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Combining larval parasitoids and an entomopathogenic fungus for biological control of Sitophilus granarius (Coleoptera: Curculionidae) in stored grain
Biological Control 40 (2007) 237–242 www.elsevier.com/locate/ybcon
Combining larval parasitoids and an entomopathogenic fungus for biological control of Sitophilus granarius (Coleoptera: Curculionidae) in stored grain Lise Stengård Hansen ¤, Tove Steenberg Danish Institute of Agricultural Sciences, Department of IPM, Danish Pest Infestation Laboratory, Skovbrynet 14, DK-2800 Kgs. Lyngby, Denmark Received 22 June 2006; accepted 25 September 2006 Available online 11 October 2006
Abstract The potential of combining diVerent natural enemies for biological control of Sitophilus granarius (L.) in grain was investigated in a laboratory study. We compared the eVect of two species of larval parasitoids, Lariophagus distinguendus Förster and Anisoptermalus calandrae (Howard), alone or in combination with a surface treatment of the grain with the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin against S. granarius in units containing 9 kg of wheat over a period of 26 weeks. In the untreated units, the pest population increased about 5000 times. The highest level of pest suppression (>99.9%) was obtained in units with L. distinguendus, followed by units with A. calandrae. In units with both parasitoids and the entomopathogen, the pest suppression level was 83–98%. Although the parasitoids were negatively aVected by the fungus, they still exerted some degree of control compared to the untreated units. © 2006 Elsevier Inc. All rights reserved. Keywords: Sitophilus granarius; Lariophagus distinguendus; Anisopteromalus calandrae; Beauveria bassiana; Biological control; Grain stores
1. Introduction The granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), is an important pest of stored cereals in cool temperate areas, e.g., northwest Europe, and in highland areas in the tropics (Rees, 1996). The juvenile stages of S. granarius develop concealed within cereal kernels, each larva consuming approximately half of a wheat kernel (Hurlock, 1965). Insect infestations in stored grain lead to production of heat and moisture, thus promoting fungal growth (Christensen and Hodson, 1960; Howe, 1961). The protected environment in grain stores and long storage time appear to be ideal for successful application of biological control. An early study described the eVect of the larval parasitoid Lariophagus distinguendus Förster (Hymenoptera: Pteromalidae) on S. granarius and S. oryzae (L.)
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(Ryabov, 1926 , referred by Brower et al., 1996). More recently, this parasitoid species has been investigated as a candidate for biological control of other species of Sitophilus in rice and maize, e.g., S. oryzae in rice (Ryoo et al., 1991). Lucas and Riudavets (2002) compared the eYcacy of L. distinguendus and another larval parasitoid, Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae), for control of S. oryzae in rice and found a pest suppression level of 98 and 79%, respectively. The potential of A. calandrae for controlling S. zeamias Motschulsky in maize was investigated by Williams and Floyd (1971), who found that the pest density was reduced to 5% in the presence of A. calandrae. Cline et al. (1985) concluded that early introductions of A. calandrae could prevent residual populations of S. oryzae in wheat from infesting adjacent commodities. Subsequently, Press (1992) found that A. calandrae exerted some degree of control of S. oryzae situated down to a depth of 1.9 m in a column of wheat. Steidle and Schöller (2002) found that L. distinguendus located and parasitized S. granarius at a distance of 4 m vertically and horizontally
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from the release point in a wheat store, whereas Reppchen et al. (2002) found that release of L. distinguendus against S. granarius at a density of 1:1 in wheat resulted in a 92–94% suppression of the weevil population after 28 weeks. In northern temperate areas, e.g., Denmark, grain temperatures are reduced by aeration to below 15 °C within a few months after harvest. This temperature may be below the lower limit for parasitoid activity, although very little information exists on their development and reproduction at temperatures below 18 °C. L. distinguendus can develop and reproduce at 16 °C, albeit very slowly (Hansen, in press). Ryoo et al. (1991) calculated a lower development threshold of L. distinguendus of 15.1 and 15.2 °C for females and males, respectively. Smith (1992) estimated a lower limit of positive population growth of 20 °C for A. calandrae. S. granarius produce progeny at temperatures down to 15 °C (Fields, 1992) or lower. In Denmark, only short time is available before the grain temperature falls below the lower temperature threshold of the parasitoids and they alone may not be able to reduce pest populations suYciently before winter. Use of an entomopathogenic fungus to reduce the initial density of adult S. granarius shortly after they colonize the grain after harvest may lead to a substantial reduction of the oVspring produced and subsequently, of the overwintering pest population. Several species of entomopathogenic fungi have been tested against adult stages of diVerent stored product pests (e.g., Lord, 2005; Batta, 2004; Rice and Cogburn, 1999). Although most studies have been carried out in the laboratory, it is likely that fungal entomopathogens may hold some biocontrol potential, especially when combined with other control measures. Our study compared the potential of two species of parasitoids, A. calandrae and L. distinguendus, alone and in combination with a surface treatment of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin (Ascomycota: Hypocreales) against S. granarius in units containing 9 kg of wheat. 2. Materials and methods 2.1. Insects Sitophilus granarius was reared on whole-wheat kernels under controlled climate conditions (25 °C, 70% RH, and L:D 16:8). A colony of L. distinguendus was kindly provided by M. Schöller (Biologische Beratung, Berlin, Germany), and A. calandrae was kindly provided by J. Steidle (Universität Hohenheim, Stuttgart, Germany). The parasitoids were reared on infested wheat kernels under the same conditions as above: 200 adult parasitoids, 0–14 days after emergence, were transferred to plastic containers with ventilated lids and 100 g of wheat kernels infested with S. granarius larvae aged up to 21 days. 2.2. Fungal pathogens Beauveria bassiana isolate 678, originally from a house Xy (Musca domestica L. (Diptera: Muscidae)) in Denmark
and deposited in the isolate collection of the Danish Pest Infestation Laboratory, was cultured on plates with 2% Sabouraud dextrose agar (SDA) for 2 weeks at 25 °C. The plates were then air-dried over night and conidia harvested into glass tubes by vacuum. Conidia in glass tubes were dried further by placing tubes over silica gel and then stored at 5 °C over silica gel until used. Prior to the experiment germination of the conidial powder was determined in the microscope after placing dry conidia on plates with SDA, incubating for 18 h at 22 °C and then adding a few drops of sterile 0.02% Tween 80. This was done to allow the dry conidia to imbibe water slowly and thus avoid the problems previously encountered when conidia were placed directly in liquid, as this in some cases prevented germination. Germination was scored on two plates of agar by observing 100 conidia on each plate, selecting 4–5 areas on each plate randomly. Positive germination was determined when the length of the germ tube measured at least that of the diameter of the conidia. This isolate has previously been shown to be virulent to S. granarius when applied to grain in high concentrations (Athanassiou et al., in press). 2.3. Experimental design The experimental plan consisted of 60 units with 9 kg of infested grain. Two species of larval parasitoids and the fungal pathogen were added, alone and in combination (Table 1), and densities of weevils and parasitoids were determined after 16, 20, or 26 weeks, three replicates each time. In addition, an extra three replicates of treatments Sg (i.e., S. granarius alone) and Bb (i.e., S. granarius and B. bassiana) were set up and investigated after 7 weeks, to determine the eVect of the entomopathogen on the initially added weevils. Each unit was a plastic container (11.5 l) with two ventilation holes (4 cm in diameter) in the bottom covered with Wne metal mesh (sieve mesh no. 60) and Wlter paper. Each container was Wlled with 9 kg organic wheat (Triticum aestivum L., variety: Vinjett) previously frozen at ¡30 °C for 5 days to eliminate pests. The moisture content of the wheat was approximately 13.2%. A Xexible tube was inserted into the lid and connected to a vacuum pump to provide moderate aeration (estimated airXow: 3l kg¡1 h¡1). The containers were placed on shelves on small wooden blocks so that air could pass via the ventilation holes in the bottom and up through the grain. At the set up of the investigation, B. bassiana was added to units Bb and BbLd (i.e., S. granarius and B. bassiana and L. distinguendus) and BbAc (i.e., S. granarius, B. bassiana, and A. calandrae). To each container 0.1 g dry conidia were mixed with a small amount of whole-wheat Xour (5 g/container) and sprinkled on top of the grain, leaving 2 cm of clean grain along the perimeter. This was done to mimic the practice of treating grain surfaces with chemical insecticides, as most adult weevils are found in the surface layer. Furthermore, attempting to simulate a practical application scenario, where some areas are
L.S. Hansen, T. Steenberg / Biological Control 40 (2007) 237–242
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Table 1 Experimental design and time plan for addition of natural enemies to units with 9 kg of wheat Units Sg Bb Ld
Untreated control: Sitophilus granarius alone S. granariusa and Beauveria bassiana S. granariusa and Lariophagus distinguendus
Ac
S. granariusa and Anisopteromalus calandrae
BbLd
S. granariusa, B. bassiana and L. distinguendus
BbAc
S. granariusa, B. bassiana and A. calandrae
a
Week No.
