Introduction
Stored grain protection based on chemical control is common, which consists of two major categories of insecticides: fumigants and contact insecticides. Presently, the use of fumigants, and especially phosphine (PH3), is probably the major measure taken for insect control at the post-harvest stages of durable commodities, including grains and related amylaceous products (Chaudhry, Reference Chaudhry2000; Benhalima et al., Reference Benhalima, Chaudhry, Mills and Price2004; Corrêa et al., Reference Corrêa, Tomé, Braga, Martins, de Olivera and Guedes2014). However, after the withdrawal of methyl bromide (CH3Br), and also the development of resistance to phosphine in many parts of the world (Bell, Reference Bell2000; Fields & White, Reference Fields and White2002; Pimentel et al., Reference Pimentel, Faroni, Tótola and Guedes2007, Reference Pimentel, Faroni, Guedes, Sousa and Tótola2009, Reference Pimentel, Faroni, Corrêa and Guedes2012), research on contact insecticides, known as grain protectants has been enhanced (Daglish, Reference Daglish2008; Hertlein et al., Reference Hertlein, Thompson, Subramanyam and Athanassiou2011; Arthur, Reference Arthur2012). Grain protectants defined as insecticides that are applied directly on the grains, are expected to protect the commodities from insect infestations as long as they remain active (Arthur, Reference Arthur1996). There are several grain protectants which have already been registered for direct application in many types of grains; most of these are organophosporous compounds (OPs) and pyrethroids that act on insects’ nervous system (Arthur, Reference Arthur1999; Arthur & Campbell, Reference Arthur and Campbell2008; Kavallieratos et al., Reference Kavallieratos, Athanassiou and Arthur2015a ). Nevertheless, some neurotoxic compounds, especially OPs, may be no longer acceptable, due to the presence of toxic residues on the final product, and the consumers’ demand for residue-free food (Arthur, Reference Arthur1996, Reference Arthur1999; Athanassiou et al., Reference Athanassiou, Kavallieratos, Arthur and Throne2013). At the same time, some of the major stored-product insect species cannot be controlled with traditional grain protectants, due to resistance development, as a result of a continuous use of certain active ingredients. For example, Rumbos et al. (Reference Rumbos, Dutton and Athanassiou2013) reported that pirimiphos-methyl, which is one of the most commonly used grain protectants globally, could not control the lesser grain borer, Rhyzopertha dominica (F.). Moreover, natural pyrethrum was unable to control five species of stored product psocid species, i.e., Lepinotus reticulatus Enderlein, Liposcelis bostrychophila Badonnel, Liposcelis entomophila (Enderlein) and Liposcelis paeta Pearman (Athanassiou et al., Reference Athanassiou, Arthur and Throne2009). This fact constitutes essentially the evaluation of novel compounds that can be used successfully as alternatives to OPs and pyrethroids. In this context, one recently newly registered compound for grain protection is the bacterial insecticide spinosad, which is based on metabolites of the actinomycete Saccharopolyspora spinosa Mertz and Yao (Hertlein et al., Reference Hertlein, Thompson, Subramanyam and Athanassiou2011). However, despite the fact that spinosad was found very effective for the control of R. dominica, it was not effective for other species, such as the red flour beetle, Tribolium castaneum (Hertz), and the confused flour beetle, Tribolium confusum Jacquelin du Val (Hertlein et al., Reference Hertlein, Thompson, Subramanyam and Athanassiou2011). Another member of the spinosyn group, spinetoram, has also been proven an efficacious grain protectant, although it is not registered yet for that purpose (Vassilakos & Athanassiou, Reference Vassilakos and Athanassiou2012a , Reference Vassilakos and Athanassiou b , Reference Vassilakos and Athanassiou2013; Athanassiou & Kavallieratos, Reference Athanassiou and Kavallieratos2014). One other registered alternative is the insect growth regulator (IGR) methoprene (methoprene containing the R and S isomers and s-methoprene containing the S isomer alone), which is non-neurotoxic, as it acts on insects’ metamorphosis (Arthur, Reference Arthur2004; Athanassiou et al., Reference Athanassiou, Arthur and Throne2010a , Reference Athanassiou, Arthur and Throne b ; Wijayaratne et al., Reference Wijayaratne, Fields and Arthur2012). Nevertheless, methoprene or s-methoprene is not effective for some major species, such as the granary weevil, Sitophilus granarius (L.), the rice weevil, Sitophilus oryzae (L.) and the stored product Psocoptera L. bostrychophila, Liposcelis decolor (Pearman), L. entomophila, L. paeta and L. reticulatus (Nayak et al., Reference Nayak, Collins and Reid1998; Edwards & Short, Reference Edwards and Short1984; Daglish, Reference Daglish2008; Athanassiou et al., Reference Athanassiou, Arthur and Throne2010b , Reference Athanassiou, Arthur, Kavallieratos and Throne2011).
