Larvicidal activity of acetone extract and green synthesized silver nanoparticles from Allium sativum L. (Amaryllidaceae) against the dengue vector Aedes aegypti L. (Diptera: Culicidae)

Mosquito vectors of major human diseases are currently controlled using chemical and biological products. Extensive insecticide use has led to resistance development and human/environmental health risks, and alternative sustainable control options are needed; in this study, activity of an extract of garlic (Allium sativum; Amaryllidaceae), and silver nanoparticles (AgNPs) synthesized from the extract, were evaluated against 2nd and 3rd instar larvae of the yellow fever mosquito, Ae. aegypti (Diptera: Culicidae). Synthesis of AgNPs was confirmed using UV–Vis spectroscopy, and characterised using powdered X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy. Larvae were exposed to five concentrations (50, 100, 150, 200, 250 ppm) of garlic extract or synthesized AgNPs, with distilled water and silver nitrate solution (1 mM) as controls. The mortality of larvae was recorded after 6, 12, 24, 36, and 48 h following addition of the respective extracts.Dose- and time-dependent toxicity were recorded in both treatment groups with no mortality in control groups. Exposure to AgNPs at 250 ppm for 48 h yielded 100% mortality for both larval instars, with corresponding LC₅₀ values of 44.77 (2nd) and 62.82 ppm (3rd). Exposure to garlic extract resulted in similar 48-hour mortality (99 ± 0.77% (2nd) and 98 ± 1.10% (3rd), but consistently higher LC₅₀ values after all exposure times compared to AgNPs (e.g. 48-hour exposure: 108.42 ppm (2nd), 129.11 ppm (3rd), suggesting that AgNPs may potentially be used at lower concentrations for Ae. aegypti control.     

Toxicity of solvents and surfactants to Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) and Dermacentor nitens (Neumann, 1897) (Acari: Ixodidae) larvae

The objective of this work was to verify the sensitivity of Amblyomma cajennense and Dermacentor nitens larvae to the solvents ethanol, methanol, acetone, xylol and dimethyl sulfoxide (DMSO) and the surfactant Tween 80. The first four solvents were tested at analytical purity while the DMSO and surfactant Tween 80 were tested at a concentration of 1%. The substances tested at analytical purity that caused high mortality were also tested at concentrations of 50%, 25% and 1%. The larval packet test was used, with 10 repetitions for each treatment. A control group was also formed with the same number of repetitions, in which the larvae were only exposed to distilled water. In the first experiment, only xylol was highly toxic at the concentrations tested, causing mortality above 90% for larvae of both species. In the second experiment, xylol at 1% and at 25% showed low toxicity to the A. cajennense and D. nitens larvae, respectively, since the percentage mortality was statistically similar to that of the control group (p>0.05).     

Insecticide enhancement with feeding stimulants in corn for western corn rootworm larvae (Coleoptera: Chrysomelidae)

Amounts of the insecticide thiamethoxam required for 50% mortality of western corn rootworm larvae, Diabrotica virgifera virgifera LeConte, were reduced 100-fold when extracts of germinating corn, Zea mays L., were used to entice neonate larvae to feed on it. In behavioral bioassays, neonate rootworm larvae fed vigorously on filter paper disks treated with liquid pressed from corn roots. Moreover, disks treated with an acetone extract of corn (dried and rewetted with water) also elicited strong feeding from larvae. Larvae wandered away from filter paper disks treated with distilled water without feeding. Dilutions of thiamethoxam were tested in the bioassay alone or with corn extract and the efficacy of this insecticide was improved by the addition of the corn extract. For solutions containing 10 ppm thiamethoxam, 95% larval mortality occurred after 30 min of exposure when corn extract was present, but only 38% mortality occurred when the same concentration of insecticide alone (no feeding stimulants) was tested. Larval mortality after 24 h was significantly higher for corn extract-treated disks with 0.01, 0.1, 1, or 10 ppm insecticide than for the same concentrations without corn extract. Thiamethoxam did not deter larval feeding on corn extract, even at the highest concentration of thiamethoxam tested.     

