Dose-mortality Bioassays Research Articles

Transcriptome mining and expression analysis of ABC transporter genes in a monophagous herbivore, Leucinodes orbonalis (Crambidae: Lepidoptera)

Insecticide resistance is a global concern and requires immediate attention to manage dreadful insect pests. One of the resistance mechanisms adopted by insects is through ATP-binding cassette (ABC) transporter proteins. These proteins rapidly transport and eliminate the insecticidal molecules across the lipid membranes (Phase III detoxification mechanism). In the present study, we investigated the potential role of ABC transporter genes in imparting insecticide resistance in field-collected insecticide resistant larvae of eggplant shoot and fruit borer (Leucinodes orbonalis; Crambidae: Lepidoptera). Dose-mortality bioassays against five insecticidal molecules revealed moderate to high levels of insecticide resistance (32.2. to 134.1-fold). Thirty-one genes encoding ABC transporter proteins were mined from the transcriptome resources of L. orbonalis. They were classified under eight sub-families (ABCA to ABCH). Phylogenetic analysis indicated ABCG is the most divergent, composed of nine genes as compared to many other insects. Transcriptome analysis of the insecticide resistant and susceptible strains of L. orbonalis revealed differential expression of 13 ABC transporter genes. The altered expression of these genes was further validated using qRT-PCR. The knockdown studies indicated the involvement of ABCD1 and ABCG2 genes in chlorantraniliprole resistance in the insecticide-resistant strain of L. orbonalis. This study unveils the additional mechanisms employed by L. orbonalis in resisting insecticide toxicity through accelerated excretion mode. These ABCD and ABCG family genes could be candidate targets in developing genome-assisted pest management strategies in the future.

Chemical Composition and Insecticidal Properties of Origanum vulgare (Lamiaceae) Essential Oil against the Stored Product Beetle, Sitophilus granarius

Although phosphides are utilized in stored pest control, efforts have been made to discover environmentally friendly insecticides. For insecticidal properties, essential oils (EOs) are considered to be novel alternatives for pesticide use. This study characterized the Origanum vulgare EO by gas chromatography–flame ionization detector (GC–FID) × gas chromatography–mass spectrometry (GC–MS) and assessed the insecticidal activities against Sitophilus granarius. Mortality, post-exposure survival, behavior, and respiration caused by this EO in S. granarius were investigated. The majority of the compounds were p-cymene, carvacrol, linalool, and thymol. In dose–mortality bioassays, the lethality of this EO (LD50 = 3.05 µg insect−1 and LD90 = 10.02 µg insect−1) was confirmed in S. granarius. The survival rate was 99.9% in adults not treated with O. vulgare EOs, reducing to 44.9% and 10.3% in weevils treated with 3.05 µg insect−1 and 10.02 µg insect−1, respectively. The O. vulgare EO alters the behavioral pattern in terms of walking distance and resting time, displaying repellency. Additionally, this EO reduced the gas exchange of weevils from 2.78 to 2.36 µL CO2 h−1 at 3.05 µg insect−1, after 3 h EO exposure. The results suggest that O. vulgare EOs affect different biological functions in the insect, and open new perspectives for controlling stored pests, representing a first step in the innovation of green pesticides.

Resistance to grain protectants and synergism in Pakistani strains of Sitophilus oryzae (Coleoptera: Curculionidae)

