Global patterns of genomic and phenotypic variation in the invasive harlequin ladybird.

BACKGROUND: The harlequin ladybird Harmonia axyridis (Coleoptera: Coccinellidae), native to Asia, has been introduced to other major continents where it has caused serious negative impacts on local biodiversity. Though notable advances to understand its invasion success have been made during the past decade, especially with then newer molecular tools, the conclusions reached remain to be confirmed with more advanced genomic analyses and especially using more samples from larger geographical regions across the native range. Furthermore, although H. axyridis is one of the best studied invasive insect species with respect to life history traits (often comparing invasive and native populations), the traits responsible for its colonization success in non-native areas warrant more research. RESULTS: Our analyses of genome-wide nuclear population structure indicated that an eastern Chinese population could be the source of all non-native populations and revealed several putatively adaptive candidate genomic loci involved in body color variation, visual perception, and hemolymph synthesis. Our estimates of evolutionary history indicate (1) asymmetric migration with varying population sizes across its native and non-native range, (2) a recent admixture between eastern Chinese and American populations in Europe, (3) signatures of a large progressive, historical bottleneck in the common ancestors of both populations and smaller effective sizes of the non-native population, and (4) the southwest origin and subsequent dispersal routes within its native range in China. In addition, we found that while two mitochondrial haplotypes-Hap1 and Hap2 were dominant in the native range, Hap1 was the only dominant haplotype in the non-native range. Our laboratory observations in both China and USA found statistical yet slight differences between Hap1 and Hap2 in some of life history traits. CONCLUSIONS: Our study on H. axyridis provides new insights into its invasion processes into other major continents from its native Asian range, reconstructs a geographic range evolution across its native region China, and tentatively suggests that its invasiveness may differ between mitochondrial haplotypes.

Transcriptional Regulation of Reproductive Diapause in the Convergent Lady Beetle, Hippodamia convergens.

Diapause is an alternate development program that synchronizes an insect's life cycle with seasonally abundant resources and ensures survival in unfavorable conditions. The physiological basis of diapause has been well characterized, but the molecular mechanisms regulating it are still being elucidated. Here, we present a de novo transcriptome and quantify transcript expression during diapause in the convergent lady beetle Hippodamia convergens. H. convergens is used as an augmentative biocontrol agent, and adult females undergo reproductive diapause that is regulated by photoperiod. We sampled females at three stages (early, mid, and late diapause) and compared transcript expression to non-diapausing individuals. Based on principle component analysis, the transcriptomes of diapausing beetles were distinct from non-diapausing beetles, and the three diapausing points tended to cluster together. However, there were still classes of transcripts that differed in expression across distinct phases of diapause. In general, transcripts involved in muscle function and flight were upregulated during diapause, likely to support dispersal flights that occur during diapause, while transcripts involved in ovarian development were downregulated. This information could be used to improve biological control by manipulating diapause. Additionally, our data contribute to a growing understanding of the genetic regulation of diapause across diverse insects.

Interactions Among Native and Non-Native Predatory Coccinellidae Influence Biological Control and Biodiversity.

Over the past 30 yr, multiple species of predatory Coccinellidae, prominently Coccinella septempunctata L. and Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) have spread to new continents, influencing biodiversity and biological control. Here we review the mechanisms underlying these ecological interactions, focusing on multi-year field studies of native and non-native coccinellids and those using molecular and quantitative ecological methods. Field data from Asia show that H. axyridis, C. septempunctata, and Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) are regularly among the most abundant predatory species but their rank varies by habitat. Studies of these species in their native Asian range, primarily related to their range in mainland China, document different patterns of seasonal abundance, species specific associations with prey, and habitat separation. Intraguild predation is well documented both in Asia and in newly invaded areas, and H. axyridis benefits most from this interaction. Harmonia axyridis also seems to rely more on cannibalism in times of prey scarcity than other species, and relatively sparse data indicate a lower predation pressure on it from natural enemies of coccinellids. Declines in the abundance of native coccinellids following the spread and increase of non-native species, documented in several multi-year studies on several continents, is a major concern for native biodiversity and the persistence of native coccinellid species. We suggest that future studies focus more attention on the community ecology of these invasive species in their native habitats.

