Combining TEMPO and methyl undecenoate to produce an effective anti-mosquito compound with convenient spin-labeling.

A general method to spin-label a fatty acid was demonstrated as well as an assay of the effectiveness of methyl 10-undecenoate and the spin-labeled version, against the larvae of Aedes aegypti. The LC50s were 66 and 58 µL/120 mL (55 and 48 ppm) respectively, and the LC90s were 108 and 90 µL/120 mL (113 and 90) ppm. This shows that the spin-label has very little effect on the larvicidal activity of the compound. This opens the possibility of the use of spin-labeling as a tool to determine mechanisms of larvicidal effectiveness, as it can be employed without altering the system under study.

Repellency and toxicity of a CO2-derived cedarwood oil on hard tick species (Ixodidae).

The repellency and toxicity of a CO2-derived cedarwood oil (CWO) was evaluated against actively questing unfed nymphs of four species of hard ticks: Amblyomma americanum (L.), Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus (Latreille). Using a vertical climb bioassay for repellency, nymphs of these species avoided a CWO-treated filter paper in proportional responses to treatment concentrations. At 60 min of exposure, I. scapularis nymphs were most sensitive with 50% repellency concentration (RC50) of 19.8 µg cm-2, compared with RC50 of 30.8, 83.8 and 89.6 µg cm-2 for R. sanguineus, D. variabilis and A. americanum, respectively. Bioassays determined the lethal concentration for 50% (LC50) and 90% (LC90) mortality of nymphs exposed to CWO in treated vials after 24- and 48-h exposure. After 24 h exposure, the LC50 values were 1.25, 3.45 and 1.42 µg cm-2 and LC90 values were 2.39, 7.59 and 4.14 µg cm-2 for D. variabilis, I. scapularis and R. sanguineus, respectively, but had minimal effect on A. americanum. After 48 h exposure, the LC50 values were 4.14, 0.78, 0.79 and 0.52 µg cm-2, and LC90 values were 8.06, 1.48, 1.54 and 1.22 µg cm-2 for A. americanum, D. variabilis, I. scapularis and R. sanguineus, respectively. The repellency of CWO on tick species decreased with time. The repellency and toxicity bioassays demonstrated concentration-dependent responses of tick nymphs to the oil, indicating the potential of the CO2-derived cedarwood oil be developed as an eco-friendly repellent and/or acaricide.

Ovicidal and Larvicidal Effects of Garlic and Asafoetida Essential Oils Against West Nile Virus Vectors.

We examined the chemical composition of garlic and asafoetida essential oils and their individual and combined toxicity against larvae of Culex pipiens Linnaeus and Culex restuans Theobald (Diptera: Culicidae). The effect of the two essential oils on egg hatch was also examined. Ten and 12 compounds, respectively, were identified in garlic and asafoetida essential oils. Allyl disulfide (49.13%) and diallyl trisulfide (31.08%) were the most abundant compounds in garlic essential oil accounting for 80.2% of the total oil. In contrast, (E)-sec-butyl propenyl disulfide (30.03%), (Z)-sec-butyl propenyl disulfide (24.32%), and disulfide, methyl 1-(methylthio)propyl (21.87%) were the most abundant compounds in asafoetida essential oil. Allyl disulfide accounted for 7.38% of the total oil in asafoetida essential oil and was one of only three compounds found in both oils. For both mosquito species, garlic essential oil was more toxic than asafoetida essential oil with Cx. restuans (LC50: garlic = 2.7 ppm; asafoetida = 10.1 ppm) being more sensitive than Cx. pipiens (LC50: garlic = 7.5 ppm; asafoetida = 13.5 ppm). When combined, the two essential oils had antagonistic effects. The majority of Culex egg rafts exposed to garlic (73.1%) or asafoetida (55.8%) essential oils failed to hatch and larvae of the few that did hatch mostly died as first instars. Allyl disulfide exhibited strong ovicidal and larvicidal activity suggesting its important contribution to the overall toxicity of the two essential oils. Thus, garlic and asafoetida essential oils are potent mosquito ovicides and larvicides but if used jointly, they could undermine vector control programs.

