Bombus Affinis Research Articles

Range-wide genetic analysis of an endangered bumble bee (Bombus affinis, Hymenoptera: Apidae) reveals population structure, isolation by distance, and low colony abundance.

Declines in bumble bee species range and abundances are documented across multiple continents and have prompted the need for research to aid species recovery and conservation. The rusty patched bumble bee (Bombus affinis) is the first federally listed bumble bee species in North America. We conducted a range-wide population genetics study of B. affinis from across all extant conservation units to inform conservation efforts. To understand the species' vulnerability and help establish recovery targets, we examined population structure, patterns of genetic diversity, and population differentiation. Additionally, we conducted a site-level analysis of colony abundance to inform prioritizing areas for conservation, translocation, and other recovery actions. We find substantial evidence of population structuring along an east-to-west gradient. Putative populations show evidence of isolation by distance, high inbreeding coefficients, and a range-wide male diploidy rate of ~15%. Our results suggest the Appalachians represent a genetically distinct cluster with high levels of private alleles and substantial differentiation from the rest of the extant range. Site-level analyses suggest low colony abundance estimates for B. affinis compared to similar datasets of stable, co-occurring species. These results lend genetic support to trends from observational studies, suggesting that B. affinis has undergone a recent decline and exhibit substantial spatial structure. The low colony abundances observed here suggest caution in overinterpreting the stability of populations even where B. affinis is reliably detected interannually. These results help delineate informed management units, provide context for the potential risks of translocation programs, and help set clear recovery targets for this and other threatened bumble bee species.

Occupancy of Bombus affinis (Hymenoptera: Apidae) in Minnesota is highest in developed areas when standardized surveys are employed.

Mounting evidence of bumble bee declines and the listing of the rusty patched bumble bee (Bombus affinis Cresson) as federally endangered in the United States in 2017 and Canada in 2012 has stimulated an interest in monitoring and conservation. Understanding the influence of land use on occupancy patterns of imperiled species is crucial to successful recovery planning. Using detection data from community surveys, we assessed land use associations for 7 bumble bee species in Minnesota, USA, including B. affinis. We used multispecies occupancy models to assess the effect of 3 major land use types (developed, agricultural, and natural) within 0.5 and 1.5 km on occupancy of 7 Bombus (Hymenoptera: Apidae) species, while accounting for detection uncertainty. We found that B. affinis occupancy and detection were highest in developed landscapes and lowest in agricultural landscapes, representing an inverse relationship with the relative landcover ratios of these landscapes in Minnesota. Occupancy of 2 bumble bee species had strong positive associations with natural landscapes within 1.5 km and 2 species had strong negative associations with agricultural landscapes within 1.5 km. Our results suggest that best practices for imperiled Bombus monitoring and recovery planning depends upon the surrounding major land use patterns. We provide recommendations for urban planning to support B. affinis based on conservation success in the metropolitan areas of Minneapolis-St. Paul. We also encourage substantial survey effort be employed in agricultural and natural regions of the state historically occupied by B. affinis to determine the current occupancy state.

Chromosome-scale genome assembly of the rusty patched bumble bee, Bombus affinis (Cresson) (Hymenoptera: Apidae), an endangered North American pollinator.

The rusty patched bumble bee, Bombus affinis, is an important pollinator in North America and a federally listed endangered species. Due to habitat loss and large declines in population size, B. affinis is facing imminent extinction unless human intervention and recovery efforts are implemented. To better understand B. affinis biology and population genetic and genomic landscapes, we sequenced and assembled the B. affinis genome from a single haploid male. Whole genome HiFi sequencing on PacBio coupled with HiC sequencing resulted in a complete and highly contiguous contig assembly that was scaffolded into a chromosomal context, resolving 18 chromosomes distributed across the 365.1 Mb assembly. All material for both HiFi and HiC sequencing was derived from a single abdominal tissue segment from the single male. These assembly results, coupled with the minimal amount of tissue destructively sampled, demonstrate methods for generating contiguous and complete genomic resources for a rare and endangered species with limited material available and highlight the importance of sample preservation. Precise methods and applications of these methods are presented for potential applications in other species with similar limitations in specimen availability and curation considerations.

