Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors

Abstract

Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. We report that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B-null Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution.DOI: http://dx.doi.org/10.7554/eLife.12241.001

Morphological and functional characterization of honey bee, Apis mellifera, hemocyte cell communities

“…Development and improvement of phagocytosis assays is an important step toward understanding the basic immune biology of honey bee hemocytes (Qi et al 2016). In addition to better understanding basic immune biology, phagocytosis assays can Figure 6: Basal honey bee adult granulocyte and plasmatocyte ROS production (a ) and larval hemocyte ROS production (b ) taken at 200× magnification.…”

“…To investigate the phagocytic activity of honey bee granulocytes, we used fluorophore-labeled K-12 strain Escherichia coli BioParticles (Alexa Fluor 488 conjugate, Thermo Fisher Scientific, Waltham, MA, USA) with a Trypan Blue quenched in vitro assay, similar to the methods of Qi et al (2016). For this, 40 μL of HyClone TNM-FH Insect Cell Culture Medium (Fisher Scientific, Hampton, NH, USA) was mixed with 10 μL of hemolymph and placed on the surface of a microscope slide.…”

“…A nitric oxide (NO) sensitive dye has been used to estimate NO production in honey bee hemocytes (Negri et al 2013, Negri et al 2014b); however, given the variety of oxidative species produced by immune cells, this method is not readily applicable to experiments concerning reactive oxygen species (ROS) such as superoxide (Fang 2004). While phagocytosis assays have been conducted using hemocytes from a breadth of arthropods (Castillo et al 2006;Fallon et al 2011;Qi et al 2016), we know of only one assay examining honey bee hemocyte phagocytosis published over 50 years ago (Gilliam and Shimanuki 1967). Given the critical immune functions of ROS production and phagocytosis in pathogen killing, cell signaling, and pathogen clearance (Haas 2007;Nunes et al 2013), further development of methods for interrogating immune function is warranted.…”

A mechanistic framework to explain the immunosuppressive effects of neurotoxic pesticides on bees

“…This strong connection between haemocytes and neurones is further supported by findings in the crayfish Procambarus clarkii , in which transdifferentiation of circulating haemocytes into functional neurones (without reverting to a pluripotent state) has recently been demonstrated (Benton et al., ). These findings are supported by the fact that haemocytes can synthesize neurotransmitters (Qi et al., ), possibly including acetylcholine (Pamminger, Basley, Goulson, & Hughes, ), and express a wide range of neuroendocrinological receptors, making them responsive to both hormonal and neurotransmitter signalling (Flatt et al., ; Rolff & Siva‐Jothy, ; Rus et al., ). These signalling pathways have been found to be essential to regulate haemocyte behaviours such as phagocytosis and their migratory behaviour, both during their maturation (differentiation) as well as during acute infections (Adamo, , ; Demas et al., ; Flatt et al., ; Hoffmann, ; Malagoli et al., ; Qi et al., ; Stefano, Cadet, & Scharrer, ; Stefano, Leung, Zhao, & Scharrer, ).…”

“…In vertebrates, complex immune functions are orchestrated by a well‐documented neuroendocrinological, innate immune‐regulatory axis involving a wide range of neurotransmitters and hormones (Sternberg, ). There is now evidence for a functionally similar axis in invertebrates utilizing at least two major insect hormones, juvenile hormone (Amdam et al., ; Flatt et al., ; Pamminger, Treanor, & Hughes, ; Rolff & Siva‐Jothy, ; Tian et al., ) and ecdysone (Rus et al., ; Sun, Shen, Zhou, & Zhang, ; Tan, Vlisidou, & Wood, ), as well as a range of neurotransmitters, including serotonin (Qi et al., ) and octopamine (Adamo, ; for a comprehensive summary see Adamo, and Sternberg, ). These findings indicate that key functions of the invertebrate innate immune system are under direct neuroendocrinological control (similar to vertebrates), not only enabling the systemic orchestration of immune responses during acute infections, but also likely facilitating immune homoeostasis and appropriate adjustments during internal (nutrient) and external (environmental) stress events (Adamo, , ; Buchon et al., ; Demas, Adamo, & French, ).…”