One of the recently evaluated insecticides for stored product insect control is chlorfenapyr which is a non-neurotoxic pyrrole that causes oxidative phosphorylation in the mitochondria, which disrupts the synthesis of adenosine triphosphate (ATP), and has low mammalian toxicity (Hunt, Reference Hunt1996; Tomlin, Reference Tomlin2000; McLeod et al., Reference McLeod, Diaz and Johnson2002). This pyrrole has been found very effective against the larger grain borer, Prostephanus truncatus (Horn) in maize at doses >0.1 ppm after 7 days of exposure (Kavallieratos et al., Reference Kavallieratos, Athanassiou, Hatzikonstantinou and Kavallieratou2011). However, so far it is registered in the USA only for structural application against urban pests (Athanassiou et al., Reference Athanassiou, Kavallieratos, Arthur and Throne2014). In this regard, members of the pyrrole group are likely to have some interesting properties that merit additional investigation as insecticides at the post-harvest stages of agricultural commodities. Recently, it was found that some new sulfanyl 5H-dihydro-pyrrole derivatives have some certain biological activities by exhibiting strong antioxidant activity (i.e., inhibition of lipid peroxidation and soybean lipoxygenase) (Georgiou et al., Reference Georgiou, Toutountzoglou, Muir, Hadjipavlou Litina and Elemes2012; Oikonomou et al., Reference Oikonomou, Georgiou, Katsamakas, Hadjipavlou Litina and Elemes2015). However, there are no published data regarding the efficacy of these compounds as insecticides so far. In the present work, we examined the insecticidal activity of two new pyrrole derivatives against two major stored-product insect species, T. confusum adults and larvae and the Mediterranean flour moth, Ephestia kuehniella Zeller larvae at different temperatures and relative humidity levels.
Materials and methods
Insects
The insects used in the tests were reared at the Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, in continuous darkness. The cultures, initially collected from Greek storage facilities, have been kept at Agricultural University of Athens since 2014. T. confusum was reared in wheat flour including 5% brewer's yeast (w/w) at 27(±0.1)°C and 60(±0.2)% relative humidity. E. kuehniella was reared in wheat flour at 26(±0.1)°C and 60(±0.2)% relative humidity. Adults <2 weeks old of T. confusum and first instar-larvae of T. confusum or E. kuehniella were used in the tests. For the first instar, T. confusum and E. kuehniella eggs were collected from flour by using a sieve of 60 mesh (250 micron, W.S. Tyler, Mentor, OH, USA), then placed in incubators at 27°C and 60% relative humidity, or 26°C and 60% relative humidity respectively, and larvae were collected after hatching.
Commodity
Untreated, clean hard wheat, Triticum durum Desf. (var. Mexa), free of infestation, was used in the bioassays. The moisture content of the tested wheat was 11%, as determined by a Dickey–John moisture meter (mini GAC plus, Dickey-John Europe S.A.S., Colombes, France) at the beginning of the tests. Before the bioassays, the tested commodity was sieved to remove impurities and dockage.
