Hot extraction and characterization of a ligninlike fraction involved in larvicidal effects of decomposed leaf litter against mosquito

Hot water-extraction was performed on decomposed leaf litter in order to solubilize the toxic fraction involved in the dietary interaction against mosquito larvae in subalpine breeding sites. The toxic fraction was partially extracted by water with an optimum temperature of 60°C and recovered in an insoluble form. Phytochemical characterization was achieved through differential enzymatic hydrolyses, using the laccase mediator delignifying system, and aluminum chloride chelation monitored by standard bioassays; comparative spectrophotometric analyses in ultraviolet light after solubilization in acetyl bromide; and comparative reversed-phase high-performance liquid chromatography of the phenolic aldehydes after alkaline nitrobenzene oxidation. The results suggested the involvement of ligninlike compounds in the toxicity of the isolated fraction. Toxicity of this fraction appeared far stronger than that of the crude leaf litter. The involvement of this ligninlike fraction in the dietary toxicity of leaf litter against larval mosquito was then investigated.     

Larvicidal Effect of a Cell-Wall Fraction Isolated from Alder Decaying Leaves

Decaying alder leaves in water from Alpine Aedes breeding sites, particularly their toxicity to larval Culicidae, were investigated and characterized with comparative toxicological and chemical methods. Bioassays that used third-instar Aedes aegypti as a reference species indicated that the larvicidal effect of crude leaf litter varied with decaying age of the litter, while no toxicity was detected from leaching water of the mosquito breeding sites. Ten-month-old leaf litter was the most toxic. Comparison of the different soluble and insoluble fractions obtained after sequential extraction of decomposed litter allowed us to localize the toxicity factor to an insoluble cell-wall fraction. The toxicity seems to be linked to phenolic activity. It is higher than that found for tannic acid solutions used as a reference to mimic the larvicidal effects of the molecules naturally occurring in decaying litter. The pattern of establishing the larvicidal effect of alder leaf litter in water of Alpine Aedes breeding sites is discussed.     

Differential Sensitivity of Mosquito Taxa to Vegetable Tannins

The sensitivity of larval Culicidae to vegetable tannins was investigated in different taxa representative of the fauna from alpine hydrosystems (Aedes rusticus, Culex pipiens) and foreign noxious fauna (Aedes aegypti, A. albopictus). Bioassays reveal that tannic acid at concentrations of 0.1–6 mM is significantly more toxic for C. pipiens than for Aedes taxa, and A. aegypti is more sensitive than A. albopictus and A. rusticus. Comparison of the rank order of sensitivity among taxa with the associated levels of cytochrome P-450, esterase, and glutathione-S-transferase activities suggests that cytochrome P-450 and esterases may be involved in the detoxification of tannins. A possible involvement of these detoxifying enzymes is also revealed in vivo by the synergistic effects of S,S,S-tributyl phosphorotrithioate (esterase inhibitor) and piperonyl butoxide (P-450 inhibitor). The differential sensitivity to tannins among taxa is discussed in terms of ecological implications within mosquito communities from alpine hydrosystems, where the acquisition of tannins–detoxifying enzymatic systems may be considered as a key innovation.     

Light-dependent effects of alpha-terthienyl in eggs,larvae, and pupae of mosquitoAedes aegypti

Alpha-terthienyl is toxic toAedes aegypti larvae in the dark, but its activity is much enhanced in the presence of ultraviolet light. The development of first-instar larvae treated with alpha-terthienyl and ultraviolet light was followed until the emergence of adults. The LC₅₀ value for first instars was about 0.002 ppm. Practically all the larvae which survived 24 hr reached adulthood. Fourth-instar larvae were also sensitive to photochemical treatment. When their development into adults was followed, the LC₅₀ value was 0.45 ppm. Contrary to earlier reports, alpha-terthienyl was also phototoxic in pupae, but not when the adults were about to emerge. The LC₅₀ value was ca. 0.06 ppm for pupae which were 1 or 2 days old. This is the first example where the activity of a photoinsecticide has been demonstrated in pupae. Alpha-terthienyl did not affect the hatching of eggs.     

