Threat to european maize production by invasive quarantine pest, western corn rootworm (diabrotica virgifera virgifera): a new sustainable crop management approach.

Simulated spreading rates for WCR (Western Corn Rootworm) in France, Germany, Italy, Austria and Belgium: France: Simulating the spreading in case of introduction via Paris, WCR (Western Corn Rootworm) would have infested about 270,000ha (100 %) of maize without any containment measures after 10 years. With measures, the spreading rate per year would result in a reduced infested maize-growing area of about 21,600ha, which would represent only 8% of the infested maize-growing area without any measure in 10 years. This would be a sustainable reduction of spread in Paris area. In case of Colmar, which is in an area with high concentration of maize, the situation is different. Without any measure, about 325,000ha would be infested with the WCR after 10 years. The reduction of spreading rate is not so enormous in the Alsace region like in Paris region after 10 years and would covered a maize area of about 137,000 ha, 42% of the infested area without any measure. Germany: The simulation model output in case of an introduction via Frankfurt am Main without containment measures showed that about 476,000ha of the maize area (100%) would be infested over a period of ten years. With containment measures applied, 23,107ha would be infested, 5% of the infested maize area over ten years. In case of Passau, the 'natural spread' calculation amounted to 358,691ha of maize (100%) infested over ten years. Containment measures could reduce the infested maize area by one third (188,419ha) of the total infested area for the same period. The results of simulation of spreading of the WCR at the two locations are different by year depending on the maize concentration in crop rotation. In the 10th year without containment measures in Frankfurt, a significant ongoing spread of 265,698ha of maize occurs. The reason for this is the high maize concentration in Northrhine-Westfalia and Lower Saxony, which supports the spreading. In Passau, the conditions for the establishment of WCR are better because the amount of maize makes it a high-risk area. Therefore, the influence of containment measures is not as strong (33%) as in Frankfurt am Main (5%) where we have only a low concentration of maize in the beginning. Italy: Both already infested locations (Venice and Como) are in the North, in areas with high maize concentrations. Therefore, the spreading rate per year of WCR would be in each case high without any containment measures and would covered in case of Venice about 832,000ha and in case of Como nearly the same with about 839,000ha in the 10th year. These infested areas would represent already about 66% of the whole maize-growing area in Italy. Remarkable is that in the 6th year in Venice and Como, the maximum infested maize-growing area would be already reached in the considered period. The infested maize-growing area in Venice would be reduced by one third (about 548,000ha) with containment measure after 10 years. In case of Como, the calculated amount of infested maize-growing area with measures would be with 471,000ha little bit more than one half (56%) of the area without any measure in one decade. Austria: The simulated spreading scenarios of WCR have as result a potentially infested maize-growing area of 178,000ha in case of Linz and about the same (176,000ha) in case of Kittsee without any measure in the 10-years-period. Nevertheless, the influence of measures could be different depending on the maize-growing situation in both regions of Austria. In case of Linz, the infested maize-growing area would cover about 65,000ha which represents 36% of the potentially infested maize-growing area without any reduction of the spreading rate. In case of Kittsee, containment measures would have much more sustainable influence on the infested maize-growing area: only 1,000ha maize would be infested by the WCR, less than 1% of the potentially infested maize-growing area without any measure. In Burgenland region, maize concentration is low and the realisation of containment measures like crop rotations is relatively easy to implement into agricultural practice. Belgium: A relatively small country, but green and silage maize are of relatively great importance, the calculated spreading rate would lead to a fast infestation of all maize-growing areas (about 202,000ha) within the 7th and 6th years in Brussels respectively Arlon (201,000ha for both locations) according to simulation scenarios. But possible containment measures would not have the same influence in case of Brussels and Arlon. In case of Brussels, an infested maize-growing area of 80% (161,000ha) was calculated for the 7th year and of 92% (185,000ha) for the 10th year. Containment measures in case of Arlon have sustainable influence on the spreading rate and would cover an infested area of 3,800ha (2%) after 6 years and 13,000 ha (6%) after 10 years. Percentages refer to the area infested when no containment was carried out.

Evidence for immigration of WCR (Western Corn Rootworm) adults in non-maize crop stands Pherocon AM traps did demonstrate WCR adult presence and activity near the ground surface in winter wheat, soybean, and sunflower. The WCR population in the project trial (mainly in 2001 and 2002), measured by catching adults on Pherocon AM traps, approached the threshold level established for economic larval damage in next years maize in USA. In each year, significantly higher numbers of WCR adults were trapped in maize compared to any other crop in the rotation system. Pherocon AM traps (that visually attractant WCR adults) were covered by dense sunflower foliage in July and August or by soybean foliage. Therefore, adults caught by the traps in these crop stands were actively flying into these plots. In the case of winter wheat, one cannot speak about plant stand from mid-July under our conditions. Since winter wheat was harvested around mid-July, the traps were visible from a distance that may have contributed to increase adult numbers on traps on harvested winter wheat plots. At the population density in the project trial, there was no distinguishable adult density shift from maize to non-maize crops in the growing period of maize or after silking of maize. Certainly, observations showed that WCR adult numbers increased in harvested winter wheat plots on traps and on volunteer wheat plants (feeding adults). This phenomena raises the important question of the possibility that harvested winter wheat fields could be potential egg laying places for WCR females in Europe. However, the proportion of egg-laying WCR females within the population after harvesting winter wheat is still a question. Since harvest time and WCR phenology vary from one region to another in Europe, this phenomena should be considered and checked on a regional basis in the future. Evidence for WCR female egg laying in non-maize crops: Although we have not sampled WCR eggs in non-maize plots (and few eggs in continuous maize plots), sampling of larvae and adult emergence in maize after non-maize crops in our trial justifies, that egg laying did happen in non-maize crops. It is expected that at higher WCR population density or with improved sampling techniques WCR egg could be sampled from non-maize crops. This method, however, is labour and time intensive therefore cannot be suggested for purposes under regular farming conditions. WCR larval development happens in maize after non-maize crops Sampling in 2002 resulted in 4 larvae (one after winter wheat and sunflower, while two after soybean). Again as in the case of egg population, this density was rather low. One should note that the L1 stage practically cannot be found with the sampling method used due to their small size or being in the root, and the same applies for the early developmental period of L2. Therefore, the small number of larvae found was not unexpected. The efficacy of this larval sampling method was effective at higher population densities in continuous maize resulting in 51 and 147 larvae, respectively. Larval activity (root damage) in maize after different non-maize pre-crops varied by year. At observed population densities, no reliable evidence can be demonstrated for the effect of pre-crop on the next years maize root damage by WCR larvae. WCR adults emerge from the soil of maize after non-maize crops: In the 2001 and 2002 growing seasons, a total of 98WCR adults emerged in maize after non-maize crops the previous year within a total of 54 emergence cages. Due to relatively high adult population in 2001, the number of emerged adults was higher in 2002 when compared to 2001. Continuous maize always maintained significantly higher adult emergence numbers compared to maize after non-maize pre-crops. For the effect of pre-crop on next years adult population, there was no difference between winter wheat and soybean, while sunflower resulted in significantly fewer adults compared to soybean. All of the above points to the fact that part of the WCR population immigrates to non-maize crop stands and lays eggs, and the subsequent maize, the larvae develop (thus damaging maize roots), and adults emerge. Various pre-crops may have different impact on the various stages of WCR. Sunflower blossoms are likely to be attractive for adults. We have also observed this type of attractiveness for adults by volunteer winter wheat. Based on correlation equations WCR adults highly prefer maize plots. In all cases, there was a strong correlation between the catches on Pherocon AM traps in pre-crops and between the catches in emergence cages in the subsequent years maize. From the standpoint of maintaining the WCR population, cereals (oat and winter wheat) and sunflower were similar, while a few WCR adults in soybean plots resulted in the highest population in the next years maize. To produce the same number of WCR adults in maize, soybean plots as the pre-crop needed the fewest number of adults.

