Emerging Hyalomma lusitanicum: From identification to vectorial role and integrated control.

In the Mediterranean basin, the tick species Hyalomma lusitanicum Koch stands out among other species of the Hyalomma genus due to its wide distribution, and there is great concern about its potential role as a vector and/or reservoir and its continuous expansion to new areas because of climate warming and human and other animal movements. This review aims to consolidate all the information on H. lusitanicum, including taxonomy and evolution, morphological and molecular identification, life cycle, sampling methods, rearing under laboratory conditions, ecology, hosts, geographical distribution, seasonality, vector role and control methods. The availability of adequate data is extremely relevant to the development of appropriate control strategies in areas where this tick is currently distributed as well as in new areas where it could become established in the near future.

Biocidal effects of Piper hispidinervum (Piperaceae) essential oil and synergism among its main components.

In this study we evaluated the effect of a pressure gradient (1-2 atm) in the extraction and composition of the essential oil (EO) of Piper hispidinervum by steam distillation. We also evaluated the insect antifeedant effects (Spodoptera littoralis, Leptinotarsa decemlineata, Myzus persicae and Rhopalosiphum padi) and nematicidal activity (Meloidogyne javanica) of the oils, their major components and their synergistic interactions. Safrole was the major component (78-81%) followed by terpinolene (5-9%). The EOs tested were effective insect antifeedants. Safrole, explained most of the insect antifeedant action of P. hispidinervum EOs. When safrole and terpinolene were tested in binary combinations, low ratios of safrole improved the antifeedant effects of terpinolene. P. hispidinervum EOs caused higher mortality of M. javanica juveniles than their major components. In binary combinations, low ratios of terpinolene increased the nematicidal effects of safrole. The EO treatment strongly suppressed nematode egg hatching and juvenile infectivity. P. hispidinervum EOs affected the germination of S. lycopersicum and L. sativa mostly at 24 h of treatment, being L. sativa the most sensitive. Safrole moderately affected germination and root growth of L. sativa, S. lycopersicum and L. perenne. Terpinolene only affected S. lycopersicum root growth.

Indole alkaloids from Geissospermum reticulatum.

Ten indole alkaloids were isolated from Geissospermum reticulatum, seven (1-7) from the leaves and three (8-10) from the bark. Seven were aspidospermatan-type alkaloids (1-3, 5-9), including four (5-8) with a 1-oxa-3-cyclopentene group in their molecule, which we named geissospermidine subtype. Compounds 1-3, 5-8, and 10 had not been reported previously as natural products, while 4 and 9 were the known alkaloids O-demethylaspidospermine and flavopereirine. Their structures were determined by spectroscopic techniques including 1D and 2D NMR experiments (COSY, NOESY, HSQC, HMBC). Additionally, X-ray crystallographic analyses of 1, 2, and 6 were performed. Antiparasitic activities of the ethanolic and alkaloidal extracts and of the pure alkaloids were tested against Trypanosoma cruzi and Leishmania infantum. In general, the extracts exhibited selective action and were more active against Leishmania than against Trypanosoma. Alkaloid 4 was also very active against L. infantum.

Diterpenoid alkaloids from Delphinium gracile.

Two highly oxygenated hetisine-type diterpenoid alkaloids, delphigraciline (1), 14-hydroxyhetisinone N-oxide (2), and the norditerpenoid alkaloid 8-methoxykarakoline (3), were isolated from a neutral extract of Delphinium gracile. Their structures were elucidated on the basis of spectroscopic data and by comparison with previously reported spectroscopic data of similar alkaloids. Their antiparasitic and insecticidal activities are also discussed.

Heliotropium huascoense resin exudate: chemical constituents and defensive properties.

From the resinous exudate of Heliotropium huascoense a new compound, rel-(8R,9R)-carrizaloic acid, (1) (3-[rel-(8R,9R-9-hydroxy-9,13,13-trimethyl-12-oxo-10-cyclohexenyl)methyl]-4-methoxybenzoic acid), and three known flavonoids, [3-methylgalangin, 3,7-dimethylgalangin, and (-)-alpinone] have been isolated. The structure of 1 was determined by spectral and chemical methods. Several plant defensive properties of 1 (insecticidal and antifungal) have been evaluated.