Increasing sampling efficiency of Bombus communities with rare and endangered species by optimizing detection probabilities: A multi-species occupancy modelling approach using roadsides as a case study

Nearly one-quarter of bumble bee species assessed by the International Union for the Conservation of Nature are considered to be in decline, necessitating monitoring to support recovery planning. A key element of robust wildlife monitoring is accounting for imperfect detection in the observation process and efforts to understand sources of detection uncertainty in bumble bee surveys began recently in response to the listing of the rusty patched bumble bee, Bombus affinis, as federally endangered in the U.S. and Canada. We investigated within-community variation in detection probabilities among eight species that vary in occupancy, including B. affinis. We sampled bumble bees along roadsides in the greater metropolitan area of Minneapolis-St. Paul, Minnesota in the United States in 2018. We generated estimates of occupancy, detection probability, and cumulative detection probability from a multispecies occupancy model using a Bayesian framework that included covariates on occupancy and detection. Covariates selected a priori included floral cover and impervious surface effects on occupancy, and date, time of day, and observer effects on detection. We found that Bombus species with low occurrence are not necessarily less detectable than common species with higher occurrence, and that species reach their peak detection probability at different dates. Detection was higher in the afternoon and varied among observers. Well-designed Bombus monitoring programs can reduce sampling effort and optimize detection by sampling on dates near species detection peaks, sampling in the afternoon, and using cumulative detection probabilities to determine optimum sampling effort.

Comparison of microscopy and metabarcoding to identify pollen used by the critically endangered rusty patched bumble bee, Bombus affinis

Abstract Taxonomic analysis of pollen collected by bees can provide insights into their host plant use, providing information about the plant species selected for targeted conservation strategies. The two main identification approaches used are morphological analysis of pollen samples affixed to microscope slides (i.e., microscopic palynology) and molecular analysis of samples. Both methods are widely used for freshly collected materials and have been compared in multiple studies, yet their application to archived samples remains to be explored. Archived samples may be particularly useful for the study of rare or protected species, particularly when historical foraging patterns are uncertain. We used both methods to analyse pollen collected by the endangered rusty patched bumble bee species, Bombus affinis Cresson, applied to museum‐archived specimens. Pollen samples were removed from the corbiculae of bumble bees originally collected in Michigan between 1914 and 1974. Samples included 24 rusty patched bumble bees each with large pollen loads on both corbiculae, allowing for pollen from the same bee to be analysed using both methods. DNA metabarcoding detected more plant taxa than light microscopy, and DNA barcoding also had higher taxonomic resolution when compared to taxa determined using light microscopy. In many instances, pollen could only be confidently identified to tribe or family with light microscopy. Discrepancy between methods decreased when taxa identified via DNA metabarcoding were binned into ecologically relevant groups corresponding to those identified using light microscopy. Although binning demonstrated smaller within‐method variance, there was still minimal correspondence between the two methods. Results indicate there are benefits and biases unique to each method and highlight the utility of binning taxonomic results to morphological or ecological groupings.

Addressing Detection Uncertainty in Bombus affinis (Hymenoptera: Apidae) Surveys Can Improve Inferences Made From Monitoring.

The U.S. Fish and Wildlife Service developed national guidelines to track species recovery of the endangered rusty patched bumble bee [Bombus affinis Cresson (Hymenoptera: Apidae)] and to investigate changes in species occupancy across space and time. As with other native bee monitoring efforts, managers have specifically acknowledged the need to address species detection uncertainty and determine the sampling effort required to infer species absence within sites. We used single-season, single-species occupancy models fit to field data collected in four states to estimate imperfect detection of B. affinis and to determine the survey effort required to achieve high confidence of species detection. Our analysis revealed a precipitous, seasonal, decline in B. affinis detection probability throughout the July through September sampling window in 2021. We estimated that six, 30-min surveys conducted in early July are required to achieve a 95% cumulative detection probability, whereas >10 surveys would be required in early August to achieve the same level of confidence. Our analysis also showed B. affinis was less likely to be detected during hot and humid days and at patches of reduced habitat quality. Bombus affinis was frequently observed on Monarda fistulosa (Lamiales: Lamiaceae), followed by [Pycnanthemum virginianum Rob. and Fernald (Lamiales: Lamiaceae)], Eutrochium maculatum Lamont (Asterales: Asteraceae), and Veronicastrum virginicum Farw. (Lamiales: Plantaginaceae). Although our research is focused on B. affinis, it is relevant for monitoring other bumble bees of conservation concern, such as B. occidentalis Greene (Hymenoptera: Apidae) and B. terricola Kirby (Hymenoptera: Apidae) for which monitoring efforts have been recently initiated and occupancy is a variable of conservation interest.