For the first time a complete life - table of WCR (Western Corn Rootworm) populations were calculated by overcoming main methodological difficulties, i.e. a) by avoiding clumped populations by using artificial infestations and b) by the ensurance of compability between experiments by using absolute counts and by using same or proportional units for all life stages of WCR. However, its comparison to other studies is limited due to very few investigations of life - tables on insects with soil inhabiting stages. Total mortalities were successfully measured for most age intervals within an WCR generation, and only estimated for the pupal stage in two cases. Reasons for mortalities within an age interval were mainly unknown. No indigenous natural enemies were found to have importance for the generational mortality. Thus, the usually important dependency between efficacy of natural enemies and clusters and density of the host population can be neglected. Also intra-specific competition is suggested to be low in this study, since infestations with 100 up to 2400 eggs per plant did not affect the total generational mortality in D. virgifera until adult emergence, and since maize plants, in this study, were infested with 80 to 100 eggs. Then, emerging adults found nearly unlimited food resources in maize fields. Finally, aggregation as a beneficial factor in survival was never reported from larvae, pupae or adults of WCR. Conclusively, mortality and survival in the life stages of WCR in this study, was mainly a result of abiotic factors, such as of climatic conditions. The major factors behind the commonly high mortality in first instar larvae, such as of about 94 % in this study, have never been clarified. The role of predators or bacterial infection is unknown for this life stage. In the life - table of WCR in Europe, a total generational mortality (K Total) of 2.48 was measured in average. It represents a total mortality of about 99 % from the egg stage to the emergence of adult females. The highest reduction of D. virgifera numbers resulted from the generational mortality in 1 st instar larvae (ca. 50%), as well as in second instar larvae and in over wintering eggs (both ca 11%). accounting for 72% of the total generational mortality. However, only the variation in mortalities between years can influence the generational mortality and thus population size. This means, the very high mortality in first instars larvae had a limited potential in causing changes total generational mortalities, since its value deviated only slightly between the three studied cases (s.d. = 1, n = 67). In contrast, a high variation of marginal death rate between fields and years was found in second instar larvae (s.d. = 31.9), in 3rd instar larvae (s.d. = 32.2) and in over wintering eggs (s.d. = 14.5). Those mortalities seem to be crucial in causing changes of the generational mortality, although, due to the limitations to two years the found correlation coefficients between the kx of the above-mentioned age intervals and the total generational mortality K Total were not significant. Finally, the life - table showed that a high fecundity could recompense high generational mortalities and lead to increasing populations. WCR was performing a high mean lifetime fecundity of 353 eggs per female in the laboratory, which can increase up to a potential fecundity of 956 eggs per female. This high reproductive potential made WCR a typical r - strategist. A failure of the population to realise their potential fecundity is the main factor that the populations remained stable from 2001 to 2002 (R0 = 0.9), or even declined from 2002 to 2003 (R0 = 0.21), and is reflected by the correlation between the k - values of the loss fecundity rate to the total generational mortality (K Total) in each field and year (R2 = 0.99, P= < 0.05). The realisation of fecundity can be often suppressed in WCR by the dryness in soil, what is a typical situation in southern Hungary due to the lack of rainfalls in July and August, and as it was the case in the study sites in 2001 and 2002. Another reason for the lack of population increase in 2001 was the relatively high mortality in diapausing eggs suggested to be a result of low quality of eggs due to the exceptionally warm autumn in 2000. For 2002, in addition to the low realised lifetime fecundity, a comparatively high mortality in second and 3 rd instar larvae due to soil dryness, lead to an over-estimated declining of the population (R0 = 0.21).

A novel, non-sticky, high capacity funnel trap type code-named VARs has been developed. The new VARs type: - Has a virtually unlimited catch capacity as compared to the limited capacity of the existing PAL and PALs sticky types; - Its maintenance is cleaner (no sticky material); - And is less expensive to use in prolonged monitoring trials. On the other hand, for proper operation the addition of an insecticide into the catch containers is absolutely necessary. VARs traps have been made available to users through the non-profit advisory extension service of the Plant Protection Institute, Hungarian Academy of Science, (Budapest, Hungary) from the beginning of 2001, as a new member of the CSALOMON® pheromone trap family. (CSALOMON® is a registered trademark of the Plant Protection Institute, Hung. Acad. Sci., Budapest, Hungary). In addition the most important characteristics of each trap type developed for monitoring of the corn rootworm were deeply studied so that precise advise for trap use in different situations and for different purposes has been made available. We conclude that: - For catching males the PAL and VARs types are optimal, PALs can also be used, if sensitivity is not a critical consideration. - For catching females the PALs and VARs types can be recommended. Usually the sticky types PAL and PALs as well IPC trap are better suited to detection situations (when the population density of the pest is low, and no frequent exchange of replacement sticky sheets is necessary). The funnel VARs type seems to be more advantageous in long term monitoring trials. Throughout the test the unbaited yellow sticky traps performed worst in all aspects. It was also possible to evaluate the important possibility of developing monitoring tools suitable at the same time for both Diabrotica and other maize soil pest. Monitoring tools are available for click beetle species (larvae and adults); in preliminary trials carried out in Hungary and Italy newly developed ground sex pheromone trap (YATLORf) for Agriotes species proved to be suitable for monitoring WCR (Western Corn Rootworm) population too. Being non-saturable the traps are potentially suitable for correlating adult captures with the level of subsequent larval populations. No negative interaction was observed placing both Agriotes and Diabrotica lures in the same YATLORf traps.

- The WCR (Western Corn Rootworm) adult population, which is near economic levels 6-8 years after the initial infestation, highly prefers maize stands for adult feeding, egg laying, and larval development. - The WCR population in continuous maize showed a continuous increase though the rate of increase can differ depending on environmental (rainfall or drought) conditions. Even under extreme dry summer and unfavourable soil conditions, the WCR population can increase in the following year. - Part of the WCR adult population can immigrate to other crop stands or to other fields (harvested). The reason for such immigration can be to feed on flowers and pollen of the crop, such as sunflower, or to feed on volunteer winter wheat in harvested wheat fields. A small portion of the WCR adult population (5-15%) is active near the ground surface of these crop stands. - Biologically, the immigration of WCR adults to other crop stands, adult feeding, and/or egg laying in non-maize pre-crops and larval feeding on the roots in the next years maize was observed in the crop rotation trial. - Although there were differences among non-maize pre-crops regarding the robustness of correlations between the adult density of immigrating individuals and the next years adult population density in maize, the differences were not enough to demonstrate pre-crop preferences by a critical number of adults to non-maize crops. - There was no clear preference for non-maize pre-crops by WCR adults and no sign of any special adaptation of the WCR population to the crop rotation system. Rather, a random spread and movement of WCR adults and subsequent egg laying happened under project trial conditions. - We assume that crop rotation systems in Europe will not result in high selection pressure on the WCR at levels that have been seen in the USA Corn Belt. Diverse landscape with a more diverse plant spectrum in Europe offers broader feeding sources and different egg laying habitats compared to the landscape in the USA Corn Belt, that primarily being a maize/soybean rotation system. We believe that diversity in space, as well as in time, may contribute to the lower possibility for the development of an adapted WCR population. Diversity in time refers to avoiding long-term, narrow, and routine rotations of crops. This may be of special importance in the typical maize growing areas in Europe where maize is cultivated on large areas, over long periods, and in narrow rotation selections (1-2 crops in rotation with maize). Existing IPM (IPPM) rules also support the above. - While the rotation of maize with other crops is a primary control method of WCR populations and is strongly prescribed (by law or other types of regulations), especially in the population build-up phase, there are still major questions concerning the long-term management of WCR. If, theoretically, all maize plots (or a majority) will be rotated to non-maize crops, we do not allow WCR population to survive! This type of full rotation may pose a very high selection pressure. Therefore, the use of refuges of continuous maize for two years within rotation areas to reduce selection pressure and preserve rotation as a means for managing the WCR population needs to be studied and policies developed, if effective.

We investigated the performance of larvae of the invasive maize pest Diabrotica virgifera virgifera LeConte (Chrysomelidae, Galerucinae) on roots of alternative host plants. During laboratory feeding trials we measured larval growth, amount of ingested food and determined the food conversion efficiency. We tested eight species of weeds (seven monocot and one dicot) and three monocot crops with regard to host plant suitability employing a newly established method. We additionally examined the C/N ratio and the phytosterol content of the different plant species as parameters to interpret larval performance. Larval growth of D. v. virgifera (Western Corn Rootworm; WCR), the amount of ingested food, and the food conversion efficiency differed significantly between plant species. Plant species with high nitrogen content were less suitable for WCR development. The phytosterol content had a significant influence on the amount of ingested food, but not on larval weight gain. The performance of WCR larvae on alternative hosts was comparable to their performance on maize. The ability to use alternative hosts for larval development may contribute to the invasion potential of WCR and has important implications for integrated pest management.

