Honey bee colony behavior and ontogeny are adversely affected when exposed to a pesticide-contaminated environment.

Honey bees exhibit age polyethism and thus have a predictable sequence of behaviors they express through developmental time. Numerous laboratory studies show exposure to pesticides may impair critical honey bee behaviors (brood care, foraging, egg-laying, etc.) that adversely affect colony productivity and survival. There are fewer studies that examine the impacts of pesticides in natural field settings, especially given the challenges of implementing treatment groups and controlling variables. This study helps address the need for impact studies on pollinators under field conditions to assess the consequences of chemical overuse and dependency in agricultural and urban landscapes. To assess the impact of systemic pesticides in a natural field setting on worker bee behavioral development, observation hives were established to monitor changes in behaviors of similarly aged workers and sister queens within 2 experimental groups: (i) colonies located near point-source systemic pesticide pollution (pesticide contaminated treatment), and (ii) colonies embedded within a typical Midwestern US agricultural environment (control). In this study, worker bees in the contaminated environment exhibited important and biologically significant behavioral differences and accelerated onset of hive tasks (i.e., precocious behavioral development) compared to similarly aged bees at the control site. Queen locomotion was largely unaffected; however, the egg-laying rate was reduced in queens at the contaminated (treated) site. These results show that environmental pesticide exposure can disrupt colony function and adversely affect worker bee behavioral maturation, leading to reduced worker longevity and decreased colony efficiency.

Resilin Distribution and Abundance in Apis mellifera across Biological Age Classes and Castes.

The presence of resilin, an elastomeric protein, in insect vein joints provides the flexible, passive deformations that are crucial to flapping flight. This study investigated the resilin gene expression and autofluorescence dynamics among Apis mellifera (honey bee) worker age classes and drone honey bees. Resilin gene expression was determined via ddPCR on whole honey bees and resilin autofluorescence was measured in the 1m-cu, 2m-cu, Cu-V, and Cu2-V joints on the forewing and the Cu-V joint of the hindwing. Resilin gene expression varied significantly with age, with resilin activity being highest in the pupae. Autofluorescence of the 1m-cu and the Cu-V joints on the ventral forewing and the Cu-V joint on the ventral hindwing varied significantly between age classes on the left and right sides of the wing, with the newly emerged honey bees having the highest level of resilin autofluorescence compared to all other groups. The results of this study suggest that resilin gene expression and deposition on the wing is age-dependent and may inform us more about the physiology of aging in honey bees.

Land Use Influences the Composition and Antimicrobial Effects of Propolis.

Honey bee propolis is a complex, resinous mixture created by bees using plant sources such as leaves, flowers, and bud exudates. This study characterized how cropland surrounding apiaries affects the chemical composition and antimicrobial effects of propolis. The chemical composition and compound abundance of the propolis samples were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) and the antimicrobial effects were analyzed using the 50% minimum inhibitory concentration (MIC50) assay against four relevant bee pathogens, Serratia marcescens, Paenibacillus larvae, Lysinibacillus sphaericus, and Klebsiella pneumoniae. Propolis composition varied significantly with apiary, and cropland coverage predicted mean sum abundance of compounds. The apiary with the highest cropland coverage exhibited significantly higher MIC50 values for S. marcescens and K. pneumoniae compared to other apiaries. These results demonstrate that agricultural land use surrounding honey bee apiaries decreases the chemical quality and antimicrobial effects of propolis, which may have implications for the impacts of land use on hive immunity to potential pathogens.

Oral and parenteral immunization of chickens (Gallus gallus) against West Nile virus with recombinant envelope protein.

West Nile virus (WNV) causes morbidity and mortality in humans, horses, and in more than 315 bird species in North America. Currently approved WNV vaccines are designed for parenteral administration and, as yet, no effectiveoral WNV vaccines have been developed. WNV envelope (E) protein is a highly antigenic protein that elicits the majority ofvirus-neutralizing antibodies during a WNV immune response. Leghorn chickens were given three vaccinations (each 2 wk apart) of E proteinorally (20 microg or 100 microg/dose), of E protein intramuscularly (IM, 20 microg/dose), or of adjuvant only (control group) followed by a WNV challenge. Viremias were measured post-WNV infection, and three new enzyme-linked immunosorbent assays were developed for quantifying IgM, IgY, and IgA-mediated immune response of birds following WNV infection. WNV viremia levelswere significantly lower in the IM group than in both oral groups and the control group. Total WNV E protein-specific IgY production w assignificantly greater, and WNV nonstructural 1-specific IgY w as significantly less, in the IM group compared to all other treatment groups.The results of this study indicate that IM vaccination of chickens with E protein is protective against WNV infection and results in a significantly different antibody production profile as compared to both orally vaccinated and nonvaccinated birds.