Characterization of Tolypocladium cylindrosporum (Hypocreales: Ophiocordycipitaceae) and Its Impact Against Aedes aegypti and Aedes albopictus Eggs at Low Temperature.

We examined the growth characteristics of Tolypocladium cylindrosporum IBT 41712 and its potential to infect eggs of Aedes aegypti and Ae. albopictus at a low temperature (15°C). When grown on Sabouraud dextrose agar supplemented with yeast extract, the IBT 41712 formed white colonies turning to a slightly darker, off-white color when mature. The mycelia bore swollen conidiophores producing smooth-walled, oblong to cylindrical conidia with varying sizes, ranging from 1.5 to 3.5 µm long. To determine the optimum temperature for the fungus, we cultured the fungus at eight temperatures (4°C, 12°C, 15°C, 21°C, 28°C, 33°C, 37°C, and 40°C) and measured the diametric growth. The optimum temperature for growth was 28°C since it had the highest diametric growth rate (2.1 ± 0.05 mm/day) and the fastest sporulation period (within 8-10 days of incubation). There was no fungal growth at the 3 highest temperatures (33°C, 37°C, and 40°C) but plates incubated at 33°C, when shifted to optimal temperature (28°C), showed visible growth indicating that following incubation at 33°C, the fungus remained viable. The IBT 41712 successfully infected mosquito eggs at 15°C. Fungal treatment induced egg hatch on moist seed-germination paper and this effect was more pronounced in Ae. aegypti compared to Ae. albopictus. When treated eggs were immersed in dH2O 21 days posttreatment, larval hatch of both Ae. aegypti (control = 91%, 1 × 107 conidia/ml, fungal treatment = 0%) and Ae. albopictus (control = 85%, fungal treatment = 28%) was significantly lower in fungal treatment compared to the controls. The ability of the strain to grow in a wide temperature range, and effectively infect mosquito eggs and induce egg hatch at a low temperature warrants further investigation for its potential as a mosquito control agent targeting eggs that overwinter or undergo long diapause.

Acinetobacter lactucae sp. nov., isolated from iceberg lettuce (Asteraceae: Lactuca sativa).

Strain NRRL B-41902T and three closely related strains were isolated from iceberg lettuce. The strain was found to consist of strictly aerobic, Gram-stain-negative rods that formed cocci in late stationary phase. 16S rRNA gene sequence analysis showed that strain NRRL B-41902T was most closely related to species within the genera Acinetobacter, and that a grouping of it and the three other closely related strains was most closely related to the type strain of Acinetobacter pittii, which was also confirmed through a phylogenomic analysis. Moreover, in silico DNA-DNA hybridization analysis revealed a substantial amount of genomic divergence (39.1 %) between strain NRRL B-41902T and the type strain of A. pittii, which is expected if the strains represent distinct species. Further phenotypic analysis revealed that strain NRRL B-41902T was able to utilize a combination of l-serine, citraconic acid and citramalic acid, which differentiated it from other, closely related Acinetobacter species. Therefore, strain NRRL B-41902T (=CCUG 68785T) is proposed as the type strain of a novel species, Acinetobacter lactucae sp. nov.

Efficacy of a granular formulation containing Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) microsclerotia against nymphs of Ixodes scapularis (Acari: Ixoididae).

Technical improvements in the production and formulation of microbial agents will increase the potential for development of biological pesticides that are able to compete with chemical insecticides in the marketplace. Here we report the efficacy of a simple granule formulation containing microsclerotia of Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) for control of unfed and fed nymphs of Ixodes scpaularis Say (Acari: Ixoididae). Microsclerotial granules of M. brunneum applied to moist potting mix produce infective conidia within 2 wk and conidia remained viable for up to 8 wk after application. Microsclerotial granules produced from 3.05 x 10(9) to 1.24 x 10(10) conidia g(-1) granules in potting mix. Both unfed and fed nymphs were susceptible to infection when exposed to treated potting soil with up to 56 and 74% mortality, respectively. M. brunneum demonstrated a transtadial infection for fed nymphs exposed to treated potting mix with signs of a fungal infection becoming apparent only after molting into adults. High conidial production rates from microsclerotial granules of M. brunneum combined with significant tick mortality support the need for additional research to evaluate the efficacy of this treatment technology as a biopesticide option for control of ticks.

