BACKGROUND
the insect immune response relies upon innate reactions and involves both cellular and humoral components  <cit> .
the immune cells, called hemocytes, exist within an open circulatory cavity called the hemocoel  <cit> , where they phagocytose foreign organisms and help coordinate the humoral response to infection  <cit> .
in mosquitoes, the immune response reduces the number of pathogens that survive inside the body  <cit>  and may either prevent the transmission of disease-causing pathogens or inadvertently maintain vectorial capacity by allowing the insect to survive long enough to transmit an infection.
thus, because of its effect on transmission dynamics, the mosquito immune response could be harnessed for the control of mosquito-borne diseases  <cit> .
in both the culicine and anopheline mosquito lineages there are several morphologically distinct classes of hemocytes: granulocytes are involved in the phagocytosis response, oenocytoids are involved in the melanization response, and prohemocytes are small cells of unknown function that have been hypothesized to serve as hematopoietic progenitors  <cit> .
while the sub-types of hemocytes that circulate within the mosquito hemocoel are known, basic aspects of hemocyte biology, such as hemocyte numbers, proliferation and spatial distribution, remain poorly understood.
for example, aside from a recent study on the interaction between hemocytes and the heart  <cit> , the location, activity and number of sessile hemocytes  remain unknown.
likewise, the number of circulating hemocytes within individual mosquitoes continues to be debated, as similar techniques have sometimes led to markedly dissimilar counts  <cit> .
nevertheless, a strong consensus indicates that naïve adult mosquitoes contain between  <dig>  and  <dig>  circulating hemocytes  <cit> , which is similar to the number of hemocytes present in the related dipteran, drosophila melanogaster <cit> .
in adult mosquitoes and other insects the number of circulating hemocytes changes in response to infection, age and physiological state  <cit> .
it has often been hypothesized that this change is due to either a release of sessile hemocytes into circulation, or the replication or differentiation of prohemocytes  <cit> .
however, in adult insects little evidence supports these hypotheses, as no hematopoietic organ has been identified in the adult stage  <cit> , and although the replication of circulating hemocytes has been visualized in insect larvae  <cit> , there is a scarcity of direct evidence showing that this also occurs in adults.
nevertheless, in mosquitoes the theory of hemocyte replication is strongly supported by bromodioxyuridine- and thymidine-based studies that have documented hemocyte proliferation somewhere in the body of adult aedes spp.
in response to blood feeding or infection with filarial nematodes, respectively  <cit> .
in the present study we scrutinize the organization of hemocytes within the entire body of the malaria mosquito, anopheles gambiae, and present the first quantitative hemocyte map of any insect.
this map describes the spatial distribution of all hemocytes and shows that approximately three quarters of mosquito hemocytes exist in circulation and one quarter exists as sessile cells.
furthermore, we show that the number of hemocytes decreases with age but increases after an immune challenge, and that the increase in hemocyte numbers following infection is primarily due to mitosis by circulating hemocytes.
RESULTS
circulating and sessile hemocyte numbers: effect of age and infection
examination of fluorescently labeled hemocytes revealed that at two days post-eclosion, naïve mosquitoes contain an average of  <dig>  circulating hemocytes and that this number decreases to  <dig>  and  <dig>  by six and sixteen days after emergence, respectively .
at two days post-eclosion, naïve mosquitoes contain an average of  <dig>  sessile hemocytes, and this number decreases to  <dig> and  <dig> by six and sixteen days after emergence, respectively .
thus, naïve mosquitoes contain an average of  <dig> ,  <dig>  and  <dig>  hemocytes at two, six and sixteen days post-eclosion .
together, these data signify that the number of hemocytes in naïve mosquitoes drops with age  analysis of variance  p < <dig> ) and that, regardless of age, sessile hemocytes comprise approximately 25% of the total hemocyte population .
