Entering diapause is a prerequisite for successful cold-acclimation in adult Graphosoma lineatum (Heteroptera: Pentatomidae)

In diapause adults of Graphosoma lineatum overwintering in a field-cage, high chill-tolerance (CT) developed gradually, within 5 months from August to December. In laboratory-acclimation experiments, the diapause state appeared to be an essential pre-condition for successful cold-acclimation and overwintering. First, diapause prevented elevation of the median supercooling point (SCP) by about 5.5°C that accompanies the onset of reproductive activity in non-diapause specimens. Second, diapause allowed subsequent physiological changes resulting in cold-acclimation during a gradual (18-day) decrease of temperature from 25 to 0°C. No, or very modest, cold-acclimation was observed in non-diapause specimens. Decrease of temperature led to a rapid loss of ca. 1/3 of the body water in both non-diapause and diapause specimens. Approximately 0.1 M of trehalose accumulated in tissues of diapause specimens only, and haemolymph osmolality rose from 347 mOsm (at 25°C) to 444 mOsm after an 18-day cold-acclimation and to 764 mOsm during further storage at 0°C for 100 days. Upon transfer of cold-acclimated diapause specimens back to 25°C for one week (de-acclimation), the high CT was lost, the SCP elevated by about 2.5-3°C, and the levels of trehalose, water content and haemolymph osmolality returned to pre-acclimation or non-diapause levels.     

Toxicity of Chromobacterium subtsugae to southern green stink bug (Heteroptera: Pentatomidae) and corn rootworm (Coleoptera: Chrysomelidae)

Diabrotica spp. (Coleoptera: Chrysomelidae) beetles and southern green stink bugs, Nezara viridula (L.) (Heteroptera: Pentatomidae), are pests on corn, Zea mays L., and soybean, Glycine max (L.) Merr., as well as on cucurbits. Control of these insects has depended on chemicals. An alternative to chemical control is the use of biologicals. Use of bacteria, fungi, viruses, pheromones, and metabolites to control these insects can potentially improve resistance management and reduce pesticide use. Other than Bacillus thuringiensis Berliner, few bacteria have been discovered that are lethal to either of these pests. Chromobacterium subtsugae Martin et al., a newly described bacterium that is known to be toxic to Colorado potato beetle, Leptinotarsa decemlineata (Say), larvae, was found to be toxic to both diabroticite adult beetles and southern green stink bug adults. In laboratory assays, toxins produced by these bacteria kill 80-100% of the adults of two species of diabroticite beetles, Diabrotica undecimpunctata howardi Barber and Diabrotica virgifera virgifera LeConte, and 100% of southern green stink bug adults within 6 d. For green stink bug, live bacteria were not needed for toxicity.     

Toxicity of insecticides in a glass-vial bioassay to adult brown, green, and southern green stink bugs (Heteroptera: Pentatomidae)

Adult brown, Euschistus servus (Say); green, Acrosternum hilare (Say); and southern green, Nezara viridula (L.), stink bugs were collected from soybean, Glycine max (L.) Merr., in fall 2001 and 2002 near Stoneville, MS, and Eudora, AR. A glass-vial bioassay was used to determine LC50 values for the three species of stink bugs for the pyrethroids bifenthrin, cypermethrin, cyfluthrin, lambda-cyhalothrin, and permethrin, and the organophosphates acephate, dicrotophos, malathion, and methyl parathion. Results confirmed findings of other researchers that the brown stink bug was less susceptible to pyrethroid and organophosphate insecticides than were green and southern green stink bugs. The susceptibility of all three stink bug species to the insecticides tested was very similar at both test locations. The study established baseline insecticide mortality data from two locations in the mid-South for three stink bug species that are pests of soybean and cotton, Gossypium spp. Data from the tests are valuable for future use in studies on resistance and in resistance monitoring programs.     

The defensive secretion of Vitellus insularis (Heteroptera: Pentatomidae)

The defensive secretion of the guava bug, Vitellus insularis Stål, from Fiji was examined by gas liquid chromatography. Tridecane, trans‐dec‐2‐enal, and trans‐4‐ketohex‐2‐enal were identified, by comparison with reference compounds and by chemical treatment, as the main components.     