The widespread use of insecticides for the management of insect pests in storage facilities and food industries have caused insecticide resistance a frequent issue worldwide. Nonetheless, this issue has been little explored in Pakistan that resulted in control failures and increased dosage of insecticides. In the present study, insecticide resistance to chlorpyrifos-methyl, pirimiphos-methyl, permethrin and spinosad was surveyed in five field strains of Sitophilus oryzae: FSD-SO, GJR-SO, DGK-SO, MTN-SO and BWP-SO, collected from five different localities of Punjab, Pakistan, and contrasted with an insecticide susceptible reference strain (Lab-SO). Dose-mortality bioassays were performed in glass vials containing insecticide-treated rice grains, and lethal doses (LD50 and LD95) were calculated and compared using the ratio tests. In comparison to the Lab-SO strain at LD50 and LD95 levels, field strains exhibited: 24.51 to 52.80 and 36.55 to 69.31 resistance ratios (RRs), respectively, for chlorpyrifos-methyl; 15.89 to 45.97 and 55.12 to 194.93 RRs, respectively, for pirimiphos-methyl; 39.76 to 108.61 and 61.33 to 130.12 RRs, respectively, for permethrin; 4.23 to 27.50 and 6.28 to 41.00 RRs, respectively, for spinosad. In the synergism experiments using the Lab-SO and the most resistant strains against each insecticide, the enzyme inhibitors (PBO and DEF) failed to synergize toxicity of insecticides in the Lab-SO strain; however, toxicity of chlorpyrifos-methyl, pirimiphos-methyl and permethrin significantly enhanced in the resistant strains of S. oryzae, suggesting possibility of metabolic mechanism of resistance. In addition, activities of detoxification enzymes (CarE, MFO and GST) were significantly higher in resistant strains compared to the Lab-SO strain. The results revealed presence of insecticide resistance in field strains of S. oryzae that necessitate the need to develop a resistance management strategy.

Variation in susceptibility to insecticides and synergistic effect of enzyme inhibitors in Pakistani strains of Trogoderma granarium

Trogoderma granarium is an important quarantine insect pest of economic importance worldwide. Insecticides are heavily used against this pest that can reduce its susceptibility levels, and if left unchecked, this could lead to misuse of insecticides. Here, the susceptibility of five field strains of T. granarium in comparison to a reference strain (Lab-TG) was determined against pirimiphos-methyl, permethrin and spinosad using dose-mortality bioassays. Based on lethal dose (LD50 and LD99) values, the Lab-TG strain was the most susceptible to all the insecticides in comparison to the field strains. In the case of field strains, LD50 values varied from: 17.68–31.97 mg/kg for pirimiphos-methyl, 20.50–42.47 mg/kg for permethrin, and 1.34–5.00 mg/kg for spinosad. Whereas LD99 values for pirimiphos-methyl, permethrin and spinosad of field strains varied from 409.07 to 730.24 mg/kg, 374.24–949.48 mg/kg, and 11.41–72.25 mg/kg, respectively. The resistance ratios (RR) at LD50 and LD99 levels were in the range of 13.71–24.78 and 39.33–70.21 fold for pirimiphos-methyl; 13.49–27.94 and 37.31–94.66 fold for permethrin; 2.35–8.77 and 3.37–21.31 fold for spinosad, respectively. Considering the overall results, spinosad proved to be the most toxic insecticide to the laboratory and field strains of T. granarium when compared with toxicity of pirimiphos-methyl or permethrin. Insecticidal bioassays implementing enzyme inhibitors revealed no effect on synergizing the toxicity of all the insecticides in the Lab-TG strain; however, enzyme inhibitors significantly synergized toxicity of pirimiphos-methyl in all the field strain suggesting the major involvement of metabolic mechanism associated with pirimiphos-methyl resistance. The study demonstrates variation in susceptibility to insecticides in T. granarium strains. The results might be helpful for monitoring future variation in susceptibility to insecticides in T. granarium and for setting field rates.

Genome Mining and Expression Analysis of Carboxylesterase and Glutathione S-Transferase Genes Involved in Insecticide Resistance in Eggplant Shoot and Fruit Borer, Leucinodes orbonalis (Lepidoptera: Crambidae).

The shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) is the major cause of low productivity in eggplant and insecticides being the mainstay of management of L. orbonalis. However, field control failures are widespread due to the evolution of insecticide resistance. Taking advantage of the whole genome sequence information, the present study investigated the level of insecticide resistance and the expression pattern of individual carboxylesterase (CE) and glutathione S-transferases (GSTs) genes in various field collected populations of L. orbonalis. Dose-mortality bioassays revealed a very high level of resistance development against fenvalerate (48.2–160-fold), phosalone (94-534.6-fold), emamectin benzoate (7.2–55-fold), thiodicarb (9.64–22.7-fold), flubendiamide (187.4–303.0-fold), and chlorantraniliprole (1.6–8.6-fold) in field populations as compared to laboratory-reared susceptible iso-female colony (Lo-S). Over-production of detoxification enzymes viz., CE and GST were evident upon enzyme assays. Mining of the draft genome of L. orbonalis yielded large number of genes potentially belonging to the CE and GST gene families with known history of insecticide resistance in other insects. Subsequent RT-qPCR studies on relative contribution of individual genes revealed over-expression of numerous GSTs and few CEs in field populations, indicating their possible involvement of metabolic enzymes in insecticide resistance. The genomic information will facilitate the development of novel resistance management strategies against this pest.

Susceptibility to indoxacarb and synergism by enzyme inhibitors in laboratory and field strains of five major stored product insects in Pakistan

Resistance to commonly used grain protectants and fumigants in stored product insect pests necessitates the need to explore alternative substances. Indoxacarb is a reduced-risk oxadiazine insecticide that is generally used in field crops, but there are limited reports of its susceptibility in stored insect pests. The objective of this study was to determine susceptibility to indoxacarb in laboratory and field strains of five major stored product insects: Rhyzopertha dominica (Fabricius), Sitophilus zeamais (Motschulsky), S. oryzae (Linnaeus), Tribolium castaneum (Herbst), and Oryzaephilus surinamensis (L.), using dose-mortality bioassays on wheat grains. In most of the cases, the susceptibility of laboratory strains of all the studied pests were significantly higher than the corresponding field strains. The LD50 and LD99 values (mg a.i./kg of grains) of field strains ranged from 0.13 to 0.38, and 3.44 to 24.76, respectively (for R. dominica), 0.26 to 0.55, and 5.26 to 19.37, respectively (for S. oryzae), 0.41 to 1.01, and 13.11 to 22.46, respectively (for S. zeamais), 0.67 to 1.37, and 15.43 to 43.44, respectively (for T. castaneum), and 0.52 to 0.92, and 18.06 to 61.63, respectively (for O. surinamensis). Synergism bioassays implementing piperonyl butoxide or S,S,S-tributyl phosphorotrithioate along with indoxacarb on selected field strains revealed enhanced susceptibility to indoxacarb. The study demonstrates relative susceptibility to indoxacarb in major stored product insects. Synergism results support the probability of metabolic-based mechanisms responsible for mitigating indoxacarb toxicity. The results might be helpful for monitoring future variation in susceptibility to indoxacarb in the selected insect species and for setting field rates.

Phosphine Resistance in North American Field Populations of the Lesser Grain Borer, Rhyzopertha dominica (Coleoptera: Bostrichidae).

Phosphine is the most widely used fumigant for stored grain insect pests, and resistance to phosphine has evolved in several species worldwide. This study was designed to determine the presence of phosphine resistance in 34 populations of Rhyzopertha dominica (F.) collected from the United States and Canada. Adult R. dominica were sampled and subjected to a discriminatory dose toxicity assay of exposure to 20 ppm of phosphine for 20 h of exposure to distinguish a susceptible R. dominica adult by death from a resistant beetle that survives the treatment. All but two of the 34 geographic populations surveyed had some beetles that were resistant to phosphine, and the frequency of resistance varied from 97% in a population from Parlier, California to 0% in beetles from both Carnduff, Saskatchewan and Starbuck, Manitoba. Probit analyses of dose-mortality bioassays with beetles from a laboratory-susceptible strain and those from five of the populations sampled were used to calculate resistance ratio factors (RRs) based on the ratio of LC50 (estimate for the concentration to kill 50% of a test group) in the sampled population to the LC50 for the susceptible strain. The highest RR for the five resistant populations was nearly 596-fold in beetles from Belle Glade, Florida, whereas the lowest RR in that group was 9-fold in Wamego, Kansas. This study revealed that phosphine resistance in R. dominica is common across North America and some populations have levels of resistance that may pose challenges for continued use of phosphine for their management.