Potential pecuniary losses due to WCR (Western Corn Rootworm) and cost of eradication measures have been calculated in detail. In case of avoidance of the potential pecuniary losses the amount represents the benefit. On the other hand there are the costs for the eradication. Both, pecuniary losses as benefit and cost of eradication have to be compared for an analysis. The avoidance of losses is over a longer period if no further introduction or the ongoing of natural spread infested the former hot spot again. Economic damage is expected in high-risk areas in the 6th year of establishment. We calculated the potential pecuniary losses for France (FR), Germany (DE), Italy (IT), Austria (AT), Belgium (BE) and Switzerland (CH) for 6, 10 and 15 years after establishment of WCR. The calculation includes the change of crop rotation (no continuous maize) without recompense for the farmers (no costs) and costs for a trapping system. Two scenarios on the basis of the spreading simulations are covered to calculate the losses of maize in the infested areas, the spreading rate with and the spreading rate without containment measures. On both infested areas we would expect damage after 5 years. However, the ongoing spread of WCR without any measures should be faster than with containment measures. Therefore, we get a difference in infested area over the considered period of one decade. The difference in infested areas leads to a difference in pecuniary losses. The comparison of the costs for containment (in our case only for the trapping system because crop rotation was not considered) and the difference of pecuniary losses as described before represent the benefit or the pecuniary loss. This was done for France (Colmar), Germany (Passau), Italy (Como), Austria (Kittsee), Belgium (Arlon) and Switzerland (Kreuzlingen). The costs for eradication are split in a low, medium and high version for one year. One year of eradication is to be likely if only a small hot spot of infestation exists (such as single specimens on a single maize field in a region with low concentration of maize). However, it is also possible that the eradication measures have to be done for more than one year like in Italy. The number has then to be multiplied by the number of years. Benefit depends on the situation in the different regions and countries. In case of Italy (Venice) with high concentration of maize, costs of eradication are relatively low with about 154,000 Euro per year (high cost version) in comparison to about 20.3 million Euro loss after 6 years (one year of economic damage). The cost/benefit ratio would be 1 to 131 for one year of eradication and 1 to 44 for three years of eradication. Furthermore, in Italy the potential loss would be about 432 million after 15 years and the cost/benefit ration would be 1 to 2,820 in case of one-year eradication and 1 to 935 in case of three years of eradication. The pecuniary difference would be in both cases more than 430 million Euro over 15 years without WCR. In Germany (Frankfurt am Main), we would have a different situation. As there is only very little maize in the Frankfurt region and in most cases no continuous maize in crop rotation, the WCR would reach areas with high concentration late. In this case the potential loss is expected late and total pecuniary loss would be 28.1 million Euro after 15 years. After 6 or 10 years, no pecuniary loss is expected and therefore we would have only the costs for eradication, which would run at about 205,000 Euro. The benefit in the period would be negative, but in the long run positive with more than 27 million Euro after 15 years. On the other hand eradication in areas with low maize in crop rotation is much more easier than in areas with high concentration of maize like in the Venice region in Italy. The analysis of 10 years shows that in most cases we could expect a benefit from eradication of WCR in the mentioned countries. After 15 years we would benefit in any case and this benefit would be significantly high.

An aerial distribution technique for MCA coated grits has been optimised over the 3-year study period. Technical problems have been eliminated and reliable techniques have been developed. The original experimental design has been modified by using regular grit distribution pattern designs covering the whole field. By this technique specific areas of irregular grit distribution in the field can be identified by frequency distribution tables. Subsequent discussions with the pilot helped to understand grit distribution problems at specific sites and to avoid them during future flights. The airplane has been calibrated appropriately to apply proper rates. An application expert from the Plant Health and Soil Conservation Station in Budapest conducted proper calibration runs. Six aerial applications during 2000/2001 allowed gaining valuable expertise in this application technology. Grit application rate has been increased by 15% after the first application in order to achieve a better coverage at the field margins. Due to his high flight velocity of 170km/h the pilot cannot close the release valves exactly at the border of the field margin. Therefore, a certain amount of up to 15% has to be added to the application rate/ha. Some degree of deviation in grit distribution patterns can reasonably be accepted. One gram of corn granulate consists of about 300 granules. When applying appr. 18kg of grits/ha, this translates to some 5.4 million granules per ha. With such minute particles one cannot expect perfectly uniform distribution all over the field. Even short wind gusts and air vortices during application can influence distribution patterns significantly. Certain discrepancies between the numbers of grits collected in plastic saucers on the ground in comparison to grits accumulated on maize plants were obvious. One strategy for the future would be to mix an adhesive material with the granules in orders to make them stick better to the plant surface. Major problems with this approach are - Reduction of release rate of MCA from the granules and - Application problems from the airplane or the high clearance tractor because the free flow characteristics of sticky particles are greatly reduced. Similar problems were encountered in pheromone applications by airplanes for the control of pink bollworm in cotton fields by Brooks et al. 1979 in California. One of their solutions was to design an apparatus mounted on a specially equipped airplane. It carried spools of long plastic tubes filled with the pink bollworm sex pheromone. During flight, these tubes were 1. Simultaneously chopped into short pieces of about 1 inch length and 2. Closed at one end with a drop of adhesive material which let the fibres settle and be glued onto the leaves. This procedure automatically resulted in the open end of the tube standing upright on the leaves and dispensing the pheromone into the surrounding air. This approach, however, needed a lot of mechanical engineering and adjustment. In Southeastern Europe we did not find the necessary infrastructure and therefore decided to try first the much simpler approach of granular application, either by tractor, by helicopter, or by small airplane.

It can be concluded after a three year survey study, that all developmental stages of D. virgifera, i.e. its host niches, are not attacked by effective natural enemies in Central Europe. Except the fungi species Beauveria bassiana and Metarhizium anisopliae attacking D. virgifera beetles on an extremely low level, no other entomopathogenic fungi, entomopathogenic nematodes, and parasitoids were found, neither on eggs, nor on larvae, pupae or adults. Both fungi are also known as common on Diabrotica species in North and South America, however, their potential for reducing corn rootworm feeding damage has not been proven in the field and very little is known about their natural impact on the populations of D. virgifera. It appears that the native natural enemy community in Europe has not adapted to the invasive alien chrysomelid. In contrast, several nematodes, fungi, predators and some parasitoids are known to attack D. virgifera in South and North America. This is a typical and not unexpected situation for an invasive species and stresses the potential to implement classical biological control strategies using specific natural enemies from the area of origin.

The aim of this subcontracted project was to monitor the occurrence and abundance of indigenous natural enemies of D. virgifera and of closely related species in their area of origin in Central and South America. Thus, field surveys were conducted to mass-collect adult Diabroticite beetles in Mexico, Argentina, and Brazil between 2000 and 2002. Adult beetles were reared to screen for natural enemies that could be used as potential biological control agents for control of D. virgifera in Europe. In Mexico, nearly 10,000 beetles of seventeen species were routinely collected in the state of Veracruz, three of them from the virgifera group. Two tachinid flies, Celatoria compressa Wulp and Celatoria bosqi Blanchard, as well as one braconid wasp, Centistes gasseni Shaw, were found and identified as parasitoids of adult Diabroticite beetles. Two other tachinid flies, Celatoria setosa (Coquillett),and Celatoria diabroticae (Shimer),and the braconid wasp, Centistes diabroticae (Gahan), were not studied because of their minor importance as potential biological control agents for the European situation. In Mexico, the adult parasitoid, Celatoria compressa was reared from 17 species in four different genera of Chrysomelidae between 2000 and 2002. In 2001, 2,436 beetles belonging to different species were collected at various locations in Veracruz, and 55 parasitoid pupae were obtained. In 2002, we collected 4,844 beetles at the same locations, and 99 tachinid puparia were found. This represents an overall parasitism of 2.3% and 2.0% respectively. In 2001, tachinid larvae emerged from six species in four genera, and in 2002 from seven species in the same four genera. Parasitism within host species ranged from 0.3% in Gynandrobrotica nigrofasciata (Say) to 16.7% in Cerotoma atrofasciata Jacoby. The most abundant species in Veracruz were Acalymma blomorum and Diabrotica balteata. The former suffered from 3.5% (2001) and 0.8% (2002) parasitism, and the latter from 4.6% parasitism in 2002. In August 2001, we collected 995 beetles of three different species in the state of Oaxaca (Acalymma trivittatum, Diabrotica balteata, D. v. zeae) from squash maize cultures, and obtained a total of 24 pupae, mainly from A. trivittatum (19), representing a 5.0% parasitism rate, and an overall parasitism rate of 2.9% for the three species at those locations In September 2002, we found only 585 beetles in the same locations, probably due to adverse environmental conditions (prolonged dry season followed by heavy rains). The parasitism rate was considerably lower, 0.8%, again mainly in A. trivittatum (1.9%) In 2001 and 2002, populations of D. v. virgifera were collected in the state of Durango, northern Mexico. Beetles were found during the maize growing season from late August through October. Numbers of D. v. virgifera in maize monocultures in Durango were nevertheless surprisingly low and comparable to numbers of other Diabroticites collected from larger maize-squash fields in Veracruz. From a total of 640 D. v. virgifera collected in maize fields in Durango, 36 parasitoid pupae were collected in 2001, which represented a mean parasitism rate of 5.6% (max = 16%). In contrast, 304 D. v. virgifera were collected in the same region in 2002, and no parasitism was observed. Larvae of Celatoria compressa (n = 34) emerged within 14.2 +/- 3.0 days (mean +/- S.D.) after collection of the hosts. Pupal period lasted 15.9 +/- 4.4 days (n = 71). Upon emergence, tachinid adults were kept in the release cage where they survived approximately 5 to 7 days (for further information see WP 3/3). No copulation of the flies or actual parasitism of the host beetles was observed. Consequently, tachinids could not yet be reared under laboratory conditions in Mexico. All other natural enemies found during the sampling periods were of minor importance.