Altering Planting Date to Manage Melanaphis sacchari (Hemiptera: Aphididae) Populations in Sweet Sorghum.

Melanaphis sacchari Zehntner is a new pest of sweet sorghum in the United States, with the potential to cause complete crop failure. In Kentucky, sweet sorghum is normally planted in early May and harvested in late August or September. Planting sweet sorghum earlier in the season may avoid damaging levels of M. sacchari that develop in late summer. In a 2-yr field study, three different planting dates separated by a month (April, May, and June) were tested for their effect on M. sacchari densities and sweet sorghum yield. April (early) planted sweet sorghum was grown in greenhouses and transplanted to the field. May (mid) and June (late) planted sweet sorghum were direct seeded in the field. Melanaphis sacchari population densities were evaluated weekly starting in June. Sweet sorghum was harvested at the onset of the hard dough stage. Plots were split into two subplots, insecticide or noninsecticide, in the second year to control for planting date effect on yield. Early-planted sweet sorghum had lower aphid densities, but had lower yield relative to mid-planting date, which had the highest yield. Insecticide drenches in 2019 reduced cumulative aphid days in mid-plantings and late plantings, but did not significantly affect yield within planting dates. Seeding sweet sorghum earlier can reduce M. sacchari densities; however, this method alone may not provide the highest yields. We documented that the recommended planting date (May) for Kentucky produced the highest yield.

Comparison of native and non-native predator consumption rates and prey avoidance behavior in North America and Europe.

Novel predator-prey interactions can contribute to the invasion success of non-native predators. For example, native prey can fail to recognize and avoid non-native predators due to a lack of co-evolutionary history and cue dissimilarity with native predators. This might result in a competitive advantage for non-native predators. Numerous lady beetle species were globally redistributed as biological control agents against aphids, resulting in novel predator-prey interactions. Here, we investigated the strength of avoidance behavior of the pea aphid (Acyrthosiphon pisum) toward chemical cues of native lady beetles and non-native Asian Harmonia axyridis and European Coccinella septempunctata and Hippodamia variegata in North America, hypothesizing that cues of non-native lady beetles induce weaker avoidance behavior than cues of co-evolved native lady beetles. Additionally, we compared aphid consumption of lady beetles, examining potential predation advantages of non-native lady beetles. Finally, we compared cue avoidance behavior between North American and European pea aphid populations and aphid consumption of native and non-native lady beetles in North America and Europe. In North America, pea aphids avoided chemical cues of all ladybeetle species tested, regardless of their origin. In contrast to pea aphids in North America, European pea aphids did not avoid cues of the non-native H. axyridis. The non-native H. axyridis and C. septempunctata were among the largest and most voracious lady beetle species tested, on both continents. Consequently, in North America non-native lady beetle species might have a competitive advantage on shared food resources due to their relatively large body size, compared to several native American lady beetle species. In Europe, however, non-native H. axyridis might benefit from missing aphid cue avoidance as well as a large body size. The co-evolutionary time gap between the European and North American invasion of H. axyridis likely explains the intercontinental differences in cue avoidance behavior and might indicate evolution in aphids toward non-native predators.

Comparative Studies of Reproductive Diapause in North American Populations of Three Hippodamia Species (Coleoptera: Coccinellidae).

