Insulin-like peptide response to nutritional input in honey bee workers

Highlights

• Peripheral expression of honey bee AmIlp1 responds to dietary inputs.

• Peripheral expression of honey bee AmIlp2 and AmIRS do not respond to dietary inputs.

• Honey bee insulin-like peptides likely have different functional roles.

• Honey bees live longest on diets high in carbohydrates and low in protein and lipids.

• Honey bees are least sensitive to sugar on a diet high in lipids.

Abstract

The rise in metabolic disorders in the past decades has heightened focus on achieving a healthy dietary balance in humans. This is also an increasingly important issue in the management of honey bees (Apis mellifera) where poor nutrition has negative effects on health and productivity in agriculture, and nutrition is suggested as a contributing factor in the recent global declines in honey bee populations. As in other organisms, the insulin/insulin-like signaling (IIS) pathway is likely involved in maintaining nutrient homeostasis in honey bees. Honey bees have two insulin-like peptides (Ilps) with differing spatial expression patterns in the fat body suggesting that AmIlp1 potentially functions in lipid metabolism while AmIlp2 is a more general indicator of nutritional status. We fed caged worker bees artificial diets high in carbohydrates, proteins or lipids and measured expression of AmIlp1, AmIlp2, and the insulin receptor substrate (IRS) to test their responses to dietary macronutrients. We also measured lifespan, worker weight and gustatory sensitivity to sugar as measures of individual physical condition. We found that expression of AmIlp1 was affected by diet composition and was highest on a diet high in protein. Expression of AmIlp2 and AmIRS were not affected by diet. Workers lived longest on a diet high in carbohydrates and low in protein and lipids. However, bees fed this diet weighed less than those that received a diet high in protein and low in carbohydrates and lipids. Bees fed the high carbohydrates diet were also more responsive to sugar, potentially indicating greater levels of hunger. These results support a role for AmIlp1 in nutritional homeostasis and provide new insight into how unbalanced diets impact individual honey bee health.

Introduction

The balanced intake of key nutrients is critical for healthy living. Achieving an optimal intake of macronutrients is a question that receives growing attention in human societies in which the food environment is highly manipulated and has undergone tremendous change in recent decades. Similar challenges may be experienced by organisms that are closely connected to the human food supply. The managed honey bee population is exposed to several potential sources of nutritional stress due to their close association with agriculture. These bees feed largely from human crops, can be moved large distances to novel food environments, have their stores of nectar and pollen regulated by beekeepers, and are often fed artificial supplements to boost productivity.

Honey bees rely on nectar and pollen collected by older workers called foragers to meet the diverse dietary requirements of members of the colony (Winston, 1987). This behavior is key to modern agriculture, where honey bees provide critical pollination services (Gallai et al., 2009). Floral nectars are the primary carbohydrate resource for honey bees, (Winston, 1987), while pollen provides dietary protein, lipids, minerals and vitamins to honey bees (Herbert and Shimanuki, 1978). The high metabolisms of foragers are fueled primarily by carbohydrates, while developing larvae and young bees require relatively protein-rich diets (reviewed in Haydak, 1970, Crailsheim, 1990). The nutritional value of bee collected nectars and pollens can vary widely by species and cultivar (Butler, 1945, Schmidt et al., 1995, Abrol, 1995; Tables S1 and S2 for nectars and pollens respectively), which may result in nutritional stresses in bees maintained in highly managed agricultural environments (Jay and Jay, 1993, Naug, 2009).

Honey bees prefer nectars with high sugar concentrations (Roubik and Buchmann, 1984), and optimal pollen protein levels for brood production and colony growth are between 20% and 30% (Kleinschmidt and Kondos, 1976, Herbert et al., 1977). However, bees do not generally consume freshly collected pollen. Instead they eat bee bread, stored pollen that has been processed by bees and microorganisms, which has a crude protein content of between 19.3% and 26.5% (Herbert and Shimanuki, 1978). Managed bees also receive artificial nutritional supplements to boost productivity including sucrose solutions, high-fructose corn syrup, protein-rich flours and yeast (Somerville, 2005). It is unclear how these supplements affect colony and individual health and how efficiently honey bees are able to process the proteins found in pollen substitutes (Barker and Lehner, 1978, De Jong et al., 2009, Sammataro and Weiss, 2013).

As in vertebrates and other insects, the insulin/insulin-like signaling (IIS) pathway is thought to be an important integrator of nutritional status, lifespan, and behavior in honey bees (Tatar et al., 2003, De Azevedo and Hartfelder, 2008, Corona et al., 2007, Ament et al., 2008, Wang et al., 2010, Nilsen et al., 2011). Various components of the IIS pathway respond to dietary manipulations, influence sucrose perception, and are correlated with foraging behaviors (Wang et al., 2010, Ament et al., 2011, Nilsen et al., 2011). Suppression of the insulin receptor substrate (IRS), which functions in post-insulin receptor signal transduction, affects foragers’ loading of nectar (carbohydrates) vs. pollen (protein; Wang et al., 2010).

Honey bees have two insulin-like peptides (Ilps; (Wheeler et al., 2006)) which are expressed in fat body but have different spatial patterns (Nilsen et al., 2011). The fat body is a loose tissue analogous to vertebrate liver and adipose, and is the primary site of energy storage in insects (Arrese and Soulages, 2010). The honey bee fat body is composed of two cell types: oenocytes which are a site of lipid and lipoprotein synthesis and trophocytes which store excess lipids, proteins and carbohydrates (Paes de Oliveira and Cruz-Landim, 2003). AmIlp1 expression is confined to the oenocytes, while AmIlp2 is expressed in both cell types (Nilsen et al., 2011).

These spatial expression patterns along with previous work suggest that AmIlp1 and AmIlp2 have different but complementary functions integrating information about nutrient availability and energy stores in honey bees. AmIlp1 has been shown to respond to high sucrose diets and amino acid supplementation, and its localization in oenocytes suggests that it may play a role in lipid metabolism (Ament et al., 2008, Nilsen et al., 2011). AmIlp2 expression is increased in the high nutrition nurse phenotype relative to the poor nutrition forager phenotype, but its expression was unaffected by dietary manipulations (Ament et al., 2011). AmIlp2 expression is also correlated with juvenile hormone titer and has been hypothesized to be a more general indicator of nutritional status, potentially involved in the metabolic shift between the high nutrient nurse and low nutrient forager phenotypes (Nilsen et al., 2011).

To investigate how upstream components of the IIS pathway; AmIlp1, AmIlp2, and AmIRS respond to different macronutrients, we designed all-liquid diets that varied in their protein, lipid and carbohydrate ratios. We hypothesized that, acting as integrators of physiological responses to nutrient status, AmIlp1 and AmIlp2 would respond to dietary macronutrient imbalances in a manner consistent with their spatial expression in the fat body, with AmIlp1 correlated with specific nutrients related to lipoprotein synthesis, and AmIlp2 reflective of global nutrient status (Paes De Oliveira and Cruz-Landim, 2003, Ament et al., 2008, Nilsen et al., 2011). We also measured life-span, worker weight, and sucrose responsiveness, a measure of individual nutrient status or “hunger” (Page et al., 1998, Pankiw et al., 2001, De Brito Sanchez, 2011), to better understand the individual response of honey bee workers to imbalance in dietary macronutrients.