Possible egg positioning and gluing behavior by ovipositing southern green stink bug, Nezara viridula (L.) (Heteroptera: Pentatomidae)

From March 2002 to January 2004, a colony of southern green stink bug, Nezara viridula (L.), was kept in the laboratory, and its oviposition behavior was observed. During oviposition, soon after the egg is expelled, the female touches the egg mass with the dorsal surface of the last tarsomere; this movement of one leg of the hind pair occurred once. This small component of the oviposition behavior of this pentatomid is little known and not yet fully understood; it may help to position and glue the newly deposited egg to the others.     

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.     

The effect of vibratory disturbance on sexual behaviour of the southern green stink bug Nezara viridula (Heteroptera, Pentatomidae)

The effect of vibratory disturbance on sexual behaviour and substrate-borne sound communication of the southern green stink bug, Nezara viridula L. was studied. Disturbance signals do not change the time N. viridula males need to locate the source of vibratory signals, but decrease the number of males responding with the calling and courtship song to calling females. Female N. viridula proceed calling during stimulation with disturbance signals but some of them change the song rhythm by skipping one or more signal intervals or emitting the repelling signals. The number of females which change the dominant frequency of the calling song decreases proportionally with increasing differences between the dominant frequency of the disturbance signals and the emitted female calling song. Variation of the song dominant frequency probably serves females to avoid interference by increasing the signal to noise ratio. Signal duration and repetition rate do not change significantly when the female is stimulated with the disturbance signals. This indicates that frequency shift by calling females is the main strategy for reducing interference by competitive signalers in N. viridula vibrational communication.     

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.     

Duration of feeding and superficial and in-depth damage to soybean seed by selected species of stink bugs (Heteroptera: Pentatomidae)

Laboratory studies were conducted to compare duration of feeding and superficial and in-depth damage to soybean (Glycine max) seeds by the Southern green stink bug, Nezara viridula (L.), the Neotropical brown stink bug, Euschistus heros (F.), the red-banded stink bug, Piezodorus guildinii (Westwood), and the green-belly stink bug, Dichelops melacanthus (Dallas). Results indicated that feeding time was significantly longer for N. viridula (≈ 133 min) compared to E. heros and D. melacanthus (≈ 70 min), but not different from P. guildinii (≈ 103 min). There was a positive correlation between feeding time and the resulting damage for E. heros, N. viridula and P. guildinii (R2 > 0.80, P < 0.0001), but not for D. melacanthus (R2 = 0.1011, P = 0.1493). The deepest seed damage (2.0 mm) was made by P. guildinii and the shallowest (0.5 mm) by D. melacanthus. The depth of the seed damage by E. heros and N. viridula (0.8, 1.2 mm, respectively) was intermediate in comparison to the other species studied. Feeding damage to the seed endosperm caused variable cell disruption and protein body dissolution, particularly when P. guildinii fed on seeds, suggesting that the deleterious action of salivary enzymes was greater for this bug compared to the others.     

Photosynthetic production of hydrogen peroxide by Ulva rigida C. Ag. (Chlorophyta)

