Prevalence and distribution of Varroa destructor and Nosema spp. in symptomatic honey bee colonies across the USA from 2015 to 2022.

USDA-ARS Bee Research Laboratory received symptomatic honey bee (Apis mellifera L.) samples across the United States for disease diagnosis. Here, we present a retrospective study and cartography of ectoparasite Varroa destructor and intracellular microsporidia parasite Nosema spp. These two major parasites were identified in the diseased honey bee samples between 2015 and 2022. Varroa infestation level (VIL) was examined by a wash technique (Mites/100 bees) and calculated as a percentage, while Nosema infection was quantified by microscopical spore count (Million Spores/Bee). Data were analyzed by month, year, state, and by nine geographical climate regions described in the U.S. Of adult bee samples (n = 4039) that were analyzed for Varroa mite infestation, the overall VIL in the U.S. ranged between 0.4 and 30.85%, with an overall national VIL and Varroa prevalence of 8.21% and 85.14%, respectively. Overall monthly data showed VIL constantly exceeded the critical level of 4% except from June to September and reached a maximum of 15% in January and December. Nationwide, VIL significantly (p < 0.001) increased from 2015 to 2018 (1.1-4.7%), plateaued from 2018 to 2021 (4.7-4.5%), followed by a significant decrease in 2022 (3.6%). Significant VIL differences (p < 0.001) were recorded among climate regions, with the highest mite infestation levels in the Upper Midwest region (13.9%) and the lowest in the West region (5.1%). Of adult bee samples (n = 2,994) that were analyzed for Nosema infection, Nosema spore count ranged between (1-16.8) million spores per bee among states, with a national average of 6.8 and a prevalence of 99.7%. The lowest and highest Nosema loads were respectively recorded in the South region (3.1) and Upper Midwest (10.5), a significant difference (p < 0.001). No statistical differences were recorded among the six other climate regions. Overall, VIL and Nosema infection correlated significantly (p < 0.001) with a regression coefficient of (R2 = 0.6). Our data, which originated from ailing bee colonies, showed significantly higher rates of maladies compared to data from healthy colonies obtained by the USDA-APHIS National Honey Bee Survey, demonstrating the role of bee diseases caused by Varroa mite and Nosema in honey bee population declines.

Non-optimal ambient temperatures aggravate insecticide toxicity and affect honey bees Apis mellifera L. gene regulation.

In this study, we conducted a transcriptional analysis of five honey bee genes to examine their functional involvement vis-à-vis ambient temperatures and exposure to imidacloprid. In a 15-day cage experiment, three cohorts of one-day-old sister bees emerged in incubators, were distributed into cages, and maintained at three different temperatures (26 °C, 32 °C, 38 °C). Each cohort was fed a protein patty and three concentrations of imidacloprid-tainted sugar (0 ppb, 5 ppb and 20 ppb) ad libitum. Honey bee mortality, syrup and patty consumption were monitored daily over 15 days. Bees were sampled every three days for a total of five time points. RT-qPCR was used to longitudinally assess gene regulation of Vg, mrjp1, Rsod, AChE-2 and Trx-1 using RNA extracted from whole bee bodies. Kaplan-Meier models show that bees kept at both non-optimal temperatures (26 °C and 38 °C) were more susceptible to imidacloprid, with significantly higher mortality (P < 0.001 and P < 0.01, respectively) compared to the control. At 32 °C, no differences in mortality (P = 0.3) were recorded among treatments. In both imidacloprid treatment groups and the control, the expression of Vg and mrjp1 was significantly downregulated at 26 °C and 38 °C compared to the optimal temperature of 32 °C, indicating major influence of ambient temperature on the regulation of these genes. Within the ambient temperature groups, both imidacloprid treatments exclusively downregulated Vg and mrjp1 at 26 °C. AChE-2 and the poorly characterized Rsod gene were both consistently upregulated at the highest temperature (38 °C) compared to the ideal temperature (32 °C) in all treatment groups. Trx-1 showed no effect to both temperature and imidacloprid treatments and was regulated in an age-related manner. Overall, our results indicate that ambient temperatures amplify imidacloprid toxicity and affect honey bee gene regulation.

