Mental health recovery narratives are increasingly used in clinical practice, public health campaigns, and as directly-accessed online resources. No instrument exists to describe characteristics of individual recovery narratives. The aims were to develop and evaluate an inventory to characterize recorded recovery narratives.A preliminary version of the Inventory of Characteristics of Recovery Stories (INCRESE) was generated from an existing theory-base. Feasibility and acceptability were evaluated by two coders each rating 30 purposively-selected narratives. A refined version was produced and a formal evaluation conducted. Reliability was assessed by four coders each rating 95 purposively-selected narratives. Inter-coder reliability was assessed using Fleiss’s kappa coefficients; test-retest reliability was assessed using intra-class correlation coefficients (ICCs).Multiple refinements to description, coding categories, and language were made. Data completeness was high, and no floor or ceiling effects were found. Intercoder reliability ranged from moderate (k=0.58) to perfect (k=1.00) agreement. Test-retest reliability ranged from moderate (ICC=0.57) to complete (ICC=1.00) agreement. The final INCRESE comprises 77 items spanning five sections: Narrative Eligibility; Narrative Mode; Narrator Characteristics; Narrative Characteristics; Narrative Content.INCRESE is the first evaluated tool to characterize mental health recovery narratives. It addresses current concerns around normative recovery narratives being used to promote compulsory wellness, e.g. by identifying narratives that reject diagnosis as an explanatory model and those with non-upward trajectories. INCRESE can be used to establish the diversity of a narrative collection and will be used in the NEON trials  to allow a recommender system to match narratives to participants. A publicly-available example fitting this definition is “Making Recovery Real in Dundee: Rona’s Story,” available at https://youtu.be/7kGMazsGDJw. Recovery narratives are also used in wider health contexts such as public health and anti-stigma campaigns.14 Yet, until recently there has been little research on the impact of recovery narratives on those who access them (hereafter narrative ‘recipients’).Recovery narratives that have been recorded by others are increasingly used to augment this individual support. Recorded recovery narratives have been defined as first-person non-fiction accounts of recovery from mental health issues, including elements of adversity or struggle, and of self-defined strengths, successes, or survival.15 A large-scale interview study found that positive impacts on recipients included feeling increased connection with others, validation of own experiences; empowerment and hopefulness about the future; greater life appreciation; changes in perspective; and a reduced sense of stigma. Negative impacts included a sense of inadequacy; disconnection from others; increased pessimism; and feeling emotionally burdened by others’ distress.16 Factors mediating whether a narrative had a beneficial or harmful impact on a recipient included recognizing shared experiences or personal similarities with the narrator, which led to positive outcomes, and noticing narrator achievements, which led to hope and empowerment for some but inadequacy and disconnection for others.17Evidence about impact on recipients is now emerging. A systematic review of five studies showed that recovery narratives can be both beneficial and harmful to recipients experiencing mental health issues.19 no existing guidance describes what kinds of recovery narratives might have positive impacts for individuals, and how potential harms may be avoided.Clearly, not all narratives help all people at all times. How then can practitioners, public health campaigners, or individuals assess which narratives may be most beneficial, while avoiding potential harms? Although research has been undertaken in related areas, for example, whether content warnings are helpful in avoiding harms for some recipients,20 This evidence replicates findings in other fields. For example, reception theory, originating in communication and media studies, suggests that recipients are not passive but bring their own readings to narratives. A narrator may encode their narrative with various meanings, but the receiver may decode and interpret the narrative in different ways, relevant to their own circumstances such as individual cultural background and life experience.21 Relatedly, narrative transportation theory suggests that recipients are more fully absorbed by narrative worlds if they can identify with aspects of the narrative or narrator.23 And the more fully absorbed or transported they are, the more likely they are to be persuaded by a narrative’s messages24 – in this case, that recovery of a meaningful life may be possible for them.Evidence is emerging, however, of the narrative characteristics that may affect a recipient’s effective connection to a narrative, such as having shared experiences or the perceived authenticity of the narrator.Since characteristics of a narrative influence whether a recipient is likely to connect effectively with its message, an approach to identifying and describing the characteristics of a recovery narrative is needed. This will enable individuals to differentiate between narratives in advance of accessing them fully, and to choose narratives with characteristics that may be most helpful at a particular time.25 or narrator coherence in the Narrative Coherence Rating Scale.26 Characterizing a narrative itself requires a different tool, drawing on methods used in other fields, such as the analysis of media content. An example is the PICMIN instrument , which assesses mental illness stigma in print media.27 PICMIN was developed using principles of content analysis, a systematic, replicable technique for compressing large amounts of text into fewer content categories based on explicit rules of coding, for use with qualitative and quantitative data.28 Content analysis has been extended to health research, for example, in investigating stigmatizing attitudes towards mental health in social media29 and exploring coping strategies for withdrawing from anti-psychotic medication.30 It provides a set of procedures for the systematic coding of data, described by Robson31 as a six-stage approach: identifying the research question; selecting the sample; defining the unit of analysis; constructing the categories for analysis; testing the categories on samples and assessing reliability; and conducting analysis. In this study, we adopted these procedures in modified form as design principles, omitting the final stage.No instrument currently exists to characterize recovery narratives. Available narrative-related instruments within mental health are designed to assess a narrator’s mental processes or emotional states through their narratives, such as a sense of self in the Scale To Assess Narrative Development (STAND)33 Connection with a narrative is a complex process, involving recipient judgements about multifaceted phenomena such as authenticity.20 Therefore, the characterization of latent as well as manifest content is needed, to ensure sufficient depth of characterization is achieved.Content analysis was originally used to identify manifest  content, but has widened to include the identification of latent content, concerning underlying patterns or deep-level inferences.32 - to provide an instrument that may assist individuals and practitioners in finding their way to stories that might be most helpful for them.Any characterization of narratives risks adopting a reductive approach to its source material, and clearly a list of characteristics cannot replace the complexity of a narrative. What is needed is a ‘condensation’ - a process of shortening an artefact while still preserving its core34 for example when only certain “highly circumscribed” or “normatively successful” kinds of stories are promoted by services,35 or when stories are used to promote “compulsory wellness.”36 Since evidence suggests that people connect with narratives they can identify with,24 and since connection is the key mechanism of impact,16 a tool that can characterize maximally diverse types of recovery narrative is required to create benefits for the greatest number of people. This study is therefore based on theory that uses the widest possible definitions of recovery narratives and which has been tested on diverse populations including marginalized groups and those currently under-served by mental health services.Critical theorists and survivor/user researchers have also raised concerns about the commodification of recovery narratives within mental health practice,The aim of this mixed methods study was to develop and evaluate an inclusive and in-depth inventory to characterize recorded recovery narratives, for use by mental health practitioners, individuals experiencing mental health distress, researchers, and curators of narrative-based collections or interventions. The objectives were to assess the inventory for its statistical reliability, and to assess its feasibility and acceptability to two groups: (1) a team of coders, as representatives of inventory target users; (2) a panel of people with lived experience of mental health difficulties, as representatives of the group whose stories would be characterized by the inventory.The research was undertaken as part of the Narrative Experiences Online (NEON) study. NEON aims to examine whether engagement with the recovery narratives of others can influence an individual’s own recovery journey. Findings will inform three future trials .Ethical Committee approval was obtained in advance for a protocol describing procedures for working with narratives and coders of narratives (London-West London REC and GTAC 18/LO/0991). The protocol for narratives described procedures for collecting consent for usage of narratives. The protocol for coders applied to all contributors not employed as NEON researchers. It described procedures for informed consent and to address the emotional and physical needs of research participants. All coders classified as research participants were provided with information sheets, and met with a member of the research team prior to study commencement to provide written informed consent. Procedures for emotional and physical wellbeing took account of emotional labour, given the potentially distressing content of some recovery narratives, and the possibility of fatigue through coding large numbers of narratives.9 change models of helpful and harmful recovery narrative impact16 and recipients’ experiences of hope and connection;20 risk factors for developing mental health issues;37 research on turning points in recovery narratives;44 research on recovery factors;45 and research on content warnings.50 Demographic item responses incorporated guidance from the UK Office of National Statistics for narrator ethnicity,51 Stonewall UK for narrator sexuality,52 NHS England for disability,53 and the Diagnostic and Statistical Manual of Mental Disorders for items characterizing diagnosis.54 This produced the preliminary inventory, INCRESE v1.A theory-based list of candidate inventory items and responses was generated through inspection of: a conceptual framework for recovery narratives;55 The acceptability of a tool to its target users is important for successful implementation.56 Use of appropriate language and a reasonable level of administrative burden have been identified as important factors in achieving this.55 Acceptability was thus defined as ‘capturing sufficient characteristics to provide an accurate summary of a recovery narrative, while representing reasonable administrative burden and using appropriate language.’ Reasonable administrative burden was defined as each narrative taking <15 minutes to code. Appropriate language was defined as sensitive, inclusive, and acceptable both to coders and people with lived experience of mental health distress.The aim of the pilot evaluation was to test the feasibility and acceptability of INCRESE v1 to coders. Feasibility was defined, modifying an existing definition, as ‘suitable for use on a routine, sustainable and meaningful basis by target users when used in a specified manner for a specified purpose.’57 selected for maximum variation in format , form  and length. Characteristics of recovery narratives may differ according to form,1 so text-based, audio and video narratives were chosen to test whether INCRESE could be applied equally to narratives across formats and forms. Narratives varied in length to test whether INCRESE sufficiently captured characteristics of both longer and shorter narratives, and whether administrative burden for longer narratives remained acceptable. Very long narratives such as documentaries and memoirs were excluded, due to limitations in coding team capacity to characterize these within the time constraints of the study. The 30 narratives for the pilot study comprised text (n=15), audio (n=5) and video-based (n=10) formats.INCRESE v1 was tested by research team coders (JLB and FN) from sociology and psychology backgrounds, each of whom independently coded 30 narratives twice, two weeks apart. The narratives were a purposive sample,58 then discussed by the wider team, including researchers with a background in interaction design (SRE) and the development of psychometric measures (MS). This identified important elements missing from the inventory, and the impacts, both physical and emotional, of coding large numbers of recovery narratives. Knowledge produced by evaluation work was used to create wellbeing training and instructions for external coders. No items from INCRESE v1 were deleted, but item descriptions were refined to produce INCRESE v2.Coders completed written evaluations of feasibility and acceptability. Findings were analyzed in the first instance by the lead author (JLB) using semantic thematic analysis,The aim of the formal evaluation was to test (a) feasibility and acceptability to coders, (b) acceptability of INCRESE to people with lived experience of mental health difficulties and (c) the reliability of INCRESE. Twenty-two of the narratives used in Stage 2 were supplemented with 73 further narratives from the NEON Collection. The 95 narratives were a purposive sample chosen for maximum variation in format, form, length, gender, ethnicity, location, diagnosis, and collection source. This was to test whether INCRESE was applicable to the characteristics of narratives from as many different contexts as possible.Four coders who had not previously used INCRESE and were not part of the NEON research team were recruited, comprising two from mental health research backgrounds and two from biomedical science and humanities research backgrounds, including coders with lived experience of mental health issues. The four coders were trained in the use of INCRESE v2, then each coded the same two narratives and discussed differences to assess concordance. The 95 recovery narratives were then independently coded by each coder using INCRESE v2 twice, two weeks apart (Round 1 and Round 2). Written evaluations were collected from the four coders on the feasibility and acceptability of the layout, items, responses, and descriptions in INCRESE v2. The acceptability of INCRESE v2 was reviewed by three NEON Lived Experience Advisory Panel (LEAP) members, comprising people with personal experience of mental health issues and recovery. INCRESE was finalized on the basis of statistical evaluation of poorly-performing items and further discussion of qualitative analysis findings within the wider research team.58 In this study, the research questions related to the feasibility of INCRESE  and the acceptability of INCRESE .Written evaluations of coders and LEAP members were analyzed using a semantic approach to thematic analysis, wherein data is searched to find repeated patterns of meaning in relation to the research questions.Data were analyzed using the following criteria: (1) data completeness, (2) response distributions for each item, including floor and ceiling effects (<10% of responses), and (3) exploring the scale score distributions at each time point (Round 1 and Round 2).59 was computed on items with nominal values (un-weighted kappa) or ordinal values (weighted kappa). Round 1 and Round 2 data were analyzed separately. Kappa value categories were defined in advance as 0.00–0.20 (None to slight); 0.21–0.40 (Fair); 0.41–0.60 (Moderate); 0.61-0.80 ; and 0.81–1.00 (Almost perfect agreement). Items with a kappa > 0.40 were identified as acceptable. To assess item-level test-retest reliability, the intra-class correlation coefficient (ICC) was calculated with a two-way random effects model. Quality of agreement was classified as: 0.00–0.19 (Poor), 0.20–0.39 (Weak), 0.40–0.59 (Moderate), 0.60–0.79 , and 0.80-1.00 (Almost complete)60. Items with ICC > 0.40 were identified as acceptable. All statistical analyses were performed using SPSS .To assess item-level inter-coder reliability for agreement among all four coders, Fleiss’s kappa coefficientINCRESE v1 comprised 68 items in five sections: Narrative Eligibility, Narrative Modality, Narrator Characteristics, Narrative Characteristics, and Narrative Content.In relation to feasibility, coders evaluated instructions and descriptions of items as clear and thorough, with most items being straightforward to identify. Items in Section Four (Narrative Characteristics) were evaluated as more subjective and therefore more difficult to code. Some items could not be applied to all narratives, for example age of narrator in some audio-based narratives, and instructions were changed to reflect this. Other refinements to improve feasibility are shown in In relation to acceptability, administrative burden was evaluated as acceptable on the whole, although some negative impacts  were reported from coding large numbers of narratives without a break. Coder wellbeing was also both positively and negatively impacted by the process of coding recovery narratives. For example, coders reported both being inspired by narratives and finding it hard to read about the painful experiences of some narrators. To address this, a section on coder wellbeing was added to INCRESE instructions, wellbeing issues were highlighted and strategies suggested in INCRESE v2 training, and debriefs were offered to coders during the formal evaluation.The language used in INCRESE v1 was found to be more appropriate for psychometric measures than an inventory of narratives and was changed to address this: for example, ‘raters’ was replaced with ‘coders’ and ‘measure’ became ‘instrument.’ Other refinements to improve acceptability are shown in http://www.researchintorecovery.com/increse.INCRESE v2 comprised 82 items in the same five sections. The sample narrative referred to in the introduction was coded as one of the 95 narratives. Worked examples of this and other narratives coded using INCRESE can be downloaded from In relation to feasibility, coders evaluated INCRESE v2 as easy to apply on the whole, due to the training provided and clear instructions. Items from Section 4 (Genre and Use of Metaphor), and some narrative formats (namely poems and images) were seen as more subjective and harder to code. Suggestions were made to amplify descriptions, for example including ‘self-help/own learning’ in the ‘education’ item of Section 5 (Narrative Content). Three sets of potentially overlapping items were reported in Section 5, between ‘death/threatened death,’ ‘injury/threatened injury,’ and ‘self-inflicted injury/self-neglect;’ between ‘formal peer support’ and ‘informal peer support;’ and between ‘hobbies/interests’ and ‘creative activities.’In relation to acceptability, coders identified fatigue as a negative effect of the administrative burden involved but reported having enough time to complete the task . Therefore, INCRESE was deemed acceptable in terms of administrative burden. Coders identified that item labels describing content warnings were stark and could in themselves be distressing to encounter. Members of the LEAP independently identified the same issue. Item names were changed in the final version to reflect this, using alternatives generated by the LEAP.In relation to statistical analysis, completeness of data was very high, with <1% missing data for each item. No ceiling or floor effects were identified. The inter-coder reliability and test-retest reliability for Sections 1 (Narrative Eligibility), 2 (Narrative Mode), 3 (Narrator Characteristics) and 4 (Narrative Characteristics) are shown in Inter-coder reliability for round 1 coding ranged from 0.58 (Moderate) to 1.00 (Almost perfect), and for round 2 coding ranged from 0.63  to 1.00 (Almost perfect), with the exception of one outlier item #75 (Previous life