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.     

Parasitism of Western Corn Rootworm Larvae and Pupae by Steinernema carpocapsae

Virulence and development of the insect-parasitic nematode, Steinernema carpocapsae (Weiser) (Mexican strain), were evaluated for the immature stages of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Third instar rootworm larvae were five times more susceptible to nematode infection than second instar larvae and 75 times more susceptible than first instar larvae and pupae, based on laboratory bioassays. Rootworm eggs were not susceptible. Nematode development was observed in all susceptible rootworm stages, but a complete life cycle was observed only in second and third instar larvae and pupae. Nematode size was affected by rootworm stage; the smallest infective-stage nematodes were recovered from second instar rootworm larvae. Results of this study suggest that S. carpocapsae should be applied when second and third instar rootworm larvae are predominant in the field.     

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.     

Assessing host specificity of a classical biological control agent against western corn rootworm with a recently developed testing protocol

Celatoria compressa (Wulp) (Diptera: Tachinidae), a parasitoid of adult chrysomelid beetles in the subtribe Diabroticina in North America, has been selected as a candidate for classical biological control of the alien invader, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae: Galerucinae), into Europe. We conducted host specificity testing to evaluate the fundamental host range of C. compressa and potential risks to native European coleopteran species. Nine potential non-target beetles were tested for host selection with D. v. virgifera in no-choice tests, sequential no-choice tests, choice tests and sequential choice tests in small experimental arenas in a quarantine laboratory. The nine representative non-target species were selected for experimentation based on (1) ecological host range information of C. compressa, (2) ecological similarities to D. v. virgifera, (3) close phylogenetic/taxonomic relationships, (4) safeguard considerations, (5) morphological similarities, geographical distributions, overlap of temporal occurrences with D. v. virgifera and C. compressa, and (6) accessibility and availability. Of the potential nine non-target hosts tested, gravid C. compressa only parasitized a few red pumpkin beetles, Aulacophora foveicollis (Chrysomelidae: Galerucinae), regardless of the presence or absence of D. v. virgifera. However, C. compressa significantly preferred D. v. virgifera (44.6% parasitized) over A. foveicollis (2.7%) in choice tests. Of the 1110 A. foveicollis tested among all experiment types, only 23 were parasitized and only one C. compressa successfully developed from the parasitism, demonstrating that A. foveicollis is a poor host. In conclusion, C. compressa has a fundamental host range restricted to the subtribes Diabroticina and Aulacophorina, and would therefore be unlikely to have a direct impact on indigenous species in Europe.     

Integrating pesticide effects with inundative biological control: interpretation of pesticide toxicity curves for Anaphes iole in strawberries

We examined pesticide residue effects on the egg parasitoid Anaphes iole Girault (Hymenoptera: Mymaridae), an inundative biological control agent for Lygus hesperus Knight (Heteroptera: Miridae) in strawberries (Fragaria × ananassa Duchesne). Our objectives were to identify compatible pesticides, determine appropriate parasitoid release timings for minimizing harmful effects, and develop an approach for interpreting pesticide toxicity curves. Six insecticides, 2 acaricides and 6 fungicides were tested, and survivorship of adult A. iole exposed to foliar residues for 48 h, at 4–6 different times after pesticide application, was examined. A logistic function was developed for incorporating control mortality at each test date. Values for LT₅₀ (Lethal Time for 50% mortality) and mortalities on day 1 (initial mortality) and day 13 (estimated maximum time parasitoid releases can be delayed under extreme summer conditions) were estimated. In the study, insecticide residues proved to be the most toxic, followed by those from acaricides while most fungicides were least toxic. Among insecticides, fenpropathrin, bifenthrin and carbaryl caused the greatest mortality (estimated mortality on day 13 >75%). Residues of naled resulted in the least mortality (LT₅₀=3.2 days) followed by methomyl (LT₅₀=8.3 days) and malathion (LT₅₀=13.2 days). Estimated mortality = 12.3% on day 13 for the acaricide propargite and <1% for abamectin. For the fungicides benomyl, captan, myclobutanil and thiram, estimated mortality on day 1 was <1%, and for iprodione it was <6%, indicating compatibility with A. iole releases. For sulfur, LT₅₀=0, but the mortality decay curve was relatively flat (estimated mortality on day 13=13.6%). These results suggest possibilities for integrating A. iole releases with certain pesticide programs by appropriate timing of pesticide applications to minimize negative impacts.     