Deformed wing virus of honey bees is inactivated by cold plasma ionized hydrogen peroxide.

Deformed wing virus (DWV) is a widespread pathogen of Apis mellifera honey bees, and is considered a major causative factor for the collapse of infected honey bee colonies. DWV can be horizontally transmitted among bees through various oral routes, including via food sharing and by interactions of bees with viral-contaminated solid hive substrates. Cold plasma ionized hydrogen peroxide (iHP) is used extensively by the food production, processing and medical industries to clean surfaces of microbial contaminants. In this study, we investigated the use of iHP to inactivate DWV particles in situ on a solid substrate. iHP-treated DWV sources were ~105-fold less infectious when injected into naïve honey bee pupae compared to DWV receiving no iHP treatment, matching injected controls containing no DWV. iHP treatment also greatly reduced the incidence of overt DWV infections (i.e., pupae having >109 copies of DWV). The level of DWV inactivation achieved with iHP treatment was higher than other means of viral inactivation such as gamma irradiation, and iHP treatment is likely simpler and safer. Treatment of DWV contaminated hive substrates with iHP, even with honey bees present, may be an effective way to decrease the impacts of DWV infection on honey bees.

Development and Evaluation of a New Effective Tool and Method for Assessing Varroadestructor (Acari: Varroidae) Mite Populations in Honey Bee Colonies.

A new device for assessing Varroa destructor (Anderson−Truman) mite infestations in honey bee colonies was designed, tested, and evaluated against the sugar roll method, a widely used method by beekeepers. The Varroa Shaker Device (VSD) is constructed of polyvinyl chloride (PVC) pipe that separates into three parts. Inside the shaker there are two mesh sizes; the larger mesh separates the bees from the mites, and the smaller mesh captures the mites. The VSD can be used by shaking bees with only water as the wash solution. The recovery of mites using the VSD is >90%, which is such as that recorded for using the sugar roll method. Our tests demonstrated that the VSD accurately assessed mite loads when fewer than 250 bees were sampled and shaken with 250 mL of water for one minute. To assure accurate mite counts are achieved with any sampling device, honey bees should be taken from frames with an open and/or capped brood where the mites are more likely located. The VSD can be used in both laboratory and field settings to accurately assess honey bee colonies for levels of mite infestation or for collecting live mites for research purposes.

The vectoring competence of the mite Varroa destructor for deformed wing virus of honey bees is dynamic and affects survival of the mite.

The ectoparasitic mite, Varroa destructor and the viruses it vectors, including types A and B of Deformed wing virus (DWV), pose a major threat to honey bees, Apis mellifera. Analysis of 256 mites collected from the same set of field colonies on five occasions from May to October 2021 showed that less than a half of them, 39.8% (95% confidence interval (CI): 34.0 - 46.0%), were able to induce a high (overt) level DWV infection with more than 109 viral genomes per bee in the pupa after 6 days of feeding, with both DWV-A and DWV-B being vectored at similar rates. To investigate the effect of the phoretic (or dispersal) stage on adult bees on the mites' ability to vector DWV, the mites from two collection events were divided into two groups, one of which was tested immediately for their infectiveness, and the other was kept with adult worker bees in cages for 12 days prior to testing their infectiveness. We found that while 39.2% (95% CI: 30.0 - 49.1%) of the immediately tested mites induced overt-level infections, 12-day passage on adult bees significantly increased the infectiousness to 89.8% (95% CI: 79.2 - 95.6%). It is likely that Varroa mites that survive brood interruptions in field colonies are increasingly infectious. The mite lifespan was affected by the DWV type it transmitted to pupae. The mites, which induced high DWV-B but not DWV-A infection had an average lifespan of 15.5 days (95% CI: 11.8 - 19.2 days), which was significantly shorter than those of the mites which induced high DWV-A but not DWV-B infection, with an average lifespan of 24.3 days (95% CI: 20.2 - 28.5), or the mites which did not induce high levels of DWV-A or DWV-B, with an average survival of 21.2 days (95% CI: 19.0 - 23.5 days). The mites which transmitted high levels of both DWV-A and DWV-B had an intermediate average survival of 20.5 days (95% CI: 15.1 - 25.9 days). The negative impact of DWV-B on mite survival could be a consequence of the ability of DWV-B, but not DWV-A to replicate in Varroa.

