Variation in Pesticide Toxicity in the Western Honey Bee (Apis mellifera) Associated with Consuming Phytochemically Different Monofloral Honeys.

Insecticide toxicity to insect herbivores has long been known to vary across different host plants; this phenomenon has been widely documented in both foliage-feeders and sap-feeders. Species-specific phytochemical content of hostplant tissues is assumed to determine the pattern of induction of insect enzymes that detoxify insecticides, but specific phytochemicals have rarely been linked to host plant-associated variation in pesticide toxicity. Moreover, no studies to date have examined the effects of nectar source identity and phytochemical composition on the toxicity of insecticides to pollinators. In this study, we compared LD50 values for the insecticide bifenthrin, a frequent contaminant of nectar and pollen in agroecosystems, in the western honey bee, Apis mellifera, consuming three phytochemically different monofloral honeys: Nyssa ogeche (tupelo), Robinia pseudoacacia (black locust), and Fagopyrum esculentum (buckwheat). We found that bifenthrin toxicity (LD50) values for honey bees across different honey diets is linked to their species-specific phytochemical content. The profiles of phenolic acids and flavonoids of buckwheat and locust honeys are richer than is the profile of tupelo honey, with buckwheat honey containing the highest total content of phytochemicals and associated with the highest bifenthrin LD50 in honey bees. The vector fitting in the ordination analysis revealed positive correlations between LD50 values and two honey phytochemical richness estimates, Chao1 and Abundance-based Coverage Estimator (ACE). These findings suggest unequal effects among different phytochemicals, consistent with the interpretation that certain compounds, including ones that are rare, may have a more pronounced effect in mitigating pesticide toxicity.

Culex pipiens and Culex restuans larval interactions shape the bacterial communities in container aquatic habitats.

Container aquatic habitats host a community of aquatic insects, primarily mosquito larvae that browse on container surface microbial biofilm and filter-feed on microorganisms in the water column. We examined how the bacterial communities in these habitats respond to feeding by larvae of two container-dwelling mosquito species, Culex pipiens and Cx. restuans. We also investigated how the microbiota of these larvae is impacted by intra- and interspecific interactions. Microbial diversity and richness were significantly higher in water samples when mosquito larvae were present, and in Cx. restuans compared to Cx. pipiens larvae. Microbial communities of water samples clustered based on the presence or absence of mosquito larvae and were distinct from those of mosquito larvae. Culex pipiens and Cx. restuans larvae harbored distinct microbial communities when reared under intraspecific conditions and similar microbial communities when reared under interspecific conditions. These findings demonstrate that mosquito larvae play a major role in structuring the microbial communities in container habitats and that intra- and interspecific interactions in mosquito larvae may shape their microbiota. This has important ecological and public health implications since larvae of the two mosquito species are major occupants of container habitats while the adults are vectors of West Nile virus.

An Aspergillus flavus strain from bee bread of the Western honey bee (Apis mellifera) displays adaptations to distinctive features of the hive environment.

Aspergillus fungi are ubiquitous inhabitants of colonies of the western honey bee (Apis mellifera), where they interact with bees in associations ranging from parasitism to possible mutualism. Aspergillus Flavi fungi are frequently found in bee bread (pollen processed for longterm storage) and are thought to contribute to food preparation, processing, preservation, and digestion. Conditions in the hive are challenging for fungi due, in part, to xeric and acidic properties of bee bread and the omnipresence of propolis, an antimicrobial product manufactured by bees from plant resins. We used quantitative and qualitative assays to determine whether A. flavus isolated from bee bread demonstrates tolerance for hive environmental conditions in terms of temperature, pH, osmotic pressure, and propolis exposure. Comparisons made use of three strains of A. flavus: a fungal biocontrol product not known from beehives (AF36), a strain isolated from bee bread (AFBB) in hives from central Illinois, and a pathogenic strain from a honey bee colony displaying symptoms of stonebrood (AFPA). Strain AFBB displayed higher tolerance of acidic conditions, low matric potential (simulating xeric substrate), and propolis exposure than did other strains. A genomic comparison between this new strain and the reference NRRL-3357 showed that AFBB, like AF36, might be blocked from carrying out aflatoxin biosynthesis. Sequence comparisons also revealed several missense variants in genes that encode proteins regulating osmotolerance and osmotic pressure in Aspergillus spp., including SakA, SskB, GfdA, and TcsB/Sln1. Collectively, results of our laboratory assays and genetic analyses are consistent with the suggestion that the strain isolated from bee bread is adapted to the bee bread environment and may have persisted due to a coevolutionary relationship between Aspergillus and A. mellifera. This finding bolsters recent concerns about the effects of fungicide use near bee colonies and broadens the ecological importance of highly adaptable fungal strains.