at six and sixteen days post-eclosion, injury induces a 32% and 33% increase in the total number of hemocytes, respectively, and e. coli infection induces a 52% and 77% increase in the total number of hemocytes, respectively .
at two days post-eclosion, however, injury and e. coli infection induce a more modest 5% reduction and 12% increase in the total number of hemocytes, respectively.
statistical comparison of total hemocyte numbers in naïve, injured and e. coli infected mosquitoes by two-way  anova showed that, across the three treatment groups, the total number of hemocytes drops with age , which is exemplified by the dramatic reduction that occurs between days two and six post-eclosion.
two-way anova also revealed that treatment affects total hemocyte numbers , and post-hoc sidak’s multiple comparisons showed that this is due to a significant infection-induced increase in the total number of hemocytes .
no significant interaction was detected between age and treatment group .
independent analysis of circulating and sessile hemocyte numbers revealed that infection has a different effect on these two cell populations .
shared between circulating and sessile hemocytes is that, across all treatment groups, there is an age-specific reduction in the number of hemocytes .
however, although treatment does not have a significant effect on the number of sessile hemocytes , treatment induces a change in the number of circulating hemocytes .
sidak’s post-hoc analysis revealed that this treatment-induced change in circulating hemocyte numbers is due to an increase in the number of circulating cells following infection, which is especially pronounced in older mosquitoes .
finally, no significant interaction was detected between age and treatment group for either the circulating  or sessile  hemocyte populations.
together, these data show that the number of circulating and sessile hemocytes dramatically drops several days after eclosion, and that the infection-induced increase in total hemocyte numbers is primarily due to an increase in the number of hemocytes that circulate throughout the hemocoel.
the majority of sessile hemocytes exist attached to the abdominal epidermis, trachea, and heart-associated periostial regions
while a considerable amount of work has investigated the biology of mosquito circulating hemocytes, the location and activity of sessile hemocytes has received little or no attention.
systemic analysis of sessile hemocytes in a. gambiae revealed that they are consistently attached to the thoracic and abdominal cuticular epidermis, the visceral organs, and inside all appendages, with the exception of the antennae and halteres .
the anatomical location that contains the vast majority of sessile hemocytes is the abdominal wall, which in two-, six- and sixteen-day-old naïve mosquitoes contains an average of  <dig>   <dig> and  <dig> hemocytes , indicating that aging results in a 50% reduction in the number of abdominal sessile hemocytes .
in relation to the entire body, abdominal sessile hemocytes represent 78%, 71% and 65% of the total sessile hemocyte population at two, six and sixteen days post-eclosion, respectively.
in naïve mosquitoes of all ages, approximately two thirds of the abdominal sessile hemocytes are located in the dorsal abdomen and one-third in the ventral abdomen .
of the dorsal abdominal hemocytes, between 11% and 16% are present in the periostial regions  and, in both the dorsal and ventral abdomen, a large proportion of abdominal sessile hemocytes exist attached to the respiratory trachea .
while the ratio of dorsal versus ventral hemocytes is similar for all age groups, their spatial distribution changes with age.
specifically, abdominal sessile hemocytes are more widely dispersed in younger insects when compared to older insects .
in response to infection, the vast majority of abdominal sessile hemocytes phagocytose gfp-expressing e. coli, indicating that their ability to sequester pathogens is similar to that of circulating hemocytes .
however, at all ages tested, injury or infection did not induce changes in abdominal sessile hemocyte numbers with one notable exception: the periostial regions.
specifically, infection for  <dig> hours led to a 251%, 377% and 204% increase in the number of periostial hemocytes in two-, six-, and sixteen-day-old mosquitoes , numbers that are in agreement with our recent report detailing the physiological interaction between the mosquito circulatory and immune systems  <cit> .
in two-day-old mosquitoes, the increase in periostial hemocyte numbers was commonly coupled with the presence of melanization products inside the pericardial cells that line the heart .
