Monitoring presence and advance of the alien invasive western corn rootworm beetle in eastern Slovenia with highly sensitive Metcalf traps

The American Chrysomelid beetle Diabrotica virgifera virgifera LeConte (D.v.v.), also called the western corn rootworm, spread from the location of its original introduction into Europe, Belgrade airport (BACA 1993), in all directions. Within a decade it occupied almost all countries of South-eastern and Central Europe. However, it reached Slovenia as late as 2003: Only 19 specimen were found in maize fields of the eastern and also western provinces. Already in the summer of 2004, their number had risen to 386 which were mainly found in the eastern provinces near the borders to Croatia, Austria and Hungary. For their monitoring, a simple trap is being described which can be acquired in high numbers at a very low price and can guaranty a most sensitive detection of beetles. The Metcalf cup trap which in Slovenia so far has been unknown performed the task of monitoring quite well at five locations. It will also facilitate the future search for new and increasing infestations. Comparing 2004 with the year 2003, D.v.v. expanded its range in eastern Slovenia by about 15 km. These new infestations will include territories in which fields of the regionally Important oil seed pumpkin Cucurbita pepo are located. With important traffic connections between South-eastern and Central Europe, Slovenia will occupy a bridgehead function in the preventive protection of maize from D.v.v. spreading into more northerly European regions including western Austria and southern Germany.     

Presence of moose (<Emphasis Type="Italic">Alces alces</Emphasis>) in Southeastern Germany

Germany is situated on the western border of the distribution area of moose (Alces alces) in Europe. In the past, the number of moose sightings has been very limited and irregular in the state of Bavaria (SE Germany). Nearby populations of moose are located in neighboring countries to the east of Germany, in the Czech Republic, and Poland. A monitoring system for moose was established following a considerable increase in moose observations in Bavaria in 2007. As no reproduction has yet been recorded, moose observation has so far been attributed only to migrating individuals. Cows are more often registered than bulls (27 of 35 observations between 2004 and 2008). The reasons for the increase in migratory activities of moose have not yet been identified.     

Genes, gene flow and adaptation of Diabrotica virgifera virgifera

1  Diabrotica virgifera virgifera has emerged as a major pest of cultivated maize, due to a combination of its high capacity to inflict economic damage, adaptability to pest management techniques and invasiveness.
2 This review presents a survey of the current state of knowledge about the genetics of D.   v.   virgifera . In addition, the tools and resources currently available to Diabrotica geneticists are identified, as are areas where knowledge is lacking and research should be prioritized.
3 A substantial amount of information has been published concerning the molecular phylogenetic relationships of D.   v.   virgifera to other chrysomelids.
4 There is a growing literature focused on the population genetics and evolution of the species. Several adaptations to anthropogenic selection pressure have been studied, with resistance to synthetic insecticides providing some particularly well-characterized examples.
5 A notable deficiency is a lack of studies directed toward the formal genetics of D.   v.   virgifera .     

International cooperation on western corn rootworm ecology research: state-of-the-art and future research

1 Invasive pest species are challenging partly because the invasion process may be highly dynamic and because of the lack of knowledge of many researchers, professionals and farmers in the newly-invaded regions. The chrysomelid Diabrotica virgifera virgifera LeConte is such an invasive pest. It has been the main pest of continuous maize in the U.S.A. for more than 60 years and is currently spreading throughout Europe.
2 In the area with a long history of this pest (Central and North America), scientific knowledge concerning the ecology of this pest has accumulated over the last decades. This resource is of great importance to both America and Europe and has to be gathered, shared and adapted to new situations. We therefore examined, both qualitatively and quantitatively, the scientific literature relating to D.   virgifera virgifera ecology.
3 The quantitative analysis suggests that research on D.   virgifera virgifera ecology is still in its infancy in Europe and suffers from geographical barriers (between Europe and North America and between linguistic areas within Europe) and that scientific communication should be strengthened both between North America and Europe and within Europe.
4 As a first solution to this problem, we introduce three companion review articles that constitute a landmark for D.   virgifera virgifera research, enabling European and American scientists and decision-makers to orient themselves and discover new opportunities for research. We also stress that international research cooperation is the most important key to successfully manage invasive species.     

