Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae)

The predatory stinkbug Podisus nigrispinus has been utilized in biological control programs. Its midgut is anatomically divided into anterior, middle and posterior regions, which play different roles in the digestive process. We describe the midgut ultrastructure and the secretion of digestive enzymes in the midgut of P. nigrispinus. Midguts were analyzed with transmission electron microscopy and the digestive enzymes amylase, cathepsin L, aminopeptidase and α-glucosidase were immunolocalized. The ultrastructural features of the digestive cells in the anterior, middle and posterior midgut regions suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, storage and excretion. The digestive enzymes have different distribution along the midgut regions of the predator P. nigrispinus. Amylase, aminopeptidase and α-glucosidase occur in three midgut regions, whereas cathepsin L occurs in the middle and posterior midgut regions. The anterior midgut region of P. nigrispinus seems to play a role in water absorption, the middle midgut may be involved in nutrient absorption and the posterior midgut region is responsible for water transport to the midgut lumen.     

Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae)

Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 μM); CAL2 (17 kDa, pHo 5.5, km 11 μM Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM l-Leucine-p-nitroanilide), α-glucosidase (90 kDa, pHo 5.0, km 5mM with p-nitrophenyl α-d-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans.     

Oviposition pattern of the predator Podisus nigrispinus (Heteroptera: Pentatomidae) under different temperatures

The daily reproductive rate of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) fed with Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) larvae was studied at constant temperatures of 20, 23, 25, 28, 30 and 33±0.2°C, relative humidity of 60±10% and photoperiod of L:D 14:10. Daily reproductive rate of P. nigripinus was affected by age of this predator. Each P. nigrispinus female laid 5.3 (20°C) to 19.9 eggs/day (28°C) which developed into 4.3–16.5 nymphs, respectively. Highest daily reproductive rate of P. nigrispinus was recorded at 28 and 30°C for 5–30-day-old females. This predator showed higher daily reproductive rate than its prey A. argillacea at 25°C. It was also able to reproduce at temperatures from 20 to 33°C with maximum daily reproductive rate between 25 and 30°C. These results are important for optimizing mass rearing of P. nigrispinus in the laboratory.     

Effect of diet on male reproductive tract of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae).

The morphology and histology of the reproductive tract of males of the predator Podisus nigrispinus (Dallas) fed on different diets were studied. P. nigrispinus was fed on diets of: larvae of Alabama argillacea (Hübner), Tenebrio molitor L., Musca domestica L., and an artificial diet. The male reproductive tract, independent of diet, showed testes with intense red coloration in a compact, circular, or slightly oval structure. The vasa deferentia were similar in color to the testes and formed long filaments, which joined with the yellow-cream colored ejaculatory duct. The morphological characteristics of the male reproductive tract were similar under all diets, except for the artificial one. The histological studies demonstrated that independent of the diet the testes of P. nigrispinus were composed of four to six follicles. The testes with six follicles generally had four developed and two atrophied follicles. The morphological and histological differences of the testes of P. nigrispinus when fed with different prey are presented and discussed.     

Morphology and preliminary enzyme characterization of the salivary glands from the predatory bug Podisus nigrispinus (Heteroptera: Pentatomidae)

Podisus nigrispinus (Dallas) is a common predator in agricultural and natural systems in Neotropical America. Its feeding strategy involves extra-oral digestion and to better understand this process its salivary glands were extracted and subjected to morphological and preliminary enzyme characterization. The salivary glands of P. nigrispinus are formed by a pair of main and accessory gland complexes. The main salivary glands are further divided into an anterior and a posterior lobe. The compartmentalization of the salivary gland complex is likely to be important for the production, activation and release of the digestive enzymes used in the extra-oral digestion of prey items. Proteases and lipase, important digestive enzymes involved in zoophagy, were detected in the salivary glands of P. nigrispinus. The prevailing trypsin-like protease activity was characterized by using the serine-protease substrate N-alpha-benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and the trypsin inhibitors tosyl-L-lysine chloromethyl ketone (TLCK) and benzamidine. The KM value obtained for trypsin-like activity was 1.57 mm and the different peaks of optimum pH and temperature activity suggest the presence of multiple forms of this enzyme in P. nigrispinus. Detection of amylase activity in the salivary glands of this predator suggests its ability to digest starch and obtain nutrients from plants, which may have adaptative value under prey scarcity.     