Bioactivity of brassica seed meals and its compounds as ecofriendly larvicides against mosquitoes.

Strategic, sustainable, and ecofriendly alternatives to chemical pesticides are needed to effectively control mosquitoes and reduce the incidence of their vectored diseases. We evaluated several Brassicaceae (mustard family) seed meals as sources of plant derived isothiocyanates produced from the enzymatic hydrolysis of biologically inactive glucosinolates for the control of Aedes aegypti (L., 1762). Five defatted seed meals (Brassica juncea (L) Czern., 1859, Lepidium sativum L., 1753, Sinapis alba L., 1753, Thlaspi arvense L., 1753, and Thlaspi arvense-heat inactivated and three major chemical products of enzymatic degradation (allyl isothiocyanate, benzyl isothiocyanate and 4-hydroxybenzyl isothiocyanate) were assayed to determine toxicity (LC50) to Ae. aegypti larvae. All seed meals except the heat inactivated T. arvense were toxic to mosquito larvae. L. sativum seed meal was the most toxic treatment to larvae (LC50 = 0.04 g/120 mL dH2O) at the 24-h exposure. At the 72-h evaluation, the LC50 values for B. juncea, S. alba and T. arvense seed meals were 0.05, 0.08 and 0.1 g/120 mL dH2O, respectively. Synthetic benzyl isothiocyanate was more toxic to larvae 24-h post treatment (LC50 = 5.29 ppm) compared with allyl isothiocyanate (LC50 = 19.35 ppm) and 4-hydroxybenzyl isothiocyanate (LC50 = 55.41 ppm). These results were consistent with the higher performance of the benzyl isothiocyanate producing L. sativum seed meal. Isothiocyanates produced from seed meals were more effective than the pure chemical compounds, based on calculated LC50 rates. Using seed meal may provide an effective method of delivery for mosquito control. This is the first report evaluating the efficacy of five Brassicaceae seed meals and their major chemical constituent against mosquito larvae and demonstrates how natural compounds from Brassicaceae seed meals can serve as a promising ecofriendly larvicides to control mosquitoes.

Susceptibility of four tick species, Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus (Acari: Ixodidae), to nootkatone from essential oil of grapefruit.

Toxicity of nootkatone was determined in laboratory assays against unfed nymphs of Amblyomma americanum L., Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus Latreille. We determined the 50% lethal concentration (LC50) and 90% lethal concentration (LC90) of nootkatone by recording tick mortality 24 h after exposure in treated glass vials. Nymphs were susceptible to nootkatone with LC50 values of 0.352, 0.233, 0.169, and 0.197 microg/cm2, and LC90 values of 1.001, 0.644, 0.549, and 0.485 microg/cm2 for A. americanum, D. variabilis, I. scapularis, and R. sanguineus, respectively. The LC50 value for R. sanquineus was not significantly different from D. variabilis or I. scapularis. Other LC50 comparisons were significantly different. The LC90 for A. americanum was higher when compared with the three other tick species, which were not significantly different. Because nootkatone is volatile, we measured the amount of nootkatone recovered from duplicate-treated vials before tick exposure and from vials after tick exposure. Nootkatone recovered from vials before exposure ranged from 82 to 112% of the expected amounts. The nootkatone recovered after the 24-h exposure period ranged from 89% from vials coated with higher concentrations of nootkatone, down to 29% from vials coated with low nootkatone concentrations. Determination of the nootkatone residue after vial coating demonstrated loss of the active compound while verifying the levels of tick exposure. Toxicity of low concentrations of nootkatone to the active questing stage of ticks reported in this study provides a reference point for future formulation research to exploit nootkatone as a safe and environment-friendly tick control.

A formulation to encapsulate nootkatone for tick control.

