbial agglutination and hemocyte aggregation22,23,24,25. Conversely, astakine is a cytokine-like molecule present in chelicerates and crustaceans and is homologous to vertebrate prokineticins26,27,28,29. Astakine also induces hemocyte proliferation and differentiation of immune cells26,27,28,29. Hemocytin and astakine were broadly expressed in the Immune 1 cluster of I. scapularis hemocytes (Fig.4a). Validating our scRNA-seq results, we confirmed the expression of hemocytin and astakine in hemocyte-enriched samples obtained from unfed ticks using RNA-FISH, illustrating that these genes serve as markers for the Immune 1 cluster (Fig.4b, Supplementary Fig.10). Furthermore, we noted an upregulation of hemocytin and astakine in hemocyte-enriched samples collected from engorged ticks, a pattern also observed in other markers of the Immune 1 cluster (Fig.4c, Supplementary Data11). These findings suggested that blood-feeding expanded this hemocyte subtype and/or upregulated the expression of its marker genes in I. scapularis ticks.

a Expression patterns of hemocytin (left) and astakine (right) on t-Distributed Stochastic Neighbor Embedding (t-SNE) plots of hemocyte-enriched samples from engorged nymphs. Their highest expression is denoted in the Immune 1 cluster (outlined). b RNA FISH image of I. scapularis hemocytes labeled for hemocytin (hmc, green), astakine (astk, red), and nuclei (DAPI). White scale bars indicate a length of 50m. Refer to Supplementary Fig.10 for control images. c RT-qPCR evaluation of hemocytin (hmc; n=9 and 6) and astakine (astk; n=9 and 6) expression in hemocyte-enriched samples collected from unfed (ivory) or engorged (dark blue) ticks (with 4080 pooled ticks per sample). di Ticks were subjected to microinjection with clodronate (CLD) or empty liposomes (Control) and subsequently fed on either (d-f, hi) uninfected or (g) A. phagocytophilum-infected mice. d Total hemocyte counts (n=9 and 9) and (e) morphotype percentages (prohemocytes, plasmatocytes and granulocytes; n=8 and 9 in all cases) were assessed in the hemolymph of individual ticks. f RT-qPCR analysis of hemocytin (hmc; n=18 and 20) and astakine (astk; n=18 and 20) expression, and (g) A. phagocytophilum load in individual ticks (n=28 and 20). Bacterial quantification was based on the expression of A. phagocytophilum 16s rRNA (Ap16S) gene. h Weight measurements of engorged nymphs (n=34 and 25). i Percentage of nymphs that molted to adults. Results are represented as (ch) meanSD or as (i) a percentage from the total. A minimum of two independent experiments were conducted. Statistical significance was evaluated by (cf) an unpaired two-tailed t test with Welchs correction, (gh) two-tailed MannWhitney U test or (i) by a Fisher exact test, and significant p values (<0.05) are displayed in the figure. Source data are provided as a Source Data file.

Hemolectin is a known marker of phagocytic plasmatocytes in Drosophila30,31,32, and phagocytic hemocytes expressing hemocytin are present in crustaceans20,33,34. Therefore, we explored the phagocytic potential of the Immune 1 cluster by employing clodronate liposomes (CLD), which have been used to deplete phagocytic hemocytes in flies, mosquitoes, and, more recently, ticks35,36,37,38. We found that CLD treatment led to a 36% reduction in the total number of hemocytes in I. scapularis nymphs (Fig.4d). We observed an increase in the proportion of granulocytes along with a decrease in the percentages of prohemocytes and plasmatocytes compared to ticks treated with empty liposomes (Fig.4e). Interestingly, the expression of both Immune 1 marker genes, hemocytin and astakine, also decreased after CLD treatment, implying the Immune 1 cluster is phagocytic (Fig.4f). Taken together, CLD treatment effectively reduced the number of phagocytic hemocytes in I. scapularis, particularly impacting prohemocytes, plasmatocytes and cells associated with the Immune 1 cluster.

Phagocytosis serves as a pivotal immune mechanism against invading microbes. Previous studies have demonstrated that the depletion of phagocytic immune cells can influence the survival of arthropods following infection with either Gram-positive or Gram-negative bacteria35,36,37,38. However, the impact of depleting phagocytic hemocytes on the acquisition of tick-borne bacteria remains unclear. Thus, we further investigated the effects of CLD treatment during A. phagocytophilum infection, as this intracellular microbe is known to interact with I. scapularis hemocytes shortly after uptake39. Our findings revealed a reduction in A. phagocytophilum load in engorged ticks after injection with CLD compared to controls (Fig.4g), with no differences in tick attachment (Supplementary Fig.11), suggesting that phagocytic hemocytes promote either the acquisition or proliferation of A. phagocytophilum during blood-feeding.

