Insects, Origin and Routine Maintenance

Adults of P. guildinii were collected in 2011 at INIA La Estanzuela, Colonia, Uruguay (S 34° 20′ W 57° 41′). They were taken to the laboratory and 20 pairs were placed in a clear plastic box (25 × 20 × 20 cm) to obtain eggs (Silva & Panizzi 2008). They were fed with green bean pods Phaseolus vulgaris (L.), dry soybean seeds Glycine max (L.) Merrill, and raw shelled peanuts Arachis hypogaea (L.). Insects were maintained at 25 ± 1 °C, 80 ± 10% RH, and 14 h photophase. Food was replaced on alternate days and distilled water was supplied every day by using moistened cotton in a plastic container (1.0 cm diameter). Egg masses were removed daily, mixed to avoid genetic effect, and divided into four groups in order to assign the treatments. To evaluate the effect of photoperiod and temperature on egg, nymph and adult biology, four different conditions (treatments) were tested: 14:10, 12:12, and 10:14 h L:D at 25 ± 1 ^°C, and 10:14 h L:D at 20 ± 1 °C. A photoperiod of 14:10 h L:D at 25 °C represents the approximate conditions recorded at the summer solstice, while a photoperiod of 10:14 h L:D at 20 °C represents the approximate conditions recorded at the winter solstice at a range of latitude between 30° to 35° S.

Egg and Nymph Biology

Eggs were held in Petri dishes (9.0 × 1.5 cm) lined with moistened filter paper and maintained at the four conditions of photoperiod and temperature (treatments) described. Relative humidity was kept constant at 80 ± 10%. Chamber temperatures were monitored daily using HOBO® data loggers, model U23-001 (Onset Computer, Pocasset, Massachusetts) at 1h intervals. During the 1st instar, nymphs were maintained only with distilled water. On the first day of the 2nd stadium, nymphs from each treatment were individualized in plastic boxes (11.0 × 11.0 × 3.5 cm) lined with moistened filter paper. They were fed the same food as adults and distilled water was supplied as described above. Nymphs were maintained at the same conditions tested for the egg stage. Instar change and mortality were recorded daily. Upon emergence, adults were separated by sex and weighed on an electronic scale (OHAUS Pioneer^™) (to the nearest 0.1 mg).

Egg and instar duration, total development time and nymph mortality were calculated for each treatment, and weight was measured at adult emergence. Treatments were set up in a completely randomized design. Each nymph that reached the adult stage was considered a replicate (n > 30). The experiment was repeated twice.

Adult Biology

Egg masses were obtained as described above, and maintained under the four conditions of photoperiod and temperature (treatments). Relative humidity was kept constant (80 ± 10%). Chamber temperatures were monitored every day using HOBO™ data loggers, model U23-001 at 1 h intervals. During the 1st instar, nymphs were maintained only with distilled water. On the first day of the 2nd instar, groups of 10 nymphs were placed in a plastic box (11.0 × 11.0 × 3.5 cm) lined with filter paper. They were supplied with the food described above. Nymphs were reared under the same photoperiod as that of the egg stage. Food was renewed on alternate days and distilled water was supplied daily. At adult emergence, more than 30 pairs were formed for each treatment. Each pair was held in a plastic box lined with moistened filter paper, and fed the same food as nymphs. They were supplied distilled water on a daily basis and food was renewed on alternate days. Fresh body weight (mg) for both sexes was taken at the moment of emergence and once a week thereafter until mortality, using an electronic scale (OHAUS Pioneer^™) (to the nearest 0.1 mg). Males and females that died were not replaced.

The reproductive performance was evaluated using the following parameters: percentage of females ovipositing, preoviposition and oviposition period, number of egg masses/female, number of eggs/female, and egg viability. Survivorship and longevity of males and females were also evaluated. Treatments were set up in a completely randomized design, and each pair (n > 30) was considered a replicate. The experiment was replicated twice.