Treatments
0 0 3 5 3 5 0 3 5 0 3 5
18 imagines (unsexed) per unit; all units Surface application of 2 £ 106 conidia per g grain 5 female and 4 male parasitoids per unit 2 female and 2 male parasitoids per unit 5 female and 4 male parasitoids per unit 2 females and 2 male parasitoids per unit Surface application of 2 £ 106 conidia per g grain 5 female and 4 male parasitoids per unit 2 female and 2 male parasitoids per unit Surface application of 2 £ 106 conidia per g grain 5 female and 4 male parasitoids per unit 2 female and 2 male parasitoids per unit
The pest, Sitophilus granarius was added to all units at the start of the investigation (week 0), as described for the Sg units.
bound to be left untreated, the perimeter of the surface was left untreated to give weevils the opportunity to avoid the inoculated area. As 1 g dry conidia contained 1.8 £ 1011 conidia, the Xour mixture added to each container contained 1.8 £ 1010 conidia, providing an overall concentration of 2 £ 106 conidia/g grain. All containers were then infested with 18 adult, unsexed S. granarius, 7– 14 days after emergence. Sex ratio was assumed to be 1:1 (Richards, 1947). They were placed on top of the grain (in the untreated perimeter of the fungus treated units). After 3 and 5 weeks parasitoids were added to units Ld (i.e,. S. granarius and L. distinguendus), Ac (i.e., S. granarius and A. calandrae), BbLd and BbAc (numbers are given in Table 1). At this time, the pest larvae are assumed to have reached the stage that is preferred by the parasitoid for parasitization. Adult parasitoids, <72 h after emergence, were transferred to a plastic tube (length 70 mm, diameter 10 mm) which was inserted into the side of the plastic container above the grain surface. The parasitoids could then walk into the container. The containers were placed in a controlled climate chamber at 19.7 § 0.07 °C (SD), 72.0 § 0.7% relative humidity and a photoperiod of 16:8 (L:D). Temperature in the grain was monitored in one unit of each type by the means of Tinyview range H loggers (Gemini Data Loggers Ltd, Chichester, West Sussex, UK). Population densities were determined in units incubated for 16, 20, and 26 weeks, respectively, by separating the grain from the adult weevils, parasitoids and dust on a laboratory grain cleaner (type LAS, Westrup A/S, Slagelse, Denmark). Live and dead insects were separated by aspiration. S. granarius were either counted or quantiWed on a weight basis. Grain moisture content in each unit was measured on a Grainmaster moisture meter (Protimeter, Bucks, UK). To determine the densities of immatures, the grain was returned to the container and incubated for an additional 64 days. During this time, any juveniles (eggs, larvae, and pupae) present at the time of sieving would have developed into adults, but their oVspring would still be immatures within the kernels. They were then quantiWed and examined as described above.
Data on the weight of the grain prior to sieving after 26 weeks, of the grain prior to the Wnal sieving 64 days later and of the material (dust, frass) removed at the Wrst sieving were used to calculate the weight loss of the grain that took place during the investigation. Dead weevils and parasitoids were screened for presence of fungus by incubating them on moist Wlter paper for 1 week. As the numbers of dead insects increased dramatically during the experiment, a sub sample of 50 specimens was taken for incubation. Furthermore, fungus prevalence in live weevils was determined by incubating 25 weevils per container in a petri dish with 7 g wheat for 14 days at 20 °C after which dead weevils were placed on moist Wlter paper and incubated as described above. This incubation of dead and live insects was made to check the cause of mortality among pests and parasitoids in the containers and also to provide an additional check of the eYcacy of the fungus over time. The pest densities after 26 weeks (immatures and imagines, live and dead, log-transformed), the weight losses (log-transformed), and the moisture contents of the grain were compared using PROC GLM (SAS Institute, 2000) and a Student’s–Newman–Keul’s test to test for diVerences. 3. Results Table 2 shows the pest densities (immatures and imagines) after 26 weeks and includes both live and dead S. granarius. These data were used for the statistical analyses as each individual represents a destroyed wheat kernel. SigniWcant diVerences were found between the pest density in the untreated controls and all treatments except units with B. bassiana alone, as well as among all treatments with parasitoids (F D 87.15; df D 5, 18; P < 0.001). In the untreated control units the S. granarius population increased almost 5000 times in 26 weeks, whereas in the units with the lowest pest density (Ld units), the pest population had only increased 4-fold. Addition of parasitoids led to a population suppression of 83 to >99%, greatest in units with L. distinguendus. Weight losses of the grain
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Table 2 Total pest densities (dead and live individuals, all stages), pest population suppression, and weight loss of grain (mean § SD)
Sitophilus granarius per kg (all stages) Suppression, % Weight loss, g per kgb
Untreated control (Sg)a
Beauveria bassiana (Bb)
B.bassiana and A. calandrae (BbAc)
B. bassiana and L. distinguendus (BbLd)
Anisopteromalus calandrae (Ac)
Lariophagus distinguendus (Ld)
9574.8 § 1251.4a
8806.8 § 2189.7a
1652.9 § 1374.3b
207.7 § 125.7c
54.8 § 4.3d
8.8 § 5.8e
— 374.4 § 35.7a
8.02 290.6 § 102.2a
82.74 113.3 § 47.5b
97.83 22.9 § 8.6c
99.43 5.1 § 4.5d
99.96 4.3 §0.1d
Initial pest density: 2 Sitophilus granarius per kg. Imagines removed after 26 weeks at 20 °C. Juveniles reared out and counted 64 days later. Weight loss based on data from the Wnal examination. Means in the same row followed by diVerent letters are signiWcantly diVerent (P < 0.001, Student’s–Newman–Keul’s test). a For treatment details see Table 1. b Weight losses have been calculated as the loss per kg grain, so they can be compared to the pest density per kg.
