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Effect of uneven distribution of spinetoram-treated wheat and rice on mortality and progeny production of Rhyzopertha dominica (F.), Sitophilus oryzae (L.) and Tribolium confusum Jacquelin du Val
Journal of Stored Products Research 50 (2012) 73e80
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Effect of uneven distribution of spinetoram-treated wheat and rice on mortality and progeny production of Rhyzopertha dominica (F.), Sitophilus oryzae (L.) and Tribolium confusum Jacquelin du Val Thomas N. Vassilakos, Christos G. Athanassiou* Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Plant Production and Rural Development, University of Thessaly, Phytokou Str., 38446 Nea Ionia, Magnesia, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 31 March 2012
Laboratory bioassays were carried out in order to evaluate the effectiveness of spinetoram on rice and wheat in layer and mixture treatment applications against three major stored-grain beetle species, Rhyzopertha dominica, Sitophilus oryzae and Tribolium confusum. Spinetoram was applied at 1 ppm (1 mg/ kg of grain). In the layer treatment the grain was placed in vials (8 cm high, 3 cm in diameter); in those vials there was five categories of grain: untreated (control), totally-treated, and with the upper 1/8, 1/4 and 1/2 treated. Also, there were two categories of insect introduction: before or after the grain placement. In the mixture treatment, the vials contained 20 g of grain divided into six categories: vials that contained untreated grain (control) and vials that contained 5, 10, 25, 50 and 100% treated grains. Mortality was assessed after 14 d for both treatments but with an intermediate 7-d assessment for the mixture treatment. After this interval, all adults were removed, and progeny production was measured 65 d later. From the species tested, R. dominica was by far the most susceptible in both treatments. Generally, in the layer treatment, mortality of R. dominica reduced with the size of the treated layer in the vials. Also, mortality was significantly lower when the exposed R. dominica adults had been placed before the introduction of the grain. For S. oryzae, mortality was lower in comparison with R. dominica and in general, mortality was lower on rice than on wheat, even in the case of totally-treated grains. Survival of T. confusum was considerably higher than both R. dominica and S. oryzae. In the mixture treatment, mortality of R. dominica was 82e100% after 14 d of exposure. Again mortality was considerably lower on rice than on wheat. For S. oryzae, mortality was higher in vials containing totally-treated or 50% treated kernels, in comparison with the other treatments. Progeny production of R. dominica in the layer treatment increased with the reduction of the treated layer for both commodities. In contrast, for S. oryzae, there were no differences in progeny production counts between treated and untreated grains, with the exception of totally-treated wheat. In the mixture treatment progeny production increased with the reduction of the percentage of the treated grains, but there were considerable differences between wheat and rice. The overall results suggest that spinetoram is very effective against R. dominica, moderately effective against S. oryzae, and not very effective against T. confusum. Uneven application of spinetoram may, under certain circumstances, provide satisfactory control of R. dominica. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Spinetoram Rhyzopertha dominica Sitophilus oryzae Tribolium confusum Layer treatment Mixture treatment Wheat Rice
1. Introduction One of the most important advantages of grain protectants over the use of fumigants in bulked grain is that grain protectants can provide long-term protection, even if they are used at low dose rates. For instance, Athanassiou et al. (2004) noted that low doses of
* Corresponding author. Tel.: þ30 2421093195; fax: þ30 2421093178. E-mail address: [email protected] (C.G. Athanassiou). 0022-474X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2012.03.005
beta-cyfluthrin on wheat could provide a satisfactory level of protection against the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) for several months. However, apart from treating the entire grain mass, doses can be reduced further by treating only a part of the substrate. Several researchers proposed the “topdressing” or “surface treatment”, which is the application of the insecticide only in the upper part of the bulk. For instance Athanassiou et al. (2009) noted that surface application was effective against the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) on wheat. According to Hagstrum (1987,
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1989) and Hagstrum and Flinn (1995), most insects in grain bulks occur in the upper part of the bulk; hence, “top-dressing” with a given insecticide can be utilized to suppress insect populations. At the same time, the application of a grain protectant to only the surface layer of grain is a more cost-effective solution that reduces the overall residue levels in the final product. Traditional grain protectants, such as organophosphorous compounds (OPs), and pyrethroids have been used extensively in the past for both grain and structural treatments (Arthur, 1992, 1996, 1998). Nevertheless, many OPs leave toxic residues on the product that may endanger human health, while their extensive use is dangerous for the environment (White and Leesch, 1995; Arthur, 1996). Newer insecticides, such as insect growth regulators (IGRs), can be used as successful alternatives to OPs. Athanassiou et al. (2011a) examined the effect of partially-treated grains with the IGR S-methoprene on the control of R. dominica, and found that surface treatment suppressed progeny production. One other reduced-risk insecticide that has been evaluated under the “topdressing” scenario is spinosad (Athanassiou et al., 2009). Spinosad is based on spinosyns A and D, which are metabolites of the bacterium Saccharopolyspora spinosa Mertz and Yao (Bacteria: Actinobacteridae). Spinosad is extremely toxic to R. dominica, even if only a part of the grain mass is treated (Getchell, 2006; Getchell and Subramanyam, 2008; Athanassiou et al., 2009). Other studies indicate that spinosad can be used effectively for the control of OP or IGR-resistant insect strains (Daglish, 2008), in conjunction with other reduced-risk compounds (Daglish, 2008; Athanassiou et al., 2011b). One new member of the spinosyn group is spinetoram, which has a similar mode of action to spinosad. It is based on two novel modified spinosyns, spinosyn J (major component) and spinosyn L (secondary component). Spinetoram has proved effective against a wide range of field pests, and in many cases at doses lower than those for spinosad (Williams et al., 2003; Seal et al., 2007; Sayed et al., 2010). For instance, Seal et al. (2007) found that spinetoram controlled the armyworm Spodoptera spp. (Lepidoptera: Noctuidae) larvae in considerably lower doses than spinosad, Moreover, spinetoram can be used as acaricide (El Kady et al., 2007), while spinosad has no acaricidal effect. Finally, several recent reports indicate that spinetoram is safer for some beneficial insects than spinosad (Srivastava et al., 2008). For instance, spinetoram is considerably less toxic than spinosad to the bumblebee, Bombus terrestris (L.) (Hymenoptera: Apidae) (Besard et al., 2011). Numerous studies have examined the efficacy of spinosad against a wide range of stored-product pests and conditions (Hertlein et al., 2011 and references therein). Spinosad has received registration for direct application on grains since 2005, and global registration is expected soon (Hertlein et al., 2011). However, so far there are no data available for the effectiveness of spinetoram against stored-product insects. In the present study, we examined the insecticidal effect of spinetoram under uneven distribution scenarios, against R. dominica, S. oryzae and the confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae). 2. Materials and methods
Thaibonett). The moisture content of the two commodities, as determined by a Multitest moisture meter (GODE Co., France), was 13.5%. 2.2. Spinetoram treatment e in layers The spinetoram formulation used contained 11.7% active intergradient (AI), as suspension concentrate (SC-NC) (obtained from Dow AgroSciences, UK). The application of the insecticide was performed by using a Mecafer AG4 artist’s airbrush (Mecafer Co., France). The spinetoram dose tested was 1 ppm. Lots of 2 kg of wheat and 1 kg of rice were sprayed with spinetoram using a volume rate of 1 ml of formulated dilution per kg of grain. Additional lots of wheat and rice were sprayed with distilled water and used as controls. Plastic cylindrical vials (3 cm in diameter, 8 cm in height) were used for the experiments, filled with treated and untreated commodities in layers, as described by Athanassiou et al. (2009). Briefly, each vial was filled with 28 g of wheat and 20 g of rice, which covered the bottom 6 cm of the vials. The vials were divided in five categories: vials containing untreated wheat, vials containing treated wheat, vials with only the upper half of the grain treated, vials with only the upper one fourth of the grain treated and vials with only the upper one eighth of the grain treated (Athanassiou et al., 2009). For each category, in half of the vials the adults were placed before the introduction of the grains, and in the other half the adults were placed after the introduction of the grains. Twenty adults were placed in each vial, with separate vials for each species; then, all vials were placed at 25 C, 65% r.h., and continuous darkness. After 14 d, the vials were opened and the number of dead adults was recorded. Then, for R. dominica and S. oryzae, all adults, dead or alive, were removed from the vials and the vials remained at the same conditions for an additional period of 65 d. After this interval, the vials were reopened and the number of emerged individuals was recorded. For each species-treatmentinsect introductionecommodity combination, there were 4 vials (sub-replications). The same procedure was repeated three times (replications) by preparing new vials each time (3 4 ¼ 12 vials for each combination in total). 2.3. Spinetoram mixture treatment In this series of bioassays, lots of 1 kg of wheat and 1 kg of rice were treated as noted above, and the vials were filled with mixtures of treated and untreated grains. As above, additional lots of grains were sprayed with distilled water served as controls. The vials contained 20 g of treated grain, and were divided into six categories: vials that contained untreated wheat (control), and vials that contained 5, 10, 25, 50 or 100% treated grains, corresponding to 1, 2, 5, 10 and 20 g of treated grains, respectively. After the introduction of the grains, the vials were shaken manually for approx. 1 min, to achieve equal distribution of the treated grain. Conditions, number of sub-replications and replications and number of adults used per vial were the same as in the layer series of bioassays. However, in this series, the adults were introduced only after the introduction of the grain. Mortality was assessed after 7 and 14 d of exposure, and after this interval, all adults were removed, and progeny production was measured 65 d later, as above. Only R. dominica and S. oryzae were used in this series of bioassays.
