Rearing the Predator Brontocoris tabidus ( Heteroptera : Pentatomidae ) with Tenebrio molitor ( Coleoptera : Tenebrionidae ) Pupa on Eucalyptus grandis in the Field

The use of natural enemies represents an important strategy in programs of Integrated Pest Management (IPM). Asopinae predators are reared with alternative prey, but supplementary plant may enhance the fecundity and longevity of their females. The objective of this research was to evaluate the development and reproduction of the predator Brontocoris tabidus Signoret, 1852 (Heteroptera: Pentatomidae) fed with Tenebrio molitor L., 1785 (Coleoptera: Tenebrionidae) pupae in a Eucalyptus grandis Hill ex Maiden (Myrtaceae) plant in the field. The duration and survival of the nymphal stage of B. tabidus were 32.07 ± 8.95 days and 77.00% ± 0.42%, respectively with 15.55 ± 8.19 egg masses, 603.00 ± 379.20 eggs and 387.45 ± 291.40 nymphs per female of this predator while longevity of its males and females was 119.80 ± 32.80 and 160.20 ± 42.13 days, respectively. Better results with E. grandis plant in the field and the fact that B. tabidus will be adapted at release time show that this predator should be reared in these conditions.


Introduction
The adoption of Integrated Pest Management (IPM) reduces problems such as pest resistance and the impact of pesticides on natural enemies [1][2][3].Biological control is an important tactic in IPM programs for handling pathogens and arthropods, including the Pentatomidae, a predatory bug species in reforested areas [4] where the defenses in plants can be beneficial [5], harmful or have no impact [6] on natural enemies.
Predators have a higher reproductive rate when they get plenty of food and this rate tends to decrease with less availability of food to benefit the survivors.When predatory bugs are fed more food to gain weight, there is an increase in the frequency of attacks on prey, and posture and development are faster [7].
Supputius cincticeps Stal, 1860 (Heteroptera: Pentatomidae) nymphal stage was shorter and its females heavier when fed with pupae of Tenebrio molitor L., 1785 (Coleoptera: Tenebrionidae) and on seedlings of Eucalyptus urophylla ST Blake (Myrtaceae) [8].Podisus nigrispinus Dallas, 1851 (Heteroptera: Pentatomidae) had shorter duration of nymphal stage with Bombyx mori L., 1758 (Lepidoptera: Bombycidae) caterpillars, except in the fourth instar, when fed on cotton and tomato plants and prey than when they were fed only with prey [9].Moreover, the availability of cotton or weeds reduced the impact of the shortage of prey in the partial duration, viability and nymphal stages of this predator, but it has not reached sexual maturation and not oviposited without prey [10,11].
Females of Podisus connexivus Bergroth, 1891 (= P. nigrispinus) (Heteroptera: Pentatomidae) had greater weight gain with E. urophylla and prey.Podisus maculiventris Say, 1832 (Heteroptera: Pentatomidae) showed higher survival rates and shorter pre-oviposition and nymphal periods when fed on prey and potato leaves [5].(Coleoptera: Tenebrionidae) Pupa on Eucalyptus grandis in the Field The longevity of P. nigrispinus was similar for both sexes, but males showed greater longevity when fed on prey and cotton plants [9].Females of P. maculiventris showed a higher survival rate when they were given potato or tomato than when they were only given water until the 35th day of life, with values of 31.9, 28.8 and 27.1 days for the potato, tomato or water diets, respectively [12].The availability of prey with cotton plants increased longevity of P. nigrispinus with 30.3 ± 10.53 days for females and 43.8 ± 17.70 for males, versus 16.0 ± 11.4 and 19.93 ± 4.56 days for males and females of this predator, respectively, only, with prey [9].The ability of predators to obtain moisture and plant nutrients can be considered an adaptation to improve survival rate at these times of scarcity of prey [13,14].
The aim of this research was to investigate the rearing of Brontocoris tabidus Signoret, 1852 (Heteroptera: Pentatomidae) fed with T. molitor pupae associated to Eucalyptus grandis Hill ex Maiden (Myrtaceae) plant in field conditions.