Nootkatone is a component of grapefruit oil that is toxic to the disease-vectoring tick, Ixodes scapularis Say, but unfortunately causes phytotoxicity to treated plants and has a short residual activity due to volatility. We prepared a lignin-encapsulated nootkatone formulation to compare with a previously used emulsifiable formulation for volatility, plant phytotoxicity, and toxicity to unfed nymphs of I. scapularis. Volatility of nootkatone was measured directly by trapping nootkatone vapor in a closed system and indirectly by measuring nootkatone residue on treated filter paper after exposure to simulated sunlight (Xenon). After 24 h in the closed system, traps collected only 15% of the nootkatone applied as the encapsulated formulation compared with 40% applied as the emulsifiable formulation. After a 1-h light exposure, the encapsulated formulation retained 92% of the nootkatone concentration compared with only 26% retained by the emulsifiable formulation. For plant phytotoxicity, cabbage, Brassica oleracea L., leaves treated with the encapsulated formulation expressed less necrosis, retaining greater leaf weight compared with leaves treated with the emusifiable formulation. The nootkatone in the emulsifiable formulation was absorbed by cabbage and oat, Avena sativa L., plants (41 and 60% recovered 2 h after application, respectively), as opposed to 100% recovery from the plants treated with encapsulated nootkatone. Using a treated vial technique, encapsulated nootkatone was significantly more toxic to I. scapularis nymphs (LC50 = 20 ng/cm2) compared with toxicity of the emulsifiable formulation (LC50 = 35 ng/cm2). Thus, the encapsulation of nootkatone improved toxicity for tick control, reduced nootkatone volatility, and reduced plant phytotoxicity.

Honeysuckle essential oil as a potential source of ecofriendly larvicides for mosquito control.

BACKGROUND: Some plant essential oils have insecticidal properties against mosquitoes and can be harnessed as ecofriendly tools for mosquito control. We conducted bioassays to determine the toxicity of Italian honeysuckle (Lonicera caprifolium) essential oil and its fractions against larvae of the yellow fever mosquito, Aedes aegypti. RESULTS: Sixteen constituents were identified in honeysuckle essential oil compared to 15, 15, 15, and 11 constituents in fractions A, B, C, and E, respectively. The chemical constituents for fraction D were not determined due to lack of enough fraction sample. The two major constituents identified were patchouli alcohol (29.3%) and 6-acetyl-1,1,2,4,4,7-hexamethyltetralin (20.6%) in whole essential oil, alpha-bulnesene (27.6%) and 6-acetyl-1,1,2,4,4,7-hexamethyltetralin (23.2%) in fraction A, unknown chemical (47.3%) and diethyl phthalate (19.5%) in fraction B, unknown chemical (38.3%) and diethyl phthalate (23.2%) in fraction C, and patchouli alcohol (58.7%) and diethyl phthalate (20.5%) in fraction E. The LC50 for whole essential oil was 34.4 ppm and significantly higher than 20.6, 19.7, 18.6, and 17.7 ppm for fractions B, C, D, and E, respectively. In contrast, fraction A was inactive. At 50 ppm, all individual constituents tested were less toxic than the whole essential oil with exception of patchouli alcohol, which caused 100% mortality. CONCLUSION: These findings suggest that patchouli alcohol is one of the chemical constituents responsible for bioactivity of honeysuckle essential oil and some of its fractions. The findings also demonstrate that honey suckle essential oil and its fractions can be exploited as a source of ecofriendly larvicides for mosquito control. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Peptidoglycan Recognition Proteins (PGRPs) Modulates Mosquito Resistance to Fungal Entomopathogens in a Fungal-Strain Specific Manner.

Fungal entomopathogens are potential tools for the control of mosquito vectors that transmit infectious agents that cause disease in humans and animals. During the infection process, effective recognition of the invading fungi by the mosquito, is a crucial step in mounting an appropriate anti-fungal response. In this study, we investigated the role of peptidoglycan recognition receptors (PGRPs) in host resistance to fungal entomopathogens at the early stages of infection. Our study identified the induction of PGRP-LA, -LB, -LD, -LE, and -S1 during infection with two different fungal entomopathogenic strains. Furthermore, our data shows temporal differences in PGRP elicitation, with most PGRPs displaying significant upregulation at 60 h post-infection. Depletion of certain PGRPs via RNAi silencing resulted in a significant increase in fungal proliferation and a reduction in mosquito survival that was fungal strain-specific. Our data indicates that PGRPs play an important role in the antifungal response and expands our understanding of the factors that determine host susceptibility to fungal entomopathogens.