2.1. Insects and commodities 2.4. Data analysis Rhyzopertha dominica and S. oryzae were reared on whole wheat at 25 C, 65% relative humidity (r.h.) and continuous darkness, while T. confusum was reared at the same conditions on wheat flour. For all species, only adults were used in the tests. The commodities tested were hard wheat (var. Simeto) and paddy rice (var.
Control mortality was low so no correction was considered necessary. For the first series of bioassays, separately for each species, the data were submitted to a three-way Analysis of Variance (ANOVA) by using the JMP software (Sall et al., 2001), with
T.N. Vassilakos, C.G. Athanassiou / Journal of Stored Products Research 50 (2012) 73e80
75
A
mortality or progeny production as response variables, and way of insect introduction (insect positioning), commodity and treatment as main effects. For the second series of bioassays, mortality data were submitted to Repeated Measures Multiple Analysis of Variance MANOVA, with mortality as the repeated measures variable and treatment and commodity as main effects. For progeny production counts, the data were submitted to a two-way ANOVA, with treatment and commodity as main effects. In all cases, means were separated by using the TukeyeKramer HSD test, at P ¼ 0.05 (Sokal and Rohlf, 1995). 3. Results 3.1. Layer treatment 3.1.1. Mortality For mortality counts of R. dominica, insect introduction and treatment were significant, but commodity was not (Table 1). Generally, mortality of R. dominica was reduced with the reduction of the treated layer into the vials, regardless of the way of insect introduction (Fig. 1). However, mortality was significantly lower when the exposed R. dominica adults had been placed before the introduction of the grain, in comparison with the adults that had been placed after the introduction of the grain. Hence, in vials with the upper half of the grains treated, mortality of insects that had been placed before and after the wheat was 78 and 99%, respectively, while the figures for rice were 77 and 99%, respectively (Fig. 1). However, in the vials on which the upper one fourth and one eighth of the grains was treated, there were no significant differences between the two categories of insect introduction, despite the fact that, generally, mortality was higher when the insects were introduced after the grains. Thus, when insects were introduced after the grain on vials in which the upper one eighth of the grain was treated, mortality reached 80 and 77% for wheat and rice, respectively. In the case of S. oryzae, mortality was lower than for R. dominica (Fig. 2). For this species, treatment and commodity were significant, but insect introduction was not (Table 1). Generally, mortality was lower on rice in comparison with wheat, even in the case of totallytreated grains. Hence, mean mortality on totally-treated wheat was 98% while mean mortality for rice did not exceed 65% (Fig. 2). On the other hand, mean mortality on vials with only the upper one eighth of treated grains was 38 and 23%, respectively. For T. confusum, the only significant variable was commodity (Table 1). Overall adult mortality of this species was considerably lower than that for R. dominica and S. oryzae (Fig. 3). Generally, as in the case of S. oryzae, overall mortality was higher on wheat than on rice, but did not exceed 34%, even for vials with all grains treated. In addition, for vials containing rice with the upper half and the upper one eighth treated, mortality was significantly higher when the adults had been placed before the introduction of the grain (Fig. 3).
B
Fig. 1. Percentage mortality (mean SE) of Rhyzopertha dominica adults in vials containing wheat (A) or rice (B) totally-treated with spinetoram (1/1) or wheat or rice with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after in the diagram) (within each insect introduction, means followed by the same lowercase letter are not significantly different; within each ratio, means followed by the same uppercase letter are not significantly different; where no letters exist, no significant differences were noted; HSD test at 0.05).
3.1.2. Progeny production For R. dominica, only treatment was significant (Table 2). Progeny production was not completely avoided, even in the totally-treated grains. Nevertheless, the reduction of the treated layer caused a significant increase on progeny production for both commodities (Tables 3, 4). Thus, for rice, in vials with only the upper one eighth treated, progeny production did not differ from that in the control vials. Similarly, for wheat, in totally-treated wheat, adult emergence was negligible, while in the other treatments progeny production did not differ from that in the control vials. Generally, overall progeny production was higher on wheat than on rice. At the same time, in vials containing adults that had been placed before the grains, progeny was higher than in vials in which adults had been placed afterwards.
Table 1 Three-way ANOVA parameters, for treatment, insect introduction and commodity, and their interactions, for mortality of the species tested, for the layer-treated commodities (error df ¼ 176). Source
df
R. dominica
S. oryzae
T. confusum
F
P
F
P
F
P
Treatment Insect introduction Commodity Treatment Insect intro Treatment Commodity Insect intro Commodity Treatment Insect intro Commodity
3 1 1 3 3 1 3
15.62 20.11 1.49 1.94 1.08 1.08 1.20
<0.01 <0.01 0.22 0.13 0.36 0.30 0.31
33.70 0.02 35.62 0.26 1.30 0.08 0.23
<0.01 0.90 <0.01 0.86 0.28 0.78 0.88
0.93 1.92 8.22 1.33 0.44 2.10 1.14
0.43 0.17 <0.01 0.26 0.73 0.15 0.34
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A
A
B
B
Fig. 2. Percentage mortality (mean SE) of Sitophilus oryzae adults in vials containing wheat (A) or rice (B) totally-treated with spinetoram (1/1) or wheat or rice with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after in the diagram) (within each insect introduction, means followed by the same lowercase letter are not significantly different; within each ratio, means followed by the same uppercase letter are not significantly different; where no letters exist, no significant differences were noted; HSD test at 0.05).
Regarding S. oryzae, only treatment, commodity and their interaction were significant (Table 2). Progeny production was high, regardless of the treatment and the way of insect introduction. Moreover, with the exception of totally-treated wheat, progeny production did not differ from that in the control vials (Table 3). In case of rice, progeny production was lower than wheat and there were no differences between treatments or between insect introduction techniques. 3.2. Mixture treatment 3.2.1. Mortality For R. dominica, all main effects and interactions were significant (Table 5). After 7 d of exposure, mortality of R. dominica adults on wheat was extremely high, regardless of the proportion of the grain that was treated, and ranged between 97 and 99% (Fig. 4). Seven days later, in the same commodity all adults were dead. Mortality on rice was considerably lower than that on wheat (Fig. 4). After 7 d of exposure, mortality on rice contained 5% treated hulls was 57%, and differed significantly in comparison with the other treatments. At the same time, R. dominica mortality in vials containing totallytreated rice was 97%. However, after 14 d of exposure, mortality was 99e100%, with the exception of vials containing 5% treated hulls, where mortality reached 82% (Fig. 4). For S. oryzae, treatment and commodity were significant (Table 5). On wheat, after 7 d of exposure, mortality reached 68% in vials containing totally-treated wheat, while mortality was decreased with the decrease of the treated wheat proportion
Fig. 3. Percentage mortality (mean SE) of Tribolium confusum adults in vials containing wheat (A) or rice (B) totally-treated with spinetoram (1/1) or wheat or rice with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after in the diagram) (within each insect introduction, means followed by the same lowercase letter are not significantly different; within each ratio, means followed by the same uppercase letter are not significantly different; where no letters exist, no significant differences were noted; HSD test at 0.05).