sessile hemocytes exist attached to the indirect flight muscles and thoracic cuticular epidermis
a large proportion of the thorax is composed of the indirect flight muscles, and sessile hemocytes were found distributed throughout them with no discernible pattern .
light refraction by the myofibers prevented high resolution imaging of thoracic sessile hemocytes, but most of these cells had clearly phagocytosed bacteria following infection.
while these cells were immunologically active, injury or infection did not impact thoracic hemocyte numbers .
aging, on the other hand, impacted thoracic hemocyte numbers : two- and sixteen-day old mosquitoes contained more thoracic hemocytes than six-day-old mosquitoes .
overall, the number of thoracic hemocytes averaged  <dig> and  <dig> in naïve and infected mosquitoes, respectively, or 14% and 13% of the total number of sessile hemocytes .
sessile hemocytes exist inside the head, maxillary palps and legs
sessile hemocytes are present in all major appendages, except for the antennae and halteres.
hemocytes were present in the head, maxillary palps and legs of all individuals, and hemocytes in all of these locations were highly phagocytic .
regardless of age or treatment, mosquitoes had approximately  <dig> hemocytes in the head, and these were most commonly found near the neck and around the base of each antenna .
the maxillary palps also contained approximately  <dig> hemocytes, which in two-day-old mosquitoes were most commonly located near the base of these sensory appendages while in older mosquitoes were randomly distributed across their entire length .
finally, all mosquitoes contained hemocytes in their legs, but the number of cells in these appendages dramatically changed with age : the legs of naïve mosquitoes at two, six and sixteen days post-eclosion contained an average of  <dig>   <dig> and  <dig> hemocytes, respectively .
this age-associated doubling of leg hemocytes is even more pronounced when considering that the total number of sessile and circulating hemocytes decreases with age .
finally, injury or infection had no effect on the number of hemocytes inside the head, palps, and legs .
few hemocytes were observed inside the wings: only 17%, 10% and 26% of two-, six- and sixteen-day-old mosquitoes contained hemocytes inside the wings, and when observed, the average number of cells was < <dig>  per mosquito .
also, while hemocytes were observed at the base of the antennae and halteres, they were not observed inside of these appendages .
small numbers of hemocytes exist attached to the midgut and malpighian tubules
the midgut and malpighian tubules had a small but variable number of sessile hemocytes, and no clear pattern was observed between any age or treatment groups .
an average of  <dig> hemocytes per mosquito were scattered along the basal surface of both of these two organs, and a high proportion of these hemocytes were phagocytic.
hemocytes may be involved in developmental processes
mosquito hemocytes have largely been studied because of their role in immunity.
however, a series of labeling experiments suggest that they may also be involved in development.
first, in vivo hemocyte staining showed that abdominal sessile hemocytes are more prevalent in newly emerged mosquitoes when compared to older mosquitoes, and that in young mosquitoes they are evenly dispersed throughout the abdominal wall .
second, muscle staining revealed that at one to two days post-emergence, the histolysis of larval swimming muscles has not been completed .
finally, co-staining of hemocytes and muscle revealed that at one to two days post-emergence some of the abdominal sessile hemocytes had internalized muscle fibers and pyknotic nuclear materials that were likely remnants of the larval swimming muscles .
muscle degradation by sessile hemocytes was not observed at days six and sixteen post-emergence, a time when all larval swimming muscles had been completely broken down.
thus, hemocytes may be involved in shaping the internal architecture of adult mosquitoes during the first few days after eclosion.
infection induces mitosis in circulating hemocytes
during this study we observed that infection induces an increase in hemocyte numbers.
the increase in hemocyte numbers is due to an increase in circulating cells, and visual examination of cm-dii/hoechst  <dig> stained hemocytes suggested that a small proportion of circulating cells were undergoing cell division.
for example, some cells contained two nuclei, and the two nuclei at times appeared to be interconnected .
to test whether hemocytes divide in circulation, we exposed hemocytes to compounds that delay or halt mitosis and directly measured cell replication using tubulin and nuclear staining.