Olfactory sensitivity of two sympatric species of rice leaf folders (Lepidoptera: Pyralidae) to plant volatiles

Electroantennograms (EAGs) were recorded from male and female moths of two sympatric leaf folder species,Cnaphalocrocis medinalis (Gue-née) andMarasmia patnalis Bradley (Lepidoptera: Pyralidae) to 91 volatile chemicals of plant origin. Responses of both leaf folder species were similar to all compounds except to three monoterpenes--myrcene, menthone, and isomenthone- and two sesquiterpenes-cis-nerolidol and isophytol. Response ofM. patnalis, an oligophagous leaf folder, to these compounds was higher compared with that of polyphagousC. medinalis. EAG responses of males to saturated and unsaturated aliphatic aldehydes were significantly higher than those of conspecific females in both species. A higher response ofC. medinalis males also was observed for 1-nonanol, 3-nonen-2-one, andtrans, trans-2,6-dimethyl-2,4,6-octatriene. In contrast, females of both species responded more to monoterpenes, borneol, isoborneol, and fenchyl alchohol. Response ofC. medinalis female was higher for terpinen-4-ol, carveol, dihydrocarveol, (–)-myrtenal, and perillaldehyde. In both species and sexes, high EAG responses were recorded for compounds of the green leaf odor complex. EAG responses to nonanal and hexanal were maximum among the aliphatic aldehydes while 1-hexanol elicited the highest response among the alcohols tested. EAG responses to terpene compounds-citronellal,-terpineol, and (–)-myrtenal-were equal to the response to 1-hexanol. While all compounds tested elicited a negative potential, thymol and carvacrol elicited a positive EAG potential. The EAG data are discussed with regard     

Rejection of host plant by larvae of cabbage butterfly: Diet-dependent sensitivity to an antifeedant

Garden nasturtium,Tropaeolum majus (Tropaeolaceae), is an acceptable host plant for the cabbage butterfly,Pieris rapae. Eggs are readily laid on the plant and hatching larvae feed and develop into normal pupae and adults. However, when second- to fifth-instar larvae were transferred from cabbage to nasturtium, they refused to feed and starved to death. Similar results were obtained when larvae were transferred from other host plants to nasturtium. However, larvae that were reared on nasturtium readily accepted cabbage as a new host plant. We have demonstrated the presence of strong antifeedants in nasturtium foliage and identified the most prominent active compound as chlorogenic acid. However, larvae reared on nasturtium had limited sensitivity, and larvae reared on a wheat germ diet were completely insensitive to the antifeedants. Larvae apparently develop sensitivity to the deterrent as a result of feeding on other host plants, whereas continuous exposure to the deterrent causes habituation or suppression of sensitivity development. The results demonstrate that dietary experience can dramatically affect the response of an insect to a potentially antifeedant compound in a plant.     

Evaluation of organic infusions and synthetic compounds mediating oviposition inAedes albopictus andAedes aegypti (Diptera: Culicidae)

Oviposition responses of gravidAedes albopictus andAe. aegypti were evaluated to natural organic infusions (hay infusion, larval rearing water, and field-collected larval water) as well as compounds isolated from hay infusion (3-methylindole, 4-methylphenol, 4-ethylphenol, indole, and phenol) known to elicit oviposition inCulex mosquitoes. In laboratory bioassays, significant oviposition responses were obtained fromAe. albopictus, but not fromAe. aegypti, to dilutions of hay infusion and field water. Oviposition responses of both species were moderate to the synthetic compounds tested in the laboratory (0.01–100 µg/liter). Only 3-methylindole (0.1 µg/liter) and 4-ethylphenol (1.0 µg/liter) elicited significantly more oviposition byAe. albopictus than did well water. Of the synthetic compounds tested withAe. aegypti, only phenol (1.0 µg/liter) and 4-ethylphenol (0.1 µg/liter) elicited significantly more oviposition than did well water. Significant repellency or oviposition deterrence for both species occurred in response to at least one high concentration of most of the compounds tested. In field cage evaluations, oviposition responses byAe. albopictus were strongest to larval water and field water, moderate to hay infusion and 3-methylindole (100 µg/liter), and low to well water. A mixture of five synthetic compounds mimicking hay infusion was no more effective than 3-methylindole alone. ForAe. aegypti, oviposition responses were greatest to larval water and least to 3-methylindole. In an olfactometer, gravid females of both species oriented more to field water than to well water and onlyAe. albopictus oriented more to larval water or hay infusion than well water. In general, gravidAe. albopictus responded more strongly to oviposition stimuli than didAe. aegypti.     