Western corn rootworm beetles collected in semiochemical-lure traps during the summer of 2000 from 23 sites in Eastern Europe were sent to the USDA-ARS laboratory in Fargo, ND (USDA-ARS; Red River Valley Agricultural Research Center, Fargo, North Dakota USA). In some cases the beetles were by trapped by aspirator and in other cases collected them by hand: Insects were shipped to Fargo, ND immediately after collection in 75% alcohol via DHL and were immediately frozen upon arrival. In Fargo the PCR analysis was done thankfully by Rich ROEHRDANZ as a substantial cooperation within this project. Collections were concentrated in Serbia, Croatia, Bosnia-Herzegovina including the Republic Srpska, Hungary, and Italy. Some collection sites were so close to each other, that they were summarized under one location. Intact DNA was recovered from individual beetles from 17 of the 23 sites. Some problems were encountered with beetles from the 6 sites with negative results (result of insects drying out in shipment), but attempts are being made to refine DNA extraction methods to successfully gather material from the beetles. The intact DNA from the 17 sites was demonstrated by successful amplification of a small PCR product using a western corn rootworm specific primer set. Results of the investigations indicate that all samples appear similar to those of laboratory reared western corn rootworms (from the Brookings, South Dakota USDA-ARS laboratory), which were collected from typical populations of insects inhabiting corn/soybean rotations. Earlier studies indicate that the Brookings lab insects are similar to most populations in the central Corn Belt. To date no samples have been shown to be related to insects ovipositing in soybean. Further studies are being conducted to refine the assay technique and to provide additional data that might be useful in pinpointing the exact origin of beetles inhabiting Europe. DNA extracts were prepared using individual insects from the samples collected in Europe and sent to Fargo, ND. Insects were sampled from 16 of the vials or containers that were received in the USA. The remainder of the insects from all of the collections have not been examined, but remain frozen and will be used in additional studies to further clarify the genetic relationships between European and USA populations. The DNA extracts are total cellular DNA, which includes both nuclear, and mitochondrial sequences. The polymerase chain reaction (PCR) was used to amplify a portion of the cytochrome oxidase I and cytochrome oxidase II genes from the mitochondrial DNA (mtDNA). This particular PCR product is about 600 base pairs long. The region spanning cyotochrome oxidase I and II includes a leucine t-RNA gene and has been widely used for phylogenetic purposes with insects. Amplification was successful for all of the tested DNA extracts, a total of 35 individuals. Attempts to amplify some of the samples with some other primers were not successful but most likely those failures were attributed to old primers that had not been used in over a year (i.e. the controls, WCR from laboratory colonies, did not work either). In a paper published on mitochondrial DNA variations in western and Mexican corn rootworm populations (Szalanski et al.), good diagnostics for population identification of WCR either using mtDNA followed by cleavage with restriction endonucleases, or by obtaining nucleotide sequence data from a portion of the nuclear genome called the ITS1 spacer are described. There is so far no indication that the European WCR differs from the WCR genotype that is wide spread in North America. Nor is there any evidence, to date, of differentiation among the European collections. As a further exploration of the possible geographical distribution of genetic variation in WCR, the DNA sequence of another nuclear region will be examined. This region is another spacer in the nuclear ribosomal gene array. Three ribosomal genes, 18S, 5.8S and 28S, are present in tandem multiple copies. The coding regions for the genes are separated by noncoding regions called spacers. The spacer between the 18S and 28S genes is about 1200 base pairs in length and is called the intergenic spacer (IGS). It is often assumed that since the spacer regions do not code for important products, that they are not as tightly constrained by evolutionary forces. Therefore, the argument goes, the spacer regions should exhibit greater genetic variability.

Although the arthropod diversity in maize is naturally very low, there is a potential to enhance natural enemies through changes in conservation and cultural practises in the European maize production. The following points could be considered: -- Leaving areas of continuous vegetation in large-scale crop rotation; -- Reducing tillage intensity where applicable; and -- Allowing a tolerable degree of non - crop plants in and around maize fields. - Discontinuity of monoculture in time through crop rotations was shown to be the major cultural practise within an IPM strategies against WCR (Western Corn Rootworm) ( WP 2/1). For not loosing the natural enemy community between the change from one crop to the other, it must be recommended to leave continuous vegetation attributes such as perennial crop components (lucerne, fallow fields), herbaceous undergrowth or at least field - surrounding grass - or bush strips within the rotated maize production areas. - Frequently disturbed crop fields by plough tillage, bare soil in winter, etc., can favour rapid colonisation and growth of herbivore populations, such as WCR, due to initially natural enemy free space (Price, 1981), even though egg over wintering of WCR might be reduced (Brust and House 1990). Thus, surface tillage or at least reduced plough tillage should be recommended rather than several plough tillages -- To reduce soil erosion, -- To enhance reproductivity, survival of natural enemies due to continuous vegetation coverage (Blumberg and Crossley 1983) and -- To more efficiently use energy (Letourneau and Altieri 1999). - Non crop plants are a major problem in maize production and many herbicides are used. Moreover some non - crop plant species could serve as pollen source for adult WCR (WP 1/3). Nevertheless, it can be recommended to leave a tolerable amount of non - crop plants in the maize field by reducing weeding frequency, reducing herbicide use, and by reducing tillage frequency because benefit of enhancing natural enemies is expected.

How to measure the food utilization of subterranean insects: a case study with the Western Corn Rootworm (Diabrotica virgifera virgifera) Studies of food conversion efficiency are used to determine the suitability of a particular food item for the development, growth or maintenance of animals. When carried out on insects these studies on food conversion efficiency were up to now always limited to aboveground mostly leaf or shoot feeding insects. Insects which feed belowground or on the roots were neglected on account of methodological difficulties in handling the insects and because direct observations were not possible. The following description provides information on an experimental design, which allows to measure feeding and to subsequently calculate food conversion efficiency for belowground feeding insect larvae of the maize pest Western Corn Rootworm (WCR) (Diabrotica virgifera virgifera). This method was developed in order to acquire knowledge on the impact of different maize varieties and possible alternative host plants on the larval development. Because this species invaded Europe in the beginning of the 1990/s, it is of vital interest to determine how suitable European maize varieties and weeds are as food sources, thus facilitating the spread and the build-up of economically relevant populations.

WCR (Western Corn Rootworm) egg laying is the behavioural step which determines the site where the larvae will feed the next year. We tested the hypothesis that primary metabolites present on the maize surface, particularly the elder growth stages, could influence host acceptance and stimulate egg laying. In a first step we localized the parts of the plants on which information could be taken. For that we observed the insect abundance in a Hungarian maize field, at three periods of the day and at two months. The different plant parts were chemically analysed. Then the organ on which the insect is the majority of the time were sprayed with water to collect metabolites from its surface and the washing were then tested on Hungarian WCR egg laying in laboratory. Pre-egg laying and egg laying behaviour were observed to define which insect body parts are used for detection and the eventual influence of washings on behaviour. We also looked for gustatory like sensilla on body parts, which are used in the perception of gustatory cues. All the free amino acids, soluble carbohydrates, and sugar alcohols are present on the two different corn hybrid surfaces tested. During the reproductive stages the metabolite quantities and ratios found on the plant surface may be due to metabolites coming from the plant tissues and also to metabolites issued from pollen metabolites and microorganisms metabolism linked to the presence of these substances. Metabolite quantities and proportions discriminate plant growth stages, organs and growth year. In general the total metabolite quantities of each chemical group increase on the leaf surfaces with the growth stage, except for soluble carbohydrates. Among the sugars, sucrose dominates on leaves; especially in the elder stages, glucose proportions progressively increase whereas sucrose ones diminishes. Sugar alcohols vary progressively from young to older stages, quebraquitol diminishes and myo-inositol increases. Among free amino acids proline and glutamine increase particularly within the growth stages. At the reproductive stages, the composition of leaf surfaces is partly influenced by the presence of pollen. The comparison between leaf surfaces washed and not washed before the collect by spraying show that generally the plants not washed have more free amino acids (all of them) and particularly GABA, proline, alanine, serine, glutamine, more sugars except sucrose and more sugar alcohols particularly myo-inositol. When looking at the washing composition in metabolites of the different stages the best correlation between single metabolite of the washings and egg laying was obtained with glutamine (R² = 0.87). When testing an artificial blend composed of 28 metabolites present in the washings of young plants the same activity as the leaf surface washing (deterrence and inhibition) was observed. PCA analyse based on the compositions of metabolites on the maize surfaces on both years discriminates the elder stages which are stimulant from the inhibitors younger ones. Later growth stages were very near to soybean, which had both a neutral effect on WCR egg laying. The maize leaf washings had an effect on WCR egg laying, varying with the maize growth stage and were similar on the hybrids tested. The activity of artificial metabolite blend revealed that there was a high probability that the activity of leaf surface washings is due to a blend of primary metabolites. Maize leaf surface water washings definitely stimulate WCR egg laying. Their activities vary with the maize growth stages. From VT to R3 stages there is a bigger egg laying stimulation than on V8. The difference is all the more big as V8 has an inhibitor effect (less acceptance and less eggs per females). The V8 inhibitor effect could be due to very low quantities of metabolites under the threshold that is needed to induce acceptance of substrate for egg laying. It is too early to define which metabolites are concerned, however there is a blend of metabolites, which is active. Few correlations with individual substances and egg laying were shown. The synergism and antagonism between substances in their activity needs more years to be demonstrated. Proline, glutamine and sugars are probably much concerned in the blend stimulant effect. The observations on insect behaviour corroborate the fact that the insect takes information on the plant by scanning the surface with its ovipositor and walking a lot on plant. The egg laying site and site where the stimulation is perceived for egg laying are different and several hours may be observed between scanning and egg laying. Deposit of eggs in the soil and at the bottom of boxes is linked to tigmotactism and negative geotropism. It is independent from the chemical cues present on the substrate. Gustatory sensilla present on ovipositor and legs confirm the possibility for WCR of taking information by contact with the plant surface.