(Fig. 5). At the same time, significantly more adults were dead in vials containing totally-treated and 50% treated kernels, in comparison with the other treatments. Seven days later, mortality in vials containing totally-treated wheat reached 97%, while in vials containing 5% treated wheat mortality did not exceed 50% (Fig. 5). In rice, after 7 d of exposure, mortality in vials containing totallytreated hulls reached 53%, but it was only 7% in the case of vials containing 5% treated hulls. Seven days later, mortality in vials containing 100% treated hulls was 74%, but did not exceed 41% in the case of the other treatments (Fig. 5). 3.2.2. Progeny production For R. dominica, only treatment was significant (Table 6). Progeny production on treated wheat was significantly lower than that in the control vials (Table 7). An average of less than 0.5
Table 2 Three-way ANOVA parameters for treatment, insect introduction and commodity, and their interactions for progeny production of the species tested, for the layertreated commodities (in all cases, total df ¼ 20). Source
df
Treatment Insect introduction Commodity Treatment Insect intro Treatment Commodity Insect intro Commodity Treatment Insect intro Commodity
4 1 1 4 4 1 4
R. dominica
S. oryzae
F
P
F
P
12.16 5.86 1.04 0.75 0.35 0.65 0.20
<0.01 0.02 0.31 0.56 0.84 0.42 0.94
6.47 0.02 93.75 0.14 7.25 0.13 0.10
<0.01 0.90 <0.01 0.97 <0.01 0.71 0.98
T.N. Vassilakos, C.G. Athanassiou / Journal of Stored Products Research 50 (2012) 73e80
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Table 3 Mean number of adult progeny of R. dominica and S. oryzae (number of adults per vial SE) on vials containing wheat totally-treated with spinetoram (1/1) or wheat with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after) (within each column, means followed by the same lowercase letter are not significantly different; HSD test at 0.05). Treatment ratio
Species/Progeny
1/1 1/2 1/4 1/8 Untreated
1.58 3.17 8.75 17.08 20.42
R. dominica (after)
R. dominica (before)
1.23c 0.92bc 2.16abc 3.91ab 6.26a
0.33 12.17 22.08 28.17 30.83
0.19a 4.83a 9.44a 13.61a 10.86a
R. dominica adults emerged per vial in vials containing 100 or 50% treated wheat. For rice, progeny production was higher than wheat except with totally-treated rice. Also, reducing the percentage of treated rice increased progeny production. For S. oryzae, as above, both commodity and treatment, as well as their interaction were significant (Table 6). Progeny production was high in all cases (>20 adults/vial) while significantly less adults were found in wheat totally-treated in comparison with the control. For rice, the number of emerged adults was lower than for wheat. At the same time, there were no differences in progeny production among vials containing different percentages of treated rice. 4. Discussion Our results indicate that uneven distribution of spinetoram can seriously affect its efficacy, and this trend is similar for both wheat and rice. This reduced efficacy was evident, to different levels, for both scenarios tested here: the layer-treated grains and the partially-treated grains. Uneven distribution of insecticides to grain bulks is a common phenomenon that is likely to occur even when high-precision sprayers are used. At the laboratory level, Athanassiou et al. (2011a) found that uneven distribution of the insect growth regulator (IGR) methoprene reduced its efficacy against R. dominica. For the same insecticide and species, Daglish and Nayak (2010) noted that this reduction in efficacy is affected by the dose and the proportion of the grain that is treated. Indirectly, through the regular application process, some parts of the grain mass may receive higher doses of a given insecticide than other parts, and this uneven distribution may seriously affect efficacy (Athanassiou et al., 2009; Daglish and Nayak, 2010). For spinosad, Daglish and Nayak (2006) found that after spraying, the actual residue was 12e36% less than the application rate. Similarly, Subramanyam et al. (2007) found that right after the application the actual residue of spinosad þ chlorpyriphos-methyl was approx. 30% less than the application rate. All these studies document that,
Table 4 Mean number of adult progeny of R. dominica and S. oryzae (number of adults per vial SE) on vials containing rice totally-treated with spinetoram (1/1) or rice with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after) (within each column, means followed by the same lowercase letter are not significantly different; HSD test at 0.05). Treatment ratio
Species/Progeny
1/1 1/2 1/4 1/8 Untreated
1.25 1.58 6.67 16.25 22.42
R. dominica (after)
1.25b 0.84b 2.06b 4.12ab 7.15a
R. dominica (before) 0.25 4.92 17.33 14.67 32.33
0.13b 1.41b 5.56ab 6.86ab 8.07a
S. oryzae (after) 16.67 12.17 12.50 11.92 13.50
3.04a 1.91a 2.45a 1.68a 2.02a
S. oryzae (before) 13.33 12.42 13.67 17.17 14.75
2.96a 2.36a 2.49a 2.64a 2.47a
S. oryzae (after) 14.50 64.50 61.42 50.67 70.58
S. oryzae (before)
3.07b 10.06a 11.86a 10.39ab 15.76a
12.42 55.50 58.33 50.00 75.75
3.42b 12.33ab 13.53ab 11.57ab 18.90a
at least in the case of some insecticides, the application rate is not achieved in the entire grain mass, which may cause the existence of infestation foci within the bulk. In the present study, for R. dominica mortality, we noted that when the insects were introduced before the grains, on vials containing grains with the one eighth treated layer, mortality was 54 and 73% for wheat and rice, respectively. Moreover, for this layer, there was no difference between the two ways of insect introduction on rice. Athanassiou et al. (2009) by following the same protocol with partially-treated wheat with spinosad, noted that when R. dominica adults were already in the vial before the introduction of the grain, mortality in layer-treated columns did not exceed 19%, regardless of the size of the treated layer. This indicates that spinetoram, under the layer-treatment scenario, is more effective than spinosad against R. dominica. However, our results suggest that R. dominica mortality was high even when adults were at the bottom of the column, which may indicate that there was an upwards movement. For methoprene, Daglish and Nayak (2010) also found that, under certain dose rates, uneven distribution was equally effective as treating the entire grain mass against R. dominica. Athanassiou et al. (2011a) found that partial treatment of methoprene on wheat and rice was less effective against R. dominica than application of methoprene through the entire grain mass. Progeny production was not totally avoided in all cases. Vardeman et al. (2007) reported that R. dominica adults could penetrate a layer treated with diatomaceous earth (DE) and produce progeny in the untreated portion of the grain. Athanassiou et al. (2009) suggested that there is a reduced negative geotaxis of R. dominica adults, which prevented adults that existed in the bottom layer from exposure to the treated upper part of the column. Spinetoram efficacy against S. oryzae was highly affected by the size of the treated layer, given that the increase of the size significantly increased mortality and reduced progeny production. Generally, mortality was high only on totally-treated wheat, while Table 5 Repeated measures MANOVA parameters for treatment and commodity, and their interactions for mortality of the species tested, in commodities containing different percentages of spinetoram (error df ¼ 110). Source
df
Between variables Intercept Treatment Commodity Treatment Commodity Within variables Time Time Treatment Time Commodity All three
9 1 4 1 4 9 1 4 1 4
R. dominica
S. oryzae
F
P
F
P
13.77 14,745.41 11.61 34.05 10.86 10.24 91.72 5.62 48.67 5.25
<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
16.21 548.86 21.52 53.82 1.51 4.35 256.65 1.16 30.46 1.02
<0.01 <0.01 <0.01 <0.01 0.20 <0.01 <0.01 0.33 <0.01 0.40
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A
A
B
B
Fig. 4. Percentage mortality (mean SE) of Rhyzopertha dominica adults in vials containing wheat (A) or rice (B) that contained different percentages of treated substrates, ranged between 5 and 100%, after 7 and 14 d of exposure (within each commodity and exposure, means followed by the same lowercase letter are not significantly different; where no letters exist. no significant differences were noted; HSD test at 0.05).
Fig. 5. Percentage mortality (mean SE) of Sitophilus oryzae adults in vials containing wheat (A) or rice (B) that contained different percentages of treated substrates, ranged between 5 and 100%, after 7 and 14 d of exposure (within each commodity and exposure, means followed by the same lowercase letter are not significantly different; HSD test at 0.05).
for layer-treated wheat ranged between 32 and 72%. For rice, mortality was notably lower than wheat, which suggests that spinetoram was less effective on rice than on wheat. For spinosad, Athanassiou et al. (2008) also found that efficacy was lower on rice than on wheat against S. oryzae. Our results stand in accordance with the above observations. Practically, in contrast with the influence of commodity, insect positioning had no effect on mortality, which clearly suggests that S. oryzae adults exhibit also a negative geotaxis. For adults of the granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), Surtees (1964) showed an upwards movement in wheat bulks under laboratory conditions. Overall, the presence of untreated layers affected S. oryzae mortality. Therefore, partially-treated bulks, such as bulks on which spinetoram has been applied as a “top-dressing”, may not be effective against S. oryzae. Nevertheless, in our study, we examined wheat and rice columns that were confined in a small vial, and the results obtained here may not be representative of insecticidal performance of spinosyns in grain bulks under field conditions. From the species tested here, T. confusum was by far the least susceptible to spinetoram, given that mortality did not exceed 35%. At the same time, the size of the layer and the type of commodity did not have any effect in T. confusum, as compared with the mortality levels for the totally-treated wheat and rice. Previous studies with spinosad clearly showed that T. confusum and the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) can be classified as among the most tolerant storedproduct insect species to spinosad (Fang et al., 2002; Vayias et al., 2008). Our results are in agreement with those reports. Hence, neither spinosad nor spinetoram is able to control T. confusum, at least at the adult stage, for the doses tested.