Predatory behaviour of Podisus nigrispinus (Heteroptera: Pentatomidae) on different densities of Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae

The functional response of the predatory bug Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) feeding on its prey, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), was studied in a greenhouse compartment. Each cage enclosed three soybean plants plus two, four, six, eight, 10, 12 or 14 prey larvae. One adult predator was released and kept inside the cages for 24h. The predation rate of adult male P. nigrispinus was highest at densities of eight or more A. gemmatalis larvae with a handling time of 5.76h and an attack rate of 0.68h^?1. Adult females had higher predation rates on plants with 10 or more A. gemmatalis larvae, with a handling time of 3.84h and an attack rate of 0.65h^?1. The maximum number of larvae consumed by males and females of this predator were 4.1 and 6.0 per day, respectively, in groups of three plants. The results suggest that P. nigrispinus may be used in biological control programmes against A. gemmatalis in soybean fields.     

Population growth rate of the depredating Podisus nigrispinus (Heteroptera: Pentatomidae) and of the Tuta absoluta (Leptoptera: gelechiidae) in wintering place

The fertility life table of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) preying either on Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) caterpillars or on alternative prey Tenebrio molitor L. (Coleoptera: Tenebrionidae) under greenhouse conditions (30 +/- 5 degrees C, 61 +/- 23% RH) were studied. The life table was also determined for the pest T. absoluta under the same conditions. The net reproductive rate (Ro) and the intrinsic rate of natural increase (rm) were higher 14.13 and 46.32 times for predators fed on T. molitor prey, however, the generation time (T) was similar between prey. The pest T. absoluta showed Ro and rm higher 2.15 and 32.10 times than those achieved for predators fed on this pest. However, females fed on a suitable prey T. molitor showed higher Ro and rm than those yielded for the pest. The survival curves were similar for P. nigrispinus females fed on both prey and classified as being type II by Weibull analysis. The results suggest that P. nigrispinus is able to maintain its population preying only on T. absoluta caterpillars; however, the life table parameters determined individually for both showed that the pest produces more generations per year and faster population natural growth than the predator.     

Ultrastructure and cytochemistry of salivary glands of the predator Podisus nigrispinus (Hemiptera: Pentatomidae)

Podisus nigrispinus Dallas (Hemiptera: Pentatomidae) is a zoophytophagous insect with a potential for use as a biological control agent in agriculture because nymphs and adults actively prey on various insects by inserting mouthparts and regurgitating the contents of the salivary glands inside the prey, causing rapid paralysis and death. However, the substances found in saliva of P. nigrispinus that causes the death of the prey are unknown. As a first step to identify the component of the saliva of P. nigrispinus, this study evaluated the ultrastructure and cytochemistry of the salivary glands of P. nigrispinus. The salivary system of P. nigrispinus has a pair of principal salivary glands, which are bilobed with a short anterior lobe and a long posterior lobe, and a pair of tubular accessory glands. The principal gland epithelium is composed of a single layer of cells enclosing a large lumen. Epithelial cells of the principal salivary gland vary from cubic to columnar shape, with one or two spherical and well-developed nuclei. Cells of the anterior lobe of the principal salivary gland have an apical surface with narrow, short, and irregular plasma membrane foldings; apical and perinuclear cytoplasm rich in rough endoplasmic reticulum; and mitochondria with tubular cristae. The basal portion of the secretory cells has mitochondria associated with many basal plasma membrane infoldings that are short but form large extracellular canals. Secretory granules with electron-dense core and electron-transparent peripheral are dispersed throughout the cytoplasm. Cells of the posterior lobe of the principal salivary gland are similar to those of the anterior lobe, except for the presence of mitochondria with transverse cristae. The accessory salivary gland cells are columnar with apical microvilli, have well-developed nucleus and cytoplasm rich in rough endoplasmic reticulum, and have secretory granules. Cytochemical tests showed positive reactions for carbohydrate, protein, and acid phosphatase in different regions of the glandular system. The principal salivary glands of P. nigrispinus do not have muscle cells attached to its wall, suggesting that saliva-releasing mechanism may occurs with the participation of some thorax muscles. The cytochemical and ultrastructural features suggest that the principal and accessory salivary glands play a role in protein synthesis of the saliva.     

Age-dependent fecundity and life-fertility tables for Podisus nigrispinus (Dallas) (Het., Pentatomidae)

Abstract: The reproductive potential and longevity of Podisus nigrispinus (Dallas) were determined under laboratory conditions at a constant temperature of 25°C. The data were used to calculate life-fertility tables and rates of increase. The fecundity plateau of P. nigrispinus lasted 18 days, and during this period mean fecundity was 8.89 eggs per female per day. It started at the eighth day of age and ended at the 18th day of age. The declining fecundity period started at 18 days of age and ended with the death of the females. The female of P. nigrispinus oviposited a mean of 188.54 eggs, and had a mean longevity of 31.21 days. The gross reproductive rate (GRR) was 104.374 females/female; the net reproductive rate ( R _o) was 31.945 females/female; the generation time was 47.390 days; the doubling time was 9.480 days; the intrinsic rate of increase ( r _m ) was 0.073, and the finite rate of increase ( λ ) was 1.076. The predator population increased by 16.61 adult progeny per female per generation in the laboratory.     

