Dryad DOI: https://doi.org/10.5061/dryad.47d7wm3t5

1. Overview

This dataset contains the raw experimental data underlying the manuscript:

Liu, Hai-Hui; Zhang, Xiao-Qing; Yu, Jia-Qing; Jin, Lin; Li, Guo-Qing.
Kr-h1 is vital for vitellogenesis in two ladybird beetle species.
Insect Molecular Biology, 2025.

The experiments investigate the role of Krüppel homolog 1 (Kr-h1) and downstream
juvenile hormone (JH) pathway components in vitellogenesis and oviposition in two
ladybird beetles:

• Henosepilachna vigintioctomaculata (Hvm)
• Henosepilachna vigintioctopunctata (Hvp)

The data include:

• Temporal and tissue-based expression profiles of JH-pathway genes;
• Effects of HvmKr-h1 RNAi on vitellogenesis and oviposition;
• Effects of HvpKr-h1 RNAi on oviposition;
• Effects of HvpKr-h1 RNAi on vitellogenin and vitellogenin receptor (Vg/VgR) expression;
• Effects of HvmHairy and HvmGroucho RNAi on Vg/VgR expression and egg production.

All data are provided in one Excel workbook:

• rawdata.xlsx

Dryad datasets are treated as independent publications; this README describes the
content of each worksheet and the main variables, so that the dataset can be
understood and reused without referring to the article.

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2. File structure

2.1 rawdata.xlsx

This workbook contains five worksheets, each corresponding to data used in one figure
of the article. Within each sheet, GraphPad Prism output tables (e.g., “Paired t test”,
“P value”, “t, df”) are included alongside the raw values. These statistical result
tables were generated from the raw data in the same sheet and can be used to reproduce
the reported significance tests.

The general organization is:

• rows with gene names (e.g., HvmKr-h1, HvpKr-h1, HvmVg, HvmVgR) and the
  label 2-△ct or 2-△△ct identify the start of a data block;
• subsequent rows contain samples (e.g., developmental stage, tissue, treatment group);
• numeric columns contain replicate measurements and/or derived relative expression
  values;
• below each block, additional rows contain summary statistics (P values, t statistics,
  etc.) from GraphPad Prism.

Below, we describe the content of each sheet and its relation to the published figures.

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Sheet: Fig1

Content: Temporal and tissue-based expression profiles of JH-pathway genes
in the two Henosepilachna species.

Related figure: Figure 1 (panels B–I).
(Figure 1A – schematic diagram of the JH pathway – is not a data figure and is
not represented here.)

This sheet contains several blocks of qRT-PCR data for four JH signaling genes
(e.g. Kr-h1, Hairy, Groucho, Met/Tai-related components) in:

• different developmental stages or days in adult females;
• a panel of tissues (head, gut segments, Malpighian tubules, fat body,
  epidermis, ovary).

Typical variables in each block:

• Sample – developmental stage (e.g., “Adult 0”, “Adult 1”, …) or tissue
  (HE, FG, MG, HG, MT, FB, EP, OV).
• 2-△ct – relative expression values (2^−ΔCt) from three biological replicates
  (one column per replicate).
• optional columns with mean and/or standard error (SE) for the plotted bars.

These data were used to generate:

• Figure 1B–E: temporal expression patterns of JH pathway genes;
• Figure 1F–I: tissue-based expression patterns in selected tissues of
  H. vigintioctomaculata (Hvm) and H. vigintioctopunctata (Hvp).

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Sheet: Fig2

Content: Effects of HvmKr-h1 RNAi on vitellogenesis, Vg/VgR expression,
preoviposition period, egg production, and hatchability in
Henosepilachna vigintioctomaculata.

Related figure: Figure 2A–N.

This sheet includes several tables:

1. Gene expression (Figure 2A–C)
   • Relative transcript levels of HvmMet, HvmVg, HvmVgR in dsKr-h1–treated
     vs. dsegfp-treated females.
   • Tables contain:
     • Treatment – dsegfp (control) or dsKr-h1.
     • replicate values of 2-△△ct or relative expression.
     • Prism t-test summary rows (P value, P value summary, t, df).
2. Preoviposition period (Figure 2M)
   • One table lists the number of days from adult eclosion to first oviposition for
     individual females.
   • Typical variables:
     • Female_ID – individual female identifier.
     • Treatment – dsegfp or dsKr-h1.
     • Preoviposition_days – days to first egg laying.
3. Egg production and hatchability (Figure 2N)
   • Data for:
     • daily egg counts per female over the lifespan;
     • total eggs per female;
     • hatchability (%) of eggs from each biological replicate.
   • Typical variables:
     • Day_after_eclosion – day number from adult eclosion.
     • Eggs_per_female – number of eggs laid per female per day.
     • Total_eggs_per_female – cumulative egg number per female.
     • Hatchability_percent – percentage of eggs hatched in each replicate.