Behavior of neonate diamondback moth larvae [Plutella xylostella (L.)] on leaves and on extracted leaf waxes of resistant and susceptible cabbages

NeonatePlutella xylostella moved more rapidly, spent more time walking, and engaged in searching behaviors more often on leaves of NY 8329, a resistant cabbage with glossy leaves, than on Round-Up, a susceptible variety with normal wax bloom. The neonates also spent significantly more time palpating and more time biting and spinning silk on the susceptible cabbage (although the latter two differences were not significant). Very similar differences in neonate behavior occurred on leaf surface wax extracts (hexane and dichloromethane) of the two cabbage genotypes. Leaf surface waxes are thus strongly implicated in eliciting reduced acceptance of the glossy cabbage by neonateP. xylostella. The chemical compositions of the leaf wax extracts were markedly different. Several compounds, including the triterpenols- and-amyrin, were found only in the glossy waxes. The percentages of some major wax constituents differed between wax extracts of the two cabbage types. These differences in wax composition may condition the plant resistance in glossy types.     

Detrimental effects ofCinchona leaf alkaloids on larvae of the polyphagous insectSpodoptera exigua

YoungCinchona ledgeriana plants contain two types of alkaloid: indole alkaloids in the leaves and quinoline alkaloids in the root. FromCinchona leaves, a crude alkaloid extract was made, containing the cinchophylline type of indole alkaloids and a small amount of 5-methoxytryptamine. The leaf alkaloid extract exerted a strong detrimental effect on the growth of larvae of the polyphagous beet armyworm,Spodoptera exigua (Lepidoptera). Feeding of larvae on an artificial diet containing the leaf alkaloids at the same concentrations as those found in the plant resulted in significant growth reduction, retardation in development, and mortality of the larvae. Cinchophyllines are composed of 5-methoxytryptamine coupled to a corynantheal unit. When incorporated into the artificial diet, 5-methoxytryptamine alone had no effect on the 5.exigua larvae. Corynantheal, however, had a strong detrimental effect on growth of the larvae, its effect being comparable to that of the leaf alkaloid extract. In contrast to the indole-type leaf alkaloids, the quinolinetypeCinchona root alkaloids did not affect growth and development of the larvae. These results suggest that the indole-type alkaloids, which inCinchona plants are present at the highest concentrations in the young, vulnerable leaflets, are involved in the chemical defense of the plant against herbivorous insects.     

Effects of cotton leaf surface alkalinity on feeding ofSpodoptera littoralis larvae

The high leaf surface pH in cotton (Gossypium hirsutum) var. Acala SJ2 was bioassayed againstSpodoptera littoralis larvae. Weight gain and leaf consumption of the larvae feeding on leaves devoid of alkalinity, due to daily washing, were recorded. Untreated cotton, with a leaf surface pH of 9.5–10.0 was used as control. The gland exudates contained potassium and magnesium cations, and the gland surface and intergland leaf areas were rich in calcium and phosphorus and low in K or Mg. The role of this plant antibiosis in the insect-host-plant relationship is discussed.Contribution No. 1885-E 1986 series, from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel.     

Tasting green leaf volatiles by larvae and adults of Colorado potato beetle,Leptinotarsa decemlineata

Larvae and adults of the Colorado potato beetle,Leptinotarsa decemlineata (Say), are shown to have galeal gustatory cells that are highly sensitive to distillate of potato leaf extracts, (E)-2-hexen-1-ol, (E)-2-hexenal, and other saturated and unsaturated six-carbon alcohols. In larvae and adults, the sensory response patterns elicited by leaf homogenate, leaf distillate and a mixture of these two extracts differ in subtle ways. Beetle larvae feed most readily on Millipore disks treated with leaf homogenate and the mixture, but they did not feed on disks treated with leaf distillate. The differences in behavioral response and sensory input are used to derive a potential gustatory code that may stimulate different levels of feeding. This code may be disrupted by compounds present in nonhost leaves, thus leading to reduced feeding. Possible interactions of sapid leaf volatiles, amino acids, sugars, and potentially deterrent plant compounds are discussed.     