Minor diterpenes from Persea indica: their antifeedant activity.

The new diterpenes anhydrocinnzeylanone, garajonone and 2,3-didehydrocinnzeylanone, and the known anhydrocinnzeylanine, have been isolated from Persea indica. The antifeedant activity of these compounds has been evaluated showing the importance of the 11-hemiketal group for the antifeedant effects of ryanodane diterpenes.

Defensive chemistry of Senecio miser.

Three eremophilanolides, 1alpha-acetoxy-8beta-methoxy-10betaH-eremophil-7(11)-en-8alpha,12-olide (1); 1alpha-angeloyloxy-6beta-hydroxy-8beta-methoxy-10betaH-eremophil-7(11)-en-8alpha,12-olide (2); and 1alpha-angeloyloxy-8betaH,10betaH-eremophil-7(11)-en-8alpha,12-olide (3), and two pyrrolizidine alkaloids, integerrimine (4) and its N-oxide (5), were isolated from bioactive fractions of Senecio miser. The structures of the new compounds 1 and 2 were established by NMR spectroscopic analysis and chemical transformation. The X-ray analysis of compound 1 was also performed. Eremophilanolides 1 and 2 and alkaloids 4 and 5 were found to be strong insect antifeedants, further supporting a proposed defensive role for these classes of compounds.

Structure- and species-dependent insecticidal effects of neo-clerodane diterpenes.

Several natural neo-clerodane diterpenoids isolated from Linaria saxatilis and some semisynthetic derivatives were tested against several insect species with different feeding adaptations. The antifeedant tests showed that the oliphagous Leptinotarsa decemlineata was the most sensitive insect, followed by the aphid Myzus persicae. The polyphagous Spodoptera littoralis was not deterred by these diterpenoids; however, following oral administration, some of these compounds did have postingestive antifeedant effects on this insect. In general terms, the antifeedant effects of these compounds were species-dependent and more selective than their toxic/postingestive effects. The study of their structure-activity relationships showed that both the decalin moiety and the chain at C-9 determined their bioactivity. Furthermore, the presence of a 4,18-epoxy/diol moiety was an important feature for both the antifeedant and the toxic/postingestive effects.

Insecticidal and mutagenic evaluation of two annonaceous acetogenins.

Annonaceous acetogenins represent a new class of bioactive compounds whose primary mode of action is the inhibition of NADH-ubiquinone oxidoreductase. Given the potential pesticidal use of such a class of compounds, we have further evaluated the antifeedant and insecticidal effects of squamocin and annonacin, two annonaceous acetogenins, on Spodoptera littoralis, Leptinotarsa decemlineata, and Myzus persicae. Additionally, to partially assess their environmental risk, we have also tested their mutagenicity in Salmonella typhimurium strains TA98, TA100, and TA102 in the presence and absence of a metabolic activation system. Among the test compounds, annonacin showed antifeedant effects on L. decemlineata, while squamocin was toxic to L. decemlineata and M. persicae. Neither acetogenin was mutagenic, although both were toxic in the absence of a metabolic activation system. We compared these results with those obtained with rotenone, a well-known respiratory inhibitor that was highly toxic to L. decemlineata and M. persicae and showed no mutagenicity/toxicity in the S. typhimurium strains tested up to a concentration of 1000 microg per plate.

Selective insect antifeedant and toxic action of ryanoid diterpenes.

In this work, we have studied the antifeedant and insecticidal effects of several natural ryanoid diterpenes. These compounds can be classified in two groups according to their chemical structures: ryanodol/isoryanodol-type (nonalkaloidal type) and ryanodine-type (alkaloidal type) ryanoids. The nonalkaloidal ryanoids were isolated from Persea indica (Lauraceae) while the alkaloidal ryanoids (ryanodines and spiganthines) were isolated from Spigelia anthelmia (Loganiaceae). The effects of these compounds on the feeding behavior and performance (with and without piperonyl butoxide pretreatment) of Spodoptera littoralis larvae and Leptinotarsa decemlineata adults indicate that some strongly deterred these insects, L. decemlineata being less sensitive than S. littoralis. Their antifeedant effects did not parallel their toxic action. Additionally, more than 60% of the nonalkaloidal ryanoids were antifeedants and/or toxic in contrast to 30% of active alkaloidal ones, supporting the hypothesis of a ryanodol-specific mode of action in insects.