Statistical Analyses

Egg and instar duration and development time (in days) were analyzed with the generalized linear model with the Poisson distribution and logarithmic function (PROC GENMOD, SAS Institute, version 9.2), as they are discrete variables and an association between means and variance was detected. The percentages of nymph survivorship in each instar and the total mortality were analyzed using generalized linear model with distribution binomial (PROC GENMOD, SAS Institute, version 9.2). In both cases, results are presented as the likelihood ratio statistics of the Chi-square distribution.

Weight at adult emergence was analyzed with the general linear model (PROC GLM, SAS Institute, version 9.2), due to the fact that the variance was homogeneous and there was no association between means and variance. Means were compared using Tukey-Kramer honestly significant (HSD) test for significance (P ≤ 0.05). The differences between male and female fresh body weight were compared using Student's t-test (P ≤ 0.05).

Data of adult survivorship over the time of the four treatments were analyzed by simple linear regression. To comply with the assumptions of linear regression, the data were transformed to LN (x +1). Slope coefficients were compared using Student's t-test (P ≤ 0.05).

The percentage of females ovipositing was analyzed using generalized linear model with distribution binomial and the link function logit (PROC GENMOD, SAS Institute, version 9.2). The results are presented as the likelihood ratio statistics of the Chi-square distribution. Preoviposition and oviposition period, number of egg masses and eggs per female, and longevity were analyzed with the generalized linear model with the Poisson distribution and logarithmic function (PROC GENMOD, SAS Institute, version 9.2), as they are discrete variables and a strong association between means and variance was detected. The results are presented as the likelihood ratio statistics of the Chi-square distribution.

Egg viability was analyzed with the general linear model (PROC GLM, SAS Institute, version 9.2), due to the fact that the variance was homogeneous and there was no association between means and variance. Means were compared using Tukey-Kramer honestly significant (HSD) test for significance (P ≤ 0.05).

Data regarding change in fresh body weight were analyzed using the mixed model (Proc MIXED, SAS Institute, version 9.2), with initial body weight measured at day one as a covariate and the subsequent weights as repeated measures in the analysis. The covariance structure that fitted the data was ANTE (1). The model statement included the interaction term (gain*treat) to test for heterogeneity of slopes.

Eggs

There was a highly significant effect (χ² = 23.39; df = 3; P < 0.0001) of treatments on the incubation period. The results indicated that there was a negative correlation between photophase duration and temperature with the incubation period. Eggs held at 14:10 h L:D (25 °C) had the shortest incubation period (4.1 days), while those held at 10:14 h L:D (20 °C) had the longest (7.0 days) (Table 1). Mourão & Panizzi (2000) working with E. heros, at 25 °C and under different photoperiods observed that the incubation period was longer under short photophase (10 h) in comparison with long photophase (14 h). In turn, Ali & Ewiess (1977) for N. viridula and Albuquerque (1993) for O. poecilus found no effect of the photoperiod on the incubation period of eggs. The results obtained in this study and those obtained by the aforementioned authors may indicate that the embryos of different species during egg incubation have different sensitivity to photoperiod. Moreover, the time required to hatch increased significantly when the temperature decreased from 25 to 20 °C under short photoperiod conditions (10:14 h L:D).

Cividanes & Parra (1994) reported that P. guildinii eggs hatched faster as temperature increased from 20 to 26 °C; however, they did not find differences within the range of 26 to 28 °C. Values obtained in this study under the longest photophase at 25 °C (4.1 days) were similar with those reported by Cividanes & Parra (1994) at 26 °C (4.2 days). The results obtained by these authors at 14 h L and 20 °C (9 days) were slightly higher than the corresponding results of this study at 10 h L and 20 °C (7 days). This difference could be attributed to the fact that the bugs of both studies were collected from two regions with different climatic regimes, subtropical and temperate (23° 18′ and 34° 20′ S, respectively). The temperature threshold for development and/or the degree day required to complete development may vary with the latitude as result of local adaptation to climatic conditions (Stacey & Fellowes 2002). Species with a lower threshold temperature for development have a greater rate of development at low temperatures (Trudgill et al. 2005). As in this study, several authors working with different species of pentatomids, found that when temperature decreased egg development time increased. Ali & Ewiess (1977) for N. viridula found that the incubation period was significantly longer at 20 °C than at 25 °C, under short photophase (10 h). Chocorosqui & Panizzi (2002) reported that no nymphs of D. melacanthus hatched when eggs were incubated at a temperature lower than 20 °C under long photophase (14 h).