as an initial assay, mosquitoes were treated with colchicine, and hemocytes were then perfused, fixed, and labeled with anti-tubulin antibody and hoechst  <dig>  because colchicine binds tubulin and interferes with microtubule polymerization, thus slowing down mitosis and enriching the number of mitotic cells, we reasoned that if hemocyte replication were occurring then treatment with this chemical would allow for the direct visualization of mitotic events.
indeed, a small percentage of hemocytes were observed undergoing mitosis, with the mitotic stages of prophase, metaphase, anaphase and telophase, as well as cytokinesis, all represented .
most mitotic events led to two daughter cells of similar size .
however, grossly asymmetric cytokinesis was also observed .
the smaller cells resulting from these asymmetric divisions were similar in size to the small hemocytes commonly referred to as prohemocytes , and together with the observation that these smaller cells are phagocytic, these data suggest that what has been previously called a prohemocyte could represent a small form of a granulocyte.
finally, the vast majority of hemocytes that were undergoing mitosis had phagocytosed bacteria, indicating that replication is induced in immunologically active cells .
while the detection of any mitotic cell in a static tissue is often considered as a sign of considerable cellular proliferation, we set out to measure more accurately the rate of hemocyte mitosis.
to quantify mitotic events, hemocytes were treated with taxol, a chemical that stabilizes microtubules, leading to the arrest of mitosis and the production of highly condensed microtubule asters.
as expected, taxol treatment confirmed that circulating hemocytes undergo replication and showed that the rate of replication increases in response to infection .
specifically,  <dig> % of circulating hemocytes from five-day-old naïve mosquitoes were observed undergoing mitosis.
this mitotic index increased to  <dig> % by nine hours post e. coli infection and then dropped to around  <dig> % by  <dig> and  <dig> hours post-infection.
at least one mitotic cell was observed in  half of the naïve mosquitoes examined,  100% of the infected mosquitoes assayed at six and nine hours post-infection, and  60% to 70% of infected mosquitoes assayed at the other time points.
the average mitotic index of all samples collected after infection was  <dig> %.
assuming that the process of hemocyte mitosis in mosquitoes occurs at roughly the same speed as mitosis in hemocyte-like drosophila s <dig> cells , autonomous cell division in mosquitoes accounts for an approximately 18% increase in hemocyte numbers by  <dig> hours of infection.
considering that our method only allowed for the reliable identification of mitotic cells from pro-metaphase onward, we estimate that the actual increase in hemocyte numbers approaches 25% per day.
cytoskeleton rearrangement occurs during hemocyte-mediated immune responses
while examining hemocytes for mitotic events, we noticed that arrangement of microtubules was different in a small proportion of the larger phagocytic cells when compared to the smaller phagocytic cells, suggesting that cytoskeleton dynamics are important during the phagocytosis response.
specifically, some large and heavily phagocytic hemocytes were more rounded and contained a dense ring of tubulin around their margins , whereas most hemocytes  from naïve and infected mosquitoes had a cytoskeleton that was crosshatched or radial in appearance .
perhaps hemocytes with dense tubulin rings are those that are engaging in the ‘pooled’ phagocytosis process previously described in aedes aegypti <cit> .
discussion
insects lack adaptive  immunity, as classically defined  <cit> .
however, with an innate immune system they have filled virtually every imaginable terrestrial and freshwater niche, and by many measures have become the most successful group of multicellular organisms  <cit> .
the insect immune system is composed of both cellular and humoral factors and relies upon the actions of several types of immune cells called hemocytes  <cit> .
these cells exist within an open circulatory cavity  <cit> , where they phagocytose and encapsulate foreign elements and help coordinate the humoral response to infection  <cit> .
because insects are of paramount economic and medical importance, it is surprising that major gaps still exist in our basic knowledge of hemocyte biology.