Reports on Dipteran model organisms have demonstrated that hemocytes play roles beyond immunity, including tissue communication, clearing apoptotic cells during molting and development, and serving as vehicles for molecules40,41,42,43,44. However, whether hemocytes have non-immune roles in ticks remains unknown. Our sequencing results revealed an enrichment in functions related to metabolism, cell proliferation, and development after a blood meal, suggesting that hemocytes may participate in the feeding or molting processes in ticks (Figs.1, 2). To investigate this further, we recorded these physiological processes after injecting ticks with CLD. Although no differences in attachment were observed (Supplementary Fig.11), engorged ticks treated with CLD weighed significantly less compared to the control treatment, indicating that phagocytic hemocytes are required for proper hematophagy (Fig.4h). Furthermore, the number of nymphs that successfully molted to adults was significantly lower in CLD-treated ticks (Fig.4i). Collectively, these results indicated that phagocytic hemocytes pleiotropically impacted tick immunity, feeding, and molting in I. scapularis. Importantly, the observed correlation between changes in physiological parameters and the reduced expression of the Immune 1 markers, hemocytin and astakine, during CLD treatment suggested a potential regulatory role for these genes in various aspects of tick physiology, prompting us to explore this hypothesis further.

We found that hematophagy induces hemocyte proliferation and differentiation (Fig.1b) while upregulating astakine expression (Fig.4c). Thus, we investigated whether astakine was directly implicated in the proliferation or differentiation of I. scapularis hemocytes. We microinjected increasing amounts of recombinant astakine (rAstk) in unfed nymphs and observed a dose-dependent increase in the total number of hemocytes (Fig.5a). Specifically, we measured a decrease in the percentage of prohemocytes and an increase in plasmatocytes (Fig.5b). These findings matched alterations observed during normal blood-feeding (Fig.1b). Interestingly, we also detected an increase in tick IDE12 cell numbers in vitro following treatment with rAstk, supporting a role of astakine in inducing cell proliferation in hemocyte-like cells (Supplementary Fig.12). To corroborate these results, we then silenced astakine by microinjecting unfed nymphs with small-interfering RNA (siRNA) before feeding on uninfected mice. We utilized siRNA to knockdown gene expression, as genome editing through CRISPR has only recently been introduced for adult germline manipulation and possesses low efficiency in ticks45. We recovered 41% less hemocytes and observed an increase in the percentage of prohemocytes with a decrease in plasmatocytes from engorged ticks when astakine was silenced (Fig.5ce). Therefore, we determined that astakine acts on hematopoietic processes in the ectoparasite I. scapularis.

a Total hemocyte counts in the hemolymph of unfed I. scapularis nymphs subjected to microinjection with increasing amounts of rAstk (orange) or BSA (grey) as a control (n=21, 14, 14 and 24). b Percentage of hemocyte morphotypes (prohemocytes, plasmatocytes and granulocytes; n=10 and 10 in all cases) in the hemolymph of unfed nymphs following microinjection with 5ng rAstk (orange) compared to BSA controls (grey). ch Ticks were subjected to microinjection with astk siRNA (si-astk; blue) or scrambled RNA (sc-astk; grey) and subsequently fed on either (cg) uninfected or (h) A. phagocytophilum-infected mice. c Efficacy of astk silencing (n=20 and 16), (d) total number of hemocytes (n=9 and 8) and (e) percentage of hemocyte morphotypes in individual ticks (n=12 and 10 in all cases). f Weight measurements of engorged nymphs (n=29 and 19). g Percentage of nymphs that molted to adults. h RT-qPCR assessment of astk silencing efficiency (n=16 and 15) and A. phagocytophilum load (n=18 and 14) in individual infected ticks. Bacterial quantification was based on the expression of A. phagocytophilum 16s rRNA (Ap16S) gene. Results are represented as (af, h) meanSD or as (g) a percentage from the total. A minimum of two independent experiments were conducted. Statistical significance was evaluated by (a) one-way ANOVA with Dunnetts multiple comparisons test; (bf, h) an unpaired two-tailed t test with Welchs correction or (g) by a Fisher exact test, and significant p-values (<0.05) are displayed in the figure. Source data are provided as a Source Data file. rAstk = recombinant astakine; BSA = bovine serum albumin.

Our previous results showed alterations in the percentage of hemocytes by the CLD treatment, which influenced bacterial infection and tick physiology. Given that astakine also alters hemocyte composition in ticks, we then investigated whether decreasing the expression of astakine affects A. phagocytophilum infection, hematophagy or ecdysis in I. scapularis. Accordingly, we observed a significant reduction in weight, molting success, and A. phagocytophilum burden in ticks silenced for astakine compared to the control treatment, without differences in tick attachment (Fig.5fh; Supplementary Fig.13). Corroborating these findings, A. phagocytophilum load was also lower in astakine-silenced tick cells in vitro (Supplementary Fig.14). Overall, we demonstrated that astakine regulates hemocyte composition in I. scapularis, which affects not only bacterial infection but also feeding and ecdysis, supporting a pleiotropic role of hemocytes in ticks.

The broad expression of hemocytin detected in I. scapularis hemocytes (Fig.4a), and its homology to a Drosophila gene used as a marker of phagocytic immune cells, prompted us to explore whether hemocytin could be involved in hemocyte proliferation or differentiation, phagocytosis or immune signaling in ticks. Surprisingly, no differences were observed in hemocyte numbers or subtype proportions between ticks injected with siRNA targeting hemocytin, nor in the phagocytic capacity of hemocytin-silenced tick cells (Supplementary Figs.15, 16). We then asked whether hemocytin could act as an immune pathway regulator in I. scapularis. We focused on the IMD and the c-Jun N-terminal kinase (JNK) pathways, as previous reports indicated the importance of these molecular networks during bacterial infection of ticks5,6.

