Pupal Cues Increase Sperm Production but Not Testis Size in an Insect

Abstract

Theoretic and empirical studies show that social surroundings experienced by male insects during their larval or adult stage can influence their testicular investment in diverse ways. Although insect pupae do not feed and crawl, they can communicate using sex-specific and/or non-sex specific cues. Yet, it is unknown, in any insect, whether and how male pupae can fine-tune their resource allocation to sperm production and testis size in response to socio-sexual environments. We investigated this question using a moth, Ephestia kuehniella, which produces fertile eupyrene sperm and unfertile apyrene sperm. We held male pupae individually or in groups with different sex ratios, and dissected adults upon eclosion, measured their testis size, and counted both types of sperm. We demonstrated that after exposure to conspecific pupal cues regardless of sex, male pupae increased production of eupyrenes and apyrenes at the same rate but kept testis size unchanged. We suggest that testis size is fixed after pupation because most morphological traits are formed during the larval stage, allowing little room for pupae to adjust testis size. Like adults, male pupae with fully grown testes have sufficient resources to produce more sperm of both types according to the perceived increase in sperm competition risk.

Juvenile socio‐sexual experience determines lifetime sperm expenditure and adult survival in a polygamous moth, Ephestia kuehniella

“…In treatments 6M‐LP, 6M‐P, and SM‐LP, the lifetime number of eupyrene sperm ejaculated (Fig. 2A ) was significantly higher than that counted at emergence ( t 50 = 4.20, P < 0.001 for 6M‐LP; t 50 = 2.52, P = 0.016 for 6M‐P; t 54 = 3.09, P = 0.004 for SM‐LP) (Liu et al ., 2021 , 2022 ).The lifetime number of apyrene sperm ejaculated (Fig. 2B ) was also significantly higher than that measured at emergence in all five treatments ( t 50 = 8.61, P < 0.001 for 6M‐LP; t 51 = 3.33, P = 0.002 for 1M5F‐LP; t 50 = 5.58, P < 0.001 for 6M‐P; t 52 = 3.21, P = 0.002 for 1M5F‐P; t 54 = 5.15, P < 0.001 for SM‐LP) (Liu et al ., 2021 , 2022 ).…”

“…The sex of the fourth instar larvae and pupae can be determined by visible testes in males’ abdomens (Fig. 1 ; Liu et al ., 2021 ). We randomly selected newly molted fourth‐instar larvae (L) and newly pupated pupae (P) from the experimental line and transferred them into glass vials (7.5 cm height × 2.0 cm diameter) to create five sociosexual enviroments for focal males (M) (Fig.…”

“…A log‐rank test (LIFETEST procedure) was applied to compare the survival probability of focal males between treatments. The relationship between the total number of eupyrene and apyrene ejaculated was analyzed by a general linear model (GLM procedure) and an analysis of covariance (ANCOVA) was used to compare the slopes of regression lines between treatments (Liu et al ., 2021 ). We used a linear mixed‐effects model with repeated measures (MIXED procedure) to test how treatment affected males’ sperm allocation in successive matings.…”

“…Because the moths used in the current study were from the same batch reared under the same conditions as in Liu et al . ( 2021 , 2022 ), we used a two‐sample t test (TTEST procedure) to compare the number of eupyrene and apyrene sperm and their ratio in lifetime ejaculates with those recorded at emergence (Liu et al ., 2021 , 2022 ).…”

“…Adult males increase their sperm allocation after exposure to rival cues during early adulthood (Esfandi et al ., 2020 ; Liu et al ., 2020 ). Adults have more sperm of both types at emergence after their pupae are exposed to higher density of conspecifics (Liu et al ., 2021 ) but if such exposure starts at the larval stage, only eupyrene sperm increases at emergence (Liu et al ., 2022 ), suggesting that larvae and pupae may respond to the same social context differently.…”