Ecdysteroid levels changed by permethrin action in female Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks

As recent studies have shown that ecdysteroids may play a major role in the regulation of vitellogenesis in Ixodidae, the present study quantified, by means of a radioimmunoassay, the levels of ecdysteroids present in the hemolymph of semi-engorged females of Rhipicephalus sanguineus ticks obtained from control females (exposed to distilled water) and those exposed to increasing concentrations of permethrin. The levels of ecdysteroids decreased significantly as the concentration of permethrin increased, suggesting that this compound could be an inhibitor of ecdysteroids secretion, and consequently interfering with the reproductive ability of these ticks, since this hormone is responsible for the synthesis and incorporation of vitellogenin by oocytes. This study complements the previous results with R. sanguineus semi-engorged females, showing that permethrin is a potent agent causing major morphological changes in tick oocytes, such as the appearance of large vacuoles in the cytoplasm, reduction in the amount of yolk granules and a decrease in oocyte size, thus culminating in cell death and consequently reducing or preventing reproduction in treated females. The findings that permethrin leads to a decrease in ecdysteroid titers could represent an entry step into this scenario.     

Comparison of two commercial formulations of Bacillus thuringiensis var. israelensis for the control of Anopheles aquasalis (Diptera: Culicidae) at three salt concentrations

Anopheles aquasalis larvae are salt water tolerant, preferring concentrations between 10 and 20 parts per thousand (ppt). The larvicidal efficacy of two formulations of Bacillus thuringiensis var. israelensis (Vectobac-12AS and Bactivec), was investigated against An. aquasalis at salinities of 0, 10, and 20 ppt. A probit analysis was used to calculate the lethal concentrations (LC50 and LC95) for each product at each salinity. The LC50 and LC95 were higher for Bactivec than Vectobac-12AS, and for Bactivec, the LC50 and LC95 increased with salinity. Vectobac-12AS should thus be preferred to Bactivec for An. aquasalis control, especially in saline breeding habitats.     

Potential of Tenebrio molitor (Coleoptera: Tenebrionidae) as a bioassay probe for Metarhizium brunneum (Hypocreales: Clavicipitaceae) activity against Ixodes scapularis (Acari: Ixodidae)

The yellow mealworm, Tenebrio molitor L., has been used to indicate qualitatively the presence of entomopathogenic fungi in the soil or as a model for evaluating stress and other factors on fungal activity. Although this beetle appears highly susceptible to many of these fungi, little quantitative information is available on the sensitivity of T. molitor to a specific fungus and, therefore, fungal presence or as an indicator for pathogenicity to other species. The purpose of this study was to establish the suitability of T. molitor larvae as a bioassay probe for Metarhizium brunneum for comparison against the blacklegged tick, Ixodes scapularis. Nine concentrations of M. brunneum strain F52 ranging from 1.0 x 10(1) to 8.4 x 10(8) conidial/ml were simultaneously tested against T. molitor larvae and I. scapularis adults. Larvae of yellow mealworm were less sensitive to M. brunneum than I. scapularis adults (LC50's 4.4 x 10(7) and 1.7 x 10(5) conidia/ml, respectively, 4-wk post-treatment). The greater sensitivity of I. scapularis to the fungus suggests that the detection of fungal mycosis in mealworms would indicate sufficient inoculum to be pathogenic to I. scapularis and make this insect a suitable probe for evaluation of the presence and activity of M. brunneum against the blacklegged tick in field applications.     

Interactions of two idiobiont parasitoids (Hymenoptera: Ichneumonidae) of codling moth (Lepidoptera: Tortricidae) with the entomopathogenic nematode Steinernema carpocapsae (Rhabditida: Steinernematidae)