TABLE 1.

MEAN (±SEM) DEVELOPMENT TIME, SURVIVORSHIP AND MORTALITY OF PIEZODORUS GUILDINII EGGS AND NYMPHS AT DIFFERENT PHOTOPERIODS AND TEMPERATURES UNDER LABORATORY CONDITIONS.

Nymphs

Nymph Survivorship and Total Mortality. There were significant effects of the treatments on nymph survivorship between 2nd-4th instars (2nd χ² = 51.25, df = 3; P < 0.0001, 3rd χ² = 9.3; df = 3; P =0.0256, 4th χ² = 9.25; df = 3; P =0.0261), whereas for the 5th instar there was not (χ²= 4.61; df = 3; P = 0.2027). The greatest differences in nymph survivorship among treatments occurred in the 2nd instar, with the temperature decrease under short photophase having a more conspicuous negative effect on survivorship than the decrease in photophase at 25 °C (Fig. 1). According to the likelihood ratio statistics of the Chi-square distribution, the lowest value of nymph survivorship was obtained under the photoperiod of 10:14 h L:D (20 °C) (55.7%), whereas in the other treatments nymph survivorship, which varied from 83.6 to 93.6% (Fig. 1). During the 3rd to 5th instars under 10:14 h L:D (20 °C), nymph survivorship was greater than that recorded for the 2nd instar. For the rest of the treatments nymph survivorship for instars 3rd to 5th was similar to that of the 2nd instar. Results obtained suggest that the 2nd instar is very sensitive to the decrease of temperature under short photophase. Panizzi & Smith (1977) also recorded high mortality in 2nd instar, but they explained that the high mortality appeared to be caused by nymphs falling onto their back and being unable to right themselves. Dichelops melacantus showed the same sensibility when was reared under long photoperiod and 20 °C (Chocorosqui & Panizzi 2002). In the 3rd and 4th instars the nymph survivorships recorded under short photophase (10 h) at 20 °C were lower than those recorded under long photophase (14 h) at 25 °C (Fig. 1).

Fig. 1.

Survivorship of Piezodorus guildinii nymphs at different instars under variable conditions of light and temperature regimes in the laboratory. ¹Means in each instar followed by the same letters are not significantly different (P < 0.05) based on likelihood ratio.

Total nymph mortality (instars 2–5) was affected by photoperiod and temperature (χ² = 46.99; df = 3; P < 0.0001) (Table 1). Total nymph mortality increased when photophase decreased at 25 °C; the value recorded at 14:10 h L:D (23.4%) was lower than those recorded at 12:12 and 10:14 h L:D (40 and 50.7%, respectively), which were similar. However, differences between treatments under the short photophase (10 h) at different temperatures were greater; when temperature decreased from 25 to 20 °C, total nymph mortality increased significantly from 50.7 to 73.8%. (Table 1). Cividanes & Parra (1994) did not find differences in nymph mortality of P. guildinii when reared at 25 °C and 20 °C under long photoperiod conditions (14:10 h L:D). These results suggest that nymphs of this insect have low adaptability to temperatures below 20 °C under short photophase. Therefore, in temperate regions, nymphs are unlikely to survive during mid-autumn and winter. Similar results were obtained by Ali & Ewiess (1977), who established that photoperiod did not exert a conspicuous effect on the mortality of nymphs of N. viridula at the same temperature, and that the nymph mortality at 20 °C was higher than at 25 °C.