The role of leaf lipids in food selection by larvae of the tobacco hornworm,Manduca sexta

The role of leaf lipids in food plant selection by larvae ofManduca sexta was investigated by measuring preference responses in a two-choice preference test using glass fiber filter paper disks laced with extract (test) or water (control). The larvae respond to the petroleum ether extract of whole leaves of the host-plantLycopersicon esculentum (tomato) extract in a concentration-dependent manner. At natural concentration it is the most strongly stimulating extract or compound yet tested using the disk test. This response is affected by food plant experience of the larvae, suggesting stimulation by plant-specific compounds in the extract. The extract contains volatile compounds that attract the larvae. In contrast, it does not promote continued feeding on an agar-cellulose diet that incorporates the extract. Also stimulating are the extracts of leaf surfaces of two hosts,L. esculentum andSolanum pseudocapsicum, and two acceptable nonhosts,Brassicae napus andVigna sinensis, indicating the presence of nonpolar feeding stimulants at the leaf surface. However, similar leaf-surface extracts of the unacceptable plantCanna generalis were inactive, although the surface extraction process renders this plant acceptable. Leaf-surface extracts ofL. esculentum, S. pseudocapsicum, andB. napus evoke feeding responses that are qualitatively comparable to those of their corresponding leaves. However, no such parallel is found for surface extracts ofV. sinensis andC. generalis. Thus, nonpolar compounds at the leaf surface of host and some acceptable non-host-plant species strongly stimulate feeding and hence must play an important role in food selection by the tobacco hornworm.     

Feeding responses of eastern spruce budworm larvae to sucrose and other carbohydrates

Spruce budworm larvae reared on an artificial diet were tested for preference of twelve carbohydrates over a water control. A strong preference for sucrose was seen, followed by fructose, inositol, and glucose. Male and female larvae do not differ in their responses to sucrose. The behavioral threshold for sucrose is at 10^–4 to 10^–3 M, with a peak response in the range of 0.01 to 0.05 M sucrose. Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae).Work leading to this publication was funded in part by a USDA Forest Service sponsored program entitled Canada/United States Spruce Budworms Program (grant 23–352), and in part by the National Sciences and Engineering Council of Canada (operating grant A9723), and in part by the Whitehall Foundation.     

Orientation ofMicroplitis croceipes (Hymenoptera: Braconidae) to green leaf volatiles: Dose-response curves

FemaleMicroplitis croceipes wasps were tested in a wind tunnel for their ability to orient to various concentrations of eight different green leaf volatile (GLV) substances [hexanal, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-3-hexen-1-ol, (E)-2-hexenyl acetate, (Z)-3-hexenyl acetate, (Z)-3-hexenyl propionate, and (Z)-3-hexenyl butyrate]. Overall, the esters elicited the greatest percentage of successful orientation flights, the alcohols elicited an intermediate response, and the aldehydes elicited a low response. The semilog dose-response curves were generally hill-shaped with high responses at medium release rates and low responses at high or low release rates. For the aldehydes, positive responses occurred at all GLV release rates between 0.01 and 100 nl/min. For some alcohols and esters, positive responses occurred at release rates as low as 1 pl/min and as high as 1μl/min. These data show thatM. croceipes wasps are strongly attracted to GLVs and are capable of orienting to GLV concentrations that would occur in nature when a caterpillar feeds on a green leaf. Hence, in nature, GLVs may be important clues, enablingM. croceipes to locate their hosts.     