Simultaneous use of parasitoids and entomopathogenic nematodes for codling moth (CM) control could produce an antagonistic interaction between the two groups resulting in death of the parasitoid larvae. Two ectoparasitic ichneumonid species, Mastrus ridibundus and Liotryphon caudatus, imported for classical biological control of cocooned CM larvae were studied regarding their interactions with Steinernema carpocapsae. Exposure of M. ridibundus and L. caudatus developing larvae to infective juveniles (IJs) of S. carpocapsae (10 IJs/cm2; approximately LC(80-90) for CM larvae) within CM cocoons resulted in 70.7 and 85.2% mortality, respectively. However, diapausing full grown parasitoid larvae were almost completely protected from nematode penetration within their own tightly woven cocoons. M. ridibundus and L. caudatus females were able to detect and avoid ovipositing on nematode-infected cocooned CM moth larvae as early as 12h after treatment of the host with IJs. When given the choice between cardboard substrates containing untreated cocooned CM larvae and those treated with an approximate LC95 of S. carpocapsae IJs (25 IJs/cm2) 12, 24, or 48h earlier, ovipositing parasitoids demonstrated a significant preference for untreated larvae. The ability of these parasitoids to avoid nematode-treated larvae and to seek out and kill cocooned CM larvae that survive nematode treatments enhances the complementarity of entomopathogenic nematodes and M. ridibundus and L. caudatus.     

Susceptibility of Diatraea saccharalis (Lepidoptera: Crambidae) to tebufenozide.

Dosage-mortality baselines were determined for first and newly molted third instars of the sugarcane borer, Diatraea saccharalis (F.), with tebufenozide incorporated into an artificial diet. Using death as an endpoint for these assays, LC50 and LC90 values were estimated to be 0.17 and 0.41 ppm for the first and 0.87 and 3.67 ppm for third instars, respectively. Developmental effects also were observed and were defined to include mortality as well as physical impairment and retarded or prolonged development with absence of feeding. Concentrations causing developmental effects in treated larvae (EC50 and EC90) were 5.8- and 3.8-fold lower than the LC50 and LC90, respectively. The critical time of exposure to tebufenozide was determined for developmentally synchronous third instars to be less than 12 h postecdysis. Mortality through adult emergence significantly decreased from 92% (treatment at 0 h postecdysis) to 30% (treatment at 12 h postecdysis), when exposed to tebufenozide at the EC50. For surviving larvae, length of development time until pupation was significantly increased and female pupal weight significantly decreased when larvae were treated at 0 h postecdysis into the third instar. In addition, to modifying the traditional ways of assessing mortality 0-3 d postapplication, this study points out the value of using other approaches to pesticide assessment, especially where insect growth regulators are involved.     

Screening of Costa Rican Trichilia species for biological activity against the larvae of Spodoptera litura (Lepidoptera: Noctuidae)

Crude methanolic extracts made from the twigs of 39 plant samples from six species of Trichilia collected in Costa Rica, were incorporated into artificial diet and fed to neonate Spodoptera litura larvae. All six plant species tested significantly reduced larval growth after 7 and 10 days. The most active species was T. americana, reducing growth, on average, to 3.9% of control at 1000ppm fresh weight. The least active, on average, was T. glabra. A twig extract of T. americana proved to be more active than wood, bark or leaf extracts, with the twig extract reducing growth of S. litura larvae by 50% (EC(50)) at a dietary concentration of 17.2ppm. When T. americana wood extract was incorporated into artificial diet (10, 25, 50 and 75ppm) and fed to S. litura larvae throughout larval development, growth was slowed and the final weight of pupae and adults was reduced. At higher extract concentrations (50 and 75ppm) larvae entered one or two supernumerary instars before pupation occurred. This was shown to be due to both starvation and to post-ingestive activity of the extract.     

Cryopreservation of infective larvae of Dipetalonema viteae.

infective larvae of Dipetalonema viteae produced infections in Mongolian jirds (Meriones unguiculatus) after storage of infected ticks (Ornithodoros tartakovskyi) in the presence of dimethyl sulfoxide (DMSO, 5%) for 7 or 595 days in liquid nitrogen (-196 C). Infectivity of these larvae was only partially impaired. Microfilaremias of test jirds were generally lower than those of control jirds given nonfrozen larvae; however, the majority of test jirds developed microfilarial counts suitable for use in infecting ticks. In contradistinction, larvae frozen free of the tick failed to retain infectivity. Apparently the tick, in conjunction with DMSO, protects the larvae during freezing and thawing.     