As noted above, the mixture of treated and untreated quantities of the grain is a more realistic scenario than the layer-treatment approach. Surprisingly, even the presence of 5% of spinetoramtreated kernels within the entire wheat mass was effective against R. dominica adults. Athanassiou et al. (2009) found that R. dominica adults were dead even after a short exposure to spinosad. For chlorpyriphos-methyl, Arthur (1992) found that a mixture of treated and untreated maize was, in some cases, equally effective against the maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) and T. castaneum, as compared with treating the entire grain mass. In our tests, mortality of R. dominica on mixtures of treated and untreated rice was lower than the respective quantities on wheat, especially in the case of the 7-d exposure interval. Mortality on rice after 14 d of exposure was 99e100% on rice that contained 10, 25, 50 and 100% spinetoram-treated kernels, while with only 5% treated, mortality was 82%. This fact is an additional indication that spinosyns are more effective on wheat than on rice. Thus, we can conclude that, for R. dominica, the “critical” percentage of spinetoram-treated kernels is 5 and 10% for wheat and rice,
Table 6 Two-way ANOVA parameters for treatment and commodity, and their interactions for progeny production of the species tested, in commodities containing different percentages of spinetoram (in all cases, total df ¼ 132). Source
Treatment Commodity Treatment Commodity
df
5 1 5
R. dominica
S. oryzae
F
P
F
P
16.10 5.96 1.20
<0.01 0.16 0.31
3.42 23.64 3.41
<0.01 <0.01 <0.01
T.N. Vassilakos, C.G. Athanassiou / Journal of Stored Products Research 50 (2012) 73e80 Table 7 Mean number of adult progeny of R. dominica and S. oryzae (number of adults per vial SE) on vials containing wheat or rice with different percentages of spinetoram-treated substrates, ranged between 5 and 100% (within each column, means followed by the same lowercase letter are not significantly different; HSD test at 0.05). Treatment ratio
Species/Progeny
100% 50% 25% 10% 5% Untreated
0.42 0.17 1.25 2.42 6.25 27.58
R. dominica (wheat)
0.19b 0.17b 0.39b 0.63b 2.29b 9.30a
R. dominica (rice) 0.17 1.08 2.25 10.25 23.25 39.92
0.17c 0.45c 0.84bc 3.65bc 5.70ab 10.68a
S. oryzae (wheat) 9.17 20.92 41.50 60.08 51.75 41.50
2.21b 6.48ab 15.06ab 7.49a 13.76a 8.52ab
S. oryzae (rice) 15.58 18.42 18.50 18.33 13.33 17.00
3.22a 3.38a 3.01a 3.37a 2.14a 3.05a
79
needed to investigate the mixture technique that ensures the equal distribution of the treated quantity in bulked grains. We evaluated spinetoram as a replacement of traditional grain protectants, under two uneven distribution scenarios. The overall results suggest that spinetoram is very effective against R. dominica, moderately effective against S. oryzae, and not very effective against T. confusum. At the same time, uneven application of spinetoram may, under certain circumstances, provide a satisfactory level of control against R. dominica. Nevertheless, one of the main drawbacks of uneven distribution is the possibility of indirect selection for resistance, and this is why uneven distribution should be evaluated further in this respect (Daglish and Nayak, 2010). Acknowledgements
respectively. It should be noted that differences in parental mortality were not expressed as differences in the subsequent progeny production counts. During the mortality counts, we observed that most of the exposed R. dominica adults were immobilized, which is likely to be the main reason for the reduced reproduction. The results of the current experiment show that even a minimal contact of R. dominica with spinetoram, can cause extremely high parental mortality. In comparison with the layer-treatment approach, the treated quantities here were lower. Thus, we estimate that, against R. dominica, the mixture of spinetoram-treated quantities of grains are much more effective than the arrangement of these quantities in layers. Arthur (1992) also concluded that the mixture of chlorpyriphos-methyl-treated and untreated maize quantities was preferable than using layers treated with chlorpyriphos-methyl, against S. zeamais and T. castaneum. Overall, treated proportions of 5e10% of the entire grain mass should be considered extremely low, as compared with other insecticides. For instance, Arthur (1992) noted that a 2:3 ratio of chlorpyriphos-methyl-treated and untreated maize provided long-term protection against S. zeamais and T. castaneum. Athanassiou et al. (2011a) for S-methoprene on wheat and rice, stated that the presence of treated and untreated quantities was less effective against R. dominica, in comparison with the application on the entire grain mass. Also, Daglish and Nayak (2010) found that when low doses of methoprene were applied on wheat, progeny production of R. dominica was higher on vials that contained a 1:1 treated and untreated ratio as compared with totally-treated wheat. For S. oryzae, on wheat, only some of the mixtures were as effective as the totally-treated wheat. Taken into account both exposure intervals, we can conclude that the “critical” percentage is 50%, which is much higher than the 5% proposed for R. dominica. However, even the lowest (5%) amount of treated kernels killed approx. 50% of the exposed S. oryzae adults on wheat, after 14 d of exposure. This indicates that this minimal presence of spinetoram on wheat had some lethal effects on parental S. oryzae individuals. Mortality on rice was considerably lower than that on wheat and, based on the results, 50% can be again considered as the “critical” level, given that this was the only mixture treatment that was comparable with totally-treated rice. On the other hand, for wheat, the decrease of the percentage of treated kernels significantly increased progeny production, which means that S. oryzae can continue grain damage in partially-treated grains. It should be noted that, the mixture of treated and untreated small grain quantities under laboratory conditions can achieve equal distribution of the treated substrate in the entire grain sample, but this equal distribution may not be practically feasible in the case of large grain bulks in the field. As a result, under field conditions, the untreated substrates may be large enough to allow rapid colonization and the continuance of grain damage. Further work is
We would like to thank Dow Agrosciences for providing the spinetoram formulations for experimentation. References Arthur, F.H., 1992. Efficacy of chlorpyriphos-methyl for control of maize weevils (Coleoptera: Curculionidae) and red flour beetles (Tenebrionidae) in mixtures of treated and untreated corn. Journal of Economic Entomology 85, 554e560. Arthur, F.H., 1996. Grain protectants: current status and prospects for the future. Journal of Stored Products Research 32, 293e303. Arthur, F.H., 1998. Residual studies with cyfluthrin wettable powder: toxicity toward red flour beetles (Coleoptera: Tenebrionidae) exposed for short intervals on treated concrete. Journal of Economic Entomology 91, 309e319. Athanassiou, C.G., Kavallieratos, N.G., Vayias, B.J., Dimizas, C.B., Papagregoriou, A.S., C.Th, Buchelos, 2004. Residual toxicity of beta cyfluthrin, alpha cypermethrin and deltamethrin against Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) on stored wheat. Applied Entomology and Zoology 39, 195e202. Athanassiou, C.G., Kavallieratos, N.G., Chintzoglou, G.J., Peteinatos, G.G., Boukouvala, M.C., Petrou, S.S., Panoussakis, E.C., 2008. Effect of temperature and grain on insecticidal efficacy of spinosad dust against Sitophilus oryzae (Coleoptera: Curculionidae) and Rhyzopertha dominica (Coleoptera: Bostrichidae). Journal of Economic Entomology 101, 976e981. Athanassiou, C.G., Arthur, F.H., Throne, J.E., 2009. Efficacy of spinosad in layertreated wheat against five stored-product insect species. Journal of Stored Products Research 45, 236e240. Athanassiou, C.G., Arthur, F.H., Throne, J.E., 2011a. Efficacy of layer treatment with methoprene for control of Rhyzopertha dominica (Coleoptera: Bostrychidae) on wheat, rice, and maize. Pest Management Science 67, 380e384. Athanassiou, C.G., Arthur, F.H., Kavallieratos, N.G., Throne, J.E., 2011b. Efficacy of spinosad and methoprene, applied alone or in combination, against six storedproduct insect species. Journal of Pest Science 84, 61e67. Besard, L., Mommaerts, V., Abdu-Alla, G., Smagghe, G., 2011. Lethal and sublethal side-effect assessment supports a more benign profile of spinetoram compared with spinosad in the bumblebee Bombus terrestris. Pest Management Science 67, 541e547. Daglish, G.J., 2008. Impact of resistance on the efficacy of binary combinations of spinosad, chlorpyriphos-methyl and s-methoprene against five stored-grain beetles. Journal of Stored Products Research 44, 71e76. Daglish, G.J., Nayak, M.K., 2006. Long-term persistence and efficacy of spinosad against Rhyzopertha dominica (Coleoptera: Bostrychidae) in wheat. Pest Management Science 62, 148e152. Daglish, G.J., Nayak, M.K., 2010. Uneven application can influence the efficacy of smethoprene against Rhyzopertha dominica (F.) in wheat. Journal of Stored Products Research 46, 250e253. El Kady, G.A., El Sharabasy, H.M., Mahmoud, M.F., Bahgat, I.M., 2007. Toxicity of two potential bio-insecticides against moveable stages of Tetranychus urticae Koch. Journal of Applied Science Research 3, 1315e1319. Fang, L., Subramanyam, B., Arthur, F.H., 2002. Effectiveness of spinosad on four classes of wheat against five stored-product insects. Journal of Economic Entomology 95, 640e650. Getchell, A.I., 2006. Efficacy of Two Spinosad Formulations on Various Commodities against Stored-product Insects. M.Sc. thesis. Department of Grain Science and Industry, College of Agriculture, Kansas State University, Manhattan, Kansas, 76 pp. Getchell, A.I., Subramanyam, B., 2008. Immediate and delayed mortality of Rhyzopertha dominica (Coleoptera: Bostrychidae) and Sitophilus oryzae (Coleoptera: Curculionidae) adults exposed to spinosad-treated grain. Journal of Economic Entomology 101, 1022e1027. Hagstrum, D.W., 1987. Seasonal variation of stored wheat environment and insect populations. Environmental Entomology 16, 77e83. Hagstrum, D.W., 1989. Infestation by Cryptolestes ferrugineus (Coleoptera: Cucujidae) of newly harvested wheat stored on three Kansas farms. Journal of Economic Entomology 82, 655e659.