Cross-resistance and synergism bioassays suggest multiple mechanisms of pyrethroid resistance in western corn rootworm populations.

Recently, resistance to the pyrethroid bifenthrin was detected and confirmed in field populations of western corn rootworm, Diabrotica virgifera virgifera LeConte from southwestern areas of Nebraska and Kansas. As a first step to understand potential mechanisms of resistance, the objectives of this study were i) to assess adult mortality at diagnostic concentration-LC99 to the pyrethroids bifenthrin and tefluthrin as well as DDT, ii) estimate adult and larval susceptibility to the same compounds as well as the organophosphate methyl-parathion, and iii) perform synergism experiments with piperonyl butoxide (PBO) (P450 inhibitor) and S,S,S-tributyl-phosphorotrithioate (DEF) (esterase inhibitor) in field populations. Most of the adult field populations exhibiting some level of bifenthrin resistance exhibited significantly lower mortality to both pyrethroids and DDT than susceptible control populations at the estimated LC99 of susceptible populations. Results of adult dose-mortality bioassays also revealed elevated LC50 values for bifenthrin resistant populations compared to the susceptible control population with resistance ratios ranging from 2.5 to 5.5-fold for bifenthrin, 28 to 54.8-fold for tefluthrin, and 16.3 to 33.0 for DDT. These bioassay results collectively suggest some level of cross-resistance between the pyrethroids and DDT. In addition, both PBO and DEF reduced the resistance ratios for resistant populations although there was a higher reduction in susceptibility of adults exposed to PBO versus DEF. Susceptibility in larvae varied among insecticides and did not correlate with adult susceptibility to tefluthrin and DDT, as most resistance ratios were < 5-fold when compared to the susceptible population. These results suggest that both detoxifying enzymes and target site insensitivity might be involved as resistance mechanisms.

Screening of tropical isolates of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) for virulence to the sweet potato weevil, Cylas formicarius (Coleoptera: Brentidae)

The sweet potato weevil (SPW),Cylas formicarius, is a serious pest of sweet potato in Australia and Papua New Guinea. Ten strains ofMetarhiziumsp. isolated from Australian soil samples were evaluated for their growth characteristics and screened for virulence to adult SPW under laboratory conditions. All isolates except QD62 (48.6%) had moderate to high germination (66–97%), and all took 2 to 4 days to sporulate at 25 °C. The optimal temperature for radial growth for the majority of isolates was 30 °C, and there was a significant interaction between isolate and temperature (P&amp;lt; 0.05). Isolate QS155 showed the fastest radial growth at 30 °C. The internal transcribed spacer sequences showed slight variations among the isolates; however, all isolates were shown to beMetarhizium anisopliae. Isolates varied greatly in their virulence. At 10 days after inoculation (DAI) by immersion in a suspension of 1 × 107conidia/ml, 9 of the 10 isolates were virulent, causing 80–100% mortality of adult SPW. Only two isolates (QS001-6 and QS155) caused more than 50% mortality at 5 DAI. In dose-mortality bioassays, isolate QS155 had the lowest 20-day LC50and LC90values; however, there were no statistically significant differences in mortality among the three most promising isolates tested (QD66, QS001-6 and QS155). These results show thatM. anisopliaeisolate QS155 has potential as a microbial control agent for SPW, and that further evaluation under glasshouse and field conditions is warranted.