ranged from 60.5% (Ld and Ac units) to 37% (Sg units); signiWcant diVerences were found between weight loss in all units with parasitoids compared to the untreated control and Bb units (F D 49.60; df D 5, 18; P < 0.001). After 22 weeks, temperatures reached almost 35 °C in the two units with the highest pest densities (Sg and Bb). An intermediate temperature increase up to 27 °C was seen in the BbAc unit after 24 weeks, whereas temperatures remained below 23 °C in the BbLd unit. Hardly any temperature increase was seen in the Ld and Ac units. The moisture content of the grain was between 13 and 15% at the examinations after 16 and 20 weeks. On the Wnal examination the grain in the Sg units was caked together with storage fungi, and the moisture content was very high, but it could not be determined. In the Bb units the moisture content was 19%, whereas in the rest of the units the moisture ranged between 13 and 14%. The pest density in the untreated controls can be related to the actual damage exerted by the pests. One kilogram of this variety of wheat contains close to 30,000 kernels; thus, with a pest population of close to 10,000/kg grain in the Sg units at the Wnal examination, one third of the kernels were destroyed, resulting in a weight loss of 37%. In comparison, in the Ld units, only 9 kernels/kg were destroyed. The densities of live adult weevils and immatures are shown in Fig. 1. In the Sg units and the Bb units, the densities of S. granarius increased with time. In units with parasitoids, both with and without B. bassiana, a decrease in the numbers of immatures was observed after 20 and/or 26 week. In the Ld units, the Wnal density of immatures was very low, 0.3 per kg as opposed to 6.9 imagines/kg in these units. Prior to the experiment, 100% germination was found in the dry conidia of B. bassiana. When the initially added granary weevils were harvested from the extra Bb units after 7 weeks, 65% were dead (N D 54). Ninety-eight percentage of these dead weevils supported sporulating fungus when placed in humid conditions, showing the initial eYcacy of the added fungus. In comparison, in the untreated containers (Sg), 6% of the weevils had died after 7 weeks (N D 53), none of which were infected by the fungus. Moist incubation of dead granary weevils and parasitoids found in the fungus treated containers showed that 100% of the incu-
Fig. 1. Densities of live imagines and immatures (larvae and pupae) of Sitophilus granarius (insects per kg grain (§SD)) in units of wheat with diVerent combinations of natural enemies. See Table 1 for explanation of the legend.
bated specimens harbored fungus until 20 weeks. After that time, lower proportions proved to be killed by the fungus: 25–50% of the weevils and 49–50% of the parasitoids. This documents that the fungus kills the pest but also both species of parasitoids, and also shows that the fungus remains viable within the cadavers accumulating over time in the experimental containers. Among live weevils collected from the containers and incubated for another 2 weeks, negligible proportions were found to be infected by the fungus (means of fungus treated containers: 6% at 16 weeks, 6% at 20 weeks, and 2% at 26 weeks). These results show that over time, the fungal conidia retain their activity, albeit at very reduced levels compared to the initial activity measured at 7 weeks. The high moisture content found in units with many S. granarius at the Wnal examination obviously did not promote the growth of B. bassiana.
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4. Discussion The objective of this study was to describe the eVects of four diVerent methods for biological control in stored wheat in a type of semi-Weld trial, i.e., units containing larger amounts of grain than a small laboratory trial would use. The units were to be kept for 6 months and the initial pest density was to be low, to simulate the situation in a grain store. The highest level of suppression was obtained by the means of the parasitoid L. distinguendus alone, followed by A. calandrae alone. In units with parasitoids in combination with B. bassiana, the pest population was signiWcantly lower than the untreated controls, but even at the very high inoculum level used, the suppression was too small to be acceptable for practical application. This may be due to factors such as long lethal time of the fungal strain, so that it took some time to kill the pest, thus allowing eggs to be deposited. Furthermore, the low persistence of fungal conidia in grain over longer periods of time plays a role. The fungus seems to have aVected the parasitoids negatively, and this was veriWed by the dead parasitoids producing fungal outgrowth. Beauveria bassiana has a wide host range (Tanada and Kaya, 1993) and it is not surprising that the parasitoids were susceptible to infection. In some cases, however, parasitoids may be susceptible to infection by entomopathogenic fungi but still continue to reproduce, thus allowing fungi and parasitoids to be used simultaneously (Nielsen et al., 2005). In contrast, our study clearly indicates that the two types of biocontrol agents cannot be integrated if the grain is treated with a surface treatment of fungus. However, the combined treatments of parasitoids and fungus produced signiWcantly lower numbers of S. granarius compared to the fungus treatment alone. This emphasizes that both species of parasitoids—despite being susceptible to fungus infection—must have been able to reproduce to some extent. If the fungus could be combined with a potent attractant in a trapping device so parasitoids are not exposed to the fungus inoculum while weevils become inoculated upon visiting the trap, it might still be possible to combine the two biocontrol agents. Such targeted treatment, where the fungus is applied in a trap rather than by treating large surfaces, might also have the advantage of reducing the exposure of humans or livestock to fungus-derived residues when feeding on treated commodities, although a substantial part of the fungus inoculum is likely to be removed during cleaning processes carried out prior to milling of grain. Cleaning procedures will also remove the parasitoids from the grain. Use of parasitoids in stored grain may even lead to a reduction in the number of insect fragments in Xour (Flinn and Hagstrum, 2000). The weight loss of the grain in the untreated (Sg) units was 374 g/kg, i.e., 37% of the original weight. This Wgure was based on the weight of the grain at the Wnal examination before weevils and frass were removed. Other studies on the weight loss caused by S. granarius in wheat report similar values. Hurlock (1965) reported that a mean of 214 weevils resulted in a weight loss of 6 g. If converted to the