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Hagstrum, D.W., Flinn, P.W., 1995. Integrated pest management. In: Subramanyam, B., Hagstrum, D.W. (Eds.), Integrated Management of Insects in Stored Products. Marcel Dekker, New York, pp. 399e408. Hertlein, M.B., Thompson, G.D., Subramanyam, B., Athanassiou, C.G., 2011. Spinosad: a new natural product for stored grain protection. Journal of Stored Products Research 47, 131e146. Sall, J., Lehman, A., Creighton, L., 2001. JMP Start Statistics. A Guide to Statistics and Data Analysis Using JMP and JMP IN Software. Duxbury Press, Belmont, CA, 491 pp. Sayed, A.A., Temerak, S.A., Lysandrou, M., 2010. The use of different insect control regimes using three green chemicals to combat Vericola livia on date pulm fruit in Egypt. Acta Horticulture (ISHS) 882, 471e479. Seal, D.R., Schuster, D.J., Klassen, W., 2007. Comparative effectiveness of new insecticides in controlling armyworms (Lepidoptera: Noctuidae) and leafminers (Diptera: Agromyzidae) on tomato. Proceedings of the Florida State Horticultural Society 120, 170e177. Sokal, R.R., Rohlf, F.J., 1995. Biometry, third ed. Freeman, New York. Srivastava, M., Bosco, L., Funderburk, J., Olson, S., Weiss, A., 2008. Spinetoram is compatible with the key natural enemy of Frankliniella species thrips in pepper. Online. Plant Health Progress. doi:10.1094/PHP-2008-0118-02-RS.
Subramanyam, B., Toews, M.D., Ileleji, K.E., Maier, D.E., Thompson, G.D., Pitts, T.J., 2007. Evaluation of spinosad as a grain protectant on three Kansas farms. Crop Protection 26, 1021e1030. Surtees, G., 1964. Laboratory studies on dispersion behaviour of adult beetles in grain. VI. Three-dimensional analysis of dispersion of five species in a uniform bulk. Bulletin of Entomological Research 55, 723e725. Vardeman, E.A., Arthur, F.A., Nechols, J.R., Cambell, J.F., 2007. Efficacy of surface application with diatomaceous earth to control Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) in stored wheat. Journal of Stored Products Research 43, 333e341. Vayias, B.J., Athanassiou, C.G., Buchelos, C.Th., 2008. Evaluation of resistance development by Tribolium confusum Du Val (Coleoptera: Tenebrionidae) to diatomaceous earth under laboratory selection. Journal of Stored Products Research 44, 162e168. White, N.D.G., Leesch, J.G., 1995. Chemical control. In: Subramanyam, B., Hagstrum, D.W. (Eds.), Integrated Management of Insects in Stored Products. Marcel Dekker, New York, pp. 287e330. Williams, T., Valle, J., Viñuela, E., 2003. Is the naturally-derived insecticide spinosad compatible with insect natural enemies? Biocontrol Science and Technology 13, 459e475.
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Effect of uneven distribution of spinetoram-treated wheat and rice on mortality and progeny production of Rhyzopertha dominica (F.), Sitophilus oryzae (L.) and Tribolium confusum Jacquelin du Val Thomas N. Vassilakos, Christos G. Athanassiou* Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Plant Production and Rural Development, University of Thessaly, Phytokou Str., 38446 Nea Ionia, Magnesia, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 31 March 2012
Laboratory bioassays were carried out in order to evaluate the effectiveness of spinetoram on rice and wheat in layer and mixture treatment applications against three major stored-grain beetle species, Rhyzopertha dominica, Sitophilus oryzae and Tribolium confusum. Spinetoram was applied at 1 ppm (1 mg/ kg of grain). In the layer treatment the grain was placed in vials (8 cm high, 3 cm in diameter); in those vials there was five categories of grain: untreated (control), totally-treated, and with the upper 1/8, 1/4 and 1/2 treated. Also, there were two categories of insect introduction: before or after the grain placement. In the mixture treatment, the vials contained 20 g of grain divided into six categories: vials that contained untreated grain (control) and vials that contained 5, 10, 25, 50 and 100% treated grains. Mortality was assessed after 14 d for both treatments but with an intermediate 7-d assessment for the mixture treatment. After this interval, all adults were removed, and progeny production was measured 65 d later. From the species tested, R. dominica was by far the most susceptible in both treatments. Generally, in the layer treatment, mortality of R. dominica reduced with the size of the treated layer in the vials. Also, mortality was significantly lower when the exposed R. dominica adults had been placed before the introduction of the grain. For S. oryzae, mortality was lower in comparison with R. dominica and in general, mortality was lower on rice than on wheat, even in the case of totally-treated grains. Survival of T. confusum was considerably higher than both R. dominica and S. oryzae. In the mixture treatment, mortality of R. dominica was 82e100% after 14 d of exposure. Again mortality was considerably lower on rice than on wheat. For S. oryzae, mortality was higher in vials containing totally-treated or 50% treated kernels, in comparison with the other treatments. Progeny production of R. dominica in the layer treatment increased with the reduction of the treated layer for both commodities. In contrast, for S. oryzae, there were no differences in progeny production counts between treated and untreated grains, with the exception of totally-treated wheat. In the mixture treatment progeny production increased with the reduction of the percentage of the treated grains, but there were considerable differences between wheat and rice. The overall results suggest that spinetoram is very effective against R. dominica, moderately effective against S. oryzae, and not very effective against T. confusum. Uneven application of spinetoram may, under certain circumstances, provide satisfactory control of R. dominica. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Spinetoram Rhyzopertha dominica Sitophilus oryzae Tribolium confusum Layer treatment Mixture treatment Wheat Rice
1. Introduction One of the most important advantages of grain protectants over the use of fumigants in bulked grain is that grain protectants can provide long-term protection, even if they are used at low dose rates. For instance, Athanassiou et al. (2004) noted that low doses of
* Corresponding author. Tel.: þ30 2421093195; fax: þ30 2421093178. E-mail address: [email protected] (C.G. Athanassiou). 0022-474X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2012.03.005
beta-cyfluthrin on wheat could provide a satisfactory level of protection against the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) for several months. However, apart from treating the entire grain mass, doses can be reduced further by treating only a part of the substrate. Several researchers proposed the “topdressing” or “surface treatment”, which is the application of the insecticide only in the upper part of the bulk. For instance Athanassiou et al. (2009) noted that surface application was effective against the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) on wheat. According to Hagstrum (1987,
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1989) and Hagstrum and Flinn (1995), most insects in grain bulks occur in the upper part of the bulk; hence, “top-dressing” with a given insecticide can be utilized to suppress insect populations. At the same time, the application of a grain protectant to only the surface layer of grain is a more cost-effective solution that reduces the overall residue levels in the final product. Traditional grain protectants, such as organophosphorous compounds (OPs), and pyrethroids have been used extensively in the past for both grain and structural treatments (Arthur, 1992, 1996, 1998). Nevertheless, many OPs leave toxic residues on the product that may endanger human health, while their extensive use is dangerous for the environment (White and Leesch, 1995; Arthur, 1996). Newer insecticides, such as insect growth regulators (IGRs), can be used as successful alternatives to OPs. Athanassiou et al. (2011a) examined the effect of partially-treated grains with the IGR S-methoprene on the control of R. dominica, and found that surface treatment suppressed progeny production. One other reduced-risk insecticide that has been evaluated under the “topdressing” scenario is spinosad (Athanassiou et al., 2009). Spinosad is based on spinosyns A and D, which are metabolites of the bacterium Saccharopolyspora spinosa Mertz and Yao (Bacteria: Actinobacteridae). Spinosad is extremely toxic to R. dominica, even if only a part of the grain mass is treated (Getchell, 2006; Getchell and Subramanyam, 2008; Athanassiou et al., 2009). Other studies indicate that spinosad can be used effectively for the control of OP or IGR-resistant insect strains (Daglish, 2008), in conjunction with other reduced-risk compounds (Daglish, 2008; Athanassiou et al., 2011b). One new member of the spinosyn group is spinetoram, which has a similar mode of action to spinosad. It is based on two novel modified spinosyns, spinosyn J (major component) and spinosyn L (secondary component). Spinetoram has proved effective against a wide range of field pests, and in many cases at doses lower than those for spinosad (Williams et al., 2003; Seal et al., 2007; Sayed et al., 2010). For instance, Seal et al. (2007) found that spinetoram controlled the armyworm Spodoptera spp. (Lepidoptera: Noctuidae) larvae in considerably lower doses than spinosad, Moreover, spinetoram can be used as acaricide (El Kady et al., 2007), while spinosad has no acaricidal effect. Finally, several recent reports indicate that spinetoram is safer for some beneficial insects than spinosad (Srivastava et al., 2008). For instance, spinetoram is considerably less toxic than spinosad to the bumblebee, Bombus terrestris (L.) (Hymenoptera: Apidae) (Besard et al., 2011). Numerous studies have examined the efficacy of spinosad against a wide range of stored-product pests and conditions (Hertlein et al., 2011 and references therein). Spinosad has received registration for direct application on grains since 2005, and global registration is expected soon (Hertlein et al., 2011). However, so far there are no data available for the effectiveness of spinetoram against stored-product insects. In the present study, we examined the insecticidal effect of spinetoram under uneven distribution scenarios, against R. dominica, S. oryzae and the confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae). 2. Materials and methods
