Walter S. Sheppard

Sub-Lethal Effects of Pesticide Residues in Brood Comb on Worker Honey Bee (Apis mellifera) Development and Longevity

Background Numerous surveys reveal high levels of pesticide residue contamination in honey bee comb. We conducted studies to examine possible direct and indirect effects of pesticide exposure from contaminated brood comb on developing worker bees and adult worker lifespan. Methodology/Principal Findings Worker bees were reared in brood comb containing high levels of known pesticide residues (treatment) or in relatively uncontaminated brood comb (control). Delayed development was observed in bees reared in treatment combs containing high levels of pesticides particularly in the early stages (day 4 and 8) of worker bee development. Adult longevity was reduced by 4 days in bees exposed to pesticide residues in contaminated brood comb during development. Pesticide residue migration from comb containing high pesticide residues caused contamination of control comb after multiple brood cycles and provided insight on how quickly residues move through wax. Higher brood mortality and delayed adult emergence occurred after multiple brood cycles in contaminated control combs. In contrast, survivability increased in bees reared in treatment comb after multiple brood cycles when pesticide residues had been reduced in treatment combs due to residue migration into uncontaminated control combs, supporting comb replacement efforts. Chemical analysis after the experiment confirmed the migration of pesticide residues from treatment combs into previously uncontaminated control comb. Conclusions/Significance This study is the first to demonstrate sub-lethal effects on worker honey bees from pesticide residue exposure from contaminated brood comb. Sub-lethal effects, including delayed larval development and adult emergence or shortened adult longevity, can have indirect effects on the colony such as premature shifts in hive roles and foraging activity. In addition, longer development time for bees may provide a reproductive advantage for parasitic Varroa destructor mites. The impact of delayed development in bees on Varroa mite fecundity should be examined further.

Honey bees (Apis mellifera) reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema (Microsporidia) infection

Nosema ceranae and pesticide exposure can contribute to honey bee health decline. Bees reared from brood comb containing high or low levels of pesticide residues were placed in two common colony environments. One colony was inoculated weekly with N. ceranae spores in sugar syrup and the other colony received sugar syrup only. Worker honey bees were sampled weekly from the treatment and control colonies and analyzed for Nosema spore levels. Regardless of the colony environment (spores+syrup added or syrup only added), a higher proportion of bees reared from the high pesticide residue brood comb became infected with N. ceranae, and at a younger age, compared to those reared in low residue brood combs. These data suggest that developmental exposure to pesticides in brood comb increases the susceptibility of bees to N. ceranae infection.

The Mediterranean fruit fly in California: evidence for multiple introductions and persistent populations based on microsatellite and mitochondrial DNA variability

Microsatellite and mitochondrial DNA (mtDNA) variability data were used to study outbreaks of Mediterranean fruit fly in California in the years 1992–94 and 1997–99. A total of 359 flies caught in monitoring traps during these years were examined at three polymorphic mtDNA restriction sites and two microsatellite loci. Composite genotypes obtained through analysis of these markers indicate at least five independent introductions of medflies into California between 1992 and 1998. Whereas the majority of specimens displayed a single mtDNA haplotype (AAA), variation of microsatellite alleles among these flies suggests at least one additional introduction in 1993 into southern California. Flies displaying the AAB haplotype sampled in 1992 both in northern and southern California shared microsatellite alleles absent in AAA flies although lacking others commonly found in AAA specimens, thus supporting the hypothesis of an independent introduction of these flies from a different source. In contrast to earlier infestations, a few specimens caught in southern California in 1993 and again in 1998 showed both mtDNA and microsatellite patterns consistent with a Hawaiian origin. Single flies collected in Santa Clara County in 1997 and in El Monte, Los Angeles County & in 1999 most likely represent a sixth and seventh distinct introduction, respectively.

Gene flow within the M evolutionary lineage of Apis mellifera: role of the Pyrenees, isolation by distance and post-glacial re-colonization routes in the western Europe