Production of hydrogen peroxide has been found in Ulva rigida (Chlorophyta). The formation of H₂O₂ was light dependent with a production of 1.2 mol·g FW^–1·h^–1 in sea water (pH 8.2) at an irradiance of 700 mol photons m^–2·s^–1. The excretion was also pH dependent: in pH 6.5 the production was not detectable (< 5 nmol·g FW^–1·h^–1) but at pH 9.0 the production was 5.0 mol·g FW^–1·h^–1. The production of H₂O₂ was totally inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). The ability of U. rigida growing in tanks (7501) under a natural light regime to excrete H₂O₂ was checked and found to be seven times higher at 08.00 hours than other times of the day. The H₂O₂ concentration in the cultivation tank (density: 2 g FW·l^–1) reached the highest value (3 M) at 11.00 hours. Photosynthesis was not influenced by H₂O₂ formation. The H₂O₂ is suggested to come from the Mehler reaction (pseudocyclic photophosphorylation). With an oxygen evolution of 120 mmol·g FW^–1·h^–1 at pH 8.2 and 90 mmol·g FW^–1·h^–1 at pH 9.0, 0.5% and 2.7% of the electrons were used for extracellular H₂O₂ production. The H₂O₂ production is sufficiently high to be of physiological and ecological significance, and is suggested to be a part of the defence against epi and endophytes.Abbreviations ACL artificial, continuous light - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - GNL greenhouse - LDC Luminol-dependent chemiluminescence - SOD Superoxide dismutase This investigation was supported by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Hierta-Retzius Foundation, Marianne and Marcus Wallenberg Foundation, the Swedish Environmental Protection Board, and CICYT Spain.     

Pheromonal cross-attraction in true bugs (Heteroptera): attraction of Piezodorus hybneri (Pentatomidae) to its pheromone versus the pheromone of Riptortus pedestris (Alydidae)

We investigated the attractiveness of a synthetic form of the pheromone of the soybean stink bug, Piezodorus hybneri (Gmelin), under field conditions, and compared it with that of (E)-2-hexenyl (E)-2-hexenoate, a pheromone component of a competitor, Riptortus pedestris (Fabricius). Many adult stink bugs were attracted to traps baited with 100 mg of the synthetic pheromone (1: 1: one mixture of β-sesquiphellandrene, (R)-15-hexadecanolide, and methyl (Z)-8-hexadecenoate), but few were attracted to 1 or 10 mg. More than twice as many females as males were attracted to this male-produced pheromone. None of the individual pheromone components (30 mg) attracted conspecifics. In summer (June-July), when field P. hybneri were not in diapause, (E)-2-hexenyl (E)-2-hexenoate was more attractive to P. hybneri than the synthetic pheromone. The sex ratio of the adults attracted to the synthetic pheromone was highly female-biased, yet almost equal numbers of both sexes were attracted to (E)-2-hexenyl (E)-2-hexenoate. Most females attracted to both attractants were mated and had mature ovaries. However, adults attracted to (E)-2-hexenyl (E)-2-hexenoate were likely to have less food in their stomach than those attracted to the synthetic pheromone. In late autumn (October-November), when the bugs were in reproductive diapause, both attractants attracted many sexually immature female and male adults that had well-developed fat body. The synthetic pheromone also attracted a large number of conspecific nymphs. These results suggest that P. hybneri pheromone and R. pedestris pheromone component, respectively, have different functions for P. hybneri. The male-produced pheromone system of P. hybneri seems to be sex-related but to have other roles.     

Suppression of Colorado potato beetle infestation by pheromone-mediated augmentation of the predatory spined soldier bug, Podisus maculiventris (Say) (Heteroptera: Pentatomidae)

1 Hardware and protocols were tested to enable individual growers and insectary operators to mass-produce predatory spined soldier bugs (SSBs), Podisus maculiventris (Say) (Heteroptera: Pentatomidae: Asopinae), for augmentative biological control. Using pheromone-based technology, an average of 1775 female SSBs (potentially as many as 1.6 million offspring) were captured each year during 2–3 weeks in early spring. 2 Data for the first 2 years of a 3-year project to use SSB for biological suppression of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), verified earlier research showing that augmentation of SSB (～5 nymphs/plant) can significantly suppress CPB infestations. In the third year, wild SSBs were transferred directly from pheromone traps to mid-plot nursery cages having a mesh size chosen to retain the adult predators but allow their offspring to escape. Pheromone dispensers were placed peripherally to promote dispersal of young predators and immigration of new wild spined soldier bug adults. Pheromone-mediated augmentation using porous nursery cages and pheromone dispensers was less labour-intensive than earlier methods, and resulted in significantly improved potato yield. 3 Trapping SSB adults early in the spring protects them from parasitization by tachinid flies and scelionid wasps that use the pheromone to facilitate host-finding. The compatibility of pheromone-mediated predator augmentation/conservation with implementation of transgenic plants, imidacloprid insecticide, and other biocontrol methods is discussed.     