circumstances) which had only Fair agreement. Coder 4 had the most variance in coding from round 1 and round 2. When controlling for coder 4, eight items showed anomalous behaviour . Overall, 126 (79%) of the 160 item-level ICCs were >= 0.75, indicating inter-coder reliability is strong.Test-retest reliability (TRR) controlling for coder ranged from 0.41 (Moderate) to 1.00 (Almost complete), with the exception of the same outlier item #75 which had only Poor agreement. TRR not controlling for coder ranged from 0.57 (Moderate) to 1.00 (Almost complete), again with the exception of item #75 which had only Weak agreement. Three items performed less well in terms of TRR: #22, #34 and #38. Overall, most items had a test-retest reliability of greater than 0.75, indicating test-retest reliability is strong.Coders had different levels of test-retest agreement, with the highest for coder 2 , followed by coder 3 , coder 1  and coder 4 . Coder 4 was the coder with least mental health-specific experience or training.All item names, responses, and descriptions were reviewed and refined, with a particular focus on items identified as problematic in the statistical analysis. For example, item #75 (Previous life circumstances) performed poorly in terms of both inter-coder and test-retest reliability so was deleted from the final version. For item #34 (Relationship with recovery), coding by the two coders with non-mental health research backgrounds was less reliable, so a definition of the specific meaning of recovery in mental health was added to the final version to address this.http://www.researchintorecovery.com/increse.All refinements made at items and response level to produce the final 77-item version of INCRESE are shown in 62INCRESE has satisfactory acceptability, feasibility, and reliability across time and between coders. It comprises 77 items across five sections: Narrative Eligibility, Narrative Mode, Narrator Characteristics, Narrative Characteristics, and Narrative Content. 71 items characterize manifest content and six characterize latent content . The inclusion of these six items links this study to other content analysis-based studies within mental health research which incorporate latent content in addition to the more traditional focus on manifest content alone.65 on vicarious traumatization among mental health professionals and researchers,68 and on the wellbeing of online content moderators.70 Organizations utilizing INCRESE should select approaches that support coder wellbeing. Future research may focus on identifying the most effective approaches to supporting coder wellbeing when using INCRESE or similar tools, for example, by experimentally comparing different approaches to reducing harms.Using INCRESE can impact on the wellbeing of coders, with both positive and negative effects being reported. This links to previous work on researcher wellbeing when exploring distressing topics,Strengths of this study include an inclusive definition of recovery narratives, a strong theoretical basis for each included item, the use of coders from diverse backgrounds, thorough ethical consideration of both coder wellbeing and the issues raised by characterizing narratives, the coding of a large number of narratives, the inclusion of a pilot stage as well as a formal evaluation, and the testing of both inter-coder and test-retest reliability.71Limitations include that INCRESE is not suitable for use with group or collective narratives. INCRESE was not tested on longer recovery narratives such as documentaries or memoirs, so validating the inventory against such material could be a future research study. No mechanism was included in the study to assess the effects of coder fatigue; future research may explore this. Lastly, some items within the inventory are more difficult to apply to image-based recovery narratives and to more symbolic forms of prose, such as poetry. Other tools may need to be developed which can characterize such narratives more meaningfully. This is particularly important as evidence suggests that more symbolic forms may better support meaning-making for particular forms of mental health distress, as a recent conceptual review on understanding psychosis through poetry suggests.Five uses for the Inventory of Characteristics of Recovery Stories (INCRESE) are envisaged.First, INCRESE can be used to create catalogues of narratives for both mental health practitioners and individuals experiencing mental health distress to draw on. This would facilitate the choosing of narratives judged likely to be most helpful at a given time, with the aim of maximising benefit and minimizing harm for the recipient.72 Future research is planned to assess the effectiveness of the instrument in assisting individuals to find helpful stories.72Second, INCRESE can characterize collections of narratives for use in narrative-based online interventions. Within the NEON study INCRESE will be used to characterize a large database of narratives called the NEON Collection, to enable participants to browse stories by categories. In addition, a machine learning algorithm will use the data produced by INCRESE to choose individual narratives to present to participants opting to be ‘matched’ to stories.Third, as its name suggests, INCRESE can be used as a tool for organizations using recovery narratives in campaigns or on websites, and for curators of recovery narrative collections to consider the level of diversity within the narrative collection. The inventory will identify gaps, enabling the scope of the collection to be established and under-represented narrators or narrative types to be targeted for inclusion as appropriate. This may increase the likelihood of a positive connection with narratives for a wider variety of recipients.www.likemind.nhs.uk/your-stories) could be evaluated in relation to whether they include narratives from across the range of recovery narrative characteristics, including people who use a non-diagnostic explanatory framework (item #30), or whose recovery trajectory is not upwards (item #36). This would allow the extent to which concerns that “personal stories from consumer/survivors have been harnessed by mental health organizations to further their interests” are valid to be empirically investigated.Fourth, INCRESE can be used as a tool for researchers to assess the extent to which narratives available from organizations span a diverse range of narrative characteristics. For example, stories on health service websites (e.g., 75 to help people avoid the perceived harms which may be caused by certain narrative content. Although some empirical research is emerging,19 there is minimal empirical evidence on whether trigger warnings are helpful or not in managing distress for people who have experienced trauma. INCRESE could be used in future research to identify particularly beneficial or harmful characteristics for particular populations and to generate evidence-based guidance on the use of narratives and their potential benefits and harms.Fifth, INCRESE can be used to provide evidence about particular characteristics that are more likely to be beneficial or to trigger distress in recipients. One way in which avoiding potential harm to recipients has been attempted in this field is the contested issue of providing content warnings, also known as trigger warnings, within collections of recovery narratives, particularly those online. Graphic description of subjects such as violence, death, or abuse are thought potentially to trigger distress similar to the original trauma in those with existing mental health conditions such as post-traumatic stress disorder. Content warnings have been widely used online and within education and professional training contextsThis study also carries implications for designers of content warning systems. Reliability of content warning items in INCRESE ranged from moderate to almost perfect/almost complete agreement but were not perfectly reliable. Other content warnings systems may thus also not be perfectly reliable; hence, designers of content warning systems should assess and manage any lack of reliability, either through use of multiple coders, or by providing recipients with the information that the categorization may not be complete.INCRESE is a standardized and reliable inventory to characterize mental health recovery narratives, which is feasible for use by coders without a narrative research background, and acceptable to both coders and people with lived experience of mental health issues. It has the potential to be used not only to help individuals and practitioners to find the stories which may be most helpful to them, but also to enable curators and researchers to identify bias or lack of diversity within collections and narrative-based campaigns or interventions.


Sex pheromones used at higher concentrations than their availability in insects have been successful in decreasing the insect population. Furthermore, botanical oils have also had negative impacts on the insect population level. In the first study, we investigated the combined effect of sex pheromones and plant oils on mating of almond moth. The second study sought the burrowing ability of almond moth larvae through different flour media. Mating declined on the presence of pheromones and botanical oils, whereas burrowing ability differed with flour type. Exposure to spinosad affected both mating success and larval penetration through flour media. The findings of this study reveal that various applications of sex pheromones, plant oils and spinosad ensure the better protection of cereal-based agricultural produce from insect infestation during storage.Cadra cautella   using its sex pheromone components, -9,12-tetradecadienyl acetate (ZETA) and (Z)-9-tetradecadien-1-yl acetate (ZTA), is successful in its population management. In addition, botanical oils have extensively been investigated in stored product pest management, but the effect of synthetic sex pheromones on the mating of C. cautella in the presence of plant volatiles is still unknown. Spinosad is used in food facilities as a contact insecticide but, if C. cautella larvae burrow into food, they may escape from spinosad. Importantly, the impact of spinosad on burrowing ability of C. cautella remains unknown. Therefore, the objectives of this study were to determine the effects of sex pheromone components ZETA and ZTA in the presence of botanical oils on the mating