Constructing life-tables for the invasive maize pest Diabrotica virgifera virgifera (Col.; Chrysomelidae) in Europe

Abstract: The western corn rootworm (Diabrotica virgifera virgifera LeConte, Col.; Chrysomelidae) is an alien invasive species in Europe. It is a univoltine species with eggs that overwinter in the soil and larvae that hatch in spring. Three larval instars feed on maize roots, which can cause plant lodging and yield loss of economic importance. Adults emerge between mid‐June and early August and can reduce yields through intensive silk feeding. In order to provide a thorough understanding of the population dynamics of this invasive pest species in the invaded European region, complete age specific life‐tables were constructed in two maize fields in southern Hungary assessing the significance of natural mortality factors acting on D. v. virgifera populations. This information provides a rational basis for devising sustainable integrated pest management programmes, in particular, by enabling the identification of vulnerable pest age intervals for the timely application of various management tools. The life‐table for D. v. virgifera in Europe resulted in a total mortality of about 99% from the egg stage in the autumn to the emergence of adult females in the following year (K_Total = 2.48), which is comparable with North America. The highest reduction of D. v. virgifera numbers resulted from the mortality in first instar larvae (94% marginal death rate) and from the unrealized fecundity (80%). However, only the variation in mortality between years can change the generational mortality and thus influence population growth. High variation in the marginal death rate between fields and years was found in the second and third instar larval stages, and in the overwintering egg stage. These mortality factors therefore have the potential to cause changes in the total generational mortality. Furthermore, the life‐table suggested that a high fecundity could compensate for a high generational mortality and would lead to population increase.     

Survey for natural enemies of the invasive alien chrysomelid, Diabrotica virgifera virgifera, in Central Europe

The western corn rootworm, Diabrotica virgifera virgifera LeConte(Coleoptera: Chrysomelidae), is the mostdestructive pest of maize (Zea mays L.)in North America, and began to successfullyinvade Central Europe in the early 1990's. Thispaper reports a three-year field surveyconducted in Hungary, Yugoslavia, and Croatia,which are currently the focal points ofinvasion, with the aim to determine theoccurrence of indigenous natural enemies ofD. v. virgifera in Europe. A total of9,900 eggs, 550 larvae, 70 pupae and 33,000adults were examined for the occurrence ofparasitoids, nematodes, and fungal pathogens. It can be concluded from the survey resultsthat effective indigenous natural enemies arenot attacking any of the life stages of D.v. virgifera in Europe. The exception is theoccurrence of the fungi Beauveriabassiana (Bals.) Vuill. (Mitosporic fungi;formerly Deuteromyces) and Metarhiziumanisopliae (Metsch.) Sorok (Mitosporic fungi)attacking adults of D. v. virgifera at anextremely low level (< 1%). However no otherentomopathogenic fungal pathogens,entomopathogenic nematodes, or parasitoids werefound on eggs, larvae, pupae or adults. Whileseveral natural enemies in North and CentralAmerica are known to attack D. v.virgifera, it is apparent that indigenousnatural enemies in Europe have not adapted tothe high population density of the alieninvasive species D. v. virgifera. Classical biological control may provide anopportunity to reconstruct the natural enemycomplex of an invading alien pest, and itsapplication to manage D. v. virgiferapopulations in Europe should be considered.     

A multifaceted hemolymph defense against predation in Diabrotica virgifera virgifera larvae

The physical and chemical aspects of Diabrotica virgifera virgifera larval hemolymph were quantitatively assessed against two predatory beetle species in the laboratory. Adult Poecilus cupreus and Harpalus pensylvanicus (Coleoptera: Carabidae) were fed pupae, second or third instar D. v. virgifera or a palatable surrogate prey, i.e., Calliphora vicina or Sarcophaga bullata larvae (Diptera: Calliphoridae, Sarcophagidae, respectively) of equivalent size. The ethanol-soluble fraction of third instar D. v. virgifera hemolymph was extracted and suspended in a 0.24 M sucrose solution and offered to H. pensylvanicus (using a sucrose only control for comparison). The mean duration until first consumption was recorded for each predator, as was the amount of time spent eating, cleaning, resting, or walking for 2 min post-attack (or 5 min for the sugar assay). Maggots and D. virgifera larvae and pupae were attacked equally by both predators. But upon attack, D. v. virgifera larval hemolymph coagulated onto the mouthparts of the predators, which they began vigorously cleaning. Predators ate the sucrose solution for significantly longer than hemolymph + sucrose solution, indicating the presence of deterrent chemicals in the hemolymph. This research suggests that D. v. virgifera larvae are defended from predation by sticky and repellent hemolymph. We hypothesize that this defense partially explains the widespread success of D. v. virgifera as an invasive pest.     