Transferrin-mediated iron sequestration suggests a novel therapeutic strategy for controlling Nosema disease in the honey bee, Apis mellifera.

Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae, is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N. ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N. ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N. ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N. ceranae infection causes iron deficiency and upregulation of the A. mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N. ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N. ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee's ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees.

Varroa destructor mites vector and transmit pathogenic honey bee viruses acquired from an artificial diet.

The ectoparasitic mite Varroa destructor is one of the most destructive pests of the honey bee (Apis mellifera) and the primary biotic cause of colony collapse in many regions of the world. These mites inflict physical injury on their honey bee hosts from feeding on host hemolymph and fat body cells/cellular components, and serve as the vector for deadly honey bee viruses, including Deformed wing virus (DWV) and the related Varroa destructor virus-1 (VDV-1) (i.e., DWV-like viruses). Studies focused on elucidating the dynamics of Varroa-mediated vectoring and transmission of DWV-like viruses may be confounded by viruses present in ingested host tissues or the mites themselves. Here we describe a system that includes an artificial diet free of insect tissue-derived components for maintaining Varroa mites for in vitro experimentation. Using this system, together with the novel engineered cDNA clone-derived genetically tagged VDV-1 and wild-type DWV, we demonstrated for the first time that Varroa mites provided an artificial diet supplemented with engineered viruses for 36 hours could acquire and transmit sufficient numbers of virus particles to establish an infection in virus-naïve hosts. While the in vitro system described herein provides for only up to five days of mite survival, precluding study of the long-term impacts of viruses on mite health, the system allows for extensive insights into the dynamics of Varroa-mediated vectoring and transmission of honey bee viruses.

Gene Expression and Functional Analyses of Odorant Receptors in Small Hive Beetles (Aethina tumida).

Olfaction is key to many insects. Odorant receptors (ORs) stand among the key chemosensory receptors mediating the detection of pheromones and kairomones. Small hive beetles (SHBs), Aethina tumida, are parasites of social bee colonies and olfactory cues are especially important for host finding. However, how interactions with their hosts may have shaped the evolution of ORs in the SHB remains poorly understood. Here, for the first time, we analyzed the evolution of SHB ORs through phylogenetic and positive selection analyses. We then tested the expression of selected OR genes in antennae, heads, and abdomens in four groups of adult SHBs: colony odor-experienced/-naive males and females. The results show that SHBs experienced both OR gene losses and duplications, thereby providing a first understanding of the evolution of SHB ORs. Additionally, three candidate ORs potentially involved in host finding and/or chemical communication were identified. Significantly different downregulations of ORs between the abdomens of male and female SHBs exposed to colony odors may reflect that these expression patterns might also reflect other internal events, e.g., oviposition. Altogether, these results provide novel insights into the evolution of SHB ORs and provide a valuable resource for analyzing the function of key genes, e.g., for developing biological control. These results will also help in understanding the chemosensory system in SHBs and other beetles.

Insights into the feeding behaviors and biomechanics of Varroa destructor mites on honey bee pupae using electropenetrography and histology.