of C. cautella and the burrowing ability of C. cautella larvae in different types of flour treated with spinosad. In the first study, male and female moths were introduced into the cubicle having botanical oils and pheromone components. The mating status of female moths and male moth attraction to the trap was determined. The control experiments had only the botanical oils or pheromones. In the second study, burrowing ability of C. cautella larvae through different flour types was evaluated over 10 d. The flour was sprayed with spinosad (treatments) or water (controls). The mating success was higher with botanical oils alone but declined with exposure to pheromone either alone or combined with botanical oils. No differences in male attraction to traps were observed in botanical only, pheromone only or pheromone + botanical oil treatments. The burrowing of C. cautella larvae differed with flour type and spinosad altered burrowing ability. Thus, we conclude that the mating and burrowing of C. cautella is influenced by its pheromone and by exposure to botanicals and spinosad.Mating disruption of  There is an increasing demand for food due to the rapid increase of world population by 83 million people every year . Post-haCadra cautella   is an important pest for storage losses in Sri Lanka [C. cautella and other stored-product insects are typically contact insecticides [ri Lanka  and otheri Lanka ,12,13. Iri Lanka ,16,17,18cticides , fumigancticides ,21,22, hcticides , and aercticides . Despitecticides , non-tarcticides ,27 and tcticides . Therefocticides ,30,31.C. cautella [C. cautella release the two-component sex pheromone, -9,12-tetradecadienyl acetate (ZETA) and (Z)-9-tetradecadien-1-yl acetate (ZTA) in a natural 14:1 ratio [C. cautella [C. cautella pheromones and its implications for behavioral manipulation [Monitoring and suppressing insect populations are important parts of integrated pest management program (IPM) ,33,34. Mcautella ,36,37. F:1 ratio . ZETA is:1 ratio . The syncautella ,40,41. Rpulation  have augC. cautella [C. cautella and the extensive amount of research conducted on different uses of pheromone components of C. cautella [C. cautella mating disruption. However, it is reported that the rise of systems-level thinking in agriculture has resulted in significant innovation in food production [Botanical compounds have extensively been investigated for application in stored-product insect pest management. Many studies have documented the ability of botanical insecticides to induce mortality, repellency, and other behavioral changes in stored-product insects ,48,49,50cautella ,52. Howecautella ,42, theroduction . Thus, iC. cautella, larvae feed on durable goods and contribute to contamination, decreasing the quality of commodities to unacceptable levels for human consumption [C. cautella frequently burrow, there has been little formal study of this behavior in food sources [C. cautella may lead to inaccurate predictions of population development after exposure to contact insecticides, as larvae may be able to escape exposure in refugia found beneath the surface of food sources.In sumption . Though  sources . Lack ofC. cautella is spinosad. Spinosad is a biorational, reduced-risk insecticide developed from the bacterium Saccharopolyspora spinosa [C. cautella [C. cautella larvae to spinosad and its effect on burrowing ability has not been investigated. Therefore, the objectives of this study were to determine the effect of sex pheromone components with or without botanical oils on the mating of C. cautella moths, and to determine the burrowing ability of C. cautella larvae in different types of flour treated with spinosad or water.One potential contact insecticide that may affect burrowing of  spinosa . It is e spinosa ,57,58,59C. cautella was established using adult moths collected from a rice milling center at Puliyankulama, Anuradhapura, Sri Lanka. The adult insects were introduced to rice flour medium in a 5 L plastic bottle with a perforated lid (50 adults on 250 g of rice flour). These C. cautella cultures were kept inside an incubator with complete darkness  at a constant 33 ± 1 °C temperature and a 60 ± 5% relative humidity.The laboratory culture of Cadra cautella adults were sexed during the pupal stage by referring to the two nodes present on the ventral side of the 8th segment of male pupa under a dissecting microscope  [, Italy) . Individ2) to allow exchange of air from inside the cubicle and the outside environment. Velcro  was used to attach the polythene and net to the cubicle.Four cubicles (1.5 m × 1.5 m × 1.5 m length:width:height) were constructed using metal frames. The top, bottom and two opposite sides of each cubicle were covered by transparent polythene (25 mm thickness). The remaining two opposite sides of each cubicle were covered using an insect proof net , sandalwood oil , neem oil (Azadirachta indica), cinnamon oil (Cinnamomum verum), citronella oil (Cymbopogon nardus) and mee oil (Madhuca longifolia) . Neat 2 mL of each botanical oil was measured by using a micropipette  and added into separate plastic bottles (3.6 cm diameter and 6.2 cm height).The following botanical oils were used: camphor oil (2)  and attached to a piece of rigiform using a needle, which was then placed on a Petri dish (80 × 15 mm). The Petri dish was located inside a monitoring trap , which was hung from the center suspended 75 cm distance from the top [The plastic bottle containing the botanical oil was placed at the middle of the bottom side of the cubicle. In the control experiment 1, the cubicle contained the botanical oil alone (no pheromone components). In the control experiment 2, the cubicle contained only the pheromone blend . A mixture consisting a total of 120 μL of ZETA and ZTA pheromone components (in a 5:1 ratio as described above) was added to a triangular proprietary, slow-release filter paper . A population size of 20 moths , which was found to be most susceptible to mating disruption from prior work , was useFollowing each experiment, the entire facility and the cubicles were washed using a biodegradable detergent  to remove any pheromone residues and was allowed to air dry before the next round of experiments. Furthermore, before the next experiment, the contaminated air inside the room was removed using an exhaust fan.The temperature and relative humidity profiles inside and outside the cubicles during the experiment were recorded by data loggers . The average values of temperature and relative humidity inside and outside of the cubicles were 30 ± 1 °C, 63 ± 1% RH and 32 ± 1 °C, 64 ± 2% RH, respectively.2-tests with Bonferroni correction to the p-value were used for multiple comparisons.The experiment was conducted as a completely randomized design (CRD). Each control or treatment experiment had four replicates. Responses consisted of females (mated or unmated) and males (captured or not), which were analyzed using a generalized linear model based on a binomial distribution with a logit link function. Explanatory variables included the presence of pheromone (yes or no), botanical oils (as above), and their interaction. Overdispersion was checked and not found to be a problem. Upon a significant result from the model, χCadra cautella larvae aged 10 d were used in this experiment. These larvae were obtained from C. cautella cultures reared as described in C. cautella larvae, as described below.The following types of flour were purchased from a local retailer and used in the experiment: rice flour, maize flour, mung bean flour, cowpea flour and atta flour (hard wheat flour). For each replicate of the types of flour, 250 g of that specific flour was weighed into separate plastic sealable bags (20 × 30 cm width:length). The commercial product ‘Success’  was used as the source of spinosad. A solution with the labeled rate of 25 ppm of spinosad was prepared by diluting the 100 µL of the commercial product in 100 mL distilled water. Next, each replicate 250 g of flour weighed above was placed on a piece of aluminium foil (approx. 1 mm thickness). A total of 3.75 mL of the spinosad solution was sprayed on to each 250 g flour sample by using an artist’s air brush  . ImmediaC. cautella larvae through a given type of flour. The treatments consisted of spinosad-treated (different types as mentioned in 2.3.2) or water-treated flour. Twenty C. cautella larvae (10 d old) were introduced into each vial. The vials were maintained inside an incubator  at 33 ± 1 °C and 60 ± 5% RH. In each vial, the daily average burrowing depth of C. cautella larvae was determined by measuring the distance burrowed from the reference points  along the vertical lines. In addition, the total cumulative burrowing depth was noted as the total cumulative distance burrowed over 10 d.Glass vials  were used to assess the burrowing behavior for the experiment. The volume level filled by 40 g of flour without compaction was marked on the circumference of the vial (approx. at 18 cm from bottom). Four vertical lines in each cardinal direction, including North (A), South (B), East (C) and West (D), were marked on the circle. These points served as reference points for noting the burrowing of C. cautella larvae in different types of flour were analyzed by using ANOVA procedures of statistical analysis system (SAS) [The experiment was conducted as a completely randomized design (CRD). Each treatment or control experiment had four replicates. Differences in the cumulative burrowing depths of em (SAS)  with meaχ2 = 38.0, df = 1, p < 0.0001), but not the botanical oils alone , significantly affected the number of mated females  , the botanical oils , nor their interaction  affected male attraction  . There wtraction .C. cautella larvae gradually increased as more time elapsed. In the water-treated controls, the burrowing depth varied from 0.36 ± 0.02 cm (atta flour) to 2.04 ± 0.15 cm (maize flour). In the spinosad-treated flour, the minimum and maximum burrowing depths were 0.69 ± 0.02 cm (atta flour) and 3.2 ± 0.14 cm (rice flour), respectively. Overall, the burrowing of C. cautella larvae was higher in the spinosad-treated flour than their water-treated controls, except for maize flour   . In maizIn rice flour, mung bean flour and cowpea flour, there was a significant difference in the burrowing depth between the spinosad-treated and water-treated flour . SpecifiPlodia interpunctella (Hübner) , the Indianmeal moth, including olive, sunflower