Derris (Lonchocarpus) urucu (Leguminosae) extract modifies the peritrophic matrix structure of Aedes aegypti (Diptera:Culicidae)

Aqueous suspension of ethanol extracts of Derris (Lonchocarpus) urucu (Leguminosae), collected in the state of Amazonas, Brazil, were tested for larvicidal activity against the mosquito Aedes aegypti (Diptera:Culicidae). The aim of this study was to observe the alterations of peritrophic matrix in Ae. aegypti larvae treated with an aqueous suspension of D. urucu extract. Different concentrations of D. urucu root extract were tested against fourth instar larvae. One hundred percent mortality was observed at 150 microg/ml (LC(50) 17.6 microg/ml) 24 h following treatment. In response to D. urucu feeding, larvae excreted a large amount of amorphous feces, while control larvae did not produce feces during the assay period. Ultrastructural studies showed tha larvae fed with 150 microg/ml of D. urucu extract for 4 h have an imperfect peritrophic matrix and extensive damage of the midgut epithelium. Data indicate a protective role for the peritrophic matrix. The structural modification of the peritrophic matrix is intrinsically associated with larval mortality.     

Efficacy of leaves extract of Calotropis procera Ait. (Asclepiadaceae) in controlling Anopheles arabiensis and Culex quinquefasciatus mosquitoes

The present study aimed to investigate, the larvicidal, adult emergence inhibition and oviposition deterrent activity of aqueous leaves extract of Calotropis procera against Anopheles arabiensis and Culex quinquefasciatus as natural mosquito larvicide. The larvicidal activity was monitored against 2nd, 3rd and 4th instar larvae of each mosquito species 24 h post-treatment. Adult emergence inhibition activity was tested by exposing 3rd instar larvae of each mosquito species to different concentrations of extracts (200, 400, 600, 800 and 1000 ppm for An. arabiensis and 100, 200, 300, 400, 500 and 600 ppm for Cx. quinquefasciatus). Probit analysis was used to analyze data from bioassay experiments. The oviposition deterrent activity was tested by using three different concentrations of extracts (1000, 500 and 200 for An. arabiensis, and 1000, 500 and 100 for Cx. quinquefasciatus) that caused high, moderate and low larval mortality in the larvicidal experiment against 3rd instar larvae. It was found that, LC50–LC90 values calculated were 273.53–783.43, 366.44–1018.59 and 454.99–1224.62 ppm for 2nd, 3rd and 4th larval instars, respectively, of An. arabiensis and 187.93–433.51, 218.27–538.27 and 264.85–769.13 ppm for 2nd, 3rd and 4th larval instars, respectively, of Cx. quinquefasciatus. Fifty percent of adult emergence inhibition (EI50) was shown at 277.90 and 183.65 ppm for An. arabiensis and Cx. quinquefasciatus, respectively. The pupal stage was not affected till a concentration of 5000 ppm. The extract showed oviposition deterrence and effective repellence against both mosquito species at different concentrations, with the observation on that maximal eggs were laid in low concentration of extract. These results suggest that the leaves extract of C. procera possess remarkable larvicidal, adult emergence inhibitor, repellent and oviposition deterrent effect against both An. arabiensis and Cx. quinquefasciatus, and might be used as natural biocides for mosquito control.     

Evaluation of larvicidal efficacy of Ricinus communis (Castor) and synthesized green silver nanoparticles against Aedes aegypti L.

Aedes mosquitoes are the most important group of vectors that transmit pathogens, including arboviruses, and cause human diseases such as dengue fever, yellow fever, Zika virus, and Chikungunya. Biosynthesis and the use of green silver nanoparticles (AgNPs) is a vital step to identify reliable and eco-friendly controls for these vectors. In this study, Aedes (Ae.) aegypti larvae (2nd and 3rd instar) were exposed to leaf extracts of Ricinus communis (Castor) and AgNPs synthesized from the extract to evaluate their larvicidal potential. Synthesized AgNPs were characterized by UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and energy-dispersive X-ray spectroscopy (XRD). Ae. aegypti larvae were treated with different concentrations (50–250 ppm) of the leaf extract and synthesized AgNPs. There were five replicates per treatment, in addition to a positive (temephos) and negative control (dechlorinated water). Mortality was recorded after 12, 24, 36, and 48 h and the data were subjected to Probit analysis. The nanoparticles were more toxic (LC₅₀ = 46.22 ppm and LC₉₀ = 85.30 ppm) than the plant extract (106.24 and 175.73 ppm, respectively). The leaf extracts of Ricinus communis were subjected to HPLC analysis to identify their chemical constituents. This study suggests that plant extracts and synthesized nanoparticles are excellent alternatives to hazardous chemical pesticides used to control vector mosquitoes. This is a potentially useful technique that can reduce aquatic toxicity from insecticide use.     

