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Specimens of the lobster Homarus gammarus, the type host species for Porospora gigantea, were collected either from the sea in Roscoff bay (France) or from commercial lobster tanks in Roscoff (Fig. 1, Table S2). A total of 35 lobsters (9 from the wild and 26 from captivity) were dissected and infection with P. gigantea was quantified (Fig. 1, Fig. S1). Overall, infection levels were significantly higher in lobsters freshly caught from the sea (prevalence of 100%, high parasitic loads) than in lobsters that had been held in captivity in lobster tanks (prevalence < 62%, low parasitic loads, see Table S2), a similar result to that reported by Van Beneden (1869) [14]. The morphology of cysts, gymnospores, zoites and trophozoites was imaged and measured (Fig. 1, Tables S3, S4 and S5). Cysts were mostly spherical but some were ovoid, with diameters ranging from ~ 104 μm to ~ 252 μm (mean ± standard deviation, 151.1 ± 45.3 μm, n = 97), and they enclosed thousands of gymnospores, that were also mostly spherical, with diameters from less than 5 μm to almost 7 μm (5.63 ± 0.69 μm, n = 265). These gymnospores were indeed composed of radially arranged zoites forming a monolayer with an optically void center. Observation of broken gymnospores by scanning electron microscopy made it possible to measure the length of the constituent zoites (1.04 ± 0.16 μm, n = 105) and their apical width (0.630 ± 0.129 μm, n = 176). Trophozoites were very thin and long, up to 2585 μm for a mean width of 41.8 ± 10.4 μm (n = 104). As previously described, the posterior of the trophozoite was slightly thinner, ~ 30 μm. The whole trophozoite surface was covered by longitudinal epicytic folds (Fig. S1.B) that are thought to be necessary for eugregarine gliding [27]. The sum of these morphological observations all accord with the species being P. gigantea from the type host H. gammarus [6, 14, 15].

Morphological characterization of Porospora cf. gigantea.
A. Trophozoite stage (Tropho #8, Lobster #12) (scale bar = 100 μm). B. Zoom on A, showing trophozoite epimerite (scale bar = 10 μm). C. Rectal ampulla showing cysts in folds (Lobster #4) (scale bar = 1 mm). D. Isolated cyst (Cyst #4, Lobster #12) (scale bar = 50 μm). E. Broken cyst packed with gymnospores (Lobster #4) (scale = 10 μm). F. Section across a cyst illustrating radial arrangement of zoites in gymnospores (JS449 = Lobster #35) (scale bar = 2 μm). G., H. Zoom on intact and broken gymnospores showing zoites (Lobster #4) (scale = 1 μm). All images are scanning electronic micrographs except F which is a transmission electronic micrograph. See also Fig. S1, Tables S2, S3, S4, S5 and S6

Gliding of isolated trophozoites was filmed. The dynamic recordings confirm that trophozoites moved uni-directionally, with the protomerite forwards, in either straight or curved lines depending on the individuals observed, with the whole body (deutomerite) following the same path as the apical protomerite (Film S1). The speed of trophozoite displacement was estimated to be ~ 60 μm/sec, as initially observed by King and Sleep (2005) [25], but was faster than 100 μm/sec in some recordings (Table S6). No syzygy was observed. A few solitary encysted trophozoites were observed, supporting the observations of Léger and Duboscq (1909) [28], who considered that encysted gymnospores correspond to a schizogonic rather than gamogonic phase of Porospora development. This hypothesis is still open since the gamogonic phase with male and female gametes and the fertilization process are not yet documented in the life cycle of P. gigantea [6].

Two highly related genomes

Four biological samples were sequenced and analyzed independently, and then assembled together (Fig. S2.A). The raw assembly produced 214,938 contigs (99.6 Mb) among which were 13,656 contigs longer than 1 kb (47.9 Mb). The scaffolds obtained were cleaned by removing contaminants such as bacterial, fungal and host sequences (Fig. S2.B), resulting in a raw assembly of 1719 contigs covering 18 Mb.

The analysis of contig coverage for each individual library revealed a bimodal distribution suggesting a mixture of genomes in differing proportions depending on the biological sample (Fig. S3). More precisely, while only one set of contigs displayed a significant coverage for the lobster tank parasite sample (JS-470, peak around 250×), the three other parasite samples from freshly captured hosts (JS-482, JS-488, JS-489) showed two distinct sets of scaffolds with similar size (~ 9 Mb) and different coverage values. The difference in coverage was used to split the whole assembled contigs into two sets that were named A for the set of contigs present in all four samples, and B for the set present only in the three lobsters freshly captured in the wild (Fig. S2.C). The percentages of genomes A and B in each biological DNA sample was estimated (Fig. S3) as 100% A for JS-470, 63.2% A and 36.8% B for JS-482, 70.5% A and 29.5% B for JS-488, and 62.4% A and 37.6% B for JS-489, based on medium coverage levels. Genome A maps to 787 contigs for a total of 8.8 Mb, whereas genome B maps to 933 contigs for a total of 9.0 Mb. Contigs from the two genomes can be aligned with each other over 7.7 Mb, with a percentage of divergence around 10.8% at the nucleotide level.

To summarize, these two genomes have a similar size (~ 9 Mb) and are syntenic with nevertheless 10.8% of divergence. These highly related genomes have been named A and B and are associated to the species name P. cf. gigantea (Fig. S2).

Genome features

Two genomes with similar coding capacities

A total of 10,631 putative genes were predicted from the raw assembly (17,930 alternative splicings), which were split into two sets of similar size: 5270 genes in genome A (8895 alternative splicings) and 5361 genes (9035 alternative splicings) in genome B (Table 1, Fig. S2). The completeness of both A and B genomes was assessed by using BUSCO software [29] on the Apicomplexa geneset (n = 446). Genomes A and B respectively showed completeness scores of 70% (n = 312) and 67.7% (n = 302) (Fig. S4).

The number of A and B orthologues was investigated. The predicted proteins of P. cf. gigantea A and B were split into 5656 orthogroups including 4443 groups (88%) which had at least one orthologous gene for both A and B. This percentage of common orthogroups between genomes A and B is higher than that observed between Plasmodium falciparum and Plasmodium berghei (70%), thought to have diverged around 33 Mya ago (TimeTree [30]), but similar to that observed between P. falciparum and Plasmodium reichenowi (86%, 3.3–7.7 Mya, TimeTree).

The percentages of shared orthogroups between P. cf. gigantea genomes and each of the reference apicomplexan species are similar (Cryptosporidium parvum, 18%; G. niphandrodes, 17%; P. falciparum, 14%; T. gondii, 14%) despite the differences in divergence, but it is higher than the percentages observed with chromerid species (Chromera velia, 8%; Vitrella brassicaformis, 10%). We can deduce from these results that the P. cf. gigantea genomes do not share significantly more orthogroups with G. niphandrodes, the only other available gregarine genome, than with any other apicomplexan (Fig. 2).

Shared apicomplexan proteins. Distribution of the orthogroups among P. cf. gigantea A and B and 4 species of apicomplexans: the gregarine G. niphandrodes, the cryptosporidian C. parvum, the coccidian T. gondii and the hematozoan P. falciparum. Orthogroups only shared by P. cf. gigantea A and B are highlighted in green, whereas orthogroups shared by all species are highlighted in pink. Only bars with more than 20 orthogroups are shown. See also Table S1

Two gene-dense genomes with small introns

The proportion of coding sequences in A and B genomes is 84%, which is particularly high compared to other reference species (with values ranging from 25 to 76%; Table 1). The genomic compaction of non-coding DNA in genomes A and B can be explained by the shortness of most introns (Fig. S5). A specific class of introns with lengths around 25–30 bp (mode at 28 bp) represents 71–72% of the introns. The donor and acceptor sites of these small introns have specific consensus patterns (Fig. S5) which are different from other Porospora introns. Specifically, these introns exhibit a strongly conserved adenine located 6 bp upstream of the 3′ acceptor site which could represent the intron branch point, as observed for the small introns (20 bp) in B. microti [31].

Loss of organellar genomes

Recent studies suggest that organellar genomes are lost in most gregarines [10, 32]. A precise protocol was set up to identify putative contigs associated with organellar genomes in P. gigantea. All the assembled contigs (assigned to P. gigantea or not) were searched for regions similar to known organellar genomes. A sensitive protocol based on TBLASTX identified 108 putative regions that were aligned to the NCBI NR library. 102 regions were discarded as bacterial contamination. The 4 contigs corresponding to the remaining 6 regions with at least one significant hit against an eukaryotic sequence were manually curated. Two contigs were assigned to host-derived contaminants whereas the two other long contigs (L = 24,892 and L = 33,594) corresponded to P. gigantea nuclear genome. Thus, our analyses did not reveal any putative contigs compatible with mitochondrial or apicoplastic genomes.

Evolutionary histories of P. cf. gigantea

Genomes A and B diverged several million years ago

We estimated the putative divergence time of A and B genomes by using the divergence between P. falciparum and P. reichenowi as a calibration point. The synonymous divergence (dS) was calculated for 1003 quartets of orthologous genes. The mean dS value observed between P. falciparum and P. reichenowi orthologues was 0.0959, similar to that calculated by Neafsey et al. [33] (0.068 substitutions per site) or Reid et al. [34] (0.086–0.11 per site). We assumed that these Plasmodium species diverged between 3.3 and 7.7 Mya (TimeTree). The mean dS value observed between the same orthologues in both P. cf. gigantea genomes was about 0.4295 substitutions per site. Assuming similar substitution rates in gregarines and Plasmodium species, we dated the split between genomes A and B to have occurred between 15.5 Mya and 37.7 Mya. This order of magnitude is similar to the estimation of when the basal splits of the mammal Plasmodium [35] (12.8 Mya) or all Plasmodium [36] (21.0–29.3 Mya) occurred, but is significantly later than the emergence of Nephropidae (lobster family) around 180 Mya [37, 38].

