Effects of the Argentine ant venom on terrestrial amphibians.

Invasive species have major impacts on biodiversity and are one of the primary causes of amphibian decline and extinction. Unlike other top ant invaders that negatively affect larger fauna via chemical defensive compounds, the Argentine ant (Linepithema humile) does not have a functional sting. Nonetheless, it deploys defensive compounds against competitors and adversaries. We estimated levels of ant aggression toward 3 native terrestrial amphibians by challenging juveniles in field ant trails and in lab ant foraging arenas. We measured the composition and quantities of toxin in L. humile by analyzing pygidial glands and whole-body contents. We examined the mechanisms of toxicity in juvenile amphibians by quantifying the toxin in amphibian tissues, searching for histological damages, and calculating toxic doses for each amphibian species. To determine the potential scope of the threat to amphibians, we used global databases to estimate the number, ranges, and conservation status of terrestrial amphibian species with ranges that overlap those of L. humile. Juvenile amphibians co-occurring spatially and temporally with L. humile die when they encounter L. humile on an ant trail. In the lab, when a juvenile amphibian came in contact with L. humile the ants reacted quickly to spray pygidial-gland venom onto the juveniles. Iridomyrmecin was the toxic compound in the spray. Following absorption, it accumulated in brain, kidney, and liver tissue. Toxic dose for amphibian was species dependent. Worldwide, an estimated 817 terrestrial amphibian species overlap in range with L. humile, and 6.2% of them are classified as threatened. Our findings highlight the high potential of L. humile venom to negatively affect amphibian juveniles and provide a basis for exploring the largely overlooked impacts this ant has in its wide invasive range.

Efectos del Veneno de la Hormiga Argentina sobre los Anfibios Terrestres Resumen Las especies invasoras tienen un impacto importante sobre la biodiversidad y son una de las causas principales del declive y extinción de los anfibios. A diferencia de otras hormigas super-invasoras que afectan negativamente a animales más grandes por medio de compuestos químicos de defensa, la hormiga argentina (Linepithema humile) no tiene unaguijón funcional. Sin embargo, esta hormiga despliega compuestos defensivos contra sus competidores y adversarios. Estimamos los niveles de agresión de las hormigas hacia tres anfibios terrestres nativos exponiendo a los anfibios juveniles en pistas de hormigas en el campo y en las arenas de forrajeo de las hormigas en el laboratorio. Medimos la composición y las cantidades de toxina que presenta L. humile por medio del análisis de las glándulas pigidiales y el contenido en el cuerpo completo. Examinamos los mecanismos de la toxicidad en los anfibios juveniles cuantificando la toxina en el tejido del anfibio, buscando daños histológicos y calculando las dosis tóxicas para cada especie de anfibio. Para determinar el alcance potencial de la amenaza para los anfibios usamos bases de datos mundiales para estimar el número, distribución y estado de conservación de las especies terrestres de anfibios con distribuciones que se solapan con la de L. humile. Los anfibios juveniles que co-ocurren temporal y espacialmente con L. humile mueren al encontrarse con esta especie de hormiga en sus pistas. En el laboratorio, cuando un anfibio juvenil entró en contacto con L. humile, las hormigas reaccionaron rápidamente rociando a estos juveniles con veneno proveniente de las glándulas pigidiales. La iridomyrmecina fue el compuesto tóxico que encontramos en las glándulas pigidiales. Después de ser absorbida por la piel del anfibio, se acumuló en el cerebro, los riñones y el hígado. La dosis tóxica para los anfibios depende de la especie. A nivel mundial, se estima que 817 especies de anfibios terrestres tienen una distribución que se solapa con la de L. humile, y el 6.2% de estas especies se encuentran clasificadas como amenazadas. Nuestros hallazgos resaltan el potencial alto del veneno de L. humile para tener efectos negativos sobre los anfibios juveniles y también proporcionan una base para la exploración de los impactos de esta hormiga en su amplio rango invasivo, los cuales generalmente son ignorados.

Reverse chemical ecology approach for the identification of an oviposition attractant for Culex quinquefasciatus.