among these gaps are the numbers of hemocytes present in mosquitoes, as well as their spatial distribution.
specifically, the number of circulating hemocytes in mosquitoes continues to be debated  <cit> , and until this study, the spatial distribution of sessile hemocytes within the hemocoel had not been addressed.
aging and immune stimuli are known to impact circulating hemocyte numbers in multiple insect species  <cit> , including mosquitoes  <cit> .
it has often been assumed that the release of hemocytes from a discrete hematopoietic organ, or the replication of a progenitor cell type known as the prohemocyte, leads to increases in circulating hemocyte numbers.
however, no hematopoietic organ has been found in any adult insect, the release of sessile hemocytes into circulation has not been reported in this life stage and there is a scarcity of data on the replication of circulating hemocytes in adult insects  <cit> .
using novel techniques, we herein present the first quantitative map of hemocyte distribution in any insect.
along with describing the spatial increases in hemocyte numbers following infection and the decreases in hemocyte numbers associated with aging, we also report direct evidence of mitosis by mosquito circulating hemocytes.
a variety of techniques have been employed to study hemocyte biology.
hemocytes are usually collected from circulating populations by some variation of hemolymph perfusion or extraction  <cit> , and most prior studies have relied on ex vivo examination of live or fixed cells, although flow cytometry has also been used  <cit> .
in vivo studies have been conducted in larval drosophila <cit> , but with one exception  <cit> , no direct in vivo examination of hemocytes has been conducted in non-drosophilids.
here, using cm-dii to stain hemocytes in vivo we quantified the number of circulating hemocytes and the distribution and numbers of sessile hemocytes within the entire mosquito.
we found that sessile hemocytes form a substantial proportion  of the total hemocyte population and are heavily phagocytic.
many of the sessile hemocytes in the abdomen, the compartment of the hemocoel where they are most abundant, are found attached to the trachea or near the ostia of the heart .
we hypothesize that aggregation near the ostia and trachea represents an adaptation for increased immune surveillance, as this places hemocytes in areas of high hemolymph flow and in areas of potential pathogen entry, respectively.
indeed, immune factors are transcribed in heart-associated tissues  <cit> , and we hypothesize that these factors are produced by immunologically active periostial hemocytes   <cit> .
finally, in addition to being a strategic site for pathogen capture, hemocyte positioning at tracheal sites could also enhance their oxygen supply, as has been hypothesized in lepidopteran larvae  <cit> .
total hemocyte numbers decline over the first six days of an adult mosquito’s life, as had been previously shown in the circulating hemocytes of aedes aegypti and anopheles gambiae <cit> .
the reason for this decline is not clear, but it may be related to development.
specifically, in d. melanogaster hemocytes are involved in the digestion of apoptotic cells during ecdysis-associated tissue remodeling  <cit> , a finding that is consistent with our observation that hemocytes degrade larval swimming muscles following eclosion.
in d. melanogaster hemocytes are known to originate from two distinct cell lineages  <cit> .
in larvae, embryonic hemocytes occur as immunologically active sentinel cells, while lymph gland derived hemocytes are deployed following an immune challenge  <cit>  and, presumably, during the process of lymph gland degeneration that occurs during eclosion.
we hypothesize that the dramatic age-specific decline in hemocyte numbers seen in this study could be due to lineage-specific hemocyte apoptosis following the completion of ecdysis-associated tissue remodeling.
circulating hemocyte counts in a. gambiae indirectly support this hypothesis, as pupae contain considerably more hemocytes than three-day-old adults  <cit> .
multiple studies have reported that circulating hemocytes increase in number following immune stimulation  <cit> .
our circulating hemocyte data are in agreement with these findings, but our data on sessile hemocytes show that the only locations where infection induces a consistent and significant increase in sessile hemocytes are the periostial regions.
we have previously shown that this increase in periostial hemocytes is due to the adhesive capture and subsequent migration of circulating hemocytes  <cit> , which suggests that hemocyte replication in adult mosquitoes occurs in circulation.
