DOI: 10.5061/dryad.qbzkh18w6
title: Early adaptation to an unusual host in the bean weevil <em>Zabrotes subfasciatus</em> is associated with changes in body size and reproductive physiology

description: Host shifts can present unique challenges and exert selective pressures
 that influence the evolutionary trajectory of phytophagous insect species.
 In Bruchinae beetles, which are highly specialized feeders on Fabaceae
 plants, host shifts are often linked to changes in life-history traits and
 are typically accompanied by reduced fitness. Since fitness is closely
 tied to reproductive success, morphophysiological traits such as body
 size, gonadal development, and the expression of vitellogenic genes may
 serve as indicators of how well a population adapts to a novel host. To
 test this hypothesis, we conducted a short-term selection experiment (12
 generations) involving the use of an atypical host, Cicer arietinum
 (chickpea), by the bean weevil Zabrotes subfasciatus (Boheman 1833)
 (Chrysomelidae: Bruchinae: Amblycerini). We estimated eight body measures,
 assessed the development of ovaries and testes, and quantified the
 expression of vitellogenin (vg) and vitellogenin receptor (vgR) genes in
 the fat body and ovary of newly emerged individuals (within 24 hours) from
 both selected populations. Our results reveal that the adaptation involves
 an increase in body size (e.g., the length of the abdomen not covered by
 elytra in females), which correlates with higher oocyte storage in the
 oviducts. This morphological change was coupled with a reduction in the
 size of the germarium in selected females and a decrease in vgR
 transcription levels. These findings provide valuable insight into the
 initial phases of phytophagous insect adaptation to an unconventional
 host, illustrating how such adaptations are accompanied by significant
 morphophysiological changes that affect crucial life-history traits and
 ultimately lead to a decline in fitness.
description: # Early adaptation to an unusual host in the bean weevil *Zabrotes
 subfasciatus* is associated with changes in body size and reproductive
 physiology Dataset DOI: [10.5061/dryad.qbzkh18w6](10.5061/dryad.qbzkh18w6)
 ## Description of the data and file structure **Body measures:** For
 measurement, individuals were positioned in dorsal and lateral views under
 a stereomicroscope (Zeiss - Stemi 305) and photographed using a 5 MP
 camera (ZEISS Axiocam 105 color) at 30x magnification. Scale bars were
 added to the photographs using the ZEN program (Zeiss, version 3.8) and
 analyzed with Photoshop software (version 25.3.1) (Adobe Inc., 2024) using
 the measurement tool. The defined scale was 341 pixels to 500 µm. The
 measurements analysed for males and females were: body length, body width,
 body thickness, elytra length, abdomen length, length of the abdomen not
 covered by elytra, pronotum length, and pronotum width. **Gonad
 measures:** Ovaries and testicles went through the DAPI-phallodin staining
 protocol. Slides were mounted in 80% glycerol and examined under a
 fluorescence microscope (Nis-Element C2 - Nikon), through which
 photographs were taken at 40x magnification. The images obtained after the
 fluorescence protocol were analyzed using Photoshop software (version
 25.3.1) (Adobe Inc., 2024) at a scale of 83 pixels to 100 µm. To assess
 the degree of activation, several parameters were recorded for each ovary:
 Average area of the last oocyte in the ovarioles, measured in each sampled
 ovary; germarium length, measured from its end to the dark dividing line
 visible in the fluorescence protocol; percentage of calyces with reserved
 oocytes, indicated by the enlarged size and visible curves of the oocytes.
