In my testing, single-note passages were accurately registered about 85-90% of the time on an acoustic piano, but chord recognition was less reliable in noisy rooms

The major differentiator is five instruments (guitar, piano, ukulele, bass, and voice) under one subscription

The trade-off lies in pedagogical depth: the scrolling-note interface enhances reflexes more than music literacy. Users who wish to read sheet music or grasp theory will ultimately need to complement their learning with a more structured tool.

The proprietary MusicSense acoustic engine listens via your device's microphone and performed well with single-note melodies in my testing. However, fast polyphonic passages and background noise caused occasional misreads (MIDI input avoids these issues).

The audio recognition on my acoustic piano performed well for two-hand passages, while the MIDI input was even more accurate. Skoove also has hardware partnerships with Roland, Kawai, and Alesis. If you purchase a qualifying keyboard, you'll receive several months of free Premium access, similar to the Flowkey-Yamaha deal. Skoove is available on both iOS and Android platforms.

The curriculum follows a method-book progression (right hand, left hand, both hands theory), but unlike a static book, it waits when you slow down and highlights errors so you can self-correct. This approach helps kids develop true musical literacy—such as reading notes, understanding rhythm, and honing technique—rather than depending on gamification rewards, giving young learners skills that transfer to real-world playing.

Skoove is the best piano learning app for beginners—both adults and kids—who want to develop true musical literacy rather than just following colored dots.

I also analyzed over 1,000 user reviews from the App Store, Google Play, and Reddit. I spent hours reading through existing comparison articles and material about these apps to identify which features and pricing claims were consistent across sources.

As a bit of a geek myself, I'd point to far more interesting things: their adaptive learning algorithms, their efficiency in audio signal processing, and how they translate raw microphone or MIDI input into meaningful, real-time feedback. Think about it: some of these apps have worked really hard to nail note detection in a pretty remarkable way. It is not easy to detect an I-V-vi-IV chord progression while the neighbor's dog is barking, your partner is shouting at you to stop and come eat, or your kid is in the next room singing Baby Shark on repeat.

How real-time audio recognition, adaptive algorithms, and machine learning are transforming adult piano learning, and which app does it best.

eLife Assessment

Muetter et al. provide an important argument that luminescence is a reliable, high-throughput alternative to colony-forming units (CFU) for super-MIC investigations, particularly when the quantity of interest is biomass. By examining 20 antimicrobials spanning 11 classes, the work shows that discrepancies between CFU and luminescence are often biological (filamentation, Viable But Not Culturable). The work provides a compelling view of how these three common measurements (luminescence, optical density, and CFU) relate to one another across a range of drug treatments, although testing on clinical isolates could be of further benefit.

Reviewer #1 (Public review):

Summary:

This study examines how luminescence can be used to measure bacterial population dynamics during antimicrobial treatment by comparing it directly with optical density and colony counts. The authors aim to determine when luminescence reflects changes in population size and when it instead captures metabolic or physiological states induced by drug exposure. By generating parallel datasets under controlled conditions, the work provides a detailed view of how these three common measurements relate to one another across a range of drug treatments.

Strengths:

The study is technically strong and thoughtfully designed. Measuring luminescence, optical density, and colony counts from the same cultures allows the authors to make clear and informative comparisons between methods. The data are compelling, and the analyses highlight both agreements and divergences in a way that is easy to interpret. The manuscript also succeeds in showing why these divergences arise. For example, the observation that filamentation and metabolic shifts can sustain luminescence even when colony counts drop provides valuable information on how different readouts capture distinct aspects of bacterial physiology. The writing is clear, the figures are effective, and the work will be useful for researchers who need high-throughput approaches to quantify microbial population dynamics experimentally.

Weaknesses:

The study also exposes some inherent limitations of luminescence-based measurements. Because luminescence depends on metabolic activity, it can remain high when cells are damaged or unable to resume growth, and it can fall quickly when drugs disrupt energy production, even if cells remain physically intact. These properties complicate interpretation in conditions that induce strong stress responses or heterogeneous survival states. In addition, the use of drug-free plates for colony counts may overestimate survival when filamented or stressed cells recover once the antibiotic is removed, making differences between luminescence and colony counts harder to attribute to killing alone. Finally, while the authors discuss luminescence in the context of clinically relevant concentration ranges, the current implementation relies on engineered laboratory strains and does not directly demonstrate applicability to clinical isolates. These limitations do not detract from the technical value of the work but should be kept in mind by readers who wish to apply the method more broadly.

Reviewer #2 (Public review):

Summary:

This preprint proposes luxCDABE-based luminescence as a high-throughput alternative (or complement) to CFU time-kill assays for estimating antimicrobial rates of population change at super-MIC concentrations, by comparing luminescence- and CFU-derived rates across 20 antimicrobials (22 assays) and attributing divergences primarily to filamentation (luminescence closer to biomass/volume than cell number) and changes in culturability/carryover (CFU undercounting viable cells).

Strengths:

The authors do not merely report discrepancies; they experimentally validate the biological causes. Specifically, they successfully attribute the slower decline of luminescence in certain drugs to bacterial filamentation (maintaining biomass despite halted division) and the rapid decline of CFU in others to loss of culturability or carryover effects.

The inclusion of 20 antimicrobials spanning 11 classes provides a robust dataset that allows for broad categorization of drug-specific assay behaviors.

The study critically exposes flaws in the "gold standard" CFU method, specifically regarding antimicrobial carryover (demonstrated with pexiganan) and the potential for CFU to overestimate cell death in the presence of VBNC (viable but non-culturable) states induced by drugs like ciprofloxacin.

The use of chromosomal integration for the lux operon to minimize plasmid copy-number effects and the validation of linearity between light intensity and cell density establish a solid technical foundation.

Weaknesses:

The study is conducted exclusively using Escherichia coli. While E. coli is a standard model organism, the paper claims to evaluate luminescence as a generalizable high-throughput tool. Many of the discrepancies observed are driven by filamentation. However, distinct morphological responses occur in other critical pathogens (e.g., Staphylococcus aureus does not filament in the same way).

The authors propose that luminescence data can be corrected using microscopy-derived volume data to better align with CFU counts. The primary appeal of luminescence is high-throughput efficiency. If a researcher must perform time-lapse microscopy to calculate cell volume changes to "correct" their luminescence data, the high-throughput advantage is lost.

The paper argues that for ciprofloxacin, CFU underestimates viability because cells remain intact and impermeable to propidium iodide. While the cells are metabolically active and membrane-intact, if they cannot divide to form a colony (even after drug removal/dilution), their clinical relevance as "living" pathogens is debatable.

Some other comments:

The use of a population dynamical model to simulate filamentation effects is excellent. The finding that light intensity tracks volume ($\psi_V$) better than cell number ($\psi_B$) is a key theoretical contribution.

The model assumes linear elongation. The authors should briefly comment on whether this holds true for the specific drug mechanisms tested (e.g., PBP inhibition vs. DNA gyrase inhibition).

The use of bootstrapping to estimate rate distributions is appropriate and robust.

Conclusion:

Muetter et al. provide a compelling argument that luminescence is a reliable, high-throughput alternative to CFU for super-MIC investigations, particularly when the quantity of interest is biomass. The paper effectively warns researchers that discrepancies between CFU and luminescence are often biological (filamentation, VBNC) rather than methodological failures.

Author response:

We are grateful for the effort and time invested in reviewing our manuscript. We find the comments and suggestions very helpful for improving the manuscript, and we will address them in a revised submission.

https://doi.org/10.1002/pmrj.13240 https://features.propublica.org/diabetes-amputations/black-american-amputation-epidemic/ https://www.mdpi.com/2673-1592/6/6/99#:~:text=The%20blue%2Dlight%20scanner%20achieved,with%20the%20use%20of%20markers

Note the impact shall be different depending on the game! In social games, take VR chat, or Second Lind, perhaps even The Sims, this is much more prominent. These aren't examined. Games like Minecraft allow more than parametric customisation, they have mods and skins... and in this sense, ethnographies on game worlds, will be exemplatory, but also limited by these constraints. Sure, white is a terrible default... but we shouldn't ask indie devs to add perfect customisation settings when their games lack basic accessibility features like high contrast or text read-aloud.

eLife Assessment

This valuable study examined how sensory adaptation supports visual perception in the presence of noise. The authors used a combination of human psychophysics, electroencephalography (EEG), and deep neural networks to show that adaptation to noise can improve perception. The results are solid but are, at present, weakened by a number of concerns, including some related to the experimental design and some regarding the interpretation of the results in terms of particular mechanisms. With these concerns adequately addressed, the study and conclusions would be likely to be of broad interest to the neuroscience community.

Reviewer #1 (Public review):

The authors sought to investigate the role of adaptation in supporting object recognition. In particular, the extent to which adaptation to noise improves subsequent recognition of objects embedded in the same or similar noise, and how this interacts with target contrast. The authors approach this question using a combination of psychophysics, electroencephalography, and deep neural networks. They find better behavioural performance and multivariate decoding of stimuli preceded by noise, suggesting a beneficial effect of adaptation to noise. The neural network analysis seeks to provide a deeper explanation of the results by comparing how well different adaptation mechanisms capture the empirical behavioural results. The results show that models incorporating intrinsic adaptation mechanisms, such as additive suppression and divisive normalisation, capture the behavioural results better than those that incorporate recurrent interactions. The study has the potential to provide interesting insights into adaptation, but there are alternative (arguably more parsimonious) explanations for the results that have not been refuted (or even recognised) in the manuscript. If these confounds can be compellingly addressed, then I expect the results would be of interest to a broad range of readers.

The study uses a multi-modal approach, which provides a rich characterisation of the phenomenon. The methods are described clearly, and the accompanying code and data are made publicly available. The comparison between univariate and multivariate analyses is interesting, and the application of neural networks to distinguish between different models of adaptation seems quite promising.

There are several concerning confounding factors that need to be addressed before the results can be meaningfully interpreted. In particular, differences in behavioural accuracy may be explained by a simple change detection mechanism in the "same noise" condition, and temporal cuing by the "adaptor" stimulus may explain differences in reaction time. Similarly, interference between event-related potentials may explain the univariate EEG results, and biased decoder training may explain the multivariate results. Thus, it is currently unclear if any of the results reflect adaptation.

My main concerns relate to how adaptation is induced and how differences between conditions are interpreted. The adaptation period is only 1.5 s. Although brief adaptors (~1 s) can produce stimulus history effects, it is unclear whether these reflect the same mechanisms as those observed with standard, longer adaptation durations (e.g., 10-30 s). Prior EEG work on visual adaptation using longer adaptors has shown that feature-specific effects emerge very early (<100 ms) after test onset in both univariate and multivariate responses (Rideaux et al., 2023, PNAS). In contrast, the present study finds no difference between same and different adaptor conditions until much later (>300 ms). These later effects likely reflect cognitive processes such as template matching or decision-making, rather than sensory adaptation. Although early differences appear between blank and adaptor conditions, these could be explained by interactions between ERPs elicited by adaptor onset/offset and those elicited by the test stimulus; therefore, they cannot be attributed to adaptation. This contradicts the statement in the Discussion that "Our EEG measurements show clear evidence of repetition suppression, in the form of reduced responses to the repeated noise pattern early in time."

A second concern is the brief inter-stimulus interval. The adaptor is shown for 1.5 s, followed by only a 134 ms blank before the target. When the "adaptor" and test noise are identical, improved performance could simply arise from detecting the pixels that change, namely, those forming the target number. Such change detection does not require adaptation; even simple motion detector units would suffice. If the blank period were longer-beyond the temporal window of motion detectors-then improved performance would more convincingly reflect adaptation. Given the very short blank, however, a more parsimonious explanation for the behavioural effect in the same-noise condition is that change detection mechanisms isolate the target.

Differences between the blank and adaptor conditions may also be explained by temporal cueing. In the noise conditions, the noise reliably signals the upcoming target time, whereas the blank condition provides no such cue. Given the variable inter-trial interval and the brief target presentation, this temporal cue would strongly facilitate target perception. This account is consistent with the reaction time results: both adaptor conditions produce faster reaction times than the blank condition, but do not differ from each other.

The decoding analyses are also difficult to interpret, given the training-testing protocol. All trials from the three main conditions (blank, same, different) were used to train the classifier, and then held-out trials - all from one condition-were decoded. Because ERPs in the adaptor conditions differ substantially from those in the blank condition, and because there are twice as many adaptor trials, the classifier is biased toward patterns from the adaptor conditions and will naturally perform worse on blank trials. To compare decoding accuracy meaningfully across conditions, the classifier should be trained on a separate unbiased dataset (e.g., the "clean" data), or each condition should be trained and tested separately using cross-fold validation.

Reviewer #2 (Public review):

Summary:

Neurons adapt to prolonged or repeated sensory inputs. One function of such adaptation may be to save resources to avoid representing the same inputs over and over again. However, it has been hypothesized that adaptation could additionally help improve the representation of sensory stimuli, especially during difficult recognition scenarios. This study sheds light on this question and provides behavioral evidence for such enhancement. The behavioral results are interesting and compelling. The paper also includes scalp electroencephalographic (EEG) data, which are noisy but point toward similar conclusions. The authors finally implement a deep convolutional neural network (DCNN) with adaptation mechanisms, which nicely capture human behavior.

Strengths:

(1) The authors introduce an interesting hypothesis about the role of adaptation in visual recognition.

(2) The authors present interesting and compelling behavioral data consistent with the hypothesis.

(3) The authors introduce a computational model that can capture mechanisms that can lead to adaptation, enhancing visual recognition.

Weaknesses:

(1) The main weakness is the scalp EEG data. As detailed below, the results are minimal at best and do not contribute to understanding the mechanisms of adaptation. The paper would be stronger without the EEG data.

(2) I wonder whether the hypothesis also holds with real-world objects in natural scenes, beyond the confines of MNIST digits.

Reviewer #3 (Public review):

Summary:

Brands and colleagues investigate how temporal adaptation can aid object recognition, and what neural computations may underlie these effects. They employed a previously published experimental paradigm to study how adaptation to temporally constant distractor input facilitates the recognition of a newly appearing target object. Specifically, they studied how this effect is modulated by the contrast of the target object.

They found that adaptation enhances the recognition of high-contrast objects more than that of low-contrast objects. This behavioral effect was mirrored by a larger effect of adaptation on the response to the high-contrast objects in relatively higher visual areas.

To investigate what neural computations can support this interaction, they implement several candidate neural mechanisms in a deep convolutional neural network: additive suppression, divisive suppression, and lateral recurrence. The authors conclude that divisive and additive suppression, which are intrinsic to the neuron, best explain the interaction between contrast and adaptation in the human data. They further show that these mechanisms, and divisive suppression in particular, show increased robustness to spatial shifts of the adaptor stimulus, hinting and potential perceptual benefits.

Strengths:

(1) Overall, this is a well-written paper, supported by thorough analyses and illustrated with clear, well-designed figures that effectively show overall trends as well as data variance. The authors tell a compelling story while responsibly steering away from overreaching conclusions.

(2) What makes this paper stand out is its comprehensive approach to understanding the behavioral benefit of neural adaptation and its mechanistic underpinnings. The authors effectively achieve this through integrating new behavioral and neural data with simulations using neural network models.

(3) The findings convincingly demonstrate that neuronally intrinsic adaptation mechanisms are sufficient to explain the observed interaction between temporal adaptation, contrast, and object recognition. Furthermore, the paper highlights that these intrinsic mechanisms offer superior robustness compared to learned lateral recurrence mechanisms, which, while being more expressive, can also be more brittle.

Weaknesses:

While the results and conclusion are well supported, there were a few major points that need clarification for me.

(1) Divisive normalization

I was confused by the author's classification of divisive normalization as a neuronally intrinsic mechanism, that is, one that operates within a single neuron, independent of interactions with other neurons.

My understanding is that divisive normalization, as originally proposed by Heeger in the early nineties, describes a mechanism where neurons integrate pooled activity from neighboring cells to mutually inhibit one another. In this form, divisive normalization is fundamentally an interneuronal mechanism involving recurrence. Adding to the confusion, the authors highlight in the introduction their interest in divisive normalization for its relation to stimulus contrast, a relation likely linked to neuronal pooling.

