DOI: 10.5061/dryad.47d7wm3t5
title: Kr-h1 is vital for vitellogenesis in two ladybird beetle species

description: Krüppel homolog1 (Kr-h1), a transcription factor with a DNA-binding motif
 of eight C2H2 zinc fingers, exerts species-specific functions by
 mediating juvenile hormone (JH) signal in certain physiological processes
 during female reproduction in insects. Nevertheless, the regulatory role
 of Kr-h1 on vitellogenesis deserves further exploration. Here, we found
 that Kr-h1 was actively expressed from 0- to
 12-day-old females and among detected tissues, including fat body and
 ovary in two potato-defoliating
 beetles, Henosepilachna vigintioctopunctata and H. vigintioctomaculata. RNA interference (RNAi) for Kr-h1 significantly lengthened the preoviposition period, dramatically impaired ovarian development, and decreased oviposition. Weaker yolk accumulation occurred in the Kr-h1 RNAi oocytes, in contrast to a larger quantity of yolk granules in controls. Correspondingly, Vg transcript levels in the fat bodies and ovaries and of VgR amounts in the ovarian tissues were substantially reduced in the Kr-h1 knockdown samples. In H. vigintioctomaculata, RNAi for the other two JH signal genes, HvmHairy and HvmGroucho, was also performed. Silencing either or both genes affected neither female reproduction nor the mRNA levels of Vg and VgR. Therefore, our findings suggest Kr-h1, rather than Hairy/Groucho, regulates vitellogenesis in the two Henosepilachna species. Moreover, targeting Kr-h1 could potentially be a new way to control potato pests.
description: Methods summary: Female beetles of
 <em>H. vigintioctomaculata</em> and <em>H.
 vigintioctopunctata</em> were injected with dsRNAs targeting Kr-h1,
 Hairy, Groucho, or control dsEGFP. qRT-PCR was used
 to quantify transcript levels of JH-pathway genes (Kr-h1, Met, Hairy,
 Groucho, Vg, VgR, and related biosynthetic genes) in whole bodies or
 selected tissues. Relative expression was calculated
 using the 2 <sup>−ΔΔCt</sup> or 2 <sup>−ΔCt</sup>
 method with appropriate reference genes.
 Preoviposition period, daily egg production, total egg number
 per female, and hatchability were recorded over time.
 Statistical comparisons between treatments were performed using
 Student’s t-test in GraphPad Prism, and the corresponding P values and
 test statistics are included in the tables where applicable.
 For full experimental details and interpretation,
 please refer to the associated article in <em>Insect Molecular
 Biology</em>.
description: # Kr-h1 is vital for vitellogenesis in two ladybird beetle species Dryad
 DOI:
 [https://doi.org/10.5061/dryad.47d7wm3t5](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. --- ## 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. --- ### 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). --- ### 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. --- ### 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. --- ### 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. --- ### 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` --- ## 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