Effects of diet on development of Podisus nigrispinus (Dallas) (Het., Pentatomidae), a predator of the cotton leafworm

Abstract:  The effects of diet on development of Podisus nigrispinus (Dallas) (Het., Pentatomidae) were studied at 25°C, relative humidity of 60 ± 10%, and photoperiod of 14 h light : 10 h dark. Development times of P. nigrispinus nymphs were similar when fed with third or fifth instar larvae of cotton leafworm ( Alabama argillacea Hübner) (Lep., Noctuidae) or Tenebrio molitor L. (Col., Tenebrionidae). When fed with housefly larvae ( Musca domestica L.) (Dipt., Muscidae) or artificial diet, the predator had a longer development time. Independent of diet, instar or sex, the females of P. nigrispinus showed a longer longevity than the males. The total survival of the nymphal stage, on the different diets, varied from 22.46 (fed with housefly) to 77.33% (fed with T. molitor larvae). P. nigrispinus males were heavier when fed with third or fifth instar cotton leafworm larvae than when fed with artificial diet. The weight of the females varied from 37.91 (with artificial diet) to 64.68 mg (with fifth instar cotton leafworm larvae). Independently of the diet, newly emerged females of P. nigrispinus were heavier than the males. Females of P. nigrispinus which were fed with fifth instar cotton leafworm larvae had heavier ovaries than those fed other diets.     

Nymphal Development and Reproduction of Podisus nigrispinus (Heteroptera: Pentatomidae) Fed With Combinations of Tenebrio molitor (Coleoptera: Tenebrionidae) Pupae and Musca domestica (Diptera: Muscidae) Larvae

The development and reproduction of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) fed on Tenebrio molitor L. (Coleoptera: Tenebrionidae) and Musca domestica L. (Diptera: Muscidae) were studied in four treatments. P. nigrispinus was permitted to feed on: (1) T. molitor pupae (T 1 ); (2) Musca domestica larvae (T 2 ); (3) both prey supplied simultaneously (T 3 ); (4) both prey supplied on alternate days (T 4 ). Duration of the nymphal period of P. nigrispinus was similar in all diets studied with nymphal viability of approximately 75%. Heavier females were obtained in T 1 and T 4 , but no correlation between this factor and the reproductive rate of the predator was found. Therefore, the use of body weight as a parameter to evaluate rearing quality should be approached with caution. However, females of this predator showed higher egg and nymph production when they received both prey. For this reason P. nigrispinus should be reared with T. molitor and M. domestica simultaneously or on alternate days.     

Record of Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) preying on Metrogaleruca obscura Degeer (Coleoptera: Chrysomelidae)

Asopines are predators of insects, with several species with potential as biocontrol agents of a number of pests. Metrogaleruca obscura (Degeer), a neotropical species of Galerucini, was introduced in Malaysia, Asia, and Mauritius, Africa, to control the spread of Cordia curassavica (Boraginaceae), a native plant of the neotropics. The occurrence of Podisus nigrispinus (Dallas) preying on M. obscura is recorded, and Cordia verbenacea (Boraginaceae) is mentioned as a host plant for M. obscura. A list of Chrysomelidae attacked by asopines in the neotropical region is also presented.     

Reproductive Strategy of Podisus rostralis (Stal) (Heteroptera: Pentatomidae) Females Under Different Feeding Intervals

The reproduction and survival of Podisus rostralis was evaluated under different feeding intervals using Tenebrio molitor larvae as food and a continuous supply of bean slices. There was a linear relationship between the pre-oviposition period and the periods without feeding. Females fed at intervals of 1-4 days laid their first egg mass 8-10 days after emergence, while those fed every 8 and 16 days did so after 13.6 and 20 days respectively. The oviposition period, number of egg masses, total number of eggs, number of eggs/egg mass, total number of nymphs and rate of egg consumption/day showed negative linear correlations with the increase in feeding interval. The longevity of P. rostralis was not affected by feeding interval (P> 0.05). The present results suggest a type of trade-off, where longevity is maintained at the expense of reproduction. This behaviour or attribute of P. rostralis can contribute to its establishment and survival in an environment of fluctuating food supply.     