 For the testicles, an average area was calculated based on the area of
 each testiole. **Transcript levels determination by RT-qPCR:** Total RNA
 was extracted using TRIzol® (Ambion) from pools (six pools each, n= 6) of
 three whole bodies and five pairs of ovaries sampled from individuals of
 the parental population (F0) and from the F3, F7, and F11 generations of
 selection. These samples included individuals reared on both chickpeas and
 beans. Relative gene expression levels were calculated using the 2-ΔΔCT
 method with efficiency correction, using a control sample for calibration
 (Livak and Schmittgen, 2001; Applied Biosystems User Bulletin #2). ###
 Files and variables #### File: gonadas.csv **Description:** This file
 holds the data from the analysis of the beetles' gonads. For females,
 the parameters include the average area of the last oocyte, the length of
 the germarium, and the percentage of calyces with reserved oocytes. For
 males, the file contains the average area of each testiole. The data was
 collected from 5 females and 5 males per population, from individuals that
 emerged within 24 hours. A column named "Adicional" contains
 values for the F0 generation to facilitate calculations in R. N/A values
 in this file indicate cells where a measurement was not applicable. #####
 Variables * Adicional: Used to analyse additional block (F0) in the ExpDes
 R package. (categorical interpretation) * Geracao: Generation (categorical
 interpretation) * Semente: Seed / Grain (categorical interpretation) *
 Populacao: Population (categorical interpretation) * AUO: Last oocyte
 area (μm^2^) * NOO: Number of oocytes per ovariole (count) * CGO:
 Germarium length (μm) * ATT: Area of testioles (μm^2^) **Categorical
 Interpretation Keys:** * **Adicional**: Used to analyse aditional block
 (F0) in ExpDes R package. The only value is F0. - **Geracao**
 (Generation): This column represents the generation of the beetle in the
 short-term selection experiment. Values: * 1: Represents the F1 generation
 * 2: Represents the F2 generation * 3: Represents the F3 generation * 8:
 Represents the F8 generation * 12: Represents the F12 generation -
 **Semente** (Seed): This column specifies the type of seed, or host, on
 which the beetles developed. Values: * F: Represents common bean
 (*Phaseolus vulgaris*), the beetle's usual host. * GB: Represents
 chickpea (*Cicer arietinum*), the unusual and less preferred host. -
 **Populacao** (Population): This column indicates the replicate group
 within the experiment. Three replicate groups were used for each seed
 type. Values: * 1: Replicate 1 * 2: Replicate 2 * 3: Replicate 3 ####
 File: Corpos.csv **Description:** This file contains the raw data from the
 body size measurements of the beetles. The dataset includes eight
 different body measurements taken from 10 females and 10 males from each
 population. The data were collected from the parental generation (F0) and
 the selection generations F1, F2, F3, F8, and F12, for both the bean and
 chickpea populations. A column named "Adicional" contains values
 for the F0 generation to facilitate calculations in R. N/A values in this
 file indicate cells where a measurement was not applicable.  #####
 Variables * Adicional: Used to analyse additional block (F0) in the ExpDes
 R package. (categorical interpretation) * Geracao: Generation (categorical
 interpretation) * Semente: Seed / Grain (categorical interpretation) *
 Populacao: Population (categorical interpretation) - CCF: Body length of
 females (μm) - CPF: Pronotum length of females (μm) - LPF: Pronotum width
 of females (μm) - CEF: Elytra length of females (μm) - LCF: Body width of
 females (μm) - ANF: Not covered abdomen of females (μm) - CAF: Adomen
 length of females (μm) - ECF: Body thickness of females (μm) - CCM: Body
 length of males (μm) - CPM: Pronotum length of males (μm) - LPM: Pronotum
 width of males (μm) - CEM: Elytra length of males (μm) - LCM: Body width
 of males (μm) - ANM: Not covered abdomen of males (μm) - CAM: Adomen
 length of males (μm) - ECM: Body thickness of males (μm) **Categorical
 Interpretation Keys:** * **Adicional**: Used to analyse aditional block
 (F0) in ExpDes R package. The only value is F0. * **Geracao**
 (Generation): This column represents the generation of the beetle in the
 short-term selection experiment. Values: * 1: Represents the F1 generation
 * 2: Represents the F2 generation * 3: Represents the F3 generation * 8:
 Represents the F8 generation * 12: Represents the F12 generation *
 **Semente** (Seed): This column specifies the type of seed, or host, on
 which the beetles developed. Values: * F: Represents common bean
 (*Phaseolus vulgaris*), the beetle's usual host. * GB: Represents
 chickpea (*Cicer arietinum*), the unusual and less preferred host. *
 **Populacao** (Population): This column indicates the replicate group
 within the experiment. Three replicate groups were used for each seed
 type. Values: * 1: Replicate 1 * 2: Replicate 2 * 3: Replicate 3 ####
 File: reservas.csv **Description:** This file provides data on the storage
 of oocytes within female ovaries. The dataset includes two main metrics
 related to this physiological change, which is a key indicator of
 reproductive fitness. One column contains the percentage of oocytes
 reserved, based on an average of 5 gonads, which reflects the overall
 level of oocyte retention. The other column contains data on the
 percentage of calyces with reserved oocytes, based on an average of 10
 calyces (2 per ovary), providing a more granular look at oocyte storage.
