Pukalide,a widely distributed octocoral diterpenoid,induces vomiting in fish

Tissue and extractable metabolites from the subtropical gorgonianLeptogorgia virgulata (Coelenterata: Anthozoa: Octocorallia: Gorgonacea) induce vomiting in a variety of fish species. To elucidate the chemical bases of this phenomenon, experiments were undertaken with purified pukalide, a cembranoid diterpene that comprises as much as 0.1–0.5% of the wet tissue weight ofL. virgulata. When incorporated into artificial foods at concentrations corresponding to the levels found inL. virgulata, pukalide induced emesis when delivered orally to killifish (Fundulus heteroclitus). The threshold dose for pukalide-induced emesis was 0.05 mg/g fish body weight. Control pellets devoid of pukalide did not induce vomiting. The emetic effect of pukalide was dose-dependent, as fish ingesting more than 0.100 mg pukalide/ g body weight exhibited a significantly higher incidence of vomiting than fish ingesting lower doses of pukalide (G=5.5,df=1,P<0.025). The elapsed time between ingestion of pukalide-containing pellets and emesis was significantly longer in fish that ingested marginally emetic doses of pukalide (Kruskal-WallisH=4.00, significant withP<0.05). Although not markedly unpalatable to fish, pukalide may function in nature as a defensive toxin by inducing emesis and learned aversion in potential octocoral predators.     

Contact chemosensory cues in egg bundles elicit male-male agonistic conflicts in the squid Loligo pealeii

Male Loligo pealeii engage in frequent agonistic bouts to gain access to female mates while aggregated at communal egg beds. Male squids are attracted to eggs in the field and in the laboratory. It was recently demonstrated that visual detection followed by physical contact with egg capsules elicited male–male aggression. We tested specific physical and chemical features of the egg capsules that may cause this strong behavioral reaction. Male squids were presented with either natural or artificial egg stimuli and scored for four selected behaviors (egg touch, egg blowing, forward-lunge grab, and fin-beating), the last two of which are highly aggressive behaviors. First, squids were presented with natural eggs versus eggs sealed in agarose-coated tubes (ESACT), which eliminated both tactile and chemical stimuli. Second, males were presented with natural eggs versus eggs sealed in agarose coated tubes containing C₁₈ Sep-Pak-purified extracts (TCPE) from squid egg capsules, which provided chemical cues from natural eggs without the physical stimulus of the egg capsules. Third, natural eggs versus heat-denatured eggs were tested to determine whether the active factor in natural eggs is heat-labile. Squids responded aggressively when contacting natural eggs and TCPE, whereas squids did not respond after touching ESACT or denatured eggs. These results suggest that aggressive behavior is elicited by a heat-labile factor that is embedded within squid egg capsules. This chemosensory cue appears to be a contact pheromone that stimulates the agonistic interactions that characterize the mating behavior of migratory squids on inshore spawning grounds.     

Purification and removal of Ascaris and Fasciola hepatica eggs from drinking water using roughing filters

The performance of a horizontal roughing filter (HRF) and a downflow roughing filter (DRF) in the removal of Ascaris and Fasciola hepatica eggs was investigated. The experiments were performed at three filtration rates of 0.5, 1.0 and 1.5 m/h and different influent concentrations of Ascaris and Fasciola hepatica eggs. Alteration of the filtration rate in the range of 0.5–1.5 m/h did not have significant influence on the effectiveness of the roughing filters. The HRF had higher efficiency in the removal of both Fasciola hepatica and Ascaris eggs in comparison with the DRF, so that the average efficiencies of the HRF for the removal of Fasciola hepatica and Ascaris eggs at filtration rate of 1.0 m/h were determined to be 89.0 and 57.3%, respectively, whereas the same values of the DRF were 77.2 and 52.5%, respectively. The straining was confirmed to be the main mechanism of helminth eggs removal by the roughing filters, because Fasciola hepatica eggs with larger size were removed more effectively than Ascaris eggs. The results of this study indicate that the roughing filters, especially the HRF, had promising performance in the removal of helminth eggs and could be used for water and secondary effluent treatment.     