Toxicity of Selected Insecticides to the Spined Soldier Bug, Podisus maculiventris (Heteroptera: Pentatomidae)

Susceptibility of Podisus maculiventris to the insect growth regulator teflubenzuron, the carbamate methomyl, the pyrethroid deltamethrin, and the bioinsecticide Bacillus thuringiensis subsp. kurstaki (Bactospeine) was evaluated in the laboratory. Newly-moulted fourth instars and seven-day-old female adults were exposed to formulated materials of each compound via ingestion for 48 h. Teflubenzuron was highly toxic to fourth instars (LC 50 = 14.7 mg a.i. l -1 ) of the predator, but proved to be harmless to female adults. Both fourth instars and females suffered the highest mortality when they were exposed to methomyl (LC 50 = 5.4 and 10.6 mg a.i. l -1 , respectively). Deltamethrin was relatively safe for the predator in either stage; however, fourth instars were more tolerant to the pyrethroid than females (LC 50 = 158.8 and 43.4 mg a.i. l -1 , respectively). Neither fourth instars nor females were affected by B. thuringiensis at 10 000 mg formulated material l -1 . Reproductive capacity of the predator was also examined when applying field concentrations of teflubenzuron (200 mg a.i. l -1 ) or deltamethrin (12.5 mg a.i. l -1 ) via ingestion. Treatment with deltamethrin only prolonged preoviposition period. A marked decline in egg hatch was observed when the insects were exposed to teflubenzuron compared with the control (7 vs 63%, respectively). Fourth and fifth instars that had ingested sublethal doses of teflubenzuron demonstrated longer developmental times than did those of the control. Ingestion experiments suggest that use of deltamethrin and B. thuringiensis may be compatible with releases of P. maculiventris . In contrast, populations of the predator may be harmed when methomyl or teflubenzuron are applied.     

Comparison of susceptibility of pest Euschistus servus and predator Podisus maculiventris (Heteroptera: Pentatomidae) to selected insecticides

Susceptibility of the brown stink bug, Euschistus serous (Say), and the spined soldier bug, Podisus maculiventris (Say), to acetamiprid, cyfluthrin, dicrotophos, indoxacarb, oxamyl, and thiamethoxam, was compared in residual and oral toxicity tests. Generally, susceptibility of P. maculiventris to insecticides was significantly greater than or not significantly different from that of E. servus. Cyfluthrin and oxamyl were more toxic to the predator than to E. servus in residual and feeding tests, respectively. Dicrotophos is the only compound that exhibited both good residual and oral activity against E. servus, but even this toxicant was more toxic to the predator than to the pest in oral toxicity tests. Feeding on indoxacarb-treated food caused high mortality for both nymphs and adults of P. maculiventris. In contrast, E. servus was unaffected by feeding on food treated with this compound. Insecticide selectivity to P. maculiventris was detected only with acetamiprid for adults in residual toxicity tests and for nymphs in oral toxicity tests. Because insecticide selectivity to P. maculiventris was limited, it is extremely important to conserve P. maculiventris in cotton fields by applying these insecticides for control of brown stink bugs only when the pest reaches economic threshold.     

Development and reproduction of Podisus distinctus (Heteroptera: Pentatomidae) fed on larva of Bombyx mori (Lepidoptera: Bombycidae).

Biological control has been reducing the use of chemical products against insect pests, especially predatory Pentatomidae. Species of this group can present high variations in their life cycle as a result of their diet. Thus, the objective of this research was to study nymph development and reproduction of Podisus distinctus (St?l, 1860) (Heteroptera: Pentatomidae) fed on Bombyx mori L., 1758 (Lepidoptera: Bombycidae) larvae (T1), compared to those fed on Tenebrio molitor L., 1758 (Coleoptera: Tenebrionidae) (T2) and Musca domestica L., 1758 (Diptera: Muscidae) larvae (T3) at a temperature of 25 +/- 0.5 degrees C, relative humidity of 70 +/- 2%, and photophase of 12 h. Predators fed on B. mori showed duration of the nymph phase (18.68 +/- 1.02) similar to those fed on T. molitor (18.32 +/- 1.49). Pre-oviposition and oviposition periods and number of egg masses, besides eggs and nymphs per female, were higher with B. mori (5.83 +/- 2.02; 15.00 +/- 7.40; 8.42 +/- 1.84; 296.69 +/- 154.75; and 228.55 +/- 141.04, respectively) while longevity of males and females of P. distinctus was 25.76 +/- 16.15 and 35.00 +/- 16.15 days with T. molitor, and 20.57 +/- 13.60 and 23.46 +/- 12.35 days with B. mori, respectively.     