Development of a new bioluminescent mutagenicity assay based on the Ames test.

A newly developed rapid mutagenicity assay based on the adenosine triphosphate (ATP)-bioluminescence technique and the Ames test is described. Salmonella typhimurium strains TA98 and TA100 were exposed in an appropriate liquid medium to the direct mutagens 4-nitroquinoline-N-oxide and methyl methanesulphonate, respectively, and to the indirect mutagen 2-aminoanthracene. Both auxotrophic and prototrophic growth were monitored throughout the incubation period as variations in the intracellular ATP levels by means of the luciferin-luciferase assay. After 9-12 h of incubation a dose-response increase in the levels of ATP was readily detected. In order to demonstrate that this increase was due to the growth of revertant bacteria, aliquots from each culture were plated on minimal agar plates. A very good correlation between the changes in ATP levels and the appearance of revertant colonies on the plates was found. Given the rapidity of this method as compared with conventional mutagenicity assays, it has potential for industrial and environmental applications. Other potential applications are also discussed.

Pyrrolizidine alkaloids from Heliotropium megalanthum.

Two pyrrolizidine alkaloids, megalanthonine (1) and lycopsamine (2), have been isolated from Heliotropium megalanthum. The structure of the novel compound 1 was determined by spectroscopic methods. The insecticidal, antifeedant, and antifungal effects of compounds 1 and 2 have been evaluated.

Genotoxicity of the insecticide rotenone in cultured human lymphocytes.

We have investigated the genotoxic activity of rotenone on three genetic endpoints, sister-chromatid exchanges (SCE), chromosome aberrations (CA) and micronuclei (MN) in human lymphocyte cultures in the presence and absence of a metabolic activation system (S9 mix). Our results indicate that rotenone increases the frequency of binucleated micronucleated (BNMN) cells and causes a delay in the cell cycle but does not increase the frequency of CA and SCE at the concentrations used. The presence of S9 mix reduces the genotoxic activity of rotenone.

Antifeedant Delphinium Diterpenoid Alkaloids. Structure-Activity Relationships.

The insect antifeedant and toxic activity of the Delphinium diterpene alkaloids 15-acetylcardiopetamine, cardiopetamine along with its amino alcohol, the beta,gamma unsaturated ketone, and the acetylated ketone derivatives were studied in Spodoptera littoralis and Leptinotarsadecemlineata. Cardiopetamine and 15-acetylcardiopetamine strongly inhibited the feeding activity of S. littoralis and L. decemlineata, respectively. Structure-activity studies with S. littoralis showed that the C13 and C15 hydroxy substituents are essential features of the active molecule, while a C13 hydroxy and/or a C15 acetate determined their effect on L.decemlineata. The C11 benzoate group enhanced the biological effect on both insect species. These alkaloids were not toxic to S. littoralis, while their toxicity on L. decemlineata was inversely correlated with their antifeedant effects, the beta,gamma unsaturated ketone derivative being the most toxic. Cardiopetamine showed little antifungal action against several species of plant pathogens and did not have any mutagenic effects on Salmonella typhimurium by means of the Ames test.

Antifeedant and toxic effects of sesquiterpenes fromSenecio palmensis to colorado potato beetle.

A bioassay-guided fractionation of the aerial parts ofSenecio palmensis resulted in the isolation of two sesquiterpenes, 2,10-bisaboladien-1-one and 11ß-acetoxy-5-angeloyloxy-silphinen-3-one. The bisabolene and the silphinene represented 0.012% and 0.024% of the plant dry weight, respectively. Both compounds showed antifeedant activity againstLeptinotarsa decemlineata larvae and adults in short-term choice and no-choice bioassays. Both compounds were also tested against different species of phytopathogenic fungi. The beetles were more sensitive to these compounds in choice than in no-choice assays, with a gradient of increasing sensitivity from second instars to adults. Bisabolene was 45 times less active as an antifeedant than juglone, which was tested as a positive control. The silphinen was more active than the bisabolene, with a range of activity similar to juglone. Furthermore, exposure of fourth instars to these compounds over a 24-hr period resulted in reduced feeding and growth rates. To distinguish between antifeedant and toxic effects, growth efficiencies were calculated as the slope of the regression of relative growth rate on relative consumption rate. The comparison of these results with those of antifeedant simulation and contact toxicity bioassays indicates that feeding inhibition is the primary mode of action of the bisabolene, while the silphinene shows both antifeedant and toxic effects.