Floral Resources Used by the Endangered Rusty Patched Bumble Bee (Bombus affinis) in the Midwestern United States

The once-common rusty patched bumble bee (RPBB, Bombus affinis) has disappeared from most of its original range in eastern North America. As a result of this dramatic population decline, RPBB was listed as federally endangered in 2017. Unlike many endangered species, remnant populations of the RPBB often occur in urban/suburban parks and natural areas. This paper summarizes photographic records of RPBB floral use collected largely by volunteer community scientists in the midwestern United States, with a focus on Wisconsin, one of the species' remaining strongholds. RPBB were documented in 37 of Wisconsin's 72 counties. We identified flowers to genus in 772 of 803 digital images (some bees were not on flowers). Although 87 plant genera were identified, 76% of the flowers represented just 13 genera. Over half of the flower records (54.6%) were from Monarda, Eutrochium, Veronicastrum, Agastache, or Solidago. Incidental surveys from other states show a similar pattern, although additional genera have been shown to be used by RPBB. Our results support existing recommendations for plantings and habitat management favoring specific plant species, generally associated with native grasslands and savannas. Because the active period of RPBB covers a large part of the growing season, however, we also provide evidence for the importance of spring ephemeral woodland wildflowers (e.g., Dicentra, Anemone, Hydrophyllum) and late-flowering species of wetlands and wet meadows (e.g., Eutrochium, Eupatorium). A landscape that includes woodland, native grassland, and wet meadows provides floral resources that support all RPBB life history stages, including early-season queens, late-season gynes, and males.

Notes from rusty patched bumble bee (Bombus affinis Cresson) nest observations

Abstract The rusty patched bumble bee, Bombus affinis, is federally endangered in the United States and Canada. Information on natural history is crucial to conservation planning, but little is known about B. affinis nesting biology, habitat, overwintering, and mating behaviours. We report observations on three B. affinis nests in Minnesota and Wisconsin found in 2020. Previous to this report, there were only four published descriptions of B. affinis nests, with the last nest recorded nearly 30 years ago. Here, we share findings on nesting habitat, nest size, mating behaviours, and nest parasites. The nests were located in between the foundation and a layer of insulation in an urban residence, in an urban backyard near a house foundation, and in a degraded semi‐wooded, riparian natural area. Colony size estimates ranged from 251 to 1341 bees. We observed males chasing gynes near a nest entrance and one mating event approximately 5 m from the nest. We documented the chalcid wasp, Melittobia acasta, as a new host association. These observations increased the known colony size range, documented a new host association, and indicate the potential importance of urban and degraded habitats for recovery of the endangered rusty patched bumble bee.

Long‐term surveys support declines in early season forest plants used by bumblebees

Abstract Populations of bumblebees and other pollinators have declined over the past several decades due to numerous threats, including habitat loss and degradation. However, we can rarely investigate the role of resource loss due to a lack of detailed long‐term records of forage plants and habitats. We used 22‐year repeated surveys of more than 262 sites located in grassland, forest, and wetland habitats across Illinois, USA to explore how the abundance and richness of bumblebee food plants have changed over the period of decline of the endangered rusty patched bumblebee Bombus affinis. We documented a decline in abundance of bumblebee forage plants in forest understories, which our phenology analysis suggests provide the primary nectar and pollen sources for foundress queens in spring, a critical life stage in bumblebee demography. By contrast, the per‐unit area abundance of food plants in primarily midsummer‐flowering grassland and wetland habitats had not declined. However, the total area of grasslands had declined across the region resulting in a net loss of grassland resources. Synthesis and applications. Our results suggest a decline in spring‐flowering forest understorey plants is a previously unappreciated bumblebee stressor, compounding factors like agricultural intensification, novel pathogen exposure and grassland habitat loss. These findings emphasize the need for greater consideration of habitat complementarity in bumblebee conservation. We conclude that the continued loss of early season floral resources may add additional stress to critical life stages of bumblebees and limit restoration efforts if not explicitly considered in pollinator conservation.

