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Summary 1.To sustain the vital ecosystem service of pollination, new methodical developments are needed for research on the underlying factors of globally observed bee losses. In particular, robust laboratory methods for assessing adverse effects on honey bee brood are required. In addition, from a statistical point of view, the shared origin of test individuals must be considered when analysing ecotoxicological data. 2. To improve honey bee in vitro rearing, we adopted a nongrafting method to collect honey bee larvae without direct manipulation. Linear mixed effects model to evaluate LD 50 , larvae survival and prepupae weights integrated the colony background of larvae as a random factor into the statistical analyses. The novel rearing approach and appropriate statistical tools for data analyses are illustrated in an in vitro case study on acute oral dimethoate toxicity. 3. We recommend our honey bee larvae collection approach for in vitro larvae-rearing applications, because of (i) a mere 3% background mortality upon the prepupae stage, (ii) a high quantitative capacity and (iii) because of robustness of performance which are great benefits for standardization. 4. The analyses indicate clear adverse effects of dimethoate by a significant survival reduction and prepupae weight reduction. For second instars, the acute 48-h LD 50 was 1AE67 lg dimethoate per larva. 5. We conclude that both our larvae collection method and the applied statistical approaches will help to improve the quality of environmental risk assessment studies on honey bees, to secure honey bee pollination and to sustain biodiversity.

Honey bees are important pollinators, requiring floral pollen and nectar for nutrition. Nectar is rich in sugars, but contains additional nutrients, including amino acids (AAs). We tested the preferences of free-flying foragers between 20 AAs at 0.1% w/w in sucrose solutions in an artificial meadow. We found consistent preferences amongst AAs, with essential AAs preferred over nonessential AAs. The preference of foragers correlated negatively with AA induced deviations in pH values, as compared to the control. Next, we quantified tradeoffs between attractive and deterrent AAs at the expense of carbohydrates in nectar. Bees were attracted by phenylalanine, willing to give up 84units sucrose for 1unit AA. They were deterred by glycine, and adding 100 or more units of sucrose could resolve to offset 1unit AA. In addition, we tested physiological effects of AA nutrition on forager homing performance. In a no-choice context, caged bees showed indifference to 0.1% proline, leucine, glycine or phenylalanine in sucrose solutions. Furthermore, flight tests gave no indication that AA nutrition affected flight capacity directly. In contrast, low carbohydrate nutrition reduced the performance of bees, with important methodological implications for homing studies that evaluate the effect of substances that may affect imbibition of sugar solution. In conclusion, low AA concentrations in nectar relative to pollen suggest a limited role in bee nutrition. Most of the 20 AAs evoked a neutral to a mild deterrent response in bees, thus it seems unlikely that bees respond to AAs in nectar as a cue to assess nutritional quality. Nonetheless, free choice behavior of foraging bees is influenced, for instance by phenylalanine and glycine. Thus, AAs in nectar may affect plant-pollinator interactions and thereby exhibit a selective pressure on the flora in the honey bee habitat.

Pollen is the main protein and lipid source for honey bees (Apis mellifera), and nutritionally impoverished landscapes pose a threat to colony development. To determine colony nutritional demands, we analyzed a yearly cycle of bee-collected pollen from colonies in the field and compared it to colony worker production and honey bee body composition, for the first time in social insects. We monitored monthly bee production in ten colonies at each of seven sites throughout Israel, and trapped pollen bi-monthly in five additional colonies at each of four of these sites. Pollen mixtures from each sampling date and site were analyzed for weight, total protein, total fatty acids (FAs), and FA composition. Compared to more temperate climates, the eastern Mediterranean allows a relatively high yearly colony growth of ca. 300,000-400,000 bees. Colonies at higher elevation above sea level showed lower growth rates. Queen egg-laying rate did not seem to limit growth, as peaks in capped brood areas showed that queens lay a prolific 2000 eggs a day on average, with up to 3300 eggs in individual cases. Pollen uptake varied significantly among sites and seasons, with an overall annual mean total 16.8kg per colony, containing 7.14kg protein and 677g fat. Overall mean pollen protein content was high (39.8%), and mean total FA content was 3.8%. Production cost, as expressed by the amount of nutrient used per bee, was least variable for linoleic acid and protein, suggesting these as the best descriptive variables for total number of bees produced. Linolenic acid levels in pollen during the autumn were relatively low, and supplementing colonies with this essential FA may mitigate potential nutritional deficiency. The essentiality of linoleic and linolenic acids was consistent with these FAs' tendency to be present at higher levels in collected pollen than in the expected nutrients in bee bodies, demonstrating a well-developed adjustment between pollinator nutritional demands and the nutritional value of food offered by pollinated plants.

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