Expanded superfamily of crustacean gregarines

To assess the position of P. cf. gigantea A and B within Apicomplexa, we constructed a genome-wide phylogeny based on 312 concatenated proteins from the datasets published by Salomaki et al., 2021 [13] and all recently published transcriptomic data from gregarines [10, 11, 13] (Fig. 3). This phylogeny grouped P. cf. gigantea A and B into one clade, placed as a sister group of other crustacean gregarines (Cephaloidophora communis, Heliospora caprellae), although having shorter branch lengths. In agreement to Salomaki et al. (2021) [13] Cryptosporidium species remain at the base of A + G (Apicomplexa + gregarines), using a LG + C60 + G + F model in maximum likelihood phylogenomic analyses. However, the bayesian analysis using classical partitioned model LG + G + F is in favor of a A + C topology (Apicomplexans + Cryptosporidium) (average standard deviation of split frequencies = 0.020977). More sampling of Cryptosporidium relatives is required to address the apicomplexan topology issue.

Phylogeny of Apicomplexa. Maximum likelihood phylogeny of apicomplexans as retrieved from a 312 proteins dataset, merged from two previously published datasets [10, 11, 13]. Final concatenated alignment comprised 93,936 sites from 80 species. Bootstrap support values (n = 1000) followed by MrBayes posterior probabilities are shown on the branches. Black spots indicate 100/1 supports. Porospora cf. gigantea A and B sequenced in this study are bolded. See also Figs. S7 and S8

The sequences of 18S small subunit ribosomal DNA, for which the largest taxonomic sampling for gregarines is available in databases, was also used to position P. cf. gigantea within the crustacean gregarines. Using a combination of amplifications with specific primers (initially based on Simdyanov et al. (2015) [39] and Schrével et al. (2016) [40] then partly redesigned (Fig. S6, Table S7)) and in silico clustering, we were able to fully reconstruct complete ribosomal loci covering 18S-ITS1–5.8S-ITS2-28S (5977 bp) for both A and B genomes. Thirty polymorphic positions were found between A and B, only one within the 18S sequence, and 29 within the 28S sequence (Fig. S6). Two phylogenetic studies were performed, one excluding environmental sequences (Fig. S7), the other including them (Fig. S8). Most environmental sequences are derived from marine sediments from a wide range of habitats but only two sequences are from the North Atlantic where European and American lobsters live.

Congruent with the concatenated phylogeny (Fig. 3), both 18S phylogenies assigned P. cf. gigantea A and B to their own clade, placed as a sister group to all other crustacean gregarines (Cephaloidophora, Heliospora, Thiriotia, and Ganymedes species), as established in Rueckert et al. (2011) [41] (Figs. S7 and S8). Five main clades constituting the superfamily Cephaloidophoroidea were retrieved. The four clades previously outlined [41], redenominated as Ganymedidae, Cephalodophoridae, Thiriotiidae (as proposed by Desportes and Schrével (2013) [6]), and Uradiophoridae, had at their base the clade Porosporidae. Historically defined as the family gathering Porospora and Nematopsis genera [6], this clade is constituted of the two sequences of P. cf. gigantea. A new putative clade was formed by the five sequences from a Slovenian karst spring published by Mulec and Summers Engel (2019) [42] (Fig. S8), and it is very well supported to be a sister group to four of the crustacean gregarine families, while the family Porosporidae retains its position as a sister group to all these other clades.

Partially conserved glideosome machinery

We conducted an inventory of the presence or absence of genes encoding proteins involved in the gliding motility based on the molecular description of the so-called glideosome machinery, grouped according to their function as established by Frénal et al. (2017) [26] (Fig. 4A, all orthologues for P. cf. gigantea are detailed in Table S8). Genes for these T. gondii and P. falciparum reference proteins were searched for in both P. cf. gigantea genomes and in the genomes of a selection of representative species, as well as the recently published gregarine transcriptomes [10, 11, 13].

Comparative analysis of glideosome components. A. Table of presence/absence of genes encoding glideosome proteins, distributed into functional groups. Glideosome components have been described mainly in T. gondii and P. falciparum. Protein sequences were searched for in the genomes of both Porospora and a selection of representative species as well as in available gregarine transcriptomes. Green indicates the presence, while white indicates the absence of an orthologous protein-encoding sequence. Light red refers to cases where only partial sequences have been retrieved. Violet indicates the presence of at least one protein in multigenic family proteins. * refers to the GAP45 3′ short conserved domain found in some gregarines species. All P. cf. gigantea orthologous proteins are detailed in Table S8. B. Schematic comparison of the canonical model of the glideosome and the elements found in P. cf. gigantea A and B. Missing proteins are shown with dotted lines

Actin in apicomplexans is characterized by a globular monomeric form (G-actin) which polymerizes as needed into short unstable filaments (F-actin) [43] using various regulators such as profilin [44,45,46], ADF cofilin [47], formin [48,49,50], cyclase-associated proteins (CAP) [51] and F-actin capping protein Cpβ [52]. The inactivation of actin or its associated regulators compromises motility and host cell invasion and egress, although motility may persist in an altered form for a few days, perhaps through alternative mechanisms [26, 53,54,55]. Overall, these proteins are well conserved among Apicomplexa. However, profilin appears to be absent in insect-infecting Gregarinoridea; CAP and Cpβ also seem to be poorly conserved in gregarine transcriptomes but present in both P. cf. gigantea.

Apicomplexan-specific glideosome proteins

The core glideosome machinery mainly comprises specialized proteins found only in apicomplexans. The single-headed short heavy chain myosin class XIV, named myosin A (MyoA), acts as a motor generating the rearward traction required for gliding motility, invasion and egress, as evidenced by various conditional depletion experiments [56,57,58]. The glideosome itself is situated between the plasma membrane and the apicomplexan-specific inner membrane complex (IMC). In the IMC, MyoA is associated with a light chain, myosin light chain 1 (MLC1) in T. gondii or MyoA tail domain-interacting protein (MTIP) in P. falciparum [59], as well as several glideosome associated proteins (GAP), GAP40, GAP45, GAP50 [60,61,62], GAP70 and GAP80 as yet only described in T. gondii [57]. GAP45 is thought to anchor the glideosome to the plasma membrane by recruiting MyoA as a bridge [62], whereas GAP40 and GAP50 are predicted to help anchor MyoA to the parasite cytoskeleton [63]. Another set of glideosome-associated proteins with multiple-membrane spans (GAPM) are believed to interact with the alveolin and subpellicular microtubules network, suggesting an indirect interaction with the IMC [26, 64]. Finally, the conoid-associated myosin H is necessary for initiating gliding motility in T. gondii [65].

Genes encoding myosins A, B, C, D and E and associated light chains were found in all species. Myosin H is also widely conserved in intracellular apicomplexans. However, among the gregarines Myosin H was only found in a few species. For glideosome associated proteins, only GAP40 was found in all species, although the sequences from gregarine transcripts and chromerids were less well conserved. Surprisingly, given the central role attributed to GAP45 in the glideosome model, no ortholog was found in gregarines except for two poorly conserved sequences in Lankesteria abotti, Lecudina tuzetae, Cryptosporidium and chromerids. However, we identified a short conserved 3′ domain (<50aa) in L. tuzetae, Pterospora schizosoma and Siedleckia nematoides. A similar domain is found in P. cf. gigantea A and B. It is however not sufficient to conclude whether it is an orthologous protein. GAP50 seems to be more conserved among apicomplexans, but is absent or only partially conserved in most of the gregarines. As expected, GAP70 and GAP80, only identified so far in T. gondii, were not found in other species, except for an orthologue of GAP80 in the coccidia Hammondia hammondi. Concerning GAPMs, we found orthologues of at least one of its variants (GAPM 1, 2 or 3) in most species. However, GAPMs seem to be totally absent in at least 7 species of gregarines (Ancora sagittata, Protomagalhaensia sp. Gyna, Protomagalhaensia wolfi, Gregarina sp. Pseudo, Pterospora schizosoma, Selenidium pygospionis, Siedleckia nematoides). Finally, GAC is overall well conserved in apicomplexans but absent from chromerids, supporting its apicomplexan-specific status. However, we were not able to identify GAC in several gregarine transcriptomes (P. sp. Gyna, P. wolfi, G. sp. Pseudo, H. caprellae, L. abotti, L. tuzetae, P. schizosoma) (Fig. 4A).

