Introduction

The family Pentatomidae comprises important predators responsible for the biological control of Lepidoptera and Coleoptera larvae (^{Soares et al. 2009}; ^{De Carvalho et al. 2020}; ^{Pires et al. 2020}; ^{Cornelius et al. 2021}). However, phytophagous species of this order are pests of wild and cultivated plants (^{Soares et al. 2022}). Podisus is the most diverse genus in the Pentatomidae, with species distributed in the Neotropical and Nearctic regions (^{Brugnera et al. 2020}; ^{Oliveira-Júnior et al. 2021}; ^{Roell 2021}).

The predatory stink bug Podisus fuscescens (Dallas, 1851) (Hemiptera: Pentatomidae) (often identified in the scientific literature as P. distinctus (Stål) in the last two decades) has generalist predatory habits and survives in the field, even when its prey populations is at low level (^{Lacerda et al. 2004}; ^{Brugnera et al. 2020}). The efficiency of this natural enemy depends on its high ability to reproduce, characterizing it as an r strategist, with high oocyte development, ovulation and oviposition rates (^{Force 1972}). Furthermore, mating and components of the ejaculation increases female reproductive output in many insect species (^{Nanfack-Minkeu and Sirot 2022}).

Polyandry, multiple mating by females, increases the genetic variability in insects (^{Dahan et al. 2022}). Females of Podisus spp. are polyandrous and can copulate in the laboratory up to 13 times during its reproductive cycle. However, three to four mating are sufficient to fertilize the eggs and to maintain the nymph hatching above 80% during the female lifetime (^{Torres et al. 1997}; ^{Soares et al. 2011}). The period necessary for the reproductive apparatus to mature is also important to maintain the females' fertility in the laboratory. Ovaries of the predator Podisus nigrispinus (Dallas, 1851) (Hemiptera: Pentatomidae) are immature when they emerge, but after seven days, their ovaries are developed with stored mature oocytes (^{Soares et al. 2011}). The period for the maturation of the ovaries of P. fuscescens is unknown.

Understanding the reproduction of P. fuscescens and determining the ideal mating age can improve the mass rearing techniques of this predator. Avoiding unnecessary copulations prevents the insects from wasting energy or becoming injured, reducing the individuals' longevity. The objective of this work was to evaluate the fecundity of females of the predator P. fuscescens with different pre-mating periods.

Materials and methods

The experiment was carried out in the laboratory at an air temperature of 25 ± 2 °C, an air relative humidity of 65 ± 10% and a photophase of 12 h. Individuals of P. fuscescens were obtained from mass rearing at the Laboratory for Biological Control of Insects (LCBio) of the Universidade Federal de Viçosa. Three hundred eggs from different egg masses were collected and separated in Petri dishes (9.0 x 1.5 cm) with a moistened cotton swab until the nymphs hatched. Nymphs were reared in Petri dishes (9.0 x 1.5 cm), in groups of 10 individuals, with a cotton swab moistened with distilled water and with Tenebrio molitor L. (Coleoptera: Tenebrionidae) pupae were fed “ad libitum” until adulthood (^{Soares et al. 2009}; ²⁰¹¹).

Females were mated at the day of emergence (T1) or at one (T2), two (T3), three (T4), four (T5), or five (T6) days after the adult's emergence. Males were selected at three days of emergence in all treatments, as they are receptive to copulation from that age onwards (^{Carvalho et al. 1994}). This homogenization of the age of the male individuals is important to standardize their fertility and the quantity and quality of seminal material transferred to the females. The males were put together with the females for 24 hours (^{Souza-Souto et al. 2006}; ^{Reis et al. 2018}) in 500 mL plastic containers, closed with a screened plastic lid to facilitate ventilation. Distilled water was supplied in tubes (anesthetic type) fitted into a hole in the top of the lid.

The eggs were collected and counted daily and were kept in Petri dishes (9.0 X 1.5 cm), with a moistened cotton swab, at 25 ± 2°C and 65 ± 10 % RH. The number of hatched nymphs, eggs, eggs laid per female, percentage of eggs hatched, longevity, pre-oviposition, oviposition and post-oviposition periods (days) of the females were evaluated. The experimental design was completely randomized, with six treatments and 15 replications, with each couple of P. fuscescens considered one replication. Data were not normally distributed and were analyzed by the non-parametric Kruskal-Wallis test, and the means compared using the Student-Newman-Keuls (SNK) method (P ≤ 0.01).

Results

The number of hatched P. fuscescens nymphs was higher in treatment T6 (105.13 ± 29.20) than in treatments T1 (3.60 ± 2.57) and T2 (10.87 ± 8.30) (H = 82.38; P < 0,001) (Fig. 1 A ).

The total number of eggs per female was lower in the T2 individuals (128.27 ± 32.16) than in the T6 individuals (307.8 ± 4.89) (H = 80.39; P < 0,001) (Fig. 1 B ). The number of egg masses per female was higher in T6 (14.80 ± 1.74) and lower in T1 (6.4 ± 1.01) and T2 (7.4 ± 1.74) (H = 79.25; P < 0,001) (Fig. 1 C ).

Figure 1 Number of nymphs/female (A), number of eggs/female (B), number of posture/female (C), proportion of hatched posture (%) (D), pre-oviposition period (days) (E), oviposition period (days) (F), Post-oviposition period (days) (G) e longevity (days) (H) of females of Podisus fuscescens (Hemiptera: Pentatomidae), mated immediately after emergence (T1) and with one (T2), two (T3), three (T4), four (T5) and five (T6) days after emergence. Means followed by the same letter do not differ by Kruskal-Wallis test, and the means were compared using the Student-Newman-Keuls (SNK) method (P ≤0.01). 

