DOI: 10.5061/dryad.6hdr7srbc
title: Fungicide consumption exacerbates the negative effects of a common gut parasite in bumble bee

description: Bumble bees face numerous environmental stressors, including gut-parasite
 infection and exposure to agricultural fungicides, which can negatively
 impact colony health. This study evaluates the interactive effects of
 these stressors on bumble bee (Bombus impatiens) microcolonies, focusing
 on colony development, worker survival, and parasite infection dynamics.
 Our aim in evaluating these interactions was to determine if bees would
 experience synergistic negative health outcomes compared to single
 stressor exposures. We reared forty queenless bumble bee microcolonies,
 and treated them with either fungicide-contaminated pollen, inoculation
 with a gut parasite, both, or neither. Contrary to original expectations,
 we did not observe significant synergistic interactions between the two
 stressors, however we found that consumption of fungicide was associated
 with higher likelihood of gut-parasite infection, and delayed recovery
 from infection. Fungicide consumption was also connected to smaller
 workers, and smaller male offspring. We also found that gut-parasite
 infection was correlated with decreased pollen consumption overall,
 decreased worker survival, and fewer developed pupae. This study provides
 insights into the impacts of co-occuring stressors affecting bumble bees
 and emphasizes the importance of sublethal effects on pollinator health.
  
description: We established 40 queenless microcolonies of <em>B.
 impatiens</em> workers between June 9<sup>th</sup> and
 July 17<sup>th</sup> of 2022, and treated them with either
 fungicide-contaminated pollen, gut parasite inefction, both, or neither.
 Each microcolony consisted of 5 callow female workers.
 To evaluate the effects of fungicide consumption and parasite
 infection, alone and in combination, on bumble bee microcolony growth and
 individual health, we established the following treatments. Starting 48 h
 after establishment, we fed half of our microcolonies (N = 20) 1 g pollen
 patties containing 15,000 ppb of the commercial fungicide Pristine® while
 the remaining microcolonies (N = 20) continued to receive untreated
 pollen. These pollen patties were replaced every 48 h, and we calculated
 pollen consumption following Runnion et al. (2024). Fourteen days after
 the establishment of each microcolony, we removed each worker from their
 microcolony and starved them for 1 h. We then randomly selected one worker
 from half of the fungicide-fed microcolonies (N = 10) and from half of the
 fungicide-free microcolonies (N = 10) and fed 1µl of 50/50 sucrose
 solution-<em>Crithidia </em>inoculum at a concentration of 600
 <em>Crithidia</em> cells/µl<sup> </sup>(see
 electronic supplementary methods for inoculation details). Individuals
 from microcolonies not fed inoculum were instead fed 1µl of 50/50 sucrose
 solution. We recorded the identity of the inoculated bee and returned all
 workers to their microcolonies within 2 h of removal. This resulted in
 four stress treatments: control (<em>-Fungicide</em>,
 -<em>Crithidia</em>), fungicide only
 (+<em>Fungicide</em>, - <em>Crithidia</em>),
 parasite only (-<em>Fungicide</em>, +<em>
 Crithidia</em>), and combined (<em>+Fungicide</em>,
 +<em> Crithidia</em>). There were ten replicates of each
 treatment, blocked by spatial location in the rearing room. All workers
 within a block were sisters, sourced from the same commercial queenright
 colony. Following treatment establishment,
 microcolonies were monitored daily for worker survival, time until first
 oviposition, and time until first offspring emergence.
 Beginning 24 h after inoculation, we collected frass from all
 workers in the experiment for 14 days, in 24 h intervals. We extracted DNA
 from all frass samples. We conducted quantitative PCR using a
 Mastercycler® RealPlex2 (Eppendorf, Hamburg, Germany) for DNA extracted
 from frass samples. Upon microcolony experiment
 completion, we placed each microcolony in a freezer until we later
 dissected each microcolony and recorded counts of total brood cells, honey
 pots, eggs, and dead and live larvae and/or pupae. All
 analyses were completed in R.