Other species of pentatomids also showed variation in total nymph mortality induced by the length of the photophase. Mourão & Panizzi (2000) for E. heros observed higher mortality under short photophase in comparison to long photophase (56.7 vs. 28.3%). Chocorosqui & Panizzi (2003) for D. melacanthus recorded the highest total nymph mortality (60%) under short photophase at 25 °C. They also found almost 100% mortality when nymphs were reared at 20 °C under long photophase (Chocorosqui & Panizzi 2002).

Nymph Development Time. There were highly significant effects of treatments on nymph development time (1st instar χ² = 18.33; df = 3; P = 0.0004) (2nd χ² = 66.70, 3rd χ² = 66.49, 4th χ² = 58.57, and 5th χ²= 115.97; df = 3; P < 0.0001). During the first three instars, no differences were recorded in the development rate under different photoperiod at 25 °C. The length of photophase only affected development of 4th and 5th instars, which were completed faster under long photophase. Similar results were obtained by Chocorosqui & Panizzi (2002) for D. melacanthus. In contrast, Mourão & Panizzi (2000) reported that 2nd and 3rd instar nymphs of E. heros exposed to short photophase needed more time to complete their development.

For both genders, the mean number of days to complete the nymphal stage varied with the environmental conditions used (females χ² = 156.24, males χ² = 155.34; df = 3; P < 0.0001)(Table 1). The decrease of photophase did not exert an important effect in total nymph development time, for the three photoperiods at 25 °C; total development time of females was similar and shorter than that recorded at 10:14 h L:D (20 °C). For males total nymph development time at 25 °C was shorter at 14:10 h L:D than at 10:14 h L:D (Table 1). Albuquerque (1993) reported that the length of photophase at 25 °C did not significantly affect the nymph development time of O. poecilus. In contrast, nymph development time of N. viridula, E. heros and D. melacanthus was reduced significantly when photophase increased (Ali & Ewiess 1977; Mourão & Panizzi 2000; Chocorosqui & Panizzi 2003). The differences found by different authors on the effect of photoperiod on nymph development of different species of pentatomids indicate that sensitivity to day length is variable and may appear at different stages of development (Saulich & Musolin 2012). This illustrates the importance of performing studies locally in order to determine the influence of the abiotic factors on the insect biology and behavior (Ali & Ewiess 1977).

The decrease in temperature significantly increased the effect of the short photophase on nymph development time (Table 1). Both genders reared at 10:14 h L:D required 40% more time to complete the nymphal stage at 20 °C than at 25 °C. The values of the total nymph development time determined by Cividanes & Parra (1994) for P. guildinii reared at 14:10 h L:D (26 °C) (20.2 days) and 20 °C (44.2 days) were similar to those obtained in this study for 14:10 h L:D (25 °C) (21.2 days) and 10:14 h L:D (20 °C) (41.9 days). This similarity may suggest that the temperature exerts a more conspicuous effect than photoperiod in total nymph development time.

Chocorosqui & Panizzi (2003) also detected effects of temperature on the nymph development time of D. melacanthus, stating that values tended to be longer when temperature decreased and no nymphs developed when temperature was lower than 20 °C. Ali & Ewiess (1977), working with a range of temperature between 20 and 30 °C, determined that the rate of nymph development time of N. viridula decreased as the temperature increased.

Fresh Body Weight at Adult Emergence. The fresh body weight of females varied with the conditions tested (F3,161 = 10.43;P < 0.0001). Photoperiod affected fresh body weight of newly emerged females. They were heaviest when reared under long photophase (14 h) (25 °C), probably because nymphs reared under these conditions fed more than those that developed under short photophase (Table 2). The fresh body weight of males was not affected by photoperiod or temperature (F3,143 = 2.2; P = 0.0908). Females were heavier than males in all conditions tested (Table 2).