Natural occurrence of insect pathogenic fungi and insect parasitic nematodes in <Emphasis Type="Italic">Diabrotica virgifera virgifera</Emphasis> populations

The Western Corn Rootworm D. virgifera virgifera Le Conte (Coleoptera: Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. In order to get information on the role of naturally occurring pathogens in the regulation of Diabrotica populations, we started an investigation in established populations in Hungary, Romania, Serbia, Austria, and Italy in 2005 and 2006. In infested maize fields in Hungary, plants and their root systems were grubbed out and larvae and pupae were collected. Adult D. v. virgifera were collected in Hungary, Austria, Romania, Serbia and Italy. Additionally, the occurrence of entomopathogenic fungi in soils of maize fields was determined using Galleria mellonella and Tenebrio molitor larvae as bait insects. The density of entomopathogenic fungi was obtained by plating soil suspension on selective medium. Metarhizium anisopliae and Beauveria spp. infections were found in 1.4% of field collected larvae, 0.2% of field collected pupae and 0.05% of field collected adults. Whereas natural infections of D. v. virgifera were rarely found, a high density of insect pathogenic fungi was recorded in Hungarian soils. M. anisopliae could be detected in every maize field either using the “bait method” or a “selective medium” method. This is the first report of a natural occurrence of entomoparasitic nematodes (Heterorhabditis sp., Steinernema sp.) in Diabrotica v. virgifera in Europe.     

Evolutionary directional asymmetry and shape variation in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae): an example using hind wings

The western corn rootworm Diabrotica virgifera virgifera LeConte is a pest of maize in the USA and Europe and especially a problem in particular regions of Croatia. In the present study, patterns of variation in hind wing shape were examined. The first objective was to examine the influence of soil type on 10 populations of D. v. virgifera sampled from three regions in Croatia that differed according to edaphic factors and climate. The second objective was to investigate the potential evolutionary presence of directional asymmetry on hind wings. Geometric morphometrics was used to examine these objectives by quantifying the morphological variation within and among individuals and populations. Overall, D. v. virgifera hind wing shape changed according to major soil type classifications in Croatia. The three hind wing morphotypes found varied because of basal radial vein differences, related to landmarks 1, 3, 7, and 14. The findings of the present study show that hind wing shape in D. v. virgifera can be used to differentiate populations based on edaphic factors and may have application as a monitoring tool in the integrated management of D. v. virgifera. In an evolutionary context, the presence of directional asymmetry in the hind wings of D. v. virgifera adds to the ever growing data on the evolution of insect wings. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 110–118.     

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.     

Comparative assessment of the efficacy of entomopathogenic nematode species at reducing western corn rootworm larvae and root damage in maize

The western corn rootworm (Diabrotica virgifera virgifera LeConte, Coleoptera: Chrysomelidae) is an invasive maize (Zea mays L.) pest in Europe. Crop yield is significantly impacted by the feeding of all three larval instars on maize roots, making them prime targets for control measures. Therefore, the control efficacy of three entomopathogenic nematodes (EPNs), Steinernema feltiae (Filipjev), Heterorhabditis bacteriophora Poinar, and H. megidis Poinar, Jackson and Klein (Nematoda: Rhabditida), was studied in four field plot experiments in southern Hungary in 2005 and 2006. All EPN species significantly reduced D. v. virgifera independently, whether applied as a row spray with a solid stream into the soil at sowing or onto the soil along maize rows in June. When applied at maize sowing, H. bacteriophora was more effective at reducing D. v. virgifera (81%) than H. megidis (49%) and S. feltiae (36%). When applied in June, H. bacteriophora and H. megidis were more effective at reducing D. v. virgifera (around 70%) than S. feltiae (32%). All tested EPN species significantly reduced damage on maize roots independently, whether they were applied at sowing or in June. Damage, however, was not totally prevented. The use of H. bacteriophora for the development of a biological control product for inundative releases against D. v. virgifera larvae is suggested.     

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.     

How to measure the food utilization of subterranean insects: a case study with the western corn rootworm (Diabrotica virgifera virgifera)

To date, nutritional studies on subterranean insects have focused on qualitative aspects due to experimental limitations. We have developed a method of studying insect–plant interactions quantitatively in subterranean environments. The initial and final weights of larvae of Diabrotica virgifera virgifera LeConte (Chrysomelidae, Galeruncinae) and those of maize roots, which served as food items were determined. The difference between initial and final weight of larvae and roots allowed for the calculation of the food conversion efficiency. This can be used to portray differences in food quality and its impact on larval performance and development.     