Lethal and sublethal effects of neem oil to the predatory mite Proprioseiopsis neotropicus (Acari: Phytoseiidae)

Lethal and sublethal toxicity of the neem oil Bioneem® to the predatory mite Proprioseiopsis neotropicus (Acari: Phytoseiidae) were evaluated by combining lethal doses (LD) with population growth and biological parameter studies. Dose-mortality bioassays were conducted to estimate the lethal doses of the neem oil to P. neotropicus using probit analyses. Afterwards, the instantaneous rate of increase (ri) was used to evaluate the sublethal effects of the neem oil to P. neotropicus based on reproduction and mortality data. The effects were further assessed by comparing some life history parameters of P. neotropicus exposed to a sublethal dose of neem oil. The dose of neem oil which kills 50% of the population (LD50) of the predatory mite P. neotropicus was 7.5 μl/ cm2. The instantaneous rate of increase of the predatory mite P. neotropicus did not vary with increases of dosages of neem oil. Similarly, the LD25 of the neem oil did not affect biological parameters of the predatory mite. Based on our lethal and sublethal approach we conclude that the neem oil Bioneem® is generally selective to the predatory mite P. neotropicus.

Lethal doses of ozone for control of all stages of internal and external feeders in stored products

Gaseous ozone (O(3)) has potential for control of insects in stored grain. Previous studies have focused on freely exposed insects. Immatures of internal pests (e.g. Sitophilus spp. and most stages of Rhyzopertha dominica F.) are protected within kernels and probably require higher doses and/or longer treatment times for full control. A laboratory study determined the doses of ozone necessary for full control of freely exposed and internal stages of eleven stored-product pest species. Test insects were three species of Sitophilus, R. dominica, Tribolium confusum Jacquelin du Val, T. castaneum Herbst, Plodia interpunctella Hübner, Sitotroga cerealella Olivier, Oryzaephilus surinamensis L., Ephestia kuehniella Zeller and Stegobium paniceum L. Insects were exposed to continuous flows of ozone in doses of 10-135 ppm and exposure times of 5-8 days. Dose-mortality bioassays were conducted on three species of Sitophilus and P. interpunctella. Freely exposed stages (with a few exceptions) were controlled with 35 ppm of ozone for 6 days. Full mortality of internal stages within kernels required exposure to 135 ppm for 8 days. This study confirms that higher doses and/or longer treatment times are necessary for control of internal stages of stored-product pests.

Characteristics and virulence of nucleopolyhedrovirus isolated from Hyposidra talaca (Lepidoptera: Geometridae), a pest of tea in Darjeeling Terai, India

Nucleopolyhedrovirus (NPV) was isolated from infected Hyposidra talaca (Walker) caterpillars. This defoliating pest was sampled from the Terai and Dooars tea plantations along the foothills of Darjeeling, India. Phase contrast and transmission electron microscopy revealed polyhedral occlusion bodies (OBs) typical of NPVs. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis showed that the molecular weight of the major protein of the OBs is 32.17 kDa. Dose-mortality bioassays conducted for OBs on the second instar caterpillars of H. talaca resulted in a median lethal concentration (LC50) of 2.8 × 103 OBs/ml. The median lethal time (LT50) was 5.45 days for 1 × 104 OBs/ml, 4.15 days for 1 × 105 OBs/ml and 4.05 days for 1 × 106 OBs/ml concentrations. These results indicate the potential of using this NPV as a microbial pesticide against H. talaca.

Insecticide Susceptibility of &lt;I&gt;Euschistus heros&lt;/I&gt; (Heteroptera: Pentatomidae) in Brazil