Pheromones and other semiochemicals play a crucial role in today's integrated pest and vector management strategies. These semiochemicals are typically discovered by bioassay-guided approaches. Here, we applied a reverse chemical ecology approach; that is, we used olfactory proteins to lead us to putative semiochemicals. Specifically, we used 7 of the top 10 odorant receptors (ORs) most expressed in the antennae of the southern house mosquito, Culex quinquefasciatus, and which are yet to be deorphanized. We expressed these receptors in the Xenopus oocyte recording system and challenged them with a panel of 230 odorants, including physiologically and behaviorally active compounds. Six of the ORs were silent either because they are not functional or a key odorant was missing. CquiOR36, which showed the highest transcript levels of all OR genes in female antennae, was also silent to all odorants in the tested panel, but yielded robust responses when it was accidentally challenged with an old sample of nonanal in ethanol. After confirming that fresh samples were inactive and through a careful investigation of all possible "contaminants" in the old nonanal samples, we identified the active ligand as acetaldehyde. That acetaldehyde is activating CquiOR36 was further confirmed by electroantennogram recordings from antennae of fruit flies engineered to carry CquiOR36. Antennae of female mosquitoes also responded to acetaldehyde. Cage oviposition and dual-choice assays demonstrated that acetaldehyde is an oviposition attractant in a wide range of concentrations and thus of potential practical applications.

Activity of Vetiver Extracts and Essential Oil against Meloidogyne incognita.

Vetiver, a nonhost grass for certain nematodes, was studied for the production of compounds active against the southern root-knot nematode, Meloidogyne incognita . In laboratory assays studying the effects on second-stage juvenile (J2) activity and viability, crude vetiver root and shoot extracts were nematotoxic, resulting in 40% to 70% J2 mortality, and were also repellent to J2. Vetiver oil did not exhibit activity against J2 in these assays. Gas chromatography-mass spectrometry analyses of three crude vetiver root ethanol extracts and a commercial vetiver oil determined that two of the major components in each sample were the sesquiterpene acid 3,3,8,8-tetramethyltricyclo[5.1.0.0(2,4)]oct-5-ene-5-propanoic acid and the sesquiterpene alcohol 6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-ol. The acid was present in higher amounts in the extracts than in the oil. These studies demonstrating nematotoxicity and repellency of vetiver-derived compounds to M. incognita suggest that plant chemistry plays a role in the nonhost status of vetiver to root-knot nematodes, and that the chemical constituents of vetiver may be useful for suppressing nematode populations in the soil.

Exocrine secretions of wheel bugs (Heteroptera: Reduviidae: Arilus spp.): clarification and chemistry.

Wheel bugs (Heteroptera: Reduviidae: Harpactorinae: Arilus) are general predators, the females of which have reddish-orange subrectal glands (SGs) that are eversible like the osmeteria in some caterpillars. The rancid odor of Arilus and other reduviids actually comes from Brindley's glands, which in the North (A. cristatus) and South (A. carinatus) American wheel bugs studied emit similar blends of 2-methylpropanoic, butanoic, 3-methylbutanoic, and 2-methylbutanoic acids. The Arilus SG secretions studied here are absolutely species-specific. The volatile SG components of A. carinatus include (E)-2-octenal, (E)-2-nonenal, (E)-2-decenal, (E,E)-2,4-nonadienal, (E)-2-undecenal, hexanoic acid, 4-oxo-nonanal, (E,E)-2,4-decadienal, (E,Z)-2,4- or (Z,E)-2,4-decadienal, and 4-oxo-(E)-2-nonenal; whereas in A. cristatus the SG secretion contains beta-pinene, limonene, terpinolene, terpinen-4-ol, thymol methyl ether, alpha-terpineol, bornyl acetate, methyl eugenol, beta-caryophyllene, caryophyllene oxide, and farnesol. Arilus spp. SG secretions may be sex pheromones, but verification of this hypothesis requires further testing.

Evaluation of a Novel User-Friendly Arthropod Repellent Gel, Verdegen.