Feeding behaviors and biomechanics of female Varroa destructor mites are revealed from AC-DC electropenetrography (EPG) recordings of mites feeding from Apis mellifera honey bee pupae and histology of mite internal ingestion apparatus. EPG signals characteristic of arthropod suction feeding (ingestion) were identified for mites that fed on pupae during overnight recordings. Ingestion by these mites was confirmed afterwards by observing internally fluorescent microbeads previously injected into their hosts. Micrographs of internal ingestion apparatus illustrate the connection between a gnathosomal tube and a pharyngeal lumen, which is surrounded by alternating dilator and constrictor muscles. Inspection of EPG signals showed the muscularized mite pharyngeal pump operates at a mean repetition rate of 4.5 cycles/s to ingest host fluids. Separate feeding events observed for mites numbered between 23 and 33 over approximately 16 h of recording, with each event lasting ~10 s. Feeding events were each separated by ~2 min. Consecutive feeding events separated by either locomotion or prolonged periods of quiescence were grouped into feeding bouts, which ranged in number from one to six. Statistical analyses of EPG data revealed that feeding events were prolonged for mites having lower pharyngeal pump frequencies, and mites having prolonged feeding events went unfed for significantly more time between feeding events. These results suggest that mites may adjust behaviors to meet limitations of their feeding apparatus to acquire similar amounts of food. Data reported here help to provide a more robust view of Varroa mite feeding than those previously reported and are both reminiscent of, as well as distinct from, some other acarines and fluid-feeding insects.

Deformed wing virus type A, a major honey bee pathogen, is vectored by the mite Varroa destructor in a non-propagative manner.

Honey bees, the primary managed insect pollinator, suffer considerable losses due to Deformed wing virus (DWV), an RNA virus vectored by the mite Varroa destructor. Mite vectoring has resulted in the emergence of virulent DWV variants. The basis for such changes in DWV is poorly understood. Most importantly, it remains unclear whether replication of DWV occurs in the mite. In this study, we exposed Varroa mites to DWV type A via feeding on artificially infected honey bees. A significant, 357-fold increase in DWV load was observed in these mites after 2 days. However, after 8 additional days of passage on honey bee pupae with low viral loads, the DWV load dropped by 29-fold. This decrease significantly reduced the mites' ability to transmit DWV to honey bees. Notably, negative-strand DWV RNA, which could indicate viral replication, was detected only in mites collected from pupae with high DWV levels but not in the passaged mites. We also found that Varroa mites contain honey bee mRNAs, consistent with the acquisition of honey bee cells which would additionally contain DWV replication complexes with negative-strand DWV RNA. We propose that transmission of DWV type A by Varroa mites occurs in a non-propagative manner.

The Dynamics of Deformed Wing Virus Concentration and Host Defensive Gene Expression after Varroa Mite Parasitism in Honey Bees, Apis mellifera.

The synergistic interactions between the ectoparasitic mite Varroa destructor and Deformed wing virus (DWV) lead to the reduction in lifespan of the European honey bee Apis mellifera and often have been implicated in colony losses worldwide. However, to date, the underlying processes and mechanisms that form the multipartite interaction between the bee, mite, and virus have not been fully explained. To gain a better understanding of honey bees' defense response to Varroa mite infestation and DWV infection, the DWV titers and transcription profiles of genes originating from RNAi, immunity, wound response, and homeostatic signaling pathways were monitored over a period of eight days. With respect to DWV, we observed low viral titers at early timepoints that coincided with high levels of Toll pathway transcription factor Dorsal, and its downstream immune effector molecules Hymenoptaecin, Apidaecin, Abaecin, and Defensin 1. However, we observed a striking increase in viral titers beginning after two days that coincided with a decrease in Dorsal levels and its corresponding immune effector molecules, and the small ubiquitin-like modifier (SUMO) ligase repressor of Dorsal, PIAS3. We observed a similar expression pattern for genes expressing transcripts for the RNA interference (Dicer/Argonaute), wound/homeostatic (Janus Kinase), and tissue growth (Map kinase/Wnt) pathways. Our results demonstrate that on a whole, honey bees are able to mount an immediate, albeit, temporally limited, immune and homeostatic response to Varroa and DWV infections, after which downregulation of these pathways leaves the bee vulnerable to expansive viral replication. The critical insights into the defense response upon Varroa and DWV challenges generated in this study may serve as a solid base for future research on the development of effective and efficient disease management strategies in honey bees.