 The data was collected from 5 females per population, from individuals
 that emerged within 24 hours. A column named "Adicional"
 contains values for the F0 generation to facilitate calculations in R. N/A
 values in this file indicate cells where a measurement was not applicable.
 ##### Variables * Adicional: Used to analyse additional block (F0) in the
 ExpDes R package. (categorical interpretation) * Geracao:
 Generation (categorical interpretation) * Semente: Seed /
 Grain (categorical interpretation) * Populacao: Population (categorical
 interpretation) * GOO: Gonads with oocytes (in %) * CCO: Calyces with
 oocytes (in %) **Categorical Interpretation Keys:** * **Adicional**: Used
 to analyse additional block (F0) in the ExpDes R package. The only value
 is F0. - **Geracao** (Generation): This column represents the generation
 of the beetle in the short-term selection experiment. Values: * 1:
 Represents the F1 generation * 2: Represents the F2 generation * 3:
 Represents the F3 generation * 8: Represents the F8 generation * 12:
 Represents the F12 generation - **Semente** (Seed): This column specifies
 the type of seed, or host, on which the beetles developed. Values: * F:
 Represents common bean (*Phaseolus vulgaris*), the beetle's usual
 host. * GB: Represents chickpea (*Cicer arietinum*), the unusual and less
 preferred host. - **Populacao** (Population): This column indicates the
 replicate group within the experiment. Three replicate groups were used
 for each seed type. Values: * 1: Replicate 1 * 2: Replicate 2 * 3:
 Replicate 3 #### File: Vg_levels.csv **Description:** This file contains
 the mRNA levels of the vitellogenin (vg) gene, as determined by RT-qPCR.
 The measurements were performed on pools of three whole bodies from the F0
 (parental) generation and the F3, F7, and F11 selection generations. The
 data includes individuals from both the chickpea and bean populations. N/A
 values in this file indicate cells where resulting values were not
 applicable. ##### Variables * Tube: the analysed replicate sample
 (categorical interpretation) * RPL32: Cycle threshold (CT) for the
 reference gene RPL32 (CT values) * Vg: Cycle threshold (CT) for the gene
 of interest Vg (CT values) * DCt: Delta CT method (subtract reference
 gene's CT from gene of interest's CT, unitless) * DDCt:
 Comparative CT method (subtract the control sample's Delta CT from
 the experimental sample's Delta CT, unitless) * 2^-(DDCt): Fold
 change (Raise two to the power of negative DDCT, unitless) * Group: Sample
 group analysed for the statistics that follow (categorical interpretation)
 * Mean: Mean for the analysed group (unitless) * SD: Standard deviation
 for the analysed group (unitless) * SEM: Standard error of mean for the
 analysed group (unitless) **Categorical Interpretation Keys:** * **Tube**
 (Replicate Sample): This column identifies the specific sample tube
 analyzed for gene expression. The values combine a grain abbreviation, the
 generation, and a replicate number to uniquely identify each sample. It
 has the format *[Grain Abbreviation] [Generation] [Replicate Number]*.