Kaolin as a management alternative for insecticide-resistant navel orangeworm (Lepidoptera: Pyralidae).

The production of almonds and pistachios in California's Central Valley employs insecticides for the management of their primary pest, navel orangeworm. The pyrethroid Bifenthrin is commonly used, and now a strain of Amyelois transitella Walker (Lepidoptera: Pyralidae) (R347) obtained from Kern County almond orchards with a history of Bifenthrin use has acquired >110-fold resistance toward pyrethroids. One method to improve control is to use additives and spray adjuvants, which are applied simultaneously with an insecticide to increase coverage and/or duration of control. We tested 2 levels of the naturally occurring clay Kaolin as an additive, alone and in combination with either Bifenthrin or the diamide Chlorantraniliprole, to determine if it could reduce feeding damage and decrease survival of pyrethroid-resistant A. transitella on almonds in the laboratory and improve the efficacy of Chlorantraniliprole in the field. Larval performance was measured for the strains R347 and ALM (34.7-fold resistance compared to susceptible lab strain) reared on treated almonds. Strain R347 had 1.9-fold greater survival and caused 1.3-fold more feeding damage than strain ALM across all treatments, although both strains were susceptible to the combination of Kaolin + insecticide. Kaolin synergized Bifenthrin for R347, decreasing survival by 10.0%. Kaolin did not reduce feeding damage for either strain. When combined with insecticide, feeding damage was similar to insecticide alone, but the addition of Kaolin to the insecticide generally decreased survival more than the insecticide alone. In the field, the addition of Kaolin to Chlorantraniprole during application helped retain activity against this challenging pest.

Effects of pesticide-adjuvant combinations used in almond orchards on olfactory responses to social signals in honey bees (Apis mellifera).

Exposure to agrochemical sprays containing pesticides and tank-mix adjuvants has been implicated in post-bloom mortality, particularly of brood, in honey bee colonies brought into California almond orchards for pollination. Although adjuvants are generally considered to be biologically inert, some adjuvants have exhibited toxicity and sublethal effects, including decreasing survival rates of next-generation queens. Honey bees have a highly developed olfactory system to detect and discriminate among social signals. To investigate the impact of pesticide-adjuvant combinations on honey bee signal perception, we performed electroantennography assays to assess alterations in their olfactory responsiveness to the brood ester pheromone (BEP), the volatile larval pheromone ß-ocimene, and the alarm pheromone 2-heptanone. These assays aimed to uncover potential mechanisms underlying changes in social behaviors and reduced brood survival after pesticide exposure. We found that combining the adjuvant Dyne-Amic with the fungicide Tilt (propiconazole) and the insecticide Altacor (chlorantraniliprole) synergistically enhanced olfactory responses to three concentrations of BEP and as well exerted dampening and compensatory effects on responses to 2-heptanone and ß-ocimene, respectively. In contrast, exposure to adjuvant alone or the combination of fungicide and insecticide had no effect on olfactory responses to BEP at most concentrations but altered responses to ß-ocimene and 2-heptanone. Exposure to Dyne-Amic, Altacor, and Tilt increased BEP signal amplitude, indicating potential changes in olfactory receptor sensitivity or sensilla permeability to odorants. Given that, in a previous study, next-generation queens raised by nurses exposed to the same treated pollen experienced reduced survival, these new findings highlight the potential disruption of social signaling in honey bees and its implications for colony reproductive success.