Effects of temperature on predation by the stinkbugs Picromerus bidens and Podisus maculiventris (Heteroptera: Pentatomidae) on noctuid caterpillars

Environmental risks associated with the use of non-indigenous organisms for augmentative biological control have received growing attention. In Europe, the native pentatomid predator Picromerus bidens (Linnaeus) has been considered a potential alternative to the North American pentatomid Podisus maculiventris (Say) for the control of lepidopteran, coleopteran and hymenopteran defoliator pests. In the current study, prey consumption and developmental duration of the predatory stages of P. bidens and P. maculiventris were investigated at three temperatures (18, 23 and 27 degrees C) in the laboratory using caterpillars of Spodoptera littoralis as prey. Development time from second to fifth instar was longer for P. bidens than for P. maculiventris, taking on average 17-44 and 14-32 days, respectively, at the different temperatures. Total nymphal consumption of fourth instar S. littoralis caterpillars indicated a greater voracity of P. bidens as compared with P. maculiventris at both the low and high temperatures tested (18 and 27 degrees C). At 23 degrees C, however, the predation rate of P. maculiventris nymphs exceeded that of P. bidens nymphs. Effect of temperature on the functional response of P. bidens to densities of fourth instar Spodoptera exigua was assessed on potted green bean plants. Female adults of P. bidens exhibited a type II functional response at 18 and 23 degrees C but a type III response at 27 degrees C. Searching efficiency was not affected by temperature but handling time decreased from 4.2 to 1.4 h as temperature increased from 18 to 23 degrees C. However, the predator spent twice as much time handling prey at 27 degrees C (2.9 h) than at 23 degrees C. This study indicates high predation rates of P. bidens at a wide range of temperatures and suggests that the species may be a valuable asset for the biological control of defoliating caterpillars, provided that obstacles to its mass production can be overcome.     

Sense organs on the antennal flagellum of the green stink bug,Nezara viridula (L.) (Heteroptera : Pentatomidae): Sensillum types and numerical growth during the post-embryonic development

The sensilla on the antennal flagellum of Nezara viridula (Heteroptera : Pentatomidae) were studied with the scanning and transmission electron microscopes. Seven morphologically distinct types of sensilla were identified in adults. The number and distribution of six types were studied. Male and female antennae bear the same numbers and types of sensilla. As in other Heteroptera, the flagellum comprises two segments. The second flagellar segment bears more sensilla than the first segment. Sensilla are evenly distributed over the flagellum surface. The total number of sensilla increases during the post-embryonic development. Individual numerical growths of each type follow simple exponential laws when expressed as functions of developmental stage. The growth in number shows positive allometry in respect to body size for sensillum types 1, 2 + 5 and 4, but negative allometry for types 3 and 6. The density of sensilla remains constant. These findings are compared with previously published data from other heterometabolous insects. Adaptation to the increase in body size during the post-embryonic development seems to be the main determinant of the growth of the olfactory system in N. viridula.     

Impact of Genetically Modified Potato Expressing Plant-Derived Insect Resistance Genes on the Predatory Bug Podisus maculiventris (Heteroptera: Pentatomidae)

The effects of the transgene proteins Galanthus nivalis agglutinin (GNA) and cowpea trypsin inhibitor (CpTI) on the predatory stinkbug Podisus maculiventris were studied under laboratory conditions. When the P. maculiventris were provided with tomato moth (L. oleracea) larvae injected with either GNA or CpTI at a dose of 10 μg day^-1, growth was significantly reduced and, as a consequence, female adult weight was also significantly reduced. For GNA-fed and CpTI-fed P. maculiventris, this weight reduction was 11.3 and 16.6%, respectively. Males, however, were not significantly affected. Female bugs that had not been exposed to the transgene proteins as nymphs, however, showed no reduction in fecundity when these adults were provided with prey injected with either GNA or CpTI at this same dose. When provided with hosts that had been reared on transgenic plants expressing either GNA or CpTI, no effects on the survival of nymphs were observed and only small, largely non-significant, reductions in weights were recorded throughout preadult development. Male nymphs fed on the GNA-fed prey did, however, exhibit a significant lengthening of preadult development of 0.8 days. The subsequent adults showed significantly reduced egg production for the GNA treatment. The results indicate that P. maculiventris may suffer some indirect adverse effects from foraging for prey in crops expressing either GNA or CpTI, due to prey being of inferior quality, rather than to direct toxicity of the transgene products themselves.