After transfection with siRNA targeting hemocytin, we found a decrease in JNK phosphorylation in hemocytin-silenced tick cells, without alteration in Relish cleavage (Fig.6a, b). To complement our findings, we overexpressed hemocytin in tick cells through CRISPR activation (CRISPRa). CRISPRa has been widely used to enhance the expression of an endogenous locus, employing a catalytically inactive Cas9 (dCas9) fused with transcription activators and single guide RNAs (sgRNAs) that direct the modified enzyme to the promoter region of a gene of interest46. However, so far none of the CRISPRa effectors have been tested in tick cell lines and no endogenous promoter has been identified for genetic expression of sgRNAs. We optimized reagents and developed a protocol to up-regulate hemocytin in ISE6 cells using two rounds of nucleofection with different expression plasmids. The first plasmid expressed dCas9-VPR paired with neomycin resistance under the control of the CMV promoter. The second plasmid expressed either a sgRNA specific to the promoter region of hemocytin (hmc-sgRNA) or a control sgRNA (ctrl-sgRNA) driven by an endogenous RNA polymerase III promoter (Fig.6c). Tick ISE6 cells were used as a platform for CRISPRa given the lower expression of hemocytin compared to IDE12 cells (Supplementary Fig.17). Strikingly, we detected a 277% increase in the expression of hemocytin in dCas9+ cells transfected with the hmc-sgRNA compared to dCas9+ cells transfected with the ctrl-sgRNA (Fig.6d). Importantly, we also noticed elevated levels of both jun (the transcription factor for the JNK pathway) and jnk expression (Fig.6d).

a, b Tick cells were transfected with either hmc siRNA (si-hmc) or scrambled RNA (sc-hmc). a Efficiency of hmc silencing in IDE12 cells (n=12 and 11). b Representative western blot (left) of N-Rel and p-JNK during treatment with sc-hmc (lane 1) or si-hmc (lane 2). N-Rel and p-JNK protein expression was quantified (right) in si-hmc (blue) or sc-hmc (grey) IDE12 cells (n=4). Data were normalized to the scrambled control, with N-Rel values relative to Actin and p-JNK values to JNK. A representative blot from four experiments is shown. Uncropped blots containing the molecular weight markers are supplied in the Source data. c Overview of CRISPRa-mediated hmc overexpression in ISE6 cells. d RT-qPCR analysis of hmc (left; n=9 and 10), jnk (middle; n=9 and 9) and jun (right; n=10 and 10) expression in dCas9+ ISE6 cells transfected with either a single guide RNA (sgRNA) specific to the promoter region of hemocytin (hmc-sgRNA, blue) or a random sgRNA (ctrl-sgRNA, grey). eh Ticks were subjected to microinjection with hmc siRNA (si-hmc; blue) or scrambled siRNA (sc-hmc; grey) and subsequently fed on either (eg) uninfected or (h) A. phagocytophilum-infected mice. e RT-qPCR analysis of hmc (left; n=19 and 18), relish (middle; n=17 and 18) and jun (right; n=17 and 18) expression in engorged ticks. f Weight measurements of engorged nymphs (n=20 and 23). g Percentage of nymphs that molted to adults. h RT-qPCR assessment of hmc silencing efficiency (n=11 and 10) and A. phagocytophilum load (n=11 and 10) in individual infected ticks. Bacterial quantification was based on the expression of A. phagocytophilum 16s rRNA (Ap16S) gene. Results are represented as meanSD. A minimum of two independent experiments were performed. Statistical significance was evaluated by (a, b, d, e) an unpaired two-tailed t-test with Welchs correction; (f, h) a two-tailed MannWhitney U test or (g) by a Fisher exact test, and significant p-values (<0.05) are displayed in the figure. Source data are provided as a Source Data file. N-Rel = cleaved Relish; p-JNK = phosphorylated JNK; JNK = c-Jun N-terminal kinase.

To corroborate our results in vivo, we microinjected unfed ticks with a scrambled control or siRNA targeting hemocytin and allowed them to feed on nave mice. Upon repletion, we measured the expression of jun and relish. Consistently, we found that reduction in hemocytin expression led to a decrease in jun levels in ticks, without affecting relish expression (Fig.6e), uncovering a role for hemocytin in the activation of the JNK pathway in I. scapularis.

Finally, building on our previous findings demonstrating that blood-feeding and infection alter the expression of hemocytin in ticks (Fig.4c, Supplementary Data9), coupled with the documented roles of hemocytin as an agglutinating factor and the JNK pathway in enhancing organismal growth and metabolism in insects47, we delved deeper into the relationship between hemocytin expression and physiological parameters in I. scapularis. We detected a decrease in weight and molting success to adulthood in hemocytin-silenced ticks that fed on uninfected mice compared to scrambled controls, with no differences in tick attachment (Fig.6f, g; Supplementary Fig.18). Additionally, we noted that silencing hemocytin increased A. phagocytophilum load both in vivo and in vitro (Fig.6h; Supplementary Fig.19), supporting an antimicrobial role for hemocytin. Collectively, our findings indicated that tick hemocyte subtypes and their associated marker genes play a critical role in I. scapularis immunophysiology.

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March 6, 2024

UPDATE

Three app creators shaping the future of education, fitness, andhealth

The creators behind Boddle, Run Legends, and Wysa all alumni of Apples Entrepreneur Camp share how theyre empowering people with their cutting-edge apps

Around the world, developers are building apps that break down barriers across gender, race, socioeconomic status, language, and physical ability.