In the EU, there are grown 7.932 million ha of maize. 4.350 million ha of them are grain maize & CCM and 3.582 million ha are silage maize (ZMP-Marktbilanz, Getreide, Olsaaten, Futtermittel, 2001, 238 pp). Except for Finland, Sweden and Ireland, all EU members grow maize. For many countries, maize is an important energy supplier as animal food. The 8 countries analysed have 6.721 million ha of maize. Except for Switzerland, the other 7 EU member states (France, Germany, Italy, Austria, the Netherlands, Belgium and Luxembourg) represent with 6.659 million ha maize 84 % of the entire maize-growing area of the EU. France has, with 3.129 million maize, the largest maize-growing area in the EU. Out of 18.3 million ha arable land 17.1 % are maize-growing area. France grows about 1.7 million ha grain maize & CCM and about 1.4 million ha silage maize. Germany as the second biggest maize producer in the EU has 1.559 million ha of maize (13.2 % of the arable land). The largest portion with about 1.2 million ha is silage maize. Italy follows Germany in maize production with 1.271 million ha of maize. About 1.1 million ha are grain maize & CCM. Maize is of great importance for Italy because about one-forth of the arable land are maize-growing areas. This is also valid for the Netherlands with 28 % maize (225,615 ha) and for Belgium with 30.5 % (about 200,000 ha maize). In contrast to the Netherlands, where silage maize amount to 91 % of the whole maize-growing area, Italy has 86 % under grain maize and CCM. The climatic conditions lead to a difference between Northern member countries with more silage maize and Southern countries with more grain maize & CCM. For the development of the WCR, it is unimportant which type of maize is grown. However, the different value of grain and silage maize causes a higher pecuniary loss in case of grain and especially seed maize.

Monitoring protocol implemented in focus and safe area of Veneto region, described above, proved to be effective. In sensitive or newly infested area the following instructions can be summarized: - Sex pheromone traps have to be used; PAL or other traps, which proved to be as effective as, PAL traps in detecting very low populations. - Instructions accompanying PAL traps can be considered reliable; the position at maize ear level can be considered suitable; lure duration is over 45 days. - Sex pheromone traps have to place out exclusively in monoculture maize fields; when the latter are few the traps have to be placed out in 1st year maize fields in any case. - Traps can be placed out both in the edge and in the inner part of monoculture maize fields. - The lower the population the higher the number of traps to be used to detect population presence; with very low populations as those observed around Veneto focus area at least 8-10 sex pheromone traps should be placed out along the edge, 30-40m far from each other, per each monoculture maize fields about 1 ha large. - In order to get data for long term monitoring each year sex pheromone traps should be placed in the same monoculture maize field plus one nearby first year maize field or in one monocolture field and one 1st year maize fields within 500m from the position of the maize field where the first detection was recorded. A qualitative information about WCR (Western Corn Rootworm) presence can be obtained by inspecting cucurbit plants (mainly flowers).

We studied the performance of larvae of Diabrotica virgifera virgifera LeConte (Chrysomelidae, Galerucinae) on 17 different maize varieties from 6 European countries. We performed food conversion efficiency studies using a newly established method. The growth of D. v. virgifera (Western Corn Rootworm, WCR) larvae and the amount of ingested food was measured and the food conversion efficiency (ECI) was calculated. In addition to this we analysed the C/N ratio and the phytosterol content of the different varieties. Significant differences between the maize varieties with regard to larval weight gain, amount of ingested food and food conversion efficiency were encountered. The efficiency of WCR in converting root biomass into own biomass was positively correlated with the amount of nitrogen in the plant tissue. Furthermore the phytosterol content had a strong influence on larval weight gain and the amount of ingested food. It was possible to group the varieties into suitable and unsuitable cultivars with regard to WCR larval performance on the basis of the phytosterol content. Our results provide first evidence of the high variability of European maize varieties with respect to WCR nutrition. The use of less suitable maize varieties is discussed with respect to integrated pest management strategies.

In the USA, corn rootworms cause one billion US$ for treatment costs and crop losses per year. Corn rootworm infestations have been shown over a longer time to decrease yields of maize by 10-13%. For Europe, the potential damage of WCR (Western Corn Rootworm) for grain maize & CCM and for silage maize in Europe was calculated separately. The average yield of grain maize & CCM and silage maize in the 7 mentioned EU member states (France, Germany, Italy, Austria, the Netherlands, Belgium and Luxembourg) and Switzerland amounted to 93.9dt/ha and 444.9dt/ha, respectively. The greatest yields were reached by Belgium/Luxembourg with 108dt/ha of grain maize & CCM and by Italy with 527.3dt/ha of silage maize. In case of the establishment of WCR, an average potential loss of 9.39dt/ha grain maize & CCM and 44.49dt/ha of silage maize would correspond with the yield. The greatest potential loss per hectare is expected in Belgium/Luxembourg (10.8dt/ha) for grain maize & CCM and in Italy (52.73dt/ha) for silage maize. For grain maize & CCM, the absolute potential loss would amount to 1,072,610 t for the 8 above-mentioned countries and more than 1 million t for the 7 EU member states. Because of the largest high-risk areas in Italy for grain maize & CCM, the potential yield loss would be the highest with 518,803t. That would 48.4% of the total potential loss in grain maize & CCM of all the countries. This amount of potential yield loss of grain maize & CCM is followed by France with 368,552 t (34.4 %) and Germany with 113,145t (10.5%). In Switzerland the potential loss would be low (899t) because of the low concentration of grain maize & CCM in crop rotation. In Luxembourg (no high-risk areas), no economic yield reduction is expected. For silage maize, the potential yield loss for the 8 mentioned countries would be twice as high (2,020744t) as that for grain maize & CCM. This is the result of higher yield and higher potential yield reduction in silage maize and not of larger high-risk areas. Also for the 7 EU member states the amount would be higher than 2 million t of silage maize. Especially for Germany, the potential yield loss of silage maize would reach 930,946t (46.1% of the total potential loss in silage maize) and so the highest reduction followed by the Netherlands with 649,877t (32.2%) and Belgium with 217,696 t (10.8%). For the same reason like for grain & CCM for Luxembourg and Switzerland, the potential yield reduction of silage maize is zero and about 15,000t, respectively.

Celatoria compressa (Diptera: Tachinidae) was selected to be further tested on its suitability as a classical biological control agent for D. v. virgifera in Europe, i.e. on its reproductive biology, on its potential host range among European target and non-target species, and on the development of an efficient rearing technique. - It has been found on many species in four different genera of Diabroticina beetles. The key hosts seem to be A. trivittata (Mannerheim), D. tibialis Baly and D. v. virgifera LeConte. - It was collected at higher altitudes in Central and northern Mexico. - It is possible to rear this species on D. virgifera in laboratory. - It is reproducing in European and American D. virgifera. The adult parasitoid Celatoria compressa was the only parasitoid, which fullfilled the four requirements, i.e. to originate from the target host, to fit into European climate conditions, to easily reproduce under quarantine conditions, and to attack and parasitise the European and American strain of WCR (Western Corn Rootworm).

