Biology, Behavior, and Pest Detection on Stored Grain

PS5-8 – 6169 Behavioural responses of confusum Jacquelin du Val (Coleoptera: Tenebrionidae) to flour previously infested or contaminated by Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) semiochemicals

C.G. Athanassiou1*, N.G. Kavallieratos2, S.N. Xyrafidis2, P. Trematerra3

Abstract after 24 h of “contamination” duration, significantly more T. confusum adults were found in the traps The captures of Tribolium confusum containing frass. After 48 h, more T. confusum Jacquelin du Val (Coleoptera: Tenebrionidae) adults were found in the control traps, where there adults in traps previously infested/contaminated were no differences in response between by Ephestia kuehniella Zeller (Lepidoptera: the flour that contained frass or saliva. After 7 d Pyralidae) were evaluated under laboratory of “contamination”, significantly more beetle adults conditions. Two series of tests were carried out, were found in the “saliva” and the control traps by using three-choice test-traps. In the first Test, than in the traps that contained frass. The results the three traps contained a) flour that have been of the present work indicate that under certain infested/contaminated by E. kuehniella larvae, exposure intervals, semiochemicals derived by E. b) flour that have been infested/contaminated by kuehniella were “activated” and “inactivated” at E. kuehniella adults, c) untreated (control) flour. certain exposure intervals. Since E. kuehniella In the second Test, the three traps contained a) and T. confusum are frequently observed in the flour that have been contaminated by E. same habitat, interspecific relations are mediated kuehniella larvae frass, b) flour that have been by semiochemical production. contaminated by E. kuehniella larvae saliva (silk) and c) untreated (control) flour. In both series of Key words: Tribolium confusum, Ephestia tests, there were three “infestation/contamination” kuehniella, semiochemicals, frass, saliva, periods: 24 h, 48 h and 7 d. In the first Test, after contaminated flour. 24 h of “contamination”, significantly more T. confusum adults were found in the control traps. After the 48 h and 7 d contamination periods, Introduction more T. confusum adults were found in traps contaminated by E. kuehniella larvae, than by Species coexistence in storage facilities forces the other two choices. In the second Test series, to regulate their biological, ecological and

1 Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, 75 Iera Odos str., 11855, Athens, Attica, Greece. 2 Laboratory of Agricultural Entomology, Department of Entomology and Agricultural Zoology, Benaki Phytopathological Institute, 8 Stefanou Delta str., 14561, Kifissia, Attica, Greece. 3 Department of , Plant and Environmental Science, University of Molise, Via de Sanctis I-86100, Campobasso, Italy. * Corresponding author. Fax: +30-2105294572. Email address: [email protected]

441 9th International Working Conference on Stored Product Protection behavioural characteristics (Trematerra et al., Materials and methods 2000; Athanassiou et al., 2006). All these variables directly affect any effort for Insects and commodity detection in stored products, especially when traps are used, since behavioural trends determine Both species were reared on wheat flour at 27 oC the possibility of an individual to be detected in a and 70 % r.h., at the Benaki Phytopathological Institute given trapping device (Subramanyam and (Kifissia, Greece). Soft wheat flour was used for Hagstrum, 1995; Trematerra et al., 2000). This experimentation. multi-species coexistence in storage facilities is probably more important than in the field, since, Bioassays apart from the capability for direct damage, the presence of an insect in the stored products is The tests were carried out in a cylindrical arena opposed to the regulations for zero tolerance. made of plexi-glass, 45 cm in diameter and 30 cm Apart from that, this coexistence makes sampling high (Trematerra et al., 2000). In this arena three 2 a more complex procedure, since each species’ modified Flit-Trak M traps (Trècè Inc, USA) were placed. In the center of the arena, 100 individuals presence must be assessed, and often detected, of T. confusum, of mixed sex and age, were in a different way. Finally, coexistence often lead released. The number of trapped individuals was insects to utilize different niches and infest different checked 24 h later. All tests were conducted in parts of the product. Hence, unless these incubators set at 27 oC, 70 % r.h. and continuous parameters are taken into account, the use of a darkness. There were two Test series: sampling protocol may be ineffective. In the first series of tests (Test 1), the three traps The mediterranean flour moth, Ephestia contained a) flour contaminated by E. kuehniella kuehniella Zeller (Lepidoptera: Pyralidae) and larvae, b) flour contaminated by E. kuehniella adults the confused , Tribolium confusum and c) untreated (control) flour. The contamination Jacquelin du Val (Coleoptera: Tenebrionidae) are was achieved by placing the flour with 5 adults or 5 among the most common and destructive insect larvae of the Mediterranean flour moth, in a plastic species in flour mills and related areas (Aitken, box (24 cm length, 11 cm width and 9 cm height). 1975; Buchelos, 1980, 1981; Rees, 1995). Both There were three “contamination” periods: 24 h, 48 species are considered as secondary colonisers, h and 7 d. which means that they cannot develop easily in In the second series of tests (Test 2) the three sound grain kernels and prefer damaged, cracked traps contained a) flour contaminated by E. or processed grains and grain products. Although kuehniella larvae frass, b) flour contaminated by both species coexist very often in the same E. kuehniella larvae saliva and c) untreated (control) facilities, there is still inadequate information about flour. The contamination was made by placing the their potential interactions. In a recent study, flour with 5 adults or 5 larvae of the Mediterranean Athanassiou et al. (2006) in a series of choice flour moth, in a plastic box (24 cm length, 11 cm width tests with floor traps with adults of T. confusum and 9 cm height). There were three “contamination” and the rice weevil, Sitophilus oryzae (L.) periods: 24 h, 48 h and 7 d. (Coleoptera: Curculionidae), found noticeable variations in trapping efficacy when both species, Data analysis or volatiles that were related with individuals of these species, were together, from the single- There were 6 replicates in each case. The data species presence. In the present work, we carried were transformed and analysed as suggested by out similar tests of E. kuehniella and T. Trematerra et al. (2000). For each Test, the confusum. transformed data were submitted to two-way ANOVA, for trap containment and contamination