 Values: * Grain Abbreviation: * F: Common bean (*Phaseolus vulgaris*) *
 GB: Chickpea (*Cicer arietinum*) * Generation: * 0F: Parental generation
 (F0) * 3F: 3rd generation of selection (F3) * 7F: 7th generation of
 selection (F7) * 11F: 11th generation of selection (F11) * Replicate
 Number: A number from 1 to 4 indicating the biological replicate pool. *
 **Group** (sample group): This column groups the analyzed tubes that share
 the same grain type and generation. It is used to analyze the expression
 patterns across different conditions. It has the format *[Grain
 Abbreviation] [Generation]*. Values: * Grain Abbreviation: * F: Common
 bean (*Phaseolus vulgaris*) * GB: Chickpea (*Cicer arietinum*) *
 Generation: * 0F: Parental generation (F0) * 3F: 3rd generation of
 selection (F3) * 7F: 7th generation of selection (F7) * 11F: 11th
 generation of selection (F11) #### File: VgR_levels.csv
 **Description:** This file contains the mRNA levels of the vitellogenin
 receptor (vgR) gene, as determined by RT-qPCR. The samples used for this
 analysis were pools of five pairs of ovaries from individuals in the
 parental (F0) generation and the F3, F7, and F11 selection generations.
 The data includes individuals from both the chickpea and bean populations.
 A column named "adicional" contains values for the F0 generation
 to facilitate calculations in R. N/A values in this file indicate cells
 where resulting values were not applicable. ##### Variables * Tube: the
 analysed replicate sample (categorical interpretation) * RPL32: Cycle
 threshold (CT) for the reference gene RPL32 (CT values) * VgR: Cycle
 threshold (CT) for the gene of interest VgR (CT values) * DCt: Delta CT
 method (subtract reference gene's CT from gene of interest's CT,
 unitless) * DDCt: Comparative CT method (subtract the control
 sample's Delta CT from the experimental sample's Delta CT,
 unitless) * 2^-(DDCt): Fold change (Raise two to the power of negative
 DDCT, unitless) * Group: Sample group analysed for the statistics that
 follow (categorical interpretation) * Mean: Mean for the analysed group
 (unitless) * SD: Standard deviation for the analysed group (unitless) *
 SEM: Standard error of mean for the analysed group (unitless)
 **Categorical Interpretation Keys:** * **Tube** (Replicate Sample): This
 column identifies the specific sample tube analyzed for gene expression.
 The values combine a grain abbreviation, the generation, the
 "ov" that indicates the samples were obtained from ovaries, and
 a replicate number to uniquely identify each sample. It has the
 format *[Grain Abbreviation] [Generation] ["ov"] [Replicate
 Number]*. Values: * Grain Abbreviation: * F: Common bean (*Phaseolus
 vulgaris*) * GB: Chickpea (*Cicer arietinum*) * Generation: * 0: Parental
 generation (F0) * 3: 3rd generation of selection (F3) * 7: 7th generation
 of selection (F7) * 11: 11th generation of selection (F11) * Replicate
 Number: A number from 1 to 4 indicating the biological replicate pool. *
 **Group** (sample group): This column groups the analyzed tubes that share
 the same grain type and generation. It is used to analyze the expression
 patterns across different conditions. It has the format *[Grain
 Abbreviation] [Generation]*. Values: * Grain Abbreviation: * F: Common
 bean (*Phaseolus vulgaris*) * GB: Chickpea (*Cicer arietinum*) *
 Generation: * 0F: Parental generation (F0) * 3F: 3rd generation of
 selection (F3) * 7F: 7th generation of selection (F7) * 11F: 11th
 generation of selection (F11) ## Code/software Although we used Microsoft
 Excel to open the archives, LibreOffice Calc is also an option. ## Access
 information Data was derived from the following sources: * [Analysed
 pictures](https://unifalmgedubr-my.sharepoint.com/:f:/g/personal/bruno_cruz_sou_unifal-mg_edu_br/EiLe7IJcMRFChPtV9YH7lhwB1ZQr7axhkUDdhQiTJ_ysqw?e=7RGUv1)