A century of pollen foraging by the endangered rusty patched bumble bee (Bombus affinis): inferences from molecular sequencing of museum specimens

In 2017 the rusty patched bumble bee (Bombus affinis) became the first bee listed under the Endangered Species Act in the continental United States due to population declines and an 87% reduction in the species’ distribution. Bombus affinis decline began in the 1990s, predating modern bee surveying initiatives, and obfuscating drivers of decline. While understood to be a highly generalist forager, little is known about the role that resource limitation or shifting floral community composition could have played in B. affinis decline. Determining which floral species support B. affinis could assist conservation efforts where B. affinis persists and identify floral species for restoration efforts. We constructed a historical foraging profile of B. affinis via DNA sequencing of pollen from museum specimens spanning seven states collected from 1913 to 2013. Molecular analysis revealed no temporal changes in the floral richness or composition of B. affinis pollen samples across our sampling period. Likewise, we found no temporal changes in the presence or proportion of native vs. introduced species in pollen samples, though we observed much greater use of introduced floral species than previously determined for B. affinis. Floral community composition was regionally dissimilar, inconsistent with patterns of B. affinis decline by state. Our results suggest B. affinis decline was unlikely to have been driven by spatial or temporal limitations of specific floral species. This work greatly expands the known forage of B. affinis and will provide managers with insight to aid the conservation of B. affinis.

A Probabilistic Co-Occurrence Approach for Estimating Likelihood of Spatial Overlap Between Listed Species Distribution and Pesticide Use Patterns.

Characterizing potential spatial overlap between federally threatened and endangered ("listed") species distributions and registered pesticide use patterns is important for accurate risk assessment of threatened and endangered species. Because accurate range information for such rare species is often limited and agricultural pesticide use patterns are dynamic, simple spatial co-occurrence methods may overestimate or underestimate overlap and result in decisions that benefit neither listed species nor the regulatory process. Here, we demonstrate a new method of co-occurrence analysis that employs probability theory to estimate spatial distribution of rare species populations and areas of pesticide use to determine the likelihood of potential exposure. Specifically, we 1) describe a probabilistic method to estimate pesticide use based on crop production patterns; 2) construct species distribution models for 2 listed insect species whose ranges were previously incompletely described, the rusty-patched bumble bee (Bombus affinis) and the Poweshiek skipperling (Oarisma poweshiek); and 3) develop a probabilistic co-occurrence methodology and assessment framework. Using the principles of the Bayes' theorem, we constructed probabilistic spatial models of pesticide use areas by integrating information from land-cover spatial data, agriculture statistics, and remote-sensing data. We used maximum entropy methods to build species distribution models for 2 listed insects based on species collection and observation records and predictor variables relevant to the species' biogeography and natural history. We further developed novel methods for refinement of these models at spatial scales relevant to US Fish and Wildlife Service (FWS) regulatory priorities (e.g., critical habitat areas). Integrating both probabilistic assessments and focusing on USFWS priority management areas, we demonstrate that spatial overlap (i.e., potential for exposure) is not deterministic but instead a function of both species distribution and land use patterns. Our work serves as a framework to enhance the accuracy and efficiency of threatened and endangered species assessments using a data-driven likelihood analysis of species co-occurrence. Integr Environ Assess Manag 2019;00:1-12. © 2019 SETAC.

Decline of bumble bees in northeastern North America, with special focus on Bombus terricola

Concerns about the rapid and severe declines of many bumble bee (Bombus spp.) species in Europe, and more recently North America, have spurred research into the extent and possible causes for these losses. Drawing conclusions has been difficult due to a lack of long-term data, especially for specific regions that may have different factors at play than the global trend. In this study, 150years of Bombus records in the state of New Hampshire from the University of New Hampshire Insect Collection were examined. This allowed for changes in abundance and distribution to be tracked over time, with focus on species designated of greatest conservation need by NH Fish & Game Department. Floral records also provided insight into the diet breadth of these species, which may affect their vulnerability. Evidence of drastic decline was found in Bombus affinis, Bombus fervidus, and Bombus terricola, as well as significant decline in Bombus vagans with data suggesting it has been ecologically replaced by Bombus impatiens over time. We suggest Bombus vagans receive future conservation consideration. Our analyses found a severe constriction of the geographic range of Bombus terricola to high elevation regions in the latter half of the 20th century, and its role as pollinator of several alpine plants necessitates immediate conservation action.