Quantifying responses of three congeneric species of lady beetles, Hippodamia parenthesis (Say), Hippodamia convergens (Guerin), and Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae), to abiotic factors that influence their seasonal biology provides an understanding of the phenology of these species in North America. The developmental response and the induction and duration of adult hibernal diapause in four North American populations of H. parenthesis, collected between 40° N and 44° N latitude, was determined when beetles were reared at four photoperiods (L:D 16:8, 14:10, 12;12, and 10:14) at 22°C. Preimaginal development of the four H. parenthesis populations reared at the photoperiods was affected by population, photoperiod, and the interaction between population and photoperiod. Fifteen to 19% of H. parenthesis females reared at L:D 16:8 entered diapause, whereas shorter photoperiods (L:D 12:12 and 10:14) induced diapause in all females. Variation in response to L:D 14:10 was observed among the four populations of H. parenthesis, similar to the response observed in H. convergens and H. variegata. In contrast to the response of H. parenthesis females, in which four individuals oviposited at L:D 12:12 or 10:14 within 120 d, the duration of reproductive diapause in H. convergens and H. variegata females at L:D 12:12 and 10:14 showed a prolonged quantitative response to photoperiod. Comparisons of the responses (days to first oviposition) to photoperiod of H. parenthesis and H. variegata from the same collection sites showed significant differences at most photoperiods. Similarly, responses at all photoperiods varied between H. parenthesis and H. convergens from similar latitudes in Iowa.

Insights from Population Genomics to Enhance and Sustain Biological Control of Insect Pests.

Biological control-the use of organisms (e.g., nematodes, arthropods, bacteria, fungi, viruses) for the suppression of insect pest species-is a well-established, ecologically sound and economically profitable tactic for crop protection. This approach has served as a sustainable solution for many insect pest problems for over a century in North America. However, all pest management tactics have associated risks. Specifically, the ecological non-target effects of biological control have been examined in numerous systems. In contrast, the need to understand the short- and long-term evolutionary consequences of human-mediated manipulation of biological control organisms for importation, augmentation and conservation biological control has only recently been acknowledged. Particularly, population genomics presents exceptional opportunities to study adaptive evolution and invasiveness of pests and biological control organisms. Population genomics also provides insights into (1) long-term biological consequences of releases, (2) the ecological success and sustainability of this pest management tactic and (3) non-target effects on native species, populations and ecosystems. Recent advances in genomic sequencing technology and model-based statistical methods to analyze population-scale genomic data provide a much needed impetus for biological control programs to benefit by incorporating a consideration of evolutionary consequences. Here, we review current technology and methods in population genomics and their applications to biological control and include basic guidelines for biological control researchers for implementing genomic technology and statistical modeling.

Optimizing Sample Size for Population Genomic Study in a Global Invasive Lady Beetle, Harmonia Axyridis.

Finding optimal sample sizes is critical for the accurate estimation of genetic diversity of large invasive populations. Based on previous studies, we hypothesized that a minimal sample size of 3-8 individuals is sufficient to dissect the population architecture of the harlequin lady beetle, Harmonia axyridis, a biological control agent and an invasive alien species. Here, equipped with a type IIB endonuclease restriction site-associated (2b-RAD) DNA sequencing approach, we identified 13,766 and 13,929 single nucleotide polymorphisms (SNPs), respectively, among native and invasive H. axyridis populations. With this information we simulated populations using a randomly selected 3000 SNPs and a subset of individuals. From this simulation we finally determined that six individuals is the minimum sample size required for the accurate estimation of intra- and inter-population genetic diversity within and across H. axyridis populations. Our findings provide an empirical advantage for population genomic studies of H. axyridis in particular and suggest useful tactics for similar studies on multicellular organisms in general.

Supplemental Foods Affect Energetic Reserves, Survival, and Spring Reproduction in Overwintering Adult Hippodamia convergens (Coleoptera: Coccinellidae).

For insects that overwinter as adults, winter food resources may affect subsequent spring reproduction and abundance. We tested if provision of food supplements to overwintering adult Hippodamia convergens (Guerin) increased energy reserves, winter survival, and spring reproduction. During 2015-2016, H. convergens adults were placed in field cages in December; adults in each cage received water, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) eggs, bee pollen, wheast protein, sugar, honey, or no food (control). In 2016-2017, treatments were reduced to sugar, bee pollen, A. pisum with E. kuehniella eggs, and no food (control). Adults were sampled to quantify weight, lipid, carbohydrate, and protein content. In 2015-2016, A. pisum and E. kuehniella eggs increased adult weight and protein content, but adult carbohydrate content was reduced by A. pisum and wheast protein treatments. Adults receiving honey and sugar supplementation had higher lipid and carbohydrate content relative to controls. The number of live individuals at the end of the experiment in March 2016 did not differ among treatments. In 2016-2017, winter prey supplements had the greatest effect on protein content, weight, and number of live adults recovered, whereas sugar supplementation increased lipid and carbohydrate content, and number of live adults recovered. Spring reproduction of surviving pairs was evaluated among treatments in March 2017. Prey supplementation in 2016-2017 increased the number of eggs laid and decreased preoviposition period, and food treatment did not affect fertility. Our results indicate that prey and sugar resources improve the overwintering success and spring reproduction of H. convergens.