In celebration of Entrepreneur Camps fifth anniversary, Apple spoke with alumni app creators whose apps are shaking things up in education, fitness, and mental health. All three women have participated in the immersive tech lab that invites developers from underrepresented groups to take their apps to the next level with one-on-one guidance from Apple experts, engineers, and leaders. Entrepreneur Camp underscores Apples ethos that apps for everyone should be made by everyone.

Edna Martinson, a 2023 Entrepreneur Camp alum based in Tulsa, Oklahoma, launched Boddle Learning during the COVID-19 pandemic, when students and teachers were adjusting to the sudden transition to learning from home. The education app for kids uses gameplay and machine learning to tailor content to their learning levels, helping them tackle learning gaps and boost their confidence in the classroom.

After graduating from MIT, 2022 Entrepreneur Camp alum Jenny Xu founded her own studio and fused two of her lifelong passions running and gaming into Run Legends. The immersive multiplayer game was designed to make walking and running more fun, encouraging players of all fitness levels to team up with friends to battle and overcome real-life anxieties.

And Jo Aggarwal, a 2019 Entrepreneur Camp alum, is the founder and CEO of Wysa, an emotionally intelligent chatbot written by therapists working with AI designers to help users talk through difficult emotions and thoughts anonymously, anytime. Based in Boston and Bengaluru, the app has helped millions of people across 95 countries learn how to feel happier.

Below, Martinson, Xu, and Aggarwal reflect on their journeys to becoming app developers, how theyve infused personalization into their apps to make them more accessible to all, key lessons theyve learned along the way and at Entrepreneur Camp, and how theyve harnessed technology to craft innovative solutions to real-world problems.

Making Space for Women in App Development

Jenny Xu (JX): When I started out in the gaming industry, I felt like I was the only one doing what I was doing. I grew up playing a lot of story-based interactive fiction artistic games that make you come away having learned something about life, and I think thats a genre thats predominantly popular with women. With Run Legends, even though its a battle game, it isnt a game that makes people feel really aggressive when theyre playing it they can feel empowered. Now that Ive raised some money and shipped some games, I try to give back and show that its possible to build games that arent just shooters, but games that actually make the world a better place.

Edna Martinson (EM): My perspective as a woman in this field has really shaped our team and how we connect with our community, especially since most elementary school teachers that we serve are women. Weve had a blast doing virtual career days on Zoom, showing kids how we made Boddle, and introducing them to game design. Its super rewarding to see the excitement on little girls faces when they find out a woman cofounded Boddle. It shows them they can be leaders in tech too.

Breaking Down Barriers Through Personalization

Jo Aggarwal (JA): We set out to solve for global mental health and give people a safe space to work through their thoughts and emotions, building resilience irrespective of stigma, race, gender, access to a therapist, or diagnosis Language can also be a barrier, and in an effort to make Wysas support more inclusive, we are launching Wysa in Spanish to create more equitable access to marginalized communities.

EM: At Boddle, we understand that every kid learns differently and at their own pace, so were using machine learning to tailor content to kids levels and help identify learning gaps through fun and interactivegameplay. We also provide teachers with the tools to be able to support their students, giving them access to kindergarten through sixth-grade assessments and videos in math and English, and a host of grading and reporting tools.

JX: We noticed that in other fitness experiences, people who were faster or more fit were often rewarded for how fit they were, so others would be intimidated. Run Legends uses core location, core motion, and accelerometer data on the Apple platform to detect how fast somebody is going and adjust the gameplay based on that. So if you want to play at a 20-minute walking pace or at an eight-minute running pace, its all the same. Theres actually no advantage to going faster or slower through our game. There are quite a few walkers who play the game and say that the games gotten them to go from walking to eventually going faster and moving up to a jog.

Finding Support and Mentorship

JA: Entrepreneur Camp was amazing, meeting so many like-minded entrepreneurs and making friends with them. I do stay in touch with some of the cohort who are in similar fields. The campus is so beautifully designed, and to learn Apple design principles within that environment was like a dream come true. We had design sessions with Apple team members who helped us understand how to position Wysa, and today, we are one of the top-ranked apps in our space.

JX: When we attended Entrepreneur Camp, it was very early on in Run Legends. We didnt even know necessarily what the game would look like. We got this opportunity to talk to the design team, who knew what was working on the App Store and would do deep dives on our app. One example was our onboarding experience our rep told us, What happens if a player isnt ready to run or go outside? You would lose so many people if you didnt provide an alternate experience that could be played indoors. So we changed our entire onboarding flow because of that feedback and realized after launch that almost half of all players used the alternate experience.

Building More Immersive Experiences

EM: Boddle is more than just a game for math or English. Were creating a learningmetaverse with different gameexperiences. If kids love basketball, they can jump into a basketball game. If theyre into pets, they can check out our pet-battling game. The cool part is they get to pick what they want to play, and their learning goes with them wherever they go in Boddles world. Theyre in charge of their own learning adventure, picking what they want to do and learning along the way.

JX: In Run Legends, we use Spatial Audio so that when you turn your head, it sounds like thebattle is actually happening around you. If you turn to the left, you might hear the monster snarling at you, but if you turn right, you could hear your teammates cheering you on. You feel the haptics as you charge up your attack and as you sprint, and you feel like the battle is happening right there. That feeling gets people to work out longer and harder. Players often tell us theyve done the fastest walks or runs in their entire life through it, and its because of that interactive audio experience.