We present a population genetic study focused on the two subspecies of the M evolutionary lineage, A. m. mellifera and A. m. iberiensis. Nuclear and mtDNA variation was analysed in 27 bee populations from the Iberian Peninsula, France and Belgium. Microsatellite data provides compelling evidence of a barrier to neutral gene flow at the Pyrenees. In addition, they suggest isolation by distance between populations of the M lineage. Mitochondrial data support the hypothesis that the Iberian Peninsula served as glacial refugia for the honeybees of western Europe. They show two paths of post-glacial re-colonization in the extremes of the Pyrenees and suggest that the western path was more significant in the post-glacial re-colonization process. Thus, we report here on three main factors for mellifera and iberiensis subspecies differentiation: the Pyrenean barrier, isolation by distance and the post-glacial re-colonization process.ZusammenfassungDie Unterarten der Honigbiene (Apis mellifera) werden in die 5 evolutionäre Linien A (African), C (northern Mediterranean), M (western Europe), O (Oriental) and Y (Yemenitica) gruppiert. Die in dieser Studie untersuchte evolutionäre Linie M enthält zwei Unterarten: A. m. mellifera wird von Frankreich bis zu den Bergen des Ural gefunden und A. m. iberiensis ist auf der iberischen Halbinsel verbreitet. Obwohl allgemein angenommen wird, dass die Pyrenäen ein bedeutendes Hindernis für den Genfluss zwischen A. m. mellifera und A. m. iberiensis darstellt, wurde ein solcher Barriereeffekt bislang nicht nachgewiesen. Die Existenz genetischer Gefälle vom Süden der Iberischen Halbinsel bis zum nördlichen Europa und eine ungewisse taxonomische Zuordnung einiger Populationen in den Pyrenäen tragen zu der Unsicherheit bezüglich der Rolle der Pyrenäen als genetische Barriere bei. Andererseits wird seit längerem angenommen, dass die Iberische Halbinsel während der Eiszeit als Refugium für die westliche Honigbiene diente (Ruttner, 1952, 1988) und eine nacheiszeitliche Wiederbesiedlung von Nordeuropa wird von mehreren Autoren unterstützt (Garnery et al., 1998a,b; Franck et al., 1998, 2000b). Gegenstand dieser Untersuchung war es, einen potentiell isolierenden Effekt der Pyrenäen nachzuweisen, neue Daten zu dem Differenzierungsprozess der zwei Unterarten beizutragen und den Ablauf der Wiederbesiedlung zu untersuchen. Wir untersuchten 1398 Völker aus 27 Populationen der Iberischen Halbinsel sowie aus Frankreich und Belgien auf Variation an 10 Mikrosatellitenloci und der COI-COII intergenischen Region der mtDNA. Wir verwendeten verschiedene Arten statistischer Analysen wie die DA genetische Distanzmatrix, neighbor-joining trees, Korrelationen erster Ordnung und partielle Korrelationen, AMOVA, Analyse räumlicher Autokorrelationen und COCOPAN für Mikrosatellitendaten. Die Ergebnisse zeigten eine Isolation zwischen den verschiedenen Populationen der M Linie durch die Entfernung auf und lieferten sehr deutliche Hinweise auf eine Barriere für den neutralen Genfluß bei den Pyrenäen. Die Verteilung der mtDNA Haplotypen bestätigte das Iberische Refugium der Westeuropäischen Honigbiene in der Eiszeit. Wir konnten zwei verschiedene Wege der nacheiszeitlichen Wiederbesiedlung von der Iberischen Halbinsel aus an den beiden Enden der Pyrenäen ableiten. Es gab deutliche Unterschiede in der Verteilung der Mitotypen zwischen den westlichen und östlichen Enden der Pyrenäen, diese legten nahe, dass der westliche Weg für den nacheiszeitlichen Widerbesiedlungsprozess wichtiger war. Nach der in dieser Untersuchung beobachteten hohen Variabilität der M Mitotypen südlich der Pyrenäen könnten diese eine nützliche genetische Ressource für die Konservation der Westeuropäischen Honigbienen darstellen. Der nacheiszeitliche Wiederbesiedlungsverlauf, die Isolation durch die Entfernung und die von den Pyrenäen gebildete Verbreitungsbarriere sind Einflüsse, die zu der Ausbildung der Unterarten A. m. mellifera und A. m. iberiensis beigetragen haben.

Identification of African-Derived Bees in the Americas: A Survey of Methods

Abstract In 1956, honey bees of the African subspecies A. m. scutellata were imported to Brazil, and their descendents subsequently spread to South, Central, and North America. This invasion sparked significant academic controversy, particularly concerning the genetic composition of the expanding population. We review the biogeography and intraspecific phylogeny of Apis mellifera in the Old World as it pertains to African-derived bees in the Americas, the methods used to study gene flow between European-derived and African-derived populations in the New World, and the techniques used in identification of African-derived bees.

Analysis of mitochondrial DNA and development of PCR-based diagnostic molecular markers for Mediterranean fruit fly (Ceratitis capitata) populations.

A 2.99 kb mtDNA fragment containing two variable restriction endonuclease sites (EcoRV and Xbal) was subcloned and sequenced from the Mediterranean fruit fly (Ceratitis capitata). This fragment represents approximately one‐fifth of the entire mitochondrial sequence. The sequence was aligned with the comparable region from Drosophila yakuba and Anopheles gambiae, resulting in 81.8% and 76.7% identity at the nucleotide level, and 77% and 67.7% identity, respectively, at the amino acid level. The sequenced region includes the complete genes for NADH dehydrogenase 4, NADH dehydrogenase 4L, NADH dehydrogenase 6, and transfer RNAs for proline, threonine and histidine, and part of the genes for NADH dehydrogenase 5 and cytochrome b. Oligonucleotide primers were designed to asymmetrically bracket each of two variable restriction endonuclease sites to allow PCR amplification and subsequent restriction endonuclease analysis of individual fly samples.