Photoperiodic Induction of Adult Diapause in North American Populations of the Convergent Lady Beetle (Coleoptera: Coccinellidae).

Characterization of intraspecific variation is required to assess the potential nontarget effects of augmentative releases of Hippodamia convergens (Guerin) (Coleoptera: Coccinellidae) from the Western United States on local populations of this species in the Eastern United States. Adults of this predatory lady beetle species overwinter in adult reproductive diapause, thus examining responses to photoperiods can characterize geographic variation influencing their seasonal biology. This laboratory study quantified the induction and duration of adult reproductive diapause in five North American populations of H. convergens in response to four constant photoperiods (16:8, 14:10, 12:12, and 10:14 [L:D] h) at 22°C. Three populations were collected over a range of latitudes (31° N to 42° N) in the central portion of the United States (Texas, Kansas, and Iowa); two populations were purchased from commercial sources in the Western United States. All populations exhibited a long-day response to photoperiods: ≤17% of females reared at 16:8 (L:D) h entered diapause, whereas shorter photoperiods (12:12 and 10:14 [L:D] h) induced diapause in 82 to 100% of females. The response to 14:10 (L:D) h showed significant variation among the populations, ranging from 0 to 89% of females in reproductive diapause. Both the phenotypic variation in response to diapause inducing photoperiods and the genetic variation in North American populations of H. convergens document the geographic variability in this widely distributed predatory species.

Reproductive Diapause in North American Populations of the Introduced Lady Beetle Hippodamia variegata (Coleoptera: Coccinellidae).

The Palearctic lady beetle species, Hippodamia variegata (Coleoptera: Coccinellidae), first collected in 1984 near Montreal, Quebec, Canada, is expanding its distribution into northeastern and north central portions of North America. Examination of responses to abiotic factors that influence the seasonal biology of H. variegata may provide insights into its potential range expansion in North America. The induction and duration of adult hibernal diapause in three North American populations of H. variegata, collected between 40°N and 44°N latitude, was determined at four constant photoperiods (L:D 16:8, 14:10, 12:12, and 10:14) at 22°C. Thirteen to twenty-one percent of females reared at L:D 16:8 entered diapause, whereas shorter photoperiods (L:D 12:12 and 10:14) induced diapause in 100% of females. Variation in the response to L:D 14:10 was observed among the three populations, 27-100% of females exhibited reproductive diapause. Pupae and young adults were sensitive to changes in constant photoperiods (L:D 16:8 ⇆ 10:14). Individuals reared at L:D 10:14 that were moved to L:D 16:8 on the day of pupation or the day of adult eclosion produced ovipositing females. Individuals reared at L:D 16:8 and transferred to L:D 10:14 on the day of pupation or the day of adult eclosion produced females that did not oviposit within 30 d of eclosion.

Demographic histories of three predatory lady beetles reveal complex patterns of diversity and population size change in the United States.