JA: What has truly been unique about Wysa has been how we iterated the product. We did not start with a clinical workbook and turn it into a chatbot. We took evidence-based techniques that worked, and then listened to users about what they needed and where some didnt work for them. We then worked with clinicians to create models that worked for users when they tried to help themselves. This created a power balance between users and clinicians, powered by AI and analytics, which had never been possible before.

Creating Innovative Solutions to Real-World Problems

JA: We wanted to make access to mental health permissionless something you could reach out to on your phone at 3 a.m. and continue to use as often as you needed. Research shows that people are 3x more likely to open up to AI than even a human therapist. This creates an opportunity to create psychological safety, but also must be followed through with privacy-first design and proven, measurable outcomes. We were able to show impact with adolescents as well as older adults with chronic pain, with blue collar workers as well as tech professionals.

JX: The narrative in Run Legends is that youre fighting real-world anxieties through your battles in the game. So youre not fighting a minion or a generic enemy: Youre fighting your critical grandma whos telling you youre not good enough, or a hyper-competitive Chad whos always trying to beat you. Weve heard our players say its a cathartic experience. Theyre actually healing from the battles and the narrative of overcoming obstacles.

Press Contacts

Cathy Park

Apple

cathypark@apple.com

Apple Media Helpline

media.help@apple.com

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Three app creators shaping the future of education, fitness, and health - Apple

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A competition described as a "global fitness race" is building up a mass following among UK gym-goers.

Hyrox, which combines running with a full-body workout, is the latest fitness craze among those who want to "go one better" than a Parkrun but have had enough of "hardcore obstacle courses" like Tough Mudder, said health writer Peta Bee in The Times. And the hype about the exercise challenge is "huge", in part because the target market isn't triathlon or marathon types, but rather "regular gym-goers seeking an outlet for a previously untapped competitive streak".

Launched in 2017 by German former athlete Christin Ttzke, Hyrox has gained a global following, said the BBC, with a "1,000% increase in participation in the past five years".

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The premise of the race is simple. Participants run 1km and then complete a functional fitness exercise. "Rinse and repeat eight times, and you have yourself a Hyrox race," said GQ.

The exercises comprise a 1km sprint on a SkiErg, a full-body cardio machine; a 50m weighted sled push; a 50m sledge pull; 80m of burpee broad jumps; 1,000m of rowing; a 200m farmer's carry (using a kettlebell); 100m of sandbag lunges; and 100 wall ball throws.

There are no qualification entry requirements or finishing time restrictions. And each Hyrox race consists of waves of starters every 10 minutes throughout the day, so "there's no fear of finishing in 'last' place", said GQ. It is an "own-pace, own-race event" where amateurs and pros take part side by side.

More than 175,000 people are due to take part in 65 Hyrox races this year, some of which are in major arenas, such as Birmingham's NEC and London ExCel.Entries to a race in May at London Olympia "sold out months ago", said Bee in The Times, with some 12,500 people expected to take part.

Hyrox founder Ttzke has even bigger ambitions for the competition, telling The Times that he wanted to see it "become the gym-goer's equivalent of the London Marathon for runners".

The hashtag '#hyroxlondon' has garnered "millions of views on TikTok and tens of thousands of Instagram posts", said the BBC.Along with social media, another key reason why Hyrox is popular is that people don't have to compete alone; many are in pairs or teams of four.

It is also within the realms of possibility for most regular gym-goers. The functional stations "aren't highly technical and don't require brute strength but are pitched so that even people with little circuit training experience can have a stab at them", said Bee.

The race has become particularly popular with 35- to 39-year-olds, with the average participant at the latest London Hyrox race aged 37. But there is no upper age limit to participate Hyrox said its oldest finisher was 74 "and still going strong".

Hyrox classes are also increasing in popularity as more gym-goers become interested in participating in the races, as well as the opportunity the classes offer to train in groups while using a range of equipment.

The Gym Group first offered Hyrox classes in March last year at one of its venues and has since expanded into 14 gyms across London. One of the chain's trainers, Jenni Tardiff, told the BBC that Hyrox classes "really quickly became the most popular class in the gym".

"It started off with members who maybe knew about Hyrox and then it just exploded into everybody," she said.

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Hyrox: the new fitness trend taking over gyms - The Week

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There is no shortage of places to exercise in Vancouver, but there is definitely a shortage of truly safe, inclusive places to do so. And soon well have one less.

Olympic Village fitness studio Cmmn Grnd announced today that it is closing down on March 28.

Despite pouring our souls into this endeavour, we must acknowledge the reality that we haven't reached a sustainable state where our business could thrive while also repaying the debts incurred during our tumultuous initiation amid the COVID-19 pandemic, Cmmn Grnd posted in an Instagram statement. To our incredible community, words cannot express the depth of our appreciation for your unwavering support and belief in our mission.

Cmmn Grnd practises radical inclusivity in every aspect of its workout offerings. Sure, they have spin classes and yoga classes, like every other gym in the city. But from their diverse instructors and staff, to their programming, to the very way they speak about fitness and movement in their classes and marketing materials, the studio has been doing things refreshingly different.

With the pillars of social wellness, body neutrality, and gender neutrality guiding the vision, Cmmn Grnd promotes the idea that movement is for everyonenot just fitspo models with six-packs. It makes exercise about feeling good and giving back to your body, not about punishing yourself.It takes the fear out of exercise studios, which are so often intimidating spaces in this city, and brings back the joy.