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same densities as in our study, this corresponds to 275 g, which is a bit lower than we found. Richards (1947) found that 193 weevils led to a loss in dry weight of 3 g, again a little lower than our study. However, the diVerences can be due a number of factors such as diVerences in experimental design, kernel size, pest density, weighing procedures, or whether or not beetles and frass were included, etc. In addition, these two studies were conducted with small amounts of grain and for short periods of time, and the results have a diVerent level of precision. Reppchen et al. (2002) used slightly higher numbers of parasitoids than in our study and achieved a lower level of pest suppression; a maximum of 94% suppression using 2.5 female parasitoids per female S. granarius. In our study the ratio between female parasitoids and female S. granarius was 0.8 (sex ratio of S. granarius 1:1). The greatest suppression in our study, when using data comparable to theirs, i.e., dead and live weevils, but not juveniles, was >99.7% found in the Ld units (2889.3 § 811.4 weevils/kg in the untreated controls, and 8.1 § 5.4 in the Ld units). Lucas and Riudavets (2002) compared the eVect of L. distinguendus and A. calandrae on S. oryzae in rice in a 6week trial at 25 °C. They found the highest suppression (98%) with L. distinguendus. Smith (1994) constructed a simulation model to evaluate diVerent strategies for using A. calandrae for biological control of S. zeamais at 25 °C. When applying a comparable number of parasitoids as in our study, Smith (1994) calculated that the pest population was reduced by 95.3% after 17 weeks. It is encouraging to note the high pest suppression obtained in our study at 19.7 °C. This condition is relatively less favorable for the parasitoids than for the cold-hardy S. granarius. This result contradicts the calculated lower threshold for positive population growth in A. calandrae of 20 °C (Smith, 1994). Even though our study was not replicated in time, the conclusions are considered to be thoroughly supported by the fact that (1) the pest population was sampled three times during the course of 6 months, (2) the variation in population densities in units with the same treatment was very small on all sampling occasions, and (3) diVerences between treatments were highly signiWcant (Table 2 and Fig. 1). Further support can be found in the following: the Wnal pest density in the untreated units in our study is surprisingly similar to the one found by Reppchen et al. (2002). That is, we had 2889.3 S. granarius/kg, dead and live adults, as opposed to their Wnding of 4349/kg with a longer test duration (28 weeks) and higher initial numbers of weevils (5 female S. granarius/kg). Finally, the degree of pest suppression exerted by the parasitoids is similar to the one found by Reppchen et al. (2002) and comparable to the suppression found by Lucas and Riudavets (2002) and Smith (1994). The diVerence between the eVects of each of the parasitoids was signiWcant. Even though the suppression level with both parasitoids was above 99%, the diVerence between the two species is important for practical application. With A. calandrae, the pest density increased more than 25 times
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from an initial density of 2 weevils/kg to 54.8/kg, compared with the 4-fold increase when L. distinguendus was used (from 2 to 8.8/kg). Furthermore, there were very few immatures in the units with L. distinguendus at 26 weeks. As increasing populations typically have a predominance of immatures (Birch, 1948), our result suggests that the pest population in the Ld units was about to crash. These diVerences may be decisive for which parasitoid species is best suited for practical application. Further studies are necessary to elucidate the potential of parasitoids for biocontrol of S. granarius under the inXuence of the low temperature regimes found in grain stores in northern Europe. Acknowledgments We thank Lars Damberg, Bodil M. Pedersen, Minna Wernegreen, and Claus Dahl for technical assistance and Henrik Skovgård for useful comments on the manuscript. The project was supported by the Danish Ministry of Food, Agriculture and Fisheries, DLG (Danish Cooperative Farm Supply) and the Association of Danish Millers. References Athanassiou, C.G., Steenberg, T., Kavallieratos, N.G., in press. Insecticidal eVect of diatomaceous earth applied alone or in combination with Beauveria bassiana and beta cyXuthrin against Sitophilus granarius in stored wheat. Bulletin IOBC/wprs. Batta, Y.A., 2004. Control of rice weevil (Sitophilus oryzae L., Coleoptera: Curculionidae) with various formulations of Metarhizium anisopliae. Crop Protect. 23, 103–108. Birch, L.C., 1948. The intrinsic rate of natural increase of an insect population. J. Anim. Ecol. 17, 15–26. Brower, J.H., Smith, L., Vail, P.V., Flinn, P.F., 1996. Biological control. In: Subramanyam, B., Hagstrum, D.W. (Eds.), Integrated management of insects in stored products. Marcel Dekker Inc. New York, New York, pp. 223–286. Christensen, C.M., Hodson, A.C., 1960. Development of granary weevil and storage fungi in columns of wheat – II. J. Econ. Entomol. 53, 375–380. Cline, L.D., Press, J.W., Flaherty, B.R., 1985. Suppression of the rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae), inside and outside of burlap, woven polypropylene, and cotton bags by the parasitic wasp Anisopteromalus calandrae (Hymenoptera: Pteromalidae). J. Econ. Entomol. 78, 835–838. Fields, P.G., 1992. The control of stored-product insects and mites with extreme temperatures. J. Stored Prod. Res. 28, 89–118. Flinn, P., Hagstrum, D.W., 2000. Augmentative releases of parasitoid wasps in stored wheat reduces insect fragments in Xour. J. Stored Prod. Res. 37, 179–186. Howe, R.W., 1961. A study of the heating of stored grain caused by insects. Ann. Appl. Biol. 50, 137–158. Hansen, L.S., in press. Biocontrol potential of Lariophagus distinguendus (Hym.: Pteromalidae) against Sitophilus granarius (Col.: Curculionidae) at low temperatures—reproduction and parasitoid induced mortality. J. Econ. Entomol.