Compound interactions effects of plant antioxidants in combination with carbaryl on performance ofTrichoplusia ni (Cabbage Looper).

Plant chemicals naturally exist in complex mixtures, which can interact either additively, synergistically, or antagonistically. We investigated the potential interactions of three naturally occurring antioxidants- nordihydroguaiaretic acid (NDGA), safrole, and α-tocopherol-with the general insecticide carbarayl to affect the performance of cabbage looper larvae (Trichoplusia ni). The cabbage looper is known to produce a mixed-function oxidase enzyme system in response to the presence of carbaryl. We proposed that plant antioxidants would interfere with enzymatic oxidation, enhancing the susceptibility of this insect to carbaryl. Insects were fed artificial diets containing each antioxidant alone or in pairwise combinations with the insecticide carbaryl to test for their effects on the insect's nutritional measurement indices. The three antioxidants tested were not equally effective individually against insect survivorship and interacted differentially in combination with the insecticide. The nutritional indices were measured on insects fed diets containing the chemicals at nonlethal doses. Insects fed 0.001 % wet wt NDGA diets grew 1.62 times less, and had gross and net conversion efficiencies reduced 3.20 and 3.63 times, respectively, compared to the control larvae. Carbaryl (0.002% wet wt) in combination with NDGA acts as an antagonist to the effects mentioned above, while safrole (1 × 10(-4) wet wt) had an additive effect when combined with the insecticide, reducing 1.76 times larval relative growth rate and efficiency of conversion of ingested food in respect to the control. The larvae fed significantly more (1.2 times) on both insecticide and safrole diets than on the controls or their combined diets. Larvae fed α-tocopherol alone or in combination with carbaryl had similar growth and conversion efficiencies as controls. We conclude that the effects of different combinations of compounds cannot be predicted a priori and must be determined experimentally.

Chemical ecology of canarian laurel forest: Toxic diterpenes fromPersea indica (Lauraceae).

The tree speciesP. indica (Lauraceae) is an important endemism in the Canary Islands laurel forest and can readily be distinguished by its defoliated appearance due to the seasonal action of wild rats (Rattus rattus), which eat the plant and become intoxicated. These observations and the phytochemical interest of this plant species led us to study the potentially toxic chemicals responsible for such action. We found that an ethanolic extract ofP. indica and its water fraction were toxic when injected into laboratory mice. The mice also died after ingestion of the stems and showed a significant preference for those extracted and rehydrated with an 8% aqueous extract solution when compared with the water control. Two compounds that have been isolated from the toxic fraction and identified by spectroscopic methods are the polyhydroxy pentacyclic diterpenes ryanodol and cinnceylanol. Possible ecological implications are discussed.

Ecological tannin assays : Evaluation of proanthocyanidins, protein binding assays and protein precipitating potential.

Estimations of condensed tannin content are generally based on calibration standard curves from Quebracho condensed tannins. We generated calibration standard curves from eight Sonoran Desert species for comparison with estimates of tannin concentrations derived from the Quebracho standard curve. Estimates of leaf tannin concentrations of each of the eight species using each species standard curve differed significantly with the estimates given by the Quebracho standard curve. Standard curves constructed from tannins from different individuals of three of the species varied significantly between, but not within, species. The efficiency of precipitation of protein bovine serum albumin (BSA) by each different tannin varied up to a factor of fifty for tannins of different species. Ordering species from highest to lowest based on tannin concentrations or binding efficiencies gave two different ranks. We argue that concentration or efficiency alone do not describe adequately tannin ecological activity. Instead, we suggest combining tannin concentrations and binding efficiencies to measure the protein precipitating potential of a leaf. Leaf protein precipitating potential is a more ecologically realistic parameter, we feel, for between-species comparisons than tannin content or binding efficiencies alone.