Evaluation of a low-cost staining method for improved visualization of sweet potato whitefly (Bemisia tabaci) eggs on multiple crop plant species.

BACKGROUND: The sweet potato whitefly (Bemisia tabaci) is a globally important insect pest that damages crops through direct feeding and by transmitting viruses. Current B. tabaci management revolves around the use of insecticides, which are economically and environmentally costly. Host plant resistance is a sustainable option to reduce the impact of whiteflies, but progress in deploying resistance in crops has been slow. A major obstacle is the high cost and low throughput of screening plants for B. tabaci resistance. Oviposition rate is a popular metric for host plant resistance to B. tabaci because it does not require tracking insect development through the entire life cycle, but accurate quantification is still limited by difficulties in observing B. tabaci eggs, which are microscopic and translucent. The goal of our study was to improve quantification of B. tabaci eggs on several important crop species: cassava, cowpea, melon, sweet potato and tomato. RESULTS: We tested a selective staining process originally developed for leafhopper eggs: submerging the leaves in McBryde's stain (acetic acid, ethanol, 0.2% aqueous acid Fuchsin, water; 20:19:2:1) for three days, followed by clearing under heat and pressure for 15 min in clearing solution (LGW; lactic acid, glycerol, water; 17:20:23). With a less experienced individual counting the eggs, B. tabaci egg counts increased after staining across all five crops. With a more experienced counter, egg counts increased after staining on melons, tomatoes, and cowpeas. For all five crops, there was significantly greater agreement on egg counts across the two counting individuals after the staining process. The staining method worked particularly well on melon, where egg counts universally increased after staining for both counting individuals. CONCLUSIONS: Selective staining aids visualization of B. tabaci eggs across multiple crop plants, particularly species where leaf morphological features obscure eggs, such as melons and tomatoes. This method is broadly applicable to research questions requiring accurate quantification of B. tabaci eggs, including phenotyping for B. tabaci resistance.

A contemporary survey of bumble bee diversity across the state of California.

Bumble bees (genus Bombus) are important pollinators with more than 260 species found worldwide, many of which are in decline. Twenty-five species occur in California with the highest species abundance and diversity found in coastal, northern, and montane regions. No recent studies have examined California bumble bee diversity across large spatial scales nor explored contemporary community composition patterns across the state. To fill these gaps, we collected 1740 bumble bee individuals, representing 17 species from 17 sites (~100 bees per site) in California, using an assemblage monitoring framework. This framework is intended to provide an accurate estimate of relative abundance of more common species without negatively impacting populations through overcollection. Our sites were distributed across six ecoregions, with an emphasis on those that historically hosted high bumble bee diversity. We compared bumble bee composition among these sites to provide a snapshot of California bumble bee biodiversity in a single year. Overall, the assemblage monitoring framework that we employed successfully captured estimated relative abundance of species for most sites, but not all. This shortcoming suggests that bumble bee biodiversity monitoring in California might require multiple monitoring approaches, including greater depth of sampling in some regions, given the variable patterns in bumble bee abundance and richness throughout the state. Our study sheds light on the current status of bumble bee diversity in California, identifies some areas where greater sampling effort and conservation action should be focused in the future, and performs the first assessment of an assembly monitoring framework for bumble bee communities in the state.

Flexibility in the Critical Period of Nutrient Sequestration in Bumble Bee Queens.