Materials and Methods
This study was conducted in an area (field) of the "Departamento de Biologia Animal (DBA)" of "Universidade Federal de Viçosa (UFV)" in the city of Viçosa, Minas Gerais State, Brazil.The mean temperature during the nymphal and adult stages of B. tabidus was 19.55˚C, with the minimum temperature of 11.70˚C recorded in July and the maximum temperature of 30˚C recorded in March.The relative humidity was 77.58% (Table 1).
Nymphs of B. tabidus were obtained at the "Laboratório de Controle Biológico de Insetos (LCBI)" of the "Instituto de Biotecnologia Aplicada à Agropecuária (BIOAGRO)" of UFV, where the referred species is reared at 25˚C  2˚C, 70%  5% of relative humidity and photoperiod of 12 hours.Egg masses and first instar nymphs of B. tabidus were maintained in Petri dishes (9.0 × 1.2 cm) with a cotton pad soaked in distilled water.One hundred second instar nymphs of B. tabidus were placed in a white organza bag (20 × 30 cm) involving the extremity of a branch of an approximately 2-year-old E. grandis plant.Nymphs were fed with T. molitor pupae from a mass rearing facility of the UFV.Water was supplied to this predator in 2.5 mL tubes (similar to those used in dental anesthesia) fastened to E. grandis branches with adhesive ribbon (Figure 1).
Mortality of nymphs, change of instars and emergence of B. tabidus adults were daily observed [15].Adults of this predator were weighed using a scale with 0.1 mg precision, approximately 24 hours after their emergence when they were sexed and individually placed in Petri dishes [16,17].Twenty B. tabidus females weighing more than 120 mg were selected to reduce the influence of this trait on female productivity and mated four days after their emergence [18].Pairs of this predator were involved in organza bags similar to those used for their nymphs and maintained on E. grandis plant in the field until the death of males and females.The males that died before their respective females were replaced by other ones reared under similar conditions.Pairs of B. tabidus were given water daily and fed on T. molitor pupae, when the duration of pre-oviposition, oviposition and post-oviposition periods, as well as the number of egg masses, eggs and nymphs per female, egg viability and longevity of females of this predator were evaluated.Egg masses of B. tabidus were daily collected and placed in Petri dishes (9.0 × 1.2 cm) with a cotton pad soaked in distilled water and its nymphs were counted 24 hours post-eclosion.

Results
Brontocoris tabidus did not prey during the first instar that lasted 3.52  0.50 days, whereas the second, third, fourth and fifth instars lasted 5.80  1.38, 4.93  1.50, 6.10  3.04 and 11.72  2.68 days (Figure 2(a)).The duration of the nymphal stage of this predator was 32.07  8.95 days on E. grandis plant (Figure 2(a)).
The survival rate of B. tabidus was 91.00%  0.28%, 92.30%  0.26%, 94.04%  0.23% and 97.46%  0.15% during the second, third, fourth and fifth instars, whereas adult emergence of this predator reached 77.00%  0.42% on the E. grandis plant with T. molitor pupae (Figure 2  The pre-oviposition, oviposition and post-oviposition periods of B. tabidus females were 50.16  The survival rate of B. tabidus females was over 50% until they were 170 days old and egg production was greater between days 60 and 160 of their life (Figure 3).This predator had 0.0 to 5.49 eggs and 0.0 to 4.72 nymphs per day per fertile female (Figure 4).Some B. tabidus findings regarding oviposition are: 15.67 to 51.21 eggs and 5.00 to 37.50 nymphs per egg mass (Figure 5), whereas the mean number of eggs and nymphs for all egg masses of each female ranged from 0.00 to 52.11 and 0.00 to 40.78, respectively (Figure 6).
The mean longevity and weight of B. tabidus males were 119.80  32.50 days and 102.70  5.91 mg, respecttively, whereas these values were 160.20  42.13 days and 138.40  11.24 mg for females of this predator with T. molitor on the E. grandis plant in the field (Table 2).