Which is all to say: this one hurts.

Those who want to show support for Cmmn Grnd can still attend classes until the 28; drop-in and five-class passes are available for 20 per cent off.

Cmmn Grnd is located at 121 West 2nd Avenue.

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Cmmn Grnd, a pillar of inclusive fitness, is closing - The Georgia Straight

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George Heaton does not consider himself an influencer. But if you live and breathe training with every thread of your being or aspire to then the co-founder of the UK

From holding a blistering 2.5 min KM pace, racing side-by-side with Hyrox World Champion Hunter McIntyre, to building a 5000 sq ft functional fitness gym at Represent HQ which he staffs with world-class coaches and opens up to all members of the Represent team for Heaton, fitness is more than just a solo pursuit, it's a mission to make his world a better place. He also has a rig that any bodybuilder would be proud of.

Taken from his exclusive interview, which you can read in the latest issue of Mens Health, Heaton shares the training advice that has helped him achieve front cover status.

I was always overweight and could never control my diet or get to a point of consistency with my training,' said Heaton. 'I want to get the message across that youve got to dial it in.

Youve got to be consistent for a long period of time to see the results you really want. You need to have patience. So many people rush in head on and expect results yesterday. It can be disheartening when they dont see them. It takes time. Lay those foundations, youll get there.

Robert Hicks is the Executive Editor at Mens Health UK. A Sport Science graduate and author of three fitness books published by Bloomsbury, Robert has written numerous articles on health, fitness and nutrition and created several documentaries, most notably Britains Steroid Epidemic and The Faces of Attempted Suicide. Robert has been working at Men's Health UK for seven years.

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Top Fitness Tips from the Founder of Represent - Men's Health UK

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Looking like a fitness model is one thing. But feeling strong, fit and energised is something very different. For George Heaton co-founder of British streetwear

Along with his brother Michael, Heaton founded Represent in 2011. But only in the past five years has he really started to take his training seriously a fact that makes him stick out among the fashion crowd, he says.

I was always a morning person I was never a heavy drinker and wouldnt take a load of drugs or whatever. That was never me, he tells MH. When you think about creatives in fashion or the arts, you have this idea of people whose outlets and hobbies arent quite as healthy... Thats something that kind of held me back for a while because I thought I had to be that way. Everyone in fashion is nocturnal and theyre up at night drinking. Not many people in fashion are extremely diligent with fitness.

But Heaton chose to do things his own way. Today, his training comprises everything from functional fitness and marathon-running to CrossFit and bodybuilding. Running, in particular, has played a significant role in his creative output.

What Id read about running is that the high and how you feel after a workout is a form of creative expression, he says. [I thought], Why cant I try to figure out if thats the way it works for me? I found an area where I could express what Im feeling on those long runs. For me, thats when Im actually thinking about things and where the best ideas come from. Youre finding that sense of stillness where the creativity can come through.

With his Represent 247 clothing range which is designed to be stylish enough for everyday wear, but functional enough for the gym Heaton believes he can help others make the transition into fitness-as-a-lifestyle, too.

A lot of our customers were that guy who was drinking at night and partying and not into their fitness. Now theyve changed their lives through the brand, he says.

Scarlett Wrench is the Senior Editor at Mens Health UK.

With more than 12 years experience as a health and lifestyle editor, Scarlett has a keen interest in new science, emerging trends, mental well-being, and food and nutrition. For Mens Health, she has carried out extensive research into areas such as wellness in the workplace, male body image, the paradoxes of modern masculinity, and mental health among school-age boys.

Her words have also appeared in Womens Health, Runners World and The Sunday Times.

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George Heaton: 'Not Many People in Fashion Are Diligent With Fitness' - Men's Health UK

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New senior fitness group 'Chatham Chiliad' to meet for the first time March 19 - New Jersey Hills

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Crossfit Salute, a health and fitness gym, relocated to Tomball after nine years in The Woodlands.

The details

The gym offers a wide range of classes catering to individuals of all fitness levels, from beginners to enthusiasts, owner Frank DeSantis said.

DeSantis said the programs provide functional fitness training with a focus on movements that benefit everyday life.

CrossFit really encompasses community, DeSantis said. It's not your typical gym where you punch in and check out.

A closer look

Before, the gym was located in a smaller facility on 28543 Kuyendahl Road, The Woodlands, but has since moved to a larger space, about 40% bigger than the previous location, DeSantis said.

The gym has moved in with Rivalry Baseball, collaborating to offer enhanced fitness and baseball training opportunities for members.

The facility offers various classes, such as:

Whats next?

Soon to come, DeSantis said he plans to host events for the area in the future and introduce:

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CrossFit Salute opens in Tomball, prioritizing community, fitness - Community Impact

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WASHINGTON The message that Joe Biden most needed to deliver Thursday night wasn't so much that the American union is strong but that the American president is fit.

It's not the highest bar, but it's one that he cleared.

Amid polls showing that most of the country believes he is too old to serve another four-year term, Biden stood for 68 minutes in the House chamber and gave a speech that was both energetic and crisp. He then set a modern presidential record for the time spent shaking hands and giving hugs in the chamber afterward.