Selection of entomopathogenic fungi for the control of the western corn rootworm Diabrotica virgifera virgifera

Abstract:  The western corn rootworm Diabrotica virgifera virgifera Le Conte (Col., Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. During a field survey in Hungary in 2005, naturally occurring entomopathogenic fungi were found to attack this pest. These novel isolates together with standard isolates were tested for virulence against D. v. virgifera larvae and adults. Twenty strains of Metarhizium anisopliae , Beauveria bassiana and Beauveria brongniartii were used in bioassays in the laboratory. Larvae and adults were dipped into a spore suspension with a concentration of 1 × 10⁷ conidia (con.)/ml. They were kept for 14 days at 22°C (±2°C) and 70% relative humidity. The number of infected larvae and adults were counted and infection rates were calculated. Adults were significantly more susceptible to entomopathogenic fungi than larvae. The most virulent isolate infected about 47% of larvae ( M. anisopliae Ma2277), whereas the infection rate in adults was up to 97% ( M. anisopliae Ma2275). Isolates of M. anisopliae caused significantly higher mortalities than isolates of B. brongniartii and B. bassiana . Most of the adult beetles were killed within 12 days. Isolates from D. v. virgifera were more virulent than those from other hosts.     

Population dynamics of a Western corn rootworm (Coleoptera: Chrysomelidae) variant in east central Illinois commercial maize and soybean fields

Three on-farm sites in Iroquois County, IL, each containing an adjacent 16.2-ha commercial production maize, Zea mays L., and soybean, Glycine max (L.) Merr., field, were monitored for western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), adults from June through September 1999-2001. Mean captures of D. v. virgifera adults as measured with Pherocon AM yellow sticky traps were significantly greater in maize than in soybean. Overall mean numbers of D. v. virgifera adults captured with vial traps were significantly greater in soybean than in maize. Emergence cage data revealed that after 50% emergence of D. v. virgifera adults occurred, peak captures of D. v. virgifera adults occurred in maize as measured with vial and Pherocon AM traps. After maize reached the R2 (blister stage, 10-14 d after silking) stage of development and 90% emergence of D. v. virgifera adults had occurred, peak captures of D. v. virgifera adults were observed in soybean by using vial and Pherocon AM traps. Also, after maize reached the R2 stage of development, numbers of females significantly increased in soybean and decreased in maize. Captures of female D. v. virgifera adults frequently exceeded published economic thresholds in soybean, regardless of trap type used. Estimated survival of variant D. v. virgifera (egg to adult) in these commercial rotated maize fields was 10.7 and 9.4% from 1999 to 2000 and from 2000 to 2001, respectively. This compares with nonvariant D. v. virgifera survival estimates in continuous maize production systems in Iowa of 6.7 and 11% from 1983 to 1984 and from 1984 to 1985, respectively.     

Assessment of establishment and persistence of entomopathogenic nematodes for biological control of western corn rootworm

Abstract:  The use of entomopathogenic nematodes (EPN) is potentially one ecological approach to control the invasive alien western corn rootworm ( Diabrotica virgifera virgifera LeConte, Col., Chrysomelidae) in Europe. This study investigated the establishment and the short- and long-term persistence of Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), Heterorhabditis megidis Poinar, Jackson and Klein (Rh., Heterorhabditidae) and Steinernema feltiae Filipjev (Rh., Steinernematidae) in three maize fields in southern Hungary, using the insect-baiting technique. All three EPN species equally established and persisted in maize fields. The timing of application (April or June) did not influence the establishment of EPN species. EPNs persisted for 2–5 months, i.e. they survived up to and throughout D. v. virgifera larval occurrence in the soil. Results demonstrate that D. v. virgifera larvae can potentially be controlled by EPNs during the same year of EPN application but no long-term control effect is expected under intensive maize cultivation practices.     

