Antheraea peptide and its analog: Their influence on the maturation of the reproductive system, embryogenesis, and early larval development in Tenebrio molitor L. beetle

Abstract

In recent years, many new immunologically active peptides from insects have been identified. Unfortunately, in most cases, their physiological functions are not fully known. One example is yamamarin, a pentapeptide isolated from the caterpillars of the Antheraea yamamai moth. This peptide has strong antiproliferative properties and is probably involved in the regulation of diapause. Additionally, antiviral activity was discovered. The results of the research presented in this paper are, to our knowledge, the first attempt to characterize the biological effects of yamamarin on the functioning of the reproductive processes and embryonic development of insects using a model species, the beetle Tenebrio molitor, a commonly known pest of grain storage. Simultaneously, we tested the possible activity of the molecule in an in vivo system. In this research, we present the multifaceted effects of yamamarin in this beetle. We show that yamamarin influences ovarian growth and development, maturation of terminal oocytes, level of vitellogenin gene transcript, the number of laid eggs, duration of embryonic development, and larval hatching. In experiments with palmitic acid-conjugated yamamarin (C16-yamamarin), we also showed that this peptide is a useful starting molecule for the synthesis of biopharmaceuticals or new peptidomimetics with gonadotropic activity and effects on embryonic development. The data obtained additionally provide new knowledge about the possible function of yamamarin in insect physiology, pointing to the important role of this pentapeptide as a regulator of reproductive processes and embryonic development in a heterologous bioassay with T. molitor.

Fig 1. Timeline of experiments.

A—Gonadotropic effects of peptides in females during the first reproduction cycle, the influence of peptides on the oocyte’s maturation process and development of follicular epithelium development, B—Influence of peptides on egg production, C—Examination of gene encoding vitellogenin expression level, D—Duration of embryogenesis and larval hatching after peptideinjection, E—Duration of embryogenesis and larval hatching after topical application of peptides.

Fig 2. Scheme of follicular epithelium and method of its analysis.

S₁–S₃ follicular cells, S₄–intercellular space.

Fig 3

Representative micrographs obtained with a stereoscopic microscope of 4-day-old T. molitor ovaries isolated from control females injected with physiological saline (B), yamamarin (C) and C16-yamamarin (D) at concentrations of 10⁻⁷ M. (A) Representative picture of ovary: red frame—single ovariole, blue arrow—terminal oocyte, orange arrow—lateral oviduct, green arrow—common oviduct. The white scale bar corresponds to 1000 μm.

Fig 4. Changes in the volume of the terminal oocytes in the 4-day-old T. molitor females, injected on the first day after eclosion with saline (control), yamamarin, and C16-yamamarin at concentrations 10⁻¹¹, 10⁻⁷ and 10⁻³ M.

Data represent the mean value ± SD for n ≥ 10. Statistically significant differences (one-way ANOVA, Dunn’s multiple comparisons test) from the control values are indicated by asterisks as indicated: p ≤ 0.0001 (****), p ≤ 0.1 (*).

Fig 5

Representative images of a properly developed follicular epithelium in ovaries isolated from 4-day-old T. molitor beetles injected on the first day after eclosion with saline (A, B), yamamarin (C, D) and C16-yamamarin (E, F) at concentrations of 10⁻³ M. The white scale bar corresponds to 100 μm.

Fig 6

Patency index (A), surface of intercellular spaces (B) and surface of follicular cells. Statistically significant differences (one-way ANOVA with Dunn’s multiple comparisons test) in comparison to the control are indicated by asterisks p ≤ 0.1 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p <0,0001 (****), n ≥26.

Fig 7. The expression level of vitellogenin in female fat body tissue 1 h, 24 h and 48 h after injection, was calculated by the ΔΔCt method (reference syntaxin-1).

The data represent mean value ± SD from 4 independent biological (each biological sample consisted of tissue isolated from ≥ 10 females) and 3 technical repetitions. Statistical significance was tested with Kruskal-Wallis H = 15.49 and Mann-Whitney test (1 h control and 48 h yamamarin U = 37.0, 48 h control and 48 h yamamarin U = 25.0). Significant differences are indicated by asterisks as indicated: p ≤ 0.1 (*).

Fig 8. Oviposition starts time and the number of eggs laid by the females injected 1 day after eclosion with saline (control), yamamarin and C16-yamamarin.

Statistically significant differences in comparison to the control (Spearman’s correlation coefficient) are indicated by asterisks p ≤ 0.001 (***).

Fig 9. Duration of embryonic development and the hatchability of the next generation larvae from eggs laid by females injected with physiological saline (control), yamamarin and C16-yamamarin.

Statistically significant differences (two-way ANOVA with Dunnett’s multiple comparisons test) in comparison to the control are indicated by asterisks p ≤ 0.001 (***), p ≤ 0.01 (**).

Fig 10. Duration of embryonic development and the hatchability of the next generation larvae from eggs, which were treated topically with physiological saline (control), yamamarin and C16-yamamarin.

Statistically significant differences (two-way ANOVA with Dunnett’s multiple comparisons test) in comparison to the control are indicated by asterisks p ≤ 0.01 (**), p ≤ 0.001 (***), p <0,0001 (****).