However, my reading of the methods section (Equations 6 and 7) suggests the authors implemented only a temporal feedback component, leaving out the pooling across neurons (Equation 5). This distinction should be disambiguated early in the paper. I recommend choosing a less ambiguous term than "divisive normalization". Even "temporal divisive normalization" is still ambiguous, as lateral neuronal interactions are also inherently temporal.

(2) Parietal electrodes

The paper's adapter-specific effects are centered around the P9/P10 electrodes, which the authors identify as "parietal." However, it is unclear to me which part of the cortex drives these electrodes, particularly whether it is actually the parietal cortex. I am no expert in EEG, but based on the topomaps in Figures 4 and 5, it appears that these electrodes cover more posterior occipito-temporal regions rather than truly parietal regions. Given the central role of P9/P10 to the main findings, the paper would be significantly improved for non-EEG readers by clarifying which cortical regions are covered by these electrodes.

(3) Interpretation of non-significant statistical results

In some places, the authors attach relatively strong claims to non-significant statistical results. For example, in Figure 5D, they claim that there is no effect of contrast on occipital electrodes, based on a non-significant p-value. P-values do not quantify evidence for the null hypothesis, so the authors should be careful with such claims. In fact, Figure 5D shows such a clear negative slope, with variance comparable to Figure 5A, that I am surprised that the p-value for the slope of Figure 5D was in fact so large. A similar issue arises in the discussion for Figure 6, where the authors claim that the effect of contrast is adapter-specific. However, this claim is based on the observation that is significant for same-noise trials, but not for different-noise or blank trials. To statistically substantiate the claims that there is an adapter-specific effect, the authors should directly compare the slope for same-noise trials with the slope for different-noise/blank trials.

(4) The match between behavior and models

The authors' claim that models with intrinsic adaptation better match the interaction between contrast and temporal adaptation observed in human behavior is not fully substantiated. This conclusion appears to be based on a qualitative assessment of Figure 8, which, in my view, does not unambiguously rule out an interaction for lateral recurrence. Furthermore, a potential confounding factor is the ceiling effect that limits higher accuracy values. Indeed, conditions where the interaction was not/less (i.e., shorter time sequences and lateral inhibition) are also the conditions where accuracy values are closer to this ceiling, which may mask a potential interaction.

eLife Assessment

This study presents a valuable and well-documented computational pipeline for the scalable analysis and spike sorting of large extracellular electrophysiology datasets, with particular relevance for high-density recordings such as Neuropixels. The authors demonstrate the pipeline's utility for benchmarking spike sorter performance and evaluating the effects of data compression, supported by thorough testing, clear figures, and openly available code. The workflow is reproducible, portable, and practical, providing concrete guidance on computational cost and runtime. Overall, the evidence supporting the pipeline's performance and output quality is compelling, and this work will be of broad interest to the systems neuroscience community.

Reviewer #1 (Public review):

Summary:

Extracellular electrophysiology datasets are growing in both number and size, and recordings with thousands of sites per animal are now commonplace. Analyzing these datasets to extract the activity of single neurons (spike sorting) is challenging: signal-to-noise is low, the analysis is computationally expensive, and small changes in analysis parameters and code can alter the output. The authors address the problem of volume by packaging the well-characterized SpikeInterface pipeline in a framework that can distribute individual sorting jobs across many workers in a compute cluster or cloud environment. Reproducibility is ensured by running containerized versions of the processing components.

The authors apply the pipeline in two important examples. The first is a thorough study comparing the performance of two widely used spike-sorting algorithms (Kilosort 2.5 and Kilosort 4). They use hybrid datasets created by injecting measured spike waveforms (templates) into existing recordings, adjusting those waveforms according to the measured drift in the recording. These hybrid ground truth datasets preserve the complex noise and background of the original recording. Similar to the original Kilosort 4 paper, which uses a different method for creating ground truth datasets that include drift, the authors find Kilosort 4 significantly outperforms Kilosort 2.5. The second example measures the impact of compression of raw data on spike sorting with Kilosort 4, showing that accuracy, precision, and recall of the ground truth units are not significantly impacted even by lossy compression. As important as the individual results, these studies provide good models for measuring the impact of particular processing steps on the output of spike sorting.

Strengths:

The pipeline uses the Nextflow framework, which makes it adaptable to different job schedulers and environments. The high-level documentation is useful, and the GitHub code is well organized. The two example studies are thorough and well-designed, and address important questions in the analysis of extracellular electrophysiology data.

Weaknesses:

The pipeline is very complete, but also complex. Workflows - the optimal artifact removal, best curation for data from a particular brain area or species - will vary according to experiment. Therefore, a discussion of the adaptability of the pipeline in the "Limitations" section would be helpful for readers.

Reviewer #2 (Public review):

Summary:

This work presents a reproducible, scalable workflow for spike sorting that leverages parallelization to handle large neural recording datasets. The authors introduce both a processing pipeline and a benchmarking framework that can run across different computing environments (workstations, HPC clusters, cloud). Key findings include demonstrating that Kilosort4 outperforms Kilosort2.5 and that 7× lossy compression has minimal impact on spike sorting performance while substantially reducing storage costs.

Strengths:

(1) Extremely high-quality figures with clear captions that effectively communicate complex workflow information.

(2) Very detailed, well-written methods section providing thorough documentation.

(3) Strong focus on reproducibility, scalability, modularity, and portability using established technologies (Nextflow, SpikeInterface, Code Ocean).

(4) Pipeline publicly available on GitHub with documentation.

(5) Clear cost analysis showing ~$5/hour for AWS processing with transparent breakdown.

(6) Good overview of previous spike sorting benchmarking attempts in the introduction.

(7) Practical value for the community by lowering barriers to processing large datasets.

Weaknesses:

No significant weaknesses were identified, although it is noted that the limitations section of the discussion could be expanded.

Reviewer #3 (Public review):

Summary:

The authors provide a highly valuable and thoroughly documented pipeline to accelerate the processing and spike sorting of high-density electrophysiology data, particularly from Neuropixels probes. The scale of data collection is increasing across the field, and processing times and data storage are growing concerns. This pipeline provides parallelization and benchmarking of performance after data compression that helps address these concerns. The authors also use their pipeline to benchmark different spike sorting algorithms, providing useful evidence that Kilosort4 performs the best out of the tested options. This work, and the ability to implement this pipeline with minimal effort to standardize and speed up data processing across the field, will be of great interest to many researchers in systems neuroscience.

Strengths:

The paper is very well written and clear in most places. The accompanying GitHub and ReadTheDocs are well organized and thorough. The authors provide many benchmarking metrics to support their claims, and it is clear that the pipeline has been very thoroughly tested and optimized by users at the Allen Institute for Neural Dynamics. The pipeline incorporates existing software and platforms that have also been thoroughly tested (such as SpikeInterface), so the authors are not reinventing the wheel, but rather putting together the best of many worlds. This is a great contribution to the field, and it is clear that the authors have put a lot of thought into making the pipeline as accessible as possible.

Weaknesses:

There are no major weaknesses. I have only a handful of very minor questions and suggestions that could clarify/generalize aspects of the pipeline or make the text more understandable to non-specialists.

(1) Could the authors please expand on the statement on line 274, that processing their test dataset serially "on a single GPU-capable cloud workstation... would take approximately 75 hours and cost over 90 USD." How were these values calculated? I was a bit surprised that this is a >4-fold slow-down from their pipeline, but only increases the cost by ~1.35x, if I understood correctly. More context on why this is, and maybe some context on what a g4dn.4xlarge is compared to the other instances, might help readers who are less familiar with AWS and cloud computing.

(2) One of the most commonly used preprocessing pipelines for Neuropixels data is the CatGT/ecephys pipeline from the developers of SpikeGLX at Janelia. It may be worth commenting very briefly, either in the preprocessing section or in the discussion, on how the preprocessing steps available in this pipeline compare to the steps available in CatGT. For example, is "destriping" similar to the "-gfix" option in catGT to remove high-amplitude artifacts?

(3) Why are there duplicate units (line 194), and how often is this an issue? I understand that this is likely more of a spike sorter issue than an issue with this pipeline, but 1-2 sentences elaborating why might be helpful for readers.

(4) It seems from the parameter files on GitHub that the cluster curation parameters are customizable - correct? If so, it may be worth explicitly saying so in the curation section of the text, as the presented recipe will not always be appropriate. A presence ratio of >0.8 could be particularly problematic for some recordings, for example, if a cell is only active during a specific part of the behavior, that may be a feature of the experiment, or the animal could be transitioning between sleep and wake states, in which different units may become active at different times.

(5) The axis labels in Figures 3d-e are too small to see, and Figure 3d would benefit from a brief description of what is shown.

(6) What is the difference between "neural" and "passing QC" in Figure 4?

(7) I understand the current paper is focused on spike data, so there may not be an answer to this, but I am curious about the NP2.0 probes that save data in wideband. Does the lossy compression negatively affect the LFP data? Is software filtering applied for the spike band before or after compression?

I found it interesting that APIs create a filtered version of reality for researchers and developers. When we analyze Reddit data through the API, it’s easy to forget that we are only seeing what the platform allows us to see, not the full picture of user behavior. This makes me wonder how data mining conclusions might change if hidden data—like deleted interactions or internal metrics—were included.

This section explains how Python uses the Reddit API through the PRAW library to request and retrieve data from Reddit. It introduces APIs as rule-based systems that control what data can be accessed and how. The passage also notes that official documentation can be difficult for beginners to understand.

What stood out to me is how much of this data is collected passively rather than intentionally shared. Actions like pausing on a post or scrolling speed feel almost subconscious, yet they can reveal a lot about a user’s interests or emotional state. This raises an ethical question about whether users truly understand how much they are communicating through behavior alone, not just through what they choose to post.

This passage explains how social media platforms and advertisers track user behavior across websites to deliver targeted ads. It also points out that platforms may collect data about non-users through photos or contact lists. Overall, the text highlights privacy concerns and how personal data is used to generate profit.

eLife Assessment

This manuscript presents a novel investigation of organizational principles governing brain activity at both global and local scales during naturalistic viewing paradigms, an important advance for theoretical neuroscience, functional neuroimaging, and neurology. The authors demonstrate that brain activity during naturalistic viewing is dominated by two anti-correlated states that toggle between each other with a third transitional state mediating between them. The evidence supporting this finding is compelling, with the successful replication across three independent datasets (StudyForrest, NarrattenTion, and CamCAN) a particular strength.

Reviewer #1 (Public review):

In this work, the authors provide a comprehensive investigation of antagonistic dynamics across large-scale brain networks. They characterize this phenomenon at the global (regional dynamics) and local (multivariate patterns of voxels within regions) levels.

Furthermore, as opposed to studying these dynamics under resting-state or explicit task conditions, the authors make use of naturalistic narratives, both auditory and visual.

Perhaps most importantly, this work provides evidence that event boundaries in narratives drive sensory responses, which, in turn, predict anticorrelated activity in task-positive networks and the default mode network. These findings open up new questions regarding the interaction across perceptual systems and these higher-order dynamics in association networks.

This work is methodologically solid and presents compelling findings that will surely invite new approaches and questions in this area.

Importantly, these data do not speak to the order or causal structure of these interactions. Time-resolved methods and direct causal interventions will be needed to understand how these interactions drive one another more precisely.

Reviewer #2 (Public review):

This manuscript presents an impressive and novel investigation of organizational principles governing brain activity at both global and local scales during naturalistic viewing paradigms. The proposed multi-scale nested structure offers valuable new insights into functional brain states and their dynamics. Importantly, investigation of global brain states in the context of a naturalistic viewing context represents an important and timely contribution that addresses unresolved issues about global signals and anticorrelations in resting-state fMRI. This manuscript presents a novel investigation of organizational principles governing brain activity at both global and local scales during naturalistic viewing paradigms. The authors demonstrate that brain activity during naturalistic viewing is dominated by two anti-correlated states that toggle between each other with a third transitional state mediating between them. The successful replication across three independent datasets (StudyForrest, NarrattenTion, and CamCAN) is a particular strength. The successful replication across three independent datasets (StudyForrest, NarrattenTion, and CamCAN) is a particular strength, and I appreciate the authors' careful documentation of both convergent and divergent findings across these samples.

Overall, this manuscript makes important contributions to our understanding of large-scale brain organization during naturalistic cognition. The multi-scale framework and robust replication across datasets are notable strengths. Addressing the concerns raised below will substantially strengthen the impact and interpretability of this work.

(1) Network Definition and Specificity

(a) The authors adopt an overly broad characterization of the Default Mode Network (DMN). The statement that "areas most active in the default mode state... consist of the precuneus, angular gyrus, large parts of the superior and middle temporal cortex, large parts of the somatomotor areas, frontal operculi, insula, parts of the prefrontal cortex and limbic areas" includes regions typically assigned to other networks. The insula is canonically considered a core node of the Salience Network/Ventral Attention Network (VAN), not the DMN. Also, not clear which limbic areas? The DMN findings reported need to be critically reassessed in this context.

(b) Given the proposed role of state switching in your framework, a detailed analysis of salience network nodes (particularly insula and dorsal ACC) would be highly informative.

(c) While you report transition-related signals in the visual and auditory cortex, the involvement of insular and frontal control systems in state transitions remains unaddressed.

(d) My recommendation is to provide a more anatomically precise characterization of network involvement, particularly distinguishing DMN from salience/VAN regions, and analyze the specific role of salience network nodes in mediating state transitions.

(2) Distinguishing Top-Down from Stimulus-Driven Effects

(a) The finding that "the superior parietal lobe (SPL) and the frontal eye fields (FEF) show the greatest overlap between their local ROI state switches and the global state switches" raises an important question: To what extent are these effects driven by overt changes in visual gaze or attention shifts triggered by stimulus features versus internally-generated state changes?

(b) Similarly, the observation that DAN areas show the highest overlap with global state changes in StudyForrest and NarrattenTion, while VAN shows the highest overlap in CamCAN, lacks sufficient anatomical detail regarding which specific nodes are involved. This information would help clarify whether insular regions and other VAN components play distinct roles in state switching.

(c) It will be important to (i) discuss potential confounds from eye movements and stimulus-driven attention shifts; (ii) provide detailed anatomical breakdowns of network nodes involved in state transitions, particularly for VAN; (iii) if eye-tracking data or any other relevant stimulus-related data are available, include analyses examining relationships between these measures and state transitions.

(3) Physiological Interpretation of the "Down" State

The linkage between the "Down" state and the Default Mode State (DMS) is intriguing but requires deeper physiological grounding. Recent work by Epp et al. (Nature Neuroscience, 2025) demonstrates that decreased BOLD signal in DMN regions does not necessarily indicate reduced metabolic activity and can reflect neurovascular coupling modes with specific metabolic profiles. It would be useful to discuss whether your "Down" state might represent a particular neurovascular coupling mode with distinct metabolic demands rather than simply reduced neural activity. Alternatively, your analytical approach might be insensitive to or unconfounded by such neurovascular uncoupling. This discussion would substantially enrich the biological interpretation of the DMS versus TPS dual mechanism framework.

(4) Statistical Validation of Bimodality Detection

The method of selecting bimodal timepoints using the Dip test followed by sign-alignment is novel and creative. However, this filter-then-align procedure could potentially introduce circularity by imposing the anticorrelated structure the authors aim to detect. It would be important to implement validation analyses to confirm that anticorrelation is an intrinsic property rather than a methodological artifact. Approaches include leave-one-subject-out cross-validation, unsupervised dimensionality reduction (e.g., PCA) applied independently to verify the anticorrelated structure, and split-half reliability analysis. Such validation would significantly strengthen the statistical foundation of findings.

(5) Quantifying Hyperalignment Contribution

The appendix notes that non-hyperaligned data show a coarser structure, but the specific contribution of hyperalignment to your findings requires more thorough quantification. Please provide a systematic comparison of results with and without hyperalignment, demonstrating that similar (even if weaker) anatomical correspondence exists in native subject space. This would establish that the mesoscale organizational principles you identify are not artifacts of the alignment procedure but reflect genuine neurobiological organization. Consider presenting correlation coefficients or overlap metrics quantifying the similarity of state structures before and after hyperalignment.