Hand sanitizers are developed as alcohol-based liquid gel formulations, generally used to decrease the amount of infectious agents on human hands. Verdegen, LLC proposed to prepare an arthropod repellent gel for public use when the recent outbreaks of Zika infection vectored through Aedes mosquitoes in the American continents prompted multi-faceted emergency measures. Four different gel formulations were developed, comprising two of the most efficacious commercial arthropod repellent active ingredients, N,N-diethyl-3-methyl benzamide (deet) and 2-(2-hydroxyethyl)-1-piperidinecarboxylic acid 1-methylpropyl ester (picaridin), each at different concentrations (20 and 33% deet, or 20 and 33% picaridin). Compliance with the use of topical arthropod repellents remains an issue among military personnel. One of the most common complaints by Soldiers is that they do not like how the repellents applied on their skin leave behind an oily or greasy residue. These new gel formulations offer a user-friendly alternative for commonly used arthropod repellents formulations for the military and civilian personnel. We tested the efficacy and protection time of these new gel formulations in comparison with the commercially available cream formulations of deet and picaridin at similar concentrations. Our data show that gel formulations have better topical attributes, and offer equal or better biting protection for up to 48 h against host-seeking Aedes aegypti (L.) (Diptera: Culicidae) female mosquitoes.

Mosquito biting behavior: statistical power and sources of variation in toxicity and repellent bioassays.

Compounds thought to be effective against mosquitoes as either 'insecticides' or 'repellents' have recently been shown to contain properties of both, or possess other behavior-modifying actions. Prompted in part by these reports, we conducted posterior analyses of our data to examine some interrelated statistical issues inherent in our bioassay system. Using a modified K&D module, the responses of over 25,000 adult Aedes aegypti (L.) females exposed to either alphacypermethrin or DEET were compared with the responses of mosquitoes exposed to untreated controls for toxicity and biting (alphacypermethrin) or biting alone (DEET). Our analyses indicated that; (1) our bioassay system has more statistical power to determine a compound's toxicity than its repellent qualities, (2) day-to-day variability is large and needs to be accommodated in analyses; there are other, potentially even larger sources of variability (e.g., mosquito heterogeneity) which invalidate statistical tests that are based on the assumption of binomially or multinomially distributed data (e.g., chi2 tests), and (3) unlike biting mosquitoes exposed to DEET, the proportions of biting mosquitoes exposed to alphacypermethrin are unrelated to the proportions of concurrently tested biting controls, even after adjusting for daily variation in toxicity. Thus, there is a clear behavioral indicator in this bioassay system that the 'repellency' of DEET (a presumed repellent) differs in a fundamental way from that of alphacypermethrin (a presumed toxicant), which may allow the differentiation between classes of compounds based on biting behavior alone.

4-oxo-aldehydes from the dorsal abdominal glands of the bed bug (Hemiptera: Cimicidae).

Analyses of the dorsal abdominal glands of fourth- and fifth-instar nymphs of the bed bud, Cimex lectularius L. (Hemiptera: Cimicidae), indicated the predominant constituents were (E)-2-hexenal and (E)-2-octenal, with lesser amounts of 4-oxo-(E)-2-hexenal and 4-oxo-(E)-2-octenal. The latter two compounds have not been reported previously as occurring in bed bugs. There were no differences in the chemical composition of the dorsal abdominal glands excised from exuviae left behind by either male or female adults, nor from glands excised from fourth-instar exuviae. Because the two oxo-aldehydes made up at least 16% of the gland contents, further study of the functional role of these chemicals seem advisable.

A high throughput screening system for determining the three actions of insecticides against Aedes aegypti (Diptera: Culicidae) populations in Thailand.

Chemicals can protect humans from the bites of hemophagous arthropods through three different primary actions: irritancy (excitation), repellency, or toxicity, actions that can be evaluated using a laboratory-based assay system. In this study, the deterrent and toxic actions of three synthetic pyrethroids and DDT were characterized on six field strains of Aedes aegypti from Thailand under laboratory-controlled conditions using the high throughput screening system. All six strains showed significant contact irritant responses to the three synthetic pyrethroids, but significantly weaker irritant responses to DDT. Marked repellency responses were seen in all six Ae. aegypti test strains exposed to DDT, whereas the synthetic pyrethroids resulted in greater toxicity than DDT under similar test conditions. Although significantly different in actions, irritancy and repellency may reflect and be influenced by the background insecticide susceptibility status of a particular mosquito population. Results from this study can be used to guide decision making regarding more effective Ae. aegypti adult control in Thailand.

