Differential expression of immunity-related genes in larval Manduca sexta tissues in response to gut and systemic infection.

Introduction: The midgut epithelium functions as tissue for nutrient uptake as well as physical barrier against pathogens. Additionally, it responds to pathogen contact by production and release of various factors including antimicrobial peptides, similar to the systemic innate immune response. However, if such a response is restricted to a local stimulus or if it appears in response to a systemic infection, too is a rather underexplored topic in insect immunity. We addressed the role of the midgut and the role of systemic immune tissues in the defense against gut-borne and systemic infections, respectively. Methods: Manduca sexta larvae were challenged with DAP-type peptidoglycan bacteria - Bacillus thuringiensis for local gut infection and Escherichia coli for systemic stimulation. We compared the immune response to both infection models by measuring mRNA levels of four selected immunity-related genes in midgut, fat body, hematopoietic organs (HOs), and hemocytes, and determined hemolymph antimicrobial activity. Hemocytes and HOs were tested for presence and distribution of lysozyme mRNA and protein. Results: The midgut and circulating hemocytes exhibited a significantly increased level of lysozyme mRNA in response to gut infection but did not significantly alter expression in response to a systemic infection. Conversely, fat body and HOs responded to both infection models by altered mRNA levels of at least one gene monitored. Most, but not all hemocytes and HO cells contain lysozyme mRNA and protein. Discussion: These data suggest that the gut recruits immune-related tissues in response to gut infection whereas systemic infections do not induce a response in the midgut. The experimental approach implies a skewed cross-talk: An intestinal infection triggers immune activity in systemic immune organs, while a systemic infection does not elicit any or only a restricted immune response in the midgut. The HOs, which form and release hemocytes in larval M. sexta, i) synthesize lysozyme, and ii) respond to immune challenges by increased immune gene expression. These findings strongly suggest that they not only provide phagocytes for the cellular immune response but also synthesize humoral immune components.

High-throughput screening of caterpillars as a platform to study host-microbe interactions and enteric immunity.

Mammalian models of human disease are expensive and subject to ethical restrictions. Here, we present an independent platform for high-throughput screening, using larvae of the tobacco hornworm Manduca sexta, combining diagnostic imaging modalities for a comprehensive characterization of aberrant phenotypes. For validation, we use bacterial/chemical-induced gut inflammation to generate a colitis-like phenotype and identify significant alterations in morphology, tissue properties, and intermediary metabolism, which aggravate with disease progression and can be rescued by antimicrobial treatment. In independent experiments, activation of the highly conserved NADPH oxidase DUOX, a key mediator of gut inflammation, leads to similar, dose-dependent alterations, which can be attenuated by pharmacological interventions. Furthermore, the developed platform could differentiate pathogens from mutualistic gastrointestinal bacteria broadening the scope of applications also to microbiomics and host-pathogen interactions. Overall, larvae-based screening can complement mammals in preclinical studies to explore innate immunity and host-pathogen interactions, thus representing a substantial contribution to improve mammalian welfare.

Characterization and mode of action analysis of black soldier fly (Hermetia illucens) larva-derived hemocytes.

With the growing importance of the black soldier fly (Hermetia illucens) for both sustainable food production and waste management as well as for science, a great demand of understanding its immune system arises. Here, we present the first description of the circulating larval hemocytes with special emphasis on uptake of microorganisms and distinguishing hemocyte types. With histological, zymographic, and cytometric methods and with a set of hemocyte binding lectins and antibodies, the hemocytes of H. illucens are identified as plasmatocytes, crystal cells, and putative prohemocytes. Total hemocyte counts (THC) are determined, and methods for THC determination are compared. Approximately 1100 hemocytes per microliter hemolymph are present in naive animals, while hemocyte density decreases dramatically shortly after wounding, indicating a role of hemocytes in response to wounding (and immune response in general). The determination of the relative abundance of each hemocyte type (differential hemocyte count, DHC) revealed that plasmatocytes are highly abundant, whereas prohemocytes and crystal cells make up only a small percentage of the circulating cells. Plasmatocytes are not only the most abundant but also the professional phagocytes in H. illucens. They rapidly engulf and take up bacteria both in vivo and in vitro, indicating a very potent cellular defense against invading pathogens. Larger bioparticles such as yeasts are also removed from circulation by phagocytosis, but slower than bacteria. This is the first analysis of the potent cellular immune response in the black soldier fly, and a first toolbox that helps to identify hemocyte (types) is presented.