Influence of corn planting date on the life stage development and phenology of Diabrotica virgifera virgifera

Under field conditions in eastern South Dakota, USA three different planting dates of corn and three times of egg infestation were used to imposed synchronous and asynchronous timing of corn growth and Diabrotica virgifera virgifera LeConte egg hatch and subsequent development. Median occurrence of each of the three larval stages and of the adult stage for each of the infestation-planting treatments was determined by relating occurrence to thermal units and to days after infestation. No significant differences were found among the treatments for median occurrence of each life stage when the thermal unit approach was used. However, significant differences were found among treatments for median occurrence when the day after infestation approach was used. Consistent parameters are needed for prediction of the occurrence of life stages of this insect. Despite the various imposed conditions, this study indicates that the least variable method of predicting life stage occurrence and adult emergence of D. v. virgifera was the use of thermal unit accumulations (base 11°C).

---

Résumé Dans les conditions de l'est du Sud Dakota (USA), 3 dates de semailles de maïs et 3 dates de contamination avec des ufs de D. virgifera virgifera LeConte ont servi à imposer ou à empêcher la synchronisation des éclosions des ufs et par conséquent des développements ultérieurs. Les dates médianes d'apparition de chacun des 3 stades larvaires et des imagos ont été déterminées en relation avec les unités de température et le nombre de jours depuis la contamination. Aucune différence significative entre les dates médianes d'apparition n'a été mise en évidence par utilisation des unités de température; par contre, ces différences deviennent significatives lorsque l'on utilise le nombre de jours depuis la contamination. Des paramètres fiables sont nécessaires pour prédire la présence des différentes stades de cet insecte. En dépit des différentes conditions imposées pour cette étude, la méthode la moins capricieuse pour prédire l'apparition d'un stade larvaire et l'émergence des adultes est la somme de températures avec une base de 11°C.

     

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.     

Wind tunnel assays of olfactory responses of femaleRhagoletis pomonella flies to apple volatiles: effect of wind speed and odour release rate

Little is known about how adults of the corn leafhopper,Dalbulus maidis (DeLong & Wolcott), and its congeners survive subfreezing temperatures at high elevations during the dry winter in Mexico. In the laboratory, duration of survival at −5°C was measured for four MexicanDalbulus species:D. maidis, D. elimatus (Ball),D. gelbus DeLong andD. quinquenotatus DeLong & Nault; and a closely related North American species,Baldulus tripsaci Kramer & Whitcomb. Adult leafhoppers reared under environmental conditions that simulated the beginning of the dry winter season during October in Mexico (‘October-reared’) were at least twice as tolerant of −5°C than adults reared under environmental conditions that simulated the beginning of the wet summer season during June (‘June-reared’).Dalbulus species found primarily at high elevations, such asD. elimatus, were seven times more tolerant of −5°C thanD. quinquenotatus, a species which overwinters at low to mid elevations on itsTripsacum hosts. October-rearedD. maidis adults survived relatively short periods at −5°C (LT₅₀=8.9h) compared to October-rearedD. elimatus adults (LT₅₀=42.3h). This suggests that in Mexico,D. maidis either overwinters in protected habitats at higher elevations or it migrates to lower, frost-free regions. October-rearedB. tripsaci adults, which overwinter in the egg stage, were intolerant of −5°C (LT₅₀=2.6h). A conditioning period for 1 h at +5°C before and after exposure to −5°C significantly improved survival forD. maidis. Supercooling points (SCPs) were between −23 and −20°C, indicating that mortality of these leafhoppers at −5°C was due to cold shock injury rather than internal ice formation.     