Predatory lady beetles (Coccinellidae) contribute to biological control of agricultural pests, however, multiple species frequently compete for similar resources in the same environment. Numerous studies have examined ecological interactions among the native North American convergent lady beetle (Hippodamia convergens) and two introduced species, the seven-spotted lady beetle (Coccinella septempunctata) and the Asian lady beetle (Harmonia axyridis), in agricultural fields and described multiyear population dynamics. However, the evolutionary dynamics of these interacting species of predatory beetles are uncharacterized. We utilize publicly available multilocus genotype data from geographically disjunct populations of these three species to estimate demography across North American populations. Coalescent analyses reveal (1) a recent (∼4-5 years) decline (>12 fold) in microsatellite effective population size of H. convergens, while expanding (mutation scaled growth rate in 1/u generations = 2910, SD = 362) over evolutionary time scales, (2) a massive (>150 fold), and very recent, effective population size decline in Ha. axyridis, and (3) population size growth (mutation scaled growth rate = 997, SD = 60) over recent and evolutionary time scales in C. septempunctata. Although these estimates are based on genetic data with different mutation rates and patterns of inheritance (mitochondrial versus nuclear), these dynamic and differing population size histories are striking. Further studies of the interactions of these predatory lady beetles in the field are thus warranted to explore the consequences of population size change and biological control activities for evolutionary trajectories in North America.

Development and application of molecular gut-content analysis to detect aphid and coccinellid predation by Harmonia axyridis (Coleoptera: Coccinellidae) in Italy.

Despite their positive effect in reducing pest populations, exotic generalist predators sometimes become invasive and contribute to the displacement of indigenous species in the same trophic level. Although laboratory experiments have linked intraguild predation (IGP) to these interactions, field evidence and quantification of IGP are still lacking for most systems. The recent establishment of the exotic Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) in Italy raises concern about the detrimental effect that the ladybird could have on native coccinellids. Here we assessed, under laboratory conditions, the acceptability and suitability of eggs of 2 native ladybirds, Adalia bipunctata L. and Oenopia conglobata (L.), as prey items for H. axyridis larvae. Then we developed primers for molecular gut-content analysis to detect predation by H. axyridis on the 2 ladybirds and on the aphid Eucallipterus tiliae L. Species-specific 16S primers were developed for the 3 species and laboratory feeding trials were conducted to quantify the rate of prey DNA breakdown in the gut of H. axyridis. Moreover, to field evaluate primers, H. axyridis 4th instars (n = 132) were systematically collected from linden trees in northern Italy and screened for the presence of prey DNA. Seventy-three percent and 7% of field collected H. axyridis were positive for aphid and coccinellid DNA, respectively. Predation upon aphid and A. bipunctata was lower than predicted if density dependent consumption was expected, while predation upon O. conglobata was significantly higher. Here, we provided the first evidence of IGP among feral populations of H. axyridis and indigenous ladybird beetles, occurring in Italy.

Do defensive chemicals facilitate intraguild predation and influence invasion success in ladybird beetles?

Egg predation and cannibalism are believed to be common phenomena among many species of aphidophagous predatory ladybird beetles despite the presence of alkaloid based defensive chemicals in all life stages. We identified defensive chemicals from eggs of three congeneric species, one introduced into North America (Coccinella septempunctata L.), and two native (C. transversoguttata richardsoni Brown, and C. novemnotata Herbst), and examined the effects of ingested defensive chemicals on first instars. Ingested congeneric alkaloids were not toxic to first instars, likely because the three congeners produce the same principal alkaloids, precoccinelline and coccinelline, in similar amounts. First instars of the three congeners accumulated alkaloids ingested through egg cannibalism and congeneric predation. Egg consumption doubled the amount of alkaloids in first instars when they fed on conspecific or congeneric eggs, in comparison to a pea aphid diet. No detrimental effects of ingested congeneric alkaloids on development or survival of first instars were observed among these congeners. Chemical defenses of eggs are therefore not likely to be important in favoring the invasive species, C. septempunctata, in interactions with these native congeneric species. Because the invasive species is the most aggressive predator, having the same types of alkaloids may facilitate disproportionate intraguild predation on native congeners by C. septempunctata thereby potentially enhancing the invasion success of this introduced species.

A population genetic signature of human releases in an invasive ladybeetle.