SYNOPSIS: Bumble bee queens undergo a nutrient storage period prior to entering diapause wherein they sequester glycogen and lipids that are metabolized during overwintering. In the laboratory under optimal food availability conditions, the majority of nutrients are sequestered during the first few days of adulthood. However, if food resources are scarce during this narrow window of time, wild queen bumble bees might be limited in their ability to obtain adequate food resources for overwintering. Here we used a laboratory experiment to examine whether queen bumble bees exhibit flexibility in the timing of pre-overwintering nutrient sequestration, by limiting their access to either nectar (artificial) or pollen, the two primary foods for bumble bees, for varying periods of time. In response to these treatments, we quantified queen survival, changes in weight, and glycogen and lipids levels. We found evidence that queens are able to recuperate almost entirely from food resource limitation, with respect to nutrient storage, especially when it is experienced for shorter durations (up to 6 days). This study sheds light on how bumble bee queens are impacted by food resource availability at a critical life stage. PORTUGUESE: As abelhas rainhas do gênero Bombus armazenam nutrientes antes de entrarem em diapausa, sequestrando o glicogênio e os lipídios que serão metabolizados durante o inverno. Em condições ideais de disponibilidade de alimento no laboratório, a maioria dos nutrientes é sequestrada nos primeiros dias de vida adulta. No entanto, em condições de escassez de alimento na natureza, as rainhas podem sofrer limitações em sua capacidade de obter recursos para o inverno. Nesse contexto, em condições controladas, examinamos se as rainhas exibem variações no sequestro de nutrientes, limitando o acesso ao néctar (artificial) ou pólen, seus principais alimentos, em diferentes intervalos de tempo. Em resposta a esses tratamentos, quantificamos a taxa de sobrevivência das rainhas, as mudanças no peso e os níveis de glicogênio e lipídios. Encontramos evidências de que as rainhas são capazes de recuperar a capacidade de armazenar nutrientes quase inteiramente, especialmente em períodos mais curtos de escassez de alimento (até 6 dias). Este estudo lança luz sobre como as rainhas são afetadas pela variação na disponibilidade de recursos alimentares em um estágio crítico da vida. SPANISH: Las abejas reinas de generó Bombus, mejor conocidas como reinas de abejorro se someten a un período de almacenamiento de nutrientes antes de entrar en diapausa, en el cual secuestran glucógeno y lípidos que se metabolizan durante el invierno. En el laboratorio, en condiciones óptimas de disponibilidad de alimentos, la mayoría de los nutrientes se secuestran durante los primeros días de la edad adulta. Sin embargo, si los recursos alimenticios son escasos durante esta estrecha ventana de tiempo, las abejas reinas silvestres podrían verse limitadas en su capacidad para obtener recursos alimenticios adecuados para pasar el invierno. Aquí utilizamos un experimento de laboratorio para examinar si las abejas reinas exhiben flexibilidad en el momento del secuestro de nutrientes antes de la hibernación, al limitar su acceso al néctar (artificial) o al polen, los dos alimentos principales de los abejorros, durante períodos variables. En respuesta a estos tratamientos, cuantificamos la supervivencia de la reina, los cambios de peso y los niveles de glucógeno y lípidos. Encontramos evidencia de que las reinas pueden recuperarse casi por completo de la limitación de los recursos alimenticios, con respecto al almacenamiento de nutrientes, especialmente cuando se experimenta por períodos más cortos (hasta 6 días). Este estudio arroja luz sobre cómo las abejas reinas se ven afectadas por la disponibilidad de recursos alimenticios en una etapa crítica de la vida.

Transcriptome analysis reveals nutrition- and age-related patterns of gene expression in the fat body of pre-overwintering bumble bee queens.

Many diapausing insects undergo a nutrient storage period prior to their entry into diapause. Bumble bee queens diapause as adults in the winter preceding their spring nest initiation period. Before diapause, they sequester glycogen and lipids, which they metabolize during the overwintering period. We used RNA sequencing to examine how age and nectar diet (specifically, the concentration of sucrose in nectar) impact gene expression in the pre-overwintering bumble bee queen fat body, the "liver-like" organ in insects with broad functions related to nutrient storage and metabolism. We found that diet on its own, and in combination with age, impacts the expression of genes involved in detoxification. Age was also a strong driver of gene expression, especially at earlier ages (up to 3 days). In addition to these molecular correlates of diet and age, we also found a putative molecular signature of diapause entry or preparation in adult queens in the oldest age group (12 days) fed the most sucrose-rich diet, based on comparisons between our data set and another transcriptome data set from bumble bee queens. This transcriptomic pattern suggests that preparation for (or entry into) diapause might be in part mediated by nutritional state in bumble bee queens. Collectively, these findings show that there are molecular processes in the fat body that are responsive to sucrose levels in the diet and/or associated with age-related maturational changes. A better understanding of these processes may shed light on important aspects of bumble bee biology, such as queen responses to nutritional and other forms of stress, and the factors that regulate their entrance into diapause.

Pollen Diet Composition Impacts Early Nesting Success in Queen Bumble Bees Bombus impatiens Cresson(Hymenoptera: Apidae).

Bumble bees are generalist pollinators that typically collect floral rewards from a wide array of flowering plant species. Among the greatest threats to wild bumble bee populations worldwide, many of which are declining, is a loss of floral resource abundance and diversity in the landscapes they inhabit. We examined how composition of pollen diet impacts early nesting success in laboratory-reared queens of the bumble bee Bombus impatiens. Specifically, we provided queens and their young nests with one of three pollen diets, each of which was dominated by a single pollen type, and explored how this diet treatment influenced the length of time until queens initiated nests, total counts of brood in the nest at the end of the experiment (8 wk later), and the size and weight of adult offspring produced. We found that the amount of later-stage brood (pupae and/or adults) produced by recently-initiated nests was strongly impacted by pollen diet. For example, on average 66% fewer later-stage brood were found in nests provided with the Cistus pollen Linnaeus (Cistaceae), relative to the predominantly Asteraceae pollen. This finding suggests that particular pollen diet compositions may delay larval growth, which delays colony development and may ultimately be detrimental for young nests. This study sheds light on how one of the leading stressors for bumble bees (nutritional stress) may negatively impact populations through its influence on brood production during the nest-founding stage of the colony cycle.

Documenting Potential Sunn Hemp (Crotalaria juncea L.) (Fabaceae) Pollinators in Florida.