Chemical cues from Murgantia histrionica eliciting host location and recognition in the egg parasitoid Trissolcus brochymenae

Host location and recognition by the egg parasitoid Trissolcus brochymenae were analyzed in terms of response to kairomones from several stages of its host, Murgantia histrionica. In a Y-tube olfactometer, parasitoid females responded by increasing residence time and/or reducing linear speed to chemical cues from gravid females, virgin females and males, fifth and third instars, and eggs. In an open arena, T. brochymenae females also responded to patches contaminated by chemicals from the host in the same stages, sexes, and/or physiological conditions as those tested in the olfactometer. The parasitoid displayed arrestment behavior, increased residence time, changed walking pattern, and intense substrate examination. When host egg clusters or glass dummies with a chemical egg extract were placed on the host-contaminated open arena, these elicited an orientation response in the parasitoid. In addition, the chemical egg extract without dummies elicited the same response, whereas dummies without extract did not influence parasitoid behavior. In a closed arena, the parasitoid females recognized and attempted to probe glass beads treated with chemical extracts of host eggs. There were no significant differences compared with their response to the host eggs, and they did not respond to untreated beads. Host recognition was elicited by chemicals from the follicular secretion used by the host to glue the eggs on the substrate. These results are discussed in relation to the level of the host selection sequence influenced by these cues.     

Kairomones Used by Trichogramma chilonis to Find Helicoverpa assulta Eggs

Chemically mediated interactions between an egg parasitoid, Trichogramma chilonis, and its host insect, Helicoverpa assulta, were studied in laboratory experiments. T. chilonis was attracted to the sex pheromone of H. assulta, and, among four components of its sex pheromone, (Z)-11-hexadecenyl acetate seemed to be most attractive. T. chilonis was also highly attracted to (E)-12-tetradecenyl acetate, a component of the sex pheromone of Ostrinia furnacalis, another host. H. assulta eggs were more parasitized by T. chilonis when the eggs were treated with male moth scale extract (MSE) of H. assulta. Parasitism was also affected by the age of the parasitoid, time of day, and MSE concentration. Silica gel chromatography and subsequent argentation chromatography for MSE fractionation indicated the activity was associated with the fraction of saturated hydrocarbons. A linear olfactometer experiment revealed that H. assulta eggs also contain a short-range attractant(s).     

Earthworm mucus stimulates oviposition in a predatory fly (Diptera: Anthomyiidae)

Coenosia tigrina larvae feed on earthworms. We hypothesized that earthworm mucus contains a kairomone that stimulates oviposition behavior in adultC. tigrina females, thus minimizing the search area in the soil required for newly eclosed larvae to find earthworms. In bioassays, adult females responded with extension of the ovipositor 25–43% of the time to earthworm-mucus-soaked filter paper disks compared to 6–7% in response to water-soaked disks. Ovipositor extension on mucus-soaked disks was followed by egg-laying 29% of the time and 0% of the time on water-soaked disks. Egg-laying byC. tigrina followed a diurnal periodicity, with most eggs laid in the latter half of the photophase even in the absence of earthworm mucus. More eggs were deposited from 1600 to 1800 hr by females given access to earthworm mucus during that period than were deposited by females not given access. There was no difference in the number of eggs deposited from 0600 to 0800 hr, by females given access to earthworm mucus or not. This is a time of day when few eggs are normally laid. This paper is the first report of an earthworm-produced kairomone in an insect-earthworm interaction. The kairomone may have potential for enhancing biological control of the onion maggot,Delia antiqua, which is a prey of adultC. tigrina.     

Responses of parasiteEdovum puttleri to kairomone from eggs of colorado potato beetle,Leptinotarsa decemlineata

When females of the eulophid parasiteEdovum puttleri were exposed to ann-hexane wash of eggs of the Colorado potato beetle,Leptinotarsa decemlineata, their responses indicated that the eggs contained a kairomone which elicited host-finding and host-acceptance behaviors. The hostfinding responses included an increased amount of time searching glass beads treated with kairomone, and drumming on the beads with the flagellum of the antennae. Host-acceptance responses included grasping of the beads with the legs, raising of the abdomen, and probing the kairomone-treated surface with the ovipositor. None of these responses were noted in females on beads treated only with hexane, or in males exposed to kairomone-treated beads. When kairomone was applied to a flat surface, filter paper disks, few episodes of drumming and no episodes of probing with the ovipositor were noted. Responses ofE.puttleri to eggs of Colorado potato beetle were similar to those on kairomone-treated beads, but females normally drilled into eggs and fed on host fluids after retracting the ovipositor. The kairomone is volatile, and responsiveness was reduced in parasites exposed to beads treated several hours previously.Hymenoptera: Eulophidae.Coleoptera: Chrysomelidae.     