(6) Functional Characterization of the Unimodal State

The observation that the brain spends approximately 34% of its time in a "Unimodal State" is presented primarily as a transition period. This is an interesting observation. However, it would be useful to characterize the functional connectivity profile of the unimodal state. Specifically, investigate whether it represents a distinct functional state with its own characteristic connectivity pattern. More detailed analysis would provide a more complete picture of temporal brain dynamics during naturalistic viewing and could yield new perspectives on how the brain reorganizes between stable states.

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Reply to the reviewers

We sincerely appreciate the feedback, attention to detail and timeliness of the referees for our manuscript. Below, we provide a point-by-point response to all comments from the referees, detailing the changes we have already made, and those that are in progress. Referee's comments will appear in bolded text, while our responses will be unbolded. Any text quoted directly from the manuscript will be italicised and contained within "quotation marks". Additionally, we have grouped all comments into four categories (structural changes, minor text changes, experimental changes, figure changes), comments are numbered 1-n in each of these categories. Please note: this response to reviewer's comments included some images that cannot be embedded in this text-only section.

1. General Statements

We appreciate the overall highly positive and enthusiastic comments from all reviewers, who clearly appreciated the technical difficulty of this study, and noted amongst other things that this study represents" a major contribution to the future advancement of oocyst-sporozoite biology" and the development of the segmentation score for oocysts as a "major advance[ment]". We apologise for the omission of line numbers on the document sent to reviewers, we removed these for the bioRxiv submission without considering that this PDF would be transferred across to Review Commons.

We have responded to all reviewers comments through a variety of text changes, experimental inclusions, or direct query response. Significant changes to the manuscript since initial submission are as follows:

1. Refinement of rhoptry biogenesis model: Reviewers requested more detail around the content of the AORs, which we had previously suggested were a vehicle for rhoptry biogenesis as we saw they carried the rhoptry neck protein RON4. To address this, we first attempted to address this using antibodies against rhoptry bulb proteins but were unsuccessful. We then developed a * berghei* line where there rhoptry bulb protein RhopH3 was GFP-tagged. Using this parasite line, we observed that the earliest rhoptry-like structure, which we had previously interpreted as an AOR contained RhopH3. By contrast, RhopH3 was absent from AORs. Reflecting these observations we have renamed this initial structure the 'pre-rhoptry' and suggested a model for rhoptry biogenesis where rhoptry neck cargo are trafficked via the AOR but rhoptry bulb cargo are trafficked by small vesicles that move along the rootlet fibre (previously observed by EM).
2. Measurement of rhoptry neck vs bulb: While not directly suggested by the reviewers, we have also included an analysis that estimates the proportion of the sporozoite rhoptry that represents the rhoptry neck. By contrast to merozoites, which we show are overwhelmingly represented by the rhoptry bulb, the vast majority of the sporozoite rhoptry represents the rhoptry neck.
3. Measurement of subpellicular microtubules: One reviewer asked if we could measure the length of subpellicular microtubules where we had previously observed that they were longer on one side of the sporozoite than the other. We have now provided absolute and relative (% sporozoite length) length measurements for these subpellicular microtubules and also calculated the proportion of the microtubule that is polyglutamylated.
4. More detailed analysis of RON11cKD rhoptries: Multiple comments suggested a more detailed analysis of the rhoptries that were formed/not formed in RON11cKD We have included an updated analysis that shows the relative position of these rhoptries in sporozoites.

2. Point-by-point description of the revisions

Reviewer #1

Minor text changes (Reviewer #1)

1. __Text on page 12 could be condensed to highlight the new data of ron4 staining of the AOR. __

We agree with the reviewer that it is a reasonable suggestion. After obtaining additional data on the contents of the AOR (as described in General Statements #1), this section has been significantly rewritten to highlight these findings. 2.

__Add reference on page 3 after 'disrupted parasites' __

This sentence has been rewritten slightly with some references included and now reads: "Most data on these processes comes from electron microscopy studies 6-8, with relatively few functional reports on gene deleted or disrupted parasites9-11. 3.

__Change 'the basal complex at the leading edge' - this seems counterintuitive __

This change has been made. 4.

__Change 'mechanisms underlying SG are poorly' - what mechanisms? of invasion or infection? __

This was supposed to read "SG invasion" and has now been fixed. 5.

__On page 4: 'handful of proteins' __

This error has been corrected. 6.

__What are the 'three microtubule spindle structures'? __

The three microtubule spindle structures: hemispindle, mitotic spindle, and interpolar spindle are now listed explicitly in the text. 7.

__On page 5: 'little is known' - please describe what is known, also in other stages. At the end of the paper I would like to know what is the key difference to rhoptry function in other stages? __

The following sentence already detailed that we had recently used U-ExM to visualise rhoptry biogenesis in blood-stage parasites, but the following two sentences have been added to provide extra detail on these findings: "In that study, we defined the timing of rhoptry biogenesis showing that it begun prior to cytokinesis and completed approximate coincident with the final round of mitosis. Additionally, we observed that rhoptry duplication and inheritance was coupled with centriolar plaque duplication and nuclear fission." 8.

__change 'rhoptries golgi-derived, made de novo' __

This has been fixed. 9.

__change 'new understand to' __

This change has been made 10.

__'rhoptry malformations' seem to be similar in sporozoites and merozoites. Is that surprising/new? __

We assume this is in reference to mention of "rhoptry malformations" in the abstract. In the RON11 merozoite study (PMID:39292724) the authors noted no gross rhoptry malformations, only that one was not formed/missing. The abstract sentence has been changed to the following to better reflect this nuance: "*We show that stage-specific disruption of RON11 leads to a formation of sporozoites that only contain half the number of rhoptries of controls like in merozoites, however unlike in merozoites the majority of rhoptries appear grossly malformed."

* 11.

__What is known about crossing the basal lamina. Where rhoptries thought to be involved in this process? Or is it proteins on the surface or in other secretory organelles? __

We are unaware of any studies that specifically look at sporozoites crossing the SG basal lamina. A review, although now ~15 years old stated that "No information is available as to how the sporozoites traverse the basal lamina" (PMID:19608457) and we don't know any more information since then. To try and better define our understanding of rhoptry secretion during SG invasion, we have added the following sentence:

"It is currently unclear precisely when during these steps of SG invasion rhoptry proteins are required, but rhoptry secretion is thought to begin before in the haemolymph before SG invasion16." 12.

__On page change/specify: 'wide range of parasite structures' __

The structures observed have been listed: centriolar plaque, rhoptry, apical polar rings, rootlet fibre, basal complex, apicoplast. 13.

__On page 7: is Airyscan2 a particular method or a specific microscope? __

Airyscan2 is a detector setup on Zeiss LSM microscopes, this was already detailed in the materials and methods sections, but figure legends have been clarified to read: "...imaged by an LSM900 microscopy with an Airyscan2 detector". 14.

__how large is RON11? __

RON11 is 112 kDa in * berghei*, as noted in the text. 15.

__There is no causal link between ookinete invasion and oocyst developmental asynchrony __

We have deleted the sentence that implied that ookinete invasion was responsible for oocyst asynchrony. This section now simply states that "Development of each oocyst within a midgut is asynchronous..." 16.

__First sentence of page 24 appears to contradict what is written in results____ I don't understand the first two sentences in the paragraph titled Comparison between Plasmodium spp __

This sentence was worded confusingly, making it appear contradictory when that was not the intention. The sentence has been changed to more clearly support what is written in the discussion and now reads: "Our extensive analysis only found one additional ultrastructural difference between Plasmodium spp."

__On page 25 or before the vast number of electron microscopy studies should be discussed and compared with the authors new data. __

It is not entirely clear which new data should be specifically discussed based on this comment. However, we have added a new paragraph that broadly compares MoTissU-ExM and our findings with other imaging methods previously used on mosquito-stage malaria parasites:

"*Comparison of MoTissU-ExM and other imaging modalities

Prior to the development of MoTissU-ExM, imaging of mosquito-stage malaria parasites in situ had been performed using electron microscopy7,8,11,28, conventional immunofluorescence assays (IFA)10, and live-cell microscopy25. MoTissU-ExM offers significant advantages over electron microscopy techniques, especially volume electron microscopy, in terms of accessibility, throughput, and detection of multiple targets. While we have benchmarked many of our observations against previous electron microscopy studies, the intracellular detail that can be observed by MoTissU-ExM is not as clear as electron microscopy. For example, previous electron microscopy studies have observed Golgi-derived vesicles trafficking along the rootlet fibre8 and distinguished the apical polar rings44; both of which we could not observe using MoTissU-ExM. Compared to conventional IFA, MoTissU-ExM dramatically improves the number and detail of parasite structures/organelles that can be visualised while maintaining the flexibility of target detection. By contrast, it can be difficult or impossible to reliably quantify fluorescence intensity in samples prepared by expansion microscopy, something that is routine for conventional IFA. For studying temporally complex processes, live-cell microscopy is the 'gold-standard' and there are some processes that fundamentally cannot be studied or observed in fixed cells. We attempt to increase the utility of MoTissU-ExM in discerning temporal relationships through the development of the segmentation score but note that this cannot be applied to the majority of oocyst development. Collectively, MoTissU-ExM offers some benefits over these previously applied techniques but does not replace them and instead serves as a novel and complementary tool in studying the cell biology of mosquito-stage malaria parasites.**"

*

__First sentence on page 27: there are many studies on parasite proteins involved in salivary gland invasion that could be mentioned/discussed. __

The sentence in question is "To the best of our knowledge, the ability of sporozoites to cross the basal lamina and accumulate in the SG intercellular space has never previously been reported."

This sentence has now been changed to read as follows: "While numerous studies have characterized proteins whose disruption inhibited SG invasion9,10,15,59-63, to the best of our knowledge the ability of sporozoites to cross the basal lamina and accumulate in the SG intercellular space has never previously been reported ."

__On page 10 I suggest to qualify the statement 'oocyst development has typcially been inferred by'. There seem a few studies that show that size doesn't reflect maturation. __

In our opinion, this statement is already qualified in the following sentence which reads: "Recent studies have shown that while oocysts increase in size initially, their size eventually plateaus (11 days pot infection (dpi) in P. falciparum4)."

__On page 16 the authors state that different rhoptries might have different function. This is an interesting hypothesis/result that could be mentioned in the abstract. __

The abstract already contains the following statement: "...and provide the first evidence that rhoptry pairs are specialised for different invasion events." We see this as an equivalent statement.

Experimental changes (Reviewer #1)

1. On page 19: do the parasites with the RON11 knockout only have the cytoplasmic or only the apical rhoptries?

The answer to this is not completely clear. We have added the following data to Figures 6 and 8 where we quantify the proportion of rhoptries that are either apical or cytoplasmic: In both wildtype parasites and RON11ctrl parasites, oocyst spz rhoptries are roughly 50:50 apical:cytoplasmic (with a small but consistent majority apical), while almost all rhoptries are found at the apical end (>90%) in SG spz. Presumably, after the initial apical rhoptries are 'used up' during SG invasion, the rhoptries that were previously cytoplasmic take their place. In RON11cKD the ratio of apical:cytoplasmic rhoptries is fairly similar to control oocyst spz. In RON11cKD SG spz, the proportion of cytoplasmic rhoptries decreases but not to the same extent as in wildtype or RON11Ctrl. From this, we infer that the two rhoptries that are lost/not made in RON11cKD sporozoites are likely a combination of both the apical and cytoplasmic rhoptries we find in control sporozoites.

__in panel G: Are the dense granules not micronemes? What are the dark lines? Rhoptries?? __

We have labelled all of Figure 1 more clearly to point out that the 'dark lines' are indeed rhoptries. Additionally, we have renamed the 'protein-dense granules' to 'protein-rich granules', as it seems we are suggesting that these structures are dense granules the secretory organelle. At this stage we simply do not know what all of these granules are. The observation that some but not all of these granules contain CSP (Supplementary Figure 2) suggests that they may represent heterogenous structures. It is indeed possible that some are micronemes, however, we think it is unlikely that they are all micronemes for a number of reasons: (1) micronemes are not nearly this protein dense in other Plasmodium lifecycle stages, (2) some of them carry CSP which has not been demonstrated to be micronemal, (3) very few of these granules are present in SG sporozoites, which would be unexpected because microneme secretion is required for hepatocyte invasion.

__Figure 2 seems to add little extra compared to the following figures and could in my view go to the supplement. __

We agree that Figure 2b adds little and so have moved that to Supplementary Figure 2, but think that the relative ease at which it can be distinguished if sporozoites are in the secretory cavity or SG epithelial cell is a key observation because of the difficulty in doing this by conventional IFA.

__On page 8 the authors mention a second layer of CSP but do not further investigate it. It is likely hard to investigate this further but to just let it stand as it is seems unsatisfactory, considering that CSP is the malaria vaccine. What happens if you add anti-CSP antibodies? I would suggest to shorten the opening paragraphs of this paper and to focus on the rhoptries. This could be done be toning down the text on all aspects that are not rhoptries and point to the open question some of the observations such as the CSP layers raise for future studies. __

When writing the manuscript, we were unsure whether to include this data at all as it is a purely incidental finding. We had no intention of investigating CSP specifically, but anti-CSP antibodies were included in most of the salivary gland imaging experiments so we could more easily find sporozoites. Given the tremendous importance of CSP to the field, we figured that these observations were potentially important enough that they should be reported in the literature even though they are not something we have the intention or resources to investigate subsequently. Additionally, after consultation with other microscopists we think there is a reasonable chance that this double-layer effect could be a product of chemical fixation. To account for this, we have qualified the paragraph on CSP with this sentence:

"We cannot determine if there is any functional significance of this second CSP layer and considering that it has not been observed previously it may well represent an artefact of chemical (paraformaldehyde) fixation."

__Maybe include more detail of the differences between species on rhoptry structure into Figure 4. I would encourage to move the Data on rhoptries in Figure S6 to the main text ie to Figure 4. __

We have moved the images of developing rhoptries in * falciparum *(previously Figure S6a and b) into figure 4, which now looks as follows:

Figure S8 (previously S6c) now consists only of the MG spz rhoptry quantification

Manuscript structural changes (Reviewer #1)

1. Abstract: don't focus on technique but on the questions you tried to answer (ie rewrite or delete the 3rd and 4th sentence)

2. 'range of cell biology processes' - I understand the paper that the key discovery concerns rhoptry biogenesis and function, so focus on that, all other aspects appear rather peripheral.

3. 'Much of this study focuses on the secretory organelles': I would suggest to rewrite the intro to focus solely on those, which yield interesting findings.

4. Page 11: I am tempted to suggest the authors start their study with Figure 3 and add panel A from Figure 2 to it. This leads directly to their nice work on rhoptries. Other features reported in Figures 1 and 2 are comparatively less exciting and could be moved to the supplement or reported in a separate study.____ Page 23: I suggest to delete the first sentence and focus on the functional aspects and the discoveries.

5. __Maybe add a conclusion section rather than a future application section, which reads as if you want to promoted the use of ultrastructure expansion microscopy. To my taste the technological advance is a bit overplayed considering the many applications of this techniques over the last years, especially in parasitology, where it seems widely used. In any case, please delete 'extraordinarily' __

Response to Reviewer#1 manuscript structural changes 1-5: This reviewer considers the findings related to rhoptry biology as the most significant aspect of the study and suggests rewriting the manuscript to emphasize these findings specifically. Doing so might make the key findings easier to interpret. However, in our view, this approach could misrepresent how the study originated and what we see as the most important outcomes. We did not develop MoTissU-ExM specifically to investigate rhoptry biology. Instead, this technique was created independently of any particular biological question, and once established, we asked what questions it could answer, using rhoptry biology as a proof of concept. Given the authors' previous work and available resources, we chose to focus on rhoptry biology. Since this was driven by basic research rather than a specific hypothesis, it's important to acknowledge this in the manuscript. While we agree that the findings related to rhoptry biology are valuable, we believe that highlighting the technique's ability to observe organelles, structures, and phenotypes with unprecedented ease and detail is more important than emphasizing the rhoptry findings alone. For these reasons, we have decided not to restructure the manuscript as suggested.

Reviewer #2

Minor text changes (Reviewer #2)

1. __The 'image Z-depth' value indicated in the figures is ambiguous. It is not clear whether this refers to the distance from the coverslip surface or the starting point of the z-stack image acquisition. A precise definition of this parameter would be beneficial. __

In the legend of Figure 1, the image Z-depth has been clarified as "sum distance of Z-slices in max intensity projection". 2.