Pyrrole derivatives
Two pyrrole derivatives, developed by the late Prof Dr Y. Elemes and his group (Oikonomou et al., Reference Oikonomou, Georgiou, Katsamakas, Hadjipavlou Litina and Elemes2015), the ethyl 3-(benzylthio)-4,6-dioxo-5-phenyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-carboxylate (3i) and the isopropyl 3-(benzylthio)-4,6-dioxo-5-phenyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrrole-carboxylate (3k), were used for experimentation (fig. 1). These are pure substances and were used as powders in the tests. The codes 3i and 3k correspond to the ones given for the certain derivatives by Oikonomou et al. (Reference Oikonomou, Georgiou, Katsamakas, Hadjipavlou Litina and Elemes2015).
Fig. 1. Chemical structures of the pyrrole derivatives tested.
Bioassays
In cylindrical glass jars 1 kg lots of wheat were placed and treated with the pyrrole derivatives at three doses: 0.1 ppm (mg pyrrole derivate powder/kg wheat), 1 ppm and 10 ppm. The jars were shaken manually for 5 min to achieve the uniform distribution of the pyrrole derivatives particles in the entire wheat mass. An additional 1 kg of untreated wheat was served as control. From each lot, three samples, of 10 g each, were taken and placed in small cylindrical glass vials (7 cm diameter, 12 cm height) with a different scoop that was inside each jar. The quantity of 10 g was weighed with a Precisa XB3200D compact balance (Alpha Analytical Instruments, Gerakas, Greece). The closure of the vials had a 1.5 cm diameter hole in the middle, which was covered by gauze, to allow sufficient aeration inside the vial. Then, 10 adults or larvae of each insect species were separately placed inside each vial. The internal ‘necks’ of the vials were covered by Fluon (Northern Products Inc., Woonsocket, USA), to prevent insects from escaping. Subsequently, six series of bioassays of each compound were placed in controlled chambers under the following conditions: 20(±0.1)°C and 55(±0.2)% relative humidity, 25(±0.1)°C and 55(±0.2)% relative humidity, 30(±0.1)°C and 55(±0.2)% relative humidity, 20(±0.1)°C and 75(±0.2)% relative humidity, 25(±0.1)°C and 75(±0.2)% relative humidity, 30(±0.1)°C and 75(±0.2)% relative humidity for the duration of the experimental period. Mortality of the treated individuals was assessed after 7, 14 and 21 days of exposure. Dead adults were determined by prodding with a brush to detect movement under an Olympus stereomicroscope (Olympus SZX9, Bacacos S.A., Athens, Greece). The brush was carefully washed after the examination of each vial. All tests were repeated three times for each species (adults or larvae), by preparing new lots each time. After the 21 days mortality counts, all (alive or dead) parental individuals were discarded. In the case of vials that contained parental T. confusum adults, they were again put inside the incubators at the same conditions for an additional period of 60 days. They were then removed and the numbers of progeny were counted as noted above. Adult and immature progeny were recorded and progeny production was expressed as number of individuals/vial. However, the majority of the total number of progeny (>87%) consisted of adults. Since control mortality was very low (<3%), no correction was necessary for the mortality data.
Statistical analysis
Data were analyzed separately for each of the tested species or stage according to the repeated-measures model (Sall et al., Reference Sall, Lehman and Creighton2001). The repeated factor was exposure interval, while mortality was the response variable. Dose, pyrrole derivative, relative humidity and temperature were the main effects. The associated interactions of the main effects were incorporated in the analysis. Progeny production counts were subjected to four-way ANOVA, with dose, pyrrole derivative, relative humidity and temperature as main effects. The associated interactions of the main effects were incorporated in the analysis. Progeny production in the control vials (0 ppm) was also included in the analysis. All analyses were conducted using the JMP 11 software (SAS Institute, SAS Institute Inc., Cary, NC, USA). Means were separated by the Tukey–Kramer honestly significant difference (HSD) test at 0.05 probability (Sokal & Rohlf, Reference Sokal and Rohlf1995).