In the experiments within the DIABROTICA project period, orientation disruption effects of MCA on WCR (Western Corn Rootworm) were visible, but variable. In general, a reduced number of beetles is attracted for a few days to different trap types in MCA treated versus untreated fields. Throughout the research in Southern Hungary it was obvious that after each MCA application there were more or less pronounced peaks of orientation disruption after MCA coated grits have been distributed. These peaks varied somewhat with trap and bait type and were also susceptible to variation in temperature and wind direction/wind speed. As to success with demonstrating mating disruption itself, we are now less optimistic than in 2000 to demonstrate this effect in addition to orientation disruption. On first sight, no impact of MCA on the mating status of females was demonstrated. Frequency of sperm deposition in females spermathecae did not differ significantly between treated fields and control. Further research during subsequent field seasons should therefore concentrate on formulation techniques and on prolonging the lifetime of granular formulations. Sticky material will be added to the MCA/acetone/granular mixture in order to coat the grit surface with an additional cover reducing the volatility of MCA and making the granule stick to plants. We assume that we can then prolong the viability of the MCA release by a factor of 2-3. The general evaluation of the granular formulation has to be done in a holistic approach. Many independent factors have to be considered to merge into the final result, a stable, inexpensive, easy to prepare and long lasting formulation. In the original proposal we promised to investigate orientation disruption of WCR. During the review and budget approval process at Brussels our proposal wording was changed to mating disruption which is of course a related concept. It is, however, much harder to prove experimentally. The difficulty of proof has been discussed by the authors in their first report at the meeting in April of 2000 at Mezohegyes. As to significantly reducing the mating of Diabrotica virgifera virgifera, great care may be needed to have the disruption tools in place and working at the very first appearance of the female beetles. Males are in the field about a week to 10 days prior to females. Males are ready to mate any receptive female appearing at the scene and releasing her sex pheromone lure. Assuming that MCA will disrupt mating, we may not be able to see the effect since 100% of the females we possibly can catch in the field may already be mated before they come in contact with the upper parts of the corn plants where the MCA is concentrated. As a tool to get a handle on these females, we established emergency cages in the corn field. Yet, based on our present experience, we would have to monitor each cage 24 hours a day in order to prevent any early mating upon emergence, a task requiring considerably more man power than we had available so far.

The food utilization of adults of the invasive maize pest western corn rootworm (WCR; Diabrotica virgifera virgifera) was studied in its newly colonized range in Southern Europe. During a period of ten weeks we collected ten beetles per field per week from six fields with a high abundance of flowering weeds and six fields with a low abundance with the aim of understanding adult feeding behaviour in Europe. Gut content analysis was performed to determine the use of maize tissue and weed pollen with regard to maize phenology. Furthermore, all pollen found within the gut was identified and quantified to plant species level. The use of maize tissue by adult WCR (Western Corn Rootworm) changed with time according to maize phenology. Furthermore, pollen resources other than maize were used more frequently as the maize matured. A more detailed pollen analysis of the beetles revealed that adults fed on a high diversity of pollen, comprising 73% of all weed species (19 different plant species from 25 in total) found within maize fields. The use of different pollen resources was not dependent on their abundance but was determined by the preference of adult WCR for specific weed pollen. Pollen other than maize was found more frequently in beetles from fields with a high abundance of weeds compared to beetles from fields with a low abundance of weeds. Female and male beetles differed significantly in their use of alternative pollen resources; total numbers of pollen were higher in females, whereas males fed on a higher diversity of host plants. The pollen resources used by adult WCR in Southern Hungary are more diverse in comparison to data from the USA. Adaptation of their feeding behaviour to more heterogeneous environmental conditions may contribute to the invasion success of WCR in Europe.

About 33% additional contribution in mortality of WCR (Western Corn Rootworm) populations would have been needed to suppress the maximum realised net reproductive rate (Rm) of WCR in this study to a level of no population growth (R m <1). In the worse case of performing its maximum potential progeny, the potential capacity of the net reproductive rate (R p) could have been suppressed by additional mortality of 68% in the life - table. Two sustainable control options could be considered, i.e. - The enhancing of indigenous natural enemies in Europe (Conservation biocontrol) and - The inundative or inoculative release of exotic biological control agents (Classical biological control). The first option has to be neglected since this study showed a low effect of indigenous natural enemies on WCR. This is mainly due to: - The exotic status of WCR in Europe, - The exotic nature of maize in Europe, and - To the reduced complexity and diversity in the managed crop system. None of the life stages of WCR were attacked by effective natural enemies. Very few numbers of adult beetles were infested by Beauveria bassiana or Metharizium anisopliae having an intensity of mortality (kx) close to zero. No predators of WCR became obvious in this study, which could significantly reduce populations. In general predation has to be considered as a less frequent key factor in reducing exotic herbivores than parasitism. Even parasitism by a single parasitoid species has been frequently identified as key factor in life - tables on exotic herbivores, whereas predation by a set of generalist predator species has been more frequently identified as a key factor in native herbivore populations and in natural habitats. Thus, the second sustainable control option by using biological control agents, such as European entomopathogenic nematodes or exotic parasitoids in inundative or inoculative biological control is highly addressed. Generally, it is difficult to make theoretical generalisations about which stage of WCR might be the most suitable as a target for biological control actions because knowledge about interactions between mortality factors is limited. Two scenarios of action of biological control agents can be simulated. First, the agents would parasites WCR, contemporaneously with other natural mortality factors in each age interval. Secondly, the agents would parasite WCR and cause mortalities independently and in addition to natural mortalities in each age interval. The second case is most desirable, and hypothised biological control agents would have highest impact in reducing progeny when occurring as additional morality to high natural mortality, such as in first instar larvae. However, since it is not likely that all mortality factors operate independently and additionally, the first scenario is more realistic. Hereby, it s is assumed that control agents would have the same chance in each of the life stage of WCR to cause an additional significant change in the survival and in reducing progeny. For example, a marginal attack rate of 10% parasitism in any age interval would have the potential to additionally reduce the progeny by 10%. Thus, entomopathogenic nematodes as inundative biological control agents would have the potential to reduce the progeny in WCR. In third instar larvae, an average parasitism of 63% by Steinernema arenarium, as shown in semi-natural trials, can potentially reduce 63% of progeny, when contemporaneously calculated in the life - table. It could cause a loss of up to 98 % when acting independently and in addition to natural mortalities. In the same two ways a parasitism of 40.7% third instar larvae by Steinernema feltiae can potentially reduce progeny by 26% and up to 63.6%. Furthermore, parasitoids of adult WCR are known from its area of origin in Central America, such as the tachinid fly Celatoria compressa. Adult WCR can be parasitised by 27% of C. compressa as an overall mean at different densities, and would cause a direct loss in progeny of up to 27% when acting before the maturation of adults. The potential impact of such a parasitoid on WCR populations is estimated to be high since they can directly reduce the realisation of fecundity in the population, which is the major factor behind population increase in WCR. Since WCR is syn-ovigenic, the parasitoids can even reduce progeny over the whole reproductive period of WCR from July to September. Thus, WCR, such as many herbivorous insects in natural or cultivated habitats seems to be a good subject to different sources of top down control. When considering the above-mentioned need of additional control to prevent population growth, parasitoids may be an important source of population regulation for the WCR, and studies on host parasitoid density dependence are required.

Provision of supplementary food (e.g. food spray, pollen, nectar): Presence of wild flowers can increase food sources for parasitoids in crops. Minor negative impact is expected due to additional pollen supply for adult WCR (Western Corn Rootworm) when maize starts drying. Provision of refugia for overwintering: Overwintering refugia such as grass strips or remaining crop plant material after harvesting can increase survival of natural enemies in maize and can increase survival of WCR eggs. Maintenance of non-economic levels of hosts or alternative prey / hosts over time to ensure survival of natural enemies: As there are no specific natural enemies known on D. v. virgifera in Europe the maintenance of a low population of this pest is not necessary as long as alternative hosts, e.g. other insect larvae or adult, are available for generalist predators. Those will be available when non - crop plants and their herbivores remain in or around fields. Composition of non - crop vegetation within and around fields, Pro and contra of weeds: Weeds are a major problem in maize production. On the other hand non - crop plants within and around fields can benefit natural enemies e.g. the presence of wild flowers increases parasitoid abundances. Some entomophagous insects are attracted or arrested by chemicals released by the herbivore hosts plant or by associated plants, which can be investigated as soon as specific natural enemies against D. v. virgifera are known. Outbreaks of crop pests are more likely to occur in weed free fields than in weed diversified crop systems, which were also shown for maize. Thus presence of tolerable levels of weeds should be achieved. Minor negative impact of weeds are expected due to additional pollen supply for adult WCR when maize starts drying (especially Ambrosia spp, and Sunflower), due to higher oviposition rates of Diabrotica beetles at higher plant coverage and due to competitions between weeds and young maize plants. Use of kairomones: No kairomone is known to attract specific natural enemies in the European maize system Increasing environmental heterogeneity and complexity of agricultural areas: Vegetation mosaics and corridors in the agricultural areas are important for enhancing natural enemies, e.g. parasitism levels of Ostrinia nubilalis in maize near wooded sites were found to be higher. Additionally high crop diversity through mixtures in time and space generally enhance natural enemies. Soil type: Soil type is difficult to change, although it has an influence on ground living predators and on the survival of D. v. virgifera larvae.

