Ovary Activation Dynamics in the Bean Weevil Zabrotes subfasciatus (Bruchinae): The Essential Roles of Seeds and Males

Abstract

Phytophagous beetles, particularly those within the superfamilies Chrysomeloidea and Curculionoidea, constitute one of the most diverse and ecologically significant groups of insect herbivores. Within this group, the subfamily Bruchinae is especially notable for its close association with leguminous plant seeds. As most Bruchinae species do not feed during the adult stage, the timing and regulation of vitellogenesis remain unclear. Previous studies suggest that vitellogenesis may be triggered by volatile organic compounds emitted by host seeds, which promote juvenile hormone (JH) synthesis. This increase in JH is hypothesized to stimulate vitellogenesis, enhance female attractiveness, and ultimately facilitate fertilization and oviposition. To explore this hypothesis, we investigated the external cues regulating reproductive physiology in the capital breeder Zabrotes subfasciatus. Specifically, we examined the effects of host seeds and male presence on oviposition dynamics, fecundity, ovary activation, and the expression of vitellogenic genes (vg and vgR) throughout adult life. Our results show that females initiate vitellogenesis during the final phases of adult development, enabling oviposition to begin as early as the first day after emergence. Oviposition remains at basal levels throughout adult life unless both host seeds and males are present (p < 0.0001). This oviposition pattern is consistent with ovary activation dynamics, which reveal that vitellogenesis peaks early in the oviposition period and is prolonged by the presence of seeds and males (p < 0.05). Notably, vg and vgR gene expression respond differentially to these cues (p < 0.05). We integrate our findings with previous literature to propose a working model for the regulation of oviposition in the Bruchinae beetle Z. subfasciatus.

Keywords: Bruchinae; VOCs; Zabrotes subfasciatus; bean weevil; host seed; juvenile hormone; ovary activation; oviposition; vitellogenesis; vitellogenin.

Figure 1

Ovary activation in Zabrotes subfasciatus occurs during the pharate-adult stage, prior to adult emergence. The ovaries (except that in (C)) were embedded in Historesin, sectioned to a thickness of 5 μm, and stained with Harris’ hematoxylin and eosin [55]. Micrographs were captured using a Nikon Eclipse 80i microscope equipped with a digital camera and Nis-Element 3.1 imaging software. For the image in (C), ovary was processed for 4′,6-diamidino-2-phenylindole (DAPI) and phalloidin conjugated to Alexa 546 and the micrograph captured in a confocal microscope C2⁺ Nikon. (A) The diagram illustrates the ovary of an adult insect. (B,C) Histological and confocal images display a telotrophic ovariole highlighting nutritive cords. (D–G): Representative images of females and ovariole sections; (D) white-eyed pupa; (E,F) final pharate-adult phase; (G) one-day-old adult female.

Figure 2

Impact of host seed availability and mate presence on oviposition behaviour and fecundity of Zabrotes subfasciatus females. Eighteen newly emerged females per experimental group were individually placed in acrylic containers. Group A: females only; Group B: females and males; Group C: females and seeds; Group D: females, males, and seeds. The number of laid eggs was registered daily as well as the number of adults emerged from each of the laid egg during the 10 days of the oviposition period. (A) Oviposition and adult emergence profiles of the females from experimental group D: The number of emerged adults represents the individuals that developed from eggs laid on each specific day during the oviposition period. Different uppercase letters on the bars indicate statistically significant differences in the mean number of eggs laid during the oviposition period and different lowercase letters indicate statistically significant differences in the mean number of emergents (comparing the means of the different days). Two-way repeated measures ANOVA, followed by Šídák’s multiple comparisons test, ** p < 0.01. (B) Total number of eggs laid per female during the oviposition period (mean + SD). Ordinary one-way ANOVA followed Tukey’s multiple comparisons test, **** p < 0.0001. (C) Heatmap depicting the mean number of eggs laid per female in each of the 10-day oviposition period. (D) Oviposition profile per female during the 10-day oviposition period (mean + SD). * p < 0.05; ** p < 0.01; *** p < 0.001. For statistical details, see Tables S2–S5.

Figure 3

Impact of host seed availability and mate presence on the ovary activation of adult Zabrotes subfasciatus females. Eighteen newly emerged females per experimental group were individually placed in acrylic containers. Group A: females only; Group B: females and males; Group C: females and seeds; Group D: females, males, and seeds. The upper images depict representative photographs of ovarioles with oocytes in different levels of development (A), (Stage I ovarioles) and an ovary with fully developed oocytes (B), (Stage II ovarioles). Ten females from each experimental group were dissected and the activation level of their ovarioles was registered as corresponding to Stage I (C) or Stage II (D). Different letters on the columns with values from the same experimental group (colour) over the time studied indicate statistically significant differences (p < 0.05). Data are presented as mean + SD. Fisher’s exact test, * p < 0.01 and *** p < 0.001. For statistical details, see Tables S6–S9.

Figure 4

Impact of host seed availability and mate presence on the expression of vitellogenic genes in Zabrotes subfasciatus females. Eighteen newly emerged females per experimental group were individually placed in acrylic containers. Group A: females only; Group B: females and males; Group C: females and seeds; Group D: females, males, and seeds. Tissues from adult females were dissected and 4 RNA pools from 5 individuals were subjected to RT-qPCR (whole body of pupae were processed). Raw data were first normalized to the reference gene (rpl32) and the relative quantities of transcripts were calculated using the 2^−ΔΔCt method with efficiency correction and a control sample for calibration [59] (Applied Biosystems User bulletin #2). Y axes: 2^−ΔΔCt values. Comparisons with the pupal stage, as well as between experimental groups and days, were performed using the Mann–Whitney and test Kruskal–Wallis’s test followed by Dunn’s multiple comparisons test. Median and interquartile range (25th–75th percentiles), with minimum and maximum values; * p < 0.05; ** p < 0.01. (A) Vg gene transcript levels in carcass tissues, which include fat body. (B) VgR gene transcript levels in carcass tissues. (C) Vg gene transcript levels in ovary tissues. (D) VgR gene transcript levels in ovary tissues. For statistical details, see Tables S10–S17.

Figure 5

Proposed working model of oviposition regulation in the seed beetle Zabrotes subfasciatus. The proposed increase in juvenile hormone (JH) synthesis following female exposure to host seeds, as well as the chemical nature of the sex pheromone involved, require experimental validation. CA = corpora allata; FB = fat body; Ov = ovaries; JH = juvenile hormone; Vg = vitellogenin; VgR = vitellogenin receptor.