Adhesins and TRAP-like candidates

The glideosome machinery, anchored in the parasite cytoskeleton, needs to interact with extracellular receptors of the host cell to propel the parasite forward over the host surface. This is made possible by the presence of extracellular adhesins secreted by the micronemes [66, 67] and connected to the glideosome through the glideosome associated connector (GAC) protein [68]. Thrombospondin adhesive protein (TRAP) [69] is a Plasmodium adhesin required for gliding, whose homologue in T. gondii is MIC2 [70]. At the end of the gliding process, rhomboid protease 4 (ROM4) cleaves the adhesins, disengaging them from receptors and, for intracellular parasites, allowing them to enter the host cell [71,72,73]. TRAP-like proteins, while highly divergent from one species to another, constitute a family of functionally homologous proteins sharing adhesive domain types, involved in parasite motility and cell penetration [74,75,76]. TRAP-like or TRAP-related proteins have been detected in various stages of Plasmodium (CTRP [77], MTRAP [78], TLP [79]) and have also been found in silico in Cryptosporidium (TRAPCs, CpTSPs [76, 80, 81]) as well as in several Babesia and Theileria species [82,83,84,85], in Neospora caninum [86] and in Eimeria [87, 88]. We first looked for the TRAP proteins which have been implicated in gliding through experimental studies (MIC2, TRAP, TPL, CTRP, MTRAP), as well as the ROM4 protein involved in adhesin cleavage. Unsurprisingly, the currently described TRAP proteins seem to be genus- or even species-specific. On the other hand, we found orthologues for ROM4 in all species, except for chromerids.

The TRAP proteins described to date all have an extracellular region containing one or more TSP1 domains and/or one or more vWA domains [74,75,76]. They are also characterized by the presence of a single transmembrane domain, a signal peptide, and, in some cases, a juxtaposed rhomboid protease cleavage site, and a short, charged C-terminal cytoplasmic domain with aromatic residues. The presence of a YXXΦ tyrosine sorting signature has also been described [75] (where X signifies any amino acid, and Φ any hydrophobic amino acid).

To evaluate the presence of TRAP-like proteins in P. cf. gigantea genomes, we inventoried all predicted proteins containing at least one TSP1 domain (Table S8), then identified potential candidates with several TRAP-like structural characteristics (Fig. 5). We identified a CpTSP2 orthologue within both P. cf. gigantea genomes, designated PgTSP2. Like CpTSP2, it is a large protein (~ 2800 aa) composed of Notch, TSP1, and Sushi domains. PgTSP2 has a localization signal, a transmembrane domain and a short, charged, basic cytoplasmic tail. This protein also has orthologues in G. niphandrodes, in chromerids and coccidia.

Structures and molecular domains of candidate TRAP-like proteins in P. cf. gigantea A and B. See also Table S8

We demonstrated the presence of genes encoding four other related protein pairs in both A and B genomes, most of which appear to be specific to P. cf. gigantea. PgTSP-1 has a TSP1 domain, a signal peptide, a transmembrane domain and a short, charged, acidic cytoplasmic tail. PgTSP-2, very similar in structure to PgTSP-1 also has a TSP1 domain, a signal peptide, a transmembrane domain, and a short, charged but basic cytoplasmic tail. PgTSP_EGF-1 has two TSP1 domains, a signal peptide, a transmembrane domain and a short, charged, acidic cytoplasmic tail, plus several extracellular EGF or EGF-like domains, as also described in C. parvum CpTSP7, CpTSP8 and CpTSP9 [80]. We identified another protein, PgTSP_EGF-2, very similar in structure.

Moving-junction associated proteins

In apicomplexans with intracellular stages such as T. gondii, invasion occurs when the extracellular tachyzoite initiates a pivotal movement known as reorientation, and a mobile junction settles into the host cell membrane allowing the parasite to enter. Gliding forces are also involved in this process [89], to which the host cell contributes [90]. A micronemal protein, AMA1, combines with rhoptry neck proteins (RON2, RON4, RON5 and RON8) to firmly secure the parasite to the host cell. In P. falciparum, another AMA-like protein, merozoite apical erythrocyte-binding ligand (MAEBL) has an important role in invasion alongside AMA1 [91].

Gregarines remain extracellular during their entire life cycle and Cryptosporodium display an intracellular but extra-cytoplasmic stage, so it was not surprising that we did not identify any orthologue of the moving-junction proteins of either these groups. We also searched for predicted proteins implicated in adherence and invasion in Cryptosporidium, such as GP15/40, GP900 and mucins, but found no equivalent in either P. cf. gigantea [92, 93].

Regulatory factors and signaling pathways

Increases in parasite intracellular calcium activate calcium-dependent protein kinases (CDPK) that regulate motility, microneme secretion, invasion and egress [94, 95]. Other proteins acting in such signaling pathways include diacylglycerol kinase 1 (DGK1) and acylated pleckstrin homology domain-containing protein (APH), which are involved in microneme secretion regulation [96, 97]; the C2 domain-containing protein DOC2.1 which mediates apical microneme exocytosis [98]; and the apical lysine methyltransferase (AKMT), which is involved in gliding motility, invasion and egress in T. gondii [99]. We were unable to identify APH in most gregarines and chromerids, and DOC2.1 could not be identified in several transcriptomes. All other regulatory factors appeared to be largely conserved.

For the scanning electron microscopy (SEM) studies, isolated trophozoites and cysts, or host intestines and rectal ampullas opened along their longitudinal axis, were washed as indicated above then fixed in 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate (pH 7.2) at 4 °C for 6 to 12 hours. After two washing steps in 0.1 M sodium cacodylate (pH 7.2), biological specimens were transferred to microporous specimen capsules (30 μm porosity, 12 mm diameter, 11 mm high, ref. #70187–20, Electron Microscopy Science) and dehydrated in a graded series of ethanol in double-distilled water (50, 70, 90, and 100%). Biological specimens in the capsules were critical point-dried in liquid CO₂ (Emitech K850, Quorum Technologies), then transferred to adhesive carbon-coated holders, and coated with 20 nm of gold (JEOL Fine Coater JFC-1200). Specimens were then examined with a Hitachi SU3500 Premium scanning electron microscope.

For the transmission electron microscopy (TEM) studies, samples were fixed for 2 h in 0.2 M sodium cacodylate buffer with 4% glutaraldehyde, 0.25 M sucrose in 0.2 M sodium cacodylate buffer pH 7.4. Cells were then washed three times in sodium cacodylate buffer containing decreasing concentrations of sucrose (0.25 M, 0.12 M, 0 M) for 15 min each time, followed by post-fixation for 1 h at 4 °C in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer. After three rinses in 0.2 M sodium cacodylate buffer, samples were dehydrated by successive transfer through an increasing ethanol series (25, 50, 70, 90%, 3 × 100%), then embedded in Spurr’s resin. Sections were cut using a diamond knife on a Leica Ultracut UCT ultramicrotome (Leica, Wetzlar, Germany) and after staining with saturated uranyl acetate for 15 min and Reynolds’ lead citrate for 3 min, were examined on grids with a Jeol 1400 transmission electron microscope (Jeol, Tokyo, Japan).

Genomic DNA (gDNA) was isolated from 4 biological samples of pooled cysts taken from 3 specimens of the host H. gammarus: sample JS-470 from Lobster #7 (~ 70 cysts), sample JS-482 from Lobster #11 (~ 50 cysts), samples JS-488 and JS-489 from Lobster #12 (~ 100 cysts each). Lobster #7 was provided by the Roscoff lobster tank facility while Lobster #11 and Lobster #12 were caught from Roscoff bay. DNA was extracted from the pooled cysts using Macherey Nagel Tissue and Cells isolation kit (ref 740,952.50) with yields of 4.1 μg (JS-470), 2 μg (JS-482), 4.5 μg (JS-488) and 6.7 μg (JS-489) of total DNA per sample, as measured by Nanodrop quantification. The protocol was used as recommended by Macherey Nagel, except that the initial lysis step at 56 °C was extended beyond the recommended to 1–3 hours with frequent microscopic (binocular) inspection to monitor cyst digestion until completion.

RNA was also isolated from 2 additional biological samples, both composed of pooled cysts taken from the rectal ampulla of their respective hosts: JS-555 (~ 35 cysts, Lobster #26, Roscoff bay) and JS-575c (~ 40 cysts, Lobster #34, Roscoff Lobster tank facility). Two distinct protocols were used to isolate total RNA from these two biological samples. For sample JS-555, we used Macherey Nagel basic RNA Isolation kit (ref 740,955.10) which yielded ~ 155 ng of total RNA in 55 μl as assessed by Qbit quantification. For sample JS-575c, we used Macherey Nagel Nucleozol-based RNA Isolation kit (refs 74,040.200 and 740,406.10) which yielded ~ 50 ng of total RNA in 55 μl as assessed by Qbit quantification.

The gDNA extracted from the 4 biological samples (JS-470, JS-482, JS-488 and JS-489) was sequenced individually using Illumina NextSeq technology (2 × 151 bp; NextSeq 500 Mid Output Kit v2; Institut du Cerveau et de la Moelle – CHU Pitié-Salpêtrière – Paris). We obtained 2 × 50 M to 2 × 70 M reads which were checked using FastQC [112] (version 0.11.5). Reads were cleaned with Trim Galore [113] (version 0.4.4) which removed remnant Nextera adaptors, clipped 15 bp at 5′-ends and 1 bp at 3′-ends and trimmed low-quality ends (phred score < 30). The assembly was carried out using SPAdes [114] (version 3.9.1; options: careful mode, automatic k-mers) with the pooled libraries (Fig. S2.A).

RNA was extracted from both samples (JS-555 and JS-575c) and treated with RNAse-free DNase. Libraries (Institut du Cerveau et de la Moelle – CHU Pitié Salpétrière – Paris) were prepared following the kit manufacturer’s recommendations (SMART-Seq v4 Ultra Low Input RNA Kit from Takara). Samples were sequenced on a NextSeq 500 Illumina device with MidOutPut cartridge to generate a total of 2 × 87 M reads of 75 bp. The read quality was checked by using FastQC and cleaned by using Trim Galore to remove remnant Nextera adaptors, clipping 15 bp at 5′-ends and 1 bp at 3′-end and trimming low-quality ends (phred score < 30). The sequence reads of both samples were merged into one library which was assembled using Trinity [115, 116].

