Male and female age affects the reproductive potential of two tephritid flies

In insects, aging produces deterioration in physiological and cellular functioning, affecting their reproductive potential. Anastrepha fraterculus and Ceratitis capitata are two fruit fly species where overwintering adults resume their reproductive activity in spring, giving old individuals the possibility of mating with young adults. Most age studies focus on male reproductive capacity; however, we lack information on how the interaction between the ages of both sexes can determine post-mating processes. Here, we studied sex and age effects on (i) female fecundity and fertility, (ii) failure to leave viable offspring (reproductive failure), and (iii) female remating behavior. We found that young pairs of both species had higher fecundity, but young C. capitata males mated with old females had the lowest fecundity. This suggests that overwintering flies in this species will not substantially contribute to the next generation. We also found in C. capitata more prevalent reproductive failure in hetero-age combinations, which could be due to age recognition between mates, resulting perhaps in differential ejaculate allocation. Copula duration was positively associated with female age, yet it was longer for older A. fraterculus females and shorter for C. capitata females. Female remating was lower when young females mated with old males in C. capitata. This would imply that males perceive young females of “good quality” and thus invest and transfer all the ejaculate possible to ensure the delay of renewal of female receptivity. Aging does not always cause a decline in reproductive potential, which may be important in species that overwinter as adults. Complex interactions between female physiology and male ejaculate senescence can impact postcopulatory behaviors that affect reproductive success for both sexes.

Methods
On the day of emergence, flies of both species were sorted by sex and placed in 14 L plastic containers with 200 adults each, provided with ad libitum access to water and a diet of sugar (57.9%), hydrolyzed yeast (14.5%), hydrolyzed corn (27.3%), and vitamin E (0.3%). The study used old flies (54 days for A. fraterculus and 35 days for C. capitata) and young flies (11-14 days for A. fraterculus and 5 days for C. capitata).

For testing, one virgin female and one virgin male were placed in individual 350 cc containers in four age combinations (♂ old x ♀ old; ♂ old x ♀ young; ♂ young x ♀ old; ♂ young x ♀ young). Copulations were recorded, and female remating was checked 24 hours post-copulation for C. capitata and 48 hours for A. fraterculus. The study included three replicates with N=540 for A. fraterculus (135 per combination) and N=680 for C. capitata (170 per combination).

For fecundity and fertility evaluations, after copulation, males were discarded, and females were kept individually with food and water. An agar-based oviposition substrate was used, replaced every 2-3 days. Eggs were collected, counted, and incubated at 25 ± 1 °C for 5 days. Hatched eggs were identified by broken chorions, while unviable eggs remained turgid. The trial lasted 18 days with 8 observation dates.

Reproductive failure was defined as successful copulation without offspring, either due to zero fecundity (no eggs laid) or zero fertility (no eggs hatched). Most reproductive failures were due to zero fecundity. Two replicates were performed for each species, with a total sample size of 180 (60 for old x old and 40 for the other combinations).