The results predict that C. compressa would have a narrow host range in Europe, being restricted to few genera among the Galerucinae. This is consistent with the host records of C. compressa in Mexico, which was only found from Diabrotica sp. and Acalymma sp. There are several lines of evidences that host specificity in Dipteran parasitoids, which attack adult insects, are more often determined by the events leading up to oviposition, rather than by the events occurring after oviposition. The specifically modified ovipositors and specialized attack behaviour of these parasitoids further reinforces its host specificity. It is likely that the more host specific nature of Celatoria species when compared to many other tachinids, is due to the elaborately modified piercing ovipositor of the females, which would be directly related to the host acceptance of adult Coleoptera by Celatoria sp. In addition, the larviposition behaviour has been thoroughly investigated in C. compressa, C. diabroticae and C. setosa. The host-selection of Diptera, especially Tachinidae, follows phylogenetic evolution much more closely than previously suspected from the existing host-lists. Aulacophora represents the New World genus equivalent of Diabrotica in the Old World, and there is a remarkable resemblance between the two genera on larval, pupal and adult structures, breeding habits and food plants. In the current tests of non-target species within Galerucinae, only A. foveicollis was found to be parasitised by C. compressa. This suggests that phylogenetically closely related non-target species would be most likely at risk when C. compressa would be selected in a classical biological control programme. Although A. foveicollis was occasionally attacked by C. compressa in the laboratory, the parasitism was extremely low compared to that of D. v. virgifera. Only a single puparia was obtained from 22 parasitised A. foveicollis and from a total of 1,110 A. foveicollis tested. Encapsulation of the tachinid larvae was found in A. foveicollis, showing an immune response of the resistant host toward C. compressa. It suggests that A. foveicollis is sub-optimal as a host for development of C. compressa, although it is possible that tachinid larvae in A. foveicollis require a longer development time than that in D. virgifera. Besides two more Aulacophora species in Russia, A. foveicollis is the only Aulacophora species present in Europe and is restricted to southern Europe. Thus it is unlikely that C. compressa would encounter A. foveicollis in the field in Central/Middle Europe if future field release is implemented in Hungary. In addition, there were only two natural enemies found attacking A. foveicollis, one is a mite Histiostoma sp. that attacked larvae in Greece and another is Rhinocoris fuscipes preying on adults in India. Therefore, the impacts of C. compressa on A. foveicollis and other organisms would be extremely low even if they are encountered in the ecosystem. Learning is very common in Hymenoptera parasitoids and learning effects could increase the acceptance of a low ranked host for Hymenopteran parasitoids. It was also suggested that learning is also likely to be common in Diptera parasitoids, although it has been only approved in one species, Drino bohemica. Our results of sequential no-choice and choice test all indicated that the parasitism pattern of C. compressa did not change in over time. No difference was found between the host acceptance of naive and experienced C. compressa. Thus increased oviposition pressure for C. compressa and the experience effects of sequential exposure had no influence on host range. Learning might play a minor role in C. compressa behaviour, and would likely further reduce the risk level to non-target species in the field.

Eradication and containment strategies were implemented in and just around a focus area near the Venice Airport since 1999, just after the first Diabrotica specimens had been captured in 1998. First strategies deployed in 1999 have been continued and improved in the framework of the EU-project. The results were checked: - In the focus and the safe area; - In areas at increasing distances from the focus area in Veneto and other Italian regions. The strategies implemented in Veneto proved to be very effective in stopping WCR (Western Corn Rootworm) populations; the population has been reduced to the minimum from the year when the first captures were recorded (1998). Differently from all the other sites in Europe where the species was detected, in five years there was no significant spread from the initial focus area and a dramatic reduction of the population levels despite the fact that the area proved to be suitable for WCR population development. Based on data from the Veneto WCR eradication program and from first observations collected in newly infested sites guidelines for a successful eradication-containment program have to be divided into prevention and actual eradication-containment strategies. A. Prevention Once the first introduction of WCR occurred the probability of successful transportation of gravid females inside Europe has been increasing because of: - Shortest distances within this area; this elevates the possibilities of adult survival and movement by plane and other modes of transportation; - The increase of WCR population and distribution in Europe makes it even more likely that transportation of WCR beetles occur. Two main factors appear associated with new introductions as observed in Veneto in 1998 (Fig. 4.40), Friuli Venezia Giulia in 2002 (around the airport is more than 90% of the cultivated area and maize monoculture fields are more than 50%), Belgrade in 1992 (maize monoculture fields next to the border of airport facility), France 2002 (Reynaud 2002, unpublished data): - An area with frequent movement of vehicles coming from infested areas; - The presence of monoculture maize fields near to the border of the area of possible infestation. Therefore prevention measures must guarantee the absence of monoculture maize fields all around selected sensitive sites and in these and other sites an effective monitoring suitable for promptly detecting newly arrived populations must be implemented. A reliable prevention procedure for Western Europes sensitive sites should include: -Prohibition of maize cultivation or maize monoculture 500 - 1000 m around selected sensitive sites such as airports, custom facilities; precise identification in the maps of the prevention zone; -Monitoring of monocolture maize fields around the prevention zone and of maize fields in the prevention zone if the interruption of maize monocolture is implemented; sex pheromone traps like PAL must be used. B. Actual eradication-containment strategies Once WCR has been promptly detected the following proved to be the key factors to implement a successful eradication/containment program: - Immediate identification of a focus area where WCR has been detected; from the beginning these areas should be at least 1-2km larger than the area from which the first specimens were captured. In each focus area: -- Imposing restrictions on the planting of maize in fields: prohibition of planting maize after maize. -- Monitoring the WCR population by using sex pheromone traps; -- Applying foliar insecticide treatments to all maize fields to control WCR adults; -- Prohibiting the movement of fresh maize or soil in which corn was grown the previous year outside of the focus area; -- Not allowing maize to be harvested before 1st of October or in any case allow plants dry to appropriate levels before removing; -- Preventing volunteer maize and grasses serving as alternative food sources for WCR larvae from being present in non maize fields (including set aside fields); - Prompt delineation of safety areas just around the focus area. In each safety area: -- Monitoring the WCR population by using sex pheromone traps; -- Applying foliar insecticide treatments to maize fields where WCR specimens have been captured and to all maize fields around the infested fields.

A simulation model for spreading scenarios of WCR (Western Corn Rootworm) has been developed using the GIS software ArcView/ArcInfo (ESRI). The calculation is based on grid processing of ARC-Macro-Language with a grid extent of 250X250m. The programme checked each grid for the kind of crop for further processing. The following spreading parameters are considered: - Spreading rates of populations per year, - Influence of possible containment measures (i.e. phytosanitary measures), - Concentration of maize (analysis of high-risk areas), and - Topography (elevation of mountains). The dispersal rate of WCR in Europe was analysed starting with the discovery of the economic WCR infestation in Serbia at the beginning of the 1990ies. Dispersal rates of the WCR differed from year to year. The spreading of the WCR ranged from 60 to 100km per year without containment measures and from 0 to 37km with (FAO programme TCP/RER/6712). The simulation model used as an average a maximum WCR population-spreading rate of 80km per year without containment measures and of 20km per year with. The maximum spreading rate is reached by WCR in the succeeding year only if continuous maize is available in the infested area. The concentration of maize in crop rotation is the main factor in the simulation model. In case of low maize concentration, multiplication factor and spreading pressure are very low. In this case, the spreading rate was reduced by a correction factor K, which is defined as follows: In case of >= 50 % of maize in crop rotation K = 1 and concentration of maize in % 2 in case of < 50 % of maize in crop rotation: K = 100 The following formula was used in the simulation model to calculate the spreading rate of the WCR: AR = FD K; where AR = spreading rate of the WCR; FD = distance of flight with containment measures (20km/year) or without (80km/year) and K = correction factor (see above). Furthermore, the topography was analysed in the infested areas of Southeast Europe. Analysis showed that the WCR is not able to fly over altitudes of 900m, which was considered in the simulation model. However, it cannot be fully excluded that in some cases single individuals are floated by wind flows over mountains of more than 900m. We assume that this has no influence on the ongoing of spreading of populations of WCR in general. The lowest mountain chain in Western Europe is up to 800m and has valleys (often with maize), which favour progressive dispersal. Only the Alps have tremendous influence on the ongoing of spread in West Europe and are a natural barrier. Any appropriate information is utilised in the WCR spreading simulation model. This simulation model was used to simulate the spreading rate in France, Germany, Italy, Austria, and Belgium and for Switzerland over ten years. For the Netherlands, the precise growing data for maize and digital maps are not suited. Therefore, simulation of possible spreading scenarios for the Netherlands was not possible. Four scenarios were chosen for each country: - Introduction via the international airport with and - Without containment measures and - Progression of ''natural spread' via the next potentially infested country with the starting point at a location near the border with and - Without containment measures. The used locations for the simulation of spreading scenarios are in Tab. 2.1. Using ArcView/ArcInfo software, different spreading scenarios of WCR were simulated for France, Germany, Italy, Austria, Belgium and Switzerland on maps and these data were also the basis for the cost/benefit analyses. Damage due to WCR is expected in the 5th to 7th year. This conforms with findings from the USA and Hungary (C. R. Edwards and J. Kiss, personal communication). Results are presented on coloured maps, showing the spreading of WCR with and without containment measures. These maps may be requested from the author.

