DOI: 10.5061/dryad.b2rbnzstv
title: A basic helix-loop-helix/Per-Arnt-Sim transcription factor Taiman is essential for metamorphosis in <em>Henosepilachna vigintioctomaculata</em>

description: Insect Taiman (Tai) is a basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS)
 transcription factor that plays a role in juvenile hormone (JH) signaling
 during the larval stage in various insect species, as well as in
 20-hydroxyecdysone (20E) signaling during the oogenesis of Drosophila
 melanogaster. It forms a heterodimer with another bHLH-PAS transcription
 factor Methoprene-tolerant (Met) mediating JH signaling, and acts as a
 steroid receptor coactivator of the functional 20E receptor heterodimer,
 Ecdysone receptor (EcR) and Ultraspiracle (USP). However, whether
 Tai plays the dual functions in other insects deserves further
 exploration. In this study, we determined the role of Tai in
 Henosepilachna vigintioctomaculata, a Coleopteran potato pest mainly
 distributed in northern China. HvTai mRNA levels were higher just
 before and right after the ecdysis within each larval instars and at early
 and late stages of prepupae than those in the intermediate days. RNA
 interference (RNAi)-aided knockdown of Tai at the second, penultimate, and
 final larval instar stages caused 100% mortalities 9,
 11, and 10 days post dsRNA administration. All Tai RNAi larvae failed to
 complete molting, they were wrapped in the old larval exuviae, gradually
 darkened and shriveled, and ultimately died. The Tai-depleted larvae did
 not complete gut clearing, the larval guts contained food residues.
 Moreover, knockdown of *Tai *disrupted both JH and 20E pathway genes. Our
 findings demonstrated that Tai was necessary for metamorphosis in H.
 vigintioctomaculata. The findings also imply that H.
 vigintioctomaculata is an ideal model for further exploration of
 how Tai contributes specifically to hormone signaling.
description: # A basic helix-loop-helix/Per-Arnt-Sim transcription factor Taiman is
 essential for metamorphosis in *Henosepilachna vigintioctomaculata* ##
 General information Related article: Wu Y-K, Yu J-Q, Jin L, Li G-Q. A
 basic helix-loop-helix/Per-Arnt-Sim transcription factor Taiman is
 essential for metamorphosis in Henosepilachna vigintioctomaculata. Insect
 Molecular Biology, forthcoming. Species: Henosepilachna
 vigintioctomaculata Main techniques: RNA interference (RNAi), qRT-PCR,
 body weight measurement, survival analysis. All data in this repository
 correspond to Figures 1–5 in the associated article. ### File list
 rawdata.xlsx Excel workbook containing all numerical data used to generate
 Figures 1–5. No image files or narrative text from the article are
 included here. All values are numeric and intended for re-analysis. ###
 Conventions and units * Time after dsRNA injection is given in: * hours
 (h) for short-term measurements (e.g., 0 h, 24 h, …); * days (d) for
 longer observations (e.g,. day 1, day 2, …). * Body weight is expressed in
 milligrams (mg). * qRT-PCR data are given as normalized relative
 expression values (2^-ΔCt or 2^-ΔΔCt, unitless). * For qRT-PCR tables: *
 the first column in each block is the sample label (stage, tissue, or
 treatment); * the next three columns are biological replicates of the
 normalized expression value (three independent pools of individuals, each
 measured once). GraphPad Prism output rows (e.g., “Paired t test”, “P
 value”, “Multiple t test”) are kept in some sheets as summary statistics
 and can be ignored if only raw measurements are needed. #### Detailed
 description of rawdata.xlsx Workbook: rawdata.xlsx\ Sheets: “Fig1”,
 “Fig2”, “Fig3”, “Fig4”, “Fig5”. All row and column references below use
 Excel conventions (columns A, B, C…). ##### Sheet “Fig1” – HvTai
 expression (Figure 1) This sheet contains two qRT-PCR tables for HvTai:
 (1) Figure 1A – Temporal expression of HvTai - Data block: rows 3–26,
 columns C–F. - Row 3 (header): * C3: “HvmTai” (gene name) * D3–F3: “2-△ct”
 (three biological replicates; unitless relative expression) - Rows 4–26: *
 Column C: developmental stage / time point label (e.g. “L1D0”, “L1D1”, …,
 “PD4”). * Columns D–F: three biological replicates of HvTai relative
 expression (2^-ΔCt) for each stage. (2) Figure 1B – Tissue expression of
 HvTai - Data block: rows 3–10, columns Q–T. - Row 3 (header): * Q3:
 “HvmTai” * R3–T3: “2-△ct” (three biological replicates; unitless) - Rows
 4–10: * Column Q: tissue name (Head, Foregut, Midgut, Hindgut, Malpighian
 tubules, Fat body, Epidermis). * Columns R–T: three biological replicates
 of HvTai relative expression (2^-ΔCt) in each tissue. There are no
 separate columns for raw Ct values in this sheet; only normalized 2^-ΔCt
 values are provided. ##### Sheet “Fig2” – RNAi at second instar (Figure 2)
 This sheet contains: (1) Figure 2A – Knockdown efficiency of HvTai - Data
 block: rows 3–5, columns C–F. - Row 3: * C3: “HvmTai” * D3–F3: “2-△△ct”
 (three biological replicates; unitless) - Rows 4–5: * Column C: treatment
 group (“dsegfp” control or “dsTai”). * Columns D–F: three biological
 replicates of HvTai relative expression (2^-ΔΔCt) in each treatment. (2)
 Figure 2B – Fresh weight of second-instar larvae after dsRNA injection -
 Data block: rows 4–10, columns L–AF. - Row 3: * L3: “Fresh weight (mg)” *
 M3: “dsegfp” * W3: “dsTai” - Rows 4–10: * Column L: time after injection
 (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h). * Columns M–V: fresh weights
 (mg) of 10 individual larvae in the dsegfp control group. * Columns W–AF:
 fresh weights (mg) of 10 individual larvae in the dsTai-treated group. (3)
 Additional rows for Figure 2B (“Paired t test”, “P value”, “Multiple t
 test”) - Rows 8–25 contain GraphPad Prism output for statistical
 comparisons between dsegfp and dsTai body weights at each time point (P
 values, significance, t ratio, degrees of freedom, etc.). - These rows are
 derived statistics and not raw measurements. (4) Figure 2C–2D – Survival
 and developmental stage distribution - Data block: starting around row 39,
 columns C–Y. - Row 40 (approximate; the exact row is labelled “Days”): *
 Column C: “Days” (days after injection). * Columns D and E: numbers or
 proportions of surviving larvae in the dsegfp and dsTai groups (“dsegfp”,
 “dsTai”). * Columns with headers such as “Normal pupa”, “Deformed larva”,
 “Deformed pupa” contain the numbers of individuals in each phenotype
 category on each day. These data correspond to the Kaplan–Meier survival
 curves and phenotypic ratios shown in Figure 2C and 2D. ##### Sheet “Fig3”
 – RNAi at third instar (Figure 3) Structure is similar to “Fig2”: (1)
 Figure 3A – Knockdown efficiency of HvTai - Data block: rows 3–5, columns
 C–F. - Row 3: * C3: “HvmTai” * D3–F3: “2-△△ct” - Rows 4–5: * Column C:
 treatment group (“dsegfp”, “dsTai”). * Columns D–F: three biological
 replicates of HvTai relative expression (2^-ΔΔCt). (2) Figure 3B – Fresh
 weight of third-instar larvae - Data block: rows 4–15, columns L–AF. - Row
 3: * L3: “Fresh weight (mg)” * M3: “dsegfp” * W3: “dsTai” - Rows 4–15: *
 Column L: time after injection (days 1–12). * Columns M–V: fresh weights
 (mg) of 10 dsegfp control larvae. * Columns W–AF: fresh weights (mg) of 10
 dsTai-injected larvae. - Subsequent rows store GraphPad Prism “Multiple t
 test” outputs for comparisons between treatments (P value, mean
 difference, etc.). (3) Figure 3C–3D – Survival and phenotypes - Data
 block: as in “Fig2”, a table with: * Column labelled “Days”: days after
 injection. * Columns “dsegfp” and “dsTai”: survivors in each group. *
 Additional phenotype columns for different developmental outcomes (e.g.,
 “Normal pupa”, “Deformed prepupa/pupa”), used to generate Figure 3C–3D.