Ovary images (Figure 2D–L) are not included in this dataset; only numeric
measurements are provided.

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Sheet: Fig3

Content: Effects of HvpKr-h1 RNAi on gene expression and oviposition in
Henosepilachna vigintioctopunctata.

Related figure: Figure 3A–H.

This sheet contains:

1. Gene expression (Figure 3A–D)
   • Relative expression of HvpKr-h1, Hvpjhamt, HvpMet and Hvphairy in
     dsKr-h1–treated vs. control females.
   • Each block includes:
     • gene name and label 2-△△ct;
     • values for dsegfp and dsKr-h1 groups;
     • Prism t-test summary (P value, P value summary, t, df).
2. Egg production (Figure 3G–H)
   • Oviposition from 0- to 14-day-old females and accumulated eggs up to 15 days
     after eclosion.
   • Typical variables:
     • Day_after_eclosion – day number;
     • Eggs_per_female – mean egg number per female on each day for each treatment;
     • Total_eggs_per_female – cumulative eggs per female.

Photographs of 8-day-old females (Figure 3E–F) are not included; only numeric data.

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Sheet: Fig4

Content: Effects of HvpKr-h1 RNAi on vitellogenin and vitellogenin receptor
expression and ovarian development in H. vigintioctopunctata.

Related figure: Figure 4A–H.

This sheet contains:

1. Gene expression in fat body and ovary (Figure 4A–D)
   • Relative expression of HvpVg and HvpVgR in the fat bodies and ovaries of
     8-day-old females injected with dsKr-h1 vs. dsegfp.
   • For each gene–tissue combination, there is a Prism-style table with:
     • Treatment – dsegfp or dsKr-h1;
     • 2-△△ct values for biological replicates;
     • P value, P value summary, t, df.
2. Ovary morphology and histology (Figure 4E–H)
   • These are image-based results (dissected ovaries, HE-stained sections); they are
     not included as files here.
   • No additional numeric measurements beyond those used for gene expression are
     stored.

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Sheet: Fig5

Content: Effects of HvmHairy, HvmGroucho, or both on Vg/VgR expression,
preoviposition, oviposition, and hatchability in the two Henosepilachna species.

Related figure: Figure 5A–N.

This sheet includes several experimental blocks:

1. Target gene knockdown (Figure 5A)
   • Relative transcript levels of HvmHairy and HvmGroucho (and combinations)
     after RNAi.
   • Tables include 2-△△ct values and Prism t-test outputs.
2. Vg / VgR expression (Figure 5B–E)
   • Relative expression of HvmVg and HvmVgR after knockdown of Hairy,
     Groucho, or both.
   • Columns follow the same pattern: treatment groups, replicate 2-△△ct values,
     and Prism statistics.
3. Preoviposition period (Figure 5F–H)
   • Days to first oviposition for individual females under different RNAi treatments.
   • Typical variables:
     • Female_ID
     • Treatment – e.g., dsegfp, dsHairy, dsGroucho, dsHairy_dsGroucho
     • Preoviposition_days
4. Oviposition and hatchability (Figure 5I–N)
   • Daily egg production, total eggs per female, and hatchability (%) under different
     treatments in the two species.
   • Variables are similar to those described for Figure 2 and Figure 3:
     • Day_after_eclosion
     • Eggs_per_female
     • Total_eggs_per_female
     • Hatchability_percent

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3. Abbreviations and gene names

• JH – juvenile hormone
• JHAMT – juvenile hormone acid methyltransferase
• MFE – methyl farnesoate epoxidase
• Met – Methoprene-tolerant
• Tai – Taiman
• Kr-h1 – Krüppel homolog 1
• Vg – vitellogenin
• VgR – vitellogenin receptor
• Hvm – Henosepilachna vigintioctomaculata
• Hvp – Henosepilachna vigintioctopunctata
• HE – head
• FG – foregut
• MG – midgut
• HG – hindgut
• MT – Malpighian tubules
• FB – fat body
• EP – epidermis
• OV – ovary
• dsRNA – double-stranded RNA
• dsKr-h1 – dsRNA targeting Kr-h1
• dsegfp – dsRNA targeting enhanced green fluorescent protein (negative control)
• SE – standard error
• Ct – cycle threshold in qRT-PCR