Sunn hemp, Crotalaria juncea L., is a warm-season legume that can be planted in rotation to cash crops to add nitrogen and organic matter to the soils, for weed growth prevention, and to suppress nematode populations. Sunn hemp flowers also provide nectar and pollen for pollinators and enhance biological control by furnishing habitat for natural enemies. Experiments were conducted in Northern and North Central Florida to evaluate bee populations that visited flowers within mixed plots of sunn hemp and sorghum-sudangrass and plots of two sunn hemp germplasm lines. Collections of bees that visited 'AU Golden' and Tillage Sunn flowers indicated that Xylocopa virginica (L.) (Hymenoptera: Apidae), Xylocopa micans Lepeletier (Hymenoptera: Apidae), Megachile sculpturalis Smith (Hymenoptera: Megachilidae), Megachile mendica (Cresson) (Hymenoptera: Megachilidae), and Megachile georgica Cresson (Hymenoptera: Megachilidae) were present in large numbers in May through July and then again in October. Although Tillage Sunn seeds planted in March flowered in May, percent bloom and number of bee visits were low. Compared with short day sunn hemp cultivars, 'AU Golden' plants produced flowers early in the season to provide food and habitat for pollinators and have the potential to produce an abundant seed crop in Northern and North Central Florida.

Diet and nutritional status during early adult life have immediate and persistent effects on queen bumble bees.

Many insects sequester nutrients during developmentally programmed periods, which they metabolize during subsequent life history stages. During these periods, failure to store adequate nutrients can have persistent effects on fitness. Here, we examined a critical but under-studied nutrient storage period in queen bumble bees: the first days of adult life, which are followed by a diapause period typically coinciding with winter. We experimentally manipulated availability of pollen (the primary dietary source of lipids and protein) and the sugar concentration of artificial nectar (the primary source of carbohydrates) for laboratory-reared queens during this period and examined three nutritional phenomena: (i) diet impacts on nutritional status, (ii) the timescale upon which nutrient sequestration occurs and (iii) the fitness consequences of nutrient sequestration, specifically related to survival across the life cycle. We found evidence that pollen and nectar starvation negatively impact lipid storage, whereas nectar sugar concentration impacts stored carbohydrates. The majority of nutrients were stored during the first ~ 3 days of adult life. Nutrients derived from pollen during this period appear to be more critical for surviving earlier life stages, whereas nutrients sequestered from nectar become more important for surviving the diapause and post-diapause periods. Negative impacts of a poor diet during early life persisted in our experiment, even when pollen and a relatively high (50%) nectar sugar concentration were provided post-diapause. Based on these findings, we posit that the nutritional environment during the early adult life of queens has both immediate and persistent impacts on fitness. These findings underscore the importance of examining effects of stage-specific nutritional limitations on physiology and life history traits in this social insect group. Moreover, the findings may shed light on how declining food resources are contributing to the decline of wild bumble bee populations.

Effects of neonicotinoid insecticide exposure and monofloral diet on nest-founding bumblebee queens.

Bumblebees are among the world's most important groups of pollinating insects in natural and agricultural ecosystems. Each spring, queen bumblebees emerge from overwintering and initiate new nests, which ultimately give rise to workers and new reproductives later in the season. Nest initiation and survival are thus key drivers of both bumblebee pollination services and population dynamics. We performed the first laboratory experiment with the model bumblebee species Bombus impatiens that explores how early nesting success is impacted by the effects of temporary or more sustained exposure to sublethal levels of a neonicotinoid-type insecticide (imidacloprid at 5 ppb in nectar) and by reliance on a monofloral pollen diet, two factors that have been previously implicated in bumblebee decline. We found that queens exhibited increased mortality and dramatically reduced activity levels when exposed to imidacloprid, as well as delayed nest initiation and lower brood numbers in the nest, but partially recovered from these effects when they only received early, temporary exposure. The effects of pollen diet on individual queen- and colony-level responses were overshadowed by effects of the insecticide, although a monofloral pollen diet alone was sufficient to negatively impact brood production. These findings speak to the sensitivity of queen bumblebees during the nest initiation phase of the colony cycle, with implications for how queens and their young nests are uniquely impacted by exposure to threats such as pesticide exposure and foraging habitat unsuitability.

Landscape Factors Influencing Stink Bug Injury in Mid-Atlantic Tomato Fields.

Landscape structure and diversity influence insect species abundance. In agricultural systems, adjacent crop and non-crop habitats can influence pest species population dynamics and intensify economic damage. To investigate the influence of landscape factors on stink bug damage in agricultural systems, we assessed stink bug damage from 30 processing tomato fields in the mid-Atlantic United States and analyzed landscape structure and geographic location. We found that forest shape and size, and geographic location strongly influenced stink bug damage. Landscapes with larger forest edge in southern portions of the mid-Atlantic region experienced the greatest damage, perhaps owing to the introduction of the invasive brown marmorated stink bug. We conclude that landscape structure will likely influence damage rates in nearby agricultural fields.