Introduction

Wheat is the most widely cultivated and consumed plant species throughout the globe (Fourar-Belaifa et al. 2011). During growing, wheat crops are heavily infested by different insect pests that cause serious losses and qualitative degradations (Cressey et al. 1987; Swallow and Every 1991; Hariri et al. 2000; Rosell et al. 2002; Salis et al. 2013). One of the major causal agents of wheat infestation at the pre-harvest stages is the occurrence of the so-called wheat bugs that belong to the genera Eurygaster (Hemiptera: Scutellaridae) and Aelia (Hemiptera: Pentatomidae). Aelia spp. and Eurygaster spp. are the most prominent on wheat damage in Europe (Rosell et al. 2002), with Eurygaster integriceps Puton being the most important in Eastern Europe, West and Central Asia, and Eurygaster maura (L.) in Central and Southern Europe and Central Asia (Vaccino et al. 2006; Trissi et al. 2006; Parker et al. 2011). In New Zeeland, the wheat bug Nysius huttoni White (Heteroptera: Lygaeidae) is considered as the most important (Rea et al. 2002), while Nezara viridula (L.) (Heteroptera: Pentatomidae) is regarded as an important pest of wheat in USA (Viator et al. 1983). According to Stavraki (1979) the wheat bug species that have been mostly reported in Greece are E. maura, Eurygaster austriaca (Schrank) and Aelia rostrata Boheman.

Durable commodities are stored in different structures and periods for future consumption. The presence of foreign materials, such as weed seeds, dust, can increase heating, moisture content and deterioration in stored grain (Sinha 1975). Insect fragments in stored products are of major concern for quality preservation and legislation reasons (Rosell et al. 2002; Perez-Mendoza et al. 2003; Hubert et al. 2018; Sharma et al. 2020). In a recent study, Georgousakis et al. (2020) examined the effect of weed seeds on wheat and barley in two stored product insects and found that the presence of weed seeds can affect the progeny production of these species. Sharma et al. (2020) identified the impact of grasshopper carcasses on different grain quality and found negative consequences such as reduction of germination and increase of fat acidity values. The effect of wheat bugs was mostly focused on biochemical properties of the wheat quantifying the bug damage effect in specific protein fractions (Sivri et al. 2004; Salis et al. 2010). For instance, Torbica et al. (2014) tested the effect of wheat infestation of Eurygaster spp. and Aelia spp. on the composition of wheat gluten proteins and found noticeable differences in gluten complex.

Post-harvest losses of grains are estimated in some regions up to 50% with molds, insects and rodents as the primary pests of infestation (Brader et al. 2002; Athanassiou and Arthur 2018). Pests, and especially insects, not only decrease stored grain quality but also contaminate the products with their metabolic by-products and body fragments (Neethirajan et al. 2007; Hubert et al. 2018). Several pest categories that infest stored products occur from the pre-harvest stages in the field, while there are other species that are present in the field but cannot continue the infestation at the post-harvest stages (Sharma et al. 2020). The Food and Drug Administration (FDA) in USA has established the so-called food defect action levels of insect contamination on amylaceous commodities, which are 32 insect-damaged kernels per 100 g of wheat and 75 insect fragments per 50 g of wheat flour (FDA 1998). In a surveillance from mills in Italy, Trematerra et al. (2011) found that 75% of semolina samples contained insect fragments. Stored product insects such as the primary pests Sitophilus oryzae (L.) (Coleoptera: Curculionidae), the rice weevil, and Rhyzopertha dominica (F.) (Coleoptera: Bostryhidae), the lesser grain borer, are considered as the main source of insects fragments in wheat flour (Campbell et al. 1976; Pedersen 1992; Perez Mendoza et al. 2005). Nevertheless, there are cases where insect contamination in stored grains and flour is related to whole bodies of insects, such as thrips, that are present in the field before harvest (Locatelli et al. 1993; Perez Mendoza et al. 2005; Trematerra et al. 2011; Bhuvaneswari et al. 2011). Other types of contaminants on wheat, such as weed seeds, may have a serious effect on certain quality characteristics of the commodity (Wrigley 1994; Wilson et al. 2016), while they also affect development of certain stored product insect species (Georgousakis et al. 2020).