Discussion
The B. tabidus had five instars on E. grandis plant, with similar results for this predator fed with an artificial diet based on beef meat and liver and/or T. molitor pupae [19].This suggests that the number of instars of B. tabidus is similar for this predator fed with prey and/or on a diet with or without plants.
The fourth instar of B. tabidus had a similar duration on the E. grandis plant with T. molitor pupae compared to this predator fed only with T. molitor pupae [19].However, the duration of the fifth instar of B. tabidus with T. molitor on E. grandis was longer than that observed for this predator with pupae of T. molitor [19].This is consistent with the longer duration of the fourth and fifth instars of P. maculiventris with prey [third instar of Leptinotarsa decemlineata Say, 1824 (Coleoptera: Chrysomelidae)] and plants [Solanum tuberosum L. and Lycopersi-con esculentum Mill.(Solanaceae)] compared to that of B. tabidus fed only on prey [12].
The nymphal stage of B. tabidus was longer on eucalyptus plant in the field than this predator in the same stage fed only with T. molitor pupae [19].The longer duration of the nymphal stage of B. tabidus fed with plant is similar to that found for S. cincticeps fed with T. molitor and Myrtaceae plants [20].Although this may delay the emergence of B. tabidus adults, the predation rate is increased, since the Podisus rostralis Stal, 1860 (Heteroptera: Pentatomidae) killed a greater number of B. mori caterpillars when these had a longer nymphal period [17].
The survival rate during the nymphal stage of B. tabidus was longer than during the stage where the predator was fed with pupae of T. molitor [19].The higher survival of B. tabidus in the field can be due to a change in temperature, relative humidity and photoperiod [21].It may affect biological traits of insects because their activities can be regulated by duration of the day [22].
The pre-oviposition and oviposition periods of B. tabidus were longer than that with Musca domestica L., 1758 (Diptera: Muscidae) larvae [23], which can be explained by the low temperature before the reproductive stage of this predator and to the decreasing photoperiod in the field.The longer interval between egg mass laying and preoviposition and oviposition periods, and the longevity of P. nigrispinus and S. cincticeps, were also reported as possibly due to the reduction on their metabolic activities at temperatures below 25˚C [15,20].The longer pre-oviposition period with Eucalyptus spp.plants may also result from the higher nitrogen content in their leaves [14,24].However, these parameters can be affected by other factors, because the females of this predator that weigh 95 to 150 mg and 160 to 220 mg had a greater pre-oviposition period (16.53 ± 3.33 and 16.09 ± 2.57 days, respectively) [25] than that observed in the present work.The oviposition period of this predator was 5.7 to 10.4 days when fed with pupae of T. molitor, M. domestica and the combination of both, respectively [23].Again, the high values for this period demonstrate that the B. mori has a higher nutritional quality as a prey for the predator species [1,17].
The number of eggs (603.00  311.20), nymphs (387.45  291.40) and longevity (160.20  42.13 days) of B. tabidus females showed higher values than that with T. molitor pupae alone [9].The females live longer when predators oviposit more often and stay on the field during a longer period of time, but may be affected by the type of food ingested, since females of P. maculiventris and Podisus sagitta F., 1794 (Heteroptera: Pentatomidae) did not lay eggs when fed only with green beans and potato leaves [13].Other predators such as P. sagitta [13], P. maculiventris [5] and S. cincticeps [8] have also shown greater longevity and higher reproduction rates when fed on plant and prey.
The number of eggs per egg mass was not influenced by the age of B. tabidus females, which is not consistent with the findings obtained for P. nigrispinus, since the older females of this species laid a lower number of eggs per egg mass [26][27][28].Females of B. tabidus had similar weight, but a variable number of eggs and egg masses, indicating that these traits may be related to genetic characteristics of this predator besides its weight [18,25].
The greater longevity and the number of eggs of B. tabidus fed with T. molitor and seedlings of E. grandis are important factors for the improvement of the potential of this predator as a biological control.However, the type of prey alone cannot affect the longevity of stinkbug predators, because males and females mated S. cincticeps showed similar longevity, 19.2 ± 3.3 and 21.2 ± 2.7 days and 29.2 ± 6.1 and 30.7 ± 4.1 days when fed with M. domestica or T. molitor, respectively [8].

Conclusion and Future Prospects
The nymphal stage of B. tabidus was longer when the predator was fed with E. grandis and, thus, this must be considered in mass rearing and in the implementation of programs involving this predator.The B. tabidus had better reproductive parameters such as longer longevity and shorter period to double its population when fed with plant and prey.Therefore, this predator should be reared on eucalyptus plants in the field where its adults would be better adapted at the moment of their release in biological control programs.

Figure 1 .
Figure 1.Organza bag used to rear Brontocoris tabidus on Eucalyptus grandis plant in the field.A-anesthesic odonthological tube; B-organza cloth.C-Eucalyptus grandis branch.

Figure 2 .
Figure 2. Duration (a) and survival (b) (mean  standard deviation) during each instar and of the nymphal stage of Brontocoris tabidus fed with Tenebrio molitor pupae on a Eucalyptus grandis plant in the field.Municipality of Viçosa, Minas Gerais State, Brazil.

Figure 3 .FemalesFigure 4 .
Figure 3. Number of eggs per period of 10 days, accumulated percentage of eggs produced and survival of Brontocoris tabidus females fed with Tenebrio molitor pupae on a Eucalyptus grandis plant in the field.Municipality of Viçosa, Minas Gerais State, Brazil.

Figure 5 .FemalesFigure 6 .
Figure 5. Number of eggs and nymphs per egg mass of Brontocoris tabidus fed with Tenebrio molitor pupae on a Eucalyptus grandis plant in the field.Municipality of Viçosa, Minas Gerais State, Brazil.