description: # Fungicide consumption exacerbates the negative effects of a common gut
 parasite in bumble bee
 [https://doi.org/10.5061/dryad.6hdr7srbc](https://doi.org/10.5061/dryad.6hdr7srbc) ## Description of the data and file structure This data set inlcudes files for analysis for brood production of bumble bee micrcolonies, worker health, pollen consumption, and detection of* Crithidia bombi* cells found in bumble bee frass collected from workers in microcolonies.  File adl.csv contains data used for measuring average infection levels on a per bee basis.  File brood.csv contains data used for analyzing brood production on a per micrcolony basis. File drones.csv contains data used for analyzing health metrics of male bees emerged within each treatment microcolony on a per-male basis. File pollen.dates.csc contains data used to analyze pollen consumption within each treatment micrcolony on a per-pollen ball basis.  File qpcr.csv contains data used to anlyze infection levels on a per-bee-per-day basis.  File qpcr_time2.csv contains data used to analyze infection levels on a per-bee-per-general time period basis.  File workers.csv contains data used to analyze worker bee health metrics on a per-bee basis.  ### Author Information *A. Principal Investigator Contact Information* Name: Emily Runnion Institution: The Ohio State University Address: Columbus, OH USA Email: [Runnion.4@osu.edu](mailto:Runnion.4@osu.edu) *B. Associate or Co-investigator Contact Information* Name: Jamie Strange Institution: The Ohio State university Address: Columbus, OH USA Name: Ellen Klinger Institution: The Ohio State university Address: Columbus, OH USA Name: Frances Sivakoff Institution: The Ohio State university Address: Columbus, OH USA ##### General Information Date of data collection (single date, range, approximate date): 2023 Geographic location of data collection: Columbus, Ohio, USA Information about funding sources that supported the collection of the data: This work was funded by Sustainable Agriculture Research and Education Projects, North-Central Region, Grant Agreement GNC21-335 (to ENR), an OSU CFAES Research & Graduate Education Internal Grant (to FSS and JPS), and a National Institute of Food and Agriculture pre-doctoral fellowship award 2024-67011-43017 (to ENR). Licenses/restrictions placed on the data: CC0 1.0 Universal (CC0 1.0) Public Domain #### DATA & FILE OVERVIEW ##### 1.     File List: a.      adl.csv b.     brood.csv c.     drones.csv d.     pollen.dates.csv e.     qpcr.csv f.      qpcr_time2.csv g.     workers.csv 2.              Relationship between files, if important: None 3.              Additional related data collected that was not included in the current data package: None 4.              Are there multiple versions of the dataset? No a.     If yes, name of file(s) that was updated: NA b.     Why was the file updated? NA c.     When was the file updated? NA \######################################################################## DATA-SPECIFIC INFORMATION FOR: adl.csv 1.         Number of variables: 10 2.         Number of cases/rows: 100 3.         Variable List: •                     avg_infc: a number reporting the average count of cells *Crithidia bombi*/ul of DNA reported for that bee’s frass using qPCR analysis over the duration of the 14-day monitoring period •                     treatment: number 1-4 signifying each of the 4 treatments, 1 = control, 2 = fungicide only, 3 = fungicide and parasite, 4 = parasite only •                     inoculate: a logical column where 1 = true yes that bee was the originally inoculated individual fed 600cells crithidia/ul sugar water of artificial inoculate at the beginning of the 14 day frass monitoring period, and 0 = false they were not •                     max_inf: a number reporting the highest amount of cells/ul *Crithida *detected in the frass of that bee using qPCR methods over the course of the 14 day monitoring period •                     total-adl: a discrete number reporting the total number of times that bee had *Crithidia *detected from its frass sample over the assay detection limit of the qPCR methods •                     round: a number designated to describe the unique identification of the round in which the microcolony that bee belonged to was inoculated. There were 3 rounds of inoculation. •                     bee_id: a unique identifier given to each bee •                     days_to_adl: the number of days until qPCR methods detected *Crithidia *cells in that bees frass above the assay detection limit •                     colony: a unique identifier to designate each of the 40 microcolonies •                     block: number signifying the unique blocking variable 4.     Missing data codes: NA (data not available) a.     “days_to_adl” NA for the following bees they never had *Crithidia *cells detected above the assay detection limit  | T3.4-26 | | :------- | | T3.4-28 | | T3.4-29 | | T3.4-50 | | T3.6-59 | | T3.6-60 | | T3.9-33 | | T4.10-63 | | T4.12-34 | | T4.4-39 | | T4.4-40 | | T4.4-52 | 5.     Specialized formats or other abbreviations used: None \######################################################################## DATA-SPECIFIC INFORMATION FOR: brood.csv 1.     Number of variables: 23 2.     Number of cases/rows: 37 3.     