All genomic contigs longer than 1 kb were analyzed by principal component analysis (PCA) based on their 5-mer composition, which classified them into 6 groups using a hierarchical clustering method (HCA) based on the Ward criterion (Fig. S2.B).

For all contigs, the putative protein coding genes were then predicted using Augustus [117] (version 3.3) and the Apicomplexa gene model for T. gondii. All the predicted proteins were thus compared with the NCBI non-redundant protein database using BLAST [118]. The analysis of the taxonomic groups corresponding to the best hits, enabled us to identify five clusters as putative bacterial contaminants whereas the sixth cluster which included 1745 contigs (18.0 Mb), was identified as organisms closely related to Apicomplexa, referred to as the “apicomplexa” cluster (Fig. S2.B).

Preliminary analysis of the “apicomplexa” cluster exhibit two sets of contigs with approximatively 10% of divergence and specific coverage values in the four libraries. The contigs of the “apicomplexa” cluster were split into genomes A and B by using the difference in coverage observed for the four gDNA libraries (Fig. S2.C, Fig. S3). Each gDNA library (JS-470, JS-482, JS-488 and JS-489) was individually mapped to the contigs using Bowtie2 [119] and the median coverage was calculated for each contig and each library using Samtools [120] and Bedtools [121] suites. This coverage information was processed by PCA and a k-means algorithm which classified the contigs into 2 clusters. Then, a linear discriminant model was trained with the coverage information and the result of this first classification before applying it to all the contigs in order to improve the classification. The linear discriminant method (training and classification) was iterated 3 times until convergence. A similar analysis was carried out with 1-kb non-overlapping windows (instead of full-length contigs) to identify putative hybrid contigs. Contigs were thus classified to different genomes depending on the windows, then divided into sub-contigs which were re-assigned to their respective genomes. A detailed protocol with R scripts is available on github (see data and code availability).

The nucleotidic divergence between genome A and genome B was estimated from the alignment of contigs built with Mummer3.0 [122]. All alignments of the syntenic regions were parsed to compute the divergence using a home-made script. Assembly metrics were assessed by using QUAST [123] (version 5.0).

The Infernal software [134] (version 1.3.3) and the Rfam database [135] (version 14.2) were used together to search for transfer RNAs, spliceosomal RNAs and ribosomal RNAs. The snoReport software [136] (version 2) was used to search C/D and H/ACA small nucleolar RNAs.

The 189-sequence phylogeny was built from the 18S SSU rDNA sequences from genomes A and B aligned with 14 from crustacean gregarines, and 154 environmental sequences from several projects described in Rueckert et al. (2011) [41] or gathered from NCBI Genbank. The sequences from the Gregarinoidae clade (n = 19) were used as the outgroup, as this clade has been placed as a sister group to the crustacean gregarine clade in recent literature [10,11,12]. A total of 1135 sites were found to be conserved after selecting conserved blocks as defined by Gblocks with the following parameters: minimum number of sequences for a conserved position, 95; minimum number of sequences for a flanking position, 95; maximum number of contiguous non-conserved positions, 8; minimum length of a block, 3; allowed gap positions, all. Maximum likelihood and Bayesian analyses were performed following the same protocol and parameters as in the previous 18S phylogeny.

For each orthogroup containing at least one of the reference proteins, the list of proteins was extracted, and the protein sequences were recovered with their respective coding sequences for both P. cf. gigantea genomes. BLASTP was performed for extracted proteins against the proteomes of P. cf. gigantea, as well as for the candidate proteins from each P. cf. gigantea genome against the 25 species reference proteomes. BLASTN was performed against NCBI NR for the coding sequences of the candidate proteins of both P. cf. gigantea genomes. The sequences thus collected for each described protein were aligned with mafft. Maximum likelihood molecular phylogeny was deduced from each alignment using RAxML. Analyses were performed using the LG model; bootstraps were estimated from 1000 replicates. Annotations of the conserved molecular domains were searched for in the automatic annotation and structure analyzed with SMART [150]. For each protein, the results of all the analyses were examined to validate the candidate proteins within the proteomes of the two P. cf. gigantea genomes. A table summarizing the presence or absence of glideosome proteins was visualized using R using the tidyverse package [151]. Putative TRAP-like proteins were identified by searching for sequences encoding the TSP1 molecular domain (IPR000884) within the two P. cf. gigantea genomes. The predicted structure of each candidate protein was studied, and if necessary partially predicted proteins were re-edited with Genewise [152]. Schematic representation of TRAP-like proteins was done using BioRender (biorender.com).

The 22 times grand slam winner Nadal is set to be last in action today on Centre Court where he will take on the No.1 Dutch men’s single-player Botic van de Zandschulp.

Plus the 2019 champion Halep will battle it out for the women’s singles against No4 seed Paula Badosa. 

Whilst Kyrgios will Americain professional Brandon Nakashima on Centre Court later today. 

So you don’t miss out on any of the action we’ve got all the information you need to know for today’s order of play. 

Wimbledon Order of Play Day 8:

Centre Court (Starting from 1.30pm)

Brandon Nakashima (USA) vs Nick Kyrgios (AUS)

Paula Badosa (ESP) vs Simona Halep (ROU)

Botic van de Zandschulp (NED) vs Rafael Nadal (ESP)

No.1 Court (Starting 1pm)

Elena Rybakina (KAZ) vs Petra Martic (CRO)

Jason Kubler (AUS) vs Taylor Fritz (USA)

Armanda Anisimova (USA) vs Harmony Tan (FRA)

No.2 Court (Starting from 11am)

Cristian Garin (CHI) vs Alex de Minaur (AUS)

Alize Cornet (FRA) vs Ajla Tomljanovic (AUS)

Yifan Xu and Zhaoxuan Yang (CHN) vs Alexa Guarachi (CHI) and Andreja Klepac (SLO)

Jack Stock and Coco Gauff (USA) vs Edouard Rodger-Vasselin and Alize Cornet (FRA)

No.3 Court (Starting from 11am)

Harriet Dart and Heather Watson (GBR) vs Lyumyla Kichenok (UKR) and Jelena Ostapenko (LAT)

Jamie Murray (GBR) and Bruno Soares (BRA) vs John Peers (AUS) and Filip Polasek (SVK)

Robert Farah (COL) and Jelena Ostapenko (LAT) vs Nikola Cacic and Aleksandra Krunic (SRB)

Mate Pavic (CRO) and Sania Mirza (IND) vs John Peers (AUS) and Gabriela Dabrowski (CAN)

Court 12 (Starting form 11am)

Alicja Rosolska (POL) and Erin Routliffe (NZL) vs Asia Muhammad (USA) and Ena Shibahara (JPN)

Jonny O’Mara and Kenneth Skupski (GBR) vs Kevin Krawietz and Andreas Mies (GER)

Mingge Xu (GBR) vs Lucia Peyre (ARG)

Court 18 (Starting from 11am)

Ella McDonald (GBR) vs Victoria Mboko (CAN)

Wesley Koolhof (NED) and Neal Skupski (GBR) vs Matthew Ebden and Max Purcell (AUS)

Talia Neilson Gatenby (GBR) vs Annabelle Xu (CAN)

Jean-Julien Rojer (NED) and Ena Shibahara (JPN ) vs Matthew Ebden and Samantha Stosur (AUS)

Court 4 (Starting at 11am)

Rose Marie Nijkamp (NED) vs Giorgia Pedone (ITA)

Edas Butvilas (LTU) and Mili Poljicak (CRO) vs Louis Bowden and Matthew Rankin (GBR)

Jack Loutit (NZL) and Edward Winter (AUS) vs Patrick Brady and William Jansen (GBR)

Liv Hovde (USA) v Anastasiya Lopata (UKR)

Court 5 (Starting 11am)

Weronika Ewald (POL) vs Nikola Bartunkova (CZE)

Hugo Coquelin and Phoenix Weir (GBR) vs Alessio Basile (BEL) and Peter Privara (SVK)

Irina Balus (SVK) vs Kayla Cross (CAN)

Luca Pow and Henry Searle (GBR) vs Tanapatt Nirundorn (THA) and Jaden Weekes (CAN)

Court 6 (Starting from 11am)

Nishesh Basavareddy (USA) and Rodrigo Pacheco Mendez (MEX) vs Coleman Wong (HKG)and Michael Zheng (USA)

Sayaka Ishii (JPN) vs Ella Seidel (GER)

Jonah Braswell (USA) and Dino Prizmic (CRO) vs Gilles Arnaud Bailly  (BEL) and Jakub Nicod (CZE)

Luca Udvardy (HUN) vs Alexis Blokhina (USA)

Court 7 (Starting from 11am)

Alexander Blockx (BEL) and Leanid Boika (USA) vs Dylan Dietrich and Kilian Feldbausch (SUI)

Joao Fonseca (BRA) and Juan Carlos Prado Angelo (BOL) vs Gonzalo Bueno and Ignacio Buse (PER)

Yu-Yun Li (TPE) vs Taylah Preston (AUS)

Peter Nad (SVK) and Martyn Pawelski (POL) vs Mika Brunold (SUI) and Nicholas Godsick (USA)

Court 8 (Starting from 11am)

Johanna Svendsen (DEN) vs Olivia Lincer (POL)

Mia Kupres (CAN) vs Celine Naef (SUI)