Biological invasions have been accelerated by a variety of human activities. Propagule pressure, the number of introduced individuals and independent introductions, is probably to be influenced by these human activities and may be an important factor for successful range expansion in new environments. We tested whether the current distribution of the predatory ladybeetle Coccinella septempunctata in the introduced range (USA) is the result of multiple historical human introductions or natural range expansion from the first established populations in the USA. To test this hypothesis, we compared historical records of propagule size, propagule number, specific introduction locations and the date of each introduction, with estimates of genetic variation in mitochondrial DNA (cytochrome oxidase I). Our results indicated that genetic diversity in the introduced range was positively correlated with historical records of propagule size and number and negatively correlated with distance to nearest introduction point, suggesting that multiple human releases were successful. Higher genetic diversity in populations found near introduction points suggest that initial founder effects were limited, but lower genetic diversity found farther from introduction points is probably the result of serial founder effects during secondary range expansion. These results suggest that the current distribution of C. septempunctata in the introduced range is the result of a combination of human releases and short-range expansion from multiple established populations in the introduced range.

Larval feeding on Bt hybrid and non-Bt corn seedlings by Harmonia axyridis (Coleoptera: Coccinellidae) and Coleomegilla maculata (Coleoptera: Coccinellidae).

Zoophytophagy is an omnivorous activity that occurs when a primarily carnivorous species feeds on plant material. Plant feeding by beneficial predators may have negative consequences if the plant material has been chemically treated, contains toxins, or was transgenically altered. Although common in predaceous Hemiptera, zoophytophagy has been rarely studied in aphidophagous coccinellids. This study examined the likelihood of feeding on Bt and non-Bt corn seedlings by third- and fourth-instar coccinellid larvae, the regularity of feeding events by fourth instars, and the effect of leaf feeding on development time and adult size. Both third- and fourth-instar Harmonia axyridis Pallas and Coleomegilla maculata DeGeer consume leaf tissue, with fourth instars being significantly more likely to feed on corn seedlings. C. maculata larvae ingested leaf tissue more frequently than H. axyridis. Furthermore, when given access to corn seedlings daily, development time of fourth-instar C. maculata increased after Bt hybrid corn treatments compared with non-Bt corn treatments. Zoophytophagous feeding behavior is thought to sustain predators during times of low prey availability, and leaf tissue feeding by coccinellids has typically been attributed to their need for water. However, in this study, tissue feeding regularly occurred even though coccinellid larvae had constant access to water and a daily ad libitum supply of aphids. We suggest that, in addition to environmental conditions, the physiological state of the zoophytophagous species will influence the probability of plant feeding.

Tracking the role of alternative prey in soybean aphid predation by Orius insidiosus: a molecular approach.

The soybean aphid, Aphis glycines (Hemiptera: Aphididae), is a pest of soybeans in Asia, and in recent years has caused extensive damage to soybeans in North America. Within these agroecosystems, generalist predators form an important component of the assemblage of natural enemies, and can exert significant pressure on prey populations. These food webs are complex and molecular gut-content analyses offer nondisruptive approaches for examining trophic linkages in the field. We describe the development of a molecular detection system to examine the feeding behaviour of Orius insidiosus (Hemiptera: Anthocoridae) upon soybean aphids, an alternative prey item, Neohydatothrips variabilis (Thysanoptera: Thripidae), and an intraguild prey species, Harmonia axyridis (Coleoptera: Coccinellidae). Specific primer pairs were designed to target prey and were used to examine key trophic connections within this soybean food web. In total, 32% of O. insidiosus were found to have preyed upon A. glycines, but disproportionately high consumption occurred early in the season, when aphid densities were low. The intensity of early season predation indicates that O. insidiosus are important biological control agents of A. glycines, although data suggest that N. variabilis constitute a significant proportion of the diet of these generalist predators. No Orius were found to contain DNA of H. axyridis, suggesting intraguild predation upon these important late-season predators during 2005 was low. In their entirety, these results implicate O. insidiosus as a valuable natural enemy of A. glycines in this soybean agroecosystem.

Trophic interactions between two herbivorous insects, Galerucella calmariensis and Myzus lythri, feeding on purple loosestrife, Lythrum salicaria, and two insect predators, Harmonia axyridis and Chrysoperla carnea.