442 Biology, Behavior, and Pest Detection on Stored Grain period, with catches as the response variable. between the flour containing frass or saliva. After 7 Means were separated by the Tukey-Kramer d of exposure, significantly more were found (HSD) test. in the saliva and the control traps than in the traps containing frass (df = 2.15; F = 15.16; P = 0.0002).

Results

Test 1

Captures are shown in Figure 1. The two main effects, as well as the associated interaction were all significant (containment df = 2, 45; F = 10.41; P = 0.0002; contamination duration df = 2, 45; F = 18.0; P < 0.0001; and containment x contamination df = 4, 45; F = 28.21; P < 0.0001). After 24 h of “contamination”, significantly more T. confusum adults were found in the control traps (> 3 times more adults) (df = 2, 15; F = 15.98; P = 0.0002). After 48 h of exposure, significant differences among traps were recorded (df = 2, 45; F = 37.80; P < 0.0001), but in this case more T. confusum adults were found in traps contaminated by E. kuehniella larvae, than in the other two choices. In addition, the traps that had been contaminated by E. Adults captured kuehniella adults were significantly more attractive than the control traps. After 7 d of exposure, similar trends as previously were recorded (df = 2, 15; F = 18.69; P < 0.0001). Significantly more beetle adults were found in the “larvae” traps than in the “adult” traps.

Test 2

Captures are shown on Figure 2. The two main effects, as well as the associated interaction were all significant, as above (containment df = 2, 45; F = 6.97; P = 0.0023; contamination duration df = 2, 45; F = 4.73; P = 0.0136; and containment x contamination df = 4, 45; F = 11.81; P < 0.0001). After 24 h of “contamination”, significantly more T. Figure 1. Number of T. confusum adults captured confusum adults were found in the traps containing (+SE) in traps containing flour that has previously E. kuehniella larvae frass (df = 2, 15; F = 4.22; P been contaminated by E. kuehniella larvae, adults, = 0.0351). After 48 h of exposure, significant or non-contaminated flour (indicated as larvae, differences among traps were recorded (df = 2, 45; adults and control), at three “contamination” periods, F = 14.26; P = 0.0003), but in this case, more T. 24 h, 48 h and 7 d (within each diagram, means confusum adults were found in the control traps, followed by the same letter are not significantly where there was no difference in beetle response different; HSD test at 0.05).