Suitability of Aphis fabae, Myzus persicae (Homoptera: Aphididae) and Aleyrodes proletella (Homoptera: Aleyrodidae) as prey for Coccinella undecimpunctata (Coleoptera: Coccinellidae)

The suitability of Aphis fabae Scopoli, Myzus persicae Sulzer and Aleyrodes proletella L. as food sources for Coccinella undecimpunctata L. was evaluated by studying the impact of prey consumption on the predator’s population growth parameters and feeding parameters. Unlike A. proletella, A. fabae and M. persicae supported the development and reproduction of C. undecimpunctata. A. fabae and M. persicae were considered to be essential prey, whereas A. proletella was considered to be an alternative prey. Aphid species showed different degrees of suitability: M. persicae significantly decreased the pre-oviposition period and increased adult longevity, fecundity and fertility compared with A. fabae. Moreover, A. fabae represents a suitable diet for larval development, but is not a suitable food source for adult reproduction. The predator’s population growth parameters, R₀, r_m and λ were increased with M. persicae, whereas T decreased. We found that the 4th instar larvae were the most voracious, particularly when fed on M. persicae; nevertheless, with this prey daily weight gain and feeding efficiency of 4th instar larvae were similar to that of individuals fed with A. fabae.     

Pathogenicity, formulation and storage of insect pathogenic hyphomycetous fungi tested against Diabrotica speciosa

Studies were conducted tosearch for fungal strains with potentialpathogenicity against Diabrotica speciosa(Germar) (Coleoptera: Chrysomelidae).Among sixteen fungal isolates screenedthe most virulent was a Beauveria bassiana(Balsamo) Vuillemin isolate (FHD13) thatcaused 70% mortality of D. speciosathird instar larvae. The LC₅₀ value ofB. bassiana isolate FHD13 was3.48 × 10¹⁰ conidia/ml.Different temperatures (4, 17 and 26 °C)and vegetable oils (corn, sunflower and canola)used for storage did not significantly affectviability of conidia. A pathogenicity trialagainst D. speciosa larvae performed withthe corn oil formulation (1 × 10⁸ conidia/mlof oil) caused 65% of mortality.     

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.     

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.     

Corn rootworm survey in North Dakota and a comparison of two sticky traps

Northern, Diabrotica barberi Smith & Lawrence, and western, D. virgifera virgifera LeConte, corn rootworms are major economic pests of corn, Zea mays L., in the United States. This research was conducted to determine the geographic distribution, abundance, and species composition of Diabrotica species in North Dakota, and to compare effectiveness of unbaited green Scentry™ Multigard and unbaited yellow Pherocon^® AM/NB sticky traps for monitoring. Fifty-one corn fields were monitored using traps from July through October of the 2013, 2014, and 2015 growing seasons for rootworm beetle activity. The overall species composition was 61% D. barberi and 39% D. v. virgifera. Both species were frequently captured, and the highest densities (i.e. >10 beetles per trap per week) were found in southeastern North Dakota. Low densities (i.e. <0.1 beetles per trap per week) of D. barberi were found in areas further north, but no D. v. virgifera were captured in those fields. The two different coloured sticky traps were not significantly different across 38 sites for D. barberi and across 21 sites for D. v. virgifera. However, green Scentry™ Multigard traps captured more D. barberi beetles than yellow Pherocon^® AM/NB traps at 68% of the 38 fields. In contrast, the yellow Pherocon^® AM/NB traps captured more D. v. virgifera beetles at 57% of the 21 fields. Findings also indicated that, although D. barberi was the predominant species in surveyed fields, populations rarely reached the economic threshold. Our study observed that economic populations of corn rootworms were infrequent among the field sites trapped in North Dakota. As a result, producers should scout fields regularly for corn rootworm populations levels to make sound pest management decisions. This knowledge can enable producers to effectively protect their crop when control is economically justified, and the information can also provide input cost savings when populations do not warrant control efforts.     