Response of larvae of invasive maize pest Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) to carbon/nitrogen ratio and phytosterol content of European maize varieties

We studied the performance of larvae of Diabrotica virgifera virgifera LeConte (Chrysomelidae, Galerucinae) on 17 different maize, Zea mays L., varieties from six European countries. Food conversion efficiency studies were performed using a newly established method. The growth of D. v. virgifera (western corn rootworm) larvae and the amount of ingested food was measured and the food conversion efficiency was calculated. In addition, we analyzed the carbon/nitrogen 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 D. v. virgifera in converting root biomass into insect biomass was positively related to the amount of nitrogen in the plant tissue. Furthermore the root phytosterol content influenced the larval weight gain and the amount of ingested food. It was possible to group the varieties into suitable and unsuitable cultivars with regard to D. v. virgifera larval performance on the basis of the phytosterol content. Our results provide the first evidence of the high variability among European maize varieties with respect to D. v. virgifera nutrition. The use of less suitable maize varieties is discussed with respect to integrated pest management strategies.     

Relationship between phenotypic traits and selected fitness components of Diabrotica virgifera virgifera

Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) is one of the most important and best-studied maize pests, yet little information is available regarding the basic relationships among its phenotypic traits, particularly those that reflect the overall fitness of this successful invader in Europe and North America. Such information is critical for studies on the invasiveness, behavioural ecology, and management of this pest. Phenotypic traits that change over the lifetime of the beetle were investigated (e.g., fresh body weight) as they can indicate changes in adult physiology or fitness of D. v. virgifera . Phenotypic traits that remain stable over the beetle's lifetime were also investigated (e.g., pronotum width, head capsule width, hind tibia length), as they most likely allow detection of genetic differences between populations. Furthermore, phenotypic traits were investigated that may best predict fecundity (e.g., fresh body weight, elytra width) and life span (e.g., elytra length), as these two factors influence the population growth of this highly invasive species. Finally, regression equations are provided for the age-specific oviposition and survival of the long-living D. v. virgifera adults, which may allow researchers to reduce the duration of their bioassays without losing information.     

Unexpected Patterns of Admixture in German Populations of Aedes japonicus japonicus (Diptera: Culicidae) Underscore the Importance of Human Intervention

The mosquito Aedes japonicus japonicus, originally restricted to temperate East Asia, is now widespread in North America and more recently has become established in Europe. To ascertain the putative number of separate introductions to Europe and examine patterns of expansion we analyzed the genetic makeup of Ae. j. japonicus populations from five cemeteries in North Rhine-Westphalia and Rhineland-Palatinate, two western German federal states, as well as of specimens from populations in Belgium, Switzerland, and Austria/Slovenia. To do so, we genotyped individual specimens at seven pre-existing polymorphic microsatellite loci and sequenced part of the nad4 mitochondrial locus. We found evidence of two different genotypic signatures associated with different nad4 mitochondrial haplotypes, indicating at least two genetically differentiated populations of Ae. j. japonicus in Europe (i.e. two distinct genotypes). Belgian, Swiss, and Austrian/Slovenian populations all share the same genotypic signature although they have become differentiated since isolation. Contrary to expectations, the German Ae. j. japonicus are not closely related to those in Belgium which are geographically nearest but are also highly inbred. German populations have a unique genotype but also evidence of mixing between the two genotypes. Also unexpectedly, the populations closest to the center of the German infestation had the highest levels of admixture indicating that separate introductions did not expand and merge but instead their expansion was driven by punctuated human-mediated transport. Critically, the resulting admixed populations have higher genetic diversity and appear invasive as indicated by their increased abundance and recent spread across western Germany.     

Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics

1 The western corn rootworm Diabrotica virgifera virgifera LeConte is a major insect pest of field maize, Zea mays L. Larvae can cause substantial injury by feeding on maize roots. Larval feeding may destroy individual roots or root nodes, and reduce plant growth, stability, and yield. Costs associated with managing corn rootworms in continuous maize are annually one of the largest expenditures for insect management in the United States Corn Belt.
2 Even though D. virgifera virgifera has been studied intensively for over 50 years, there is renewed interest in the biology, ecology, and genetics of this species because of its ability to rapidly adapt to management tactics, and its aggressive invasive nature.
3 This article provides a comprehensive review of D. virgifera virgifera population dynamics, specifically: diapause, larval and adult development, seasonality, spatial and temporal dynamics at local and landscape scales, invasiveness in North America and Europe, and non-trophic interactions with other arthropods.
4 Gaps in current knowledge are identified and discussed especially within the context of challenges that scientists in North America and Europe are currently facing regarding pest dynamics and the need to develop appropriate management strategies for each geographic area.     

European populations of Diabrotica virgifera virgifera are resistant to aldrin,but not to methyl‐parathion

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of cultivated corn in North America and has recently begun to invade Europe. In addition to crop rotation, chemical control is an important option for D. v. virgifera management. However, resistance to chemical insecticides has evolved repeatedly in the USA. In Europe, chemical control strategies have yet to be harmonized and no surveys of insecticide resistance have been carried out. We investigated the resistance to methyl‐parathion and aldrin of samples from nine D. v. virgifera field populations originating from two European outbreaks thought to have originated from two independent introductions from North America. Diagnostic concentration bioassays revealed that all nine D. v. virgifera field populations were resistant to aldrin but susceptible to methyl‐parathion. Aldrin resistance was probably introduced independently, at least twice, from North America into Europe, as there is no evident selection pressure to account for an increase of frequency of aldrin resistance in each of the invasive outbreaks in Europe. Our results suggest that organophosphates, such as methyl‐parathion, may still provide effective control of both larval and adult D. v. virgifera in the European invasive outbreaks studied.     

Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) larval feeding behavior on transgenic maize (MON 863) and its isoline

Diabrotica species (Coleoptera: Chrysomelidae) larval behavior studies have posed a challenge to researchers because of the subterranean life cycle of this pest. To fully understand how the western corn rootworm, Diabrotica virgifera virgifera LeConte, injures the maize, Zea mays L., root system, its behavior must be studied. For example, larvae that can detect an area of the root that has a lower amount of toxin, whether from an insecticide or a transgenic maize plant, have an increased chance of survival. This study assessed D. v. virgifera larval feeding behavior on rootworm-susceptible maize and maize containing a biotechnology-derived trait (MON 863) with resistance to D. v. virgifera first instar feeding. Maize plants were grown in a medium that allowed for direct observation and measurements during feeding of larval stadia. Neonates were placed on maize seedlings, and data were taken at 3, 6, 9, and 12 d postinfestation on resistant and susceptible maize. On rootworm-susceptible maize, neonate larvae aggregated at the root tips and began actively feeding, and then they moved to older root tissue. Conversely, some larvae that ingested Cry 3Bb1 from the resistant maize exhibited no movement. Other larvae on the resistant maize moved continuously, sampling root hairs or root tissue but not actively feeding. The continuously moving larvae had visibly empty guts, suggesting possible nonpreference for the resistant root. This study contributes to our understanding of D. v. virgifera larval behavior and provides insight into questions surrounding the potential evolution of behavioral and biochemical resistance to Cry3Bb1.     

A core set of microsatellite markers for western corn rootworm (Coleoptera: Chrysomelidae) population genetics studies

Interest in the ecological and population genetics of the western corn rootworm, Diabrotica virgifera virgifera LeConte, has grown rapidly in the last few years in North America and Europe. This interest is a result of a number of converging issues related to the increasing difficulty in managing this pest and the need to characterize and understand gene flow in the context of insect resistance management. One of the key components needed for successful population genetics studies is the availability of suitable molecular markers. Using a standard group of microsatellite markers enables researchers from different laboratories to directly compare and share their data, reducing duplication of effort and facilitating collaborative work among laboratories. We screened 22 candidate microsatellite loci against five criteria to create a core set of microsatellite markers for D. v. virgifera population genetics studies. The criteria for inclusion were moderate to high polymorphism, unambiguous readability and repeatability, no evidence of null alleles, apparent selective neutrality, and no linkage between loci. Based on our results, we recommend six microsatellite markers to be included as a core set in future population genetics studies of D. v. virgifera along with any other microsatellite or genetic markers. As more microsatellites are developed, those meeting the criteria can be added to the core set. We encourage other groups of researchers with common interests in a particular insect species to develop their own core sets of markers for population genetics applications.     