Hurlock, E.T., 1965. Some observations on the loss in weight caused by Sitophilus granarius (L.) (Coleoptera, Curculionidae) to wheat under constant conditions. J. Stored Prod. Res. 1, 193–195. Lord, J.C., 2005. Low humidity, moderate temperature, and desiccant dust favor eYcacy of Beauveria bassiana (Hyphomycetes: Moniliales) for the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bruchidae). Biol. Control 34, 180–186. Lucas, É., Riudavets, J., 2002. Biological and mechanical control of Sitophilus oryzae (Coleoptera: Curculionidae) in rice. J. Stored Prod. Res. 38, 293–304. Nielsen, C., Skovgård, H., Steenberg, T., 2005. EVect of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) on survival and reproduction of the Wlth Xy parasitoid, Spalangia cameroni (Hymenoptera: Pteromalidae). Environ. Entomol. 34, 133–139. Press, J.W., 1992. Comparative penetration eYcacy in wheat between the weevil parasitoids Anisopteromalus calandrae and Choetospila elegans (Hymenoptera: Pteromalidae). J. Entomol. Sci. 27, 154–157. Rees, D.P., 1996. Coleoptera. In: Subramanyam, B., Hagstrum, D.W. (Eds.), Integrated management of insects in stored products. Marcel Dekker Inc., New York, New York, pp. 1–39. Reppchen, A., Schöller, M., Prozell, S., Adler, C., Reichmuth, C., Steidle, J., 2002. The granary weevil Sitophilus granarius is suppressed by the parasitoid Lariophagus distinguendus (Hymenoptera: Pteromalidae). In: Credland, P.F., Armitage, D.M., Bell, C.H., Cogan, P.M., Highly, E. (Eds.), Advances in Stored Product Protection. Proceedings of the 8th International Working Conference on Stored Product Protection. 22– 26 July 2002, York, UK. CAB International, Oxon UK, pp. 230–232. Rice, W.C., Cogburn, R.R., 1999. Activity of the entomopathogenic fungus Beauveria bassiana (Deuteromycota: Hyphomycetes) against three coleopteran pests of stored grain. J. Econ. Entomol. 92, 691–694. Richards, O.W., 1947. Observations on grain weevils, Calandra (Col. Curculionidae). I. General biology and oviposition. Proc. Zool. Soc. Lond. 117, 1–43. Ryabov, M.A., 1926. The possibilities of applying the parasitic method of control in the case of granary pests. Bull. N. Caucasian Plant Prot. Sts. 1, 19–54. Ryoo, M.I., Hong, Y.S., Yoo, C.K., 1991. Relationship between temperature and development of Lariophagus distinguendus (Hymenoptera: Pteromalidae), an ectoparasitoid of Sitophilus oryzae (Coleoptera. Curculionidae). J. Econ. Entomol. 84, 825–829. SAS Institute. 2000. SAS Users’ Guide, release 8.01. SAS Institute, Cary, NC, USA. Smith, L., 1992. EVect of temperature on life history characteristics of Anisopteromalus calandrae (Hymenoptera: Pteromalidae) parasitizing maize weevil larvae in corn kernels. Environ. Entomol. 21, 877–887. Smith, L., 1994. Computer simulation model for biological control of maize weevil by the parasitoid Anisopteromalus calandrae. In: Highley, E.J., Wright, E.J., Banks, H.J., Champ, B.R. (Eds.), Proceedings of the 6th International Working Conference on Stored Product Protection. 17–23 April, 1994. Canberra, Australia. CAB International, Oxon, UK, pp. 1147–1151. Steidle, J., Schöller, M., 2002. Fecundity and ability of the parasitoid Lariophagus distinguendus (Hymenoptera: Pteromalidae) to Wnd larvae of the granary weevil Sitophilus granarius (Coleoptera: Curculionidae) in bulk grain. J. Stored Prod. Res. 38, 43–53. Tanada, Y., Kaya, H.K., 1993. Insect Pathology. Academic Press, Inc., San Diego, CA. Williams, R.N., Floyd, E.H., 1971. EVect of two parasites, Anisopteromalus calandrae and Choetospila elegans, upon populations of the maize weevil under laboratory and natural conditions. J. Econ. Entomol. 64, 1407–1408.
Combining larval parasitoids and an entomopathogenic fungus for biological control of Sitophilus granarius (Coleoptera: Curculionidae) in stored grain Lise Stengård Hansen ¤, Tove Steenberg Danish Institute of Agricultural Sciences, Department of IPM, Danish Pest Infestation Laboratory, Skovbrynet 14, DK-2800 Kgs. Lyngby, Denmark Received 22 June 2006; accepted 25 September 2006 Available online 11 October 2006
Abstract The potential of combining diVerent natural enemies for biological control of Sitophilus granarius (L.) in grain was investigated in a laboratory study. We compared the eVect of two species of larval parasitoids, Lariophagus distinguendus Förster and Anisoptermalus calandrae (Howard), alone or in combination with a surface treatment of the grain with the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin against S. granarius in units containing 9 kg of wheat over a period of 26 weeks. In the untreated units, the pest population increased about 5000 times. The highest level of pest suppression (>99.9%) was obtained in units with L. distinguendus, followed by units with A. calandrae. In units with both parasitoids and the entomopathogen, the pest suppression level was 83–98%. Although the parasitoids were negatively aVected by the fungus, they still exerted some degree of control compared to the untreated units. © 2006 Elsevier Inc. All rights reserved. Keywords: Sitophilus granarius; Lariophagus distinguendus; Anisopteromalus calandrae; Beauveria bassiana; Biological control; Grain stores
1. Introduction The granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), is an important pest of stored cereals in cool temperate areas, e.g., northwest Europe, and in highland areas in the tropics (Rees, 1996). The juvenile stages of S. granarius develop concealed within cereal kernels, each larva consuming approximately half of a wheat kernel (Hurlock, 1965). Insect infestations in stored grain lead to production of heat and moisture, thus promoting fungal growth (Christensen and Hodson, 1960; Howe, 1961). The protected environment in grain stores and long storage time appear to be ideal for successful application of biological control. An early study described the eVect of the larval parasitoid Lariophagus distinguendus Förster (Hymenoptera: Pteromalidae) on S. granarius and S. oryzae (L.)
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(Ryabov, 1926 , referred by Brower et al., 1996). More recently, this parasitoid species has been investigated as a candidate for biological control of other species of Sitophilus in rice and maize, e.g., S. oryzae in rice (Ryoo et al., 1991). Lucas and Riudavets (2002) compared the eYcacy of L. distinguendus and another larval parasitoid, Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae), for control of S. oryzae in rice and found a pest suppression level of 98 and 79%, respectively. The potential of A. calandrae for controlling S. zeamias Motschulsky in maize was investigated by Williams and Floyd (1971), who found that the pest density was reduced to 5% in the presence of A. calandrae. Cline et al. (1985) concluded that early introductions of A. calandrae could prevent residual populations of S. oryzae in wheat from infesting adjacent commodities. Subsequently, Press (1992) found that A. calandrae exerted some degree of control of S. oryzae situated down to a depth of 1.9 m in a column of wheat. Steidle and Schöller (2002) found that L. distinguendus located and parasitized S. granarius at a distance of 4 m vertically and horizontally
