Lethal and Sublethal Effects of Methyl Benzoate on the Predatory Bug Nesidiocoris tenuis.

Benzoates (naturally occurring plant toxins) produce pesticidal effects on various pest insects and mites, but their effects on non-target insects are poorly understood. In this study, we evaluate the lethal and sublethal toxicity of methyl benzoate (MB) to adults of the generalist predatory bug Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae). To assess lethal effects, N. tenuis was exposed to plant surfaces treated with 0.25%, 0.5% and 1% MB, as well as negative and positive controls (water and the neonicotinoid acetamiprid, respectively). Exposure to 1% MB resulted in the highest corrected mortality of 17.8% and 13.3% under laboratory and greenhouse conditions, respectively. Thus, 1% MB can be classified as harmless to N. tenuis according to the International Organization for Biological Control rating scheme. At the sublethal level, MB exposure did not significantly affect the consumption of eggs of the whitefly Bemisia tabaci by N. tenuis relative to negative control feeding rates. In contrast, acetamiprid at the manufacturer's recommended concentration reduced N. tenuis feeding activity by 45.4%. Furthermore, in a Y-tube olfactometer assay, there were no significant differences between the olfactory responses of N. tenuis to MB concentrations and the negative control (water). This study therefore suggests that MB could be used safely for pest control in combination with N. tenuis.

Identification of begomoviruses from different cryptic species of Bemisia tabaci in Bangladesh.

Bemisia tabaci is a global species complex consisting of at least 40 cryptic species. It is also a vector for at least 100 species of begomovirus, many of which cause severe crop damage. The relationship between begomoviruses and cryptic species of the B. tabaci species complex, however, remains unclear. Our previous study [13] was identified four cryptic species (Asia I, Asia II 1, Asia II 5, and Asia II 10) of B. tabaci from Bangladesh. Using those 110 whitefly samples, vector-based PCR analysis identified 8 different begomovirus species: BYVMV, BGYVV, OELCV, SLCCV, SLCV, TbCSV, ToLCBV, and ToLCNDV. The overall rate of virus infection was 26.4%, and BYVMV and ToLCNDV were the most frequently detected in the B. tabaci vector. Virus infection rates for Asia I, Asia II 1, Asia II 5, and Asia II 10 were 22.4% (15/67), 35% (7/20), 27.3% (6/22), and 100% (1/1), respectively. Each cryptic species infected multiple virus species, but SLCCV, TbCSV, and BGYVV were each only detected in, Asia I, Asia II 1, and Asia II 5, respectively. This study demonstrates the geographic distribution of various begomoviruses in Bangladesh and their relationships with cryptic species of B. tabaci.

Acaricidal effects of methyl benzoate against Tetranychus urticae Koch (Acari: Tetranychidae) on common crop plants.

BACKGROUND: Methyl benzoate (MB) is a small, hydrophobic organic compound isolated from the freshwater fern Salvinia molesta (Salviniales: Salviniaceae). It is used as a fragrance and flavor enhancer owing to its pleasant smell. It has also demonstrated potential as a green pesticide for various groups of insects. However, its effects on mites are yet to be studied. RESULTS: Here, we assessed the acaricidal and repellent effects of MB against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), under laboratory and greenhouse conditions. MB demonstrated concentration-dependent contact toxicity against eggs and adults of the mite. A leaf-dipping assay using 1% MB prevented the hatching of 92.7% of eggs and killed 100% of adults within 48 h of treatment. Concentration-mortality statistics were subjected to probit analysis, and the median lethal concentration (LC50 ) values for eggs and adults were 0.25% and 0.5%, respectively. Treatment with 1% MB showed the highest mortality (100%), with a median lethal time (LT50 ) estimated of 8.1 h. The efficacy of MB against adults of T. urticae on tomato plants under greenhouse conditions was 97.5% within 96 h post-treatment. Further, MB showed significant repellent activity against adult females of T. urticae, although this declined with time. Spraying with 1% MB (three times per plant) was not phytotoxic to bean, cucumber, pepper, or tomato plants. CONCLUSION: MB is highly acaricidal and repellent, but not phytotoxic, and is a promising green pesticide.

Methyl Benzoate Is Superior to Other Natural Fumigants for Controlling the Indian Meal Moth (Plodia interpunctella).

The Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), is an insect pest that commonly affects stored and postharvest agricultural products. For the control of insect pests and mites, methyl benzoate (MBe) is lethal as a fumigant and also causes contact toxicity; although it has already been established as a food-safe natural product, the fumigation toxicity of MBe has yet to be demonstrated in P. interpunctella. Herein, we evaluated MBe as a potential fumigant for controlling adults of P. interpunctella in two bioassays. Compared to the monoterpenes examined under laboratory conditions, MBe demonstrated high fumigant activity using a 1-L glass bottle at 1 µL/L air within 4 h of exposure. The median lethal concentration (LC50) of MBe was 0.1 µL/L air; the median lethal time (LT50) of MBe at 0.1, 0.3, 0.5, and 1 µL/L air was 3.8, 3.3, 2.8, and 2.0 h, respectively. Compared with commercially available monoterpene compounds used in pest control, MBe showed the highest fumigant toxicity (toxicity order as follows): MBe > citronellal > linalool > 1,8 cineole > limonene. Moreover, in a larger space assay, MBe caused 100% mortality of P. interpunctella at 0.01 µL/cm3 of air after 24 h of exposure. Therefore, MBe can be recommended for use in food security programs as an ecofriendly alternative fumigant. Specifically, it provides another management tool for curtailing the loss of stored food commodities due to P. interpunctella infestation.

Insecticidal efficacy of three benzoate derivatives against Aphis gossypii and its predator Chrysoperla carnea.

Plant-derived benzoates are known to have insecticidal and repellent activities, however, these effects have been evaluated thoroughly in only a few groups of insects. In this study, the insecticidal activities of three commercially available benzoates, methyl benzoate (MB), ethyl benzoate (EB), and vinyl benzoate (VB), were assessed against the cotton aphid, Aphis gossypii Glover, and its lacewing predator, Chrysoperla carnea Stephens. MB showed the highest contact toxicity against nymphs and adults of A. gossypii, as compared to those of EB and VB. Treatments with 1% MB, EB, and VB were 100.0%, 93.0% and 60.0% effective, respectively, against third-instar nymphs and 100.0%, 69.0%, and 39.0% effective, respectively, against adults of A. gossypii, as evaluated 24 h after application. A mixture of MB + EB showed higher efficacy than other benzoate combinations against A. gossypii. The efficacy of MB, EB, and VB against A. gossypii on cucumber plants under greenhouse conditions was 93.7%, 68.5%, and 56.6%, respectively. In addition, treatments with 1% MB, EB, and VB were 20.0%, 24.0%, and 12.0% effective, respectively, against first-instar larvae and 6.7%, 13.3%, and 6.7% effective, respectively, against adults of C. carnea at 24 h after treatment. Our results showed that the tested benzoates were less toxic to the predator C. carnea than to the pest A. gossypii. In addition, the aphid population was significantly lower in a treatment combining predation by C. carnea followed by use of 1% MB, compared with treatments of either 1% MB or predators alone. This study suggests that plant-derived benzoates are potential biopesticides for aphid control and are compatible with natural enemies in integrated pest management.

Differential responses between a vector species Bemisia tabaci and a nonvector species Trialeurodes vaporariorum following ingestion of tomato yellow leaf curl virus.

In transmitting plant viruses, insect vectors undergo physiological and behavioral alterations. The whitefly Bemisia tabaci is a vector of tomato yellow leaf curl virus (TYLCV), causing severe damages to various horticultural crop plants. To determine whether whitefly alteration is specific to vector species, the responses to TYLCV ingestion were compared between B. tabaci and Trialeurodes vaporariorum, a nonvector for TYLCV. The two species were reared on TYLCV-infected and noninfected tomato, a host of TYLCV, and their longevity and fecundity were determined while rearing in either tomato or eggplant, a nonhost of TYLCV. TYLCV-ingested B. tabaci increased their developmental rates but reduced fecundity when they were reared in either tomato or eggplant compared with those of TYLCV-free ones. In contrast, TYLCV-ingested T. vaporariorum did not show any of the aforementioned changes when reared on both plant species. In addition, TYLCV-ingested B. tabaci increased their levels of three heat shock protein genes ( hsp20, hsp70, and hsp90) against thermal stress, whereas TYLCV-ingested T. vaporariorum did not. The presence of TYLCV virions was identified in two colonies of both species via polymerase chain reaction analysis. TYLCV was detected in the whole body, saliva, and eggs of B. tabaci, while TYLCV was detected only in the whole body but not in the saliva and eggs of T. vaporariorum. The present results strongly indicated that TYLCV specifically manipulate physiological processes of the vector species, B. tabaci.