Euschistus heros (F.) (Heteroptera: Pentatomidae) is the most prevalent stink bug pest in Brazil, and populations can be difficult to manage using organophosphates or endosulfan. Because E. heros is difficult to rear in the laboratory, no baseline insecticide susceptibility data have been published. Therefore, we conducted dose-mortality studies using field-collected insects, and, using the results from the dose-mortality studies, we surveyed susceptibility to acephate, methamidophos, and endosulfan in populations from southern and central Brazil. In addition, esterase activity was evaluated among populations as these enzymes have been related to organophosphate resistance. Finally, newer chemistries were evaluated for E. heros population management in small-plot field studies. In the dose-mortality bioassays, variation in susceptibility to methamidophos and endosulfan among populations was significant, but the variation in susceptibility to acephate was not. The population from Londrina (at the Embrapa Soybean Research Station, northern Parana state) was among the most susceptible, whereas the population from Pedrinhas Paulista (southwestern Sao Paulo state) was among the least susceptible. Significant variation among populations was observed in esterase activity with the population from Pedrinhas Paulista having the highest esterase levels and the population from Londrina having among the lowest levels. To survey populations, we used the dose-mortality data for the Londrina population (because it was, consistently, one of the most susceptible populations) to estimate the insecticide concentration that killed 99% of the population (LC99). The corresponding doses were 5.9 microg (AI) of acephate adult(-1), 0.7 microg (AI) methamidophos adult(-1), and 1,600 microg (AI) endosulfan adult(-1). The survey data confirmed the data from the dose-mortality bioassays in that the population from Pedrinhas Paulista was identified as one of the least susceptible and the population from Londrina was one of the most susceptible. The field tests confirmed that older chemistries provided minimal control (< 50% control), whereas the products containing mixtures of pyrethroids and neonicotinoids provided better control (> 60%).

In vivo production, stabilization, and infectivity of baculovirus preoccluded virions

Wild-type and polyhedrin-negative isolates of Autographa californica nuclear polyhedrosis virus were replicated in fifth-instar Trichoplusia ni larvae. Insect tissues infected with wild-type virus contained two types of virions that are highly infectious when ingested, those occluded in polyhedra and preoccluded virions. Tissue infected with the polyhedrin-negative virus contained only preoccluded virions. The relative potencies of the two types of infected tissue were determined by dose-mortality bioassays by using the neonate droplet feeding procedure. On a fresh weight basis, preparations of tissues infected with the polyhedrin-negative virus were approximately four times more potent than equivalent preparations of tissue infected with wild-type virus. Approximately half of the observed potency of the wild-type-virus preparations was due to polyhedra, and the remaining activity was due to preoccluded virions present in the tissue. The potency of the polyhedrin-negative preparations was not reduced significantly by lyophilization. The polyhedrin-negative isolate produced about 60% more infectious virus per unit of larval weight than did the wild-type isolate. The ability to produce large amounts of high-potency viral preparations in larvae and the convenience of being able to lyophilize the preparations for long-term storage shows promise for the use of preoccluded virus preparations as biopesticides.

Responses of Populations of the Tobacco Budworm (Lepidoptera: Noctuidae) from Northwest Mexico to Pyrethroids

Dose-mortality bioassays were begun in 1984 to determine the susceptibility of the tobacco budworm, Heliothts virescens (F.), to commonly used pyrethroids. Responses of different populations from the northwestern region of Mexico were compared with responses of two susceptible strains. The LD50’s indicated that the populations have been and are being selected for resistance to varying degrees. A population from Valle de Mexicali had the highest LD50 and resistance ratio for deltamethrin. The highest LD50’s for permethrin were obtained from 1987 populations from Valle del Yaqui and Valle de Mexicali (resistance ratios of 55.4 and 48.7, respectively). Resistance ratios from Valle del Yaqui for cypermethrin indicated that the 1987 tobacco budworm population was 32 times more resistant than the susceptible colony. Although control failures have not been reported from these areas, the dose-mortality data suggest that control failures may occur. Permethrin resistance appears to be increasing in Valle del Yaqui, Costa de Hermosillo, and Valle de Mexicali. Resistance to cypermethrin and cyfluthrin seems to be increasing in Valle del Yaqui and Valle de Mexicali. On the basis of LD50’s, deltamethrin resistance remains low except for Valle de Mexicali but should be carefully monitored.