Taste Sensitivity of Insect Herbivores to Deterrents is Greater in Specialists Than in Generalists: A Behavioral Test of the Hypothesis with Two Closely Related Caterpillars

Sensitivity of caterpillars of Heliothis virescens, a generalist, and H. subflexa, a specialist, to eight different plant secondary compounds was examined behaviorally. The compounds were nicotine hydrogen tartrate, hordenine, caffeine, sinigrin, linamarin, arbutin, chlorogenic acid, and salicin. All compounds deterred feeding, at least at the higher concentrations, but the generalist was less affected than the specialist. Thus the hypothesis that specialists have greater sensitivity to deterrents than generalists was supported. In most cases deterrence occurred on first encounter, indicating that the response was sensory; in some cases short-term postingestive effects also appeared to play a role. The larger quantities of deterrent-containing food ingested by H. virescens sometimes resulted in measurable postingestive effects during the second control test. This did not occur in H. subflexa, which more commonly rejected the deterrent-containing food on first contact. The contrast between the species is discussed in relation to tradeoffs involved in different diet breadths.     

Arrestant responses of southwestern corn borer larvae to free amino acids: Structure-activity relationships

The leaf-feeding resistance of corn or maizeZea mays L. to the southwestern corn borer, SWCB,Diatraea grandiosella Dyar has been attributed at least in part to decreased protein, increased crude fiber, and increased hemicellulose in the whorls of resistant genotypes. In this study, individual amino acids and sugars were evaluated as arrestants, with the objective of identifying those that gave weak or negative responses. Several structure-activity relationships were identified. Larvae responded to three-carbonn alkyl alpha amino acids more than to two-, four-, five-, and six-carbon compounds. Amino acids with terminal isopropyl functions gave decreased responses relative to theirn-alkyl counterparts. Dicarboxylic acids and their amides gave the lowest responses of all classes of amino acids. The normally occurring basic amino acids were all good arrestants. The guanido [HN:C(NH₂)NH-] function was somewhat important to an arrestant response, as was the number of methylenes between the alpha and omega amino functions of diaminon-alkyl amino acids. Hydroxy amino acids were generally good arrestants unless the hydroxyl was located on a ring system. The two sugars present in expressed corn whorl juice, glucose and fructose, gave poor responses. However, two other sugars, mannose and arabinose, whose C-2 hydroxyls are conformationally in the axial position, were strongly arrestant. Formulated amino acid mixtures based on their content in whorl juice were as strong arrestants as whorl juice. However, the relative contributions of amino acids and sugars that are weak arrestants to the resistance of corn to SWCB larvae is uncertain because amino acid analyses did not reveal significantly higher contents of these amino acids in the whorl juices of resistant lines.     

Oxidative responses in soybean foliage to herbivory by bean leaf beetle and three-cornered alfalfa hopper

Variation in induced responses in soybean is shown to be dependent, in part, upon herbivore species. Herbivory by the phloem-feeding three-cornered alfalfa hopper caused increases in the activities of several oxidative enzymes including lipoxygenases, peroxidases, ascorbate oxidase, and polyphenol oxidase. Bean leaf beetle defoliation caused increased lipoxygenase activity, but had little effect upon peroxidase, polyphenol oxidase, ascorbate oxidase, or trypsin inhibitor levels in either field or greenhouse studies. In one field experiment, prior herbivory by the bean leaf beetle subsequently reduced the suitability of foliage to the corn earwormHelicoverpa zea. The contribution of these findings to emerging theories of insect-plant interactions is discussed.     

Inhibition of salvinia (Salvinia molesta Mitchell) by parthenium (Parthenium hysterophorus L.). I. Effect of leaf residue and allelochemicals

Parthenium (Parthenium hysterophorus L.) leaf residue (LP, leaf powder) inhibited salvinia (Salvinia molesta Mitchell) biomass and the number of healthy fronds at 0.25% (w/v) and killed the treated plants at and above 0.75% (w/v) in about 5–15 days, depending on the quantity of the residue. At the lethal dose, the LP caused an abrupt desiccation of above-water plant parts, probably due mainly to root dysfunction. This was concurrent with the loss of dehydrogenase activity in, and an increase in solute leakage from, the roots and loss of chlorophylla, b, and total chlorophyll contents in the fronds, resulting in death of the treated plants. The LP appears inhibitory to salvinia through affecting macromolecules—proteins, lipids, and nucleic acids. The inhibitory activity of LP at the lethal dose suspended in water was completely lost when allowed to stand for 30 days under outdoor conditions and promoted growth of the salvinia plants placed in it. The standard allelochemicals, including those present in parthenium LP, except parthenin andp-hydroxybenzoic acid, did not inhibit growth up to 100 ppm. However, parthenin andp-hydroxybenzoic acid killed salvinia plants at 100 and 50 ppm, respectively. Sincep-hydroxybenzoic acid is unlikely to be present at such a high concentration, parthenin appears to be one of the main allelochemicals responsible for the inhibitory effect of parthenium leaf residue on salvinia.A portion of this work was presented at the International Symposium on Weed Management for Sustainable Agriculture held at C.C.S. Haryana Agricultural University, Hissar, India, November 18–20, 1993.     