Susceptibility of Delia radicum to steinernematid nematodes

Isolates of different Steinernema species (S. affine, S. bicornutum, S. feltiae and Steinernema C1) were used in mortality assays with third instar larvae of Delia radicum (L.) (Diptera: Anthomyiidae). The nematode isolates had been obtained by baiting soil regularly grown with cabbage. One isolate (S. feltiae) was the result of a natural infection of a D. radicum puparium. The highest mortality (77%) was obtained with an isolate of S. feltiae (DK1). The isolate DK1 was also used in tests with all larval stages of D. radicum. Mortality around 60% was observed for second and third instar larvae, while first instar larvae showed very low or no susceptibility. Maximum mortality of second and third instar larvae was reached applying only 25 nematodes per larva. Observations of larvae that pupated revealed that some of these puparia contained nematodes. Experiments with hatching puparia showed that a high proportion was infected by nematodes if the flies were prevented from leaving nematode-containing soil. In addition to mortality, the ability of the nematodes to successfully reproduce in the insects was studied. It was found that the species S. feltiae and S. bicornutum reproduced in D. radicum larvae and adults with S. feltiae being the most successful.     

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis (Lepidoptera: Crambidae)

Photorhabdus temperata is an entomopathogenic bacterium that is associated with nematodes of the Heterorhabditidae family in a symbiotic relationship. This study investigated the effects of P. temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis. Histopathology of the infection was also investigated using scanning electron microscopy. Groups of 20 larvae were infected by injection of approximately 50 bacterial cells directly into the hemocoel. After different periods of infection, larvae were dissected and different tissues were used for bacterial cell quantification. P. temperata was highly virulent with an LD₅₀ of 16.2 bacterial cells at 48 h post-infection. Infected larvae started dying as soon as 30 h post-infection with a LT₅₀ value of 33.8 h (confidence limits 32.2–35.6) and an LT₉₀ value of 44.8 h (CL 40.8–51.4). Following death of the larvae, bacteria from the midgut did not invade the hemocoel. In the midgut epithelium, P. temperata occupied the space underneath the basal lamina. The cultivable intestinal bacterial populations decreased as soon as 1 h post-infection and at 48 h post-infection, 90% of the gut microbiota had died. The role of P. temperata in control of the midgut microbiota was discussed.     

Virulence of entomopathogenic nematodes to larvae of the guava weevil, Conotrachelus psidii (Coleoptera: Curculionidae), in laboratory and greenhouse experiments

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil and causes severe reduction in fruit quality. This weevil is difficult to control with insecticides because adults emerge over a long period, and larvae develop to the fourth-instar inside the fruit and move to the soil for pupation. We assessed the virulence of entomopathogenic nematodes to fourth-instar larvae in soil by comparing their susceptibility to nine species or strains: Heterorhabditis bacteriophora HP88, H. baujardi LPP7, and LPP1, H. indica Hom1, Steinernema carpocapsae All and Mexican, S. feltiae SN, S. glaseri NC, and S. riobrave 355. In petri dish assays with sterile sand at a concentration of 100 infective juveniles (IJs) of a given nematode species/strain, larval mortality ranged from 33.5 to 84.5%, with the heterorhabditids being the most virulent. In sand column assays with H. baujardi LPP7, H. indica Hom1, or S. riobrave 355 at concentrations of 100, 200, and 500 IJs, mortality was greater than the control only for H. baujardi (62.7%) and H. indica (68.3%) at the highest concentration. For H. baujardi LPP7 in a petri dish assay, the time required to kill 50 and 90% of the larvae (LT₅₀ and LT₉₀) for 100 IJs was 6.3 and 9.9 days, whereas the lethal concentration required to kill 50 and 90% of the larvae (LC₅₀ and LC₉₀) over 7 days was 52 and 122.2 IJs. In a greenhouse study with guava trees in 20-L pots, 10 weevil larvae per pot, and concentrations of 500, 1000 or 2000 IJs, H. baujardi LPP7 caused 30 and 58% mortality at the two highest concentrations. These results show that H. baujardi is virulent to fourth-instar larvae and has potential as a biological control agent in IPM programs.     