Ali & Ewiess (1977) did not find great variation in body weight for newly emerged N. viridula when nymphs were reared under different photophases, but they reported that adults were heavier at 20 °C than at 25 °C. Other authors studying other species of pentatomids obtained different results. Mourão & Panizzi (2000) for E. heros, and Chocorosqui & Panizzi (2003) for D. melacanthus established that the weight of females tended to be higher with long photophase, although no statistically significant differences were recorded.

Adults

Adult Survivorship and Longevity. Adult survivorship gradually decreased over time in all treatments. The effects of the treatments were different on both genders (Fig. 2). The decrease of survivorship of females under different photophases at 25 °C was similar and higher than the decrease recorded under short photophase (10 h) (20 °C) (Table 3). In contrast, the decrease of survivorship of males was affected by photoperiod and temperature. The slope obtained under long photophase (14 h) (25 °C) was higher than those obtained at 12 h and 10 h (25 °C), whereas under short photophase (10 h) the decrease of survivorship was higher at 25 °C than at 20 °C (Table 3).

TABLE 2.

MEAN (± SEM) FRESH BODY WEIGHT AT ADULT EMERGENCE OF PIEZODORUS GUILDINII AT DIFFERENT PHOTOPERIODS AND TEMPERATURES UNDER LABORATORY CONDITIONS.

Fig. 2.

Survivorship up to 126 days of adult Piezodorus guildinii, expressed as logarithm (LN) (%+1). Females right figures; males left figures.

Total mean longevity was affected by the photoperiod and the temperature, with a similar response for both genders (females χ² = 21.73, males χ² = 29.69; df = 3; P < 0.0001) (Table 3). Adults lived longer under short photoperiod conditions (10:14 h L:D) (20 °C) (females = 107.0 days; males 94.1 days); rather, adults reared with long photoperiod (14:10 h L:D) (25 °C) had the shortest lifespan (females = 46.1 days; males= 31.2 days) (Table 3). When adults of P. guildinii were reared in greenhouse in summer and winter in Curitiba - Brazil, Panizzi & Smith (1977) reported similar values of total longevity to those obtained in this study. Females held under summer and winter conditions lived 41.2 and 96.6 days, respectively; while males lived 34.0 and 112.5 days, respectively. In contrast, Cividanes & Parra (1994) found that under long photophase (14 h) the longevity of adults of P. guildinii at 20 °C was less than at 26 °C. The results obtained for longevity in this study and those reported by Panizzi & Smith (1977) and Cividanes & Parra (1994) may likely suggest the occurrence of an interaction in the response of this variable to both, photoperiod and temperature. The greater longevity under winter conditions (shorter photoperiod and lower temperature) may be due to a decrease in neuro-hormonally mediated metabolic activity (Tauber et al. 1986). Particularly, females spend less reserve in egg production by decreasing their reproductive activity (Slansky & Panizzi 1987).

TABLE 3.

SLOPE (B) OF DE SIMPLE LINEAR REGRESSION OF SURVIVORSHIP AND MEAN (± SEM) LONGEVITY OF PIEZODORUS GUILDINII ADULTS AT DIFFERENT PHOTOPERIODS AND TEMPERATURES UNDER LABORATORY CONDITIONS.

Adult Reproduction. Photoperiod and temperature affected the percentage of ovipositing females (χ² = 27.19; df = 3; P < 0.0001) (Table 4). Temperature decrease had a more conspicuous negative effect on the percentage of ovipositing females than the decrease in photophase at 25 ^°C. The maximum value was obtained at 14:10 h L:D (25 °C) (84.5%), and the minimum at 10:14 h L:D (20 °C) (24.2%). Similar values were recorded at 12:12 h L:D (25 °C) (61.5%) and 10:14 h L:D (25 °C) (62.1%), which were different from the other two treatments. Cividanes & Parra (1994) evaluating the percentage of ovipositing P. guildinii females under long photophase (14 h) at 20 °C, reported similar values (24%) than those obtained in this work under the same temperature and short photophase (10 h). These results suggest that the temperature has a more important role than photoperiod on percentage of females ovipositing.