Fipronil-induced cell death in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females

The tick Rhipicephalus sanguineus is currently considered an urban plague. For this reason many studies are intended to find methods to control these ectoparasites. Thus, the present study analyzed the ultrastructural modifications of the salivary glands cells of semi-engorged females of R. sanguineus resulting from their exposition to Fipronil (active ingredient of Frontline®). The studied individuals were divided into four groups. Group 1 was exposed to distilled water (control) and groups 2, 3 and 4 were exposed to 1, 5 and 10 ppm of Fipronil, respectively. The salivary gland of ticks subjected to the acaricide showed accelerated process of cell death by atypical apoptosis, as well as augmented cell damages as the concentration of the chemical compound was increased. The acaricide toxicity at cellular level was demonstrated by remarkable changes of elements of the cytoskeleton and spherocrystals (extremely hard inorganic structures). However, tick defense mechanisms, such as the observed autofagic vacuoles proved the cells attempt to preserve their integrity and minimize the devastating action of this chemical compound on the salivary glands.     

Underwater survival of Rhipicephalus sanguineus (Acari: Ixodidae)

Rhipicephalus sanguineus (Acari: Ixodidae) is a worldwide distributed tick, also due to its adaptability to different environmental conditions. In order to assess its ability to survive and to lay eggs after water immersion, 150 engorged females from southern Italy were water immersed for 1-15 days whereas eggs were flooded for 1-5 days. All females survived water immersion for 48 h, some of them up to 72 h, but egg hatch rate was negatively correlated with female submersion period. All eggs flooded for up to 120 h hatched successfully. These findings suggest that R. sanguineus is able to survive underwater for some days without loosing any biological activity. This feature should be considered in relation to its potential to spread to new areas and to its role as a vector of pathogens also in consideration of changes in climate the Earth is currently experiencing.     

Determination of LC50 of permethrin acaricide in semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Currently, the most efficient and widely used method of tick control still is the treatment with acaricides, especially permethrin (active ingredient of the Advantage^® Max3, Bayer), a pyrethroid with neurotoxic action. Due to the wide use of this acaricide in the control of the tick Rhipicephalus sanguineus, this study carried out laboratorial procedures to determine the LC₅₀ (lethal concentration fifty) of permethrin in semi-engorged females of R. sanguineus. Based on the result of 14 dilutions of permethrin in distilled water and later Probit analysis, the LC₅₀ of permethrin for R. sanguineus was 2062 ppm (1549-2675 ppm). This work can be used as a protocol with other chemicals, to determinate the LC₅₀, basic procedure for studies of control, resistance and behavior of ticks treated with acaricides, especially the brown dog tick R. sanguineus. Also, the knowledge of the LC₅₀ provides information on the potency of chemicals, the sensitivity of Arthropods to them and even estimates on pest control.     

Laboratory evaluation of the toxicity of systemic insecticides for Control of Anoplophora glabripennis and Plectrodera scalator (Coleoptera: Cerambycidae)