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from the release point in a wheat store, whereas Reppchen et al. (2002) found that release of L. distinguendus against S. granarius at a density of 1:1 in wheat resulted in a 92–94% suppression of the weevil population after 28 weeks. In northern temperate areas, e.g., Denmark, grain temperatures are reduced by aeration to below 15 °C within a few months after harvest. This temperature may be below the lower limit for parasitoid activity, although very little information exists on their development and reproduction at temperatures below 18 °C. L. distinguendus can develop and reproduce at 16 °C, albeit very slowly (Hansen, in press). Ryoo et al. (1991) calculated a lower development threshold of L. distinguendus of 15.1 and 15.2 °C for females and males, respectively. Smith (1992) estimated a lower limit of positive population growth of 20 °C for A. calandrae. S. granarius produce progeny at temperatures down to 15 °C (Fields, 1992) or lower. In Denmark, only short time is available before the grain temperature falls below the lower temperature threshold of the parasitoids and they alone may not be able to reduce pest populations suYciently before winter. Use of an entomopathogenic fungus to reduce the initial density of adult S. granarius shortly after they colonize the grain after harvest may lead to a substantial reduction of the oVspring produced and subsequently, of the overwintering pest population. Several species of entomopathogenic fungi have been tested against adult stages of diVerent stored product pests (e.g., Lord, 2005; Batta, 2004; Rice and Cogburn, 1999). Although most studies have been carried out in the laboratory, it is likely that fungal entomopathogens may hold some biocontrol potential, especially when combined with other control measures. Our study compared the potential of two species of parasitoids, A. calandrae and L. distinguendus, alone and in combination with a surface treatment of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin (Ascomycota: Hypocreales) against S. granarius in units containing 9 kg of wheat. 2. Materials and methods 2.1. Insects Sitophilus granarius was reared on whole-wheat kernels under controlled climate conditions (25 °C, 70% RH, and L:D 16:8). A colony of L. distinguendus was kindly provided by M. Schöller (Biologische Beratung, Berlin, Germany), and A. calandrae was kindly provided by J. Steidle (Universität Hohenheim, Stuttgart, Germany). The parasitoids were reared on infested wheat kernels under the same conditions as above: 200 adult parasitoids, 0–14 days after emergence, were transferred to plastic containers with ventilated lids and 100 g of wheat kernels infested with S. granarius larvae aged up to 21 days. 2.2. Fungal pathogens Beauveria bassiana isolate 678, originally from a house Xy (Musca domestica L. (Diptera: Muscidae)) in Denmark
and deposited in the isolate collection of the Danish Pest Infestation Laboratory, was cultured on plates with 2% Sabouraud dextrose agar (SDA) for 2 weeks at 25 °C. The plates were then air-dried over night and conidia harvested into glass tubes by vacuum. Conidia in glass tubes were dried further by placing tubes over silica gel and then stored at 5 °C over silica gel until used. Prior to the experiment germination of the conidial powder was determined in the microscope after placing dry conidia on plates with SDA, incubating for 18 h at 22 °C and then adding a few drops of sterile 0.02% Tween 80. This was done to allow the dry conidia to imbibe water slowly and thus avoid the problems previously encountered when conidia were placed directly in liquid, as this in some cases prevented germination. Germination was scored on two plates of agar by observing 100 conidia on each plate, selecting 4–5 areas on each plate randomly. Positive germination was determined when the length of the germ tube measured at least that of the diameter of the conidia. This isolate has previously been shown to be virulent to S. granarius when applied to grain in high concentrations (Athanassiou et al., in press). 2.3. Experimental design The experimental plan consisted of 60 units with 9 kg of infested grain. Two species of larval parasitoids and the fungal pathogen were added, alone and in combination (Table 1), and densities of weevils and parasitoids were determined after 16, 20, or 26 weeks, three replicates each time. In addition, an extra three replicates of treatments Sg (i.e., S. granarius alone) and Bb (i.e., S. granarius and B. bassiana) were set up and investigated after 7 weeks, to determine the eVect of the entomopathogen on the initially added weevils. Each unit was a plastic container (11.5 l) with two ventilation holes (4 cm in diameter) in the bottom covered with Wne metal mesh (sieve mesh no. 60) and Wlter paper. Each container was Wlled with 9 kg organic wheat (Triticum aestivum L., variety: Vinjett) previously frozen at ¡30 °C for 5 days to eliminate pests. The moisture content of the wheat was approximately 13.2%. A Xexible tube was inserted into the lid and connected to a vacuum pump to provide moderate aeration (estimated airXow: 3l kg¡1 h¡1). The containers were placed on shelves on small wooden blocks so that air could pass via the ventilation holes in the bottom and up through the grain. At the set up of the investigation, B. bassiana was added to units Bb and BbLd (i.e., S. granarius and B. bassiana and L. distinguendus) and BbAc (i.e., S. granarius, B. bassiana, and A. calandrae). To each container 0.1 g dry conidia were mixed with a small amount of whole-wheat Xour (5 g/container) and sprinkled on top of the grain, leaving 2 cm of clean grain along the perimeter. This was done to mimic the practice of treating grain surfaces with chemical insecticides, as most adult weevils are found in the surface layer. Furthermore, attempting to simulate a practical application scenario, where some areas are
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Table 1 Experimental design and time plan for addition of natural enemies to units with 9 kg of wheat Units Sg Bb Ld
Untreated control: Sitophilus granarius alone S. granariusa and Beauveria bassiana S. granariusa and Lariophagus distinguendus
Ac
S. granariusa and Anisopteromalus calandrae
BbLd
S. granariusa, B. bassiana and L. distinguendus
BbAc
S. granariusa, B. bassiana and A. calandrae
a
Week No.
Treatments
0 0 3 5 3 5 0 3 5 0 3 5
18 imagines (unsexed) per unit; all units Surface application of 2 £ 106 conidia per g grain 5 female and 4 male parasitoids per unit 2 female and 2 male parasitoids per unit 5 female and 4 male parasitoids per unit 2 females and 2 male parasitoids per unit Surface application of 2 £ 106 conidia per g grain 5 female and 4 male parasitoids per unit 2 female and 2 male parasitoids per unit Surface application of 2 £ 106 conidia per g grain 5 female and 4 male parasitoids per unit 2 female and 2 male parasitoids per unit
The pest, Sitophilus granarius was added to all units at the start of the investigation (week 0), as described for the Sg units.
bound to be left untreated, the perimeter of the surface was left untreated to give weevils the opportunity to avoid the inoculated area. As 1 g dry conidia contained 1.8 £ 1011 conidia, the Xour mixture added to each container contained 1.8 £ 1010 conidia, providing an overall concentration of 2 £ 106 conidia/g grain. All containers were then infested with 18 adult, unsexed S. granarius, 7– 14 days after emergence. Sex ratio was assumed to be 1:1 (Richards, 1947). They were placed on top of the grain (in the untreated perimeter of the fungus treated units). After 3 and 5 weeks parasitoids were added to units Ld (i.e,. S. granarius and L. distinguendus), Ac (i.e., S. granarius and A. calandrae), BbLd and BbAc (numbers are given in Table 1). At this time, the pest larvae are assumed to have reached the stage that is preferred by the parasitoid for parasitization. Adult parasitoids, <72 h after emergence, were transferred to a plastic tube (length 70 mm, diameter 10 mm) which was inserted into the side of the plastic container above the grain surface. The parasitoids could then walk into the container. The containers were placed in a controlled climate chamber at 19.7 § 0.07 °C (SD), 72.0 § 0.7% relative humidity and a photoperiod of 16:8 (L:D). Temperature in the grain was monitored in one unit of each type by the means of Tinyview range H loggers (Gemini Data Loggers Ltd, Chichester, West Sussex, UK). Population densities were determined in units incubated for 16, 20, and 26 weeks, respectively, by separating the grain from the adult weevils, parasitoids and dust on a laboratory grain cleaner (type LAS, Westrup A/S, Slagelse, Denmark). Live and dead insects were separated by aspiration. S. granarius were either counted or quantiWed on a weight basis. Grain moisture content in each unit was measured on a Grainmaster moisture meter (Protimeter, Bucks, UK). To determine the densities of immatures, the grain was returned to the container and incubated for an additional 64 days. During this time, any juveniles (eggs, larvae, and pupae) present at the time of sieving would have developed into adults, but their oVspring would still be immatures within the kernels. They were then quantiWed and examined as described above.