Methyl benzoate exhibits insecticidal and repellent activities against Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae).

Methyl benzoate (MB) is a plant-derived volatile organic compound with insecticidal properties, but such activity has not been evaluated against the sweetpotato whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), a major crop pest. In this study, we tested methyl benzoate control efficacy on B. tabaci infecting tomato plants in a greenhouse, specifically measuring contact and fumigant toxicity, as well as repellent activity. For direct spray applications of 0% (control), 0.1%, 0.25%, 0.5%, 1%, 2% MB onto tomato leaves infested with adults of B. tabaci (< 5-d-old), 2% MB showed the highest corrected mortality (100%) at 24 h post-treatment. For residual toxicity in which the same MB solutions were sprayed onto tomato leaves and allowed to dry for 2 h before < 5-d-old adults were released, the 2% MB also showed the highest corrected mortality (100%) at 48 h post-treatment. The lethal median concentration (LC50) for eggs, fourth-instar nymphs, and adults were 0.3%, 0.2%, and 0.2%, respectively. In pot culture experiments, 1% MB concentration was found more effective at killing nymphs and preventing adult eclosion than all other concentrations, and gave 100 percent population reduction compared with the control. MB repelled adult whiteflies and caused 96.5% fumigant toxicity within 10 h post-treatment. Repellency and anti-oviposition rates against B. tabaci had median effective doses of 0.24% and 0.16%, respectively. Our results suggest that MB has strong potential as an environmentally friendly biopesticide for control of B. tabaci but field trials and further greenhouse studies are required to establish efficacy under more natural conditions.

Tomato yellow leaf curl virus (TYLCV-IL): a seed-transmissible geminivirus in tomatoes.

Tomato yellow leaf curl virus (TYLCV) is one of the most well-known tomato-infecting begomoviruses and transmitted by Bemisia tabaci. Seed transmission has previously been reported for some RNA viruses, but TYLCV has not previously been described as a seed-borne virus. In 2013 and 2014, without whitefly-mediated transmission, TYLCV was detected in young tomato plants germinated from fallen fruits produced from TYLCV-infected tomato plants in the previous cultivation season. In addition, TYLCV-Israel (TYLCV-IL) was also detected in seeds and their seedlings of TYLCV-infected tomato plants that were infected by both viruliferous whitefly-mediated transmission and agro-inoculation. The seed infectivity was 20-100%, respectively, and the average transmission rate to seedlings was also 84.62% and 80.77%, respectively. TYLCV-tolerant tomatoes also produced TYLCV-infected seeds, but the amount of viral genome was less than seen in TYLCV-susceptible tomato plants. When tomato plants germinated from TYLCV-infected seeds, non-viruliferous whiteflies and healthy tomato plants were placed in an insect cage together, TYLCV was detected from whiteflies as well as receiver tomato plants six weeks later. Taken together, TYLCV-IL can be transmitted via seeds, and tomato plants germinated from TYLCV-infected seeds can be an inoculum source of TYLCV. This is the first report about TYLCV seed transmission in tomato.

Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella.

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in naïve insects and in insects manipulated with bacterial and hormonal treatments.

Upregulation of heat shock protein genes by envenomation of ectoparasitoid Bracon hebetor in larval host of Indian meal moth Plodia interpunctella.

Effect of envenomation of ectoparasitoid Bracon hebetor was determined on the heart rate and the expression of shsp, hsc70 and hsp90 of the lepidopteran host Plodia interpunctella. Envenomated host larvae were promptly immobilized but heart rate was not changed until 4 days after envenomation. Northern hybridization showed that each hsp gene was differentially influenced by envenomation: continued high induction of shsp, gradual strong induction of hsc70, but no induction of hsp90. Our results suggest that upregulation of both shsp and hsc70 may produce potent factors that have important roles in the mechanism of host-parasitoid relationship.

Molecular cloning of the heat-shock cognate 70 (Hsc70) gene from the two-spotted spider mite, Tetranychus urticae, and its expression in response to heat shock and starvation.