__Paragraph 3 of the introduction - line 7, "handful or proteins" should be handful of proteins __

This has been corrected. 3.

__Paragraph 5 of the introduction - line 7, "also able to observed" should be observe __

This has been changed. 4.

__In the final paragraph of the introduction - line 1, "leverage this new understand" should be understanding __

This has been fixed. 5.

__The first paragraph of the discussion summary contains an incomplete sentence on line 7, "PbRON11ctrl-infected SGs." __

This has been removed. 6.

__The second paragraph of the discussion - line 10, "until cytokinesis beings" should be begins __

This mistake has been corrected. 7.

__One minor point that author suggest that oocyst diameter is not appropriate for the development of sporozoite develop. This is not so true as oocyst diameter tells between cell division and cell growth so it is important parameter especially where the proliferation with oocyst does not take place but the growth of oocyst takes place. __

We agree that this was not highlighted enough in the text. The final sentence of the results section about this now reads:

"While diameter is a useful readout for oocyst development in the early stages of its growth, this suggests that diameter is a poor readout for oocyst development once sporozoite formation has begun and highlights the usefulness of the segmentation score as an alternative.", and the final sentence of the discussion section about this now reads "Considering that oocyst size does not plateau until cytokinesis begins4, measuring oocyst diameter may represent a useful biological clock specifically when investigating the early stages of oocyst development." 8.

__How is the apical polarity different to merozoite as some conoid genes are present in ookinete and sporozoite but not in merozoite. __

Our hypothesis is that apical polarity is established by the positioning and attachment of the centriolar plaque to the parasite plasma membrane in both forming merozoites and sporozoites. While the apical polar ring proteins are obviously present at the apical end, and have important functions, we think that they themselves are unlikely to regulate polarity establishment directly. Additionally, it seems that the apical polar rings are visible in forming sporozoites far before the comparable stages of merozoite formation. An important note here is that at this point, this is largely inferences based on observational differences and there is relatively little functional data on proteins that regulate polarity establishment at any stage of the Plasmodium 9.

__Therefore, I think that electron microscopy remains essential for the observation of such ultra-fine structures __

We have added a paragraph in the discussion that provides a more clear comparison between MoTissU-ExM and other imaging modalities previously applied on mosquito-stage parasites (see response to Reviewer#1 (Minor text changes) comment #17). 10.

__The author have not mentioned that sometimes the stage oocyst development is also dependent on the age of mosquito and it vary between different mosquito gut even if the blood feed is done on same day. __

In our opinion this can be inferred through the more general statement that "development of each oocyst within a midgut is asynchronous..."

Figure changes (Reviewer #2)

1. __Fig 3B: stage 2 and 6 does not show the DNA cyan, it would-be good show the sate of DNA at that particular stage, especially at stage 2 when APR is visible. And box the segment in the parent picture whose subset is enlarged below it. __

We completely agree with the reviewer that the stage 2 image would benefit from the addition of a DNA stain. Many of the images in Figure 3b were done on samples that did not have a DNA stain and so in these * yoelii samples we did not find examples of all segmentation scores with the DNA stain. Examples of segmentation score 2 and 6 for P. berghei, and 6 for P. falciparum* can be found with DNA stains in Figure S8. 2.

__For clarity, it would be helpful to add indicators for the centriolar plaques in Figure 1b, as their locations are not immediately obvious. __

The CPs in Figure 1a and 1b have been circled on the NHS ester only panel for clarity. +

__Regarding Figure 1c, the authors state that 'the rootlet fiber is visible'. However, such a structure cannot be confirmed from the provided NHS ester image. Can the authors present a clearer image where the rootlet fibre is more distinct? Furthermore, please provide the basis for identifying this structure as a rootlet fiber based on the NHS ester observation alone. __

The image in Figure 1c has been replaced with one that more clearly shows the rootlet fibre.

Based on electron microscopy studies, the rootlet fibre has been defined as a protein dense structure that connects the centriolar plaque to the apical polar rings (PMID: 17908361). Through NHS ester and tubulin staining, we could identify the apical polar rings and centriolar plaque as sites on the apical end of the parasite and nucleus that microtubules are nucleated from. There is a protein dense fibre that connects these two structures. Based on the fact that the protein density of this structure was previously considered sufficient for its identification by electron microscopy, we consider its visualisation by NHS ester staining sufficient for its identification by U-ExM.

__Fig 1B - could the tubulin image in the hemispindle panel be made brighter? __

The tubulin staining in this panel was not saturated, and so this change has been made.

__Fig 4A - the green text in the first image panel is not visible. Also, the cyan text in the 3rd image in Fig 1A is also difficult to see. There's a few places where this is the case __

We have made all microscopy labels legible at least when printed in A4/Letter size.

__Fig 6A - how do the authors know ron11 expression is reduced by 99%? Did they test this themselves or rely on data from the lab that gifted them the construct? Also please provide mention the number of oocyst and sporozoites were observed. __

The way Figure 6a was previously designed and described was an oversight, that wrongly suggested we had quantified a >99% reduction in *ron11 * The 99% reduction has been removed from Figure 6a and the corresponding part of the figure legend has been rewritten to emphasise that this was previously established:

"(a) Schematic showing previously established Ron11Ctrl and Ron11cKD parasite lines where ron11 expression was reduced by >99%9."

As to the second part of the question, we did not independently test either protein or RNA level expression of RON11, but we were gifted the clonal parasite lines established by Prof. Ishino's lab in PMID: 31247198 not just the genetic constructs.

__Fig 6E - are the data point colours the wrong way round on this graph? Just looking at the graph it looks as though the RON11cKD has more rhoptries than the control which does not match what is said in the text. __

Thank you for pointing out this mistake, the colours have now been corrected.

__Fig S8C, PbRON11 ctrl, pie chart shows 89.7 % spz are present in the secretory cavity while the text shows 100 %, 35/35 __

The text saying 100% (35/35) only considered salivary glands that were infected (ie. Uninfected SGs were removed from the count. The two sentences that report this data have been clarified to reflect this better:

"Of *PbRON11ctrl SGs that were infected (35/39), 100% (35/35) contained sporozoites in the secretory cavity (Figure S8c). Conversely of infected PbRON11cKD SGs (59/82), only 24% (14/59) contained sporozoites within the secretory cavity (Figure S9d)."

*

__Fig S9D shows that RON11 ckd contains 17.1% sporozoites in secretory cavity while the text says 24%. __

Please see the response to Reviewer#2 Figure Changes Comment #8 where this was addressed.

Experimental changes (Reviewer #2)

1. __Why do the congruent rhoptries have similar lengths to each other, while the dimorphic rhoptries have different lengths? Is this morphological difference related to the function of these rhoptries? __

We hypothesise that this morphological difference arises because the congruent rhoptries are 'used' during SG invasion, while the dimorphic rhoptries are utilized during hepatocyte invasion. It is not straightforward to test this functionally at this point, as no protein is known to have differential localization between the two. Additionally, RON11 is likely directly involved in both SG and hepatocyte invasion through a secreted portion of the protein (as seen in RBC invasion). Therefore, RON11cKD sporozoites may have combined defects, meaning we cannot assume any defect is solely due to the absence of two rhoptries. Determining this functionally is of high interest to our research groups and remains an area of ongoing study, but it is beyond the scope of this study. 2.

Would it be possible to show whether RON11 localises to the dimorphic rhoptries, the congruent rhoptries, or both, by using expansion microscopy and a parasite line that expresses RON11 tagged with GFP or a peptide tag?

__ __We do not have access to a parasite line that expresses a tagged copy of RON11, or anti-PbRON11 antibodies. Based on previously published localisation data, however, it seems likely that RON11 localises to both sets of rhoptries. Below are excerpts from Figure 1c of PMID: 31247198, where RON11 (in green) seems to have a more basally-extended localisation in midgut (MG) sporozoites than in salivary gland (SG) sporozoites. From this we infer that in the MG sporozoite you're seeing RON11 in both pairs of rhoptries, but only the one remaining pair in the SG sporozoite.

__The knockdown of RON11 disrupts the rhoptry structure, making the dimorphic and congruent rhoptries indistinguishable. Does this suggest that RON11 is important for the formation of both types of rhoptries? I believe that it would be crucial to confirm whether RON11 localises to all rhoptries or is restricted to specific rhoptries for a more precise discussion of RON11's function. __

Based on our analysis, it does indeed seem that RON11 is important for both types of rhoptries as when RON11 isn't expressed sporozoites still have both apical and cytoplasmic rhoptries (ie. Not just one pair is lost; see Reviewer #1 Experimental changes comment #1).

__The authors state that 64% of RON11cKD SG sporozoites contained no rhoptries at all. Does this mean RON11cKD SG sporozoites used up all rhoptries corresponding to the dimorphic and congruent pairs during SG invasion? If so, this contradicts your claims that sporozoites are 'leaving the dimorphic rhoptries for hepatocyte invasion' and that 'rhoptry pairs are specialized for different invasion events'. If that is not the case, does it mean that RON11cKD sporozoites failed to form the rhoptries corresponding to the dimorphic pair? A more detailed discussion would be needed on this point and, as I mentioned above, on the specific role of RON11 in the formation of each rhoptry pair. __

We do not agree that this constitutes a contradiction; instead, more nuance is needed to fully explain the phenotype. As shown in the new graph added in response to Reviewer#1 Figure changes comment #1 in RON11cKD oocyst sporozoites, 64% of all rhoptries are located at the apical end. Our hypothesis is that these rhoptries are used for SG invasion and, therefore, would not be present in RON11cKD SG sporozoites. Consequently, the fact that 64% of RON11cKD sporozoites lack rhoptries is exactly what we would expect. Essentially, we predict three slightly different 'pathways' for RON11cKD sporozoites: If they had 2 apical rhoptries in the oocyst, we predict they would have zero rhoptries in the SG. If they had 2 cytoplasmic rhoptries in the oocyst, we predict they would have two rhoptries in the SG. If they had one apical and one cytoplasmic rhoptry in the oocyst, we predict they would have one rhoptry in the SG. In any case, we expect the apical rhoptries to be 'used up,' which appears to be supported by the data.

__Out of pure curiosity, is it possible to measure the length and number of subpellicular microtubules in the sporozoites observed in this study using expansion microscopy? __

We have performed an analysis of subpellicular microtubules which is now included as Supplementary Figure 2. We could not always distinguish every SPMT from each other and so have not quantified SPMT number. We have, however, quantified their absolute length on both the 'long side' and 'short side', their relative length (as % sporozoite length) and the degree to which they are polyglutamylated.

A description of this analysis is now found in the results section as follows: "*We quantified the length and degree of polyglutamylation of SPMTs on the 'long side' and 'short side' of the sporozoite (Figure S2). 'Short side' SPMTs were on average 33% shorter (mean = 3.6 µm {plus minus}SD 1.0 µm) than 'long side' SPMTs (mean = 5.3 µm {plus minus}SD 1.5 µm) and extended 17.4% less of the total sporozoite length. While 'short side' SPMTs were significantly shorter, a greater proportion of their length (87.9% {plus minus}SD 11.2%) was polyglutamylated compared to 'long side' SPMTs (69.4% {plus minus}SD 13.8%)." *

Supplementary Figure 2: Analysis of sporozoite subpellicular microtubules. Isolated P. yoelii salivary gland sporozoites were prepared by U-ExM and stained with anti-tubulin (microtubules) and anti-PolyE (polyglutamylated SPMTs) antibodies. SPMTs were defined as being on either the 'long side' (nucleus distant from plasma membrane) or 'short side' (nucleus close to plasma membrane) of the sporozoite as depicted in Figure 1f. (a) SPMT length along with (b) SPMT length as a proportion of sporozoite length were both measured. (c) Additionally, the proportion of the SPMT that was polyglutamylated was measured. Analysis comprises 25 SPMTs (11 long side, 14 short side) from 6 SG sporozoites. ** = p The following section has also been added to the methods to describe this analysis: * "Subpellicular microtubule measurement

• To measure subpellicular microtubule length and polyglutamylation maximum intensity projections were made of sporozoites stained with NHS Ester, anti-tubulin and anti-PolyE antibodies, and SYTOX Deep Red. The side where the nucleus was closest to the parasite plasma membrane was defined as the 'short side', while the side where the nucleus was furthest from the parasite plasma membrane was defined as the 'long side'. Subpellicular microtubules were then measured using a spline contour from the apical end of the sporozoite to the basal-most end of the microtubule with fluorescence intensity across the contour plotted (Zeiss ZEN 3.8). Sporozoite length was defined as the distance from the sporozoite apical polar rings to the basal complex, measuring through the centre of the cytoplasm. The percentage of the subpellicular microtubule that was polyglutamylated was determined by assessing when along the subpellicular microtubule contour the anti-PolyE fluorescence intensity last dropped below a pre-defined threshold."

*

__In addition to the previous point, in the text accompanying Figure 7a, the authors claim that "64% of PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries". Could this data be used to infer which rhoptry pair are missing from the RON11cKD oocyst sporozoites? Can it be inferred that the 64% of salivary gland sporozoites that had no rhoptries in fact had 2 congruent rhoptries in the oocyst sporozoite stage and that these have been discharged already? __

Please see the response to Reviewer #2 Experimental Changes Comment #4.

__Is it possible that the dimorphic rhoptries are simply precursors to the congruent rhoptries? Could it be that after the congruent rhoptries are used for SG invasion, new congruent rhoptries are formed from the dimorphic ones and are then used for the next invasion?____ Would it be possible to investigate this by isolating sporozoites some time after they have invaded the SG and performing expansion microscopy? This would allow you to confirm whether the dimorphic rhoptries truly remain in the same form, or if new congruent rhoptries have been formed, or if there have been any other changes to the morphology of the dimorphic rhoptries. __

In theory, it is possible that the dimorphic rhoptries are precursors to the uniform rhoptries, specifically how the larger one of the two in the dimorphic pair might be a precursor. Maybe the smaller one is, but we have no evidence to suggest that this rhoptry lengthens after SG invasion. We are interested in isolating sporozoites from SGs to add a temporal perspective, but currently, this isn't feasible. When sporozoites are isolated from SGs, they are collected at all stages of invasion. Additionally, we don't know how long each step of SG invasion takes, so a time-based method might not be effective either. We are developing an assay to better determine the timing of events during SG invasion with MoTissU-ExM, but this is beyond the scope of this study.

__In the section titled "Presence of PbRON11cKD sporozoites in the SG intercellular space", the authors state that "the majority of PbRON11cKD-infected mosquitoes contained some sporozoites in their SGs, but these sporozoites were rarely inside either the SG epithelial cell or secretory cavity". - this is suggestive of an invasion defect as the authors suggest. Could the authors collect these sporozoites and see if liver hepatocyte infection can be established by the mutant sporozoites? They previously speculate that the two different types of rhoptries (congruent and dimorphic) may be specific to the two invasion events (salivary gland epithelial cell and liver cell infection). __

It has already been shown that RON11cKD sporozoites fail hepatocyte invasion (PMID: 31247198), even when isolated from the haemolymph and so it seems very unlikely that they would be invasive following SG isolation. As mentioned in the discussion, RON11 in merozoites has a 'dual-function' where it is partially secreted during merozoite invasion in addition to its rhoptry biogenesis functions. Assuming this is also the case in sporozoites, using the RON11cKD parasite line we cannot differentiate these two functions and therefore cannot ascribe invasion defects purely to issues with rhoptry biogenesis. In order to answer this question functionally, we would need to identify a protein that only has roles in rhoptry biogenesis and not invasion directly.

Reviewer #3

Minor text changes (Reviewer #3)

1. __Page 3 last paragraph: ...the molecular mechanisms underlying SG (invasion?) are poorly understood. __

This has been corrected 2.

__The term "APR" does not refer to a tubulin structure per se, but rather to the proteinaceous structure to which tubulin anchors. Are there any specific APR markers that can be used in Figure 1C? If not, I recommend avoiding the use of "APR" in this context. __

The text does not state that the APR is a tubulin structure. Given that it is a proteinaceous structure, we visualise the APRs through protein density (NHS Ester). It has been standard for decades to define APRs by protein density using electron microscopy, and it has previously been sufficient in Plasmodium using expansion microscopy (PMIDs: 41542479, 33705377) so it is unclear why it should not be done so in this study. 3.