Variable List: •                     treatment: number 1-4 signifying each of the 4 treatments, 1 = control, 2 = fungicide only, 3 = fungicide and parasite, 4 = parasite only •                     block: number signifying the unique blocking variable •                     fungicide: true/false column where 0 = false, no fungicide, and 1 = true, yes fungicide •                     crithidia: true/false column where 0 = false no crithidia/parasite, and 1 = true, yes crithidia/parasite •                     colony: unique identifier for each of the 40 microcolonies •                     brood_cells = the total count of pupae, larvae, egg sacs, and honey pots per microcolony at the conclusion of the experiment, following microcolony dissection •                     live_larvae: total count of live larvae per microcolony at the end of the experiment following microcolony dissection •                     dead_larvae: total count of dead larvae per microcolony at the end of the experiment following microcolony dissection •                     total_larvae: live and dead larvae columns added together •                     live_pupae: total count of live pupae per microcolony at the end of the experiment following microcolony dissection •                     dead_pupae: total count of dead pupae per microcolony at the end of the experiment following microcolony dissection •                     total_pupae: live and dead pupae counts added together •                     total_larv_pup: totals of total pupae and total larvae counts added together •                     total_dead_larv_pup: totals of dead larvae and dead pupae columns added together •                     total_live_larv_pup: totals of live larvae and live pupae columns added together •                     eggs = total count of eggs per microcolony at the end of the experiment following microcolony dissection •                     honey_pot = total count of honey pots per microcolony. Honey pot determined as a open pula cell containing nectar. •                     total_drones: total amount of male offspring emerged during the experiment timeframe within that microcolony •                     emerge_days: the amount of days from microcolony start date until the first male offspring successfully emerged from that microcolony •                     duration: the amount of days a microcolony had living workers •                     workers_alive: the count of living workers in the microcolony when the microcolony was placed in the freezer marking the experiment end date for that microcolony •                     avg_pollen: the average amount of pollen consumed by that microcolony ever 48 hours of the experiment •                     qro: the unique identifier assigned to the queenright original colony the 5 workers used to supply the microcolony came from 4.     Missing data codes: NA (data not available) a.     “emerge_days”  NA for the following microcolonies because male offspring never emerged from these microcolonies | T1.1 | | :---- | | T1.6 | | T2.11 | | T2.6 | | T3.11 | | T3.12 | | T3.6 | | T3.7 | | T4.12 | | T4.6 | | T4.8 | | T4.11 |   5.     Specialized formats or other abbreviations used: None \######################################################################## DATA-SPECIFIC INFORMATION FOR: drones.csv 1.         Number of variables: 25 2.         Number of cases/rows: 281 3.         Variable List: •                     colony: a unique identifier to designate each of the 40 microcolonies •                     treatment: number 1-4 signifying each of the 4 treatments, 1 = control, 2 = fungicide only, 3 = fungicide and parasite, 4 = parasite only •                     block: the unique number assigned to the blocking variable •                     fungicide: true/false column where 0 = false, no fungicide, and 1 = true, yes fungicide •                     crithidia: true/false column where 0 = false no crithidia/parasite, and 1 = true, yes crithidia/parasite •                     number: the numerical order that bee was counted as a successfully emerged male across the entire experiment for all microcolonies, serves to double as a unique identifier •                     id: a unique identifier given to each bee •                     emerge_date: the day that male was found emerged from the brood cell within the microcolony •                     live_weight: the weight of that male immediately (withing 24hrs) of emergence from the brood cell •                     radial_um: the length of the male’s radial cell in the right wing reported in micrometers •                     radial_mm:  the length of the male’s radial cell in the right wing reported in millimeters •                     alive_on_emerge: a logical column reporting if the male was successful in fully emerging alive from the brood cell (1) or of the male half emerged/ or was found dead immediately after emergence (within 24hrs) (0). •                     Dry_weight: weight in g of the entire male body after being placed in an oven for 72hrs at 60C •                     Dry_abdomen: the weight of only the dried abdominal portion in g of the male •                     Abdomen_post_ee: the weight of the male's abdomen, in grams, after being removed following the initial 72 hour drying, soaked in ethyl ether for 24 hours, and dried again •                     fat_content: the fat content of the male's abdomen, calculated as the difference between the "abdomen_dry" value and the "abdomen_post_ethyl" value reported as g •                     fat_mg: the fat_content reported in mg •                     