Inhibitory effect of parthenium (Parthenium hysterophorus L.) residue on growth of water hyacinth (Eichhornia crassipes mart solms.) I. Effect of leaf residue

The allelopathic effect of parthenium (Parthenium hysterophorus L.) leaf residue (dry leaf powder, DLP) on water hyacinth (Eichhornia crassipes Mart Solms.) was studied. The treatment caused wilting starting from the margins of the older leaves and desiccation of above-water plant parts (shoot). Appearance, persistence, and disappearance of symptoms depended on the level and duration of the treatment and recovery of the treated plants, if it occurred. The treatment drastically reduced the number of healthy leaves (HLN) and the plant biomass at 0.25% (w/v) DLP; the treated plants recovered in about one month. At and above 0.50% (w/v) DLP, the plants were killed in about one month, resulting in sinking of the dead mass in water. Physiological effects of the treatment included deterioration of membrane integrity, loss of dehydrogenase activity with concurrent drastic reduction or total failure of water absorption by the roots, and reduction of chlorophyll contents in the leaves. The results indicate that the inhibitors leached out of the DLP affected the water hyacinth plants through changes in macromolecules: protein, lipid, and nucleic acid, resulting in root dysfunction and other inhibitory activities both in the root and shoot. Phenolic and other inhibitors including those found in the parthenium plant (except sesquiterpene lactones which have not been tested) at 50 ppm, exceptp-hydroxybenzoic acid, did not affect the treated plants. Such a high concentration of the allelochemicals is unlikely to be present in the medium at the lethal dose (0.50% w/v) of the DLP. Even withp-hydroxybenzoic acid, the plants recovered subsequently and grew normally. Thus, it appears that other allelochemicals including sesquiterpene lactones were mainly responsible for the inhibitory activity of the DLP on water hyacinth plants.     

Measurement of monoterpene hydrocarbon levels in vapor phase surrounding single-leaf pinyon (Pinus monophylla Torr. & Frem.: Pinaceae) understory litter

A headspace air-sampling experiment was performed in the laboratory to determine the identity and concentrations of monoterpene hydrocarbons that could be attained in the vapor phase surrounding single-leaf pinyon pine (Pinus monophylla Torr. & Frem.) understory litter using controlled air collection conditions at a simulated field temperature of 37.8°C. The total monoterpene hydrocarbon content in 21 sequential samples of air collected from a sealed glass carboy packed with 1.44 kg of single-leaf pinyon litter equivalent to a bulk density of 0.15 g/cm³ averaged 3.56 ± 1.04 mg/liter. The monoterpenes-pinene and camphene were present in the vapor phase at the highest concentrations, averaging 2.40±0.64 and 0.68±0.22 mg/ liter, respectively. Myrcene,-pinene, 3-carene,-phellandrene, and-terpinene were all present at average concentrations below 0.30 mg/liter. The first two traps of the sequential air samples yielded the highest concentrations for the monoterpene hydrocarbons; however, the average total levels were relatively stable throughout the remaining 19 traps. Therefore, the data indicate that these hydrocarbons volatilize from the source pinyon litter and maintain an equilibrium in the vapor phase. Although this analysis was conducted using an artificial system, combined with results from our previous studies, mounting evidence indicates that monoterpene hydrocarbons present in the vapor phase of the single-leaf pinyon understory may be toxic to a variety of native plant species and thus further implicates allelopathy as a significant contributor to the observed patterning of associated vegetation in these forests.