Results
Mortality and progeny of T. confusum adults
Between and within exposure intervals, the main effects and the associated interactions are presented in table 1. In the case of the pyrrole derivative 3i, after 7 days of exposure, mortality was very low (<8%) in all tested combinations (table 2). Seven days later, mortality increased further in the combinations of 55% relative humidity, but it did not exceed 24.4%, whereas in combinations of 75% relative humidity, mortality remained in very low levels (<3.5%). Similar trends were noted after 21 days of exposure. Mortalities were higher at 55% relative humidity than those at 75% relative humidity. Yet, mortality was low and it did not exceed 32.2% in wheat treated with 10 ppm 3i at 30°C and 55% relative humidity. Regarding progeny production, main effects and the associated interactions are presented in table 3. Progeny production was very low (<1 individual/vial) in all combinations of 55% relative humidity, including control (table 4). No offspring emerged in 1 ppm at 20°C or 10 ppm at 20 and 25°C and 55% relative humidity. The increase of temperature significantly increased adult emergence in 0.1 ppm at 75% relative humidity. Significant differences of progeny production between control and treatments were noted only at 25°C and 75% relative humidity where at 10 ppm significantly fewer offsprings were emerged compared with 0 ppm.
Table 1. Repeated measures MANOVA parameters for main effects and associated interactions for mortality levels of T. confusum adults or larvae and E. kuehniella larvae between and within exposure intervals (error d.f. = 384 for all species).
*P ≤ 0.05.
**P ≤ 0.01.
Table 2. Mean mortality (%±SE) of T. confusum adults after 7, 14 and 21 days in wheat treated with the pyrrole derivative 3i at three doses under three temperatures and two relative humidity levels.
Within each column, exposure and relative humidity, means followed by the same lowercase letter are not significantly different; d.f. = 3, 35. Within each row, exposure and relative humidity, means followed by the same uppercase letter are not significantly different; d.f. = 2, 26, Tukey–Kramer (HSD) test at P = 0.05. Where no letters exist, no significant differences were recorded. Where dashes exist, no analysis was performed.
*P ≤ 0.05.
Table 3. ANOVA parameters for progeny production of T. confusum adults (total d.f. = 431).
**P ≤ 0.01.
Table 4. Progeny production of T. confusum (individuals/vial ± SE) in wheat treated with the pyrrole derivative 3i at four doses (inclusive of 0 ppm) under three temperatures and two relative humidity levels 60 days after the removal of parental adults.
Within each column and relative humidity, means followed by the same lowercase letter are not significantly different; d.f. = 3, 35. Within each row and relative humidity, means followed by the same uppercase letter are not significantly different; d.f. = 2, 26, Tukey–Kramer (HSD) test at P = 0.05). Where no letters exist, no significant differences were recorded.
*P ≤ 0.05.
**P ≤ 0.01.
In the case of the pyrrole derivative 3k, after 7 days of exposure, the highest mortality (18.9%) was observed in wheat treated with 10 ppm at 25°C and 55% relative humidity while at 25 and 75% relative humidity the mortality was 1.1% (table 5). At 75% relative humidity, mortalities were very low and they did not exceed 6.7%. No mortality was recorded at 20 and 25°C and 75% relative humidity at 0.1 and 1 ppm 3k. After 14 days of exposure, mortality was further increased. At 55% relative humidity, mortality ranged from 13.3 to 37.8% while at 75% relative humidity the mortality did not exceed 12.2%. Finally, after 21 days of exposure, mortality reached 67.8% at 30°C and 55% relative humidity in wheat treated with 10 ppm. The overall mortality was lower at 75% than that at 55% relative humidity and ranged from 2.2 to 25.6%. Progeny production was low in all tested combinations (<0.7 individuals/vial) (table 6). No offspring emerged in 1 or 10 ppm 3k at 20 and 25°C and 55% relative humidity and in 1 ppm 3k at 20°C or in 10 ppm 3k at 20 and 25°C and 75% relative humidity. The progeny production in all treatments was significantly lower compared with control at 25 and 30°C and 75% relative humidity.
