DOI: 10.5061/dryad.vmcvdnd1m
title: Data from: Evaluation of a low-cost staining method for improved visualization of sweet potato whitefly (Bemisia tabaci) eggs on multiple crop plant species

description: The sweet potato whitefly (Bemisia tabaci) is a damaging insect pest that
 feeds on hundreds of crop plants. Oviposition rate is a useful metric to
 screen plants for whitefly resistance. Whitefly eggs are small and
 translucent, and can therefore be hard to count on the leaves of some
 crops. In this research, we tested a selective egg staining process on
 five crop species to determine if egg staining can improve the
 visualization and quantification of whitefly eggs. By comparing the egg
 counts before and after staining using two-sample Wilcoxon signed-rank
 tests (a non-parametric test for paired analyses). Two individuals counted
 the eggs, and for both these counters we found a significant increase in
 the number of visible eggs after staining on melon, tomato, and cowpea.
 This method could be applied to improve phenotyping for whitefly
 resistance in plant breeding applications.
description: # Evaluation of a Low-Cost Staining Method for Improved Visualization of
 Sweet Potato Whitefly (*Bemisia tabaci*) Eggs on Multiple Crop Plant
 Species Dryad DOI:
 [https://doi.org/10.5061/dryad.vmcvdnd1m](https://doi.org/10.5061/dryad.vmcvdnd1m) Article DOI: [https://doi.org/10.1186%2Fs13007-024-01209-z](https://doi.org/10.1186%2Fs13007-024-01209-z) This data was collected to analyze the ease of counting whitefly (*Bemisia tabaci* MEAM1) eggs on cassava, cowpea, melon, tomato, and sweet potato before and after a selective egg staining process. Five female whiteflies were confined in clip cages to a small section of the underside of each leaf for four days to allow them to oviposit. Then the leaf was removed from the plant, whiteflies were removed from the clip cage, and the area of leaf where the clip cage was attached was cut out using a round cookie cutter. The whitefly eggs on the leaf disc were then counted by two individuals using a stereo microscope, these are the "unstained counts". Whitefly eggs were then stained by submerging the leaf discs in McBryde’s stain (acetic acid, ethanol, 0.2% aqueous acid Fuchsin, water; 20:19:2:1) for three days, and then clearing under heat and pressure for 15 min in clearing solution (LGW; lactic acid, glycerol, water; 17:20:23). The whitefly eggs on the stained and cleared leaf disc were then counted by the same two individuals using a stereo microscope, these are the "stained counts". Counting time for each leaf disc was also recorded to help analyze the labor requirements of this process. Datasets contain stained and unstained egg counts for "counter one" (BVR) and "counter two" (RL). The counting time in minutes and seconds is also included. For some datasets where surfactants were tested, the number of bubbles on the leaf disc after staining was also recorded. The data here includes egg counts both before and after staining for each crop species and each counting individual. An R script and R Markdown document are also included. This data and code together allow for replication of all analyses performed for the associated publication in Plant Methods. Whitefly survival data ("whitefly_checks") is also included here and can be used to determine oviposition rates for these crops. ## Description of the data and file structure ### RMarkdown file The final RMarkdown (.Rmd) file used for analysis while writing the manuscript is included for reference (generated on 12 December, 2023). ### Egg count data The included datasets are labeled as: egg_counts_stain_unstain_"crop"_"replicate number".csv. The replicate number is an internal identifier based on how many total experiments have been conducted on that crop, and are not relevant to this analysis. These datasets contain the following headings:\ leaf_num = the ID number assigned to each leaf species = crop species stained = "yes" or "no" for if this count is taking place before ("no") or after ("yes") staining surfactant = optional descriptor included if a surfactant was tested on the stained leaves counter = counter one is Ben van Raalte (BVR), counter two is Ricky Le (RL) count_date = date that the egg counting took place egg_count = counted number of whitefly eggs time_m = minutes of counting (sum with time_s to get the total counting time) time_s = seconds of counting bubbles = number of bubbles counted on the leaf disc. This was only included for some of the experiments where surfactants were tested. Number of bubbles also only applies for stained leaf discs; for unstained leaf discs, "NA" is included to represent "not applicable". For stained leaf discs where the number of bubbles was not counted, "NR" is included to represent "not recorded". notes = other relevant information collected. If there are no notes for a counted leaf disc, "NA" is included in under the notes ### Whitefly survival data Whitefly survival information is not required for analysis of the egg staining method. These datasets are included because they allow calculation of oviposition rate in terms of whitefly eggs per female whitefly per day. This information could be useful for anyone replicating this method for whitefly resistance phenotyping application, as these experiments can provide a baseline oviposition rate for each of the crops used in these experiments. These datasets are labeled as: wf_checks_"crop"_"replicate number".csv. If there are no notes, "NA" is included. ## Sharing/Access information The manuscript associated with this data is available in the journal Plant Methods. ## Code/Software The file "combining_crops_stainunstain_analysis.R" is used to source and format all the data sets used in this analysis. All analyses were performed in R using an RMarkdown file for data handling, analysis, and figure creation. This code was run on 19 May 2024 and confirmed to function using "R version 4.4.0 (2024-04-24)" for Apple "aarch64-apple-darwin20" platform. An Apple MacBook Air (M1, 2020) with 16 GB of memory running macOS Monterey (Version 12.4) was used for this test. Non-base R packages used were: rstatix (0.7.2), lme4 (1.1-35.3), Matrix (1.7-0), coin (1.4-3), survival (3.5-8), kableExtra (1.4.0), ggsignif (0.6.4), ggtext (0.1.2), cowplot (1.1.3), ggplot2 (3.5.1), dplyr (1.1.4), and tidyr (1.3.1).