Julián F. Hillyer

Characterization of hemocytes from the yellow fever mosquito, Aedes aegypti.

Abstract. Mosquitoes are the most important arthropod disease vectors, transmitting a broad range of pathogens that cause diseases such as malaria, lymphatic filariasis, and yellow fever. Mosquitoes and other insects are able to mount powerful cellular and humoral immune responses against invading pathogens. To date, most studies have concentrated on the humoral response. In the current study we describe the hemocytes (blood cells) of the yellow fever mosquito, Aedes aegypti, by means of morphology, lectin binding, and enzyme activity and immunocytochemistry. Our light and electron microscopic studies suggest the presence of four distinct hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. We believe granulocytes and oenocytoids are true circulating hemocytes, but adipohemocytes and thrombocytoids are likely adhered to fixed tissues. Granulocytes, the most abundant cell type, have acid phosphatase and alpha-naphthyl acetate esterase activity, and bind the exogenous lectins WGA, HPA, and GNL. Phenoloxidase, an essential enzyme in the melanotic encapsulation immune response, was detected inside oenocytoids. This is, to our knowledge, the first report that has detected phenoloxidase inside mosquito hemocytes at the ultrastructural level. These results have begun to form a knowledge base for our ongoing studies on the function of Ae. aegypti hemocytes, and their involvement in controlling infections.

RAPID PHAGOCYTOSIS AND MELANIZATION OF BACTERIA AND PLASMODIUM SPOROZOITES BY HEMOCYTES OF THE MOSQUITO AEDES AEGYPTI

Mosquitoes are vectors of many deadly and debilitating pathogens. In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections. After challenge, mosquitoes mounted strong phagocytic and melanization responses. Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles. Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles. Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity. Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization. Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized. Similar to E. coli, most Plasmodium sporozoites were phagocytosed. The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation. The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.

Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers.

Mosquitoes vector pathogens. One aspect that has been overlooked in mosquito–pathogen relationships is the effect of host age on immune competence. Here, we show that there is age‐associated mortality following immune challenge with Escherichia coli. This mortality correlates with a decrease in haemocyte numbers (blood cells) and a decreased ability to kill E. coli. Although the number of haemocytes decreases, the available haemocytes retain their phagocytic ability regardless of age, and we estimate that individual granulocytes can phagocytose approximately 1500 E. coli. Moreover, transcription profiles for cecropin, defensin and gambicin in E. coli challenged mosquitoes do not change with age, indicating that the increased susceptibility is not attributed to fewer humoral antimicrobial peptides. These results suggest that a contributing factor for the age‐associated mortality is the decrease in circulating haemocytes, which reduces the overall phagocytic capacity of mosquitoes. To our knowledge, this is the first report detailing an age‐associated decline in the immunological capabilities of mosquitoes following challenge with an infectious agent. These data also call for caution in the analysis and interpretation of experimental results when mosquito age has not been closely monitored. Lastly, a model for haemocyte function is presented.

Complex effects of temperature on mosquito immune function

Over the last 20 years, ecological immunology has provided much insight into how environmental factors shape host immunity and host–parasite interactions. Currently, the application of this thinking to the study of mosquito immunology has been limited. Mechanistic investigations are nearly always conducted under one set of conditions, yet vectors and parasites associate in a variable world. We highlight how environmental temperature shapes cellular and humoral immune responses (melanization, phagocytosis and transcription of immune genes) in the malaria vector, Anopheles stephensi. Nitric oxide synthase expression peaked at 30°C, cecropin expression showed no main effect of temperature and humoral melanization, and phagocytosis and defensin expression peaked around 18°C. Further, immune responses did not simply scale with temperature, but showed complex interactions between temperature, time and nature of immune challenge. Thus, immune patterns observed under one set of conditions provide little basis for predicting patterns under even marginally different conditions. These quantitative and qualitative effects of temperature have largely been overlooked in vector biology but have significant implications for extrapolating natural/transgenic resistance mechanisms from laboratory to field and for the efficacy of various vector control tools.

Spatial and temporal in vivo analysis of circulating and sessile immune cells in mosquitoes: hemocyte mitosis following infection

BackgroundMosquitoes respond to infection by mounting immune responses. The primary regulators of these immune responses are cells called hemocytes, which kill pathogens via phagocytosis and via the production of soluble antimicrobial factors. Mosquito hemocytes are circulated throughout the hemocoel (body cavity) by the swift flow of hemolymph (blood), and data show that some hemocytes also exist as sessile cells that are attached to tissues. The purpose of this study was to create a quantitative physical map of hemocyte distribution in the mosquito, Anopheles gambiae, and to describe the cellular immune response in an organismal context.ResultsUsing correlative imaging methods we found that the number of hemocytes in a mosquito decreases with age, but that regardless of age, approximately 75% of the hemocytes occur in circulation and 25% occur as sessile cells. Infection induces an increase in the number of hemocytes, and tubulin and nuclear staining showed that this increase is primarily due to mitosis and, more specifically, autonomous cell division, by circulating granulocytes. The majority of sessile hemocytes are present on the abdominal wall, although significant numbers of hemocytes are also present in the thorax, head, and several of the appendages. Within the abdominal wall, the areas of highest hemocyte density are the periostial regions (regions surrounding the valves of the heart, or ostia), which are ideal locations for pathogen capture as these are areas of high hemolymph flow.ConclusionsThese data describe the spatial and temporal distribution of mosquito hemocytes, and map the cellular response to infection throughout the hemocoel.