Compound and Dose-Dependent Effects of Two Neonicotinoid Pesticides on Honey Bee (Apis mellifera) Metabolic Physiology.

Use of neonicotinoid pesticides is now ubiquitous, and consequently non-targeted arthropods are exposed to their residues at sub-lethal doses. Exposure to these neurotoxins may be a major contributor to poor honey bee colony health. Few studies have explored how sub lethal exposure to neonicotinoids affects honey bee metabolic physiology, including nutritional and energetic homeostasis, both of which are important for maintaining colony health. Reported here are results from a study of chronic oral exposure of honey bees to two sub lethal concentrations of clothianidin and imidacloprid. Neonicotinoids altered important aspects of honey bee nutritional and metabolic physiology in a compound and dose-dependent manner; both compounds at low doses reduced honey bee body weight. Low-dose clothianidin exposure resulted in bees having protein, lipids, carbohydrates, and glycogen levels similar to newly emerged bees. High-dose clothianidin exposure lowered lipids and glycogen content of bees. High-dose imidacloprid exposure resulted in bees having depressed metabolic rate. Low-dose imidacloprid exposure resulted in bees consuming low and high levels of protein and carbohydrate rich foods, respectively. Results suggest neonicotinoids interfere with honey bee endocrine neurophysiological pathways. Compound and dose-dependent effects might represent respective chemical structural differences determining an observed effect, and thresholds of compound effects on honey bee physiology.

Insights into the metabolism and behaviour of Varroa destructor mites from analysis of their waste excretions.

Varroa destructor mites (Acari: Varroidae) are harmful ectoparasites of Apis mellifera honey bees. Female foundresses of wax-capped pupal host cells and their daughters feed on host fluids from open wounds on the host's integument. Details of V. destructor mite nutrition are forthcoming, and little is known about the potential physical effects on hosts from mite feeding. Chemical analysis of waste excretions can infer details of animals' nutrition. Here, chemical analysis by high-performance liquid chromatography/mass spectrometry (HPLC-MS/MS) of mite excretions showed that the purine content of V. destructor waste consists of guanine with traces of hypoxanthine. Traces of uric acid and caffeine were also detected. Concentrations of guanine attenuated over time and excretions collected from senescing mites did not contain detectable guanine. Non-reproducing individual female mites maintained in vitro, housed in gelatin capsules and provided a honey bee pupa, deposited an average of nearly 18 excretions daily, mostly on the host's integument rather than on the capsule wall. The weight and volume of excretions suggest mites can consume nearly a microlitre of host fluids each day. Compounded over 10 days, this together with open wounds, could lead to substantial water loss and stress to developing pupae.

Genome of the small hive beetle (Aethina tumida, Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory.

Background: The small hive beetle (Aethina tumida; ATUMI) is an invasive parasite of bee colonies. ATUMI feeds on both fruits and bee nest products, facilitating its spread and increasing its impact on honey bees and other pollinators. We have sequenced and annotated the ATUMI genome, providing the first genomic resources for this species and for the Nitidulidae, a beetle family that is closely related to the extraordinarily species-rich clade of beetles known as the Phytophaga. ATUMI thus provides a contrasting view as a neighbor for one of the most successful known animal groups. Results: We present a robust genome assembly and a gene set possessing 97.5% of the core proteins known from the holometabolous insects. The ATUMI genome encodes fewer enzymes for plant digestion than the genomes of wood-feeding beetles but nonetheless shows signs of broad metabolic plasticity. Gustatory receptors are few in number compared to other beetles, especially receptors with known sensitivity (in other beetles) to bitter substances. In contrast, several gene families implicated in detoxification of insecticides and adaptation to diverse dietary resources show increased copy numbers. The presence and diversity of homologs involved in detoxification differ substantially from the bee hosts of ATUMI. Conclusions: Our results provide new insights into the genomic basis for local adaption and invasiveness in ATUMI and a blueprint for control strategies that target this pest without harming their honey bee hosts. A minimal set of gustatory receptors is consistent with the observation that, once a host colony is invaded, food resources are predictable. Unique detoxification pathways and pathway members can help identify which treatments might control this species even in the presence of honey bees, which are notoriously sensitive to pesticides.