Genetics of tolerance in honeybees to the neonicotinoid clothianidin.

The effects of neonicotinoid insecticides (NNIs) on honeybee health are intensely debated, with numerous studies showing negative effects of exposure, while others report no such effects. We carried out experiments to study the genetic and molecular basis of NNI tolerance in honeybees, which may underlie the discrepancies observed in the literature. We discovered that worker survival post-exposure to an acute oral dose of clothianidin is heritable (H 2 = 37.8%). Tolerance to clothianidin was not associated with differences in the expression of detoxification enzymes in our experiments. Instead, mutations in the primary neonicotinoid detoxification genes CYP9Q1 and CYP9Q3 were strongly associated with worker survival post-clothianidin exposure. In some instances, the strong association between CYP9Q haplotypes and worker survival was associated with the protein's predicted binding affinity for clothianidin. Our findings have implications regarding future toxicological studies utilizing honeybees as a model pollinator.

Degree of Dietary Specialization on Furanocoumarin-Containing Hostplants in a Newly Invasive Web Building Caterpillar.

The genus Depressaria (Lepidoptera: Depressariidae) mostly comprises specialist herbivores with varying capacity for detoxification of defensive phytochemistry. Depressaria depressana, a Eurasian moth recently introduced into North America, is a family-level specialist of the Apiaceae, whose hosts include more than a dozen species in multiple tribes; Depressaria radiella is a super-specialist of Eurasian origin that feeds exclusively on species in the genera Pastinaca and Heracleum throughout its native and introduced range. In eastern North America, it feeds upon Pastinaca sativa, an invasive European species, and Heracleum maximum, a native species. We determined whether differences in furanocoumarin metabolism exist between D. depressana and two isolated populations of D. radiella, feeding exclusively on either P. sativa or H. maximum. We also compared gravimetric estimates of feeding efficiency to assess D. depressana larval performance on different diets. Both populations of D. radiella metabolized furanocoumarins at a greater rate than D. depressana. Although there was no difference in rates of metabolism of linear furanocoumarins in the two populations of D. radiella, individuals collected from H. maximum metabolized angular furanocoumarins more rapidly. The gravimetric assessments of feeding efficiencies revealed that D. depressana exhibited highest efficiencies consuming Daucus carota; moreover, this species survived to pupation consuming fruits of Zizia aurea, an apiaceous species native to North America. Our preliminary phylogenetic analysis, building on an earlier morphological analysis, incorporates mitochondrial cytochrome oxidase subunit 1 data from the BOLD database and revealed that the presence or absence of furanocoumarins is not a strong predictor of species-level evolution in Depressaria.

Genetic Structure and Colonization of North America by Depressaria depressana (Fabricius 1775) (Lepidoptera: Depressariidae) over 15 Years; Contrasts with Westward Expansion of Depressaria radiella (Goeze, 1783) over 160 Years.

Depressaria depressana, the purple carrot seed moth, is a Eurasian species first reported in North America in 2008 and currently undergoing range expansion. This invasion follows that of its Eurasion congener Depressaria radiella (parsnip webworm), first documented in North America 160 years ago. Unlike D. depressana, which utilizes hostplants across multiple tribes of Apiaceae, Depressaria radiella is a "superspecialist" effectively restricted in its native and non-indigenous ranges to two closely related apiaceous genera. We investigated the genetic structure of D. depressana populations across latitudinal and longitudinal gradients in the eastern United States by constructing COI haplotype networks and then comparing these with haplotype networks constructed from available COI sequence data from contemporary European D. depressana populations and from European and North American D. radiella populations. Haplotype data revealed higher genetic diversity in D. depressana, indicating high dispersal capacity, multiple introductions, and/or a genetically diverse founding population. Museum and literature records of D. radiella date back to 1862 and indicate that range expansion to the West Coast required more than 50 years. Higher levels of genetic diversity observed in D. depressana compared to its congener may indicate a greater propensity for dispersal, colonization and establishment in its non-indigenous range.