Gerard Campana Lee (KOR) and Jeremy Jin (AUS) vs Aidan Kim and Cooper Williams (USA)

Nikola Daubnerova (SVK) v Isabella Kruger (RSA)

Court 14 (Starting from 1pm)

Santiago Gonzalez (MEX) and Andres Molteni (ARG) vs Denis Kudla and Jack Sock (USA)

Jasmine Conway (GBR) vs Ela Nala Milic (SLO)

Matej Dodig (CRO) and Borys Zgola (POL) vs Gabriel Debru and Paul Inchauspe (FRA)

Linda Klimovicova (CZE) vs Isabelle Lacy (GBR)

Elise Mertens (BEL) and Shuai Zhang (CHN) vs Nadiia Kichenok (UKR) and Raluca Olaru (ROU)

Qavia Lopez (USA) vs Hayu Kinoshita (JPN)

Connor Henry Van Schalkwyk  (NAM) and Martin Antonio Vergara Del Puerto (PAR) vs Sebastian Gorzny and Alex Michelsen (USA)

Court 17 (Starting from 11am)

Martin Landaluce and Pedro Rodenas (ESP) vs Juan Manuel La Sernaand Lautaro Midon (ARG)

Alexander Frusina (USA) and Hayden Jones (AUS) vs Ozan Colak and Learner Tien (USA)

Lennon Roark Jones and Hayato Matsuoka (JPN) vs Jakub Mensik (CZE) and Olaf Pieczkowski (POL)

Bor Artnak (SLO) and Hynek Barton (CZE) v Paul Barbier Gazeu and Arthur Gea (FRA)

What time does Wimbledon start?

Coverage for Day 8 of Wimbledon starts at 11am with BBC 2 hosting the live sporting event all today until 7pm. 

What time does Wimbledon finish? 

The last matches of the day starts at 5.30pm with a scheudled end time of 7pm but as we all know tennis matches can go on longer than expected. 

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Uncle Sam is having a good slam.

🇺🇸 @Taylor_Fritz97 beats Alex Molcan 6-4, 6-1, 7-6(3)

There are now four American men in the fourth round at #Wimbledon for the first time since 1999 pic.twitter.com/kjocwxkNjo

— Wimbledon (@Wimbledon)

4.39pm BST

Swiatek* 3-4 Cornet

Better from Swiatek, if still a bitty showing from her. A break is really required in this next match.

On Centre Court, it’s Petra Kvitova, the 2011 and 2014 champion taking on Spain’s Paula Badosa, it’s the Czech who has the early advantage at 3-1 up.

4.34pm BST

Swiatek 2-4 Cornet*

A rather lazy lob reflects Swiatek’s continuing discomfort, but a swatting backhand takes her to 30-40 and another break point. Those rather tedious chants of “let’s go, Iga, let’s go” ring out but their heroine is stymied when Cornet stuns a volley at the net, and then balloons a service return out of bounds.

Cornet had points for 4-0, got broken but now saves a break point to hold for 4-2 vs. Swiatek.

Very interesting one on Court One.

— José Morgado (@josemorgado)

Updated at 4.35pm BST

4.29pm BST

Swiatek* 2-3 Cornet

Better from Swiatek, and she’s back in it but must now try and get another break back from her opponent.

4.25pm BST

Swiatek 1-3 Cornet*

The world No 1’s radar is not working. She’s made 11 unforced errors so far. Still, you don’t go on such a long winning run without overcoming some adversity or other, and she breaks back.

Simona Halep, meanwhile, is through, having beaten Magdalena Fręch 6-4 6-1.

4.16pm BST

Swiatek* 0-3 Cornet

A shock on our hands here? Cornet was once someone who Serena Williams had problems with.

Updated at 4.37pm BST

4.11pm BST

Swiatek* 0-2 Cornet

A decent hold from Cornet and Swiatek doesn’t look too happy on the grass surface.

Updated at 4.15pm BST

4.07pm BST

Iga Swiatek, the tournament favourite, in the women’s singles, is currently in the first game of her match with Alizé Cornet on Court No 1. And she suffers a break straight away.

Simona Halep, meanwhile, is a set up on Magdalena Fręch of Poland.

Updated at 4.35pm BST

4.04pm BST

Other news: Last year’s quarter-finalist Ajla Tomljanovic rallied from a set down to complete a 2-6 6-4 6-3 victory over 2021 French Open champion Barbora Krejcikova. American Brandon Nakashima powered past Daniel Elahi Galan 6-4 6-4 6-1. Cristian Garin defeated Jenson Brooksby 6-2 6-3 1-6 6-4. Jason Kubler, of Australia, beat American Jack Sock, 6-2 4-6 5-7 7-6 6-3

3.52pm BST

Amanda Anisimova says: “I think this is the most special day in my career. Winning today is so special, especially in front of a full crowd.” Harmony Tan awaits in the last 16.

3.48pm BST

Anisimova beats Gauff 6-7 6-2 6-1

Two big serves and Anisimova is 30-0 up. Then a cute drop volley cannot be returned, and she has three match points. The first is taken, as Gauff can only lay up her compatriot for the winner. She was devastating in the second and third set.

Updated at 3.54pm BST

3.46pm BST

Anisimova is in control now against Gauff, at 5-1 up in the third set, with Gauff serving and looking in trouble, and coughing up two break points, the first of which is seized. Anisimova will now serve for a place in the last 16.

3.42pm BST

Richard Gasquet, the French veteran, is out, having been beaten in three sets by Botic van de Zandschulp, the Dutchman. Sounds an eventful match.

Gosh Gasquet just hit 4 double faults in a game when serving for the set…

— José Morgado (@josemorgado)

Botic van de Zandschulp ➡️ fourth round

The Dutchman is enjoying the grass court season, seeing off Richard Gasquet 7-5, 2-6, 7-6(7), 6-1#Wimbledon pic.twitter.com/UCv1Bw82ww

— Wimbledon (@Wimbledon)

3.37pm BST

Cristian Garin, the Chilean, is the next opponent for De Minaur, who admits his “just relief. It was harder than what I wanted it to be. More than anything, it’s a relief to be in the second week of Wimbledon.”

Since you ask, and we got lost in that heightened excitement, Gauff and Anisimova are 1-1 on sets, and Anisimova has just broken in the third, and she’s 3-1 up.

3.33pm BST

De Minaur beats Broady 6-3, 6-3 7-5

De Minaur serves with zest, trying to do better than his last service game, but he does make an error for 15-15. Then comes another for 30-30. The nerves jangling? Perhaps, as he is caught out by an odd bounce, and break point is presented. No such nerves when he cranks home an overhead to level it at deuce. Then, he nets again, with the court at his mercy for another break point to Broady. That’s saved by a disguised forehand winner, down the line and beyond. Then comes a double fault, the challenge of the call not paying off. That’s saved by some composed hitting when Broady is charging all over the court. And another break point as Broady soaks up the pressure in a long rally and De Minaur cracks. Again, saved, with a volley at the net. Finally, a huge serve gets him to his first match point, but an overhit backhand nixes that. Then comes another huge serve, and the second match point. And that’s also saved. Broady refuses to lie down but then commits an error in overhitting. A third match point, also saved, by some smart hitting and then a punched volley at the net. A fourth, and at last, Broady has to accept defeat, as he is unable to return another big serve.

Updated at 3.39pm BST

3.19pm BST

De Minaur 6-3, 6-3 6-5 Broady*

A long baseline rally is lost by Broady when he makes an error to hand over a 15-30 deficit. He then challenges, unsuccessfully, and has two break points to face. The first is taken by De Minaur, who whips the ball right to the toes of Broady, and now the Australian can serve once again to win the match.

3.14pm BST

*De Minaur 6-3, 6-3 5-5 Broady

Broady’s alive! Finally, he breaks. A glimmer at 0-30 when De Minaur nets from the baseline, and lots of roars from the home fans. But some fierce hitting a soft, deadening volley takes it to 15-30. Then comes two break points for Broady when De Minaur blams a forehand out. The first is saved by a punching forehand winner. The second is not, as the forehand goes out of bounds.

Updated at 3.22pm BST

3.09pm BST

De Minaur 6-3, 6-3 5-4 Broady*

An ace takes Broady to 40-15, but then a poor second serve offers up a chance for De Minaur to make it to 40-30, then comes deuce. De Minaur edges closer but makes a returning error at deuce, and the Broady sees out his service hold.

Anisimova now leads Gauff 4-1 in that second set.

Updated at 3.09pm BST

3.04pm BST

De Minaur* 6-3, 6-3 5-3 Broady

An easy hold for De Minaur, and now Broady must serve to save himself.

3.03pm BST

De Minaur 6-3, 6-3 4-3 Broady*

Broady relief as his forehand takes him to 40-15, when it looked to be heading out. He’s rather less happy when he’s sent round the house and can only clank the post of the net. Still, he holds, and his exit from the tournament is postponed for now.

Gauff breaks back and is 1-2 down, and serving against Anisimova.

Meanwhile, in fetching jacket, Cliff Richard is pictured in the crowd.

2.58pm BST

*De Minaur 6-3, 6-3 4-2 Broady

De Minaur being made to work on his serve. A forehand miss means it’s 30-30 but still he gets the job done.

Anisimova has broken Gauff, and it’s 2-0 in the second set.