Photoperiod and temperature affected the reproductive performance of females (Table 4). There were highly significant effects of treatments for preoviposition period, egg masses and number of eggs/female (χ² = 62.86; χ² = 60.58; χ² = 29.92; df = 3; P < 0.0001), and viability (F3, ₇₇ = 14.99; P < 0.001) (Table 4).

The preoviposition period was affected by photophase and temperature, the effect of temperature being greater under short photophase (Table 4). Minimum preoviposition values occurred when females were maintained at 14:10 h L:D (25 °C) (8.0 days), and the maximum value was obtained at 10:14 h L:D (20 °C) (50.1 days). No differences were recorded for female preoviposition period at 25 °C under 12:12 h L:D (11.3 days) and 10:14 h L:D (14.5 days) (Table 4).

Cividanes & Parra (1994) determined that the preoviposition period for P. guildinii females under long photophase (14 h) was longer at 20 °C (47 days) than at 26 °C (13 days). Hodek & Hodková (1993) for D. baccarum reported longer preoviposition period for bugs reared at shorter photophase. Musolin & Numata (2003) established that photoperiod had more pronounced effects on the preoviposition period at 20 °C.

Photoperiod and temperature had different effects on the egg masses and number of eggs/female. The number of egg masses was affected only by photoperiod. Females maintained under 14:10 h L:D (25 °C) and 12:12 h L:D (25 °C) produced a similar and greater number of egg masses (8.8 and 6.9, respectively) than females reared under 10:14 h L:D (25 and 20 °C), (3.6 and 3.1 egg masses, respectively) (Table 4). All treatments had a significantly different number of eggs/female, with the greatest value corresponding to 14:10 h L:D (25 °C) (196.2), and the lowest recorded under 10:14 h L:D (20 °C) (21.7) (Table 4). Cividanes & Parra (1994) observed under long photophase that temperature affected the number of eggs/female of P. guildinii, with means values significantly higher at 26 °C (310 eggs/female) than at 20 °C (28 eggs/female).

TABLE 4.

REPRODUCTIVE PERFORMANCE OF FEMALE PIEZODORUS GUILDINII AT DIFFERENT PHOTOPERIODS AND TEMPERATURES UNDER LABORATORY CONDITIONS.

Chocorosqui & Panizzi (2003) reported that females of D. melacanthus reared under long photoperiods (14:10 and 13:11 h L:D) had a better reproductive performance, high percentage of females ovipositing, more number of eggs masses and number of eggs, in comparison to females under short photoperiods (12:12 and 10:14 h L:D). Similar results were obtained by Mourão & Panizzi (2002), studying the biology of E. heros; they indicated that females reared under 10:14 h L:D produced infertile eggs. For another pentatomid, D. baccarum, Hodek & Hodková (1993) reported greater fecundity for bugs reared at longer than those reared at shorter photophase. However, Ali & Ewiess (1977) did not observe significant differences in the reproductive parameters of N. viridula at different photoperiods (10:14 h L:D and 14:10 h L:D).

Egg viability was not significantly affected by different photoperiod conditions at 25°C. However, under 10 h of photophase, it decreased significantly when temperature decreased from 25 °C to 20 °C (Table 4). On the other hand, under long photophase (14 h), Cividanes & Parra (1994) did not find an effect on egg viability of P. guildinii when temperatures decrease from 26 'C to 20 °C. The results of this study and those obtained by Cividanes & Parra (1994) may likely suggest the occurrence of an interaction in the response of egg viability with photoperiod and temperature.

Adult Body Weight Gain. Both males and females of P. guildinii gained weight during their first week of adult life (Fig. 3 A, B). However, changes in fresh body weight thereafter were similar for all tested conditions (interaction gain weight*treatment F3, 949 = 1.48; P = 0.2180). Females gained significantly more weight than males. Chocorosqui & Panizzi (2003) reported no significant differences in weight gain/ week for both sexes of D. melacanthus under different photoperiods at 25 °C, during a 4-wk evaluation period.