__I politely disagree with the bold statements ‚ Little is known about cell biology of sporozoite formation.....from electron microscopy studies now decades old' (p.3, 2nd paragraph); ‚To date, only a handful of (instead of ‚or') proteins have been implicated in SG invasion' (p. 4, 1st paragraph). These claims may overlook existing studies; a more thorough review of the literature is recommended. __

This study includes at least 50 references from papers broadly related to sporozoite biology, covering publications from every decade since the 1970s. The most recent review that discusses salivary gland invasion cites 11 proteins involved in SG invasion. We have replaced "handful" with a more precise term, as it is not the best adjective, but it is hardly an exaggeration.

Figure changes (Reviewer #3)

1. __The hypothesis that Plasmodium utilizes two distinct rhoptry pairs for invading the salivary gland and liver cells is intriguing but remains clearly speculative. Are the "cytoplasmic pair" and "docked pair" composed of the same secretory proteins? Are the paired rhoptries identical? How does the parasite determine which pair to use for salivary gland versus liver cell invasion? Is there any experimental evidence showing that the second pair is activated upon successful liver cell invasion? Without such data this hypothesis seems rather premature. __

We are unaware of any direct protein localisation evidence suggesting that the rhoptry pairs may carry different cargo. However, only a few proteins have been localised in a way that would allow us to determine if they are associated with distinct rhoptry pairs, so this possibility cannot be ruled out either. It seems unlikely that the parasite 'selects' a specific pair, as rhoptries are typically always found at the apical end. What appears more plausible is that the "docked pair" forms first and immediately occupies the apical docking site, preventing the cytoplasmic pair from docking there. Regarding any evidence that the second pair is activated during liver cell invasion, it has been well documented over decades that rhoptries are involved in hepatocyte invasion. If the dimorphic rhoptries are the only ones present in the parasite during hepatocyte invasion, then they must be used for this process. 2.

__The quality of the "Roolet fibre" image is not good and resembles background noise from PolyE staining. Additional or alternative images should be provided to convincingly demonstrate that PolyE staining indeed visualizes the Roolet fibre. It is puzzling that the structure is visible with PolyE staining but not with tubulin staining. __

This is a logical misinterpretation based on the image provided in Figure 1c. Our intention was not to imply that PolyE staining enables us to see the rootlet fibre but that PolyE and tubulin allow us to see the APR to which the rootlet fibre is connected. There is some PolyE staining that likely corresponds to the early SPMTs that in 1c appears to run along the rootlet fibre but this is a product of the max-intensity projection. Please see Reviwer#2 Figure Changes Comment #3 for the updated Figure 1c. 3.

__More arrows should be added to Figures 6b and 6c to guide readers and improve clarity. __

We have added arrows to Figure 6b and 6c which point out what we have defined as normal and aberrant rhoptries more clearly. These panels now look like this: 4.

__Figure 2a zoomed image of P. yoelii infected SG is different than the highligted square. __

We agree that the highlighted square and the zoomed area appear different, but this is due to the differing amounts of light captured by the objectives used in these two panels. The entire SG panel was captured with a 5x objective, while the zoomed panel was captured with a 63x objective. Because of this difference, the plane of focus of the zoomed area is hard to distinguish in the whole SG image. The zoomed image is on the 'top' of the SG (closest to the coverslip), while most of the signal you see in the whole SG image comes from the 'middle' of the SG. To demonstrate this more clearly, we have provided the exact region of interest shown in the 63x image alongside a 5x image and an additional 20x image, all of which are clearly superimposable.__

__ 5.

__Figure 3 legend: "P. yoelii infected midguts harvested on day 15" should be corrected. More general, yes, "...development of each oocyst within a single midgut is asynchronous." but it is still required to provide the dissection days. __

We are unsure what the suggested change here is. We do not know what is wrong with the statement about day 15 post infection, that is when these midguts were dissected. __ Experimental Changes (Reviewer #3)__

1. __The proposed role of AOR in rhoptry biogenesis appears highly speculative. It is unclear how the authors conclude that "AORs carry rhoptry cargo" solely based on the presence of RON4 within the structure. Inclusion of additional markers to characterize the content of AOR and rhoptries will be essential to substantiate the hypothesis that this enigmatic structure supports rhoptry biogenesis. __

It is important to note that the hypothesis that AORs, or rhoptry anlagen, carry rhoptry cargo and serve as vehicles of rhoptry biogenesis was proposed long before this study (PMID: 17908361). In that study, it was assumed that structures now called AORs or rhoptry anlagen were developing rhoptries. Although often visualised by EM and presumed to carry rhoptry cargo (PMID: 33600048, 26565797, 25438048), it was only more recently that AORs became the subject of dedicated investigation (PMID: 31805442), where the authors stated that "...AORs could be immature rhoptr[ies]...". Our observation that AORs contain the rhoptry protein RON4, which is not known to localize to any other organelle, we therefore consider sufficient to conclude that AORs carry rhoptry cargo and are thus vehicles for rhoptry biogenesis. 2.

__The study of RON11 appears to be a continuation of previous work by a collaborator in the same group. However, neither this study nor the previous one adequately addresses the evolutionary context or structural characteristics of RON11. Notably, the presence of an EF-hand motif is an important feature, especially considering the critical role of calcium signaling in parasite stage conversion. Given the absence of a clear ortholog, it would be interesting to know whether other Apicomplexan parasites harbor rhoptry proteins with transmembrane domains and EF-hand motifs, and if these proteins might respond similarly to calcium stimulation. Investigating mutations within the EF-hand domain could provide valuable functional insights into RON11. __

We are unsure what suggests that RON11 lacks a clear orthologue. RON11 is conserved across all apicomplexans and is also present in Vitrella brassicaformis (OrthoMCL orthogroup: OG7_0028843). A phylogenetic comparison of RON11 across apicomplexans has previously been performed (PMID: 31247198), and this study provides a structural prediction of PbRON11 with the dual EF-hand domains annotated (Supplementary Figure 9). 3.

__The study cannot directly confirm that membrane fusion occurs between rhoptries and AORs. __

This is already stated verbatim in the results "Our data cannot directly confirm that membrane fusion occurs between rhoptries and AORs..." 4.

__It is unclear what leads to the formation of the aberrant rhoptries observed in RON11cKD sporozoites. Since mosquitoes were not screened for infection prior to salivary gland dissection, The defect reports and revisited of RON11 knockdown does not aid in interpreting rhoptry pair specialization, as there was no consistent trend as to which rhoptry pair was missing in RON11cKD oocyst sporozoites. The notion that RON11cKD parasites likely have ‚combinatorial defects that effect both rhoptry biogenesis and invasion' poses challenges to understand the molecular role(s) of RON11 on biogenesis versus invasion. Of note, RON11 also plays a role in merozoite invasion. __

We are unclear about the comment or suggestion here, as the claims that RON11cKD does not help interpret rhoptry pair specialization, and that these parasites have combined defects, are both directly stated in the manuscript. 5.

__Do all SG PbRON11cKD sporozoites lose their reduced number of rhoptries during SG invasion as in Figure 7a (no rhoptries)? __

Not all RON11cKD SG sporozoites 'use up' their rhoptries during SG invasion. This is quantified in both Figure 7a and the text, which states: "64% of *PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries."

* 6.

Different mosquito species/strains are used for P. yoelii, P. berghei, and P. falciparum. Does it effect oocyst sizes/stages? Is it ok to compare?

__ __We agree that a direct comparison between for example * yoelii and P. berghei *oocyst size would be inappropriate, however Figure 3c and Supplementary Figure 4 are not direct comparisons between two species, but a summation of all oocysts measured in this study to indicate that the trends we observe transcend parasite/mosquito species differences. Our study was not set up with the experimental power to determine if mosquito host species alter oocyst size. 7.

__While I acknowledge that UExM has significantly advanced resolution capabilities in parasite studies, the value of standard microscopy technique should not be overlooked. Particularly, when discussing the function of RON11, relevant IFA and electron microscopy (EM) images should be included to support claims about RON11's role in rhoptry biogenesis. This would complement the UExM data and substantially strengthen the conclusions. Importantly, UExM can sometimes produce unexpected localization patterns due to the denaturation process, which warrants caution. __

The purpose of this study is not to discredit, undermine, or supersede other imaging techniques. It is simply to use U-ExM to answer biological questions that cannot or have not been answered using other techniques. Please refer to Reviewer # 1 Minor text changes comment#17 to see the new paragraph "Comparison of MoTissU-ExM and other imaging modalities" that addresses this

Both conventional IFA and immunoEM have already been performed on RON11 in sporozoites before (PMID: 31247198). When assessing defects caused by RON11 knockdown, conventional IFA isn't especially helpful because it doesn't allow visualization of individual rhoptries. Thin-section TEM also doesn't provide the whole-cell view needed to draw these kinds of conclusions. Volume EM could likely support these observations, but we don't have access to or expertise in this technique, and we believe it is beyond the scope of this study. It's also important to note that for the defect we observe-missing or abnormal rhoptries-the visualization with NHS ester isn't significantly different from what would be seen with EM-based techniques, where rhoptries are easily identified based on their protein density.

The statement that "UExM can sometimes produce unexpected localisation patterns due to the denaturation process..." is partially correct but lacks important nuance in this context. Based on our extensive experience with U-ExM, there are two main reasons why the localisation of a single protein may look different when comparing U-ExM and traditional IFA images. First, denaturation: in conventional IFAs, antibodies need to recognize conformational epitopes to bind to their target, whereas in U-ExM, antibodies must recognize linear epitopes. This doesn't mean the target protein's localisation changes, only that the antibody's ability to recognize it does. Second, antibody complexes seem unable to freely diffuse out of the gel, which can result in highly fluorescent signals not related to the target protein appearing in the image, as we have previously reported (PMID: 36993603). Importantly, neither of these factors applies to our phenotypic analysis of RON11 knockdown. All phenotypes described are based solely on NHS Ester (total protein) staining, so the considerations about changes in the localisation of individual proteins are not relevant.

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Referee #3

Evidence, reproducibility and clarity

Overall, the manuscript is well-written and -structured. However, I would like to raise several major points for consideration:

1. While I acknowledge that UExM has significantly advanced resolution capabilities in parasite studies, the value of standard microscopy technique should not be overlooked. Particularly, when discussing the function of RON11, relevant IFA and electron microscopy (EM) images should be included to support claims about RON11's role in rhoptry biogenesis. This would complement the UExM data and substantially strengthen the conclusions. Importantly, UExM can sometimes produce unexpected localization patterns due to the denaturation process, which warrants caution.
2. The proposed role of AOR in rhoptry biogenesis appears highly speculative. It is unclear how the authors conclude that "AORs carry rhoptry cargo" solely based on the presence of RON4 within the structure. Inclusion of additional markers to characterize the content of AOR and rhoptries will be essential to substantiate the hypothesis that this enigmatic structure supports rhoptry biogenesis.
3. The hypothesis that Plasmodium utilizes two distinct rhoptry pairs for invading the salivary gland and liver cells is intriguing but remains clearly speculative. Are the "cytoplasmic pair" and "docked pair" composed of the same secretory proteins? Are the paired rhoptries identical? How does the parasite determine which pair to use for salivary gland versus liver cell invasion? Is there any experimental evidence showing that the second pair is activated upon successful liver cell invasion? Without such data this hypothesis seems rather premature.
4. The study of RON11 appears to be a continuation of previous work by a collaborator in the same group. However, neither this study nor the previous one adequately addresses the evolutionary context or structural characteristics of RON11. Notably, the presence of an EF-hand motif is an important feature, especially considering the critical role of calcium signaling in parasite stage conversion. Given the absence of a clear ortholog, it would be interesting to know whether other Apicomplexan parasites harbor rhoptry proteins with transmembrane domains and EF-hand motifs, and if these proteins might respond similarly to calcium stimulation. Investigating mutations within the EF-hand domain could provide valuable functional insights into RON11.
5. The study cannot directly confirm that membrane fusion occurs between rhoptries and AORs.
6. It is unclear what leads to the formation of the aberrant rhoptries observed in RON11cKD sporozoites. Since mosquitoes were not screened for infection prior to salivary gland dissection, The defect reports and revisited of RON11 knockdown does not aid in interpreting rhoptry pair specialization, as there was no consistent trend as to which rhoptry pair was missing in RON11cKD oocyst sporozoites. The notion that RON11cKD parasites likely have ‚combinatorial defects that effect both rhoptry biogenesis and invasion' poses challenges to understand the molecular role(s) of RON11 on biogenesis versus invasion. Of note, RON11 also plays a role in merozoite invasion. I like the introduction of a segmentation score to Plasmodium oocyst maturation.

Minor comments:

1. The term "APR" does not refer to a tubulin structure per se, but rather to the proteinaceous structure to which tubulin anchors. Are there any specific APR markers that can be used in Figure 1C? If not, I recommend avoiding the use of "APR" in this context.
2. The quality of the "Roolet fibre" image is not good and resembles background noise from PolyE staining. Additional or alternative images should be provided to convincingly demonstrate that PolyE staining indeed visualizes the Roolet fibre. It is puzzling that the structure is visible with PolyE staining but not with tubulin staining.
3. Figure 2a zoomed image of P. yoelii infected SG is different than the highligted square.
4. Figure 3 legend: "P. yoelii infected midguts harvested on day 15" should be corrected. More general, yes, "...development of each oocyst within a single midgut is asynchronous." but it is still required to provide the dissection days.
5. More arrows should be added to Figures 6b and 6c to guide readers and improve clarity.
6. Do all SG PbRON11cKD sporozoites lose their reduced number of rhoptries during SG invasion as in Figure 7a (no rhoptries)?
7. Different mosquito species/strains are used for P. yoelii, P. berghei, and P. falciparum. Does it effect oocyst sizes/stages? Is it ok to compare?
8. I politely disagree with the bold statements ‚ Little is known about cell biology of sporozoite formation.....from electron microscopy studies now decades old' (p.3, 2nd paragraph); ‚To date, only a handful of (instead of ‚or') proteins have been implicated in SG invasion' (p. 4, 1st paragraph). These claims may overlook existing studies; a more thorough review of the literature is recommended.
9. Page 3 last paragraph: ...the molecular mechanisms underlying SG (invasion?) are poorly understood.

Significance

In this study, the authors explore Ultrastructure Expansion Microscopy (U-ExM) in Plasmodium-infected mosquito tissue with the aim to enhance the visualization of parasite ultrastructure. For this purpose, they revisit sporogony, the maturation of sporozoites inside oocysts, and sporozoite invasion of salivary glands, which has been studied both by cell biological methods and experimental genetics over four decades. They focus their analysis on the biogenesis and function of key secretory organelles, termed rhoptries, which are central to parasite invasion and, again, have been studied extensively.

This study is a follow-up of a previous study by the same authors (Ref. 19). In the former study the authors showed that U-ExM allows to visualize subcellular structures in sporozoites, including the nucleus, rhoptries, Golgi, apical polar rings (APR), and basal complex, as well as midgut-associated oocysts with developing sporozoites. Here, the authors claim a new finding by stating that sporozoites possess two distinct rhoptry pairs. Supposedly, only one pair is utilized during salivary gland invasion. The authors suggest specialization of rhoptries for different cell invasion events. The authors also revisit a RON11 knock-down parasite line, which was previously shown to be deficient in salivary gland invasion, host cell attachment, gliding locomotion, and liver invasion (Ref. 14).

I find it difficult to estimate the significance. Obviously, attention will be limited to Plasmodium researchers only, as this study is descriptive and revisits a well-studied aspect of the Plasmodium life cycle in the Anopheles vector.

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Referee #2

Evidence, reproducibility and clarity

The manuscript by Liffner et al have used the modified expansion microscopy as they term Mosquito Tissue Ultrastructure Expansion Microscopy (MoTissU-ExM) to study a cell biology of temporal development of malaria parasite sporozoite biogenesis within mosquito host. They employed three different malaria parasite models Plasmodium yoelii, P.beghei and P falciparum and infected them in mosquito host.

The application of MoTissU-ExM to infected mosquito tissues is a significant technical advance, enabling visualizations previously only achievable with electron microscopy.