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) is one of the most serious nonnative invasive forest insects discovered in North America in recent years. A. glabripennis is regulated by federal quarantines in the United States and Canada and is the subject of eradication programs that involve locating, cutting, and chipping all infested trees. Other control methods are needed to aid in eradication and to form an integrated management program in the event eradication fails. We conducted laboratory bioassays to determine the toxicity of two systemic insecticides, azadirachtin and imidacloprid, for potential control of A. glabripennis and the cottonwood borer, Plectrodera scalator (F.) (Coleoptera: Cerambycidae), a closely related native cerambycid. Larvae of both cerambycid species were fed artificial diet with dilutions of azadirachtin or imidacloprid for 14 wk. Both insecticides exhibited strong antifeedant effects and some toxicity against A. glabripennis and P. scalator larvae. For A. glabripennis, the highest larval mortality at the end of the bioassay was 60% for larvae fed artificial diet treated with azadirachtin (50 ppm) or imidacloprid (1.6 ppm). For P. scalator, the highest larval mortality at the end of the bioassay was 100% for larvae fed artificial diet treated with azadirachtin (50 ppm) or imidacloprid (160 ppm). At 14 wk, the LC50 values for P. scalator were 1.58 and 1.78 ppm for azadirachtin and imidacloprid, respectively. Larvae of both species gained weight when fed diet treated with formulation blanks (inert ingredients) or the water control but lost weight when fed diet treated with increasing concentrations of either azadirachtin or imidacloprid. In a separate experiment, A. glabripennis adults were fed maple twigs treated with high and low concentrations of imidacloprid. A. glabripennis adult mortality reached 100% after 13 d on twigs treated with 150 ppm imidacloprid and after 20 d on twigs treated with 15 ppm imidacloprid. There was no visible feeding by A. glabripennis adults on twigs treated at the higher imidacloprid rate, and feeding was significantly reduced for adults placed on twigs treated at the low imidacloprid rate compared with adults on untreated twigs. In summary, imidacloprid and azadirachtin had both antifeedant and toxic effects against A. glabripennis and P. scalator and have potential for use in management programs. Based on our results, the delivery of high and sustained insecticide concentrations will be needed to overcome the antifeedant effects and lengthy lethal time for both larvae and adults exposed to these insecticides.     

Comparative activity of three repellents against the ticks Rhipicephalus sanguineus and Argas persicus

The residual repellent activity of N,N-diethylphenyl-acetamide (DEPA), N,N-diethyl-3-methyl benzamide (DEET) and dimethylphthalate (DMP) against the hard tick Rhipicephalus sanguineus (Latreille) and the soft tick Argas persicus (Oken) from topical application on rabbits and hens respectively in the laboratory have been studied. It was found that a 25% concentration of DEET gave strong repellency (greater than 90%) against larvae and nymphs of R. sanguineus for 15 and 7 days respectively and against A. persicus larvae and nymphs greater than 90% for 11 and 5 days respectively. Strong repellency (greater than 90%) against adults of the hard and soft ticks was observed for 9 and 7 days respectively with 40% DEET. DEPA at 25% concentration showed strong repellency (greater than 90%) against larvae and nymphs of R. sanguineus and A. persicus for 11 and 5 days respectively. Adults of hard and soft ticks were strongly repelled (greater than 90%) for 9 and 5 days respectively by 40% DEPA. DMP was found to be the least effective against all stages of both species of tick.     

First report of amitraz and cypermethrin resistance in Rhipicephalus sanguineus sensu lato infesting dogs in Mexico

Engorged female Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) were collected from dogs in the state of Yucatán, Mexico. Fourteen tick populations were collected from dogs at seven veterinary clinics, four residential homes and three cattle farms. The larval immersion test was used in the progeny of collected adult females to test susceptibility to amitraz and cypermethrin. Dose–mortality regressions, 50% lethal concentrations (LC₅₀), confidence intervals and slope were estimated by probit analysis. For amitraz, 12 tick populations (85.7%) were classified as resistant and low inter‐population variation in the phenotypic level of resistance was evident [resistance ratios (RRs) at LC₅₀: 1.0–13.0]. For cypermethrin, 12 tick populations (85.7%) were classified as resistant and substantial inter‐population variation in the phenotypic level of resistance was evident (RRs at LC₅₀: 1.0–104.0). Thus, amitraz resistance in R. sanguineus s.l. is common, but generally occurs at low levels; however, alarmingly high levels of cypermethrin resistance are present in R. sanguineus s.l. populations in dogs in Yucatán, Mexico. The intensive use of both acaricides to control ectoparasites on dogs is likely to lead to more serious resistance problems that may cause high levels of control failure in the future.