Larvicidal efficacy ofBacillus thuringiensis var. israelensis andBacillus sphaericus onAnopheles arabiensis in Ethiopia

The second instar larvae of the malaria vector mosquito,Anopheles arabiensis,were more susceptible to Bacillus thuringiensis var. israelensis (IPS-82) and B. sphaericus (SPH-88) than the third instar larvae. The LC ₅₀ values were 1.0 Μg^I-1 and 1.8 Μg_I-1 for IPS-82 against second and third instar larvae respectively, after 48 h of exposure. The LC₅₀ values for SPH-88 were 3.6 Μg {siI-1} against the second instar larvae and 7.6 Μg_I-1 against the third instar larvae of An. arabiensis. The larvicidal efficacy of SPH-88 was significantly less than IPS-82. The potential of IPS-82 for the control of An. arabiensis in malaria endemic areas is promising.     

Avidin expressed in transgenic tobacco leaves confers resistance to two noctuid pests,Helicoverpa armigera and Spodoptera litura

Fertile transgenic tobacco plants with leaves expressing avidin in the vacuole have been produced and shown to halt growth and cause mortality in larvae of two noctuid lepidopterans, Helicoverpa armigera and Spodoptera litura. Late first instar H. armigera larvae and neonate (<12-h-old) S. litura larvae placed on leaves excised from T₀ tobacco expressing avidin at 3.1–4.6M (moles/kg of fresh leaf tissue) had very poor growth over their first 8 days on the leaves, significant numbers had died by days 11 or 12 and all were dead by day 22 (H. armigera) or day 25 (S. litura). Similar results were obtained when late first instar H. armigera larvae were placed on leaves from T₁ plants expressing avidin at six different average concentrations, ranging from 3.7 to 17.3M. Two larvae on the lowest expressing leaves survived to pupation, but there was total mortality among the other groups and no relationship between avidin concentration and the effects on the larvae. Synergistic effects between avidin-expressing tobacco plants and a purified Bt toxin, Cry1Ba, were demonstrated. Late instar H. armigera larvae fed with leaves from T₂ plants expressing avidin at average concentrations of either <5.3 or >12.9M, and painted with Cry1Ba protein at a rate equivalent to an expression level of 0.5% of total leaf protein, died significantly faster than larvae given either of the two treatments alone. Larvae fed with avidin-expressing leaves painted with the protease inhibitor, aprotinin, at a rate equivalent to 1% of total leaf protein had mortality similar to those given avidin-leaves alone. There was no evidence of antagonism between these two proteins.     

A review of the natural enemies of beetles in the subtribe Diabroticina (Coleoptera: Chrysomelidae): implications for sustainable pest management

Diabroticina is a speciose subtribe of New World Chrysomelidae (Subfamily Galerucinae: Tribe Luperini) that includes pests such as corn rootworms, cucumber beetles and bean leaf beetles (e.g. Diabrotica, Acalymma, Cerotoma species). The evolution and spread of pesticide resistance, the European invasion of Diabrotica v. virgifera LeConte, and possible development of resistance due to the large-scale deployment of Diabrotica-active Bt maize in North America have generated a sense of urgency in developing biological control options against Diabroticina pests. In the present study, we review available knowledge on biological control options, including 290 publications on natural enemy–Diabroticina associations in the New World. Several natural enemy species or groups appear to be promising candidates for control strategies with different ecological rationales. We propose that future research should pursue: (1) development of inundative biological control products, particularly mass-produced entomopathogenic nematodes and fungi, (2) understanding of specific natural enemies of Diabroticina larvae throughout the Americas and of adults particularly in higher altitudes of Central America or northern South America including potential classical biological control agents against D. v. virgifera; (3) enhancement of natural enemies through cultural practices, i.e., reduced tillage, reduced weed control, cover crops, diversified crop rotations or soil amendments. Research and action must be coordinated to accelerate the exploration of biological control options.     

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.