Biden spoke with fluency about the stakes in the presidential race, never mentioning former President Donald Trump by name but making more than a dozen references to his "predecessor." The first came just three minutes into the speech, when Biden skewered Trump for inviting Russian President Vladimir Putin to attack NATO allies who don't contribute enough money to the alliance.

Drawing a distinction, Biden said: "My message to President Putin is simple. We will not walk away. We will not bow down. I will not bow down."

Biden flubbed a few prepared lines and stumbled a bit during ad-libs notably when he said drug prices in Moscow are lower than in the U.S. but he belied the GOP caricature of him as an enfeebled old man who needs to retire.

No ones going to talk about cognitive memory now, Rep. Jerry Nadler, D-N.Y., told the president after he finished the speech and made his way out of the House chamber.

That was, of course, the idea when Biden's speechwriters worked on the draft. The prepared text included 80 exclamation points cuing Biden to when he needed to raise his voice and project strength. By contrast, there were no exclamation points in last year's text.

It's too soon to tell whether Biden's address will make an enduring impression on voters who may have been watching. The Biden campaign is likely to repurpose snippets of the speech in which he was his most eloquent, while Republicans may showcase instances when he tripped over a word.

What didn't happen Thursday night, though, may be the biggest takeaway. Biden, 81, didn't stumble as he walked through the chamber as some of his allies feared might happen. Nor did he freeze as he read the teleprompter or get rattled when Republicans jeered him. Indeed, at times he seemed to goad Republicans into unscripted back-and-forth.

Urging Congress to revive a border security bill that House Republicans scuttled, Biden gazed out at Republican members and said: "You're saying no. Look at the facts. I know you know how to read."

Heckling can test a presidents wits and alacrity in its own way. Aides were thrilled last year as Biden turned Republican derision to his advantage. When Republicans hooted that they didnt intend to cut Social Security and Medicare, as Biden had claimed, he ad-libbed that the boos must mean they, too, wanted to protect the entitlement programs.

Biden's challenge Thursday night was to "convince people that hes got the strength, energy, vigor and focus to be an effective president," Rep. Adam Smith, D-Wash., said in an interview in the hours before the speech.

Gone is the era when a State of the Union speech served as a unifying national event. Over the years it has devolved into a televised drama in which the president and the opposing party, arrayed before him on the House floor, grasp for ways to gain ground at each others expense.

Jeff Shesol, a speechwriter in Bill Clintons White House, said in an interview, Part of the game really is to make sure that youre writing some lines and sections that are forcing the other party, against its better instincts, to stand and applaud or to force them to remain seated while the rest of you applaud apple pie and the American flag."

The night had a partisan feel from the first. Biden looked taken aback when he entered the chamber and made eye contact with Rep. Marjorie Taylor Greene, R-Ga., wearing a bright red "Make America Great Again" hat. (House rules prohibit wearing caps in the chamber.)

At the lectern, he suggested he was on the side of democratic freedoms while Trump was among those who wanted to "bury the truth" about the riot at the Capitol on Jan. 6, 2021, when Trump supporters nearly prevented the peaceful transfer of power.

After he said his core values include honesty, decency, dignity and equality, Biden added: Now, some other people my age see a different story: an American story of resentment, revenge and retribution. Thats not me. In one breath, he sought to neutralize questions about his age while painting Trump as a menace to a functioning democracy.

The speech came later in the year than usual, and it came close to being pushed back even more.

House Republican leaders were prepared to move the date of the speech in case Congress failed to reach a budget deal, forcing a partial government shutdown, a House GOP leadership aide said.

GOP leaders didnt want to give Biden a chance to berate them about a shutdown as millions of Americans watched from home, the aide said. It turned out that Congress reached a budget deal averting a shutdown, so the date wasnt changed.

We were thinking about moving the speech because you dont want to give him [Biden] something to beat up on us for an hour, the House aide said, speaking on condition of anonymity to discuss internal strategy.

With the campaign now fully engaged, the White House sought to wring maximum advantage from the speech. Aides stocked the gallery with people symbolizing the key components of the Democratic coalition. Guests invited to sit with first lady Jill Biden included Kate Cox of Texas, who needed to leave the state to get a doctor-recommended abortion after the Supreme Court overturned Roe v. Wade; Shawn Fain, the president of the United Auto Workers labor union; and Bettie Mae Fikes, a civil rights advocate who was part of the Bloody Sunday march in Selma, Alabama, in 1965.

Biden made sure to name-check them during the address.

Whether those sorts of gestures are remembered in the fall is by no means certain. Terry Szuplat, a speechwriter for President Barack Obama, pointed to the irony of State of the Union addresses. For all the work that goes into them, they're often quickly forgotten.

"It's the greatest platform a president has, and it often becomes the least memorable speeches of his presidency," he said.

This article was originally published on NBCNews.com

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Biden stares down questions about his age and fitness for office with spirited speech - Yahoo! Voices

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The fitness franchise industry is growing fast, with high interest from investors and consumers alike as new modalities emerge

The fitness franchise industry has seen remarkable transformations over the years, with 2024 marking a pivotal point in its evolution.

Amidst a landscape of innovation, sustainability and personalized wellness, fitness franchises are redefining what it means to be fit, both physically and mentally. With high CAGR growth, the Fitness Franchise industry is flushed with interest amongst new franchise investors and new founders. At the heart of this transformation is a cadre of visionary leaders who exemplify the drive and innovation steering the industry forward.