Bees of Maine, with a State Species Checklist

We present a new county checklist developed from bee research in Maine since the 1800s. The list contains 278 bee species in 37 genera and 6 families, of which all but 8 are native, with ≥50 taxa each in Andrena and Lasioglossum. Data for 16 counties from publications, museum collections, and recent surveys varied in number of species from 8 (Androscoggin) to 197 (Hancock). Research since 1930 on Vaccinium angustifolium (Lowbush Blueberry) led to many records. Twenty-one species are considered unusual, including 3 first recorded in 2016: Epeoloides pilosulus, Melitta melittoides, and Holcopasites calliopsidis. Maine records provide evidence of declines in Bombus affinis, decline in B. terricola followed by partial recovery, and increase in B. impatiens. Crops that should be studied regarding associated bees are Malus pumila (Apple), Vaccinium corymbosum (Highbush Blueberry), Vaccinium macrocarpon (American Cranberry), and Curcurbitaceae (cucurbits). Montane, sandy, and island habitats were identified as priorities for future sampling. We discuss records of bee species from New England relevant to understanding the Maine fauna, bee diversity, changes in abundance, cleptoparasitism, pesticide impacts, habitat requirements, and climate change.

Bumble bees of Kanawha County, WV, with statewide trends over the last century.

The goal of our research is to assess the overall diversity of and to evaluate the presence and relative abundance of declining bumble bee species (e.g., Bombus affinis, B. pensylvanicus and B. terricola), in Kanawha County, WV. We collected bees locally from a variety of field sites to establish local population numbers and community structure. Additionally, to determine whether the community structure of bumble bees has changed over time, we compiled museum collection data to calculate community structure data from the last 95 years. Our local data indicated suggested that the bumble bee community of Kanawha County is dominated (~95% of total specimens collected) by three species (B. impatiens, B. bimaculatus and B. griseocollis); a pattern of diversity similar to that found in other regions of North America that have been similarly surveyed. Additionally, our data suggested very weak populations of bumble bees in the subgenera Thoracobombus and Bombus (sensu strictu). Our statewide data indicated that species of bees that are experiencing declines in other parts of North America are also declining in WV and that the bees that are replacing those declining species are predominantly those in the subgenera Pyrobombus and Cullumanobombus.This study undoubtedly contributes to our understanding of the local diversity of these insects, but perhaps more importantly, it will provide valuable data regarding the decline of these pollinators and perhaps provide insights to the locations of refugia where otherwise declining species may occur in higher, sustainable densities.

A Transmission Right-of-Way as Habitat for Wild Bees (Hymenoptera: Apoidea: Anthophila) in Connecticut

Abstract Transmission line corridors in forested landscapes provide important early successional habitats for a taxonomically rich array of invertebrates. In this study, we investigated the wild bee diversity at 19 sites along a transmission line right-of-way in southeastern Connecticut over a 2-yr period. One hundred sixty-three species representing 31 genera—roughly 50% of the state's bee species—were captured over the course of the study. Richness estimates suggest total faunas of 152, 180, and 204 bee species for the bee bowl, net, and combined samples, respectively. One globally rare bee, Epeoloides pilosula (Cresson) (Apidae: Osirini), formerly thought to be extirpated in the United States, was recovered. Two rarely collected species, Melitta melittoides (Viereck) (Melittidae) and Colletes productus Robertson (Colletidae), were associated with maleberry (Lyonia ligustrina (L.) de Candolle) flowers. Four Bombus species known to have declined regionally, Bombus affinis Cresson, Bombus ashtoni (Cresson), Bombus pensylvanicus (DeGeer), and Bombus terricola Kirby, were not among the eight bumble bee (Bombus) species found. Our results underscore the importance of transmission line corridors as managed early successional habitat for wild bees, including rare species, in the largely forested landscapes of New England.

Do pathogen spillover, pesticide use, or habitat loss explain recent North American bumblebee declines?

AbstractSeveral North American bumblebee species have recently undergone dramatic declines. The use of managed, pathogen‐carrying bumblebees for pollination of greenhouse crops began shortly before these declines, and wild bumblebees near greenhouses now have high pathogen loads. This has led to speculation that pathogen spillover from commercial bumblebees caused declines of these species. We test this hypothesis using a large dataset of bumblebee occurrence records and agricultural census data. We find support for the pathogen spillover hypothesis for two species but no evidence that pathogen spillover caused the near disappearance of the previously widespread Bombus affinis. Furthermore, we show that pesticide use and habitat loss are unlikely to be major causes of decline for any of the Bombus species examined. Collectively, our analyses underscore that there remains an urgent need to identify causes of pollinator population losses.