Prothoracic gland semiochemicals of green lacewings.

Adult chrysopids have paired prothoracic glands (PG) that are thought to produce defensive secretions (allomones). We analyzed PG extracts of the following green lacewings from North and South America, Australia, and China: Ceraeochrysa cubana (Brazil); Chrysopa (= Co.) oculata, Co. nigricornis, Co. incompleta, Co. quadripunctata (USA), and Co. septempunctata (China); Chrysoperla (= Cl.) rufilabris (USA) and Cl. sp. (Brazil); Plesiochrysa ramburi and Mallada spp. (Australia). PG secretions are characteristic for species within a genus, except for Chrysopa spp. (Z)-4-Tridecene is ubiquitous, but (Z,Z)-4,7-tridecadiene is a major PG constituent in some Chrysopa spp. and in P. ramburi. Earlier reports that Co. oculata and Co. nigricornis produce 1-tridecene were shown to be in error. Chrysopa PG secretions are distinguished by the presence or absence of N-3-methylbutylacetamide, plus skatole (3-methylindole). Skatole is also identified for the first time from the Plesiochrysa and Ceraeochrysa. The PG secretion in Plesiochrysa ramburi is characterized by the presence of (Z)-4-undecene instead of (Z)-4-tridecene, and N-3-methylbutylpropanamide instead of the acetamide, resembling the PG secretions of Chrysopa nigricornis, Co. septempunctata and Co. incompleta. The chemotaxonomic value of PG semiochemicals is discussed, including evidence for subgroups within the genus Chrysopa as it now stands.

Characterization of spatial repellent, contact irritant, and toxicant chemical actions of standard vector control compounds.

A previously described modular high-throughput screening system was used to characterize the spatial repellent, contact irritant, and toxicant chemical actions of 14 compounds historically used or under investigation for vector control. The response of F1-F4 Aedes aegypti (Thailand strain) to various concentrations of 4 organochlorines (chlordane, DDT, dieldrin, methoxychlor); 4 pyrethroids (alphacypermethrin, cypermethrin, deltamethrin, permethrin); 3 organophosphates (chlorpyrophos methyl, fenitrothion, malathion); 2 carbamates (bendiocarb, propoxur); and 1 pyrazole (chlorfenapyr) were evaluated. Results show chemicals exert different combinations of contact irritant, spatial repellent, and toxic actions. This is true even within the same chemical class. These actions can be ordered for each chemical based on the testing dose at which the specific response is elicited. Data also indicate that behavioral responses to spatial repellent and contact irritant actions are separate (or independent) from the toxic action of a compound. Results from pyrethroid and DDT assays also show chemicals can induce behavior-modifying actions, such as contact irritancy and spatial repellency, which will reduce man-vector contact, despite evidence of insecticide resistance within the test population. These findings support previous laboratory and field studies showing man-vector contact and disease transmission are routinely interrupted by spatial repellent and contact irritant actions of common public health insecticides. Studies similar to that presented here can be used as baseline evidence for expected vector responses and support best approaches for more detailed behavioral research.

Behavioral responses of catnip (Nepeta cataria) by two species of mosquitoes, Aedes aegypti and Anopheles harrisoni, in Thailand.

An investigation of the biological effect of catnip oil (Nepeta cataria L.) on the behavioral response of field collected Aedes aegypti and Anopheles harrisoni was conducted using an automated excitorepellency test system. Aedes aegypti showed significantly higher escape rates from the contact chamber at 5% catnip oil compared to other concentrations (P < 0.05). With Anopheles harrisoni, a high escape response was seen at 2.5% catnip oil from the contact chamber, while in the noncontact chamber a higher escape response was observed at a concentration of 5%. Results showed that this compound exhibits both irritant and repellent actions.