Table 5. Mean mortality (% ±SE) of T. confusum adults after 7, 14 and 21 days in wheat treated with the pyrrole derivative 3k at three doses under three temperatures and two relative humidity levels.
Within each column, exposure and relative humidity, means followed by the same lowercase letter are not significantly different; d.f. = 3, 35. Within each row, exposure and relative humidity, means followed by the same uppercase letter are not significantly different; d.f. = 2, 26, Tukey–Kramer (HSD) test at P = 0.05). Where no letters exist, no significant differences were recorded.
*P ≤ 0.05.
**P ≤ 0.01.
Table 6. Progeny production of T. confusum (individuals/vial ± SE) in wheat treated with the pyrrole derivative 3k at four doses (inclusive of 0 ppm) under three temperatures and two relative humidity levels 60 days after the removal of parental adults.
Within each column and relative humidity, means followed by the same lowercase letter are not significantly different; d.f. = 3, 35. Within each row and relative humidity, means followed by the same uppercase letter are not significantly different; d.f. = 2, 26, Tukey–Kramer (HSD) test at P = 0.05. Where no letters exist, no significant differences were recorded.
*P ≤ 0.05.
**P ≤ 0.01.
Mortality of T. confusum larvae
Between and within exposure intervals, the main effects and the associated interactions are presented in table 1. In the case of the pyrrole derivative 3i, after 7 days of exposure, significantly more larvae died at 25°C than at 30°C and 55% relative humidity in wheat treated with 1 ppm but the mortality did not exceed 26.7% (table 7). Similar trend was also observed in 10 ppm but without significant differences. Still, mortality was low (<37%). At 75% relative humidity, mortality remained at very low levels (<12%) in 1 ppm 3i. However, in 10 ppm 3i it arose to 36.7% at 20°C which was significantly higher than that at 25 and 30°C. Similar trends were noted after 14 days of exposure, where mortality was significantly higher in 0.1 and 1 ppm at 25°C and 55% relative humidity, reaching 35.6 and 44.4% respectively, than at 30°C and 55% relative humidity. In 10 ppm, mortality was 60% at 25°C and 55% relative humidity. The overall mortality was lower at 75% relative humidity than at 55% relative humidity. After 21 days of exposure, mortality was further increased. At the highest dose it was 82.2 at 25°C and 55% relative humidity and significantly higher than that at the other two temperatures. Yet, mortality was lower at 75% than at 55% relative humidity and it did not exceed 55.6 at 20°C and 75% relative humidity in 10 ppm 3i.
Table 7. Mean mortality (%±SE) of T. confusum larvae after 7, 14 and 21 days in wheat treated with the pyrrole derivative 3i at three doses under three temperatures and two relative humidity levels.
Within each column, exposure and relative humidity, means followed by the same lowercase letter are not significantly different; d.f. = 3, 35. Within each row, exposure and relative humidity, means followed by the same uppercase letter are not significantly different; d.f. = 2, 26, Tukey–Kramer (HSD) test at P = 0.05). Where no letters exist, no significant differences were recorded.
*P ≤ 0.05.
**P ≤ 0.01.
In the case of 3k, after 7 days of exposure, mortality was low in 0.1 and 1 ppm at all temperatures and relative humidity levels and it did not exceed 26.7% (table 8). In 10 ppm, mortality was significantly higher at 25°C than that at 30°C and 55% relative humidity. After 14 days of exposure, mortality increased further and reached 67.8 and 52.2% at 55 and 75% relative humidity at 25°C in 10 ppm, respectively. After 21 days of exposure, mortality increased to 77.8% at 25°C and 55% relative humidity. At 75% relative humidity, mortality was lower than at 55% in all combinations tested and reached 65.6% at 25°C except in 1 ppm at 25 °C after 21 days of exposure.
Table 8. Mean mortality (%±SE) of T. confusum larvae after 7, 14 and 21 days in wheat treated with the pyrrole derivative 3k at three doses under three temperatures and two relative humidity levels.