Kairomones and their use for management of entomophagous insects

Volatile chemicals emanating from an excretion (apparently meconium) and abdominal tips of femaleHeliothis zea (Boddie) moths mediated increased rates of parasitization ofH. zea eggs byTrichogramma pretiosum Riley. A blend of synthetic chemicals, consisting of hexadecanal, (Z)-7-hexadecenal, (Z)-9-hexadecenal, and (Z)-11-hexadecenal, which has been identified as the sex pheromone of and from the abdominal tip of femaleH. zea moths, also increased rates of parasitization ofH. zea eggs byT. pretiosum in greenhouse experiments. In addition, parasitization ofH. zea eggs by wildTrichogramma spp., in field plots of cotton,Gossypium hirsutum L., treated with a similar blend of chemicals, in Conrel fibers, was more than double that in untreated plots.Hymenoptera: Trichogrammatidae.Lepidoptera: Noctuidae.In cooperation with the University of Georgea College of Agriculture Experiment Stations, Coastal Plain Station, Tifton, Georgia 31793.Mention of a proprietary product does not constitute endorsement by the USDA.     

Mortality and inhibition ofHelicoverpa zea Egg parasitism rates byTrichogramma in relation to trichome/methyl ketone-mediated insect resistance ofLycopersicon hirsutum f. glabratum,accession PI 134417

The glandular trichome/methyl ketone-mediated insect resistance of the wild tomato,Lycopersicon hirsutum f. glabratum C.H. Mull, accession PI 134417, toManduca sexta (L.) andLeptinotarsa decemlineata (Say) was shown to affect adverselyTrichogramma pretiosum Riley. AdultT. pretiosum were killed by direct contact with PI 134417 foliage and by exposure to its volatiles. This effect was greatly reduced or eliminated by removing the exudate of the glandular trichomes from the foliage. 2-Tridecanone, a principal consituent of the foliar glandular trichomes of PI 134417, was toxic to adultT. pretiosum at concentrations similar to those associated with PI 134417 foliage. Incubation of parasitizedHelicoverpa (=Heliothis) zea (Boddie) eggs on PI 134417 foliage or 2-tridecanone-treated filter paper significantly reduced the proportion of eggs producing adult parasitoids. Similarly, incubation of parasitizedH. zea eggs on filter paper treated with 2-undecanone, another constituent of the glandular trichomes of PI 134417, caused an increase in the percentage of host eggs containing dead parasitoid pupae.     

Zinc-mediated hatching of eggs of soybean cyst nematode,Heterodera glycines

Egg hatching of the soybean cyst nematode,Heterodera glycines, was not affected by millimolar concentrations of calcium sulfate or calcium chloride. However, zinc chloride and zinc sulfate caused strong and moderate increases in hatching, respectively. The inhibitors of calcium transport, ruthenium red and lanthanum chloride, and calcium ionophore A23187 had no effect on hatching in the presence or absence of 3 mM zinc chloride. Selected chelators decreased the zinc-induced hatching ofH. glycines eggs. Eggs exhibited a formation constant with zinc between 5.5 and 11.2. The addition of zinc chloride after chelation with EDTA and rinsing caused expected hatching rates. Concentrations of calcium chloride, manganese chloride, and magnesium chloride had no effect on hatching of eggs in zinc chloride, but reduced hatching at higher concentrations, possibly by osmotic influences. Hatching of eggs was increased as the time of exposure to zinc chloride was increased and was maximal at 28 °C and a pH of 5.3. Picrolonic acid, a known hatching stimulant, increasedH. glycines hatching, while sodium metavanadate had no effect. Analysis of seasonal hatching during 1981–1982 in untreated control eggs indicated that hatching was most pronounced in May.     