relative_fat: the relative fat content of the male's abdomen, calculated as the value of "abdomen_post_ethyl" divided by "radial" •                     rf_mg/mm: the relative_fat value reported in value of milligrams per millimeter •                     qro: the unique identifier assigned to the queenright original colony the 5 workers used to supply the microcolony came from •                     qro: the unique identifier assigned to the queenright original colony the 5 workers used to supply the microcolony came from •                     workers_surviving: the count of living workers in the microcolony when the microcolony was placed in the freezer marking the experiment end date for that microcolony •                     avg_pollen: the average amount of pollen consumed by that microcolony ever 48 hours of the experiment •                     start date: the date the microcolony was initiated with the 5 female worker bees •                     emerge: the number of days from the start date for that male to emerge from the brood cells •                     workers_alive: a redundant column, equivalent to workers_surviving 4.     Missing data codes: none 5.     Specialized formats or other abbreviations used: None \######################################################################## DATA-SPECIFIC INFORMATION FOR: pollen.dates.csv 1.              Number of variables: 18 2.              Number of cases/rows: 734 3.              Variable List: •         colony: a unique identifier for each of the 40 microcolonies •         treatment: number 1-4 signifying each of the 4 treatments, 1 = control, 2 = fungicide only, 3 = fungicide and parasite, 4 = parasite only •         fungicide: true/false column where 0 = false, no fungicide, and 1 = true, yes fungicide •         crithidia: true/false column where 0 = false no crithidia/parasite, and 1 = true, yes crithidia/parasite •         block: the spatial blocking variable •         pollen.ball.id: a number that designates a unique identifier for every single individual pollen ball over the course of the experiment •         pollen.start: the date that pollen ball was placed in the microcolony •         start.time: the time that pollen ball was placed in the micrcolony •         start.weight: the weight in g of the pollen ball immediately before placement in the microcolony •         pollen.end: the date the pollen ball was removed from the microcolony •         end.time: the time the pollen ball was removed from the microcolony •         end.weight: the weight in g of the pollen ball immediately upon removal from the micrcolony  •         difference: the difference between the pollen start weight and the pollen end weight in g •         ec_diff: the calculated water loss based on evaporative control pollen (g) •         whole_dif: the difference between the pollen start weight and the pollen end weight with the calculated water loss based on evaporative control pollen analysis added back in (g) •         workers_alive: the number of workers that were alive in the microcolony over the 48hr period that pollen ball was inside the microcolony •         qro: the original queenright colony the workers in that microcolony were sourced from. •         pollen.time: a number used to describe how many days between the microcolony start date and the date the pollen ball was placed inside the microcolony, this variable was used to track time as a factor variable in pollen consumption analysis 4.              Missing data codes: none 5.              Specialized formats or other abbreviations used: None \######################################################################## DATA-SPECIFIC INFORMATION FOR: qpcr.csv 1.              Number of variables: 34 2.              Number of cases/rows: 1387 3.              Variable List: •                     bee_id: a unique identifier for each bee •         fungicide: true/false column where 0 = false, no fungicide, and 1 = true, yes fungicide •         crithidia: true/false column where 0 = false no crithidia/parasite, and 1 = true, yes crithidia/parasite •                     qro: a unique identifier for the original queenright colony each worker in the microcolony was sourced from •                     bee_count: a unique identifier for the original queenright colony each worker in the microcolony was sourced from, different from "bee*id" because these numbers are based on 1-5 while bee* •                     inoculate_round: a number designated to describe the unique identification of the round in which the microcolony that bee belonged to was inoculated. There were 3 rounds of inoculation. •                     start live weight: the live weight of the callow worker immediately before being put in the microcolony •                     inoculate: a t/f column column where TRUE = true yes that bee was the originally inoculated individual fed 600cells crithidia/ul sugar water of artificial inoculate at the beginning of the 14 day frass monitoring period, and FALSE = false they were not •                     inoculate_01: inoculate: a logical column where 1 = true yes that bee was the originally inoculated individual fed 600cells crithidia/ul sugar water of artificial inoculate at the beginning of the 14 day frass monitoring period, and 0 = false they were not •                     premature_death: a t/f column where TRUE = that bee died before the colony was artificially ended as the end of the