Thaibonett). The moisture content of the two commodities, as determined by a Multitest moisture meter (GODE Co., France), was 13.5%. 2.2. Spinetoram treatment e in layers The spinetoram formulation used contained 11.7% active intergradient (AI), as suspension concentrate (SC-NC) (obtained from Dow AgroSciences, UK). The application of the insecticide was performed by using a Mecafer AG4 artist’s airbrush (Mecafer Co., France). The spinetoram dose tested was 1 ppm. Lots of 2 kg of wheat and 1 kg of rice were sprayed with spinetoram using a volume rate of 1 ml of formulated dilution per kg of grain. Additional lots of wheat and rice were sprayed with distilled water and used as controls. Plastic cylindrical vials (3 cm in diameter, 8 cm in height) were used for the experiments, filled with treated and untreated commodities in layers, as described by Athanassiou et al. (2009). Briefly, each vial was filled with 28 g of wheat and 20 g of rice, which covered the bottom 6 cm of the vials. The vials were divided in five categories: vials containing untreated wheat, vials containing treated wheat, vials with only the upper half of the grain treated, vials with only the upper one fourth of the grain treated and vials with only the upper one eighth of the grain treated (Athanassiou et al., 2009). For each category, in half of the vials the adults were placed before the introduction of the grains, and in the other half the adults were placed after the introduction of the grains. Twenty adults were placed in each vial, with separate vials for each species; then, all vials were placed at 25 C, 65% r.h., and continuous darkness. After 14 d, the vials were opened and the number of dead adults was recorded. Then, for R. dominica and S. oryzae, all adults, dead or alive, were removed from the vials and the vials remained at the same conditions for an additional period of 65 d. After this interval, the vials were reopened and the number of emerged individuals was recorded. For each species-treatmentinsect introductionecommodity combination, there were 4 vials (sub-replications). The same procedure was repeated three times (replications) by preparing new vials each time (3 4 ¼ 12 vials for each combination in total). 2.3. Spinetoram mixture treatment In this series of bioassays, lots of 1 kg of wheat and 1 kg of rice were treated as noted above, and the vials were filled with mixtures of treated and untreated grains. As above, additional lots of grains were sprayed with distilled water served as controls. The vials contained 20 g of treated grain, and were divided into six categories: vials that contained untreated wheat (control), and vials that contained 5, 10, 25, 50 or 100% treated grains, corresponding to 1, 2, 5, 10 and 20 g of treated grains, respectively. After the introduction of the grains, the vials were shaken manually for approx. 1 min, to achieve equal distribution of the treated grain. Conditions, number of sub-replications and replications and number of adults used per vial were the same as in the layer series of bioassays. However, in this series, the adults were introduced only after the introduction of the grain. Mortality was assessed after 7 and 14 d of exposure, and after this interval, all adults were removed, and progeny production was measured 65 d later, as above. Only R. dominica and S. oryzae were used in this series of bioassays.
2.1. Insects and commodities 2.4. Data analysis Rhyzopertha dominica and S. oryzae were reared on whole wheat at 25 C, 65% relative humidity (r.h.) and continuous darkness, while T. confusum was reared at the same conditions on wheat flour. For all species, only adults were used in the tests. The commodities tested were hard wheat (var. Simeto) and paddy rice (var.
Control mortality was low so no correction was considered necessary. For the first series of bioassays, separately for each species, the data were submitted to a three-way Analysis of Variance (ANOVA) by using the JMP software (Sall et al., 2001), with
T.N. Vassilakos, C.G. Athanassiou / Journal of Stored Products Research 50 (2012) 73e80
75
A
mortality or progeny production as response variables, and way of insect introduction (insect positioning), commodity and treatment as main effects. For the second series of bioassays, mortality data were submitted to Repeated Measures Multiple Analysis of Variance MANOVA, with mortality as the repeated measures variable and treatment and commodity as main effects. For progeny production counts, the data were submitted to a two-way ANOVA, with treatment and commodity as main effects. In all cases, means were separated by using the TukeyeKramer HSD test, at P ¼ 0.05 (Sokal and Rohlf, 1995). 3. Results 3.1. Layer treatment 3.1.1. Mortality For mortality counts of R. dominica, insect introduction and treatment were significant, but commodity was not (Table 1). Generally, mortality of R. dominica was reduced with the reduction of the treated layer into the vials, regardless of the way of insect introduction (Fig. 1). However, mortality was significantly lower when the exposed R. dominica adults had been placed before the introduction of the grain, in comparison with the adults that had been placed after the introduction of the grain. Hence, in vials with the upper half of the grains treated, mortality of insects that had been placed before and after the wheat was 78 and 99%, respectively, while the figures for rice were 77 and 99%, respectively (Fig. 1). However, in the vials on which the upper one fourth and one eighth of the grains was treated, there were no significant differences between the two categories of insect introduction, despite the fact that, generally, mortality was higher when the insects were introduced after the grains. Thus, when insects were introduced after the grain on vials in which the upper one eighth of the grain was treated, mortality reached 80 and 77% for wheat and rice, respectively. In the case of S. oryzae, mortality was lower than for R. dominica (Fig. 2). For this species, treatment and commodity were significant, but insect introduction was not (Table 1). Generally, mortality was lower on rice in comparison with wheat, even in the case of totallytreated grains. Hence, mean mortality on totally-treated wheat was 98% while mean mortality for rice did not exceed 65% (Fig. 2). On the other hand, mean mortality on vials with only the upper one eighth of treated grains was 38 and 23%, respectively. For T. confusum, the only significant variable was commodity (Table 1). Overall adult mortality of this species was considerably lower than that for R. dominica and S. oryzae (Fig. 3). Generally, as in the case of S. oryzae, overall mortality was higher on wheat than on rice, but did not exceed 34%, even for vials with all grains treated. In addition, for vials containing rice with the upper half and the upper one eighth treated, mortality was significantly higher when the adults had been placed before the introduction of the grain (Fig. 3).
B
Fig. 1. Percentage mortality (mean SE) of Rhyzopertha dominica adults in vials containing wheat (A) or rice (B) totally-treated with spinetoram (1/1) or wheat or rice with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after in the diagram) (within each insect introduction, means followed by the same lowercase letter are not significantly different; within each ratio, means followed by the same uppercase letter are not significantly different; where no letters exist, no significant differences were noted; HSD test at 0.05).
3.1.2. Progeny production For R. dominica, only treatment was significant (Table 2). Progeny production was not completely avoided, even in the totally-treated grains. Nevertheless, the reduction of the treated layer caused a significant increase on progeny production for both commodities (Tables 3, 4). Thus, for rice, in vials with only the upper one eighth treated, progeny production did not differ from that in the control vials. Similarly, for wheat, in totally-treated wheat, adult emergence was negligible, while in the other treatments progeny production did not differ from that in the control vials. Generally, overall progeny production was higher on wheat than on rice. At the same time, in vials containing adults that had been placed before the grains, progeny was higher than in vials in which adults had been placed afterwards.
Table 1 Three-way ANOVA parameters, for treatment, insect introduction and commodity, and their interactions, for mortality of the species tested, for the layer-treated commodities (error df ¼ 176). Source
df
R. dominica
S. oryzae
T. confusum
F
P
F
P
F
P
Treatment Insect introduction Commodity Treatment Insect intro Treatment Commodity Insect intro Commodity Treatment Insect intro Commodity
3 1 1 3 3 1 3
15.62 20.11 1.49 1.94 1.08 1.08 1.20
<0.01 <0.01 0.22 0.13 0.36 0.30 0.31
33.70 0.02 35.62 0.26 1.30 0.08 0.23
<0.01 0.90 <0.01 0.86 0.28 0.78 0.88
0.93 1.92 8.22 1.33 0.44 2.10 1.14
0.43 0.17 <0.01 0.26 0.73 0.15 0.34
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T.N. Vassilakos, C.G. Athanassiou / Journal of Stored Products Research 50 (2012) 73e80
A
A
B
B
Fig. 2. Percentage mortality (mean SE) of Sitophilus oryzae adults in vials containing wheat (A) or rice (B) totally-treated with spinetoram (1/1) or wheat or rice with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after in the diagram) (within each insect introduction, means followed by the same lowercase letter are not significantly different; within each ratio, means followed by the same uppercase letter are not significantly different; where no letters exist, no significant differences were noted; HSD test at 0.05).