Infection-Induced Interaction between the Mosquito Circulatory and Immune Systems

Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insects open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito, Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and β-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems.

Structural mechanics of the mosquito heart and its function in bidirectional hemolymph transport.

SUMMARY The insect circulatory system transports nutrients, signaling molecules, wastes and immune factors to all areas of the body. The primary organ driving circulation is the dorsal vessel, which consists of an abdominal heart and a thoracic aorta. Here, we present qualitative and quantitative data characterizing the heart of the mosquito, Anopheles gambiae. Visual observation showed that the heart of resting mosquitoes contracts at a rate of 1.37 Hz (82 beats per minute) and switches contraction direction, with 72% of contractions occurring in the anterograde direction (toward the head) and 28% of contractions occurring in the retrograde direction (toward the tip of the abdomen). The heart is tethered to the midline of the abdominal tergum by six complete and three incomplete pairs of alary muscles, and propels hemolymph at an average velocity of 8 mm s−1 by sequentially contracting muscle fibers oriented in a helical twist with respect to the lumen of the vessel. Hemolymph enters the heart through six pairs of incurrent abdominal ostia and one pair of ostia located at the thoraco-abdominal junction that receive hemolymph from the abdominal hemocoel and thoracic venous channels, respectively. The vessel expels hemolymph through distal excurrent openings located at the anterior end of the aorta and the posterior end of the heart. In conclusion, this study presents a comprehensive revision and expansion of our knowledge of the mosquito heart and for the first time quantifies hemolymph flow in an insect while observing dorsal vessel contractions.

Nitric oxide is an essential component of the hemocyte-mediated mosquito immune response against bacteria

Nitric oxide is a signaling and immune effector molecule synthesized by the enzyme nitric oxide synthase. In mosquitoes, nitric oxide functions as a parasite antagonist in the midgut but little is known about its function in the hemocoel. Here, we characterized the temporal and spatial expression of the Anopheles gambiae nitric oxide synthase gene and explored the role nitric oxide plays in the antibacterial response in the mosquito hemocoel. Quantitative PCR and Western blot analyses showed that nitric oxide synthase is expressed in hemocytes and fat body, and is upregulated in response to systemic infection with Escherichia coli and Micrococcus luteus. Diaphorase staining and immunofluorescence showed that nitric oxide synthase is abundant in the granulocyte subpopulation of hemocytes, and both the staining intensity and the percentage of cells that stain for nitric oxide synthase significantly increase after a bacterial challenge. When nitric oxide production was inhibited, the mosquitos ability to kill E. coli was significantly reduced. Accordingly, inhibiting nitric oxide production increased the mortality rate of mosquitoes with systemic E. coli infections. Taken altogether, these data show that nitric oxide is a crucial player in the antibacterial immune response in the mosquito hemocoel.

A novel lectin with a fibrinogen-like domain and its potential involvement in the innate immune response of Armigeres subalbatus against bacteria

Mosquitoes have an efficient cellular innate immune response that includes phagocytosis of microbial pathogens and encapsulation of metozoan parasites. In this study, we describe a novel lectin in the mosquito, Armigeres subalbatus (aslectin or AL‐1). The 1.27 kb cDNA clone for the AL‐1 gene (AL‐1) encodes a 279 deduced amino acid sequence that contains a C‐terminal fibrinogen‐like domain. AL‐1 is transcribed in all life stages. AL‐1 mainly exists in the haemolymph of adult female mosquitoes, and is upregulated following both Escherichia coli and Micrococcus luteus challenge. AL‐1 specifically recognizes N‐acetyl‐d‐glucosamine and is able to bind both E. coli and M. luteus. These results suggest that AL‐1 might function as a pattern recognition receptor in the immune response in Ar. subalbatus.

Correlative Instrumental Neutron Activation Analysis, Light Microscopy, Transmission Electron Microscopy, and X-ray Microanalysis for Qualitative and Quantitative Detection of Colloidal Gold Spheres in Biological Specimens

: Colloidal gold, conjugated to ligands or antibodies, is routinely used as a label for the detection of cell structures by light (LM) and electron microscopy (EM). To date, several methods to count the number of colloidal gold labels have been employed with limited success. Instrumental neutron activation analysis (INAA), a physical method for the analysis of the elemental composition of materials, can be used to provide a quantitative index of gold accumulation in bulk specimens. Given that gold is not naturally found in biological specimens in any substantial amount and that colloidal gold and ligand conjugates can be prepared to yield uniform bead sizes, the amount of label can be calculated in bulk biological samples by INAA. Here we describe the use of INAA, LM, transmission EM, and X-ray microanalysis (EDX) in a model to determine both distribution (localization) and amount of colloidal gold at the organ, tissue, cellular, and ultrastructural levels in whole animal systems following administration. In addition, the sensitivity for gold in biological specimens by INAA is compared with that of inductively coupled plasma-mass spectrometry (ICP-MS). The correlative use of INAA, LM, TEM, and EDX can be useful, for example, in the quantitative and qualitative tracking of various labeled molecular species following administration in vivo.