Specific phytochemicals in floral nectar up-regulate genes involved in longevity regulation and xenobiotic metabolism, extending mosquito life span.

During nectar feeding, mosquitoes ingest a plethora of phytochemicals present in nectar. The ecological and physiological impacts of these ingested phytochemicals on the disease vectors are poorly understood. In this study, we evaluated the effects of three nectar phytochemicals-- caffeine, p-coumaric acid, and quercetin--on longevity, fecundity, and sugar-feeding behavior of the Asian tiger mosquito (Aedes albopictus). Adult females of Ae. albopictus were provided continuous access to 10% sucrose supplemented with one of the three phytochemicals and their fecundity, longevity, and the amount of sucrose consumed determined. Transcriptome response of Ae. albopictus females to p-coumaric acid and quercetin was also evaluated. Dietary quercetin and p-coumaric acid enhanced the longevity of female Ae. albopictus, while caffeine resulted in reduced sugar consumption and enhanced fecundity of gravid females. RNA-seq analyses identified 237 genes that were differentially expressed (DE) in mosquitoes consuming p-coumaric acid or quercetin relative to mosquitoes consuming an unamended sucrose solution diet. Among the DE genes, several encoding antioxidant enzymes, cytochrome P450s, and heat shock proteins were upregulated, whereas histones were downregulated. Overall, our findings show that consuming certain nectar phytochemicals can enhance adult longevity of female Asian tiger mosquitoes, apparently by differentially regulating the expression level of genes involved in longevity and xenobiotic metabolism; this has potential impacts not only on life span but also on vectorial capacity and insecticide resistance.

Differential regulation of cytochrome P450 genes associated with biosynthesis and detoxification in bifenthrin-resistant populations of navel orangewom (Amyelois transitella).

Pyrethroid resistance was first reported in 2013 for the navel orangeworm, Amyelois transitella, but the genetic underpinnings of pyrethroid resistance are unknown. We investigated the role of cytochrome P450 monooxygenases (P450s) belonging to the CYP3 and CYP4 clans using colonies derived from individuals collected in 2016 from almond orchards in two counties. One colony (ALM) originated from an almond orchard in Madera County with no reported pyrethroid resistance and the second colony (R347) originated from the same Kern County orchard where pyrethroid resistance was first reported. We used high-throughput quantitative real-time PCR (qRT-PCR) analyses of 65 P450s in the CYP3 and CYP4 clans of A. transitella to identify P450s induced by bifenthrin and associated with pyrethroid resistance. Nine P450s were constitutively overexpressed in R347 compared to ALM, including CYP6AE54 (11.7-fold), belonging to a subfamily associated with metabolic pesticide detoxification in Lepidoptera and CYP4G89 (33-fold) belonging to a subfamily associated with cuticular hydrocarbon (CHC) synthesis and resistance via reduced pesticide penetrance. Cuticular hydrocarbons analysis revealed that R347 produced twice as many total CHCs in the egg and adult stages as ALM. Topical toxicity bioassays for R347 determined that egg mortality was reduced at low bifenthrin concentrations and larval mortality was reduced at high concentrations of bifenthrin compared to ALM. Our discovery of both changes in metabolism and production of CHCs for R347 have implications for the possible decreased efficacy of other classes of insecticide used to control this insect. The threat of widespread pyrethroid resistance combined with the potential for cross-resistance to develop through the mechanism of reduced penetrance warrants developing management strategies that facilitate insecticide passage across the cuticle.

No buzz for bees: Media coverage of pollinator decline.