Anthraquinones in different developmental stages ofGaleruca tanaceti (Coleoptera,Chrysomelidae)

The overwintering eggs and the larvae of the leaf beetleGaleruca tanaceti (L.) contain hydroxylated anthraquinones. In both developmental stages, l,8-dihydroxy-3-methylanthraquinone (= chrysophanol) and 1,8-di-hydroxyanthraquinone (= chrysazin) were detected by GC-MS and GC-FTIR analyses. In the eggs, chrysazin was found only in traces. Anthraquinones were also present in ovaries and hemolymph of gravid females, which were investigated in order to examine the incorporation of these substances into the eggs. Neither in acidified nor in nonacidified extracts of the host plantsTanacetum vulgäre L. andAchillea millefolium L. were anthraquinones found. The activity of these anthraquinones as chemical defense substances was proved in bioassays with the antMyrmica ruginodis NYL. Further possible biological significances of anthraquinones are discussed.     

Influence Of Diet-Related Chemical Cues from Predators on the Hatching of Egg-Carrying Spiders

Previous studies have shown that animals may make adaptive adjustments in response to chemical cues from predators, but hatching responses to diet-related chemical cues from predators have not been previously demonstrated. In the system studied here, the predator is an araneophagic jumping spider (Salticidae), Portia labiata, and the prey organism is a subsocial spitting spider, Scytodes pallida (Scytodidae). The spitting spider carries its eggs in its chelicerae, and carrying eggs is known to make it more vulnerable to predators. It is also known from an earlier study that the prior diet of the predator alters how dangerous the individual predator is to the spitting spider. In the experiments reported here, incubation time was shorter when volatile cues from the predator were present and longer in control tests when no chemical cues from the predator were present. The previous predators diet also influenced incubation time: when in the presence of volatile cues from individuals of P. labiata that had previously fed on individuals of S. pallida, incubation time was shorter than when in the presence of volatile cues from individuals of P. labiatathat had been feeding instead on house flies.     

Tetrodotoxin levels in eggs of the rough-skin newt,Taricha granulosa,are correlated with female toxicity

We quantified the amount of the neurotoxin tetrodotoxin (TTX) present in females and newly deposited eggs of the rough-skin newt, Taricha granulosa, to examine the relationship between the toxicity of an individual female and the toxicity of her eggs. We found high levels of TTX in individual eggs as well as substantial variation among clutches. Variation in the amount of TTX per egg within individual clutches was extremely low. Female skin toxicity was positively correlated with the mean egg toxicity of her clutch. Neither egg volume nor female size was significantly correlated with egg TTX levels. Tetrodotoxin stereoisomer–analog profiles were identical for females and their eggs. The presence of high levels of TTX in individual eggs coupled with the relationship between levels of TTX in female skin and levels of TTX in her eggs suggests that the TTX present in eggs of T. granulosa is maternally derived. The lack of correlation between egg size and TTX levels in individual eggs, as well as the low levels of within clutch variation, may indicate that deposition of TTX in eggs of T. granulosa is not linked to the deposition of other egg resources (e.g., lipids or other yolk components).     

Chemical ecology and the search for marine antifoulants

The gorgonian octocoralLeptogorgia virgulata (Phylum Coelenterata, Class Anthozoa) is rarely overgrown by fouling organisms and is avoided by most predators. Laboratory experiments suggest that secondary metabolites and calcium carbonate spicules interact synergistically to provideL. virgulata with an effective defense against predatory fish. In spite of these defenses,L. virgulata is consumed by the symbiotic, trophically specialized gastropodNeosimnia uniplicata, which closely mimics the appearance of the gorgonian.Neosimnia uniplicata is readily eaten by fish and appears not to acquire an effective antipredator defense from its gorgonian host. Extracts ofLeptogorgia virgulata andNeosimnia uniplicata strongly inhibited the settlement of the barnacleBalanus atnphitrite. Bioassay-directed purification of the more potent antifouling agents fromL. virgulata led to the isolation of two previously described, diterpenoid hydrocarbons, known as pukalide and epoxypukalide. A third inhibitor of barnacle settlement, whose structure is presently unknown, was obtained from bothL. virgulata andN. uniplicata. When assayed for ability to inhibit barnacle settlement, these three compounds possessed EC₅₀ values ranging from 19 to 55 ng/ml. These secondary metabolites may prevent the overgrowth ofL. virgulata by fouling organisms in nature. The allelochemicals ofL. virgulata, N. uniplicata, and other marine organisms may provide nonpolluting alternatives to existing, commercial antifoulants based on derivatives of tri-n-butyltin.     