High costs of infection: Alphavirus infection reduces digestive function and bone and feather growth in nestling house sparrows (Passer domesticus).

Increasingly, ecoimmunology studies aim to use relevant pathogen exposure to examine the impacts of infection on physiological processes in wild animals. Alphaviruses are arthropod-borne, single-stranded RNA (ssRNA) viruses ("arboviruses") responsible for millions of cases of human illnesses each year. Buggy Creek virus (BCRV) is a unique alphavirus that is transmitted by a cimicid insect, the swallow bug, and is amplified in two avian species: the house sparrow (Passer domesticus) and the cliff swallow (Petrochelidon pyrrhonota). BCRV, like many alphaviruses, exhibits age-dependent susceptibility where the young are most susceptible to developing disease and exhibit a high mortality rate. However, alphavirus disease etiology in nestling birds is unknown. In this study, we infected nestling house sparrows with Buggy Creek virus and measured virological, pathological, growth, and digestive parameters following infection. Buggy Creek virus caused severe encephalitis in all infected nestlings, and the peak viral concentration in brain tissue was over 34 times greater than any other tissue. Growth, tissue development, and digestive function were all significantly impaired during BCRV infection. However, based on histopathological analysis performed, this impairment does not appear to be the result of direct tissue damage by the virus, but likely caused by encephalitis and neuronal invasion and impairment of the central nervous system. This is the first study to examine the course of alphavirus diseases in nestling birds and these results will improve our understanding of age-dependent infections of alphaviruses in vertebrate hosts.

SUSCEPTIBILITY AND ANTIBODY RESPONSE OF VESPER SPARROWS (POOECETES GRAMINEUS) TO WEST NILE VIRUS: A POTENTIAL AMPLIFICATION HOST IN SAGEBRUSH-GRASSLAND HABITAT.

West Nile virus (WNV) spread to the US western plains states in 2003, when a significant mortality event attributed to WNV occurred in Greater Sage-grouse ( Centrocercus urophasianus ). The role of avian species inhabiting sagebrush in the amplification of WNV in arid and semiarid regions of the North America is unknown. We conducted an experimental WNV challenge study in Vesper Sparrows ( Pooecetes gramineus ), a species common to sagebrush and grassland habitats found throughout much of North America. We found Vesper Sparrows to be moderately susceptible to WNV, developing viremia considered sufficient to transmit WNV to feeding mosquitoes, but the majority of birds were capable of surviving infection and developing a humoral immune response to the WNV nonstructural 1 and envelope proteins. Despite clearance of viremia, after 6 mo, WNV was detected molecularly in three birds and cultured from one bird. Surviving Vesper Sparrows were resistant to reinfection 6 mo after the initial challenge. Vesper sparrows could play a role in the amplification of WNV in sagebrush habitat and other areas of their range, but rapid clearance of WNV may limit their importance as competent amplification hosts of WNV.

Immunoglobulin detection in wild birds: effectiveness of three secondary anti-avian IgY antibodies in direct ELISAs in 41 avian species.

Immunological reagents for wild, non-model species are limited or often non-existent for many species.In this study, we compare the reactivity of a new anti-passerine IgY secondary antibody with existing secondary antibodies developed for use with birds. Samples from 41 species from the following six avian orders were analysed: Anseriformes (1 family, 1 species), Columbiformes (1 family, 2 species), Galliformes (1 family, 1 species), Passeriformes (16 families, 34 species), Piciformes (1 family, 2 species) and Suliformes (1 family, 1 species). Direct ELISAs were performed to detect total IgY using goat anti-passerine IgY, goat anti-chicken IgY or goat anti-bird IgY secondary antibodies.The anti-passerine antibody exhibited significantly higher IgY reactivity compared to the anti-chicken and/or anti-bird antibodies in 80% of the passerine families tested. Birds in the order Piciformes (woodpeckers) and order Suliformes (cormorants) were poorly detected by all three secondary antibodies. A comparison of serum and plasma IgY levels was made within the same individuals for two passerine species (house finch and white-crowned sparrow), and serum exhibited significantly more IgY than the plasma for all three secondary antibodies. This result indicates that serum may be preferred to plasma when measuring total antibody levels in blood.This study indicates that the anti-passerine IgY secondary antibody can effectively be used in immunological assays to detect passerine IgY for species in most passerine families and is preferred over anti-chicken and anti-bird secondary antibodies for the majority of passerine species. This anti-passerine antibody will allow for more accurate detection and quantification of IgY in more wild bird species than was possible with previously available secondary antibodies.