Decline of bumble bees ( Bombus) in the North American Midwest

Declines in many bumble bee species have been documented in Europe raising several ecological and economic concerns. The nature and extent of bumble bee decline in North America is poorly understood due mainly to a lack of baseline and long-term data. Museum collections provide excellent sources of information on past and current species distributions, which can be used to infer changes in the composition of insect communities. Using the Illinois Natural History Survey’s electronic database of Hymenoptera and a recent biodiversity survey of historically sampled localities, we were able to examine changes in the richness and distribution of the bumble bee fauna of Illinois over the last century. We found that bumble bee species richness declined substantially during the middle of the century (1940–1960). Four species were locally extirpated: Bombus borealis, Bombus ternarius, Bombus terricola and Bombus variabilis. The ranges of Bombus affinis, Bombus fraternus, Bombus pensylvanicus and Bombus vagans have also decreased in Illinois. Our analyses also indicated that current bumble bee diversity is highest in northern Illinois, where conservation efforts would be most productive. Our study demonstrates that half of the bumble bee species found historically in Illinois have been locally extirpated or have suffered declines, supporting observations of broader declines in North America. Major declines in the bumble bee fauna coincided with large-scale agricultural intensification in Illinois between 1940 and 1960. Attempts to conserve bumble bees in Illinois should involve wildlife-friendly approaches to agriculture, such as increasing agricultural land set-asides and hedgerows, and employing integrated pest management.

Evidence for decline in eastern North American bumblebees (Hymenoptera: Apidae), with special focus on Bombus affinis Cresson

Bumblebees (Bombus spp.) have been declining rapidly in many temperate regions of the Old World. Despite their ecological and economic importance as pollinators, North American bumblebees have not been extensively surveyed and their conservation status is largely unknown. In this study, two approaches were used to determine whether bumblebees in that region were in decline spatially and temporally. First, surveys performed in 2004–2006 in southern Ontario were compared to surveys from 1971 to 1973 in the same sites to look at changes in community composition, in one of the most bumblebee diverse areas of eastern North America. Second, the extent of range decline for a focal species (Bombus affinis Cresson) was estimated by surveying 43 sites throughout its known native range in eastern Canada and the United States. Our study documents an impoverishment of the bumblebee community in southern Ontario over the past 35 years. Bombus affinis in particular was found to have declined drastically in abundance not only in southern Ontario but throughout its native range. The loss of any bumblebee species may result in cascading impacts on native fauna and flora and reduce agricultural production. Implications for the conservation of this important group of pollinators are discussed.

Enzyme gene variation in five species of bumble bees (Hymenoptera: Apidae)

Genetic variability at eight enzyme loci was assessed in five bumble bee species from southern Ontario: Bombus affinis Cresson, B. fervidus (Fabricius), B. bimaculatus Cresson, B. impatiens Cresson, and B. vagans Smith. Average expected heterozygosity (Hexp ± SE) across all species was 0.089 ± 0.023 (range 0.046–0.139). These values are higher than any previously reported estimates of average expected heterozygosities for bumble bees.

Estimation of the fructose diphosphatase–phosphofructokinase substrate cycle in the flight muscle of Bombus affinis

1. Substrate cycling of fructose 6-phosphate through reactions catalysed by phosphofructokinase and fructose diphosphatase was estimated in bumble-bee (Bombus affinis) flight muscle in vivo. 2. Estimations of substrate cycling of fructose 6-phosphate and of glycolysis were made from the equilibrium value of the (3)H/(14)C ratio in glucose 6-phosphate as well as the rate of (3)H release to water after the metabolism of [5-(3)H,U-(14)C]glucose. 3. In flight, the metabolism of glucose proceeded exclusively through glycolysis (20.4mumol/min per g fresh wt.) and there was no evidence for substrate cycling. 4. In the resting bumble-bee exposed to low temperatures (5 degrees C), the pattern of glucose metabolism in the flight muscle was altered so that substrate cycling was high (10.4mumol/min per g fresh wt.) and glycolysis was decreased (5.8mumol/min per g fresh wt.). 5. The rate of substrate cycling in the resting bumble-bee flight muscle was inversely related to the ambient temperature, since at 27 degrees , 21 degrees and 5 degrees C the rates of substrate cycling were 0, 0.48 and 10.4mumol/min per g fresh wt. respectively. 6. Calcium ions inhibited fructose diphosphatase of the bumble-bee flight muscle at concentrations that were without effect on phosphofructokinase. The inhibition was reversed by the presence of a Ca(2+)-chelating compound. It is proposed that the rate of fructose 6-phosphate substrate cycling could be regulated by changes in the sarcoplasmic Ca(2+) concentration associated with the contractile process.