Data on the weight of the grain prior to sieving after 26 weeks, of the grain prior to the Wnal sieving 64 days later and of the material (dust, frass) removed at the Wrst sieving were used to calculate the weight loss of the grain that took place during the investigation. Dead weevils and parasitoids were screened for presence of fungus by incubating them on moist Wlter paper for 1 week. As the numbers of dead insects increased dramatically during the experiment, a sub sample of 50 specimens was taken for incubation. Furthermore, fungus prevalence in live weevils was determined by incubating 25 weevils per container in a petri dish with 7 g wheat for 14 days at 20 °C after which dead weevils were placed on moist Wlter paper and incubated as described above. This incubation of dead and live insects was made to check the cause of mortality among pests and parasitoids in the containers and also to provide an additional check of the eYcacy of the fungus over time. The pest densities after 26 weeks (immatures and imagines, live and dead, log-transformed), the weight losses (log-transformed), and the moisture contents of the grain were compared using PROC GLM (SAS Institute, 2000) and a Student’s–Newman–Keul’s test to test for diVerences. 3. Results Table 2 shows the pest densities (immatures and imagines) after 26 weeks and includes both live and dead S. granarius. These data were used for the statistical analyses as each individual represents a destroyed wheat kernel. SigniWcant diVerences were found between the pest density in the untreated controls and all treatments except units with B. bassiana alone, as well as among all treatments with parasitoids (F D 87.15; df D 5, 18; P < 0.001). In the untreated control units the S. granarius population increased almost 5000 times in 26 weeks, whereas in the units with the lowest pest density (Ld units), the pest population had only increased 4-fold. Addition of parasitoids led to a population suppression of 83 to >99%, greatest in units with L. distinguendus. Weight losses of the grain
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Table 2 Total pest densities (dead and live individuals, all stages), pest population suppression, and weight loss of grain (mean § SD)
Sitophilus granarius per kg (all stages) Suppression, % Weight loss, g per kgb
Untreated control (Sg)a
Beauveria bassiana (Bb)
B.bassiana and A. calandrae (BbAc)
B. bassiana and L. distinguendus (BbLd)
Anisopteromalus calandrae (Ac)
Lariophagus distinguendus (Ld)
9574.8 § 1251.4a
8806.8 § 2189.7a
1652.9 § 1374.3b
207.7 § 125.7c
54.8 § 4.3d
8.8 § 5.8e
— 374.4 § 35.7a
8.02 290.6 § 102.2a
82.74 113.3 § 47.5b
97.83 22.9 § 8.6c
99.43 5.1 § 4.5d
99.96 4.3 §0.1d
Initial pest density: 2 Sitophilus granarius per kg. Imagines removed after 26 weeks at 20 °C. Juveniles reared out and counted 64 days later. Weight loss based on data from the Wnal examination. Means in the same row followed by diVerent letters are signiWcantly diVerent (P < 0.001, Student’s–Newman–Keul’s test). a For treatment details see Table 1. b Weight losses have been calculated as the loss per kg grain, so they can be compared to the pest density per kg.
ranged from 60.5% (Ld and Ac units) to 37% (Sg units); signiWcant diVerences were found between weight loss in all units with parasitoids compared to the untreated control and Bb units (F D 49.60; df D 5, 18; P < 0.001). After 22 weeks, temperatures reached almost 35 °C in the two units with the highest pest densities (Sg and Bb). An intermediate temperature increase up to 27 °C was seen in the BbAc unit after 24 weeks, whereas temperatures remained below 23 °C in the BbLd unit. Hardly any temperature increase was seen in the Ld and Ac units. The moisture content of the grain was between 13 and 15% at the examinations after 16 and 20 weeks. On the Wnal examination the grain in the Sg units was caked together with storage fungi, and the moisture content was very high, but it could not be determined. In the Bb units the moisture content was 19%, whereas in the rest of the units the moisture ranged between 13 and 14%. The pest density in the untreated controls can be related to the actual damage exerted by the pests. One kilogram of this variety of wheat contains close to 30,000 kernels; thus, with a pest population of close to 10,000/kg grain in the Sg units at the Wnal examination, one third of the kernels were destroyed, resulting in a weight loss of 37%. In comparison, in the Ld units, only 9 kernels/kg were destroyed. The densities of live adult weevils and immatures are shown in Fig. 1. In the Sg units and the Bb units, the densities of S. granarius increased with time. In units with parasitoids, both with and without B. bassiana, a decrease in the numbers of immatures was observed after 20 and/or 26 week. In the Ld units, the Wnal density of immatures was very low, 0.3 per kg as opposed to 6.9 imagines/kg in these units. Prior to the experiment, 100% germination was found in the dry conidia of B. bassiana. When the initially added granary weevils were harvested from the extra Bb units after 7 weeks, 65% were dead (N D 54). Ninety-eight percentage of these dead weevils supported sporulating fungus when placed in humid conditions, showing the initial eYcacy of the added fungus. In comparison, in the untreated containers (Sg), 6% of the weevils had died after 7 weeks (N D 53), none of which were infected by the fungus. Moist incubation of dead granary weevils and parasitoids found in the fungus treated containers showed that 100% of the incu-
Fig. 1. Densities of live imagines and immatures (larvae and pupae) of Sitophilus granarius (insects per kg grain (§SD)) in units of wheat with diVerent combinations of natural enemies. See Table 1 for explanation of the legend.
bated specimens harbored fungus until 20 weeks. After that time, lower proportions proved to be killed by the fungus: 25–50% of the weevils and 49–50% of the parasitoids. This documents that the fungus kills the pest but also both species of parasitoids, and also shows that the fungus remains viable within the cadavers accumulating over time in the experimental containers. Among live weevils collected from the containers and incubated for another 2 weeks, negligible proportions were found to be infected by the fungus (means of fungus treated containers: 6% at 16 weeks, 6% at 20 weeks, and 2% at 26 weeks). These results show that over time, the fungal conidia retain their activity, albeit at very reduced levels compared to the initial activity measured at 7 weeks. The high moisture content found in units with many S. granarius at the Wnal examination obviously did not promote the growth of B. bassiana.