and corn oils. Previous work has found that plant volatiles stimulate the release of the female ermine moth’s sex pheromones to attract males  [C. cautella successfully mated in the presence of camphor oil, citronella oil and neem oil, which supports prior findings that plant volatiles may modulate mating success among moths. These studies have shown that plant volatiles may synergize attraction to their natural sex pheromones [C. cautella is 14:1; addition of botanical oils could have changed the effective concentration of the lure in the cubicles, with ramifications for mate-finding. In other systems, there may be reduced responses to pheromone after exposure to plant or non-host volatiles some non-Lepidopteran taxa, as was shown to be the case with Dendroctonus frontalis LeConte (Coleoptera: Scolytidae), the southern pine beetle, following exposure to 4-allyl anisole in Loblolly pine (Pinus taeda), and for some other bark beetle species [C. cautella following exposure to plant volatiles, considering multiple tactics together may have important implications for their implementation for food facilities.The mate-finding behavior of lepidopterans mainly depends on males following sex pheromone plumes to their point source  ,68. Nansnomeuta) . In thiseromones ,71,72,73 species ,75,76. NC. cautella is also emitted by the unrelated taxon Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), the beet armyworm. The plant volatiles linalool and myrcenebenzaldehyde have been shown to increase the response of male S. exigua to their natural sex pheromone [Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) [The chemical -9,12-tetradecadienyl acetate secreted by heromone . Howeverhilidae) . It may C. cautella readily burrowed in different types of flour. Prior to this work, little information was available on the burrowing ability of C. cautella larvae through different commodities. Bell [C. cautella larvae will wander in the food and may bore through packaging material. It has also been documented that C. cautella larvae can penetrate through a variety of packing materials, such as cellophane, polyethylene, paper material, polyvinylchloride and polypropylene films [P. interpunctella, a closely related lepidopteran species that shares certain behavioural characteristics with C. cautella [P. interpunctella and Trogoderma variabile Ballion (Coleoptera: Dermestidae) cannot penetrate through methoprene-treated foil packaging [T. variabile larvae that were able to penetrate methoprene-treated foil experienced deformities in the adult stage [C. cautella may be worth considering in combination with behaviorally based tactics, such as mating disruption and other biorational strategies in future research.The current study revealed that es. Bell  and Mulles. Bell  showed tne films ,81,82. Pcautella  is also cautella ,84, but cautella  and the cautella . By contlt stage . The useC. cautella larvae compared with water-treated controls. This suggests that burrowing behavior by C. cautella is flexible and inducible, and may result in behavioral resistance to the use of contact insecticides, whereby larvae will escape by seeking out refugia deeper in commodities. This may severely curtail the actual efficacy of contact or residual insecticides when used to treat sites of spillage or bulk commodities with existing infestations of C. cautella. Furthermore, unless commodities are thoroughly examined during cleaning and remedial actions, C. cautella infestation may continue unabated until the population increases to higher levels and flying adults emerge. Furthermore, burrowing might negatively affect the pheromone and kairomone-baited trapping of conspecific C. cautella when contact insecticides are used in a food facility, because their application will push individuals further into commodities. However, adult moths will likely not be affected, so monitoring in long-standing infestations or for flight activity traps may not be impacted. Future experiments need to investigate the interaction between contact insecticides and monitoring for C. cautella.Our study has shown that spinosad-treated flour results in elevated burrowing activity for C. cautella larvae is affected by spinosad. It was also found that once adults emerged, their mating can be disrupted by exposure to pheromone and botanical oils used either alone or in combination. Previous work has shown how C. cautella larvae can be controlled by spinosad [C. cautella larvae seek refuge in flour treated with spinosad, and thus escape monitoring, their population can still be managed once they emerge as adults by using pheromones, botanicals or their combination as highlighted in the current work. Overall, this work illuminates the behavior and mating of C. cautella, and particularly how it is influenced by biorational pest management strategies. This information may be used for protecting stored food from infestation by this devastating pest. The current study found how the burrowing behavior of spinosad . Even ifC. cautella in the presence of the synthetic sex pheromone. The burrowing depth of C. cautella larvae varies with the type of flour, but generally is increased in spinsoad-treated flour compared to controls for rice, mung bean, and cowpea flour.In conclusion, camphor oil, citronella oil and neem oil increase the mating success of


Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment. These vesicles are passaged through the endo-lysosomal system and their contents digested and absorbed.16 A major conceptual challenge is to understand how cups are shaped and close. This requires F-actin to be organized in space over a scale of microns, first to make a cup, and then to bring its lips together for closure, all in the absence of guidance from a solid particle or coat. However the signaling lipid phosphatidylinositoltrisphosphate (PIP3) plays a key, but poorly understood, role in organizing macropinocytosis across species.19Macropinocytosis is driven by actin polymerization beneath the plasma membrane, which in different cells produces cups or sometimes flaps of membrane as the primary macropinocytic structures.Dictyostelium amoebae are prolific macropinocytes, as well as phagocytes, and can imbibe their own volume of medium in a few hours.21 Macropinosomes form around discrete domains of PIP3 (an ether-linked lipid in Dictyostelium22) in the plasma membrane, which can be microns across24 and are genetically essential for macropinocytosis.19 Domains also include active-Ras, which activates PI3-kinase, and active-Rac, which activates actin polymerization25 and are regulated by RasGAPs such as (NeuroFibromatosis 1 (NF1) and RGBARG .2825 which they shape by attracting a ring of dendritic actin polymerization to their periphery. This ring is visualized by reporters of the Scar/WAVE complex—an activator of the Arp2/3 complex—which form an irregular necklace around PIP3 domains in all cases investigated. Ringed actin polymerization helps explain how cups are shaped but not necessarily how they close.We have proposed that PIP3 domains are templates for macropinocytic cups,Dictyostelium or any other cell type, and although we previously obtained static images of Scar/WAVE rings, we could not track them over the lifetime of cups to discover how they participate in expansion and closure.Limitations in microscopy have hindered investigation of PIP3 domains and macropinocytic cups. Although cups are readily visible, they are difficult to follow over time and in 3D due to their size, dynamism, and the light sensitivity of cells. It is not known how cups close in 29 To match this advance in microscopy, image-analysis methods need extending to 3D time series, to segment cells and PIP3 domains and to measure fluorescent intensities along the plasma membrane.30Light-sheet microscopy gives a new approach to these problems, allowing macropinocytic structures to be followed in 3D from birth to closure.Dictyostelium model with unprecedented detail. We describe how macropinocytic cups are structured and show that they have two distinct ways of closing: at lip or base. We find that PIP3 domains maintain a ring of Scar/ WAVE throughout expansion and closure and that cups close when their PIP3 domain stops expanding. We propose a conceptual model for the evolution of macropinocytic cup shape over time, whereby stalled expansion of domains causes cups to close. This “stalled wave” hypothesis is tested through a simple physical model.Here, using lattice light-sheet microscopy and custom computational analysis, we follow macropinocytosis and PIP3 domains in the Dictyostelium cells for periods of 5–10 min in 3D, at around 0.3 Hz, with two fluorescent channels. Cells were observed at low density in a simple medium, where they maintain macropinocytosis for at least a day.31 Using strain Ax2, with paired reporters for PIP3 (PkgE-PH-GFP), marking the membrane of macropinocytic cups,25 and F-actin (lifeAct-mCherry) revealing the actin cytoskeleton,32 we gained an overview of how macropinosomes form.Using lattice light-sheet microscopy, we followed macropinocytosis in 33 These cups expand and close, with maximum-intensity projections showing macropinosomes sealing and being released from the plasma membrane , which do not produce macropinosomes unless they escape up the side of the cell. We regard these as frustrated macropinosomes.Cups form on any exposed part of cells and frequently move over the surface as they develop. On average, cells have 2.71 ± 1.71 PIP3 domains at any moment and produce 1.8 ± 1.2 macropinosomes per min , rangingDictyostelium cells resemble macrophages in forming macropinosomes from cups,14 but differ in that we do not observe them forming from linear ruffles or flaps, nor is there a major involvement of F-actin “tentpoles.”16Overall, Unexpectedly, we found that cups can close in two different ways—lip and basal closures—of which lip closure is roughly twice as common  and S3. In basal closure, cups deepen and narrow, then constrict locally before pinching off a macropinosome near the base. A large proportion of the PIP3 domain remains in the plasma membrane, where it can yield further macropinosomes. This sequence generally terminates by closure at the lip and extinction of the PIP3 domain.Macropinosomes formed by either route frequently remain attached to the cell surface by a thin tether, which can persist for 10 s or more, before rupturing  and S1G.Cups can also fail. Most commonly, the