Mitochondrial DNA variation in honey bee (Apis mellifera L.) populations from Turkey

Summary We have studied mitochondrial (mt) DNA variation in 334 honey bee colonies from 7 different geographic regions of Turkey. We have evaluated Dra I restriction profiles of the CO-I CO-II intergenic region, Hinf-I and Taq-I restriction profiles of the CO-I gene and EcoR-I restriction profiles of the whole mtDNA. We obtained three different mtDNA patterns by EcoR-I digestion. The pattern typical for A. m. carnica/A. m. ligustica predominated throughout Turkey (327 colonies, 97.9%). We observed the pattern common in African subspecies only in Hatay province (6 colonies, 1.8%) and a previously unreported pattern in one colony from Balıkesir province (0.3%). Dra-I restriction analysis of the CO-I CO-II intergenic region yielded seven haplotypes. Haplotype 1 (TrDra-1) was the most common one found in Turkey, whereas haplotype 2 (TrDra-2) was widely distributed in Eastern Anatolia. Based on mitochondrial ND2 sequences taken from two samples collected in each region, bees from Hatay clustered with A. m. lamarckii and A. m. meda (morphological A and O lineages), while bees from central Anatolia clustered within the C morphological lineage group.

Optimum Timing of Miticide Applications for Control of Varroa destructor (Acari: Varroidae) in Apis mellifera (Hymenoptera: Apidae) in Washington State, USA

Abstract Seven treatments for the control of Varroa destructor (Anderson & Trueman) were tested to determine the optimum timing of miticide application. Threshold mite levels indicating miticide application were determined for three possible treatment dates: April, August, and October. The treatments were as follows: (1) fluvalinate in April, (2) fluvalinate in August, (3) fluvalinate in October, (4) fluvalinate in April and October, (5) fluvalinate applied continuously (except during honey flow) with replacement every 42 d, (6) control (no treatment), and (7) coumaphos in April. The number of miticide applications in a season had no effect on brood area or colony bee population a year after initiating the experiment. However, the absence of any treatment significantly reduced brood area and colony bee population and significantly increased colony mite population. Date of treatment had significant effects on colony mortality rates, mite levels, and brood area the following spring. When coupled with sampling and threshold recommendations, a single, late-season application of fluvalinate is as effective for the control of V. destructor as semiannual or continuous miticide applications. Treatment thresholds were recommended for ether roll and 48-h sticky board sampling methods in April (three and 24 mites, respectively) and August (14 and 46 mites, respectively) and for ether rolls in October (three mites) in cold climates.

Nosema ceranae in age cohorts of the western honey bee (Apis mellifera)

Nosemaceranae intensity (mean spores per bee) and prevalence (proportion of bees infected in a sample) were analyzed in honey bees of known ages. Sealed brood combs from five colonies were removed, emerging bees were marked with paint, released back into their colonies of origin, and collected as recently emerged (0-3 days old), as house bees (8-11 days old), and as foragers (22-25 days old). Fifty bees from each of the five colonies were processed individually at each collection date for the intensity and prevalence of N. ceranae infection. Using PCR and specific primers to differentiate Nosema species, N. ceranae was found to be the only species present during the experiment. At each collection age (recent emergence, house, forager) an additional sample from the inner hive cover (background bees=BG) of each colony was collected to compare the N. ceranae results of this sampling method, commonly used for Nosema spore quantification, to the samples comprised of marked bees of known ages. No recently emerged bees exhibited infection with N. ceranae. One house bee out of the 250 individuals analyzed (prevalence=0.4%) tested positive for N. ceranae, at an infection level of 3.35×10(6) spores. Infection levels were not statistically different between the recently emerged (mean=0 spores/bee) and house bees (mean=1.34×10(4) spores/bee) (P=0.99). Foragers exhibited the highest prevalence (8.3%) and infection intensity (mean=2.38×10(6) spores/bee), with a range of 0-8.72×10(7) spores in individual bees. The average infection level across all foragers was significantly higher than that of recently emerged bees (P=0.01) and house bees (P=0.01). Finally, the prevalence of Nosema in infected bees was found to be positively correlated with the infection intensity in the sample.

Genetic characterization of commercial honey bee (Hymenoptera: Apidae) populations in the United States by using mitochondrial and microsatellite markers

ABSTRACT Genetic diversity levels within and between the two commercial breeding areas in the United States were analyzed using the DraI restriction fragment length polymorphism of the COI-COII mitochondrial region and 10 polymorphic microsatellite loci. The western commercial breeding population (WCBP) and the southeastern commercial breeding population (SCBP) were sampled in 1993–1994 and again in 2004–2005. The goal of this study was to characterize the genetic composition of these populations and to measure potential changes in genetic diversity and composition across the sampling period. The mitochondrial DNA haplotypes C1 and C2, characteristic of the most popular bee strains (Italians and Carniolans, respectively) sold in the United States, were the dominant haplotypes at both sample dates. The frequency of Apis mellifera mellifera M haplotypes, M4, M7, and M7′, decreased during the 10-yr span. An A1 haplotype characteristic of Africanized bees was found in the SCBP from 2005. Microsatellite analysis showed there was a loss of alleles in both the WCBP and SCBP, but the losses were not significant due to simultaneous gains of new alleles into these populations between 1993 and 2005. Genetic differences that occurred between the 1993–1994 WCBP and SCBP were still detectable in these populations sampled a decade later, suggesting that these populations could be useful sources of diversity for each other in the future.