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.     

Field responses of stink bugs to the natural and synthetic pheromone of the Neotropical brown stink bug, Euschistus heros (Heteroptera: Pentatomidae)

Abstract.The synthetic racemic mixture of methyl 2,6,10‐trimethyltridecanoate, a component of the male produced pheromone of Euschistus heros (F.) (Heteroptera, Pentatomidae), was attractive to pentatomid species in a field test, using homemade pheromone trap designs. The pentatomid Piezodorus guildinii was caught in high numbers in field traps, during two field experiments, indicating a consistent response of this species to the E. heros pheromone. A correlation was found between the range of insects caught in the pheromone‐baited traps and a random sampling method. The synthetic stereoisomeric mixture of methyl 2,6,10‐trimethyldodecanoate, a minor component of E. heros pheromone, was also field tested and caught no pentatomids. Egg parasitoids were caught in traps baited with E. heros pheromone, indicating that this pheromone can be exploited as a kairomone. A synchrony in the periodicity of trap catch, between the egg parasitoids and their host, was also recorded.     

Short communication: Purification and properties of acid phosphatase (EC 3.1.3.2) secreted by strain 74A of the mould Neurospora crassa

Both P_i-repressible acid phosphatases, IIb (mycelial) and IIc (extracellular), synthesized by Neurospora crassa and purified to apparent homogeneity by 7.5% PAGE, are monomers, are inhibited by 2 mm ZnCl₂ and are non-specifically stimulated by salts. However, the IIc form is activated by p-nitrophenylphosphate (in a negative co-operativity effect with a K_0.5 of 2.5 mm) whereas form IIb shows Michaelis kinetics, with a K_m of 0.5 mm. Thus, since both enzymatic forms may be expressed by the same gene (pho-3), it is possible that post-translational modifications lead to the excretion of an enzymatic form with altered Michaelis kinetics compared with the enzymatic form retained by the mycelium.     

Small edge populations at risk: genetic diversity of the green lizard (<Emphasis Type="Italic">Lacerta viridis viridis</Emphasis>) in Germany and implications for conservation management

Edge and central populations can show great differences regarding their genetic variation and thereby also in their probability of extinction. This fact might be of great importance for the conservation strategies of endangered species. In this study we examine the level of microsatellite variability within three threatened edge populations of the green lizard subspecies Lacerta viridis viridis (Laur.) in Brandenburg (Germany) and compare the observed variation to other edge and central populations within the northern species range. We demonstrate that the northernmost edge populations contain less genetic variation in comparison to the central population. However, there were no observable significant differences to the other edge population included in this study. Surprisingly, we observed a high genetic differentiation in a small geographical range between the three endangered populations in Brandenburg, which can be explained by processes like fragmentation, isolation, genetic drift and small individual numbers within these populations. We also detected unique genetic variants (alleles), which only occurred in these populations, despite a low overall genetic variation. This study demonstrates the potential of fast evolving markers assessing the genetic status of endangered populations with a high resolution. It also illustrates the need for a comparative analysis of different regions within the species range, achieving a more exact interpretation of the genetic variation in endangered populations. This will aid future management decisions in the conservation of genetic diversity in threatened species.     

Mesocestoides corti (syn. vogae, cestoda): characterization of genes encoding cysteine-rich secreted proteins (CRISP)

With the aim of identifying genes involved in development and parasite adaptation in cestodes, four coding sequences were isolated from the cyclophyllidean Mesocestoides corti larval stage (tetrathyridium). Genes showed significant similarity to the cysteine-rich secreted protein (CRISP) encoding genes, a large family that includes stage and tissue-specific genes from diverse organisms, many associated with crucial biological processes. The full-length McCrisp2 cDNA encodes a predicted protein of 202 residues in length, containing 10 cysteines and a putative signal peptide. The expression level of McCrisp2 was estimated by Real-time PCR, relative to GAPDH, showing an increase of 75% in segmented worms compared to tetrathyridia. By in situ hybridization, McCrisp2 expression was localized mainly at the larvae apical region of tetrathyridia and in the proglottids of segmented worms. Taken together our results suggest a possible role for M. corti CRISP proteins as ES products, potentially involved in differentiation processes as proposed for homologs in other organisms.     