To get a pulse on the fitness franchise category growth and forecast in 2024, we dove into the details with helpful insight from a well-known leader in the space.

Franchise Growth by The Numbers

Franchises like F45 Training and Orangetheory Fitness have rapidly expanded their global footprint, with F45 Training boasting over 1,700 locations and Orangetheory Fitness over 1,500 locations worldwide. These numbers underscore the scalability and consumer appeal of these fitness models, particularly in offering high-intensity interval training (HIIT) and heart-rate-based workouts that promise effective and efficient fitness results.

Another growth player in the industry yielding significant growth in locations is Fit Body Boot Camp, with nearly 300 locations system wide as of 2024. The organization has twice been listed among Entrepreneur Magazines 500 fastest-growing franchises and four times listed on Inc. Fit Body is a great example of high growth within the category, showing potential future growth as a strong indicator for the industry as a whole.

Strategic Considerations for Franchisees

For outsiders looking to enter the fitness franchise industry, understanding trends and consumer behaviors is crucial. The growth in online fitness, the importance of catering to older demographics and the success of specialized fitness franchises offer valuable insights into where the market is headed.

Additionally, the resilience shown by the industry in the face of economic challenges suggests a strong underlying demand for health and fitness services, making this an opportune time to invest in or expand within the fitness franchise sector. As the fitness industry continues to evolve, staying informed about these trends and leveraging them in business planning and strategy will be key to success in the competitive landscape of 2024 and beyond.

At Fit Body Boot Camp, we believe that the key to success lies not just in adapting to trends, but in anticipating them, ensuring that we provide an unmatched fitness experience that meets the diverse needs of our evolving clients, says Bryce Henson CEO of Fit Body Boot Camp. There will always be a need for preventive health and wellness services and fitness franchising has become a leading business solution for this ever-growing societal demand.

Its an opportune time for entrepreneurs to invest in a sector where impact and profitability go hand in hand and a growing industry shows indicators of continued bright horizons, Henson adds.

Technology Integration

The integration of technology into fitness routines has significantly transformed the industry, making workouts more accessible and personalized. Wearable devices now track everything from steps to sleep, offering insights that help users optimize their health routines.

Fitness apps have grown smarter, employing AI to create tailored workout and nutrition plans, adjusting to feedback and progress in real-time. Virtual and augmented reality technologies have introduced immersive exercise experiences, turning routine workouts into engaging adventures. These advancements allow users to explore virtual landscapes or participate in gamified fitness challenges, making exercise more enjoyable and varied.

AI extends beyond personalization, utilizing predictive analytics to prevent injuries by recommending adjustments in workout intensity or form. It also facilitates seamless integration between various devices and platforms, ensuring that users have a holistic view of their health data.

Moreover, the shift towards online and hybrid fitness offerings has democratized access to fitness, enabling users to join classes from anywhere, at any time. This flexibility caters to todays diverse lifestyles, ensuring that fitness is within reach for everyone, regardless of their schedule or location.

The integration of AI has been able to bridge the gap between providing personalized recommendations and scalability, Henson says. Now being able to both help more people and deliver better client results.

Community & Corporate Social Responsibility

The fitness franchise industry in 2024 has witnessed a significant evolution, driven not just by the goal of improving physical health but also by a profound commitment to enriching community life and embracing corporate social responsibility (CSR). This transformative approach underscores a broader, more integrated vision of wellness that extends beyond individual fitness goals to encompass societal well-being and environmental sustainability.

Franchises are increasingly active in local communities, leveraging their platforms to foster a sense of unity and engagement. From organizing health awareness events to participating in local clean-ups, the industrys efforts are aimed at building stronger, healthier communities.

This engagement also extends to supporting charitable causes, with many franchises aligning with organizations that tackle a wide range of issues from mental health to environmental conservation. Such partnerships not only amplify the impact of non-profits but also resonate with consumers who prioritize businesses that contribute positively to society.

Inclusivity has become a cornerstone of the industrys CSR initiatives. Recognizing the diverse needs of their clientele, fitness franchises are striving to create welcoming environments for all individuals, regardless of their background, ability, or financial status. This includes offering tailored programs for underserved populations and ensuring facilities are accessible to everyone. Such efforts reflect a commitment to breaking down barriers to fitness and wellness, fostering a culture of inclusivity and support.

The industrys embrace of corporate social responsibility and community engagement reflects a deeper recognition of our role beyond the gym walls, Henson says. By actively participating in local initiatives, were not just advocating for individual health but for the collective well-being of our communities. This approach is about forging connections, breaking down barriers to access and ensuring that our contributions leave a lasting positive impact on society.

Final Thoughts

In 2024, the fitness franchise industry is poised to redefine wellness through a blend of innovative technology, enhanced community engagement and a robust commitment to inclusivity and environmental sustainability. This year is set to witness fitness franchises leveraging advanced digital tools to offer personalized workout experiences, making fitness more accessible to a wider audience.

The emphasis on corporate social responsibility is expected to strengthen ties with local communities, highlighting the industrys role in promoting societal well-being. As fitness franchises continue to evolve, leaders are not only catering to the changing needs of todays health-conscious consumers but are also laying the groundwork for a future where fitness is an integral part of a holistic approach to health.

This forward-thinking approach promises to shape the landscape of the fitness industry, ensuring that it remains at the forefront of promoting wellness, inclusivity and sustainability.

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