Within each column, exposure and relative humidity, means followed by the same lowercase letter are not significantly different; d.f. = 3, 35. Within each row, exposure and relative humidity, means followed by the same uppercase letter are not significantly different; d.f. = 2, 26, Tukey–Kramer (HSD) test at P = 0.05. Where no letters exist, no significant differences were recorded.
*P ≤ 0.05.
**P ≤ 0.01.
Mortality of E. kuehniella larvae
Between and within exposure intervals, the main effects and the associated interactions are presented in table 1. In the case of the pyrrole derivative 3i, after 7 days of exposure, the highest mortality (20%) was observed in 10 ppm at 25°C and 55% relative humidity (table 9). The overall mortality at 75% relative humidity was very low and ranged from 1.1 to 5.6%. After 14 and 21 days of exposure, mortality reached 41.1 and 44.4%, respectively, in the same combination as at 7 days of exposure. In contrast, mortality at 75% relative humidity remained very low and did not exceed 11.1 and 13.3% after 14 and 21 days of exposure.
Table 9. Mean mortality (%±SE) of E. kuehniella larvae after 7, 14 and 21 days in wheat treated with the pyrrole derivative 3i at three doses under three temperatures and two relative humidity levels.
Within each column, exposure and relative humidity, means followed by the same lowercase letter are not significantly different; d.f. = 3, 35. Within each row, exposure and relative humidity, means followed by the same uppercase letter are not significantly different; d.f. = 2, 26, Tukey–Kramer (HSD) test at P = 0.05. Where no letters exist, no significant differences were recorded.
*P ≤ 0.05.
**P ≤ 0.01.
In the case of 3k, after 7 days of exposure in wheat treated with 0.1 or 1 ppm, mortality did not exceed 12.2% at 25°C and 55% relative humidity (table 10). Significantly higher mortality was recorded at 25°C (42.2%) than at 20 or 30°C in 10 ppm 3k. At 75% relative humidity, no significant differences among temperatures were noted in any dose. The maximum mortality (30%) was recorded in 10 ppm at 20°C and 75% relative humidity. After 14 and 21 days of exposure, mortality was further increased and reached 52.2 and 63.3% in 10 ppm 3k at 25°C and 55% relative humidity, respectively, as at 7 days of exposure. The overall mortality at 75% relative humidity was lower and it did exceed 45.6 and 50% in 10 ppm 3k at 20°C and 75% relative humidity at 14 and 21 days of exposure, respectively. As at 7 days of exposure, no significant differences were observed among temperatures in all doses either after 14 or 21 days of exposure.
Table 10. Mean mortality (%±SE) of E. kuehniella larvae after 7, 14 and 21 days in wheat treated with the pyrrole derivative 3k at three doses under three temperatures and two relative humidity levels.
Within each column, exposure and relative humidity, means followed by the same lowercase letter are not significantly different; d.f. = 3, 35. Within each row, exposure and relative humidity, means followed by the same uppercase letter are not significantly different; d.f. = 2, 26, Tukey–Kramer (HSD) test at P = 0.05. Where no letters exist, no significant differences were recorded.
*P ≤ 0.05.