Genetics and physiology of Varroa mites.

Varroa destructor is the primary biological threat to domesticated honey bee colonies in much of the world, impacting host fitness both directly and by transmitting RNA viruses. Genomic, proteomic, and functional-genetic resources provide a framework for Varroa biology. When coupled with physiological analyses of development, host finding, and reproduction, these resources reveal general traits of arthropods and offer new strategies for mite control. Efforts to develop novel controls are focused on efficacy, efficient delivery, and the avoidance of both host impacts and the swift evolution of resistance by mites.

Chronic Nosema ceranae infection inflicts comprehensive and persistent immunosuppression and accelerated lipid loss in host Apis mellifera honey bees.

Nosema ceranae is an intracellular microsporidian parasite of the Asian honey bee Apis cerana and the European honey bee Apis mellifera. Until relatively recently, A. mellifera honey bees were naïve to N. ceranae infection. Symptoms of nosemosis, or Nosema disease, in the infected hosts include immunosuppression, damage to gut epithelium, nutrient and energetic stress, precocious foraging and reduced longevity of infected bees. Links remain unclear between immunosuppression, the symptoms of nutrient and energetic stress, and precocious foraging behavior of hosts. To clarify physiological connections, we inoculated newly emerged A. mellifera adult workers with N. ceranae spores, and over 21 days post inoculation (21 days pi), gauged infection intensity and quantified expression of genes representing two innate immune pathways, Toll and Imd. Additionally, we measured each host's whole-body protein, lipids, carbohydrates and quantified respirometric and activity levels. Results show sustained suppression of genes of both humorally regulated immune response pathways after 6 days pi. At 7 days pi, elevated protein levels of infected bees may reflect synthesis of antimicrobial peptides from an initial immune response, but the lack of protein gain compared with uninfected bees at 14 days pi may represent low de novo protein synthesis. Carbohydrate data do not indicate that hosts experience severe metabolic stress related to this nutrient. At 14 days pi infected honey bees show high respirometric and activity levels, and corresponding lipid loss, suggesting lipids may be used as fuel for increased metabolic demands resulting from infection. Accelerated lipid loss during nurse honey bee behavioral development can have cascading effects on downstream physiology that may lead to precocious foraging, which is a major factor driving colony collapse.

Effects of Imidacloprid and Varroa destructor on survival and health of European honey bees, Apis mellifera.

There has been growing concern over declines in populations of honey bees and other pollinators which are a vital part to our food security. It is imperative to identify factors responsible for accelerated declines in bee populations and develop solutions for reversing bee losses. While exact causes of colony losses remain elusive, risk factors thought to play key roles are ectoparasitic mites Varroa destructor and neonicotinoid pesticides. The present study aims to investigate effects of a neonicotinoid pesticide Imidacloprid and Varroa mites individually on survivorship, growth, physiology, virus dynamics and immunity of honey bee workers. Our study provides clear evidence that the exposure to sublethal doses of Imidacloprid could exert a significantly negative effect on health and survival of honey bees. We observed a significant reduction in the titer of vitellogenin (Vg), an egg yolk precursor that regulates the honey bees development and behavior and often are linked to energy homeostasis, in bees exposed to Imidacloprid. This result indicates that sublethal exposure to neonicotinoid could lead to increased energy usage in honey bees as detoxification is a energy-consuming metabolic process and suggests that Vg could be a useful biomarker for measuring levels of energy stress and sublethal effects of pesticides on honey bees. Measurement of the quantitative effects of different levels of Varroa mite infestation on the replication dynamic of Deformed wing virus (DWV), an RNA virus associated with Varroa infestation, and expression level of immune genes yields unique insights into how honey bees respond to stressors under laboratory conditions.

Summer and fall ants have different physiological responses to food macronutrient content.