2.51pm BST

De Minaur 6-3, 6-3 3-2 Broady*

Broady challenges a call when he thinks he’s landed an ace. He’s proved wrong, and has one challenge left. He ends up over the net when a delicate drop catches him out for 30-30. A decent serve arrives us at 40-30, and when taken to deuce, he holds his serve. He hasn’t, then, lost heart.

2.46pm BST

De Minaur* 6-3, 6-3 3-1 Broady

Broady’s roar getting louder and louder, and there’s still life in him. But De Minaur’s speed catches him out and he misses an overhead to be pulled back to 30-30. Then there’s some tomfoolery with the line judge who gets two calls wrong on the De Minaur serve, both of which are challenged. And after all that, Broady lands a break point at 30-40. But, from the baseline, with De Minaur set in motion, he can only net a backhand. And then De Minaur closes him down to serve out.

Gauff has taken the first set in that tie-break with Anisomova, the first set taking an hour and four minutes.

I’ve watched tennis for 35 years and I’ve never seen anyone as fast around a court as Alex De Minaur. #Wimbledon

— Calvin Betton (@Calvbetton)

2.39pm BST

De Minaur 6-3, 6-3 2-1 Broady*

Broady double faults at 30-0 up but then gets to 40-15 with a topped backhand he clatters down the line for 40-15. De Minaur then cracks a winner from the baseline for 40-30, which seems to distract Broady who then nets for deuce. Still, he recovers himself and celebrates winning the game with a loud roar.

Anisimova and Gauff are now in a tie-break, their first set being something of an epic.

Updated at 2.40pm BST

2.33pm BST

De Minaur* 6-3, 6-3 2-0 Broady

De Minaur smells blood and gets a bit excited when spotting a chance to punch a winner home, and finds himself pegged back to deuce. Then Broady lands his first break point of the whole game when sending De Minaur round the houses. It comes and goes, as he is caught by De Minaur retrieving what looked like a lost cause and can only scoop the ball out. The call is challenged to no avail, and De Minaur serves out. A chance of an unlikely comeback is snuffed out.

2.28pm BST

De Minaur 6-3, 6-3 1-0 Broady*

Broady, sadly, seems to be losing heart. No disgrace in losing to an opponent who looks to have the tools to become a next big thing, particularly on grass. Three break points soon arrive, and though the first two are saved, Broady makes a mistake on a smash and ends up out of bounds. A shame.

Gauff has broken back, so it’s back with serve in that game with Anisimova at 5-4.

2.21pm BST

De Minaur* 6-3, 6-3 Broady

A brisk backhand takes it to 30-0, and then Broady goes for a winner, but misses the angle. Three set points, and the first is taken when Broady again overhits. That’s a long road back for the Brit.

2.18pm BST

De Minaur 6-3, 5-3 Broady*

Better serving from Broady now, and he has made De Minaur serve for the second set.

Anisimova has won four straight games and now leads the first set 4-3 and is serving.

2.14pm BST

De Minaur* 6-3, 5-3 Broady

Suddenly, Broady is bringing the noise, and he’s up to 0-30, working De Minaur’s backhand. But then De Minaur, pulling himself from the ground, somehow digs out a volley at the net for 15-30. Then comes a big serve for 30-30, then Broady overcooks from the baseline, and then does the same to hand control back. He’s yet to get to break point.

2.09pm BST

De Minaur 6-3, 4-3 Broady*

Big, gusty hold from the Stockport lad. A crashing, whipped overhead from De Minaur takes him to 30-0 up on his opponent’s serve, then Broady nets from the baseline to present three break points. The first is saved when Broady prevails in a rally by coming to the net. De Minaur’s powers of recovery take some grinding down, though he can’t return Broady’s next serve. Then, Broady sends De Minaur running, and then chops a backhand drop to go to deuce. Two fine serves cannot be returned and Broady punches the air in delight.

Anisomova, meanwhile, has broken back, and it’s back with serve at 2-3 with Gauff.

Updated at 2.14pm BST

2.04pm BST

De Minaur* 6-3, 4-2 Broady

Broady have De Minaur something of a scare there, only for the Australian to hold his serve in the end.

2.02pm BST

Good afternoon, all. Thanks, Will and Gregg, for their sterling service. I join the throng with both Liam Broady and Amanda Anismova in a bit of trouble. Some housekeeping: the denotes next server thing I shall abandon due to it confusing me with every change of server. *now denotes current server.

Updated at 2.10pm BST

2.00pm BST

De Minaur* 6-3, 3-2 Broady (* denotes next server)

Ah, hard luck Broady. De Minaur has broken again. And, with that, I’ll hand over to tennis aficionado John Brewin. Enjoy!

Updated at 2.20pm BST

1.58pm BST

Elsewhere, Coco Gauff has charged into a 3-0 lead against her fellow American Amanda Anisimova. Botic van de Zandschulp leads Richard Gasquet 2-1 in the third set, having won the first but lost the second, while Jason Kubler is 4-3 up in the fourth set against Jack Sock and Cristian Garín is 5-4 up in the fourth against Jenson Brooksby. Both Sock and Garín currently lead two sets to one.

1.53pm BST

De Minaur 6-3, 2-2 Broady* (* denotes next server)

De Minaur is a bit good, isn’t he? He holds to love, leaving Broady stranded with several thumping serves and ending on his eighth ace of the match for good measure.

1.50pm BST

De Minaur* 6-3, 1-2 Broady (* denotes next server)

De Minaur earns two break points but Broady defends them both, the latter with his first ace of the match. At deuce, Broady loses a stinging rally after being manoeuvred into the corner of the court, but hits back with another wicked drop shot which eludes De Minaur and sees out the game from there.

1.44pm BST

De Minaur 6-3, 1-1 Broady* (* denotes next server)

De Minaur powers to 40-0, but Broady puts up some resistance and wins a couple of points. He can’t quite take it to deuce, however, wafting an attempted backhand into the net.

Updated at 1.46pm BST

1.43pm BST

De Minaur* 6-3, 0-1 Broady (* denotes next server)

Broady fires some big first serves down the court, dropping a single point on the way to a hold. De Minaur ended the first set with six aces to Broady’s nil, so the British No 5 could do with more where that came from.

Updated at 1.45pm BST

1.38pm BST

First set: De Minaur 6-3 Broady

It’s a simple defence for De Minaur, who wins the game to love. Broady’s path to victory is now considerably longer.

Updated at 1.50pm BST

1.35pm BST

De Minaur* 5-3 Broady (* denotes next server)

Broady regains his composure on serve, but it may be too late to salvage the first set. De Minaur has the chance to serve it out.

1.32pm BST

De Minaur 5-2 Broady* (* denotes next server)

De Minaur races into a 40-0 lead and looks set for a love hold, but an exchange of venemous forehands ends with Broady lashing a winner down the line. It’s a temporary reprieve, however. The British hopeful tries an ambitious slice, but it sails beyond the baseline.

1.28pm BST

De Minaur* 4-2 Broady (* denotes next server)

Suddenly De Minaur ups the tempo and Broady is under severe pressure. The Australian earns three break points, wrapping up the game with a cross-court winner which his opponent can only spoon wide.

1.24pm BST

De Minaur 3-2 Broady* (* denotes next server)

De Minaur drops a single point on the way to another straightforward hold. Time for a drinks break.

Updated at 1.26pm BST

1.20pm BST

Over to De Minaur and Broady, then. Broady wins his second service game to love, finishing off with a gorgeous drop shot which leaves his opponent stretching in vain and almost stumbling into the net. The pair are level at 2-2 in the first set.

Updated at 1.21pm BST

1.17pm BST

Tomljanovic beats Krejcikova 2-6, 6-4, 6-3!

Krejcikova battles hard in the final game, earning a break point but failing to take it. Tomljanovic earns advantage and then, after a brief rally, wins match point, slumping to the ground and grinning in a mixture of triumph and relief.

Updated at 1.31pm BST

1.08pm BST

Thanks, Gregg. I’m still trying to shake off the sugar rush after necking an entire punnet of strawberries and half a pint of double cream in preparation, so let’s start with the match report from Harmony Tan’s demolition of Katie Boulter earlier on.

Alex de Minaur and Liam Broady have just emerged to warm up on Court No 1. I’m going to hand over to Will Magee to bring you updates for the next hour or so. Bye.

1.00pm BST

Tomljanovic battles hard to hold serve and plays some lovely tennis to bring up two break points in the next game. A delicious backhand slice across court is too low for Krejcikova to retrieve. Tomljanovic has the break and leads 4-2 in the deciding set.

12.58pm BST

Gary Naylor has a view on the low attendances at Wimbledon.

Though you won’t hear it from the government or its fawning media, Covid, suspected Covid and fear of Covid must be eating into these attendances @GreggBakowski.

If you invested in home tech during lockdown, why not stay home and not pay astronomical sums for travel and hotels?

— Gary Naylor (@garynaylor999)

12.53pm BST

Krejcikova’s movement looks good after her medical timeout and the Czech holds serve after being pushed hard by her Australian opponent. There’s some heavy-hitting going on in this match now. It’s 2-2 on serve in the deciding set.

12.49pm BST

Here’s a flavour of the craft and skill on display from Harmony Tan as she routed Katie Boulter in straight sets earlier on.

Harmony Tan was having a lot of fun in her 51-minute victory over Katie Boulter ⚡

The Frenchwoman’s brilliant run continues and she’s into the second week of #Wimbledon pic.twitter.com/Lj9weEAhL9

— Wimbledon (@Wimbledon)

12.46pm BST

Krejcikova is receiving a medical timeout. It looks like she may have blisters as she appears to be having strapping and a big old plaster attached to her instep. Or maybe there’s a strain in that area. It’s 2-1 on serve and the 13th seed appears to be fine to continue, which is good news for this absorbing match.