Regarding S. oryzae, only treatment, commodity and their interaction were significant (Table 2). Progeny production was high, regardless of the treatment and the way of insect introduction. Moreover, with the exception of totally-treated wheat, progeny production did not differ from that in the control vials (Table 3). In case of rice, progeny production was lower than wheat and there were no differences between treatments or between insect introduction techniques. 3.2. Mixture treatment 3.2.1. Mortality For R. dominica, all main effects and interactions were significant (Table 5). After 7 d of exposure, mortality of R. dominica adults on wheat was extremely high, regardless of the proportion of the grain that was treated, and ranged between 97 and 99% (Fig. 4). Seven days later, in the same commodity all adults were dead. Mortality on rice was considerably lower than that on wheat (Fig. 4). After 7 d of exposure, mortality on rice contained 5% treated hulls was 57%, and differed significantly in comparison with the other treatments. At the same time, R. dominica mortality in vials containing totallytreated rice was 97%. However, after 14 d of exposure, mortality was 99e100%, with the exception of vials containing 5% treated hulls, where mortality reached 82% (Fig. 4). For S. oryzae, treatment and commodity were significant (Table 5). On wheat, after 7 d of exposure, mortality reached 68% in vials containing totally-treated wheat, while mortality was decreased with the decrease of the treated wheat proportion
Fig. 3. Percentage mortality (mean SE) of Tribolium confusum adults in vials containing wheat (A) or rice (B) totally-treated with spinetoram (1/1) or wheat or rice with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after in the diagram) (within each insect introduction, means followed by the same lowercase letter are not significantly different; within each ratio, means followed by the same uppercase letter are not significantly different; where no letters exist, no significant differences were noted; HSD test at 0.05).
(Fig. 5). At the same time, significantly more adults were dead in vials containing totally-treated and 50% treated kernels, in comparison with the other treatments. Seven days later, mortality in vials containing totally-treated wheat reached 97%, while in vials containing 5% treated wheat mortality did not exceed 50% (Fig. 5). In rice, after 7 d of exposure, mortality in vials containing totallytreated hulls reached 53%, but it was only 7% in the case of vials containing 5% treated hulls. Seven days later, mortality in vials containing 100% treated hulls was 74%, but did not exceed 41% in the case of the other treatments (Fig. 5). 3.2.2. Progeny production For R. dominica, only treatment was significant (Table 6). Progeny production on treated wheat was significantly lower than that in the control vials (Table 7). An average of less than 0.5
Table 2 Three-way ANOVA parameters for treatment, insect introduction and commodity, and their interactions for progeny production of the species tested, for the layertreated commodities (in all cases, total df ¼ 20). Source
df
Treatment Insect introduction Commodity Treatment Insect intro Treatment Commodity Insect intro Commodity Treatment Insect intro Commodity
4 1 1 4 4 1 4
R. dominica
S. oryzae
F
P
F
P
12.16 5.86 1.04 0.75 0.35 0.65 0.20
<0.01 0.02 0.31 0.56 0.84 0.42 0.94
6.47 0.02 93.75 0.14 7.25 0.13 0.10
<0.01 0.90 <0.01 0.97 <0.01 0.71 0.98
T.N. Vassilakos, C.G. Athanassiou / Journal of Stored Products Research 50 (2012) 73e80
77
Table 3 Mean number of adult progeny of R. dominica and S. oryzae (number of adults per vial SE) on vials containing wheat totally-treated with spinetoram (1/1) or wheat with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after) (within each column, means followed by the same lowercase letter are not significantly different; HSD test at 0.05). Treatment ratio
Species/Progeny
1/1 1/2 1/4 1/8 Untreated
1.58 3.17 8.75 17.08 20.42
R. dominica (after)
R. dominica (before)
1.23c 0.92bc 2.16abc 3.91ab 6.26a
0.33 12.17 22.08 28.17 30.83
0.19a 4.83a 9.44a 13.61a 10.86a
R. dominica adults emerged per vial in vials containing 100 or 50% treated wheat. For rice, progeny production was higher than wheat except with totally-treated rice. Also, reducing the percentage of treated rice increased progeny production. For S. oryzae, as above, both commodity and treatment, as well as their interaction were significant (Table 6). Progeny production was high in all cases (>20 adults/vial) while significantly less adults were found in wheat totally-treated in comparison with the control. For rice, the number of emerged adults was lower than for wheat. At the same time, there were no differences in progeny production among vials containing different percentages of treated rice. 4. Discussion Our results indicate that uneven distribution of spinetoram can seriously affect its efficacy, and this trend is similar for both wheat and rice. This reduced efficacy was evident, to different levels, for both scenarios tested here: the layer-treated grains and the partially-treated grains. Uneven distribution of insecticides to grain bulks is a common phenomenon that is likely to occur even when high-precision sprayers are used. At the laboratory level, Athanassiou et al. (2011a) found that uneven distribution of the insect growth regulator (IGR) methoprene reduced its efficacy against R. dominica. For the same insecticide and species, Daglish and Nayak (2010) noted that this reduction in efficacy is affected by the dose and the proportion of the grain that is treated. Indirectly, through the regular application process, some parts of the grain mass may receive higher doses of a given insecticide than other parts, and this uneven distribution may seriously affect efficacy (Athanassiou et al., 2009; Daglish and Nayak, 2010). For spinosad, Daglish and Nayak (2006) found that after spraying, the actual residue was 12e36% less than the application rate. Similarly, Subramanyam et al. (2007) found that right after the application the actual residue of spinosad þ chlorpyriphos-methyl was approx. 30% less than the application rate. All these studies document that,
Table 4 Mean number of adult progeny of R. dominica and S. oryzae (number of adults per vial SE) on vials containing rice totally-treated with spinetoram (1/1) or rice with the upper half (1/2), one fourth (1/4), or one eighth (1/8) layer treated with spinetoram. Adults were introduced in the vials before or after the grain (before vs after) (within each column, means followed by the same lowercase letter are not significantly different; HSD test at 0.05). Treatment ratio
Species/Progeny
1/1 1/2 1/4 1/8 Untreated
1.25 1.58 6.67 16.25 22.42
R. dominica (after)
1.25b 0.84b 2.06b 4.12ab 7.15a
R. dominica (before) 0.25 4.92 17.33 14.67 32.33
0.13b 1.41b 5.56ab 6.86ab 8.07a
S. oryzae (after) 16.67 12.17 12.50 11.92 13.50
3.04a 1.91a 2.45a 1.68a 2.02a
S. oryzae (before) 13.33 12.42 13.67 17.17 14.75
2.96a 2.36a 2.49a 2.64a 2.47a
S. oryzae (after) 14.50 64.50 61.42 50.67 70.58
S. oryzae (before)
3.07b 10.06a 11.86a 10.39ab 15.76a
12.42 55.50 58.33 50.00 75.75
3.42b 12.33ab 13.53ab 11.57ab 18.90a
at least in the case of some insecticides, the application rate is not achieved in the entire grain mass, which may cause the existence of infestation foci within the bulk. In the present study, for R. dominica mortality, we noted that when the insects were introduced before the grains, on vials containing grains with the one eighth treated layer, mortality was 54 and 73% for wheat and rice, respectively. Moreover, for this layer, there was no difference between the two ways of insect introduction on rice. Athanassiou et al. (2009) by following the same protocol with partially-treated wheat with spinosad, noted that when R. dominica adults were already in the vial before the introduction of the grain, mortality in layer-treated columns did not exceed 19%, regardless of the size of the treated layer. This indicates that spinetoram, under the layer-treatment scenario, is more effective than spinosad against R. dominica. However, our results suggest that R. dominica mortality was high even when adults were at the bottom of the column, which may indicate that there was an upwards movement. For methoprene, Daglish and Nayak (2010) also found that, under certain dose rates, uneven distribution was equally effective as treating the entire grain mass against R. dominica. Athanassiou et al. (2011a) found that partial treatment of methoprene on wheat and rice was less effective against R. dominica than application of methoprene through the entire grain mass. Progeny production was not totally avoided in all cases. Vardeman et al. (2007) reported that R. dominica adults could penetrate a layer treated with diatomaceous earth (DE) and produce progeny in the untreated portion of the grain. Athanassiou et al. (2009) suggested that there is a reduced negative geotaxis of R. dominica adults, which prevented adults that existed in the bottom layer from exposure to the treated upper part of the column. Spinetoram efficacy against S. oryzae was highly affected by the size of the treated layer, given that the increase of the size significantly increased mortality and reduced progeny production. Generally, mortality was high only on totally-treated wheat, while Table 5 Repeated measures MANOVA parameters for treatment and commodity, and their interactions for mortality of the species tested, in commodities containing different percentages of spinetoram (error df ¼ 110). Source
df
Between variables Intercept Treatment Commodity Treatment Commodity Within variables Time Time Treatment Time Commodity All three
9 1 4 1 4 9 1 4 1 4
R. dominica
S. oryzae
F
P
F
P
13.77 14,745.41 11.61 34.05 10.86 10.24 91.72 5.62 48.67 5.25
<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
16.21 548.86 21.52 53.82 1.51 4.35 256.65 1.16 30.46 1.02
<0.01 <0.01 <0.01 <0.01 0.20 <0.01 <0.01 0.33 <0.01 0.40
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A
A
B
B
Fig. 4. Percentage mortality (mean SE) of Rhyzopertha dominica adults in vials containing wheat (A) or rice (B) that contained different percentages of treated substrates, ranged between 5 and 100%, after 7 and 14 d of exposure (within each commodity and exposure, means followed by the same lowercase letter are not significantly different; where no letters exist. no significant differences were noted; HSD test at 0.05).