Most studies for Aelia and Eurygaster focus on the effects of their feeding activity on the wheat fields, while there is scarce information regarding the effect of the presence of wheat bugs on stored product species. Fourar and Fleurât-Lessard (1997) reported that development of S. oryzae on wheat that had been infested in the field by the wheat bug, Aelia germani Kuster, was seriously affected, due to changes on key properties of the commodity. However, to our knowledge, there are no published data available so far about the effect of the actual presence of wheat bug individuals in stored wheat on the longevity and fecundity of stored product insects. In this context, the aim of the current study was to evaluate in laboratory conditions if the presence of two different wheat bugs, i.e., Aelia spp. and Eurygaster spp., can affect the development and progeny production capacity of three major product beetle species, i.e., the khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae), S. oryzae and R. dominica.

Materials and methods

Collection of the bugs, insects and commodity

Eurygaster spp. and Aelia spp. adults were collected from newly harvested wheat in the area located in Thessaly, Central Greece (Polydameio, region of Farsala), in June 2020. The insects were collected alive from the wheat bulks at the day of harvest and were separated in the laboratory as Aelia or Eurygaster, but were not identified up to the species level. Then, the wheat bugs were frozen at −18 °C for a week. Subsequently, the bugs were held in room temperature for 24 h and then were used for experimentation.

Adults of T. granarium, S. oryzae and R. dominica were taken from already existing insect cultures from the Laboratory of Entomology and Agricultural Zoology (LEAZ), Department of Agriculture, Crop Production and Rural Environment, University of Thessaly. All species were reared in whole wheat kernels in incubators set at 25 ± 1 °C, 60 ± 5% relative humidity (r.h.) and continuous darkness.

Untreated, clean and infestation-free organic soft wheat was used in the tests. The moisture content of the tested grains, as determined by a moisture meter (Multitest, Gode SAS, Le Catelet, France), was approx. 13.0%.

Bioassays

For the experiments, plastic cylindrical vials (3 cm in diameter, 8 cm in height, Rotilabo Sample tins Snap on lid, Carl Roth, Germany) were used, filled with 20 g of wheat. Twenty insects of each species were placed inside the vial either alone or in combination with 1, 2, 3 or 5 adults of each wheat bug (Aelia spp. or Eurygaster spp.), with separate series of vials for each beetle species and for each bug genus. Then, all vials were placed in incubators set at 30 °C, with 65% r.h. and continuous darkness. Each experiment was repeated three times, with three vials for each combination (3 replicates × 3 subreplicates = 9 vials for each combination). Beetle mortality was recorded after 14 and 65 days later, and the vials were opened and examined for progeny production, ratio of damaged kernels and weight of frass as described by Sakka and Athanassiou (2018).

Statistical analysis

For each species the data were submitted to a two-way ANOVA for bug species and combinations of wheat bugs. Progeny production and grain parameters (e.g., grain damage and frass) were analyzed separately for each species by using a two-way ANOVA with treatment and combinations as main effects. Means were separated by the HSD test. For differences between combinations of wheat bug genera Student’s t test at 0.05 was performed. All tests were performed using JPM 8 software (SAS Institute Inc., Cary, NC).

Results

Adult mortality

For T. granarium, only the interaction of bug species and containment was found to be significant (Table 1). Trogoderma granarium showed the highest mortality rate among all species tested after 14 days for all combinations tested (Table 2). Mortality was more than 79% for all combinations of T. granarium with Eurygaster spp. For all combinations with Aelia, mortality was more than 83%. Significant differences were noted in mortality levels between all vials containing Eurygaster and those containing Aelia.