Screening of entomopathogenic nematodes for virulence against the invasive western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) in Europe

Entomopathogenic nematode species available in Europe were screened for their efficacy against both the root-feeding larvae and silk-feeding adults of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Laboratory screening tests were aimed at the selection of candidate biological control agents for the management of this invasive alien pest in Europe. Steinernema glaseri, S. arenarium, S. abassi, S. bicornutum, S. feltiae, S. kraussei, S. carpocapsae and Heterorhabditis bacteriophora were studied to determine their virulence against third instar larvae and adults of D. v. virgifera in small-volume arenas (using nematode concentrations of 0.5, 0.8, 7.9 and 15.9 infective juveniles cm-2). All nematode species were able to invade and propagate in D. v. virgifera larvae, but adults were rarely infected. At concentrations of 7.9 and 15.9 cm-2, S. glaseri, S. arenarium, S. abassi and H. bacteriophora caused the highest larval mortality of up to 77%. Steinernema bicornutum, S. abassi, S. carpocapsae and H. bacteriophora appeared to have a high propagation level, producing 5970+/-779, 5595+/-811, 5341+/-1177 and 4039+/-1025 infective juveniles per larva, respectively. Steinernema glaseri, S. arenarium, S. feltiae, S. kraussei and H. bacteriophora were further screened at a concentration of 16.7 nematodes cm-2 against third instar larvae in medium-volume arenas (sand-filled trays with maize plants). Heterorhabditis bacteriophora, S. arenarium and S. feltiae caused the highest larval mortality with 77+/-16.6%, 67+/-3.5%, and 57+/-17.1%, respectively. In a next step, criteria for rating the entomopathogenic nematode species were applied based on results obtained for virulence and propagation, and for current production costs and availability in Europe. These criteria were then rated to determine the potential of the nematodes for further field testing. Results showed the highest potential in H. bacteriophora, followed by S. arenarium and S. feltiae, for further testing as candidate biological control agents.     

Intercontinental effect on sexual shape dimorphism and allometric relationships in the beetle pest Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae)

Hind wing shape variation was examined in 686 adult Diabrotica virgifera virgifera collected from maize plants in Europe and the USA Corn Belt, using geometric morphometric techniques. Sexual dimorphism at an Intercontinental scale was assessed using canonical variates analysis, a multivariate statistical method used to find the shape characters that best distinguish among groups of specimens. Our results showed that each of the populations of D. v. virgifera investigated in this study showed high levels of sex based hind wing shape dimorphism. In particular a stronger and more obvious pattern of hind wing shape variation was found in the USA than in Europe. These results support previous studies on D. v. virgifera wing shape that show that female D. v. virgifera have more elongated wings than males. These differences raise the question of whether sexual dimorphism may be modulated by natural selection.     

Basic biology and small-scale rearing of Celatoria compressa(Diptera: Tachinidae), a parasitoid of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