PIP3 domain simply fades away, followed by its associated F-actin. Or there can be an abortive closure in which the cup constricts but does not close .D. discoideum isolate NC4, and parent to Ax2.36 Ax2 was selected from DdB for growth in standard liquid medium, which is largely due to deletion of the RasGAP NF1, resulting in Ras activation and massively upregulated macropinocytosis.26To test the generality of our observations, we examined macropinocytosis in strain DdB, which is an early derivative of the original Dictyostelium macropinocytosis (DdB cells (with intact NF1) were grown in enriched liquid medium to stimulate macropinocytosis and examined using the PIP3/F-actin reporter combination. They produce generally smaller PIP3 domains and macropinosomes than Ax2 cells. Both lip and basal closures, as well as tethers are observed, showing that these are common features of ocytosis .To understand how macropinocytic cups are structured and how they change during closure, we devised computational tools to map their individual components over space and time and to combine these into composite maps. For microscopy, a reporter of interest was combined with a constant PIP3 reporter and domains defined by first segmenting the cell surface using both reporters and then the domain itself, using only the PIP3 reporter . Intensi37) falls just inside the lip, with PIP3 levels elevated over the entire inner surface of the cup but falling off beyond the lip .The flexible plasma membrane requires mechanical support to shape it into a cup. To understand how this is achieved, we mapped F-actin and some of its binding proteins in cups. F-actin forms a continuous scaffold, extending from top to bottom of cups and some distance beyond the rim , and S4.40 It is strongly recruited to cups, concentrating toward the lip. Single-headed myosins link F-actin to membranes,41 with several strongly recruited to macropinocytic cups. These localize differentially, with myosin-1B recruited toward the periphery of cups, where its staining is often fragmented, whereas the PIP3-binding myosin-1E is evenly concentrated toward the base, closely matching the distribution of PIP3, as previously described.43 The abundance of myosin-1 proteins in cups suggests that the plasma membrane is more firmly attached to the actin cytoskeleton in cups than elsewhere. Carmil44 is only modestly enriched in cups.F-actin-binding proteins are recruited in distinct spatial patterns, broadly confirming previous work  and S3. Although microtubules  moved actively within the cytoplasm and make fleeting contacts with macropinocytic structures, we did not detect any prolonged association between them suggestive of a structural role in cups (not shown). We did not notice any appreciable differences between cups that close at lip or rim, and both were included in the analysis. Nor were any acute changes detected in the patterns of any of the reporters found before cups closed, except possibly in F-actin, whose signal intensified about 20 s before closure as shown by space-time plots –2G.25 we again found that Scar/WAVE forms striking rings around PIP3 domains, which could now be followed through the entire lifetime of cups  subunit instead of HSPC300 as used previously, of cups . This sh of cups  and 3G.  of cups , as alsoThese results show that macropinocytic cups are organized around PIP3 domains, which continuously activate a ring of actin polymerization at their periphery. This creates a cup-shaped F-actin scaffold, including specialist F-actin-binding proteins. In the concave parts of the cup, the membrane must be linked to the scaffold strongly enough to prevent it from detaching (delaminating). Myosin-1, and other cross-linking proteins, may perform this role. The general structure of the cup does not appear to change as it switches from expansion to closure, implying a biophysical rather than molecular mechanism for closure.15We examined cups closing at the lip in en face views, using the Scar/WAVE reporter. This clearly shows that these cups close by concerted inward movement of the lips, until the orifice is sealed . We rare46 However, we could not detect myosin-II at the lip, or elsewhere in cups , we made detailed measurements of domain area to test the correlation.First, we analyzed a set of simple domains that did not split and could be followed from their origin to their closure at the lip . All ori2 s−1 (n = 11) and 0.30 ± 0.34 μm2 s−1 (n = 27), respectively  Dendritic actin polymerization occurs in a ring around the perimeter of PIP3 domains. It is protrusive and pushes against the membrane. (2) Macropinocytic cups are supported by a continuous F-actin scaffold. (3)The plasma membrane adheres to this scaffold via cross-linking molecules, such as myosin-1, and therefore resists delamination. (4) Cups close when expansion of the PIP3 domain stalls.T). Dendritic actin polymerization is assumed to occur only at the domain boundary with its force given by the parameter (kP). Polymerization creates a reaction force that drives a retrograde flow of F-actin inward, potentially applying a stretching force to the membrane of the cup. We explored its importance by varying how strongly this force is coupled to the membrane of the cup. Actin polymerization within the cup due to formins49 and the signaling dynamics sustaining PIP3 domains are not considered.The main components of the model are summarized in 34 The zone of actin polymerization moves outward and does not remain under the same area of membrane for long, so that the cup remains shallow. As the domain stalls, actin continues to polymerize under the same ring of membrane but does not capture more membrane.In the first phase of its life , the PIPP; T) constricts the cup, elongating the PIP3 domain and pushing its boundary toward the lip, keeping pace with the protruding lip and maintaining the position of the polymerization zone. Tubes form when parameter values lie just outside the range giving lip closure, whereas values just within this range provides the largest vesicles for a given membrane tension, although these cups take longer to close  throughout the life of macropinocytic cups, we find that it is recruited around PIP3 domains at all stages and whatever the size of the domain. The Arp2/3 complex, which is activated by Scar/WAVE is similarly recruited in rings  as is vasodilator stimulated phosphoprotein (VASP), which interacts with Scar/WAVE and may accelerate actin polymerization.53This study strongly supports the hypothesis that macropinocytic cups are shaped by a ring of dendritic actin polymerization formed around PIP3 domains.55 Our work provides an alternative mechanism. We find that macropinocytic cups can close at the base, as well as at the lip. Vesicles forming from the base of ruffles have previously been reported in macrophages,16 although whether they also undergo delamination remains to be seen.When a macropinocytic cup closes, the opposing membranes must be brought together, although they are initially microns apart. A purse-string mechanism, driven by myosin-II contraction at the lip of the cup, has been proposed for phagocytocytic cups.In our unified model, continued actin polymerization at the lip drives both types of closure: lip closure results when the lip turns inward, and basal closure results when it continues to elongate the cup, stretching the membrane until it delaminates from the scaffold and pinches off a macropinosome.Although we observe no clear evidence for a role of actomyosin contraction or microtubule motors, we cannot completely dismiss the possibility that they contribute to the inward stretching of the cup into the cell body. Importantly in our model, the force is simply imposed along the membrane and can apply equally to any mechanism. The key point is that any force that pulls the cup base away from the lip helps promote closure under a broader range of biophysical parameter space.50 although enacted differently, also incorporates the propositions of actin polymerization around PIP3 domains and stalled expansion. It, likewise, reproduces cup formation and closure at the lip and base, indicating that this is a robust consequence of the initial assumptions.Our modeling treats macropinocytic cups in 2D and only considers mechanical forces. Yet, even within these limitations, it readily reproduces an expanding cup that closes at the lip, and one that does not close at the lip, but elongates, as in base closure. The theoretical model of Saito and Sawai,35 They are likely based on a reaction-diffusion system, with auto-catalysis and cross-inhibition of diffusing molecules, including Ras, PIP3, and Rac. PIP3 recruits and activates proteins genetically required for fluid uptake by macropinocytosis, including the protein kinases Akt and serum glucocorticoid regulated kinase 1 (SGK), Leep1 (a regulator of Scar/WAVE), C2GAP2 (a RasGAP), and myosin-1,57 but these are not essential to form PIP3 domains. Active-Ras, which activates PI3-kinase, may be at the core of the domain-forming system, because Ras domains form in the absence of PIP3 domains,25 whereas, conversely, PIP3 domains are slaves to Ras activity because they are greatly expanded when Ras activity is increased by deletion of the RasGAPs NF1 or RGBARG.27PIP3 domains are complex structures that form spontaneously in the plasma membrane, including at the base of cells.58 This is consistent with PI3-kinases producing PIP3 within domains from PI4,5P2, and the PIP3 diffusing out for reversion by PTEN, whereas PI4,5P2 is replenished in the domain by diffusion from the surrounding membrane. This spatial separation of PIP3 production and destruction may be one aspect of the reaction-diffusion system that helps limit domain size. Finally, the ability of PIP3 domains to attract actin polymerization to their periphery may be due to the creation of an annulus, where Rac activation outweighs its inhibition.27We find that domains produce opposing gradients of PIP3 and PI4,5P2, as also do basal waves.59 Other cupped structures organized by PIP3 domains could include large phagosomes in Dictyostelium60 and circular dorsal ruffles in mammalian cells.61Extending our work, we propose that cupped structures of the plasma membrane are more generally created by ringed actin polymerization around PIP3 domains. Mammalian macropinocytic cups also feature a central domain of PIP3, which may similarly organize the cup and guide its closure.Dictyostelium and more widely.In summary, we have described macropinocytosis in greater detail than hitherto and gained mechanistic insights, leading to a unified model for how cups close at lip or base. The stalled wave model provides a framework for understanding macropinocytosis both in KEY RESOURCES TABLE⚬Lead contact⚬Materials availability⚬Data and code availabilityRESOURCE AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS⚬Dictyostelium transformation⚬Lattice light-sheet microscopy⚬Deconvolution and registration⚬Cell segmentation⚬Fluorescence projection⚬Annotation⚬PIP3 domain segmentation⚬PIP3 domain grading⚬Fluorescence measurements⚬PIP3 domain geometry⚬3D maximum intensity projections⚬Surfaces⚬Modelling⚬Base closure⚬Quantification and statistical analysisMETHOD DETAILS Detailed methods are provided in the online version of this paper and include the following: jason.king@sheffield.