Verification of Argentine ant defensive compounds and their behavioral effects on heterospecific competitors and conspecific nestmates.

The invasive Argentine ant (Linepithema humile) has become established worldwide in regions with Mediterranean or subtropical climates. The species typically disrupts the balance of natural ecosystems by competitively displacing some native ant species via strong exploitation and interference competition. Here we report that Argentine ants utilize glandular secretions for inter and intra-specific communications during aggressive interactions with a heterospecific competitor, California harvester ant (Pogonomyrmex californicus). Chemical analyses indicated that Argentine ants deploy glandular secretions containing two major volatile iridoids, dolichodial and iridomyrmecin, on the competitor's cuticular surface during aggressive interactions. Bioassays indicated that the glandular secretions function as a defensive allomone, causing high levels of irritation in the heterospecific. Furthermore, the same glandular secretions elicited alarm and attraction of conspecific nestmates, potentially enabling more rapid/coordinated defense by the Argentine ants. Two major volatile constituents of the glandular secretion, dolichodial and iridomyrmecin, were sufficient to elicit these responses in conspecifics (as a mixture or individual compounds). The current study suggests that invasive Argentine ants' superior exploitation and interference competition may rely on the species' effective semiochemical parsimony.

Evaluation of the Constituents of Vetiver Oil Against Anopheles minimus (Diptera: Culicidae), a Malaria Vector in Thailand.

The development of resistance by mosquitoes to current synthetic compounds has resulted in reduced effectiveness of prevention and control methods worldwide. An alternative nonchemical based control tools are needed to be evaluated particularly plant-derived essential oils. Several components of vetiver oil have been documented as insect repellents. However, detailed knowledge of those components action against insect remains unknown. In this study, behavioral response of Anopheles minimus to four constituents of vetiver oil (valencene, terpinen-4-ol, isolongifolene, vetiverol) was evaluated by using the high-throughput screening assay system. Vetiverol and isolongifolene exhibited strong contact irritancy action at 1.0% (80.2% escaping) and 5.0% (81.7% escaping) concentration, respectively, while moderate action was found in both valencene and terpinen-4-ol at 5.0% (57.6% escaping). Only at 1.0% (0.7 spatial activity index [SAI]) and 5.0% (1.0 SAI) of valencene and 0.5% (0.7 SAI) of isolongifolene showed spatial repellency activity. High mortality (58.9-98.2%) was recorded in all concentration of vetiverol and isolongifolene. Meanwhile, valencene exhibited high mortality only at 5.0%, terpinen-4-ol showed very low toxic action (0-4.3%) in all concentration. These proved that valencene in vetiver oil is the promising constituent that can be developed as an alternative mosquito control mean in efforts to prevent disease transmission.

Female goldeneyed lacewings (Neuroptera: Chrysopidae) approach but seldom enter traps baited with the male-produced compound iridodial.

Earlier, we identified (1R,2S,5R,8R)-iridodial as a male-specific compound of the goldeneyed lacewing, Chrysopa oculata Say (Neuroptera: Chrysopidae), but traps baited with this compound caught almost exclusively males. In the present report, we demonstrated by sweep-net sampling and observation in the vicinity of pheromone lures that C. oculata females, and males, are strongly attracted to iridodial. Aggregation activity of C. oculata adults occurred between dusk and dawn. This research demonstrates that iridodial may be useful to induce goldeneyed lacewings to lay eggs in targeted plant patches for biological pest control.

New Nematotoxic Indoloditerpenoid Produced by Gymnoascus reessii za-130.