Biological evidence of an oviposition-deterring pheromone inLobesia botrana Den. et Schiff. (Lepidoptera,Tortricidae)

Females of the European grapevine moth (Lobesia botrana Den. et Schiff.) usually deposit isolated eggs on flowers and berries of the grapevine (Vitis vinifera L.). We have investigated whether an epideictic pheromone could be present on the egg surface to explain this spacing behavior. About 21,000 eggs ofL. botrana were washed in cold methanol, and the biological activity was tested in a two-choice bioassay offering treated and nontreated areas. Different dilutions of the extract were tested in methanol, which was inactive alone. At the dose of four egg equivalents per microliter of extract, the number of eggs laid by theL. botrana females was reduced by as much as 57% on the treated areas compared to nontreated areas. A longer-term suppression of oviposition (at least 24 hr) following an exposure to the extract occurred for two doses (0.6 and 4.0 eggs/l) of egg extract. Our results strongly suggest the occurrence of an oviposition-deterring pheromone (ODP) on the eggs ofL. botrana. The ecological value of these results is discussed.     

Induction of Plant Synomones by Oviposition of a Phytophagous Insect

Earlier investigations of host habitat location in the egg parasitoid Oomyzus gallerucae have shown that oviposition of the elm leaf beetle (Xanthogaleruca luteola) induces the field elm (Ulmus minor) to emit volatiles that attract the egg parasitoid. In this study we investigated the mechanism of this induction by testing the effects of differently treated elm leaves on O. gallerucae in a four-arm olfactometer. First we investigated which sequence of the herbivore oviposition behavior is necessary for the synomone induction. The following major sequences were observed: (1) Prior oviposition, the gravid female gnawed shallow grooves into the leaf surface. (2) After gnawing upon the leaf surface, the female attached about 20–30 eggs with oviduct secretion in the grooves. We experimentally mimicked the shallow grooves on the leaf surface by scratching the leaf surface with a scalpel (= scratched leaves). Volatiles from such scratched leaves did not attract the egg parasitoid. However, as soon as eggs with oviduct secretion, or only oviduct secretion, was applied to these scratched leaves, they emitted attractive volatiles. Application of oviduct secretion and eggs on undamaged leaves did not elicit release of attractive synomones. Thus, an elicitor is located in the oviduct secretion, but becomes active only when the leaf surface is damaged. Jasmonic acid is known as a mediator of plant responses induced by feeding of herbivorous arthropods, and we demonstrate that it mediates production of elm synomones that attract O. gallerucae. The plant's reaction to oviposition was systemic, and leaves without eggs near leaves with eggs emitted attractants.     

Kairomones and their use for management of entomophagous insects: II. Mechanisms causing increase in rate of parasitization byTrichogramma spp.

When the effect of the kairomone, tricosane, on parasitization byTrichogramma achaeae Nagaraja and Nagarkatti of eggs ofHeliothis zea (Boddie) was studied in petri dish tests, the greatest percentage parasitization ( = 64%) was obtained if the entire filter paper was treated. Treatment of smaller areas (about the eggs) resulted in decreased parasitism. In the greenhouse, highest parasitization ( = 71%) byT. pretiosum (Riley) ofH. zea eggs placed on pea seedlings grown in pie pans was obtained if the whole pan was treated; lowest parasitism ( = 29%) occurred when the pans were untreated. Parasitization was intermediate ( = 52%) in other pans treated only at selected spots. Dissections ofH. zea eggs collected from kairomonetreated and untreated field plots revealed that eggs ofTrichogramma spp. were more efficiently distributed (less superparasitism) among host eggs in treated plots. These kairomones increase parasitization ofTrichogramma spp. by releasing and continuously reinforcing an intensified searching behavior rather than by attracting and guiding the parasite directly to the host.Hymenoptera: Trichogrammatidae.In cooperation with the University of Georgia College of Agriculture Experiment Station, Coastal Plain Station, Tifton, Georgia.Mention of a proprietary product in this paper does not constitute an endorsement of this product by the USDA.     