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4. Discussion The objective of this study was to describe the eVects of four diVerent methods for biological control in stored wheat in a type of semi-Weld trial, i.e., units containing larger amounts of grain than a small laboratory trial would use. The units were to be kept for 6 months and the initial pest density was to be low, to simulate the situation in a grain store. The highest level of suppression was obtained by the means of the parasitoid L. distinguendus alone, followed by A. calandrae alone. In units with parasitoids in combination with B. bassiana, the pest population was signiWcantly lower than the untreated controls, but even at the very high inoculum level used, the suppression was too small to be acceptable for practical application. This may be due to factors such as long lethal time of the fungal strain, so that it took some time to kill the pest, thus allowing eggs to be deposited. Furthermore, the low persistence of fungal conidia in grain over longer periods of time plays a role. The fungus seems to have aVected the parasitoids negatively, and this was veriWed by the dead parasitoids producing fungal outgrowth. Beauveria bassiana has a wide host range (Tanada and Kaya, 1993) and it is not surprising that the parasitoids were susceptible to infection. In some cases, however, parasitoids may be susceptible to infection by entomopathogenic fungi but still continue to reproduce, thus allowing fungi and parasitoids to be used simultaneously (Nielsen et al., 2005). In contrast, our study clearly indicates that the two types of biocontrol agents cannot be integrated if the grain is treated with a surface treatment of fungus. However, the combined treatments of parasitoids and fungus produced signiWcantly lower numbers of S. granarius compared to the fungus treatment alone. This emphasizes that both species of parasitoids—despite being susceptible to fungus infection—must have been able to reproduce to some extent. If the fungus could be combined with a potent attractant in a trapping device so parasitoids are not exposed to the fungus inoculum while weevils become inoculated upon visiting the trap, it might still be possible to combine the two biocontrol agents. Such targeted treatment, where the fungus is applied in a trap rather than by treating large surfaces, might also have the advantage of reducing the exposure of humans or livestock to fungus-derived residues when feeding on treated commodities, although a substantial part of the fungus inoculum is likely to be removed during cleaning processes carried out prior to milling of grain. Cleaning procedures will also remove the parasitoids from the grain. Use of parasitoids in stored grain may even lead to a reduction in the number of insect fragments in Xour (Flinn and Hagstrum, 2000). The weight loss of the grain in the untreated (Sg) units was 374 g/kg, i.e., 37% of the original weight. This Wgure was based on the weight of the grain at the Wnal examination before weevils and frass were removed. Other studies on the weight loss caused by S. granarius in wheat report similar values. Hurlock (1965) reported that a mean of 214 weevils resulted in a weight loss of 6 g. If converted to the
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same densities as in our study, this corresponds to 275 g, which is a bit lower than we found. Richards (1947) found that 193 weevils led to a loss in dry weight of 3 g, again a little lower than our study. However, the diVerences can be due a number of factors such as diVerences in experimental design, kernel size, pest density, weighing procedures, or whether or not beetles and frass were included, etc. In addition, these two studies were conducted with small amounts of grain and for short periods of time, and the results have a diVerent level of precision. Reppchen et al. (2002) used slightly higher numbers of parasitoids than in our study and achieved a lower level of pest suppression; a maximum of 94% suppression using 2.5 female parasitoids per female S. granarius. In our study the ratio between female parasitoids and female S. granarius was 0.8 (sex ratio of S. granarius 1:1). The greatest suppression in our study, when using data comparable to theirs, i.e., dead and live weevils, but not juveniles, was >99.7% found in the Ld units (2889.3 § 811.4 weevils/kg in the untreated controls, and 8.1 § 5.4 in the Ld units). Lucas and Riudavets (2002) compared the eVect of L. distinguendus and A. calandrae on S. oryzae in rice in a 6week trial at 25 °C. They found the highest suppression (98%) with L. distinguendus. Smith (1994) constructed a simulation model to evaluate diVerent strategies for using A. calandrae for biological control of S. zeamais at 25 °C. When applying a comparable number of parasitoids as in our study, Smith (1994) calculated that the pest population was reduced by 95.3% after 17 weeks. It is encouraging to note the high pest suppression obtained in our study at 19.7 °C. This condition is relatively less favorable for the parasitoids than for the cold-hardy S. granarius. This result contradicts the calculated lower threshold for positive population growth in A. calandrae of 20 °C (Smith, 1994). Even though our study was not replicated in time, the conclusions are considered to be thoroughly supported by the fact that (1) the pest population was sampled three times during the course of 6 months, (2) the variation in population densities in units with the same treatment was very small on all sampling occasions, and (3) diVerences between treatments were highly signiWcant (Table 2 and Fig. 1). Further support can be found in the following: the Wnal pest density in the untreated units in our study is surprisingly similar to the one found by Reppchen et al. (2002). That is, we had 2889.3 S. granarius/kg, dead and live adults, as opposed to their Wnding of 4349/kg with a longer test duration (28 weeks) and higher initial numbers of weevils (5 female S. granarius/kg). Finally, the degree of pest suppression exerted by the parasitoids is similar to the one found by Reppchen et al. (2002) and comparable to the suppression found by Lucas and Riudavets (2002) and Smith (1994). The diVerence between the eVects of each of the parasitoids was signiWcant. Even though the suppression level with both parasitoids was above 99%, the diVerence between the two species is important for practical application. With A. calandrae, the pest density increased more than 25 times
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from an initial density of 2 weevils/kg to 54.8/kg, compared with the 4-fold increase when L. distinguendus was used (from 2 to 8.8/kg). Furthermore, there were very few immatures in the units with L. distinguendus at 26 weeks. As increasing populations typically have a predominance of immatures (Birch, 1948), our result suggests that the pest population in the Ld units was about to crash. These diVerences may be decisive for which parasitoid species is best suited for practical application. Further studies are necessary to elucidate the potential of parasitoids for biocontrol of S. granarius under the inXuence of the low temperature regimes found in grain stores in northern Europe. Acknowledgments We thank Lars Damberg, Bodil M. Pedersen, Minna Wernegreen, and Claus Dahl for technical assistance and Henrik Skovgård for useful comments on the manuscript. The project was supported by the Danish Ministry of Food, Agriculture and Fisheries, DLG (Danish Cooperative Farm Supply) and the Association of Danish Millers. References Athanassiou, C.G., Steenberg, T., Kavallieratos, N.G., in press. Insecticidal eVect of diatomaceous earth applied alone or in combination with Beauveria bassiana and beta cyXuthrin against Sitophilus granarius in stored wheat. Bulletin IOBC/wprs. Batta, Y.A., 2004. Control of rice weevil (Sitophilus oryzae L., Coleoptera: Curculionidae) with various formulations of Metarhizium anisopliae. Crop Protect. 23, 103–108. Birch, L.C., 1948. The intrinsic rate of natural increase of an insect population. J. Anim. Ecol. 17, 15–26. Brower, J.H., Smith, L., Vail, P.V., Flinn, P.F., 1996. Biological control. In: Subramanyam, B., Hagstrum, D.W. (Eds.), Integrated management of insects in stored products. Marcel Dekker Inc. New York, New York, pp. 223–286. Christensen, C.M., Hodson, A.C., 1960. Development of granary weevil and storage fungi in columns of wheat – II. J. Econ. Entomol. 53, 375–380. Cline, L.D., Press, J.W., Flaherty, B.R., 1985. Suppression of the rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae), inside and outside of burlap, woven polypropylene, and cotton bags by the parasitic wasp Anisopteromalus calandrae (Hymenoptera: Pteromalidae). J. Econ. Entomol. 78, 835–838. Fields, P.G., 1992. The control of stored-product insects and mites with extreme temperatures. J. Stored Prod. Res. 28, 89–118. Flinn, P., Hagstrum, D.W., 2000. Augmentative releases of parasitoid wasps in stored wheat reduces insect fragments in Xour. J. Stored Prod. Res. 37, 179–186. Howe, R.W., 1961. A study of the heating of stored grain caused by insects. Ann. Appl. Biol. 50, 137–158. Hansen, L.S., in press. Biocontrol potential of Lariophagus distinguendus (Hym.: Pteromalidae) against Sitophilus granarius (Col.: Curculionidae) at low temperatures—reproduction and parasitoid induced mortality. J. Econ. Entomol.
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