Temperature is one of the most important factors for the establishment of the WCR (Western Corn Rootworm). Egg mortality depends on winter temperature and could reach 50% at -10°C for four weeks and at -15°C for one week. Infested areas in the USA show great differences in January long-term mean temperatures. January long-term mean temperatures range from 4.4 to -14.1°C and reach an average of -4.8°C. The coldest January long-term mean temperatures, up to -14.1°C, are registered in the USA in North Dakota where the WCR is established. The above-mentioned temperatures are air temperatures and the eggs over wintering in soil at a depth of 15 to 30cm, in these regions probably at 30 cm or more. For Germany for example, January long-term mean temperatures range from 2.8 to -3.8°C and are on average -0.5°C. A comparison of winter conditions between the USA and Germany concluded that winter conditions are not the limiting factor. Furthermore, the WCR is established in Canada (Ontario and Quebec) where cold winters are common. In Western Europe, long-term winter temperatures are not as cold as in most part of the USA and of Canada because Western Europe is influenced by maritime climatic conditions. Winter conditions in Western Europe are not the limiting factor for establishment. Temperatures in the growing season are responsible for the successful finishing of a generation, increase of population and finally the establishment of the species. Because this species is more adapted to continental climate with hot summer time in the growing season, this season has a stronger influence on the development, abundance and finally on the damage potential of WCR. There are great differences in the calculated development time of the single stages of WCR between the southern (e. g. Italy), and northern countries (e. g. the Netherlands and Germany) considered. In Italy, development conditions are good because of the warmer temperatures in the growing season. In the temperature sum model, the first 50% of egg hatching would be expected on 24th of April in the south and in average on 28th of May for Italy. But in the south, there could be a mortality in the development of eggs because of lower temperatures for diapause are missing. On the other hand, maize growing in the south of Italy is not so important as in the north with high maize concentration areas (e. g. Lombardia, Piemonte and Veneto). In contrast to Italy, the model calculated for the Netherlands and Germany averaged 50% egg hatching for 10th of July beginning on 5th of July and 20th of June and ending on 20th of July and 30th of July, respectively. In France, the emergence of 50% of first instar has a wide range from 21st of May in the south to 25th of July in the north. The third instar as the most damaging stage of WCR is expected to hatch between 16th of June in Italy and 29th of July in the Netherlands. The earliest emergence for the third instar was calculated for the south of Italy (14th of April) and latest for the north of Germany (28th of August). Beside Italy (14th of May to 27th of July), France would cover a wide range from 11th of June in south to 19th of August in the north. For the emergence of the short pupa stage the relations would be the same (e. g. France). First adults of WCR would appear in Italy between 27th of May and 10th of August and on average on 28th of June. In Italy, with optimal climatic conditions for the development of WCR, there exist first data for the catching of adults in pheromone traps from 2001. In contrast to Italy, in Germany the adults would appear between 16th of July and 7th of October and on average on 10th August. In the Rhine valley (Southwest of Germany, federal land Baden-Wurttemberg), there are also favourable conditions for development in the growing season and in addition a high concentration of maize. In this area the development of high abundance and economic loss is probable. In the north of Germany (e. g. federal land Schleswig-Holstein), hatching of the beetle would be very late so that the conditions for feeding and egg laying already would be unfavourable. In extremely cold years in the north of Germany (Flensburg), populations would not be able to finish the generation. The north of Germany (Flensburg region) and furthermore Denmark could form the climatic barrier to build up a damaging abundance of WCR. In France, which has a wide temperature spectrum from Mediterranean to moderate climate, the emergence of the adult beetle would range from 23rd of June to 9th of September. With the exception of some unfavourable areas where maize growing is not favoured, most areas of France provide good conditions for the development of WCR as do Austria, Switzerland and Luxembourg, too. In the Netherlands and Belgium not optimal conditions for WCR development are expected.

France, Germany, Italy, Austria, the Netherlands, Belgium, Switzerland and Luxembourg have 1.646 million ha of high-risk maize areas. This is about one-fourth of the maize-growing areas of these countries. The 7 above-mentioned EU member states reflect 1.642 million ha high-risk and 20.7% of the entire maize-growing area of the EU. Considering absolute figures, Italy has the largest high-risk area with 551,918ha. Considering relative figures, this makes up 43.4% of its whole maize-growing area. Thus it ranks second after the Netherlands with a percentage of 69.2. In the Netherlands, missing crop rotation in maize-growing areas is very suitable for the development and multiplication of the WCR. In future, the concentration in maize growing will lead to high damage or intensive control of WCR (Western Corn Rootworm). France (467,956ha) and Germany (347,620ha) have also large high-risk areas in continuous maize. In Germany for example, the four federal lands Lower Saxony, Northrhine-Westfalia, Bavaria, and Baden-Wurttemberg have high-risk areas of 133,509ha, 114,113ha, 86,992ha, and 13,006ha, respectively. In Austria, the percentage of 21.1 maize high-risk areas is comparable with the situation in Germany. The spatial distribution of the high-risk areas in Austria, which could be probably infested in 2002, is shown in. The situation in East Austria is characterised by low maize in concentration (>= 15%), which reduces the spreading rate of WCR. In Belgium, one-third of the maize-growing areas show a high concentration of maize. The situation is much better in Switzerland (7.1%) and in Luxembourg (no high-risk area). The percentage of high-risk areas in grain maize & CCM and silage maize reflects the percentage of the relevant growing areas in the 7 countries except for France. In France, the percentage of high-risk areas in grain maize & CCM is higher (89.9%) than the percentage in grain maize & CCM (55.9%). The largest high-risk area in grain maize & CCM amounts to 551,918 ha and is situated in Italy. The largest high-risk area in silage maize amounts to 217,867ha and is situated in Germany. Referring to all of the 8 countries under analysis, the high-risk areas in grain maize & CCM amounted to about 1.178 million ha and in silage maize to about 0.469 million ha. In Italy, which has one-third of the total maize high-risk areas of the 8 countries, the situation is difficult. Lombardia, Piemonte and Veneto regions are infested by WCR. Veneto is the only region allowing successful eradication. Lombardia region, the largest maize-growing region in Italy, has 251.893ha maize high-risk areas. These areas cover 76.9% of the Lombardia maize-growing region. Furthermore, Lombardia region has 45.6% of the total maize high-risk areas of Italy and 15.3% of maize high-risk areas of the 8 countries analysed. In Lombardia region, out of 1,163 maize-growing municipalities 796 municipalities have maize high-risk areas. 176 municipalities in Lombardia region have exclusively continuous maize (100%) and 459 municipalities have at least or more than 75% maize in crop rotation. The conditions for multiplication and further spreading of WCR in Lombardia region are very favourable. This will have an enormous influence on the spread ongoing in Italy.

In the Veneto focus area WCR (Western Corn Rootworm) beetles were captured exclusively in maize fields. In 2000 PAL, PALs, Pherocon CRW, Pherocon AM (2-14 per each type per field) were also placed out in fields planted with different crops included in different crop rotations, preferably close to monoculture maize fields where WCR specimens had been captured. No WCR specimens were captured in crops different from maize also if very close to the small continuous maize fields where specimens had been caught. In 2001 at least 2 PAL and 4 Pherocon AM traps were placed out in non maize-crops all around maize fields where WCR captures had been recorded. As observed in 2000 no specimens were caught. In 2002 only two specimens were captured by two PAL traps placed out in two different maize fields despite the fact that hundreds of traps had been deployed. Therefore no useful information on this issue can be inferred. In the Veneto focus area WCR proved to be tightly linked to maize monoculture and main movements seem to occur from monoculture fields to 1st year maize fields so that WCR can spread more quickly and intensively if most of the 1st year fields (and obviously monoculture fields) are planted with maize in the subsequent year.

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