Table 1 ANOVA parameters for adult mortality of T. granarium, S. oryzae and R. dominica after 14 d on wheat contained different numbers of either Eurygaster spp. or Aelia spp. adults (error df = 64)
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Table 2 Mean mortality (% ± SE) of T. granarium, S. oryzae and R. dominica adults after 14 days on wheat contained different numbers of either Eurygaster spp. or Aelia spp. (error df = 1,17); within each column and wheat bug species 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
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For S. oryzae, only bug containment was found to be significant (Table 1). The highest mortality level for S. oryzae was recorded in vials that contained 5 Eurygaster adults (24.4%), while mortality was lower in the vials that contained Aelia, which did not exceed 10% (Table 2). Similarly, as in the case of S. oryzae, for R. dominica only bug containment was significant (Table 1). For this species, the highest mortality (13.3%) was recorded in vials containing 5 Eurygaster adults, but the overall adult mortality was extremely low (Table 2).

Progeny production

No significant effects were recorded in the case of progeny production of T. granarium (Table 3). The highest progeny production for T. granarium was recorded in the vials that contained 3 Aelia adults (58.8 adults/vial) and the lowest in the vials that contained 3 Eurygaster adults (50.8 adults/vial (Table 4).

Table 3 ANOVA parameters for total progeny production of T. granarium, S. oryzae and R. dominica after 65 days on wheat contained different numbers of either Eurygaster spp. or Aelia spp. adults (error df = 64)
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Table 4 Progeny production (mean number of individuals per vial ± SE) of T. granarium, S. oryzae and R. dominica adults after 65 days (error df = 1,17); within each column and wheat bug species means followed by the same uppercase letter are not significantly different; where no letters exist no significant differences are noted; HSD test at 0.05
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Only containment was significant in the case of S. oryzae (Table 3), while progeny production was generally higher in the vials that contained Aelia adults, as compared with those that contained Eurygaster adults (Table 4). Similarly, only containment was significant for R. dominica (Table 3), while, as above, progeny production was higher in the vials that contained Aelia adults (Table 4).

Grain damage

In the case of ratio of damaged grains the two-way interaction of bug species × containment was significant only for S. oryzae. In the case of weight of frass the interaction of bug species × containment was significant only for R. dominica (Table 5). Grain damage and weight of dust were similar between Eurygaster and Aelia for T. granarium for all combinations (Table 6). The levels of frass production were found to be low for all species (Table 6). The ratio of damaged kernels was higher for R. dominica with Aelia than that with Eurygaster. Significant differences were noted for all combinations of wheat bugs between Eurygaster and Aelia for S. oryzae (Table 6). Finally, in the case of R. dominica significant differences were noted only within each of the combinations tested with Aelia.

Table 5 ANOVA parameters for ratio of damaged grains and frass of the three species (T. granarium, S. oryzae and R. dominica) on wheat with different containments of (Eurygaster spp. and Aelia spp. (error df = 64)
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Table 6 Mean ratio of damaged grains/vial and frass (in g/vial) produced; within each column and wheat bug species 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
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Discussion

Apparently, the wheat bug containment that was examined here can be considered as high and cannot be easily recorded in newly harvested grains. Nevertheless, wheat bugs are often recorded in high numbers before harvest on wheat plants and can be found in high numbers on wheat grains after harvest (Reisig et al. 2013; Blandino et al. 2015). While these contaminants are removed from the product before processing, certain interactions with some grain properties may be unavoidable. For instance, Sharma et al. (2020) reported that grasshopper carcasses on wheat can increase the presence of fungi that may endanger human health, given that these carcasses may host fungal species and, indirectly, may contribute to the increase of the moisture content of the grain. However, there were no data available so far for the direct effect of wheat carcasses on the development of stored product beetle species. The results of the present study illustrate that in some cases mortality, progeny production and infestation patterns, expressed as damaged kernels and frass, can be affected by the presence of wheat bugs. Moreover, there were some differences between wheat containing Eurygaster and wheat containing Aelia, but we are unaware for the causes of these differences. Morphologically, Aelia individuals were smaller than those of Eurygaster, so size might have played a role in the space occupied in our experimental vials or the contribution to the increase of moisture content.