The number of hatched eggs was lower for couples with females of P. fuscescens mated on the emergence day (T1) (8.89 ± 6.88), with one day (T2) (9.91 ± 5.06) or two days (T3) (14.39 ± 7.40) after emergence, than for females at five days after emergence (T6) (51.48 ± 9.58) (H = 73.68; P < 0,001) (Fig. 1 D ).

The pre-oviposition period was longer for couples with females from treatment T1 (22.80 ± 1.61) than for couples with T5 (13.07 ± 1.84) and T6 (11.07 ± 1.17) (H = 78.56; P < 0,001) (Fig. 1 E ). However, the oviposition period was similar between treatments, ranging from 29.00 ± 6.47 to 39.73 ± 4.78 (H = 14.73; P = 0.0115) (Fig. 1 F ). The post-oviposition period was longer for T1 females (13.86 ± 3.92) (H = 68.89; P < 0,001) (Fig. 1 G ). Longevity was similar among treatments, ranging from 60.33 ± 5.72 to 69.93 ± 6.69 days (H = 9.30; P = 0.097) (Fig. 1 H ).

Discussion

The higher number of nymphs hatched in the T6 treatment shows that females of the species P. fuscescens need approximately five days to mature their reproductive system. This has previously been reported for other species of the genus Podisus. The predator P. nigrispinus presents immature ovaries at emergence and, after seven days, mature ovaries, evidenced by the presence of oocytes (^{Soares et al. 2011}). The ovaries of Podisus maculiventris (Say, 1832) (Hemiptera: Pentatomidae) are fully mature up to 21 days after the emergence of its female (^{De Clercq and Degheele 1997}). Predatory stink bugs have meroistic-type ovaries (^{Lemos et al. 2005}), with nutritive cells known as guard cells or trophocytes, which supply the oocytes with metabolites and organelles through cytoplasmic bridges. Insects with this ovary type need some days to complete vitellogenesis, unlike insects such as parasitoids of the order Hymenoptera, which emerge with mature oocytes (^{Jervis et al. 2001}). Vitellogenesis is an important control point in oogenesis that integrates hormonal cues to match female physiological conditions (i.e., nutrition, mating status, etc.) (^{Zhang et al. 2022}).

In addition, females of Podisus sp. will have their spermatheca filled with the sperm during copulation. This organ is responsible for storing and maintaining viable sperm for later fertilization (^{Rodrigues et al. 2008}). However, the low viability and absence of fertile eggs in some situations may be due to insufficient transfer of viable sperm (^{Souza-Souto et al. 2006}). Sperm and other ejaculate components are often limited by quantity, quality, and timing of production (^{Perry et al., 2013}; ^{Jarrige et al. 2016}). Thus, the species' reproductive rate increases with mating success (^{Nason and Kelly 2020}). Females of P. fuscescens in non-fertile age, at the emergence and up to four days after, could reject copulation. Insects perceive and integrate a hierarchy of visual, chemical, and tactile cues for reproductive purposes (^{Benelli and Lucchi 2021}), even while in confinement. Courtship with males can result in important energy losses, increased injuries and predation risk.

The larger oviposition and offspring yield of P. fuscescens females in the treatment T6 can be explained by the stimulation caused by copulation. This hypothesis is reinforced by observing P. nigrispinus females, which start egg production and maturation soon after emergence, whether they are mated or not. However, mating is important to maintain this predator's egg production and oviposition rates (^{Soares et al. 2011}).

The lower offspring production in the treatments T1, T2 and T3 can also be due to the P. fuscescens females not being able to produce the necessary secretions to maintain the viability of the sperm in their spermatheca. Such secretions are produced in an accessory gland of the spermatheca (^{Rodrigues et al. 2008}). This gland may not be completely mature before the fifth day after the female emergence. Thus, the increased number of egg masses, eggs, the proportion of hatched eggs and the number of nymphs for females of treatment T6 suggests that five days after the emergence of the female is the ideal period necessary before the initial contact between the sex partners. The sexual maturation of this predator is a progressive process that, until completed, will not allow the females to reach their maximum fertility potential (^{Carvalho et al. 1994}).

The oviposition period and longevity did not differ among treatments. However, the values observed in this work were higher than those of P. fuscescens females (reported as P. distinctus) mated throughout their life cycle (^{Pires et al. 2009}). The uninterrupted contact between sexual partners, with successive copulations, and the physiological costs of this habit (^{Vellnow et al. 2020}), may have caused the reduction of the reproductive cycle and the premature death of the females in the work of ^{Pires et al. (2009)}. The longer post-oviposition period in the treatment T1 may be due to lower physiological stress, given the smaller egg masses produced during its reproductive cycle.

The production of many offspring is essential when planning the mass rearing of predators in the laboratory. It also plays an important role in population growth in the field, increasing the survival rate and the probability of a greater number of these insects reaching the adult stage (^{Lacerda et al. 2004}). After emergence in the laboratory, females of P. fuscescens must be kept separated from males for five days before being mated and released into the field. For those females intended to maintain mass rearing in the laboratory, mating should also occur only from the fifth day of emergence, allowing for the perfect maturation of the reproductive apparatus. Hence, the control of the ideal mating age and the presence of males in the laboratory mass rearing can benefit the fitness of P. fuscescens females and the biological control programs using this predator.

Conclusions

The improvement of the reproductive parameters of P. fuscescens females mated at five days after the emergence, evidenced by an increased number of egg masses, eggs, the proportion of hatched eggs and the number of nymphs, indicate that the sexual maturation of P. fuscescens is progressive. The sexual contact between partners before this period does not bring benefits for the fitness of this predator.