Chemical investigation of the fungal strain Gymnoascus reessii za-130, which was previously isolated from the rhizosphere of tomato plants infected by the root-knot nematode Meloidogyne incognita, led to the isolation and identification of a new indoloditerpenoid metabolite designated gymnoascole acetate. Its structure was established by spectroscopic methods including 1D- and 2D-NMR and MS analyses. Gymnoascole acetate demonstrated strong adverse effects on M. incognita second-stage juvenile (J2) viability; exposure to 36 µg/mL for 24 h induced 100% paralysis of J2 (EC50 = 47.5 µg/mL). Gymnoascole acetate suppressed M. incognita egg hatch relative to controls by >90% at 133 µg/mL after 7 days of exposure. The numbers of root galls and J2 in both soil and roots were significantly reduced (p = 0.05) by treatment with 2-200 µg/mL gymnoascole acetate/kg soil, compared to untreated control plants; nematode suppression increased with gymnoascole acetate concentration. This study demonstrated the nematotoxicity of gymnoascole acetate and indicates that it might be a potential biobased component in integrated management of M. incognita.

Feeding deterrent effects of catnip oil components compared with two synthetic amides against Aedes aegypti.

Recently, catnip, Nepeta cataria L. (Lamiaceae), essential oil has been formulated and marketed as an alternative repellent for protection against biting arthropods by several vendors. We isolated the major active components of catnip oil, E,Z- and Z,E-nepetalactone, and quantitatively measured their antibiting efficacy compared with the repellents N,N-diethyl-3-methylbenzamide (deet) and chiral (1S,2'S)-2-methylpiperidinyl-3-cyclohexene-1-carboxamide (SS220) against the yellowfever mosquito, Aedes aegypti (L.), by using an in vitro assay and human volunteers at 24 nmol compound/cm2 (cloth or skin). Of all compounds tested in an in vitro assay, SS220 ranked as the most effective, whereas catnip oil and the nepetalactone compounds did not differ significantly from each other or from deet. However, in human volunteer bioassays, neither E,Z and Z,E-nepetalactone nor racemic nepetalactone deterred mosquito biting as effectively as SS220 or deet. All compounds differed significantly from the control. We conclude that catnip oil and nepetalactone isomers are significantly less effective than deet or SS220 in deterring the biting of Ae. aegypti.

A novel high-throughput screening system to evaluate the behavioral response of adult mosquitoes to chemicals.

A modular and novel assay system for rapid mass screening of chemical compounds for contact irritant and spatial repellent actions against adult mosquitoes is described. The responses of Aedes aegypti to various concentrations of 3 topical repellents, deet, Bayrepel, and SS220, were evaluated. At treatment concentrations > or = 25 nmol/cm2 of SS220, mosquitoes exhibited significant contact irritant (escape) and spatial repellent (movement away from the chemical source) responses, whereas, a 10-fold increase in the treatment concentration of deet and Bayrepel was required to produce similar responses. The novel bioassay system detected contact irritancy and spatial repellency activity with reproducible results and provided baseline data for determining minimum effective concentrations for other chemicals. The system is compact in size, easy to decontaminate, and requires only a minute quantity of chemical compound.

Arthropod Deterrents from Artemisia pallens (Davana Oil) Components.

Davanone, a key sesquiterpene component of davana oil, has been synthesized in five convenient steps. Oxygenated sesquiterpenes have been linked to insect deterrent properties. Based on initial screening of davana oil, davanone and its hydroxy precursors have been generated and are being evaluated as arthropod deterrents against disease vectors.

Simple and short synthesis of trans-(R)-nerolidol, a pheromone component of fruit spotting bug.

A three-step synthesis of enantiomerically pure (R) and (S)-trans nerolidol from commercially available E,E-fanesol is described. Trans nerolidol is an abundant sesquiterpene in many plant species, almost enantiomerically pure; however, the configuration of chirality is S. There is no natural resource for R-trans nerolidol, which has recently been identified as a pheromone component of the fruit spotting bug Amblypelta lutescens. The simple syntheses reported here will make enantiomerically pure R- and S-trans nerolidol and homologues available for further research and ultimately for use in integrated pest management strategies comprising pheromones.

Biobased lactams as novel arthropod repellents.

Enanatiomerically pure 4aS,7S,7aR and 4aS,7S,7aS-nepetalactams and their analogs have been prepared in just two steps from 4aS,7S,7aR and 4aS,7S,7aS-nepetalactones, major components of catnip oil. Lactams or cyclic amides from iridoid monoterpenes are generated and being evaluated as a new class of compounds as arthropod deterrents against disease vectors.