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jason King  under the persistent URL: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.till.bretschneider@warwick.ac.uk).Requests for information on image processing and computation should be directed to Till Bretschneider (Dictyostelium discoideum strains used in this study derive from the axenic laboratory strain Ax2(Kay),26 with the exception of the non-axenic isolate Ddb. Reporter strains are listed in key resources table and derive from Ax2(Kay)26 unless otherwise indicated. They were grown at 22°C either in HL5 axenic medium  in shaken suspension at 180 rpm, or on tissue-culture plates; or in association with Klebsiella aerogenes on SM agar plates. Non-axenic or poorly growing axenic strains, such as DdB, were grown in HL5 reinforced with 10% dialysed FCS (Gibco).All 5 cells/cm2 in 6-well tissue culture dishes in SUM low fluorescent medium .31Cells for microscopy were grown for several days in HL5 medium to maximally up-regulate macropinocytosis. They were prepared for microscopy by incubation for 2-24 hr at approximately 1065 generally to express two reporters, one of which was for PIP3 (PH-PkgE25). The PI45P2 reporter used was Nodulin,62 PI34P2 was PH-TAPP124 and PI4P was P4M.66 Microtubules were imaged using GFP-a-tubulin67 and F-actin by LifeAct.32 Cells were transformed by electroporation and selected on pre-grown bacteria with the appropriate drug.64 Plasmids are available from Addgene, or from the authors by request.Cells were transformed with plasmids from the Paschke/Veltman series,LLSM was carried out using a 3i Lattice Light Sheet instrument with a 0.71 NA long-working-distance water-immersion excitation objective and a 1.1 NA water immersion emission objective with a total magnification of 62.5x. Images were captured on two Hamamatsu ORCA-Flash 4.0 v3 sCMOS cameras (single channel each). The sheet pattern was a Bessel lattice of 50 beams, with an inner and outer numerical aperture of 0.493 and 0.550 respectively. The thermoelectric cooler system was set to 17°C to achieve a temperature of 22°C in the sample chamber.Before imaging, 10 ml of TetraSpeck beads (Thermofisher) were dried to a 5 mm coverslip  and mounted on the sample holder using vacuum grease . 10 ml of medium (SUM) was added to the bath and the sample left to equilibrate in the microscope for 30 min before being used for alignment and PSF measurement. Cameras were aligned by exciting the beads at both 488 nm and 560 nm and a PSF measurement taken for localization.Dictyostelium cells in 6-well plates were detached by pipetting up and down and allowed to settle for at least 15 min onto 5 mm coverslips added to the wells. A coverslip was mounted on the sample holder and transferred to the microscope containing 10 ml SUM in the bath. Samples were allowed to equilibrate before recording. Images were recorded in two channels using 1-2% and 5-10% laser powers for 488 nm and 560 nm respectively, depending on the reporter. 3D volumes were recorded at 0.3 μm step size (0.163 μm deskewed) for 120-150 planes with a 5-10 ms exposure. This resulted in an overall speed of 2.24-3.12 seconds per volume, depending on the configuration, and an -resolution of 0.104 μm/pixel. Volumes were recorded at this rate for 3–10 min. All Videos and images are maximum intensity projections.68 (50 iterations).Because the same microscope was used for all experiments, deconvolution was made more efficient by condensing one PSF for each channel and applying it to all videos for deconvolution. Comparisons between these deconvolved images and a sample of videos deconvolved using PSFs generated immediately prior to the videos showed no substantial change in intensity but did yield a small translation of the image. This translation was accounted for by measuring cross-correlations of the channels within a range of 5 voxels (twice the largest observed translation) and selecting the translation that yielded the maximum average cross-correlation for the whole video. Deconvolution was performed using the Richardson-Lucy method30 The segmentation method itself requires only two parameters. However, some pre- and postprocessing is often required to enable accurate segmentation, which increases the number of parameters.All videos were segmented using the curvature-enhanced random walker.To identify parameter configurations for the segmentation, videos assigned one of three groups: (1) those with PIP3 in the red channel, (2) those with PIP3 in the green channel without LifeAct, and (3) those with PIP3 and LifeAct. A parameter configuration that yielded accurate segmentation for most videos in each group was identified. Segmentations were manually verified, and any errors to segmentation of PIP3 patches were excluded from subsequent analysis. Parameter configurations for videos with a low segmentation accuracy of PIP3 patches were adjusted to improve the accuracy.Videos in group (1), which contains the majority of the videos, were segmented using similar parameter configurations, with 70% using a single configuration. Similarly, group (2) mostly used the same parameter configuration (80%), with small variation from this configuration otherwise. Group (3) had the highest variation in parameter configurations, due to the fact that neither PIP3 nor actin have a strong presence in the cytoplasm leading to segmentation errors. However, there was a large overlap in parameter configurations, with at most four distinct values for any given parameter.The segmentation error rate for each PIP3 domain was calculated by manually grading the segmented surfaces (further details below). An average of 6.97% +/- 0.83% (standard error) of frames were identified as having a segmentation error and were excluded from subsequent analysis.v on the surface, the image voxel coordinates along a line l normal to the surface through v were calculated. Line scans were truncated to avoid including voxels containing membrane at another part of the cell surface; if a point p in l is closer to another point on the surface than to v,then l is truncated at p and any points beyond p are discarded from l. A minimum distance of 2 voxels (approx. 0.2 μm) was imposed on the truncation to prevent zero-length line scan in highly curved parts of the surface. Lines had a maximum length of 1 μm (1.5 μm for actin fluorescence). Each vertex was assigned the maximum fluorescence value along its line scan. Background subtraction of the fluorescence was applied to the image prior to projection using a top hat filter of radius 10 voxels for all images.Triangulated surface meshes were obtained from the segmented videos using Matlab’s isosurface algorithm. These meshes take the form of a set of vertices and a set of triangular faces that connect the vertices. In order to measure the fluorescence on the membrane, the fluorescence values in the image were projected onto the surface. This was performed using line scans along the direction normal to the surface. For a given vertex For the purpose of computing line scans, all surfaces were smoothed using Laplacian smoothing. Projecting actin fluorescence onto the surface additionally required shrinking the surface areas of high mean curvature. This is because the actin cortex lies slightly below the membrane, and therefore the peak fluorescence is farther from the surface than other markers. Given that highly curved areas introduce extreme truncation of line scans, this would lead to the actin at the lip of the macropinocytic cups being reduced. We solved this problem by shrinking the surface under mean curvature flow, which forces the surface to shrink in highly curved regions. In order to avoid simultaneously shrinking the cups during this process, mean curvature flow was only applied in regions of positive mean curvature. Positive mean curvature flow was applied until any point on the surface had moved up to a maximum of 5 μm from its original position.63 Markers were placed on the surface using the marker placement tool in MiCellAnnGELo at locations within PIP3 domains in each video. For de novo domain formation events, markers were placed in frames prior to this event at the approximate location of the forming domain. Similarly, after domain elimination events markers were placed in subsequent frames at the approximate location of the extinguished domain. When a single domain contained more than one cup, separate markers were placed in each cup. Markers were grouped in sequences by first measuring the distance from markers in each frame to markers in the following frame and pairing each marker to the closest marker in the following frame. This pairing process was repeated in reverse, and two markers were grouped together if they paired in both directions. Occasionally, markers moved large distances between frames, for example during a closure event, leading to them no longer pairing with the corresponding marker in the previous frame. These markers we regrouped later in the pipeline by comparing manual annotations of sequences and joining sequences with no overlapping frames, but with the same manual annotations in each frame.Surfaces were annotated using MiCellAnnGELo, a VR software for rapid annotation of surfaces.37 of the PIP3 fluorescence values on the surface. Threshold values were computed for each marker using the histogram from vertices nearest to the given marker in the geodesic sense. In cases where structures such as cup lips or ruffles present as thin protrusions with PIP3 on only one side of the structure, a low level of PIP3 will