Updated at 1.04pm BST

12.42pm BST

Garin has taken a two-set lead over the young American 29th seed Brooksby on Court 3. The Chilean world No 44 made it into the last 16 here last year and looks like he’s going to repeat that feat.

12.38pm BST

Barbora Krejcikova has just taken a very long toilet break, which suggests she may have a problem after losing the second set. She left Tomljanovic waiting far too long and the crowd appear to have sided with the Australian as a result, who holds to 30 in the opening game of the deciding set. As suspected, Krejcikova does have a problem. She calls for the trainer but will first have to serve.

Updated at 12.38pm BST

12.33pm BST

The old-timer Richard Gasquet has clearly oiled his joints because he has started like a train against 21st seed Botic van de Zandschulp on Court 2, breaking his younger Dutch opponent and racing into a 3-0 lead. Gasquet is 36 and still ranked at a respectable 69.

Updated at 12.33pm BST

12.28pm BST

Tomljanovic wins the second set 6-4 to level the match against 13th seed Krejcikova at 1-1! The Australian blitzes a forehand away to break her opponent and make a real match of this. The world No 44 reached the last eight at SW19 last year. She is no pushover.

12.24pm BST

Sock holds to love to win the second set against Kubler 6-4 and level the match at 1-1. Sock has had an awful time with injuries and it’s hard to know how deep he might go here. He has been ranked as high as No 8 in his career and is still only 29. He’s now ranked 103 and certainly has the pedigree to beat Kubler (world No 99).

12.17pm BST

The crowds at Wimbledon look thin again this afternoon but, of course, they will likely swell when play gets under way on the show courts. The lower than expected attendances may be having a knock-on effect on temporary staff at the tournament. Here’s the story from Tobi Thomas.

12.11pm BST

Right, so what are the scores on the board elsewhere? In the men’s singles Garin leads Brooksby 6-2, 3-1. Sock is a break up at 5-3 in the second set against Kubler, having lost the first set 6-2. And in the women’s singles Barbora Krejcikova is a set up against Tomljanovic but it’s on serve at 3-3 in the second set.

12.05pm BST

“I don’t believe [how well] I’m playing,” says Tan. “ It was emotional against Serena [in the first round] but I’ve just been playing match by match. Today was pretty good tennis. I don’t know why.” Tan will play either Coco Gauff or Amanda Anisimova, who play on Centre Court at 1.30pm, and neither of those will fancy that one. “Tan’s probably quite glad she can concentrate on this, rather than being distracted by the doubles,” writes Matt Dony, referring to the story below. Maybe Tan made a very good call after all.

Tan beats Boulter 6-1, 6-1 in 51 minutes!

Everything Tan hits appears to be landing on the line. Boulter looks discombobulated out there. She must feel like she’s playing a grand slam winner, not a player ranked only three places ahead of her. Boulter is made to stretch in vain to reach a cross-court backhand and Tan has a match point. Boulter double-faults. Oh dear.

Updated at 12.37pm BST

11.56am BST

I thought this would be a close game. Tan holds to love and Boulter has to serve to stay in the match. Second set: Tan 5-1 Boulter.

11.56am BST

Boulter is broken again. And what a point to win it for Tan, who is having a fine time out there. At 30-40, Tan retrieves a Boulter lob with a “tweener”, follows it up with a delicate volley and then whips a cross-court backhand towards the tramlines that Boulter can only get her racket frame to. This isn’t going to last much longer. Second set: Tan 4-1 Boulter.

11.52am BST

In the other singles matches that started early today, 13th seed Barbora Krejcikova has won the first set against Ajla Tomljanovic 6-2, Australia’s Jason Kubler has won the first set against America’s Jack Sock 6-2 and Chile’s Cristan Garin has a one-set lead over 29th seeded American Jenson Brooksby after winning it, also 6-2.

11.48am BST

Boulter stays in the opening rally long enough to force Tan into an error, the Frenchwoman hitting long. But two unforced errors in a row means the opportunity to ratchet up the pressure is spurned. Tan struggles with her first serve here, though, and Boulter is back in the game at 30-30 when Tan slices a backhand wide. A vicious forehand from Boulter brings up deuce but Tan finds her groove again to hold serve. Second set: Tan 3-1 Boulter.

Updated at 12.16pm BST

11.44am BST

That’s better from Boulter. A hold to love after some varied and sharp serving. Now, can she put pressure on Tan’s serve? Second set: Tan 2-1 Boulter

11.42am BST

This is just painful for Boulter. She can’t win a point, never mind a game. It’s an easy hold to love for Tan, with Boulter slashing wildly to give her the game. Second set: Tan 2-0 Boulter.

Updated at 11.43am BST

11.40am BST

The crowd is really trying to get behind Boulter but the Brit can’t turn the tide. She misses an easy volley at the net at 0-15 to give Tan some extra pep in her step and then Tan hits an extraordinary cross-court forehand that is called out. She challenges it and, yes, it just nicked the line. It’s 0-40 and Boulter can’t get back into the game. Tan breaks and leads 1-0 in the second set.

11.35am BST

Tan holds to 15 to win the first set 6-1. The French world 115 doesn’t even look out of breath. She rattles out three aces in that game.

11.33am BST

Boulter is broken again. Tan’s returns are incredibly sharp, exquisitely placed at the feet of Boulter who can’t get them back. It’s soon 15-40 and Boulter slams a frustrated forehand into the net. She has some thinking to do. It’s 5-1 to Tan in just 24 minutes.

11.30am BST

It’s another comfortable hold to 15 for Tan. Boulter is still struggling with her tactics, unsure whether the best approach is to trade shots from the back of the court or come to the net and use her volleying prowess to good effect. Tan 4-1 Boulter.

11.25am BST

Tan is threatening to race away with this first set. She’s got great variety in her game and is sending Boulter racing back and forth around the court. She holds her own serve comfortably to take a 3-0 lead and then pushes Boulter to the limit in the fourth game, with the Brit having to battle with everything she has to hold. Tan 3-1 Boulter.

Updated at 12.38pm BST

11.19am BST

I will also be keeping my eye on 12th seed Barbora Krejcikova v Ajla Tomljanovic out on Court 12. It’s 2-1 to the Czech 2021 French Open champion and on serve.

11.16am BST

Boulter is down an early break after a nervy opening service game, in which she double-faults and has a little misfortune when Tan enjoys two net cords. Then Boulter misjudges a Tan lob, leaving it only to watch it land in the court with room to spare behind her. It’s 2-0 to Tan.

11.10am BST

We’re under way on Court 2. Tan serves first in an entertaining game which features a delicate Boulter lob and a “tweener” from her French opponent who holds to 15 to take a 1-0 lead in the first set.

11.05am BST

Katie Boulter has made her way out on to Court 2 to warm up for her third-round match against Harmony Tan. It’s a lovely day for tennis, a very pleasant 20c at SW19 with a few fluffy clouds scattered around that shouldn’t threaten rain.

Updated at 1.16pm BST

10.47am BST

So what has caused the resurgence in British tennis at SW19? Simon Cambers reports that the team spirit forged in lockdown, plus Andy Murray’s advice and Emma Raducanu’s achievements, has given everyone a lift.

Order of play for Saturday

(All times BST, seeds in brackets)

Centre Court
13:30: (11) Cori Gauff (USA) v (20) Amanda Anisimova (USA), (4) Paula Badosa Gibert (Spa) v (25) Petra Kvitova (Cze), (27) Lorenzo Sonego (Ita) v (2) Rafael Nadal (Spa)

Court 1
13:00: (19) Alex De Minaur (Aus) v Liam Broady (Gbr), (1) Iga Swiatek (Pol) v Alize Cornet (Fra), Nick Kyrgios (Aus) v (4) Stefanos Tsitsipas (Gre)

Court 2
11:00: Harmony Tan (Fra) v Katie Boulter (Gbr), Richard Gasquet (Fra) v (21) Botic Van de Zandschulp (Ned), Magdalena Frech (Pol) v (16) Simona Halep (Rom)

Court 3
11:00: Christian Garin (Chi) v (29) Jenson Brooksby (USA), Alex Molcan (Svk) v (11) Taylor Harry Fritz (USA), Petra Martic (Cro) v (8) Jessica Pegula (USA)

Court 4
11:00: (4) Gonzalo Bueno (Per) v Juan Manuel La Serna (Arg), Jeremy Jin (Aus) v (3) Mili Poljicak (Cro), Kristyna Tomajkova (Cze) v (10) Annabelle Xu (Can), (8) Edas Butvilas (Lit) v Mika Brunold (Swi), Alessio Basile (Bel) v (11) Rodrigo Pacheco Mendez (Mex)

Court 5
11:00: Mia Kupres (Can) v Angella Okutoyi (Ken), Linda Klimovicova (Cze) v Michaela Laki (Gre), Paul Barbier Gazeu (Fra) v Connor Henry Van Schalkwyk (Nam), Julie Struplova (Cze) v Ella McDonald (Gbr), Aysegul Mert (Tur) v (3) Nikola Bartunkova (Cze)

Court 6
11:00: Patrick Brady (Gbr) v (15) Martyn Pawelski (Pol), Phoenix Weir (Gbr) v Alexander Blockx (Bel), Sarah Tatu (Gbr) v Celine Naef (Swi), Juan Carlos Prado Angelo (Bol) v William Jansen (Gbr), Talia Neilson Gatenby (Gbr) v Nina Vargova (Svk)