Effects of in vivo exposure to DEET on blood feeding behavior and fecundity in Anopheles quadrimaculatus (Diptera: Culicidae)

This study determined the effects of contact with DEET on guinea pig skin on mortality, probing time, blood feeding rate, engorgement time, and fecundity responses in female Anopheles quadrimaculatus Say. Exposure, in this manner, to 10% DEET (in ethanol) for 5 min, resulted in 98% mortality in mosquitoes after 24h. The median probing time (PT(50)) required by females, when exposed to 0.1%, 1.0%, and 10% DEET, was significantly (P<0.0001) longer (12.5, 12.1, and 19.1s, respectively) than the 6.8s required by females to probe ethanol-treated skin (control). Similarly, mean blood feeding rates in populations of females exposed to 1.0% DEET for < or = 5 min (14.4%) was 6x lower (P<0.001) (85.5%) than in females exposed to ethanol-treated skin, whereas the mean engorgement time on skin treated with 1.0% DEET (66.3s) was significantly shorter (P<0.0001) than for females feeding on the control guinea pigs (105.9s). The mean number of mature o?cytes per female (fecundity) in treatment (1.0% DEET) and control mosquitoes was not significantly different. The responses to DEET observed in this study suggest that repeated exposure of female A. quadrimaculatus populations to this repellent, in laboratory bioassays, could result in confounding of toxicant and repellent effects and inaccurate estimates of DEET repellency.     

Males of the seed bug Togo hemipterus (Heteroptera: Lygaeidae) use accessory gland substances to inhibit remating by females

In species in which females mate repeatedly, males can adopt several strategies to reduce the risk of sperm competition with future males. The refractory period of females significantly increased as the mating duration increased in the seed bug Togo hemipterus (Heteroptera: Lygaeidae). To elucidate the mechanisms by which mated females are inhibited from remating, we investigated the effects of male-derived substances on the inhibition of mating receptivity of virgin females by injecting the substances into their abdomens. The length of time from injection to mating in virgin females was significantly longer for females injected with accessory gland B solution than for those injected with seminal vesicle, accessory gland A, or control solutions. This is the first report showing that heteropteran males inhibit female remating by using substances from an accessory gland. We discuss and consider the adoption and evolution of this strategy by T. hemipterus males by focusing on female genitalia structures, oviposition habit, and paternity and comparing these traits with those of other heteropterids.     

The metabolism of photosynthetically fixed carbon by isolated chloroplasts from Codium fragile (Chlorophyta: Siphonales) and by Elysia viridis (Mollusca: Sacoglossa)

The metabolism of photosynthetically fixed ¹⁴C by isolated chloroplasts from Codium fragile is compared with that by Elysia viridis (which contains C. fragile chloroplasts). There are marked differences between the two in the formation and subsequent metabolism of both soluble and insoluble products. Less than 6% of the carbon fixed by the isolated chloroplasts during a 15 min pulse of ¹⁴C-bicarbonate in the light is released into the medium over the succeeding 24 h. During photosynthesis glycollate and glucose monophosphate are the only labelled compounds released; after the pulse very little glycollate is released and over 24 h only glucose monophosphate and an unidentified compound are found in the medium. In E. viridis photosynthetically fixed carbon can be recovered from compounds of all major classes found in animals. Soon after the pulse, hexoses are the most heavily labelled compounds, but two hours later amino acids are more heavily labelled than hexoses. The unidentified compound is not found in the animals. E. viridis can absorb and metabolize exogenous glycollate and glucose. Earlier authors' suggestions that glucose is the compound which moves from the chloroplast to the animal cell are discussed, and it is proposed that both glucose monophosphate and glucose are formed outside the chloroplast from triose phosphate exported from it.