**P ≤ 0.01.
Discussion
The results of the present study show that the substances tested have a certain insecticidal effect, but this effect is moderated by the exposure interval, the target species, the temperature and the relative humidity prevailing. Generally, these compounds had low toxicity against T. confusum adults, which means that they are poor adulticides. Furthermore, in all combinations tested here, progeny production was very low. For IGRs, a group of insecticides that disorganize the normal development of insects, there is no or low adult mortality, but progeny production is suppressed through immature mortality (Cogburn, Reference Cogburn1988; Daglish et al., Reference Daglish, Erbacher and Eelkema1993; Kostyukovsky et al., Reference Kostyukovsky, Chen, Atsmi and Shaaya2000; Daglish & Wallbank, Reference Daglish and Wallbank2005; Daglish, Reference Daglish2008; Athanassiou et al., Reference Athanassiou, Arthur, Kavallieratos and Throne2011; Kavallieratos et al., Reference Kavallieratos, Athanassiou, Vayias and Tomanović2012b ). Just as, Kavallieratos et al. (Reference Kavallieratos, Athanassiou, Vayias and Tomanović2012b ) tested eight IGR formulations against P. truncatus and found that mortality of adults was low, but the progeny suppression was satisfactory (86.4–100%), indicating that the larvicidal or ovicidal effect was high. In the present study, we found that the pyrrole derivatives tested gave a noticeable level of larval mortality, at least in some of the combinations used. Although we did not take any quantitative data towards this direction, we observed that the survived larvae were able to develop normally, which means that there was no effect on molting. The suppression of the offspring production of T. confusum is related to the biology of this species since, as a secondary colonizer, it cannot develop easily on sound grains (Brewer & Ferrell, Reference Brewer and Ferrell1992), a fact that may explain the low progeny that was observed in the control sound kernels of wheat we used. Thus, the tested ingredients may not act as IGRs. For the pyrrole chlorfenapyr, Kavallieratos et al. (Reference Kavallieratos, Athanassiou, Hatzikonstantinou and Kavallieratou2011) found that, as an admixture with the grains, mortality of P. truncatus adults was 98.3% in maize treated with 1 ppm, after 14 days of exposure. Nevertheless, at the same conditions, mortality of T. confusum adults was 47.8%. In general, adults of T. confusum cannot be easily controlled with several insecticides, such as alpha-cypermethrin, deltamethrin, abamectin, spinosad, chlorfenapyr, diatomaceous earths (DEs), and spinetoram (Athanassiou et al., Reference Athanassiou, Kavallieratos, Vayias, Papagregoriou, Dimizas and Buchelos2004; Kavallieratos et al., Reference Kavallieratos, Athanassiou, Vayias, Mihail and Tomanović2009; Hertlein et al., Reference Hertlein, Thompson, Subramanyam and Athanassiou2011; Kavallieratos et al., Reference Kavallieratos, Athanassiou and Boukouvala2012a , Reference Kavallieratos, Athanassiou, Korunic and Mikeli b ; Athanassiou & Kavallieratos, Reference Athanassiou and Kavallieratos2014). For instance, spinosad can control T. confusum adults at doses that largely exceed the recommended dose (1 ppm) (Hertlein et al., Reference Hertlein, Thompson, Subramanyam and Athanassiou2011). However, the larvae of this species were very susceptible to spinosad, suggesting that adult control will eventually occur through increased larval mortality (Hertlein et al., Reference Hertlein, Thompson, Subramanyam and Athanassiou2011). In our case, T. confusum larval mortality reached 82.2 and 77.8% in wheat treated with 3i and 3k, respectively after 21 days of exposure, indicating that high immature control can contribute to the concomitant adult control. The same holds for E. kuehniella larvae, as morality was 63.3% after 21 days of exposure in wheat treated with 3k.