experiment and FALSE = that bee did not die before the artificial end of the microcolony •                     end date: the day that bee died •                     days_alive: the number of days that bee was alive starting from the day the microcolony was initiated •                     end wet weight: the ending weight prior to being dried in the oven of that bee on the day they were removed from their microcolony in g •                     dry: the weight in g of that bee after removal from the microcolony and being dried in an oven at 60C for 72 h •                     avg_pollen: the average amount of pollen consumed by that microcolony every 48hrs •                     treatment: number 1-4 signifying each of the 4 treatments, 1 = control, 2 = fungicide only, 3 = fungicide and parasite, 4 = parasite only •                     replicate: the spatial blocking variable •                     start: the day that microcolony was initiated •                     colony: the unique identifier given to each of the 40 microcolonies •                     inoculation_date: the date that microcolony had a single worker fed an artificial inoculum of 600 cells crithidia/ul of sugar water •                     time: the number of days since the initiation of that microcolony •                     days_since_innoculation: the number of days since the date the artificial inoculum was fed to the single inoculated bee of that microcolony. Each bee has 14 rows associated with it for 14 days of frass monitoring. •                     date: the day that the frass sample was collected. Each date corresponds to a frass sample that would have been taken from the associated bee, there are 14 dates per bee. •                     alive_or_dead: there are 14 rows per bee associated with frass sampling, but some bees died before the 14 days were up. For data analysis purposes, the data still has 14 rows per bee, but it is necessary to note of that bee does not have a frass sample that day because the sample is missing, or because the bee is dead. If the value in this column = alive, then the bee was alive this day and should have an associated value in the “cells” column. If the bee was alive, and the cells column = 0 then we simply didn’t get a sample that day. If the bee was dead and the cells column = 0, it’s because there was no bee to sample. •                     cycles: the number of cycles the masterplex cycler needed to run to determine the quantity of *Crithidia *cells for that frass sample •                     cells: the number of *Crithidia *cells detected in the associated bee frass sample •                     round: this is a redundant column, equivalent to “inoculate_round” above. A number designated to describe the unique identification of the round in which the microcolony that bee belonged to was inoculated. There were 3 rounds of inoculation. •                     adl: a logical column where 1 = yes that frass sample tested above the assay detection limit of 3.75cells/ul and 0 = no the value determined via qPCR was less than the assay detection limit •                     detected: a logical column were 1 = yes qPCR detected any value above 0 for *Crithidia *cells in that frass sample and 0 = no cells detected at all •                     cell_include: a column where values are designated 1 or 2 to determine if we included the cells value in analysis, 1 = we did not include that value because it was less than the assay detection limit and 2 = yes we did include that value because values were above the assay detection limit •                     cells_assumed: the actual value of *Crithidia *cells/ul DNA as measured by qPCR used in analysis, with value = 0 assigned to any cell count value less than the assay detection limit 4.              Missing data codes: NA For bee_id = T3.11-99 column dry = NA because that bee was coated in sugar water and couldn’t be properly weighed For any NAs associated with cycles, cells, or any subsequent columns, NA exists either because that bee died or we were unable to procur a frass sample that day. Check column alive_or_dead to determine. 5.              Specialized formats or other abbreviations used: None \######################################################################## DATA-SPECIFIC INFORMATION FOR: qpcr_time2.csv 1.              Number of variables: 17 2.              Number of cases/rows: 1079 3.              Variable List: •                     bee_id: a unique identifier for each bee •               fungicide: true/false column where 0 = false, no fungicide, and 1 = true, yes fungicide •               crithidia: true/false column where 0 = false no crithidia/parasite, and 1 = true, yes                 crithidia/parasite •                     qro: a unique identifier for the original queenright colony each worker in the microcolony was sourced from •                     inoculate_round: a number designated to describe the unique identification of the round in which the microcolony that bee belonged to was inoculated. There were 3 rounds of inoculation. •                     inoculate: a t/f column column where TRUE = true yes that bee was the originally inoculated individual fed 600cells crithidia/ul sugar water of artificial inoculate at the beginning of the 14 day frass monitoring period, and FALSE = false they were not •                     treatment: number 1-4 signifying each of the 4 treatments, 1 = control, 2 = fungicide