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affect the behaviour of another species that visits this commodity, in comparison with a non- contaminated commodity. For instance, Athanassiou et al. (2006) by using traps containing filter papers previously “contaminated” by S. oryzae or T. confusum adult semiochemicals, found that S. oryzae capture was seriously influenced by the presence of filter papers, while capture of T. confusum was unaffected. In the present study, both Test series indicated that there is a noticeable interaction between E. kuehniella and T. confusum semiochemicals, but this effect is primarily affected by the contamination interval. After 24 h, in Test 1, the “contamination” of the flour was very little. On the other hand, with the increase of flour exposure period the E. Adults captured kuehniella-contaminated flour was more attractive to beetles, particularly the flour that had been in contact with larvae. Consequently, from the 48 h interval and on, the potential volatiles are “activated”, or this may be the “minimum” interval for moth adults and larvae to “contaminate” the flour. We also conclude that the previous presence of E. kuehniella larvae is more important than that of adults, at least as far as the flour “contamination” in concerned. Similarly, both trap containment and “contamination” affected capture rate in Test 2. After 24 h, frass of E. kuehniella larvae seems to have the most important influence on T. confusum capture rate. In other words, the presence of frass on flour Figure 2. Number of T. confusum adults captured after only 24 h stimulates adult confused flour beetles (+SE) in traps containing flour that has been to move towards this flour (as kairomonal effect). previously contaminated by E. kuehniella larvae However, at higher exposures (= 48 h) this response frass, saliva, or non-contaminated flour (indicated is notably changed, since the attractiveness of the as frass, saliva and control), at three “contamination” frass is reduced. At the same time, the attractiveness periods, 24 h, 48 h and 7 d (within each diagram, of saliva increased gradually. Consequently, frass is means followed by the same letter are not attractive to beetles in newly-“contaminated” flour, significantly different; HSD test at 0.05). while the reverse was noticed for saliva. Phillips et al. (1993) reported that some grain- derived volatiles that attracted S. oryzae adults were repulsive or had no effect to the , Discussion Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Moreover, Trematerra et al. (2000) The simultaneous presence of two species in the noted that kernels damaged by primary pests, same area is expected to cause an alteration in their especially by S. oryzae, were generally preferred response. Similarly, a given commodity that is by the secondary pests Oryzaephilus surinamensis previously “contaminated” by a given species may L. (Coleoptera: Cucujidae), T. castaneum and T.

444 Biology, Behavior, and Pest Detection on Stored Grain confusum, in comparison with artificially damaged Buchelos, C.Th., 1980. Moth population at a or whole kernels. Also, Athanassiou et al. (2006) typical flour mill. Annales de l’ Institut found that damaged seeds by S. oryzae increased Phytopathologique Benaki 12, 188-197 the response of other S. oryzae individuals. Trematerra et al. (1996) noted that the presence of Buchelos, C.Th., 1981. Coleoptera populations at live T. castaneum adults increased captures of adults flour mills and related areas. Annales de l’ of the same species. This behaviour could be Institut Phytopathologique Benaki 13, 6-29. attributed to the production of aggregation pheromone. In the present study, 24 h of flour Phillips, T.W., Jiang, X.L., Burkholder, W.E., “contamination” should be considered as a “critical” Phillips, J.K., Tran, H.Q., 1993: Behavioural interval affecting beetle response. responses to food volatiles by two species of The succession of insects in stored products is a stored-product Coleoptera, Sitophilus very complex phenomenon that needs additional oryzae (Curculionidae) and Tribolium attention. Each species that play a role in this castaneum (Tenebrionidae). Journal of infestation chain, is affected by, and to some extend Chemical Ecology 19, 723-734. utilise, any semiochemicals derived by the previous species in the same chain. E. kuehniella and T. Rees, D.P., 1995, Coleoptera. In: Subramanyam, confusum in flour mills are direct competitors, and Bh., Hagstrum, D.W. (Eds), Integrated each of these species can be the first coloniser on Management of Insects in Stored Products. the commodity. In our study, by examining the Marcel Dekker, New York, pp. 3-11. scenario of E. kuehniella as the first coloniser, it is clearly evident that T. confusum adults seems to be Subramanyam, Bh., Hagstrum, D.W., 1995, quite sensitive to potential kairomones derived by Sampling. In: Subramanyam, Bh., Hagstrum, moth adults and larvae. D.W. (Eds), Integrated Management of Insects in Stored Products. Marcel Dekker, New York, pp. 142-188. References Trematerra, P., Fontana, F., Mancini, M., 1996: Aitken, A.D., 1975. Insect Travelers, I: Effects of accumulate dead and live insects in Coleoptera. Technical Bulletin 31. H. M. S. trap on the capture of Tribolium castaneum O., London. (Herbst). Anzeiger für Schadlingskunde Pflanzenschutz Umweltschutz 69, 3-9. Athanassiou, C.G., Kavallieratos, N.G., Trematera, P., 2006. Responses of Sitophilus Trematerra, P., Sciarretta, Á., Tamasi, Å., 2000, oryzae (Coleoptera: Curculionidae) and Behavioural responses of Oryzaephilus Tribolium confusum (Coleoptera: surinamensis, Tribolium castaneum and Tenebrionidae) to traps baited with pheromone Tribolium confusum to naturally and and food volatiles. European Journal of artificially damaged durum wheat kernels. Entomology 103, 371-378. Entomologia Experimentalis et Applicata 94, 195-200.

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