One of the key findings is that the effects of wheat bugs were different among the three beetle species tested. For instance, in the case of S. oryzae progeny production was higher with all combinations of wheat containing Aelia in contrast with wheat containing Eurygaster, while wheat bug containment also played a role in progeny production capacity of R. dominica. These two beetle species are considered as primary colonizers of grains and their immature development occurs within the grain kernel (Athanassiou et al. 2005; Edde 2012), so the effects of wheat bug carcasses may only indirectly affect their progeny production capacity. On the other hand, T. granarium is considered as a “dirty feeder” and can develop in insect species’ carcasses (Kavallieratos et al. 2017; Athanassiou et al. 2019), so theoretically, the presence of wheat bugs was expected to have a beneficial effect on the development of this species. Kavallieratos et al. (2017) found that T. granarium could outcompete S. oryzae and R. dominica at elevated temperatures, and when the numbers of T. granarium were high, there were no individuals of the other two species, indicating that the larvae of T. granarium were fed upon the individuals of the primary colonizers. Also, the presence of frass seems to have a beneficial effect on the development of T. granarium larvae, especially in the case of young larvae, which are more prone to develop in cracked materials than whole kernels (Athanassiou et al. 2019).

According to Jian and Zhang (2022) dockage is “any material that can be removed from the grain by using cleaning equipment such as mechanical dockage tester or sieve”. Sinha et al. (1983) tested the quality of clean wheat and wheat plus dockage that was infested by the saw-toothed grain beetle, Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) and the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae), and found differences in fat acidity values and seed germination, germ and endosperm damage, as well as fungal and bacterial infestation. Moreover, high levels of infestation by stored product insects resulted in increased presence of fungal infections by Penicillium and the occurrence of bacteria (Sinha 1983; Hubert et al. 2018). Moreover, Sinha (1975) determined the effect of different percentages of dockage in wheat on stored product insect species and reported that O. surinamensis and the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) prefer to feed on broken kernels and dockage. Nevertheless, interspecific interactions in bulked grains are not necessarily negative and may exhibit a considerable beneficial effect (Nansen et al. 2009; Kavallieratos et al. 2017). For example, Nansen et al. (2004) reported a positive commensal relationship between R. dominica and T. castaneum, that were often detected simultaneously present in the same sampling units, in contrast with C. ferrugineus, which was less likely to be present together with T. castaneum individuals. The present study showed that, in some combinations, progeny production was positively affected by the presence of Aelia, for reasons that may be related to commensal interactions, but also with the size of the Aelia, which might have allowed more space within the vial, as compared with Eurygaster, which is more large-bodied. The presence of dockage and foreign materials can seriously affect insect distribution in bulked wheat, as certain stored product beetle species aggregate on areas with increased dockage containment (Athanassiou and Buchelos 2001, 2020). For instance, in vertical grain silos, Athanassiou and Buchelos (2020) found that certain species tended to concentrated in the central zone of the bulk that contained more dockage, resulting in increased infestation patterns in that zone, but also to more vigorous changes in the temperature and moisture content levels.

Not surprisingly, beetle parental mortality was not affected from the increase in the number of wheat bugs inside the vials and most of the treatments gave similar results. For instance, parental mortality of R. dominica, for both wheat bugs, was similar for all combinations, incl. the control vials. As noted above, both S. oryzae and R. dominica infest the internal part of the grain kernels, so no direct effects with wheat bug containment were expected. In contrast, the increased parental mortality of T. granarium was expected, as this species is short-lived at the adult stage, and usually most of the adults are dead within 14 d (Athanassiou et al. 2019; Gourgouta et al. 2021). In this context, mortality of this particular species, but eventually for the other two species examined, was not affected by the presence of wheat bugs.

In summary, this work is an experimental proof that the presence of wheat bugs can affect the survival and population growth of some species of stored product insects. In fact, we found that the presence of wheat bugs may even support stored product beetle development, such as S. oryzae or R. dominica. Although the wheat bug containment was high and realistically cannot reach the numbers examined here, our results illustrate that there are certain interactions between these carcasses and stored product beetles, that can be taken into account, at the post-harvest stages of wheat, and probably on other grains that are infested by wheat bugs.