Fig. 5. Percentage mortality (mean SE) of Sitophilus oryzae adults in vials containing wheat (A) or rice (B) that contained different percentages of treated substrates, ranged between 5 and 100%, after 7 and 14 d of exposure (within each commodity and exposure, means followed by the same lowercase letter are not significantly different; HSD test at 0.05).
for layer-treated wheat ranged between 32 and 72%. For rice, mortality was notably lower than wheat, which suggests that spinetoram was less effective on rice than on wheat. For spinosad, Athanassiou et al. (2008) also found that efficacy was lower on rice than on wheat against S. oryzae. Our results stand in accordance with the above observations. Practically, in contrast with the influence of commodity, insect positioning had no effect on mortality, which clearly suggests that S. oryzae adults exhibit also a negative geotaxis. For adults of the granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), Surtees (1964) showed an upwards movement in wheat bulks under laboratory conditions. Overall, the presence of untreated layers affected S. oryzae mortality. Therefore, partially-treated bulks, such as bulks on which spinetoram has been applied as a “top-dressing”, may not be effective against S. oryzae. Nevertheless, in our study, we examined wheat and rice columns that were confined in a small vial, and the results obtained here may not be representative of insecticidal performance of spinosyns in grain bulks under field conditions. From the species tested here, T. confusum was by far the least susceptible to spinetoram, given that mortality did not exceed 35%. At the same time, the size of the layer and the type of commodity did not have any effect in T. confusum, as compared with the mortality levels for the totally-treated wheat and rice. Previous studies with spinosad clearly showed that T. confusum and the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) can be classified as among the most tolerant storedproduct insect species to spinosad (Fang et al., 2002; Vayias et al., 2008). Our results are in agreement with those reports. Hence, neither spinosad nor spinetoram is able to control T. confusum, at least at the adult stage, for the doses tested.
As noted above, the mixture of treated and untreated quantities of the grain is a more realistic scenario than the layer-treatment approach. Surprisingly, even the presence of 5% of spinetoramtreated kernels within the entire wheat mass was effective against R. dominica adults. Athanassiou et al. (2009) found that R. dominica adults were dead even after a short exposure to spinosad. For chlorpyriphos-methyl, Arthur (1992) found that a mixture of treated and untreated maize was, in some cases, equally effective against the maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) and T. castaneum, as compared with treating the entire grain mass. In our tests, mortality of R. dominica on mixtures of treated and untreated rice was lower than the respective quantities on wheat, especially in the case of the 7-d exposure interval. Mortality on rice after 14 d of exposure was 99e100% on rice that contained 10, 25, 50 and 100% spinetoram-treated kernels, while with only 5% treated, mortality was 82%. This fact is an additional indication that spinosyns are more effective on wheat than on rice. Thus, we can conclude that, for R. dominica, the “critical” percentage of spinetoram-treated kernels is 5 and 10% for wheat and rice,
Table 6 Two-way ANOVA parameters for treatment and commodity, and their interactions for progeny production of the species tested, in commodities containing different percentages of spinetoram (in all cases, total df ¼ 132). Source
Treatment Commodity Treatment Commodity
df
5 1 5
R. dominica
S. oryzae
F
P
F
P
16.10 5.96 1.20
<0.01 0.16 0.31
3.42 23.64 3.41
<0.01 <0.01 <0.01
T.N. Vassilakos, C.G. Athanassiou / Journal of Stored Products Research 50 (2012) 73e80 Table 7 Mean number of adult progeny of R. dominica and S. oryzae (number of adults per vial SE) on vials containing wheat or rice with different percentages of spinetoram-treated substrates, ranged between 5 and 100% (within each column, means followed by the same lowercase letter are not significantly different; HSD test at 0.05). Treatment ratio
Species/Progeny
100% 50% 25% 10% 5% Untreated
0.42 0.17 1.25 2.42 6.25 27.58
R. dominica (wheat)
0.19b 0.17b 0.39b 0.63b 2.29b 9.30a
R. dominica (rice) 0.17 1.08 2.25 10.25 23.25 39.92
0.17c 0.45c 0.84bc 3.65bc 5.70ab 10.68a
S. oryzae (wheat) 9.17 20.92 41.50 60.08 51.75 41.50
2.21b 6.48ab 15.06ab 7.49a 13.76a 8.52ab
S. oryzae (rice) 15.58 18.42 18.50 18.33 13.33 17.00
3.22a 3.38a 3.01a 3.37a 2.14a 3.05a
79
needed to investigate the mixture technique that ensures the equal distribution of the treated quantity in bulked grains. We evaluated spinetoram as a replacement of traditional grain protectants, under two uneven distribution scenarios. The overall results suggest that spinetoram is very effective against R. dominica, moderately effective against S. oryzae, and not very effective against T. confusum. At the same time, uneven application of spinetoram may, under certain circumstances, provide a satisfactory level of control against R. dominica. Nevertheless, one of the main drawbacks of uneven distribution is the possibility of indirect selection for resistance, and this is why uneven distribution should be evaluated further in this respect (Daglish and Nayak, 2010). Acknowledgements
respectively. It should be noted that differences in parental mortality were not expressed as differences in the subsequent progeny production counts. During the mortality counts, we observed that most of the exposed R. dominica adults were immobilized, which is likely to be the main reason for the reduced reproduction. The results of the current experiment show that even a minimal contact of R. dominica with spinetoram, can cause extremely high parental mortality. In comparison with the layer-treatment approach, the treated quantities here were lower. Thus, we estimate that, against R. dominica, the mixture of spinetoram-treated quantities of grains are much more effective than the arrangement of these quantities in layers. Arthur (1992) also concluded that the mixture of chlorpyriphos-methyl-treated and untreated maize quantities was preferable than using layers treated with chlorpyriphos-methyl, against S. zeamais and T. castaneum. Overall, treated proportions of 5e10% of the entire grain mass should be considered extremely low, as compared with other insecticides. For instance, Arthur (1992) noted that a 2:3 ratio of chlorpyriphos-methyl-treated and untreated maize provided long-term protection against S. zeamais and T. castaneum. Athanassiou et al. (2011a) for S-methoprene on wheat and rice, stated that the presence of treated and untreated quantities was less effective against R. dominica, in comparison with the application on the entire grain mass. Also, Daglish and Nayak (2010) found that when low doses of methoprene were applied on wheat, progeny production of R. dominica was higher on vials that contained a 1:1 treated and untreated ratio as compared with totally-treated wheat. For S. oryzae, on wheat, only some of the mixtures were as effective as the totally-treated wheat. Taken into account both exposure intervals, we can conclude that the “critical” percentage is 50%, which is much higher than the 5% proposed for R. dominica. However, even the lowest (5%) amount of treated kernels killed approx. 50% of the exposed S. oryzae adults on wheat, after 14 d of exposure. This indicates that this minimal presence of spinetoram on wheat had some lethal effects on parental S. oryzae individuals. Mortality on rice was considerably lower than that on wheat and, based on the results, 50% can be again considered as the “critical” level, given that this was the only mixture treatment that was comparable with totally-treated rice. On the other hand, for wheat, the decrease of the percentage of treated kernels significantly increased progeny production, which means that S. oryzae can continue grain damage in partially-treated grains. It should be noted that, the mixture of treated and untreated small grain quantities under laboratory conditions can achieve equal distribution of the treated substrate in the entire grain sample, but this equal distribution may not be practically feasible in the case of large grain bulks in the field. As a result, under field conditions, the untreated substrates may be large enough to allow rapid colonization and the continuance of grain damage. Further work is
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