The major conclusion and advances are as following

• The establishment of a "segmentation score" as a great tool for staging asynchronous oocyst development.
• The location of Centriolar plaques, rootlet and other structures which are difficult to analyse
• The first detailed timeline for sporozoite rhoptry biogenesis.
• Clear quantification showing that sporozoites possess four rhoptries and utilise two during salivary gland (SG) invasion.
• A characterization of the RON11 knockout phenotype, linking it to defects in rhoptry biogenesis and a specific block in SG epithelial cell invasion. The following points are intended to further strengthen the paper for publication.

Points for Revision

1. For clarity, it would be helpful to add indicators for the centriolar plaques in Figure 1b, as their locations are not immediately obvious.
2. The 'image Z-depth' value indicated in the figures is ambiguous. It is not clear whether this refers to the distance from the coverslip surface or the starting point of the z-stack image acquisition. A precise definition of this parameter would be beneficial.
3. Regarding Figure 1c, the authors state that 'the rootlet fiber is visible'. However, such a structure cannot be confirmed from the provided NHS ester image. Can the authors present a clearer image where the rootlet fibre is more distinct? Furthermore, please provide the basis for identifying this structure as a rootlet fiber based on the NHS ester observation alone.
4. Why do the congruent rhoptries have similar lengths to each other, while the dimorphic rhoptries have different lengths? Is this morphological difference related to the function of these rhoptries?
5. Would it be possible to show whether RON11 localises to the dimorphic rhoptries, the congruent rhoptries, or both, by using expansion microscopy and a parasite line that expresses RON11 tagged with GFP or a peptide tag?
6. The knockdown of RON11 disrupts the rhoptry structure, making the dimorphic and congruent rhoptries indistinguishable. Does this suggest that RON11 is important for the formation of both types of rhoptries? I believe that it would be crucial to confirm whether RON11 localises to all rhoptries or is restricted to specific rhoptries for a more precise discussion of RON11's function.
7. The authors state that 64% of RON11cKD SG sporozoites contained no rhoptries at all. Does this mean RON11cKD SG sporozoites used up all rhoptries corresponding to the dimorphic and congruent pairs during SG invasion? If so, this contradicts your claims that sporozoites are 'leaving the dimorphic rhoptries for hepatocyte invasion' and that 'rhoptry pairs are specialized for different invasion events'. If that is not the case, does it mean that RON11cKD sporozoites failed to form the rhoptries corresponding to the dimorphic pair? A more detailed discussion would be needed on this point and, as I mentioned above, on the specific role of RON11 in the formation of each rhoptry pair.
8. Out of pure curiosity, is it possible to measure the length and number of subpellicular microtubules in the sporozoites observed in this study using expansion microscopy?
9. Is it possible that the dimorphic rhoptries are simply precursors to the congruent rhoptries? Could it be that after the congruent rhoptries are used for SG invasion, new congruent rhoptries are formed from the dimorphic ones and are then used for the next invasion? Would it be possible to investigate this by isolating sporozoites some time after they have invaded the SG and performing expansion microscopy? This would allow you to confirm whether the dimorphic rhoptries truly remain in the same form, or if new congruent rhoptries have been formed, or if there have been any other changes to the morphology of the dimorphic rhoptries.
10. In addition to the previous point, in the text accompanying Figure 7a, the authors claim that "64% of PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries". Could this data be used to infer which rhoptry pair are missing from the RON11cKD oocyst sporozoites? Can it be inferred that the 64% of salivary gland sporozoites that had no rhoptries in fact had 2 congruent rhoptries in the oocyst sporozoite stage and that these have been discharged already?
11. In the section titled "Presence of PbRON11cKD sporozoites in the SG intercellular space", the authors state that "the majority of PbRON11cKD-infected mosquitoes contained some sporozoites in their SGs, but these sporozoites were rarely inside either the SG epithelial cell or secretory cavity". - this is suggestive of an invasion defect as the authors suggest. Could the authors collect these sporozoites and see if liver hepatocyte infection can be established by the mutant sporozoites? They previously speculate that the two different types of rhoptries (congruent and dimorphic) may be specific to the two invasion events (salivary gland epithelial cell and liver cell infection).

There are a few typing errors in the document:

1. Paragraph 3 of the introduction - line 7, "handful or proteins" should be handful of proteins
2. Paragraph 5 of the introduction - line 7, "also able to observed" should be observe
3. In the final paragraph of the introduction - line 1, "leverage this new understand" should be understanding
4. The first paragraph of the discussion summary contains an incomplete sentence on line 7, "PbRON11ctrl-infected SGs."
5. The second paragraph of the discussion - line 10, "until cytokinesis beings" should be begins

Some suggestions for figures

Fig 1B - could the tubulin image in the hemispindle panel be made brighter?

Fig 3B: stage 2 and 6 does not show the DNA cyan, it would-be good show the sate of DNA at that particular stage, especially at stage 2 when APR is visible. And box the segment in the parent picture whose subset is enlarged below it.

Fig 4A - the green text in the first image panel is not visible. Also, the cyan text in the 3rd image in Fig 1A is also difficult to see. There's a few places where this is the case

Fig 6A - how do the authors know ron11 expression is reduced by 99%? Did they test this themselves or rely on data from the lab that gifted them the construct? Also please provide mention the number of oocyst and sporozoites were observed.

Fig 6E - are the data point colours the wrong way round on this graph? Just looking at the graph it looks as though the RON11cKD has more rhoptries than the control which does not match what is said in the text.

Fig S8C, PbRON11 ctrl, pie chart shows 89.7 % spz are present in the secretory cavity while the text shows 100 %, 35/35

Fig S9D shows that RON11 ckd contains 17.1% sporozoites in secretory cavity while the text says 24%.

Some point to discuss

1.One minor point that author suggest that oocyst diameter is not appropriate for the development of sporozoite develop. This is not so true as oocyst diameter tells between cell division and cell growth so it is important parameter especially where the proliferation with oocyst does not take place but the growth of oocyst takes place.<br /> 2. The author have not mentioned that sometimes the stage oocyst development is also dependent on the age of mosquito and it vary between different mosquito gut even if the blood feed is done on same day. 3. How is the apical polarity different to merozoite as some conoid genes are present in ookinete and sporozoite but not in merozoite.

Significance

The following aspects are important:

This is novel and more cell biology approach to study the challenging stage of malaria parasite within mosquito. By using MoTissU-ExM, the authors have enabled the three-dimensional observation of ultrastructures of oocyst-sporozoite development that were previously difficult to observe with conventional electron microscopy alone. This includes the developmental process and entire ultrastructure of oocysts and sporozoites, and even the tissue architecture of the mosquito salivary gland and its epithelia cells.

Advances:

By observing sporozoites formation within the oocyst and the overall ultrastructure of the sporozoite with MoTissU-ExM, the authors have provided detailed descriptions of the complete structure and three-dimensional spatial relationships of the rhoptries, rootlet fibre, nucleus, and other organelles. Furthermore, their detailed localisation analysis of sporozoites within the salivary gland is also a great achievement. Considering that such observations were technically and laboriously very difficult with conventional electron microscopy, enabling these analyses with higher efficiency and relatively lower difficulty represents a major contribution to the future advancement of oocyst-sporozoite biology. The development of the 'segmentation score' for sporozoite formation within the oocyst is another major advance. I think this will enable detailed descriptions of structural changes at each developmental stage and of the molecular mechanisms involved in the development of oocysts-sporozoites This has its advantages if antibodies can be used and somewhat reduces the need for immuno-EM. Secondly, in terms of sporozoite rhoptry biology, the Schrevel et al Parasitology 2007 seems to only focus on oocyst sporozoite rhoptries as they say that the sporozoites have 4 rhoptries. This study on the other hand also looks at salivary gland sporozoites and shows that there are potentially important differences between the two - namely the reduction from 4 rhoptries to two. This also leads to further questions about the different types of rhoptries in oocyst sporozoites and whether they're adapted to invasion of different cell types (sal gland epithelial cells or liver hepatocytes)

Limitation

It would be that expansion microscopy alone still has its limits when it comes to observing ultra-fine structures. For example, visualising the small vesicular structures that Schrevel et al. observed in detail with electron microscopy, or seeing ultra-high resolution details such as the fusion of membrane structures and their interactions with structures like the rootlet fibre and microtubules. Therefore, I think that electron microscopy remains essential for the observation of such ultra-fine structures The real impact of this work is mostly cell biologist working with malaria parasite and more in mosquito stages. But the approaches can be applied to any material from any species where temporal dynamics need to be studied with tissue related structures and where UExM can be applied. I am parasite cell biologist working with parasites stages within mosquito vector host.

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Referee #1

Evidence, reproducibility and clarity

In this paper the authors use ultrastructure expansion microscopy to investigate the mosquito stages of the malaria parasite, specifically the stage called oocyst and the process of sporozoite development. They report a number of observations of which the ones concerning rhoptries are the most interesting. There are four of these organelles in the first form of sporozoites in the oocyst and only two in the mature form in the salivary gland. Using a gene knockout of a protein that was reported to be important for rhoprty formation in merozoites, the parasites invading into human red blood cells, they found that fewer rhoptries are formed also in sporozoites and that these cannot enter into the salivary gland cells any more. The presented data are in my view conclusive and no additional experiments are needed for this work to be published. The described experiments should be readily reproducible and have a high statistical power. The text is mostly clearly written but could be improved to make it more concise and more precise and to avoid overstatements. Some references could be added. It would have helped to have line numbers in the manuscript. My suggestions are as following:

Abstract: don't focus on technique but on the questions you tried to answer (ie rewrite or delete the 3rd and 4th sentence)

Add reference on page 3 after 'disrupted parasites' Change 'the basal compelx at the leading edge' - this seems counterintuitive Change 'mechanisms underlying SG are poorly' - what mechanisms? of invasion or infection? On page 4: 'handful of proteins' 'range of cell biology processes' - I understand the paper that the key discovery concerns rhoptry biogenesis and function, so focus on that, all other aspects appear rather peripheral. what are the 'three microtubule spindle structures'? 'Much of this study focuses on the secretory organelles': I would suggest to rewrite the intro to focus solely on those, which yield interesting findings. On page 5: 'little is known' - please describe what is known, also in other stages. At the end of the paper I would like to know what is the key difference to rhoptry function in other stages? change 'rhoptries golgi-derived, made de novo' change 'new understand to' 'rhoptry malformations' seem to be similar in sporozoites and merozoites. Is that surprising/new? What is known about crossing the basal lamina. Where rhoptries thought to be involved in this process? Or is it proteins on the surface or in other secretory organelles? On page change/specify: 'wide range of parasite structures' On page 7: is Airyscan2 a particular method or a specific microscope? what are the dark lines in panel E? in panel G: Are the dense granules not micronemes? What are the dark lines? Rhoptries?? On page 8 the authors mention a second layer of CSP but do not further investigate it. It is likely hard to investigate this further but to just let it stand as it is seems unsatisfactory, considering that CSP is the malaria vaccine. What happens if you add anti-CSP antibodies? I would suggest to shorten the opening paragraphs of this paper and to focus on the rhoptries. This could be done be toning down the text on all aspects that are not rhoptries and point to the open question some of the observations such as the CSP layers raise for future studies. Figure 2 seems to add little extra compared to the following figures and could in my view go to the supplement. On page 10 I suggest to qualify the statement 'oocyst development has typcially been inferred by'. There seem a few studies that show that size doesn't reflect maturation. Page 11: I am tempted to suggest the authors start their study with Figure 3 and add panel A from Figure 2 to it. This leads directly to their nice work on rhoptries. Other features reported in Figures 1 and 2 are comparatively less exciting and could be moved to the supplement or reported in a separate study. Text on page 12 could be condensed to highlight the new data of ron4 staining of the AOR. Maybe include more detail of the differences between species on rhoptry structure into Figure 4. I would encourage to move the Data on rhoptries in Figure S6 to the main text ie to Figure 4. On page 16 the authors state that different rhoptries might have different function. This is an interesting hyopthesis/result that could be mentioned in the abstract. how large is RON11? On page 19: do the parasites with the RON11 knockout only have the cytoplasmic or only the apical rhoptries? Page 23: I suggest to delete the first sentence and focus on the functional aspects and the discoveries. There is no causal link between ookinete invasion and oocyst developmental asynchrony First sentence of page 24 appears to contradict what is written in results I don't understand the first two sentences in the paragraph titled Comparison between Plasmodium spp On page 25 or before the vast number of electron microscopy studies should be discussed and compared with the authors new data. First sentence on page 27: there are many studies on parasite proteins involved in salivary gland invasion that could be mentioned/discussed. Maybe add a conclusion section rather than a future application section, which reads as if you want to promoted the use of ultrastructure expansion microscopy. To my taste the technological advance is a bit overplayed considering the many applications of this techniques over the last years, especially in parasitology, where it seems widely used. In any case, please delete 'extraordinarily'

Significance

This interesting study investigates the development of malaria parasites in the mosquito using ultrastructure expansion microscopy adapted to mosquito tissue. It provides new and beautiful views of the process of sporozoite formation. The authors discovered that four secretory vesicles called rhoptires are formed in the sporozoites with two pairs being important for distinct functions, one pair functions during invasion of the salivary glands of the mosquito and the other in liver infection, although the latter is not shown but inferred from prior data.

This study will thus be of interest to scientists investigating malaria parasites in the mosquito as well as to scientist working on vesicle secretion and invasion in these parasites.

The authors use a previously generated parasite line that lack a protein to investigate its function in rhoptry biogenesis and find that its absence leads to fewer rhoptries which impacts the capacity of the parasite to enter into salivary gland cells. This is a nice functional addition to an otherwise largely descriptive study, but mimics largely the previously reported results from the blood stages. It is not clear to this reviewer how much the study advances the field over the many previous electron microscopy studies. This could be better elaborated in the text.

Strength of the study: beautiful microscopy, new insights into rhoptry formation and function, new technique to study malaria parasites in the mosquito

Weakness of the study: Some loose ends in the description of spindles and CSP layers, text could be more focussed on the key advancements reported

eLife Assessment

This valuable study uses a computer vision pipeline to infer the motor control of cephalopod skin, revealing that individual chromatophores exhibit anisotropic deformations and can be associated with multiple putative motor units. The evidence supporting these claims is solid, although the study's conclusions are limited to stationary or sedated animals, and the analyses of motor unit characteristics and electrophysiological validation remain incomplete. This work will be of significant interest to biologists studying cephalopod behavior and motor control.

Reviewer #1 (Public review):

Summary:

Renard, Ukrow et al. applied their recently published computational pipeline (CHROMAS) to the skin of Euprymna berryi and Sepia officinalis to track the dynamics of cephalopod chromatophore expansion. By segmenting each chromatophore into radial slices and analyzing the co-expansion of slices across regions of the skin, they inferred the motor control underlying chromatophore groups.

Strengths:

The authors demonstrate that most motor units of cephalopod skin include a subregion of multiple chromatophores, creating "virtual chromatophores" in between the fixed chromatophores. This is an interesting concept that challenges prevailing models of chromatophore organization, and raises interesting possibilities for how chromatophore arrays may be patterned during development.

This study introduces new analyses of cephalopod skin that will be valuable for the quantitative study of cephalopod behavior.

Weaknesses:

The authors chose to image spontaneous skin changes in sedated animals, rather than visually-evoked skin changes in awake, freely-moving animals. Spontaneous chromatophore changes tend to be small shimmers of expansion and contraction, rather than obvious, sizable expansions. This may make it more challenging to distinguish truly co-occurring expansions from background activity. The authors don't provide any raw data (videos) of the skin, so it is difficult to independently assess the robustness of the inferred chromatophore groupings.

The patch-clamp experiments in E. berryi are used to test the validity of their approach for inferring motor units. The stimulations evoke expansions of sub-regions of each chromatophore, creating "virtual chromatophores" as predicted from the behavioral analysis. However, the authors were not able to predict these specific motor units from behavioral analysis before confirming them with patch-clamp, limiting the strength of the validation. It would be informative to quantify the results of the patch-clamp experiments - are the inferred motor units of similar sizes to those predicted from behavior?

The authors report testing multiple experimental conditions (e.g., age, size, behavioral stimuli, sedation, head-fixation, and lighting), but only a small subset of these data are presented. It is difficult to determine which conditions were used for which experiments, and the manuscript would benefit from pooling data from multiple experiments to draw general conclusions about the motor control of cephalopod skin.