 ##### Sheet “Fig4” – RNAi at fourth instar (Figure 4) (1) Figure 4A –
 Knockdown efficiency of HvTai - Data block: rows 3–5, columns C–F. - Row
 3: * C3: “HvmTai” * D3–F3: “2-△△ct” - Rows 4–5: * Column C: treatment
 group (“dsegfp”, “dsTai”). * Columns D–F: three biological replicates of
 HvTai relative expression (2^-ΔΔCt). (2) Figure 4B – Fresh weight of
 fourth-instar larvae - Data block: rows 4–13, columns L–AF. - Row 3: * L3:
 “Fresh weight (mg)” * M3: “dsegfp” * W3: “dsTai” - Rows 4–13: * Column L:
 time after injection (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h,
 192 h, 216 h). * Columns M–V: fresh weights (mg) of 10 dsegfp control
 larvae. * Columns W–AF: fresh weights (mg) of 10 dsTai-injected larvae. -
 Rows 16–27: “Multiple t test” summary (Time, Significant?, P value, means,
 mean difference, SE, t ratio, df). (3) Figure 4C–4D – Survival and
 phenotypes - Similar structure to sheet “Fig2” and “Fig3”: * A table with
 “Days”, “dsegfp”, “dsTai”, and phenotype columns indicating numbers of
 individuals at different developmental outcomes used to draw Figure 4C and
 4D. ##### Sheet “Fig5” – JH and 20E pathway genes after Tai RNAi (Figure
 5) This sheet contains qRT-PCR data for multiple genes, each with the same
 structure: treatment vs. control with three biological replicates of
 2^-ΔΔCt values. (1) Figure 5A–5D – JH signaling genes - Data block: rows
 4–6, columns F–H, M–O, T–V, AB–AD. - Row 4 (headers): * F4: “HvmMet” *
 G4–H4: “2-△△ct” * M4: “Hvmkr-h1” * N4–O4: “2-△△ct” * T4: “HvmHairy” *
 U4–V4: “2-△△ct” * AB4: “HvmGroucho” * AC4–AD4: “2-△△ct” - Rows 5–6: * For
 each gene: - First column: treatment (“dsegfp”, “dsTai”). - Next three
 columns: three biological 2^-ΔΔCt replicates. (2) Figure 5E–5H – 20E
 signaling genes - Data block: rows 21–23, columns F–H, M–O, T–V, AB–AD,
 etc. - Row 21 (headers, example): * F21: “HvmEcR” * G21–H21: “2-△△ct” *
 M21: “HvmUSP” * N21–O21: “2-△△ct” * T21: “HvmBrC” * U21–V21: “2-△△ct” *
 AB21: “HvmE74” * AC21–AD21: “2-△△ct” * Further genes (HvmE75, HvmE93,
 HvmHR4, HvmFtz-f1) are organized in the same way. - Rows 22–23: * For each
 gene: - First column in the block: treatment (“dsegfp”, “dsTai”). - Next
 three columns: three 2^-ΔΔCt replicate values. (3) Additional rows (e.g.,
 “Paired t test”, “P value”, “P value summary”) - Rows 9–14 and 26–31
 contain GraphPad Prism outputs for statistical comparisons between dsegfp
 and dsTai for each gene (P values, significance, t, and df). - These are
 summary statistics, not raw qRT-PCR measurements. 1. Variable definitions
 (summary) Across all sheets, the variables can be summarized as follows: *
 SampleID / Stage / Tissue / Time: * Text labels identifying developmental
 stage, tissue, treatment, or time point (e.g., “L1D0”, “Head”, “0h”, “Day
 5”, “dsegfp”, “dsTai”). * 2-△Ct (three replicate columns): * Unitless
 relative expression values for HvTai (Figure 1). * Three biological
 replicates per sample. * 2-△△Ct (three replicate columns): * Unitless
 relative expression values for each target gene (HvTai, HvMet, HvKr-h1,
 HvHairy, HvGroucho, HvEcR, HvUSP, HvBrC, HvE74, HvE75, HvE93, HvHR4,
 HvFtz-f1) normalized to reference genes and to a control group. * Three
 biological replicates per treatment. * Fresh weight (mg): * Body weight of
 individual larvae at a given time point after dsRNA injection. * For each
 time point, 10 individuals per treatment group (dsegfp and dsTai). *
 Survival and phenotype counts: * Number (or proportion) of larvae alive on
 each day after injection in each treatment group. * Number of individuals
 in different developmental categories (e.g., normal pupa, deformed larva,
 deformed pupa, prepupa), used to calculate survival curves and phenotype
 ratios.