only, 3 = fungicide and parasite, 4 = parasite only •                     replicate: the spatial blocking variable •                     start: the day that microcolony was initiated •                     colony: the unique identifier given to each of the 40 microcolonies •                     days_since_innoculation: the number of days since the date the artificial inoculum was fed to the single inoculated bee of that microcolony. Each bee has 14 rows associated with it for 14 days of frass monitoring. •                     date: the day that the frass sample was collected. Each date corresponds to a frass sample that would have been taken from the associated bee, there are 14 dates per bee. •                     alive_or_dead: there are 14 rows per bee associated with frass sampling, but some bees died before the 14 days were up. For data analysis purposes, the data still has 14 rows per bee, but it is necessary to note of that bee does not have a frass sample that day because the sample is missing, or because the bee is dead. If the value in this column = alive, then the bee was alive this day and should have an associated value in the “cells” column. If the bee was alive, and the cells column = 0 then we simply didn’t get a sample that day. If the bee was dead and the cells column = 0, it’s because there was no bee to sample. •                     cycles: the number of cycles the masterplex cycler needed to run to determine the quantity of *Crithidia *cells for that frass sample •                     qpcr_cells: the number of *Crithidia *cells detected in the associated bee frass sample •                     cells_assumed: the actual value of *Crithidia *cells/ul DNA as measured by qPCR used in analysis, with value = 0 assigned to any cell count value less than the assay detection limit •                     days_factor: a redundant column equivalent to days_since_innoculation, included in the data to make coding easier when analyzing time as either a factor or a number •                     time_group: a factor assigned to designate early, middle, and end time period of the 14 day frass monitoring period. 1 = early, the first 1-4: days, 2 = middle, the 5-9 days: 3 = end, the 10-14 days. 4.              Missing data codes: NA 5.              Specialized formats or other abbreviations used: None \######################################################################## DATA-SPECIFIC INFORMATION FOR: qpcr_time2.csv 6.              Number of variables: 17 7.              Number of cases/rows: 1079 8.              Variable List: •                      treatment: number 1-4 signifying each of the 4 treatments, 1 = control, 2 = fungicide only, 3 = fungicide and parasite, 4 = parasite only •               fungicide: true/false column where 0 = false, no fungicide, and 1 = true, yes fungicide •               crithidia: true/false column where 0 = false no crithidia/parasite, and 1 = true, yes                 crithidia/parasite •                     block: the spatial blocking variable •                     colony: the unique identifier given to each of the 40 microcolonies   •                     qro: a unique identifier for the original queenright colony each worker in the microcolony was sourced from •                     id: a unique identifier for each bee •                     inoculate_round: a number designated to describe the unique identification of the round in which the microcolony that bee belonged to was inoculated. There were 3 rounds of inoculation. •                     inoculate: a logical column where 1 = true yes that bee was the originally inoculated individual fed 600cells crithidia/ul sugar water of artificial inoculate at the beginning of the 14 day frass monitoring period, and 0 = false they were not •                     premature_death: : a t/f column where TRUE = that bee died before the colony was artificially ended as the end of the experiment and FALSE = that bee did not die before the artificial end of the microcolony •                     days_alive: the number of days that bee was alive starting from the day the microcolony was initiated •                     day14_survival: of the 14 days of frass monitoring for that bee, how many of those days did the bee survive •                     inoc_censor: used for the cox proportional hazards analysis of the entire experimental timeframe, any bee that did not survive the entire experiment (i.e. premature_death = TRUE) was censored (1) and any bee that did survive was not censored (0) •                     day14_censor: used for a cox proportional hazards analysis of the 14 day frass monitoring period, any bee that did not survive the entire 14 day frass monitoring period was censored (1) and any bee that did survive was not censored (0) 9.              Missing data codes: NA                                                i.     The following bees have NA for columns survival over the 14 day frass monitoring period and number of days after inoculation of the microcolony because these bees all died before inoculation of the microcolony occurred | T4.8-4 | | :------ | | T4.5-1 | | T3.11-4 | | T3.1-1 | 10.           Specialized formats or other abbreviations used: None ## Code/software File DRYAD-Code-Disease-Dynamics.html is an html R Markdown file with the option to download the code as a link in the upper right corner. Descriptions of each chunk can be found in the table of contents of this file.