The authors use a different clustering algorithm for E. berryi and S. officinalis, but do not discuss why different clustering approaches were required for the two species.

Impact:

The authors use their computational pipeline to generate a number of interesting predictions about chromatophore control, including motor unit size, their spatial distribution within the skin, and the independent control of subregions within individual chromatophores by putatively distinct motor neurons. While these observations are interesting, the current data do not yet fully support them.

The CHROMAS tool is likely to be valuable to the field, given the need for quantitative frameworks in cephalopod biology. The predictions outlined here provide a useful foundation for future experimental investigation.

Reviewer #2 (Public review):

Summary:

Overall, this is an excellent paper, making use of a newly developed system for monitoring the behaviour of chromatophores in the skin of (mostly) free-swimming bobtail squid and European cuttlefish. The manuscript is very well-written, clearly presented and very well-structured. The central finding, that individual chromatophores are connected to multiple motor neurones, is not new. Novelty instead comes from the ability to measure the actuation of chromatophore sections across wide areas of skin in free-swimming animals, showing the diversity of local motor units and reinforcing the notion that individual chromatophores are not necessarily the individual units of colour change, but rather local motor units that cover multiple neighbour and near-neighbour chromatophore muscles. This is an excellent finding and one that will shape our understanding of the neural control of cephalopod skin colour.

Strengths:

The methodological approach to collecting large amounts of data about local variations in the expansion of sections of chromatophores is exciting, and the analysis pipeline for clustering sections of chromatophores whose spontaneous activity correlated over time is powerful and exciting.

Weaknesses:

Some minor edits and typographical errors need correcting. I also had some concerns that the preparation for the electrophysiological section of the manuscript complies with the journal's ethical requirements, so I would urge that this be carefully checked.

Reviewer #3 (Public review):

Summary:

This study uses high-resolution videography and a custom computer-vision pipeline to dissect the motor control of cephalopod chromatophores in Euprymna berryi and Sepia officinalis. By quantifying anisotropic chromatophore deformations and applying dimensionality reduction methods, the authors infer that individual chromatophores can be a part of multiple motor units. Clustering analyses reveal putative motor units that often span multiple chromatophores, with diverse and overlapping geometries. Chromatophore expansion dynamics are faster and more stereotyped than relaxation, consistent with active neural contraction followed by passive recoil. Together, the results show that chromatophores function not as uniform pixels but as fractionated, coordinately controlled elements that enable flexible pattern generation

Strengths:

The authors present compelling, direct evidence that a). chromatophore deformations are anisotropic, and indirect evidence that b) individual chromatophores can be split across multiple putative motor units. This evidence is provided through data collected over large spatial scales, but also at a sub-chromatophore resolution. This combination of scale and resolution is not possible using traditional neuroanatomical and physiological approaches alone.

The authors also develop a new non-invasive, image analysis approach to extract information about chromatophore deformation across large spatial scales on the organism's body. In principle, this approach is applicable across species and may allow for further comparative characterization of chromatophore motor control. It is therefore a promising new tool and useful resource for the community.

Weaknesses:

An important weakness of the work is that the methods the authors develop can only be applied during resting, spontaneous 'flickering' activity of chromatophores. The inability to reliably apply their technique during any kind of realistic camouflage is a large limitation, as it means this method cannot be used to study the dynamics of motor control during realistic camouflage behaviors.

Another weakness of this paper is the rather limited electrophysiological validation of the computational findings. The authors present only one electrophysiology experiment in E. berryi, the species that they used only for 'methodological development' and not for detailed characterization. A complementary electrophysiological experiment in S. officinalis, or some visualization of neuron morphology confirming that motor neurons do indeed project to multiple chromatophores, would strengthen the generalizability of their computational analysis. This would be particularly pertinent to validate the author's claim that some motor units contain chromatophores that are quite distant from one another on the animal.

Overall, the authors' technical contributions and method development are an important advance. This work serves as an excellent proof of concept that their method can extract useful information about chromatophore motor control. Further validation of their method is needed to fully trust the fine-scale conclusions drawn about the distribution and composition of multi-innervated chromatophores. Furthermore, the authors raise many interesting ideas about developmental constraints on circuit wiring and potential adaptive significance of multi-innervated chromatophores for certain features of camouflage patterning. Their method may be able to help resolve some of these questions in the future if it is refined and applied across developmental stages, regions of the animal, and across species

Low Band (Sub-1 GHz) - Covers a wide area, including cities, suburbs, and rural areas. - Good for IoT devices and deep indoor coverage. - Speed is lower: up to ~100 MB/s.

Mid Band (1–6 GHz) - Balances speed and coverage. - Includes frequencies like 3.3–3.8 GHz, 1800 MHz, 2.3 GHz, 2.6 GHz. - Wireless speed is 5–10 times faster than 4G. - This is usually the first spectrum sold in auctions.

High Band (Above 6 GHz, e.g., 26 GHz) - Extremely fast and low-latency (up to ~1 GB/s). - Covers short distances only. - Enables advanced services like vehicle-to-vehicle communication and remote surgery.

In short: low band = wide coverage, mid band = good mix, high band = super fast but short range

Market Share by Subscribers

• Shows the percentage of total customers in a market (Consumer, Enterprise, SMB) that use your service.
• Example: If 1 million people use your network out of 10 million total users, your market share = 10%. Helps track how competitive your company is.

Total Revenues - The total money earned from all services sold in each market (Consumer, Enterprise, SMB). - Higher revenue usually means more sales or higher-paying customers.

Customer Satisfaction (NPS – Net Promoter Score) - Measures how happy customers are and whether they would recommend your service. - NPS ranges from -100 to +100; higher = more satisfied and loyal customers.

Free Cash Flow - Money left after paying for: Capex (capital investments, like network upgrades) Opex (operating costs, like salaries, maintenance) - Formula: Free Cash Flow = Revenues – Capex – Opex - Shows how much cash the company can use freely, e.g., to pay debts or invest.

Cumulative Profits - Total profits accumulated over time from all markets. - Helps see overall financial health and growth of the business

In this entire thing, truth and the public good are taken to be worth protecting for their own sake. This is refreshing, after all the post-modernism of 2026, where so many are stuck discussing the meta-politico-strategic considerations of any political stance.

"Brace for the shitstorm. People on all sides will predictably forget the basic principles of cohabitation."

This entire paragraph is relevant as-is in 2026.

This is the most important part imo, and the core of TBC

Do we reflect and make choices? Or do we resign ourselves to suffering from fate and "Might is right"?

Interesting that "inefficiency" is the first problem mentioned

I had previously known that intentionally poisoning datasets was possible, and that unintentionally doing so was also possible, but I wasn't aware that something like a TikTok video was able to make that big of an impact. It's interesting to see how one influencer's video and her audience were able to make such a big impact and effectively ruin most of the researcher's data- considering her audience is mainly women in their 20s.

Data poisoning highlights that data is never neutral. Whether through unintentional bias or intentional sabotage, poisoned data can undermine research, distort automated decision-making, and be used as a form of resistance or political action in data-driven systems.

This section is an important reminder that data quality is critical before drawing insights or conclusions. If the data is biased or low quality, it can lead to misleading results that affect decision-making. In some cases, this could harm certain groups by reinforcing unfair patterns or promoting incorrect assumptions.

I believe data poisoning cause great harm often such as idea that misinformation and disinformation. This can cause corporations, governments, institutions, and ordinary individuals to make great blunders based this data poisoning, whether it was intended or not.

This statement reminds us that data poisoning is not just a technical problem; it's also a social action strategy. By creating a large number of fake applications, the attack targets the "data entry points" of a company's screening and decision-making processes, causing the system to lose its ability to distinguish between genuine and fraudulent applications. While it can change action costs and slow down replacement processes in the short term, it also raises ethical concerns and risks: could it inadvertently harm genuine job applicants and lead to a broader collapse of trust? Therefore, when discussing this issue, it's necessary to simultaneously evaluate the motives, the affected parties, and the long-term consequences.

This statement highlights a crucial point: when data quality is poor to a certain extent, continuing the analysis is not "working harder," but rather "more dangerous." This is because biases and missing data can systematically push the model or conclusions in the wrong direction, and it's difficult to detect this from the results alone. In such situations, the most important step is to conduct a data audit: checking sample representativeness, missing data mechanisms, and whether abnormal distributions are explainable. If necessary, one must acknowledge that "this dataset cannot answer this question," rather than forcing a conclusion.

This example shows how data poisoning can be used deliberately as a form of collective action, where misleading data is introduced to disrupt a system’s ability to function as intended. It also raises ethical questions about whether manipulating data is justified when it is used to counteract perceived corporate power and support labor rights, rather than for personal gain.

Social media data is used to maximize user engagement and profit, primarily through targeted advertising, but this same system can be exploited to manipulate vulnerable populations and undermine democratic processes.

1. As someone who's on social media pretty often, I can 100% attest to the fact that any sort of interaction with an ad or sponsored post will cause your timeline to be filled with other ads relating to a similar product, if not the same one. Sometimes ads do get my attention and I look at the item to see the price, and when I return to my homepage I manage to see the same post 3 or 4 times in the same hour. I also do recognize that gambling advertisements are often shown to children, which I think should 100% monitored, considering children are both not able to grasp the concept of money at their age, and they're children, the idea that it's legal to show these ads to children when they're using electronics is beyond me.****

Social media platforms use our data to keep us hooked and maximize their profits, showing that 'free' services often come at the cost of our time and attention. While targeted ads can be helpful, they also give platforms the power to manipulate users or even influence major events like elections.

Description of Clawdbot/ now named openclaw, as a PA

wut? racking up API costs to AI models like that is bizarre

clawdbot for 'small tasks across your digital life', vgl [[Aazai 2025 aantekeningen]] wrt such tasks.

clawdbot as additional attack surface

clawdbot is persistent across time / tasks.

'by default' or can do it out of the box, if switched on?

the whole point of the article no doubt.

again this is arriving at database queries you already had, but through a way more convoluted way. When 'reasoning' is deterministic then you don't need a probabilistic layer at all, no?

Congrats you just recreated a pre-existing tab in your existing support ticket system, by vibecoding a sparql query that was likely already in your system's manual even.

another telling example. Is the number of 'open support tickets' 'critical' to your business?. Most companies would say no. The article is way too vague in specifying which organisations are the mental audience here. Or worse, thinking all companies are like it. Why would you need AI for this even? Any ticketing system will have a tab for this.

ontology here is an information management ontology, mapping variables and their properties across applications ('domains' used above is a confusing term then). The mention of SAP here is a tell, in the sense of what type of scale of enterprise we're talking about.

bit hyperbolic phrasing. deterministic layers in front of an llm makes sense. Also a form of prompt engineering in a way. Seem to see that regularly: better inputs to generate better outputs. At what point are you spending so much on inputs, you might as well make the output yourself that way? Is the assumption most companies can actually formulate their processes in terms of 'business logic'? What would an example look like?

schema-rag as improvement (formal ontology added up front before a query for rag is formulated).

knowledge graphs represent semantic context and relationships / constraints. K-graphs are a 1980s thing, I know we added them into the reference architecture for systems of digital twins I cowrote. But have no understanding more recent than the 1990s. - [ ] spend #30mins collecting current state of the art on #knowledgegraphs #pkm

criticism of retrieval augmented generatio (RAG): fails in cross domain settings, finds similar text not relations between facts or meaning

author selfdescribed as "CEO @ digetiers I Building d.AP - the semantic foundation for real enterprise AI. Passionate about turning fragmented data into contextual, usable knowledge." so has a business interest in the topic. What's 'real enterprise AI' and what is his def of enterprise (99% of all companies are SME)

Google has me listed as single even though I am married. Maybe they don't understand how polyamory works and assumes that me being in multiple committed relationships must mean "single". Earlier in this book they discussed the possible problems with date collection and categorization, with the gender binary as an example. Clearly, this is another instance of something similar.

This I find interesting because many people share the sentiment that I have, which posits that it is better to get ads tailored to your wants than ads devoid of interesting material. As long as Google uses my data only for ads, then I think its okay. Although, if it were to violate that order, I would have no way of knowing...

If evidence can be found for any conclusion then the data itself is extremely flawed and oversaturated with misinformation. Often time this can lead to dangerous conclusions being draw or bad actors using the the data as evidence to further their aims at the cost of others. I wonder how we might be able to refine data to remove such impurities and draw more accurate answers from it.

I haven't seen this example before. The example I've seen for this concept is that ice cream sales go up at the same time as drowning deaths, because most people get ice cream during the summer, which is also the time more people are swimming.

It's fascinating how data mining can reveal stories from simple numbers, like the connection between COVID-19 surges and candle reviews. However, the section on spurious correlations is a great reminder that just because two trends line up—like margarine consumption and divorce rates—it doesn't mean one actually causes the other.

This raises an interesting connection for me because hedge fund "quants" also use this strategy: finding seemingly useless or irrelevant data to game the market. Somehow, both are effective, if not only for a short period

This highlights the classic problem of spurious correlation, where two trends move together without a direct causal link. For example, ice cream sales and drowning incidents are correlated because both increase in the summer, but eating ice cream does not cause drowning, seasonal factors drive both patterns.

Cisneros wants her father to recognize her as a writer, not just as a teacher. She wishes he would introduce her proudly as “my only daughter, the writer,” which would show how much his approval means to her.

This part really played with my emotions because at first I thought her father was supportive, but later I realized he only saw college as a tool for her to find someone to marry instead of getting an education.

Her brothers are honestly weird for acting like that. Instead of making her feel uncomfortable for being the only daughter.

In this paragraph, Cisneros expresses how her identity and background shape who she is. When she lists the 3 different ways to describe herself, she talks about her gender, culture, and social class.

Deception is okay and most of the time necessary for Social Psychological experiments in order to collect information about people without their personal bias. That said, deception is only valid to be used for research purposes as long as the persons being deceived are informed after the experiment.

this makes me think about how there are issues with how widespread ChatGPT has become in popular culture that some of the data it trains on is content it wrote itself. I wonder if this will become an issue in the future, especially in data science and any research having to do with the digital sphere. Will future research papers about AI have to take into account a certain orobouros character in the AI, in that its consuming itself and tainting its own processes? are there broader ramifications for the internet writ large?

oh okay im getting the hang of it.

I feel like this process results in a specific feeling to usi9ng these platforms which mine user data moreso than is already being mined online. Especially on platforms like TikTok, where the algorithm incorporates so much data in service of retention time, I feel like even if we are not constantly thinking about it, our biology being used in service of retention time leaves us with a feeling of alienation. I wonder if there is any research on this relationship

Blessing and a curse all in the same. However, it depends on how one utilizes it. Afterall, we are the only ones in control of our own actions.

All of them depend on what I am learning. #1 -3 I've learned about being motivated and interested in what I'm learning which is one of the major aspects of my learning. A preferred learning style is just what it says, how one PREFERS to learn something. I agree that a preference on learning something shouldn't be categorized in a box, however it should just be looked at a a preference by that individual. 4- Somewhat, I believe that teachers are a big part, but the most significant aspect is me.

I think people want to have a sense of belonging to something.

This setting is in ollama desktop interface. Does it set it for the terminal too? Or are these two separate instances?

Default ollama context length is 4k. Recommended minimum for websearch, agents and coding tools (like Claude Code or Open code) is 64k. I've seen 128k recommendations for Claude Code

how to set context length in ollama?

where? and in ollama or claude code? this reads like in claude code

Did this using qwen3-code, but then Claude Code just keeps spinning, even with the smallest prompt. Not sure about the cause (first load of model??)

primary source 1

It didn't occur to me that AI could be addictive but I can totally see why it would be.

It's scary that these guardrails were not stress tested to prevent this from happening, seems pretty important to me.

I feel like AI is amplifying an issue that has been around for awhile but now we will see it on a much more dramatic scale as AI evolves and more and more people use it.

I agree, this is not the answer.

This is a lot of people over the course of just a month, that's terrifying. We need to do more to help people with mental health struggles instead of dismissing the issue.

Just because the number is small doesn't make it insignificant, I am disappointed in this response to the issue.

30/100 mental health professionals experiencing issues with AI fueled emergencies is a scary number. Mental health is so important and can be so fragile at times. I wonder if anyone is doing any large scale studies on this at the national level yet.