The effects of two herbivorous insects, Galerucella calmariensis Duftschmid and Myzus lythri L. (Coleoptera: Chrysomelidae), feeding on purple loosestrife, Lythrum salicaria L. (Myrtiflorae: Lythraceae), were measured in the presence of two insect predators, Harmonia axyridis Pallas (Coleoptera: Coccinellidae) and Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae). A greenhouse cage experiment examined the direct effects of these predators on these herbivores, and indirect effects of predation on aboveground biomass, defoliation, number of leaves, and internode length. Eight treatment combinations with G. calmariensis, M. lythri, H. axyridis and C. carnea were applied to caged L. salicaria. The experiment ended when G. calmariensis adults were observed, 11 to 13 days after release of first instar G. calmariensis. G. calmariensis larvae alone removed significant amounts of leaf tissue and reduced the number of L. salicaria leaves. Predators did not reduce levels of defoliation by G. calmariensis. C. carnea had no effect on G. calmariensis survival, but H. axyridis reduced G. calmariensis survival in the presence of M. lythri. Both predators reduced the survival of M. lythri. This short duration greenhouse study did not demonstrate that predator-prey interactions altered herbivore effects on L. salicaria.

Uptake of Bt endotoxins by nontarget herbivores and higher order arthropod predators: molecular evidence from a transgenic corn agroecosystem.

The planting of transgenic crops expressing Bacillus thuringiensis endotoxins is widespread throughout the world; the prolific increase in their application exposes nontarget organisms to toxins designed to control pests. To date, studies have focused upon the effects of Bt endotoxins on specific herbivores and detritivores, without consideration of their persistence within arthropod food webs. Here, we report the first quantitative field evaluation of levels of Bt endotoxin within nontarget herbivores and the uptake by higher order arthropods. Antibody-based assays indicated significant quantities of detectable Cry1Ab endotoxin within nontarget herbivores which feed on transgenic corn (including the corn flea beetle, Chaetocnema pulicaria, Japanese beetle, Popillia japonica and southern corn rootworm, Diabrotica undecimpunctata howardi). Furthermore, arthropod predators (Coccinellidae, Araneae, and Nabidae) collected from these agroecosystems also contained significant quantities of Cry1Ab endotoxin indicating its movement into higher trophic levels. This uptake by predators is likely to have occurred by direct feeding on plant material (in predators which are facultatively phytophagous) or the consumption of arthropod prey which contained these proteins. These data indicate that long-term exposure to insecticidal toxins occurs in the field. These levels of exposure should therefore be considered during future risk assessments of transgenic crops to nontarget herbivores and arthropod predators.

Field deposition of Bt transgenic corn pollen: lethal effects on the monarch butterfly.

We present the first evidence that transgenic Bacillus thuringiensis (Bt) corn pollen naturally deposited on Asclepias syriaca; common milkweed, in a corn field causes significant mortality of Danaus plexippus L. (Lepidoptera: Danaidae) larvae. Larvae feeding for 48 h on A. syriaca plants naturally dusted with pollen from Bt corn plants suffered significantly higher rates of mortality at 48 h (20±3%) compared to larvae feeding on leaves with no pollen (3±3%), or feeding on leaves with non-Bt pollen (0%). Mortality at 120 h of D. plexippus larvae exposed to 135 pollen grains/cm(2) of transgenic pollen for 48 h ranged from 37 to 70%. We found no sub-lethal effects on D. plexippus adults reared from larvae that survived a 48-h exposure to three concentrations of Bt pollen. Based on our quantification of the wind dispersal of this pollen beyond the edges of agricultural fields, we predict that the effects of transgenic pollen on D. plexippus may be observed at least 10 m from transgenic field borders. However, the highest larval mortality will likely occur on A. syriaca plants in corn fields or within 3 m of the edge of a transgenic corn field. We conclude that the ecological effects of transgenic insecticidal crops need to be evaluated more fully before they are planted over extensive areas.