The tachinid Celatoria compressa Wulp has been evaluated as a candidate biological control agent for the western corn rootworm, Diabrotica virgifera virgiferaLeConte, in Europe, where it is an invasive alien pest of maize. Special emphasis has been placed on understanding aspects of the parasitoid basic biology and on developing a rearing technique for a small-scale production of C. compressapuparia. The age of C. compressa adults was found to be the most crucial factor in achieving mating. Only newly emerged, 1-h-old females, mated successfully with 2- to 5-day-old males, achieving a success rate of 74%. After mating, a prelarviposition period of 4 days occurred. The 5-day-old C. compressa females inserted their eggs containing fully-developed first instars directly into adults of D. v. virgifera. Total larval and pupal developmental time, including a pre-larviposition period of 4 days, was 29 days under quarantine laboratory conditions (25 degrees C daytime, 15 degrees C at night, L:D 14:10, 50% +/- 10% r.h). Females of C. compressa were capable of producing on average 30 puparia throughout a female's mean larviposition period of 15 days. A large number of host attacks by C. compressa were unsuccessful, resulting in a mean larviposition success rate of 24% per female. Parasitoid females appear to have difficulties inserting the egg through the intersegmental sutures or membranes around leg openings of the host adults. Although the small-scale rearing technique of C. compressa presented is both time and labour intensive, C. compressa has been reared successfully for at least 20 successive generations without shifting the 1 male : 1 female sex ratio using a non-diapause strain of D. v. virgifera.     

First Assessment for the Presence of Phlebotomine Vectors in Bavaria,Southern Germany,by Combined Distribution Modeling and Field Surveys

Leishmaniasis is caused by protozoa of the genus Leishmania and transmitted by sand flies from mammalian reservoirs to humans. In recent years, a northward spread of L. infantum from highly endemic Mediterranean countries into previously non-endemic Central European areas has been suspected based on presumed sporadic cases of autochthonous leishmaniasis. Here, we investigated whether sand flies are prevalent in Bavaria in Southern Germany, a federal state in which autochthonous cases have previously been reported. Considering the present and future climatic conditions, we determined whether Bavaria is suitable for five sand fly species with assumed spreading tendencies towards Central Europe: Phlebotomus ariasi, P. neglectus, P. perfiliewi and P. perniciosus that are known vectors for Leishmania in Europe, and P. mascittii, a suspected but not proven vector. Within Bavaria we defined sampling regions based on their climatic suitability and their spatial distance to the sites of the autochthonous cases and/or to areas of reported sand fly detection in states adjacent to Bavaria. At 155 locations in 7 sampling regions, CDC light traps were placed during 38 nights in the summers of 2009 and 2010, resulting in 202 trap-nights. All traps were negative for sand flies. The results suggest that Bavaria is not yet endemic for sand flies, but do not exclude the possibility of sporadic cases of autochthonous human or zoonotic Leishmania infections. This study, which combined methodological approaches from different disciplines, serves as reference for future surveys and risk analyses of sand flies and leishmaniasis in so far non-endemic areas of Europe.     

Comparative biogeography of the cytotypes of annual Microthlaspi perfoliatum (Brassicaceae) in Europe using isozymes and cpDNA data: refugia, diversity centers, and postglacial colonization

In the last few years, the biogeography of many European plant species has been analyzed using molecular markers, and some consistent patterns of Pleistocenic differentiation and range fluctuations have been established. These studies mostly focused on perennial herbs or woody species, rarely considering annual taxa. This study focused on the annual Microthlaspi perfoliatum, which is distributed all over Europe and comprises three cytotypes. Morphologically, these cytotypes are hard to distinguish, although, based on molecular markers, they should be treated as two different species. Diploid and polyploid cytotypes had a different biogeographical history, with distinct glacial refugia. For the polyploids, a well-known distribution pattern of relict areas was confirmed, with Iberia, Italy, and the Balkans serving as primary Pleistocene refugia. Secondary refuge areas have been detected in southeastern France and neighboring Switzerland, with closer affinity to the Iberian refugium than to any other region based on allozyme and cpDNA haplotype data. For the diploids, two refugia have been characterized, one of which is congruent to the secondary refugium of the polyploids in France and Switzerland. The second refuge of diploid populations is located in unglaciated lowland areas of East Austria and Croatia. Isozyme and cpDNA haplotype data favor a postglacial colonization of diploid populations into Germany from Austrian lowland areas along the Danube River as well as from Switzerland. This scenario is also true for polyploids in Germany, Belgium, and Sweden.