Methods for quantifying gene expression in ecoimmunology: from qPCR to RNA-Seq.

Historically, the use of cutting-edge molecular techniques to study immunological gene expression and related cellular pathways has been largely limited to model organisms. Few studies have been performed that quantify the molecular immunological responses of non-model species, especially in response to environmental factors, life-history events, or exposure to parasites. This dearth of information has largely occurred due to the lack of available non-model species-specific gene sequences and immunological reagents and also due to prohibitively expensive technology. However, with the rapid development of various sequencing and transcriptomic technologies, profiling the gene expression of non-model organisms has become possible. Technologies and concepts explored here include an overview of current technologies for quantifying gene expression, including: qPCR, multiplex branched DNA assays, microarrays, and profiling gene expression (RNA sequencing [RNA-Seq]) based on next-generation sequencing. Examples of the advancement of these technologies in non-model systems are discussed. Additionally, applications, limitations, and feasibility of the use of these methodologies in non-model systems to address questions in ecological immunology and disease-ecology are specifically addressed.

Buggy Creek virus (Togaviridae: Alphavirus) upregulates expression of pattern recognition receptors and interferons in House Sparrows (Passer domesticus).

Birds serve as reservoirs for at least 10 arthropod-borne viruses, yet specific immune responses of birds to arboviral infections are relatively unknown. Here, adult House Sparrows were inoculated with an arboviral alphavirus, Buggy Creek virus (BCRV), or saline, and euthanized between 1 and 3 days postinoculation. Virological dynamics and gene expression dynamics were investigated. Birds did not develop viremia postinoculation, but cytopathic virus was found in the skeletal muscle and spleen of birds 1 and 3 days postinoculation (DPI). Viral RNA was detected in the blood of BCRV-infected birds 1 and 2 DPI, in oral swabs 1-3 DPI, and in brain, heart, skeletal muscle, and spleen 1-3 DPI. Multiple genes were significantly upregulated following BCRV infection, including pattern recognition receptors (TLR7, TLR15, RIG-1), type I interferon (IFN-α), and type II interferon (IFN-γ). This is the first study to report avian immunological gene expression profiles following an arboviral infection.

Immune responses of a native and an invasive bird to Buggy Creek Virus (Togaviridae: Alphavirus) and its arthropod vector, the swallow bug (Oeciacus vicarius).

Invasive species often display different patterns of parasite burden and virulence compared to their native counterparts. These differences may be the result of variability in host-parasite co-evolutionary relationships, the occurrence of novel host-parasite encounters, or possibly innate differences in physiological responses to infection between invasive and native hosts. Here we examine the adaptive, humoral immune responses of a resistant, native bird and a susceptible, invasive bird to an arbovirus (Buggy Creek virus; Togaviridae: Alphavirus) and its ectoparasitic arthropod vector (the swallow bug; Oeciacus vicarius). Swallow bugs parasitize the native, colonially nesting cliff swallow (Petrochelidon pyrrhonota) and the introduced house sparrow (Passer domesticus) that occupies nests in cliff swallow colonies. We measured levels of BCRV-specific and swallow bug-specific IgY levels before nesting (prior to swallow bug exposure) and after nesting (after swallow bug exposure) in house sparrows and cliff swallows in western Nebraska. Levels of BCRV-specific IgY increased significantly following nesting in the house sparrow but not in the cliff swallow. Additionally, house sparrows displayed consistently higher levels of swallow bug-specific antibodies both before and after nesting compared to cliff swallows. The higher levels of BCRV and swallow bug specific antibodies detected in house sparrows may be reflective of significant differences in both antiviral and anti-ectoparasite immune responses that exist between these two avian species. To our knowledge, this is the first study to compare the macro- and microparasite-specific immune responses of an invasive and a native avian host exposed to the same parasites.