The larval haematopoietic organs of Manduca sexta (Insecta, Lepidoptera): An insight into plasmatocyte development and larval haematopoiesis.

Haematopoietic organs (HOs) in Lepidoptera are widely recognised as the source for at least two haemocyte types. With new specific markers for oenocytoids and spherule cells and a method to identify prohaemocytes, the haemocytes formed in and released by the HOs of Manduca sexta are characterised. Differentiation of HO-cells to haemocytes other than plasmatocytes and prohaemocytes neither occurs in the organ itself nor in cells released in vitro by the HOs. Differential labelling patterns evidence the existence of plasmatocyte subpopulations and prohaemocytes, which might represent a gradual differentiation of haemocytes within the organs. Prohaemocytes can be identified by PNA-labelling of the cell membrane. These prohaemocytes are found in circulation and in the HOs and are released by the organs. Circulating prohaemocytes possess characteristics for granular cells, plasmatocytes or oenocytoids while HO derived prohaemocytes share characteristics only with plasmatocytes. Ablation of the HOs diminishes the plasmatocyte and prohaemocyte number, indicating a true larval haematopoietic function.

A novel site of haematopoiesis and appearance and dispersal of distinct haemocyte types in the Manduca sexta embryo (Insecta, Lepidoptera).

With a set of haemocyte specific markers novel findings on haematopoiesis in the Manduca sexta embryo are presented. We identify a hitherto unknown paired haematopoietic cluster, the abdominal haemocyte cluster in abdominal segment 7 (A7-HCC). These clusters are localised at distinct positions and are established at around katatrepsis. Later in embryogenesis, the A7-HCCs disintegrate, thereby releasing numerous embryonic plasmatocytes which disperse both anteriorly and posteriorly. These cells follow stereotypic migration routes projecting anteriorly. The thoracic larval haematopoietic organs are established at around midembryogenesis. We identify embryonic oenocytoids in the M. sexta embryo for the first time. They appear in the head region roughly at the same time as the A7-HCCs occur and successively disperse in the body cavity during development. Localisation of the prophenoloxidase (proPO) mRNA and of the proPO protein are identical. Morphological, cytometric and antigenic traits show three independently generated haemocyte types during embryogenesis.

Neurons of self-defence: neuronal innervation of the exocrine defence glands in stick insects.

BACKGROUND: Stick insects (Phasmatodea) use repellent chemical substances (allomones) for defence which are released from so-called defence glands in the prothorax. These glands differ in size between species, and are under neuronal control from the CNS. The detailed neural innervation and possible differences between species are not studied so far. Using axonal tracing, the neuronal innervation is investigated comparing four species. The aim is to document the complexity of defence gland innervation in peripheral nerves and central motoneurons in stick insects. RESULTS: In the species studied here, the defence gland is innervated by the intersegmental nerve complex (ISN) which is formed by three nerves from the prothoracic (T1) and suboesophageal ganglion (SOG), as well as a distinct suboesophageal nerve (Nervus anterior of the suboesophageal ganglion). In Carausius morosus and Sipyloidea sipylus, axonal tracing confirmed an innervation of the defence glands by this N. anterior SOG as well as N. anterior T1 and N. posterior SOG from the intersegmental nerve complex. In Peruphasma schultei, which has rather large defence glands, only the innervation by the N. anterior SOG was documented by axonal tracing. In the central nervous system of all species, 3-4 neuron types are identified by axonal tracing which send axons in the N. anterior SOG likely innervating the defence gland as well as adjacent muscles. These neurons are mainly suboesophageal neurons with one intersegmental neuron located in the prothoracic ganglion. The neuron types are conserved in the species studied, but the combination of neuron types is not identical. In addition, the central nervous system in S. sipylus contains one suboesophageal and one prothoracic neuron type with axons in the intersegmental nerve complex contacting the defence gland. CONCLUSIONS: Axonal tracing shows a very complex innervation pattern of the defence glands of Phasmatodea which contains different neurons in different nerves from two adjacent body segments. The gland size correlates to the size of a neuron soma in the suboesophageal ganglion, which likely controls gland contraction. In P. schultei, the innervation pattern appears simplified to the anterior suboesophageal nerve. Hence, some evolutionary changes are notable in a conserved neuronal network.