Temperature plays a key role in the efficacy of several insecticides that are applied as grain protectants. For DEs, Vayias & Athanassiou (Reference Vayias and Athanassiou2004) noted that efficacy against T. confusum larvae was increased with the increase of temperature. Similar observations were made by Badii et al. (Reference Badii, Adarkwash, Ulrichs and Obeng Ofori2013) for the pulse beetle, Callosobruchus maculatus (F.) in DE-treated Kersting's groundnut (Macrotyloma geocarpum Harms) seeds. Moreover, for the neonicotinoid insecticide thiamethoxam Arthur et al. (Reference Arthur, Yue and Wide2004) reported that mortality of the saw-toothed grain, Oryzaephilus surinamensis (L.), R. dominica, the maize weevil, Sitophilus zeamais Motschulsky and T. castaneum were higher at 32 than at 22°C. Similar results have also been reported for spinosad and spinetoram as grain protectants against P. truncatus in treated maize and R. dominica, S. oryzae and T. confusum in treated wheat (Athanassiou & Kavallieratos, Reference Athanassiou and Kavallieratos2014). The rationale for this increased effect is that, at elevated temperatures, insect major metabolic activities are increased, which means that insects are more vulnerable to the toxic agent (Athanassiou & Kavallieratos, Reference Athanassiou and Kavallieratos2014). Moreover, insects are usually more mobile at increased temperatures, which means that they are likely to have a greater contact with the treated substrate (Kavallieratos et al., Reference Kavallieratos, Athanassiou, Korunic and Mikeli2015b ). Nevertheless, temperature may not play a key role on all stored product insects. For larvae of the Indianmeal moth, Plodia interpunctella (Hübner), Jenson et al. (Reference Jenson, Arthur and Nechols2009) noted that temperature did not affect the insecticidal effect of s-methoprene as surface treatment. Moreover, Rumbos et al. (Reference Rumbos, Dutton and Athanassiou2013) found that in grains treated with the OP insecticide pirimiphos-methyl, mortality of R. dominica and T. confusum was not generally affected by temperature ranging between 25 and 30°C. In that study, the authors found that relative humidity was the factor that determined the effect of temperature, as in high relative humidity values, mortality was generally lower. This could be attributed to the fact that at high relative humidity, water loss and, as a result, stress, may occur more rapidly (Arthur, Reference Arthur2000; Badii et al., Reference Badii, Adarkwash, Ulrichs and Obeng Ofori2013). Our results stand in accordance with this observation, as high relative humidity moderated larval and adult mortality of T. confusum. However, a humid environment, apart from the effect on the insect per se, may have a certain effect on the insecticide, i.e., increased dissipation, or trigger certain interactions between the insects and the active ingredient, such as reduced contact. For DEs, Vayias & Athanassiou (Reference Vayias and Athanassiou2004) reported a decrease in efficacy against larvae of T. confusum with the increase of relative humidity, due to the fact that DE dust particles are partially ‘inactivated’ at elevated humidity/moisture conditions. Our results showed that both pyrrole derivatives performed better at 25°C than at 20 or 30°C at both relative humidity levels against both insect species. This finding is important given that E. kuehniella develops quicker from egg to adult at 25°C than at lower or upper temperatures (Jacob & Cox, Reference Jacob and Cox1977). Furthermore, the level of 25°C could be critical for the potential application of the tested compounds in terms of optimization of their efficacy against E. kueniella and T. confusum.
For the pyrrole chlorfenapyr, the data about stored-product insect larvae are disproportionally fewer in comparison with those for adults. Arthur & Fontenot (Reference Arthur and Fontenot2012) found that although mortality of the 4-week old larvae of T. castaneum was low on surfaces which contained wheat flour and were partially treated with chlorfenapyr, most of the surviving larvae died later as adults. Hence, it is likely that further mortality occurred at later periods. Chlorfenapyr is generally considered as a slow-acting insecticide (Arthur & Fontenot, Reference Arthur and Fontenot2012), which means that increased mortality may not occur during initial exposure, and that exposed insects may escape from the treated substrate and colonize untreated parts of the commodity or the facility (Daglish & Nayak, Reference Daglish and Nayak2010). The results of the present study show that larvae of T. confusum were affected more than adults of T. confusum, however, we did not estimate possible pupal or adult mortality after the larval observation period, an issue that merits more investigation.
Pyrroles are novel insecticides that can be used with success against many insect pests in crop protection, but also at the post-harvest and urban environment. Our study is the first attempt to assess the insecticidal value of novel pyrrole derivatives. The data presented here illustrate that, under certain conditions, some of these ingredients can be further evaluated as insecticides, under a wider range of scenarios (i.e., biotic and abiotic factors). In this regard, the mode of insecticidal action of these compounds should be determined and characterized, as this constitutes the key element in any further assessment.
Acknowledgements
This study was partially supported by the project 4100 of Research Committee of the University of Thessaly and the scholarship “Athanassios Sotiroudas” provided by the Hellenic Entomological Society.