Court 7
11:00: Kalin Ivanovski (Mkd) v Luca Pow (Gbr), Henry Searle (Gbr) v (2) Jakub Mensik (Cze), Renata Jamrichova (Svk) v Jasmine Conway (Gbr), Joelle Steur (Ger) v Isabelle Lacy (Gbr), Benjamin Gusic Wan (Gbr) v Peter Privara (Svk)

Court 9
11:00 Amelia Waligora (Bel) v Anastasiya Lopata (Ukr)

Court 10
11:00: Irina Balus (Svk) v Andrea Obradovic (Ser), Peter Nad (Svk) v Jaden Weekes (Can), Ella Seidel (Ger) v Lucciana Perez Alarcon (Per), (4) Nikola Daubnerova (Svk) v Dominika Salkova (Cze)

Court 11
11:00 Sara Saito (Jpn) v Olivia Lincer (USA), (14) Lucija Ciric Bagaric (Cro) v Rose Marie Nijkamp (Ned)

Court 12
11:00: Ajla Tomljanovic (Aus) v (13) Barbora Krejcikova (Cze), Brandon Nakashima (USA) v Daniel Elahi Galan (Col)

Court 14
11:00 Hynek Barton (Cze) v Arthur Gea (Fra)

Court 15
11:00 Olaf Pieczkowski (Pol) v Ozan Colak (USA)

Court 16
11:00 Pedro Rodenas (Spa) v Matej Dodig (Cro)

Court 17
11:00 Dylan Dietrich (Swi) v (5) Nishesh Basavareddy (USA), Giorgia Pedone (Ned) v Amelie Van Impe (Bel)

Court 18
11:00: Jack Sock (USA) v Jason Kubler (Aus), Qinwen Zheng (Chn) v (17) Elena Rybakina (Kaz)

Good morning. It’s already day six at Wimbledon – where did the week go? The show courts don’t get going until a bit later on, with Coco Gauff v Amanda Amisova on Centre Court at 1.30pm and Alex De Minar v Liam Broady on Court No 1 at 1pm.

It’s been a great week for British tennis, with Heather Watson and Cameron Norrie breaking new ground by reaching the fourth round of a grand slam for the first time in their careers and Broady and Katie Boulter have hopes of doing the same today. Boulter is not on a show court so I’ll be paying particular attention to her match against Frances’s Harmony Tan on Court 2 at 11am. It promises to be extremely tight, with Tan ranked 115 in the world and Boulter 118. Like her British opponent, Tan is looking to make the last 16 in a slam for the first time, too.

I’m looking forward to 4th seed Paula Badosa v 25th seed Petra Kvitova on Centre Court later, too. Kvitova has won Wimbledon twice and despite slipping down the rankings in the last couple of years, I feel she can go deep in 2022. And expect fireworks – and lots of entertaining chat – when Nick Kyrgios takes on Stefanos Tsitsipas on Court 1 later. Kyrgios just wants to be loved, you know.

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Gloria Smiled by Hazel Pacheco This is book 3 of the Henry and Friends series. In this book, Gloria’s simple misunderstanding leads to a startling surprise!

In today’s rapidly evolving world, businesses must adapt to technological advances to stay competitive and sustain growth. An essential aspect of this adaptation is optimizing processes, methodologies, and tools that drive business growth. In this article, we’ll explore key optimization techniques that focus on enhancing tech-driven business growth. We’ll also discuss practical steps that businesses can take to implement these techniques.

Table of Contents

1. Understanding Optimization in Tech-driven Businesses
2. Optimization Techniques for Tech-driven Growth
3. Selecting the Right Platforms
4. Implementing Optimization Techniques
5. Monitoring Progress and Refining Strategies
6. Frequently Asked Questions (FAQs)

Understanding Optimization in Tech-driven Businesses

Modern businesses employ various technological solutions and tools—such as software, hardware, and data analytics—to improve their operations. Applied correctly, these tech tools provide companies with the resources they need to innovate, expand their reach, and increase overall efficiency. The process of optimization, in this context, involves identifying and enhancing specific aspects of the business to achieve faster growth.

Benefits of Optimization

Implementing optimization techniques in a tech-driven business offers several benefits:

• Increased efficiency: Businesses can streamline their operations and identify areas for improvement by leveraging technology.
• Enhanced decision-making: Tech-driven optimization supports data-driven decision-making, enabling businesses to make more informed choices.
• Scalability: By identifying bottlenecks and improving processes, businesses can efficiently scale without negatively impacting overall operations.

Optimization Techniques for Tech-driven Growth

Data Analytics

Collection, analysis, and interpretation of data are crucial for understanding current trends, customer behaviors, and potential opportunities for growth. Companies can harness the power of data analytics to make better-informed decisions, set priorities, and shape their strategies for success.

Automation

Automation reduces manual work, streamlines business processes, and enables organizations to handle repetitive tasks more efficiently. It helps businesses save time and resources, allowing them to focus more on innovation and growth strategies.

Cloud Computing

Cloud computing enables businesses to store and access data and software through the internet, rather than relying on physical infrastructure. Embracing cloud technology provides businesses with increased flexibility, scalability, security, and opportunities for collaboration.

Integration

Integration of various technologies within a business environment can help create seamless workflow processes, reduce data silos, and improve overall operational efficiency. Integration can be achieved through the use of APIs, middleware, or all-in-one software platforms.

Selecting the Right Platforms

Choosing the right platforms, tools, and technologies is critical for a business aiming to optimize its processes for growth. Careful evaluation and selection help ensure compatibility with existing systems, maximize benefits, and reduce the risk of potential pitfalls.

Considerations When Selecting a Platform

1. Compatibility: Ensure that the chosen platform seamlessly integrates with your existing tools and systems.
2. Scalability: Select a platform that can easily adapt to your business’s growth and evolving needs.
3. Cost-effectiveness: Compare the initial investment and ongoing costs of the platform against the potential benefits and savings it can provide.
4. Usability: Choose user-friendly platforms that your team can quickly learn and use effectively.
5. Support: Prioritize platforms that offer comprehensive support, including documentation, training, and customer service.

Implementing Optimization Techniques

Successfully implementing optimization techniques involves a systematic approach and careful planning. Businesses should consider the following steps to promote tech-driven growth:

Determine Priorities

Identify which areas of your business stand to benefit the most from optimization. This process may involve assessing current inefficiencies, analyzing market trends, and determining potential opportunities for growth.

Create a Detailed Plan

Develop a comprehensive plan outlining specific goals, resources, timelines, and milestones for executing optimization initiatives. This plan should include risk assessments, contingency plans, and a clear communication strategy to keep stakeholders informed throughout implementation.

Assign Roles and Responsibilities

Define clear roles and responsibilities for team members involved in the implementation process, ensuring a well-coordinated effort that maximizes efficiency and minimizes potential setbacks.

Train and Educate

Provide team members with adequate training and education on the chosen platforms, tools, and techniques to ensure they have the necessary skills and expertise to execute the implementation effectively.

Review and Iterate

Regularly review the progress and outcomes of the implementation process, identify areas for improvement, and adjust the plan as required to ensure ongoing success and growth.

Monitoring Progress and Refining Strategies

Once optimization techniques have been implemented, ongoing monitoring and evaluation are crucial to ensuring long-term success. Businesses should consider the following tactics to measure progress and refine strategies:

Key Performance Indicators (KPIs)

Establish KPIs to gauge the effectiveness of implemented optimization techniques. These indicators should be tied to specific business objectives and help measure success or identify areas for improvement.

Data Analysis

Analyze data collected throughout the optimization process to identify trends, patterns, and opportunities for improvement. Using this information, businesses can make data-driven decisions to refine strategies and continue driving growth.

Regular Reviews

Conduct regular reviews of implemented strategies, techniques, and tools to assess their ongoing effectiveness and identify potential changes or enhancements. These reviews can help inform future planning and ensure that optimization efforts continue to yield results.

Frequently Asked Questions (FAQs)

1. What is optimization in a tech-driven business environment?

Optimization in a tech-driven business environment involves leveraging technology to identify and enhance specific aspects of a business to achieve more rapid growth.

2. Why is optimization important for businesses?

Optimization is important for businesses because it helps them increase operational efficiency, better adapt to market trends and demands, and drivesustainable growth through improved decision-making, scalability, and resource allocation.

3. What are some key optimization techniques for tech-driven growth?

Key optimization techniques for tech-driven growth include data analytics, automation, cloud computing, and integration of various technologies within the business environment.

4. How should businesses select the right platforms to enable optimization?

When selecting platforms to enable optimization, businesses should consider factors such as compatibility, scalability, cost-effectiveness, usability, and support.

5. How can businesses effectively implement optimization techniques?

To effectively implement optimization techniques, businesses should determine their priorities, create detailed plans, assign roles and responsibilities, provide training and education, and continually review and iterate their processes.

6. How can businesses monitor and refine their optimization strategies?

Businesses can monitor and refine their optimization strategies by using key performance indicators (KPIs), data analysis, and regular reviews to assess the effectiveness of implemented techniques, identify areas for improvement, and adapt and refine their approaches accordingly.

In conclusion, mastering optimization techniques for tech-driven business growth requires a strategic approach, a thorough understanding of the available technologies, and a commitment to continuous improvement. By addressing the key considerations discussed in this article and following the outlined steps, businesses can successfully implement optimization strategies that drive growth, innovation, and long-term success.