Comparative efficacy assessment of fungi,nematodes and insecticides to control western corn rootworm larvae in maize

All three larval instars of Diabrotica virgifera virgifera LeConte (western corn rootworm, Coleoptera: Chrysomelidae) feed on the roots of maize, Zea mays (L.). We assessed the efficacies of the following four agents in controlling these larvae: (1) the entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Hypocreales: Clavicipitaceae), (2) the nematode Heterorhabditis bacteriophora Poinar (Nematoda: Rhabditida), (3) a tefluthrin-based soil insecticide and (4) clothianidin-coated seeds. The agents were applied in field plot experiments in southern Hungary in 2006 and 2007. Efficacy was assessed by comparing the number of emerging D. v. virgifera adults and corresponding root damage among treatments and untreated controls. All agents significantly reduced D. v. virgifera numbers and root damage, but the relative success of each treatment was variable. On average across fields and years, the nematode and the two insecticides reduced D. v. virgifera by 65 ± 34% SD, while the fungus reduced D. v. virgifera by 31 ± 7%. According to the node injury scale, the agents prevented 23–95% of potential root damage. Large-scale commercialisation of these biological agents could offer viable and practical control options against D. v. virgifera.     

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.     

Assessing larval rootworm behaviour after contacting maize roots: impact of germplasm,rootworm species and diapause status

Current methods of screening maize (Zea mays L.) germplasm for susceptibility or resistance to corn rootworms (Coleoptera: Chrysomelidae) rely primarily on information from large‐scale field experiments. Due to labour and cost constraints associated with field trials, alternative evaluation methods are desirable. We used a previously developed behavioural bioassay to: (1) investigate the host search behaviour of rootworm larvae after contact with 14 maize genotypes, (2) compare the behaviour of non‐diapausing Diabrotica virgifera virgifera LeConte, diapausing D. v. virgifera, and diapausing D. barberi Smith & Lawrence and (3) determine if this technique can be used to separate susceptible vs. resistant maize genotypes. The majority of rootworm larvae engaged in intensive (local search) behaviour after exposure to maize roots, whereas larvae continued to exhibit extensive (ranging) behaviour after contact with negative controls. Even though a transgenic hybrid with resistance to D. v. virgifera was included in analyses, quantitative path measurements were similar among genotypes and only differed between specific maize lines and controls. Notably, there were differences in host search behaviour among rootworm groups, with non‐diapausing D. v. virgifera having more convoluted paths and engaging in intensive search more frequently than diapausing rootworms. Correlations between larval path measurements and historic root damage ratings were not significant, although there were weak positive correlations between historic adult emergence densities and measures of path linearity. However, due to the lack of significant behavioural differences among maize lines with a range of susceptibility levels, we concluded that this bioassay is not useful in screening maize germplasm for rootworm resistance.     

Microbial Symbionts in Insects Influence Down-Regulation of Defense Genes in Maize

Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the down-regulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect.     

Comparative susceptibility of larval instars and pupae of the western corn rootworm to infection by three entomopathogenic nematodes

As a first step towards the development of an ecologically rational control strategy against western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte, Coleoptera: Chrysomelidae) in Europe, we compared the susceptibility of the soil living larvae and pupae of this maize pest to infection by three entomopathogenic nematode (EPN) species. In laboratory assays using sand-filled trays, Heterorhabditis bacteriophora Poinar and H. megidis Poinar, Jackson & Klein (both Rhabditida: Heterorhabditidae) caused comparable mortality among all three larval instars and pupae of D. v. virgifera. In soil-filled trays, H. bacteriophora was slightly more effective against third larval instars and pupae, and H. megidis against third larval instars, compared to other developmental stages. In both sand and soil, Steinernema feltiae (Filipjev) (Rh.: Steinernematidae) was least effective against second instars. In conclusion, all larval instars of D. v. virgifera show susceptibility to infection by all three nematodes tested. It is predicted that early application against young larval instars would be most effective at preventing root feeding damage by D. v. virgifera. Applications of nematodes just before or during the time period when third instars are predominant in the field are likely to increase control efficacy. According to our laboratory assays, H. bacteriophora and H. megidis appear to be the most promising candidates for testing in the field. I. Hiltpold similarly contributed to this paper as the first author.     

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 .     

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.     

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.     

Establishment and persistence of the entomopathogenic fungus Metarhizium anisopliae in maize fields

The entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) was applied in maize fields to control the Western Corn Rootworm Diabrotica virgifera virgifera Le Conte (Coleoptera: Chrysomelidae). Establishment and persistence of two strains of M. anisopliae were investigated after application as ‘fungal colonized barley kernels’ (FCBK) into the soil and as a spore suspension on maize leaves and on the soil surface in 2006 and 2007 at two locations in Hungary. The applied fungal strains were able to establish at both locations and a long‐term persistence of at least 15 months could be recorded in the soil. A positive correlation between density of colony forming units (CFU) in the soil and the soil inhabiting stages of the host insect D. v. virgifera could be found. M. anisopliae spores applied on maize leaves were able to survive for no longer than 3 days after application, whereas on the soil surface a noticeably increase of fungus densities were found after treatments. Molecular markers were used to identify the applied M. anisopliae strains before and after application of FCBK into the soil of the maize field.