Seasonally, long-lived animals exhibit changes in behavior and physiology in response to shifts in environmental conditions, including food abundance and nutritional quality. Ants are long-lived arthropods that, at the colony level, experience such seasonal shifts in their food resources. Previously we reported summer- and fall-collected ants practiced distinct food collection behavior and nutrient intake regulation strategies in response to variable food protein and carbohydrate content, despite being reared in the lab under identical environmental conditions and dietary regimes. Seasonally distinct responses were observed for both no-choice and choice dietary experiments. Using data from these same experiments, our objective here is to examine colony and individual-level physiological traits, colony mortality and growth, food processing, and worker lipid mass, and how these traits change in response to variable food protein-carbohydrate content. For both experiments we found that seasonality per se exerted strong effects on colony and individual level traits. Colonies collected in the summer maintained total worker mass despite high mortality. In contrast, colonies collected in the fall lived longer, and accumulated lipids, including when reared on protein-biased diets. Food macronutrient content had mainly transient effects on physiological responses. Extremes in food carbohydrate content however, elicited a compensatory response in summer worker ants, which processed more protein-biased foods and contained elevated lipid levels. Our study, combined with our previously published work, strongly suggests that underlying physiological phenotypes driving behaviors of summer and fall ants are likely fixed seasonally, and change circannually.

Variability in Small Hive Beetle (Coleoptera: Nitidulidae) Reproduction in Laboratory and Field Experiments.

Experiments were conducted to examine how several key factors affect population growth of the small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae). Laboratory experiments were conducted to examine effects of food quantity and temperature on reproduction of cohorts of young A. tumida adults (1:1 sex ratio) housed in experimental arenas. Daily numbers and total mass of larvae exiting arenas were highly variable within treatment. Either one or two cohorts of larvae were observed exiting the arenas. Food quantity, either 10 g or 20 g, did not significantly affect the number of larvae exiting arenas at 32°C, but did at 28°C; arenas provided 20 g food produced significantly more larvae than arenas provided 10 g. Temperature did not affect the total mass of larvae provided 10 g food, but did affect larval mass provided 20 g; beetles kept at 28°C produced more larval mass than at 32°C. Field experiments were conducted to examine A. tumida reproductive success in full strength bee colonies. Beetles were introduced into hives as egg-infested frames and as adults, and some bee colonies were artificially weakened through removal of sealed brood. Efforts were unsuccessful; no larvae were observed exiting from, or during the inspection of, any hives. Possible reasons for these results are discussed. The variability observed in A. tumida reproduction even in controlled laboratory conditions and the difficulty in causing beetle infestations in field experiments involving full colonies suggest that accurately forecasting the A. tumida severity in such colonies will be difficult.

Seasonality directs contrasting food collection behavior and nutrient regulation strategies in ants.

Long-lived animals, including social insects, often display seasonal shifts in foraging behavior. Foraging is ultimately a nutrient consumption exercise, but the effect of seasonality per se on changes in foraging behavior, particularly as it relates to nutrient regulation, is poorly understood. Here, we show that field-collected fire ant colonies, returned to the laboratory and maintained under identical photoperiod, temperature, and humidity regimes, and presented with experimental foods that had different protein (p) to carbohydrate (c) ratios, practice summer- and fall-specific foraging behaviors with respect to protein-carbohydrate regulation. Summer colonies increased the amount of food collected as the p:c ratio of their food became increasingly imbalanced, but fall colonies collected similar amounts of food regardless of the p:c ratio of their food. Choice experiments revealed that feeding was non-random, and that both fall and summer ants preferred carbohydrate-biased food. However, ants rarely ate all the food they collected, and their cached or discarded food always contained little carbohydrate relative to protein. From a nutrient regulation strategy, ants consumed most of the carbohydrate they collected, but regulated protein consumption to a similar level, regardless of season. We suggest that varied seasonal food collection behaviors and nutrient regulation strategies may be an adaptation that allows long-lived animals to meet current and future nutrient demands when nutrient-rich foods are abundant (e.g. spring and summer), and to conserve energy and be metabolically more efficient when nutritionally balanced foods are less abundant.