Oviposition response ofLobesia botrana females to long-chain free fatty acids and esters from its eggs

Avoidance of occupied ovisposition sites supposes that females perceive information related to their own progency. Fatty acids identified from egg extracts have been reevaluated using a different extraction method, and we have investigated the dose-dependent oviposition response of European grape vine moths (Lobesia botrana) to myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, methyl palmitate, methyl oleate, and ethyl palmitate; all except ethyl palmitate have been identified from eggs ofL. botrana. A methylene dichloride extract of eggs fromL. botrana revealed the presence of saturated free fatty acids (myristic, palmitic, and stearic) and unsaturated acids (palmitoleic, oleic, linoleic, and linolenic) in amounts ranging from 3.9 ng/egg equivalent for myristic acid to 30 ng/egg equivalent for palmitic and oleic acids. The extract also contained traces of methyl palmitate and methyl stearate. The greatest avoidance indexes were observed in response to palmitic, palmitoleic, and oleic acids. All the other compounds tested caused weaker responses. A reduction in the number of eggs laid was observed when moths were exposed to each of the esters applied at 0.3 µg per application spot. Reduction in eggs laid was also observed at a 10-fold higher dose of oleic acid. The present results confirm that general and simple molecules can be involved in the regulation of oviposition site selection and that they may participate in chemical marking of the eggs.     

Responses ton-dipropyl disulfide by ovipositing onion flies: Effects of concentration and site of release

Onion fly females,Delia antiqua (Diptera: Anthomyiidae) laid the most eggs on ovipositional dishes havingn-dipropyl disulfide (Pr₂S₂) release rates of 1–6 ng/sec from polyethylene capsules placed beneath a sand substrate. When dipropyl disulfide was released from the wax coating of surrogate foliage rather than from the substrate, ovipositing females again responded differentially to various concentrations, laying more eggs around stems containing 0.075 and 0.089 mg/stem. Factorial combinations of several concentrations released from surrogate foliage and substrate showed that releases from surrogate foliage stimulated four times more egg-laying than releases from the substrate. Females tended to lay more eggs around surrogate stems having Pr₂S₂ at the base rather than on the upper half of foliage. Observations of individual females performing preovipositional examining behaviors on Pr₂S₂-treated surrogate stems indicated that females tended to land on the upper portions of the foliage, but after landing, spent most of their time examining areas of soil and surrogate within 1 cm of the soil-surrogate foliage interface. Surrogate stems provide a realistic context for investigating effects of plant chemicals on host-acceptance behaviors.Journal article No. 12034 of the Michigan Agricultural Experiment Station.This work was supported by USDA Competitive Grant 5901-0410-9-0229-0.     

Cucurbitacins

Four species of diabroticites with different host specificities are shown for the first time to sequester cucurbitacins. While all beetles fed on an artificial diet (no cucurbitacins) were readily consumed by Chinese praying mantids, a significant proportion of adultDiabrotica balteata (72%),D. undecimpunctata howardi (46%), andD. virgifera virgifera (24%) fed on squash fruit containing cucurbitacins B and D were rejected. Moreover, even when adults did not feed on cucurbitacins, 21–24% ofAcalymma vittatum were rejected by the mantids which is consistent with larval sequestration of cucurbitacins. The mantids failed to learn to avoid any of the beetle species despite adverse effects associated with ingestion, i.e., uncoordination, regurgitation, etc. A cucurbitacin D metabolite accumulated and was sequestered for extended periods of time in the hemolymph of all four species. In addition, female beetles that had ingested cucurbitacins laid eggs containing substantial amounts of cucurbitacins.