We isolated a heat shock cognate 70 (hsc70) gene from the two-spotted spider mite, Tetranychus urticae, a serious agricultural pest. The hsc70 cDNA is 2275 bp and contains a 1962 bp open reading frame. The translated amino acid sequence consists of 654 residues with a calculated molecular mass of 71,275 Da and an isoelectronic point (pI) of 5.52. It also contains the highly conserved functional motifs of the Hsp70 family. A comparison of the deduced amino acid sequence shows a high identity (81-84%) with Hsp70s/Hsc70s of insects but the highest identity is with mussel Hsc71 (86%). Northern blot hybridization indicates that the hsc70 transcript level of female adults is higher than that of male adults. We evaluated the response of hsc70 gene to stresses from temperature and starvation. The level of hsc70 mRNA was not significantly changed by heat and cold shocks nor by recovery after the shocks. However, the hsc70 mRNA level was decreased by food restriction of female mites. Analysis of nucleotide and deduced amino acid sequences of hsc70 gene from T. urticae suggests that it is a member of heat shock cognate 70 gene in the highly conserved Hsp70 family but that its expression is influenced by food restriction rather than thermal stress. This is the first molecular analysis of a heat shock protein gene in an acarid.

Elevated expression of bisecting N-acetylglucosaminyltransferase-III gene in a human fetal hepatocyte cell line by hepatitis B virus.

BACKGROUND AND AIM: UDP-N-acetylglucosamine: alpha-D-mannoside beta-1,4 N-acetylglucosaminyltransferase III (GnT-III) is a key enzyme in N-glycan biosynthesis. Human GnT-III enzyme activity was found to be elevated in the serum of patients with hepatomas and liver cirrhosis and in hepatocellular carcinoma tissues. Therefore, to understand the relationship between the elevation in GnT-III activity and hepatitis B viral (HBV) hepartocarcinogenesis, we investigated GnT-III gene expression in the HBV-infected cells. METHODS: A cell line, HFH-T1, producing HBV was produced by natural infection of human fetal hepatocytes. A 170-bp band corresponding to the pre-S1 region of HBV was detected in the culture medium by polymerase chain reaction. Virions were also isolated from the culture medium by sucrose density gradient centrifugation. The synthesis of both alpha-fetoprotein and albumin as an indicator that these cells were functional hepatocytes and the extent of differentiation was examined. Polymerase chain reaction and Western blot analysis using a monoclonal antibody, GT273, which was prepared using human aglycosyl recombinant GnT-III were used for HBV DNA and GnT-III detection. RESULTS: Two types of HBV-related particles were secreted into the culture medium; one was a Dane particle (40 nm in size) containing HBV DNA and the other was a subviral hepatitis B surface antigen particle (20 nm in size) that did not contain the viral genome. The secretion from the cell line was diminished by the number of passages and, thus, this cell was renamed as HFH-T2. A decreased level of the HBV was secreted from the cells after a rest period. HFH-T2 cells showed a weak staining for alpha-fetoprotein and a moderate staining for albumin in the cytoplasm around the nucleus. High levels of a 0.7 kb DNA fragment originating from GnT-III DNA were detected in HFH-T2 cells. Western blot analysis using a monoclonal antibody, GT273, which was prepared using human aglycosyl recombinant GnT-III showed a single band, corresponding to Mr 63 kDa, whereas aglycosyl GnT-III showed a band at Mr 53 kDa, with a molecular weight difference of about 10 kDa. This indicates that HFH-T2 cells express glycosylated GnT-III. GnT-III activities were 347.2 +/- 53.6 pmol/mg of protein/h in HFH-T2, 276 +/- 26.3 in Hep3B, 252.5 +/- 23.3 in HepG2 and 30.7 +/- 3.4 in NIH-3T3. GnT-III activity was higher in HFH-T2 cells than in the hepatoma cell lines, Hep3B and HepG2. CONCLUSION: A human fetal hepatocyte cell line was transformed by infection with HBV and the cell line expressed high levels of GnT-III as the levels of secretion of HBV decreased. The decrease in HBV secretion from HFH-T2 cells could be due to a high level of expression of GnT-III. Such a cell line could be used to investigate relationships between HBV infection and glycosyltransferase gene expression. Furthermore, this cell line will be useful in future studies on the effect of the expression of GnT-III on other glycosyltransferase.