Although widespread declines in insect biomass and diversity are increasing concerns within the scientific community, it remains unclear whether attention to pollinator declines has also increased within information sources serving the general public. Examining patterns of journalistic attention to the pollinator population crisis can also inform efforts to raise awareness about the importance of declines of insect species providing ecosystem services beyond pollination. We used the Global News Index developed by the Cline Center for Advanced Social Research at the University of Illinois at Urbana-Champaign to track news attention to pollinator topics in nearly 25 million news items published by two American national newspapers and four international wire services over the past four decades. We found vanishingly low levels of attention to pollinator population topics relative to coverage of climate change, which we use as a comparison topic. In the most recent subset of ∼10 million stories published from 2007 to 2019, 1.39% (137,086 stories) refer to climate change/global warming while only 0.02% (1,780) refer to pollinator populations in all contexts, and just 0.007% (679) refer to pollinator declines. Substantial increases in news attention were detectable only in US national newspapers. We also find that, while climate change stories appear primarily in newspaper "front sections," pollinator population stories remain largely marginalized in "science" and "back section" reports. At the same time, news reports about pollinator populations increasingly link the issue to climate change, which might ultimately help raise public awareness to effect needed policy changes.

Cytochrome P450-mediated mycotoxin metabolism by plant-feeding insects.

Mycotoxins are secondary metabolites produced primarily by filamentous fungi that when consumed cause pathological responses in animal hosts or consumers. Defined functionally rather than structurally, mycotoxins derive from numerous primary metabolic pathways. Through opportunistic or mutualistic associations, insect herbivores inflict damage that can predispose plants to infection by mycotoxin-producing phytopathogens, resulting in economically significant contamination. The few cytochrome P450 subfamilies implicated in mycotoxin detoxification by insects, including CYP6 and CYP9, are also known to detoxify phytochemicals. Some insect P450s bioactivate, rather than detoxify, mycotoxins, suggestive of an 'escalation' in arms-race interactions between these herbivores and fungi. Characterizing insect P450s that detoxify mycotoxins can be useful for developing biological remediation technologies and for ensuring the safety of insects reared for human or livestock consumption.

Honey as a Functional Food for Apis mellifera.

Although nectar is consumed, primarily as a supplemental food, by a broad range of insects spanning at least five orders, it is processed and stored by only a small number of species, most of which are bees and wasps in the superfamily Apoidea. Within this group, Apis mellifera has evolved remarkable adaptations facilitating nectar processing and storage; in doing so, this species utilizes the end product, honey, for diverse functions with few if any equivalents in other phytophagous insects. Honey and its phytochemical constituents, some of which likely derive from propolis, have functional significance in protecting honey bees against microbial pathogens, toxins, and cold stress, as well as in regulating development and adult longevity. The distinctive properties of A. mellifera honey appear to have arisen in multiple ways, including genome modification; partnerships with microbial symbionts; and evolution of specialized behaviors, including foraging for substances other than nectar. That honey making by A. mellifera involves incorporation of exogenous material other than nectar, as well as endogenous products such as antimicrobial peptides and royal jelly, suggests that regarding honey as little more than a source of carbohydrates for bees is a concept in need of revision.

Selective Sweeps in a Nutshell: The Genomic Footprint of Rapid Insecticide Resistance Evolution in the Almond Agroecosystem.

Among the most familiar forms of human-driven evolution on ecological time scales is the rapid acquisition of resistance to pesticides by insects. Since the widespread adoption of synthetic organic insecticides in the mid-twentieth century, over 500 arthropod species have evolved resistance to at least one insecticide. Efforts to determine the genetic bases of insecticide resistance have historically focused on individual loci, but the availability of genomic tools has facilitated the screening of genome-wide characteristics. We resequenced three contemporary populations of the navel orangeworm (Amyelois transitella), the principal pest of almond orchards in California, differing in bifenthrin resistance status to examine insecticide-induced changes in the population genomic landscape of this species. We detected an exceptionally large region with virtually no polymorphisms, extending to up to 1.3 Mb in the resistant population. This selective sweep includes genes associated with pyrethroid and DDT resistance, including a cytochrome P450 gene cluster and the gene encoding the voltage-gated sodium channel para. Moreover, the sequence along the sweep is nearly identical in the genome assembled from a population founded in 1966, suggesting that the foundation for insecticide resistance may date back a half-century, when California's Central Valley experienced massive area-wide applications of DDT for pest control.