We don't need more possible triggers for people dealing with eccentric thoughts, especially when the consequences can be so detrimental to themselves and others.

It's scary to me that someone could go from 0-100 with improper use of an LLM/AI. I haven't really heard of this being a problem much until recently with the developments in AI and it is very concerning to me. It makes me worried about how others are using AI and how it is impacting them mentally.

primary source 2 continued

primary source 2

It seems that the hidden curriculum is not helpful to a ne student, I get that it exists, but should it? Can it be avoided?

In my personal life, I most often use the “check in” method from the four categories of microintervention strategies. When I believe something may have hurt someone, I try to be present and create space for them to talk through the situation, express their feelings, and articulate any specific pain they are experiencing. I especially appreciate the question “What do you need?” because it centers the person who was impacted and prioritizes their needs rather than assumptions. This is a strategy I plan to intentionally use in future situations.

A connection I made was with the chart’s definition of microinvalidation. A few weeks ago, my roommate was on FaceTime with a male friend I had never met, and we began casually talking. During the conversation, he asked about my ethnicity, and when I shared that I am Hispanic and of Mexican descent, he immediately began asking about my family’s political views, specifically regarding ICE. Although I do not believe his question was intentionally meant to be harmful, it reduced my identity and cultural background to a political stereotype. In that moment, it felt as though my heritage was being framed solely through politics rather than as a lived experience. The comment was especially isolating because it placed me in an uncomfortable position where any response could have led to conflict. Notably, he did not ask my other roommate, who is a white woman with blonde hair and blue eyes, similar questions about her political views. Understanding the concept of microinvalidation helped me put this experience into perspective, as it highlighted how certain identities are often singled out.

This description of “slipper, slimy words and behaviors” has left me wondering where the line is drawn between harm and intent, and how we can better recognize microaggressions in everyday interactions so that we can respond with accountability rather than defensiveness.

This would always happen with my siblings. My older brother wanted to hug my younger brother and he would exaggerate and tell my mother that my older brother wanted to hit him and so that’s where my mom would ask my brother what happened? And he would respond with, “I just wanted to give him a hug”, but in this case they would both get in trouble. One for being exaggerated and the other one for “bothering”.

Usually when someone is "othering" another person they use microaggression. The use of microaggression is a subtle jab of discrimination towards someone. The othering contained microaggression that while I was younger, I wasn't that familiar with. Now that I am older and able to understand the sounds of microaggression, it makes me feel sad knowing people use this as a tactic of discrimination.

An experience that I had was with a cop. Since he knew he had the power to do so, even though it was wrong to do what he did, yet he still did it to intimidate me. I got stopped, and just because of my color and ethnicity I got cussed at and started interrogating me to intimidate and get me nervous. I see this as a coercive power. Also I do not agree with what the cop did, it happens daily in colored people’s life sadly.

A connection that I made with this is , when we’re all young we usually get in trouble by our parents. although when for example, my family and I would visit a friends house, my parents would tell us to behave because they did not want to get on us in front of their friends. When we were in that friends house just by my mom’s look or my dads action I knew it was time for me to behave. So I connect these actions as being an unspoken rule. We see often how parents don’t even have to say nothing to their kids, but just by giving them a look, that child knows to behave. This shows how much “power” or “respected” parents can and have .

Reading this reminded me of Taylor Swift's song "Death by a Thousand Cuts" where she perfectly captures the feeling of the breaking point that microaggressions can lead to. Popular culture has the unique ability to convey lasting messages through art. Taylor Swift has undoubtedly played an integral role in this. Her music gives words to feelings we often can't put words to. While both Taylor Swift and I are part of many privileged demographics, we can also relate to microaggressions in the sense that we are women.

Simply the way our society is designed to confine women to their traditional role instead of encouraging them to thrive is an example of this. Taylor says in her song "My heart, my hips, my body, my love//Trying to find a part of me that you didn't touch." Society both subtly and unsubtly places expectations and policies on every aspect of women's lives. Like we discussed last class, just because things have gotten dramatically better for women, that does not mean they are equitable. Taylor describes the feeling that accompanies the exhaustion of this fight by saying "Gave you too much but it wasn't enough//But I'll be all right, it's just a thousand cuts." All of the small stabs women take on a daily basis might just lead to "death by a thousand cuts" if they are not addressed, and still we will persist as we bleed out.

I often ask myself when learning something; how will I apply this in my future career?

Also, having a rough draft and asking the professor or colleague to proof read it, and give pointers, is a good idea of catching mistakes and accepting constructive criticism.

As a natural people-pleaser , this resonates with me. Also, having someone that is holding you accountable is a strong motivator to keep going.

what happened to me this afternoon, because of a silly mistake I made on an assignment to miss a point.

What is the full meaning? please

Exactly, not bulking one self with unnecessary infromations.

The absence of body language online makes written communication riskier, because intent can easily be misread without tone or facial cues.

“Cultures-of-use” reminds us that language learning is incomplete without learning the communication style of the community using that language.

This section supports students learning by having one on one time with the professor gaining a better understanding.

The weekly logs help students by tracking their learning time.

The critique of reductionism resonates with how identity works today. Many people belong to multiple cultural communities at once (online spaces, workplaces, immigrant experiences), so reducing them to one national label feels outdated.

I find the idea of essentialism especially important because it reveals how stereotypes often disguise themselves as “cultural knowledge.” Saying “they’re just like that culturally” can sound neutral, but it can actually deny people agency and adaptability.

Observation: Bossuet warns kings that they will be judged by God for how they use their power

Interpretation: This shows that although kings had power, they were still expected to rule with their power responsibly escpecially with the religious standards they were being held up to.

Connection: This connects to the tertiary sourse, which explains that absolutist ideology still made a moral and ethical responsibility, even though political authority was the main rule.

Consequence: When abusing the power that they have, there are consequences that can take place if rulers failed to live up to moral and religious expecations.

Observation: The author says that the king represents everything and multiple, and is not just one thing.

Interpretation: It says that political power was centralized in the monarch and that citizens were expected to see the king as a ruler or leader as something they would be able to look up to as a nation

Connection: This connects to the visual source of Versailles, which shows how Louis XIV used symbolism and scale to represent himself as the center of the French state

Continuity: This shows the continuation of centralized authority in France as monarchs worked to control people in the area and government.

Observation: Bossuet says that the king is sacred and that harming or going against him is considered a crime.

Interpretation: This shows that people who beleived in divine rights from their God saw opposing the king as morally and religiously wrong, but not politically illegal.

Connection: I think this connects to the tertiary source, which says that absolutist rulers justified their authority by saying that it was directly in line with God, in order to prevent people from rebelling.

Cause: The belief was that the king was sacred and helped cause obedience throughout the population and caused people to less likely act out against authority.

Observation: Bossuet says that the Royal power is absolute and that the king doesn't answer to anyone on earth

Interpretation: This shows that people who may have supported absolutionism believed the king should not be limited by institutions like parliaments or assemblies, because his authority came from God, so that should be who is asked upon.

Connection: This connects to the tertiary source, which says that French absolutism places all political authority in the hands of the monarch.

Context: When you understand the contect of 17th century France it helps understand and interpret how these arguments were played out, since some monarchs were trying to justify centralized power and prevent changed to their rule.

Culture as “normal human existence”: This reminds us that culture is embedded in ordinary life, not just in special traditions or national customs—it shapes how we interpret the world in subtle ways.

Teaching the opportunity is good, but teaching the lesson is better.

This entire paragraph explains how AI is either a great tool or detrimental to learning. Giving multiple examples throughout the paragraph

Posing a question to be thought about and considered.

States the pros and cons of AI, shows more cons to out weight the pros and add a negative connotation.

The main point the article is going to be about

This shows the beginning of the problem

The writer explains that proposals begin by understanding the rhetorical situation, highlighting how important context, audience, and purpose are before drafting.

Yes! I took the test 3 different times and each time was the same result.

The passage points out that while technology helps us stay connected, it can also weaken our social ties and make real conversation harder. When so many of our interactions happen through screens, we lose important habits like listening closely, disagreeing respectfully, and seeing each other as an actual human being. Online platforms usually strengthen our existing views instead of encouraging real discussion or empathy, so we end up talking past each other instead of truly connecting. As a result, people can become emotionally distant and only engage with important topics in a shallow way, since complex debates often get reduced to quick comments, likes, or shares.

The passage also suggests that civility means more than just being polite. It is about creating a shared space where people can disagree without showing contempt. When technology encourages quick reactions and outrage, it becomes harder to slow down, ask honest questions, or admit mistakes. This can lead to more mistrust, and small misunderstandings may quickly turn into bigger social conflicts or even civil strife. It is easy for people to say what ever they want to whoever they want when they don't have to see their faces or fully interact with someone. Things can also be misinterpreted based on the "tone of voice" someone may read it in, even if that is not the tone intended. I think that makes people feel more inclined and quick to make their point, regardless of how it may make people feel. I believe it is important to be aware of the impact of words, even just written, and how it can make others feel and I hope more people will start to take that into consideration when reacting and responding online.

The first thing that came to mind would be the collection of specific location (such as I.P. addresses and specific addresses where the device is being used). While this may help in some cases (there's a case I know of where police found the location of an online bully for severe bullying and threats through pinging their IP address and pinpointing a specific house address.), there are cases currently where specific location pinging is used by ICE to track people down using their devices. It's something that can be used or abused.

Definitely a weak spot for me. I always have my phone with me for music usually but i easily get distracted and take too many breaks. If i could focus longer i would stay more consistent with the material at hand.

The steely library is a huge resource that i use constantly. Being in engineering everything i write must be a reliable recourse so having a paperback library is so important. Often times we get lost searching the internet for sources and stumble onto one that may not be reliable.

I think this is a very important note to keep in mind. When looking through text with information literacy in mind you have to be open to ideas about the topic. Otherwise we really are not doing what we set out to do.

!!! There is more harm than good being done, which would make this app unethical.

The fiscal crisis is a recurring pattern in capitalist economies like NYC. Similar breakdowns will continue to happen when economic expansion is continuously prioritized. Instead of addressing the causes of a fiscal crisis, service and budget are cut, and the government divests in poor communities. Only the government can choose to prioritize social needs over market desires, and only then will this cycle become less popular.

The passage focuses on addressing how using welfare recipients and workers served as a political strategy, therefore misleading many on the actual causes of the fiscal crisis. NY was spending the same amount of money on its working and lower class as other big cities. This includes wages, pensions, and other welfare benefits. Things went wrong when big corporations wanted to abruptly transform the city into a big, capitalist center.

What‽ Really‽ That's really rad.

What is going to happen to those that are put out of work by Ai?

Sets the issue of who should control a rapidly evolving technology that can change the world in any ways for better or for worse.

Looking to Provide a solution or an initial strategy to some of the early problems ai is causing.

Fearful and urgent tone for the article. Persuading readers/ congress to act quickly.

AI is advancing and evolving too quickly to be ignored by our regulatory agencies.

It reassures you that even though sharing your writing can feel intimidating, everyone involved is working toward improving together. As the reader they can let you know if your writing is lacking something.

sharing your work with a partner gives you a fresh perspective that can catch mistakes you might miss on your own.

The fingers may move faster than the mind, leading to typing errors.

This inspires me to create more

Shift in art focus correlated to what we "believed" in as a society at the time

These problems have existed, and still exist today, but in a much more complex manner. The normalization of depersonalized traits and alienation from one another is the scariest threat to humanity at this time with the increasing prevalence of technology stripping away our fundamental human values.

Accelerationism, technology, and AI are widening the wealth gap between the working class and billionaires, resulting in the erosion of communal values in place of hedonistic and individualistic values.

Capitalism -> the modern concept of a "9-5" and the shift of the working class towards wage labor.

Businesses and manufacturing companies had upheaved the wealthy aristocrats that dominated the preceding era.

Industrialization had rapidly advanced society in the Western world, marking the "modern" era from the mid-19th to mid-20th century.

You either have to eat a somewhat balanced meal or fast food all the time for a quick option.

call to action- with the electronic transaction systems, there should be no excuse not to do research for better dietary options

But what happens when people think that the models that they use are benign? What happens when people "assume best intentions."

Should we then look to use models that can only be scaled up? Or should that we can adjust to the different needs of different communities?

The fact that this race blindness happened in Texas is rather shocking.

!!! the bias, this is why we need peer review!!

the same is done for life insurance policies

yes!!!! However, they don't always work out. I personally love when stats can back up something, like stealing bases, but I know that although stealing bases is statistically not a smart choice, it adds drama to the game. The same can be said with boxing, but when we lose the integrity of sport, it just becomes numbers and playing in the favor of whomever get to benefit from those numbers

This final stanza now reflects how in this generation we live in, generation before may try to cut the legs we stand us but to question themselves, they act like they know everything similar to the white men from the previous poem but we got to question them in what they do, not on what we do.

It's still showing in his tone while writting this that, this generation has become one where many of us can't express ourselves freely without some type of pushback from people who generations who lived way before us

Disarticulation is the sugerical procedure of seperation of a limb, so is tying back the comparison of the rain forest.

This an interesting comparison due to the rainforest being a beautiful natural thing, where cutting it down is seen as breakign from that nature into the more futuristic/dsytopian, where they compared to their voice in this world

Okay, so let's continue our discussion of how the internet works. The next important concept is that protocols that manage the internet are govern. The Internet organized into distraction levels. Here's one representation of this model. This is called a TCP IP model, so you see, there's four layers in this model application layer, transport layer, Internet layer, and link layer each plays a distinct role, and each has its Associated protocols that manage traffic and manage processing of data at that layer. Here's an example well, well, first of all, an important

8 hops, where each hop is a router on the Internet.

This connection is the impression that is given by the technology you're using, which makes it look that way. Let's now look at another network utility called Trace route that lets us Trace how packets are routed through the internet. You can also get to this through the network tools site, and this time you want to select the trace tool and suppose I want to trace packets as they travel between Network tools and MIT. I put in MIT, I click go MIT.edu, and it shows me MIT's, IP address.

Is packet switching like a landline telephone connection between A and B? One misconception that frequently found about the internet is when you're browsing a website or sending an email or talking on the phone through your smartphone. It might seem to you like, you have a channel between the between you and your destination. Well, that's not the case package switching is not like what's called circuit switching, which is the kind of switching that was done when we used land lines to communicate in circuit switching. You actually do have a channel that remains constant throughout the call between the receiver and the caller. That's a dedicated channel. Only this caller and receiver are on that channel in a packet switch Network. There is no such sustained connection, and each individual packet each little voice packet. When you're talking on, the phone is being shipped through the internet along multiple routes. What seems to you to be a continuous connection is the impression that is given by the technology you're using, which makes it look that way. Include packet vs. circuit switching picture

delete

I would also consider deleting this section.

Possibly delete?

I would centre this as a strong intro on the page.

Can you possibly incorporate your photo into the header so it doesn't take up extra space on the sign-up page?

I would drop this paragraph down in a new visual section. Centre the heading, and add 3 columns underneath with each idea.

in my 10th year participating

I would add another button here that takes people to the last bottom section of the page: Yes, I want one!

a double up from the heading?

replace with ...

...

:

This updated version of the Companion

will love this companion if...

everything we learn is a process we learn anything in a different way and learn strategies and be successful.

you can always write what you think there will be no right or wrong answers I mean this is the reason we are here to learn.

A literacy narrative is a story about a significant experience with reading or writing. the experience should have been important for the learning, and enough time should have passes so you can think deeply about what it taught you.

narrative tells about an important experience with reading or writing, and the writer should choose an event that helped them learn and grow.

when we talk about writing narratives and plotting the first thing that comes to my mind is that we have learned to write since we were younger. that's where we start to learn how to write narratives or just random writings that means something.

Section 5f says that recording "particularity relevant or pithy quotations" and their page numbers will help you in the long run because you will be able to differentiate your words from the author's during the summary. I should create a place where I will record all my quotes with page numbers, so I do not get mixed up or confused when I need to write the data I observed in my paper.

5f defines digestion as a form of translation that assesses your own thoughts, ideas, and observations. This tells me I need to explain how the sources I use help me understand my film community more. I will ask questions such as "How does this theory of ... explain what I saw in the communities Reddit page?" This will hopefully help me to go beyond summary.