be projected onto the other side in the surface representation due to spreading of the fluorescence during image capture. In order to avoid including the opposite side of such structures in the domain, mean curvature is used to separate the two sides, while also providing geometric definitions for cups and lips.Domains were identified around each marker using Otsu thresholdingcup is defined as a connected area on the surface about the marker, where all vertices have PIP3 intensity above the Otsu threshold and mean curvature less than 0 (concave). The lip is defined as the band of vertices, grouped by geodesic distance from the cup boundary, with the highest average mean curvature (highly convex). The domain about the given marker is the set of all vertices within the lip contour with PIP3 value above the Otsu threshold.For a given marker, a cups, domains,or lips for two markers intersect, it is necessary to further segment these structures to maintain correspondence with the markers. This segmentation was performed by dividing the structures along the line of equal geodesic distance between the two markers. Domains that required segmentation in this manner were automatically graded as being split domains.In cases where domain for ease of processing. The frame when this false domain changes to a true domain were automatically marked as domain formation events, and the reverse as domain elimination events.For cases where the PIP3 intensity was below the threshold value at the marker, the marker vertex and all vertices sharing a face with that vertex were marked as the cups, domains, and lips were additionally smoothed by dilating , filling (adding connected components surrounded by the vertices of the given feature in the mesh graph), and eroding (the reverse of the dilation operation).Each of the De novo domain formation, domain elimination, lip closure, base closure, failed closure, and domain splitting. Domains were also assessed for cell segmentation and domain segmentation errors, which were noted and excluded from subsequent analysis. The automated grading above provided an initial set of gradings, which were adjusted where necessary.Each domain was subsequently graded manually for each frame to mark the events of 69 This notebook allows the user to change surface colours between fluorescence and domains, as well as providing controls for opacity and colour scaling. Buttons for grading allow the marking of base, lip, and failed closures, as well as domain splitting, segmentation and domain detection errors, and noting the start and end of the domain.Gradings were made by visual comparison of the surface and the 3D maximum intensity projection of the video. A custom Jupyter notebook was designed to allow rapid grading of domains, using ITKWidgets with PyVista to render the surfaces.For a given marker, frames between domain start and end gradings are taken to be the times when the domain is present. For domains with no start frame (either formed prior to the start of the video or formed from splitting), the domain is assumed to start from the first frame that the corresponding marker is present. Similarly, domains with no end frame are assumed to end when the marker is no longer present. Frames where the domain was not automatically detected for a given marker (see previous section) but manually marked as having a domain (and vice versa) were automatically graded as having a domain segmentation error.In the subsequent analysis, measurements of a domain graded as split or to have an error in a given frame were excluded for that frame. Where time-dependent measurements were taken, domains which were graded as split at any time in the interval being measured were excluded.Fluorescence measurements were made relative to the domain boundary. Additionally, the mean curvature of the surface was computed and processed in this manner, without the background subtraction step. The surface outside and inside this boundary was partitioned by geodesic distance to the boundary, where bands outside the domain were equally spaced at 0.2 μm, and bands inside the domains were equally spaced to 0.1*domain depth (defined below). Using a normalized distance inside the domain allows comparison between domains of different sizes. The average fluorescence values were taken in each band, giving a 1-dimensional profile of the fluorescence values for each time point. For cells with multiple domains, bands around a domain were restricted to points on the surface closest to the given domain’s boundary in the geodesic sense.For each domain, the background fluorescence measurement in each frame was taken to be the average fluorescence 5 μm outside the domain. The median background fluorescence over the lifetime of the domain was taken to be the overall background. Fluorescence values were normalized by dividing by this background value .To give an overall profile of fluorescence and curvature, the average normalized 1-dimensional profile was taken for each domain, and these averages aggregated to give the plots shown in For time-dependent analysis, closure events were taken to be the temporal reference points. The number of domain sequences was reduced to include only those that contained the closure event being investigated (either lip or base closure), and the required time interval preceding the event. Due to variable time steps of the videos and some frames being rejected in the quality control step above, fluorescence values were linearly interpolated at every 2.5 seconds prior to the closure time. The average interpolated 1-dimensional fluorescence profiles for each time point were thus computed and plotted as colour values on a 2D image. The image was distorted to account for distance normalization of the profiles by scaling the part of the image representing points inside the domain by the average domain depth along the distance axis.Geometric measurements for each domain were made using the surface geometry: the perimeter was taken to be the total length of edges along the boundary of the domain; the area was given by the total area of all faces in the domain, and the depth was taken to be the maximum distance of any point in the domain to the domain boundary.de novo formation, lip closure, and elimination were aggregated by normalizing in both time and the geometric measurement, allowing the observation of general trends in the geometry -plane) in the top left, the projection in y -plane) in the bottom left, and the projection in x -plane) in the top right. This yielded a view where the projections with lower resolution (x and y) could be easily compared to the high-resolution projection (z) due to the alignment of the common axes. These projections allowed easier identification of closure events than the projection in z alone because they provide some of the 3D information lacking in the single projection.Surface rendering for purely visualization purposes was performed in Matlab (see above for annotation using VR and grading using ITKWidgets in Jupyter). The surfaces were preprocessed by applying Laplacian smoothing (3 iterations), to provide a smoother surface to view. A customized Matlab live script was used to allow fast rendering of the surfaces. Surface colour and opacity can be linked independently to fluorescence, curvature, or PIP3 patches. These values can be scaled to allow better visualization of the surface for the length of the video.A mathematical model is used to simulate the behaviour of cells during macropinocytosis. Our aim with this model is to show the feasibility of cup formation and closure resulting from a polymerization force acting at the boundary of a PIP3 domain, given that the domain area expansion stalls at some time point.70 The total force, FT, at a given vertex is tension is the force due to membrane tension, modelled as elastic stress induced by stretching the contour, Fbending is the force due to bending of the membrane, Farea is a force preserving the area inside the contour, and Fpoly is the normal force due to actin polymerization. The first two of these forces are calculated in the same manner as previously,70 Farea=kA(A/A0-1) for area inside the contour A with initial value A0 and force coefficient kA, and Fpoly is applied along the normal direction at the domain boundary as described below. Bending and area force coefficients are kept constant in all simulations.The model is based on a previous contour model used to study blebbing.While a contour model is 2D in nature, we may interpret it as approximating a slice through a cell with rotational symmetry, allowing us to calculate values for the perimeter  from 2D P/d for a constant value vP. This gives a slowing rate of expansion in length, while the domain area, calculated using a surface of rotation, stops expanding when approaching lip closure due to the shape changes of the domain.We define a PIP3 domain on the contour as a portion of the contour with length d, expanding at a rate vpoly is applied as a constant multiple, kP, of a Gaussian function centered at the domain boundary on both sides with standard deviation 0.25 μm. A domain expansion speed of 0.1 / d μm s-1 for domain width d is used for all experiments.The contour is initiated as a circle with radius 5 μm, discretized using 1000 line segments, and PIP3 patch of initial width 0.02 μm. Throughout the simulation, the force FThis 2D model can produce range of behaviors of cups . If we a71 when setting the length of the delamination zone, allowing the membrane to continue to move inwards until closure. It is conceivable that smaller initial delamination lengths could also lead to base closure, since tension at the edges of the delaminated region would be increased, leading to further detachment from the actin cytoskeleton. A similar effect can also occur during lip closure, but is not essential for closure in this case.If a portion of the membrane in the cup, approximating a cylinder of length l and width w, delaminates from the actin cytoskeleton, then it will start to move under membrane tension. Here, we model the tension force as proportional to mean curvature, while assuming all other forces are negligible. If the aspect ratio l/w < 0.66 (approx.), then the membrane in the cylinder can attain a minimum surface area without closure. We therefore enforce an aspect ratio greater than 0.66Statistical results are presented in Figs S1-S5Vid S1Vid S2Vid S3Vid S4Vid S5Vid S6Vid S7Vid S8Vid S9