in the present study, tubulin staining of hemocytes showed that a small proportion of circulating hemocytes continuously undergo mitosis, and that the proportion of mitotic hemocytes increases following infection.
as stated by wieder  <cit> , tubulin-based mitotic assays represent the ‘one true direct measure of cellular proliferation’, although this procedure often underestimates the rate of mitosis.
therefore, the mitotic indices reported here should be viewed as a conservative measure of mitosis.
regardless, our results suggest that hemocytes are capable of proliferating by autonomous division.
furthermore, because all phases of mitosis are observed in circulation, the data strongly suggest that the increases in circulating hemocyte numbers following infection are primarily due to mitosis by already circulating cells.
finally, based on the likely division time of hemocytes  and our measured mitotic indices, proliferation of circulating hemocytes in infected mosquitoes explains a substantial proportion of the proliferation seen in our systemic measures.
whether replication also occurs within the sessile hemocyte population remains unknown, but no hematopoietic organ was identified during the course of this study.
in adult insects it has been hypothesized that new hemocytes arise from the replication and differentiation of a hemocyte morphotype known as the prohemocyte  <cit> .
hemocytes matching this physical description  were commonly seen during this study.
however, our observation of these prohemocytes ‘budding’ from larger ones  suggests that this hypothesis is incorrect and, furthermore, that the opposite might be true: we hypothesize that small granulocytes  that are immunologically active  are produced from mature granulocytes .
the phagocytic nature of the small hemocytes observed in this study is in conflict with the classical definition of prohemocytes  <cit> , but our finding is loosely in agreement with the view that prohemocytes are not multipotent stem cells but are instead fate restricted  <cit> .
ultrastructural evidence supports our hypothesis by showing that the cell types are very similar in subcellular structure  <cit> .
furthermore, hemocytes are bathed in a nutrient rich medium and are known to increase in size upon immune stimulus  <cit> .
therefore, it would not be surprising if these smaller daughter cells are produced with a minimum of sacrifice to the mother cell but are able to mature into capable immune cells in a relatively short period of time.
recent data from mammalian systems show that asymmetric partitioning of cellular components between daughter cells is common and purposeful  <cit> .
the authors propose that certain types of asymmetric mitosis might represent a physiological mechanism for producing a ‘pristine’ daughter cell from a mother cell that contains abundant waste.
we speculate that asymmetric mitosis could play a role in maintaining a healthy hemocyte population following a phagocytic response to infection.
CONCLUSIONS
in conclusion, the data presented herein represent three advancements in our understanding of insect hemocyte biology.
first, by creating the first insect quantitative hemocyte map, we describe the anatomical distribution of hemocytes and show that sessile hemocytes form a major component of the mosquito immune system.
second, by performing qualitative and quantitative analyses in different physiological states, we show that the number of hemocytes changes significantly with age and in response to infection.
lastly, we present direct evidence of mitosis in the circulating hemocytes of an adult insect, showing that hemocyte proliferation in adult mosquitoes does not require a discrete organ or progenitor cell type.

 the insect immune response relies upon innate reactions and involves both cellular and humoral components. the immune cells, called hemocytes, exist within an open circulatory cavity called the hemocoel, where they phagocytose foreign organisms and help coordinate the humoral response to infection. in mosquitoes, the immune response reduces the number of pathogens that survive inside the body and may either prevent the transmission of disease-causing pathogens or inadvertently maintain vectorial capacity by allowing the insect to survive long enough to transmit an infection. thus, because of its effect on transmission dynamics, the mosquito immune response could be harnessed for the control of mosquito-borne diseases. in both the culicine and anopheline mosquito lineages there are several morphologically distinct classes of hemocytes: granulocytes are involved in the phagocytosis response, oenocytoids are involved in the melanization response, and prohemocytes are small cells of unknown function that have been hypothesized to serve as hematopoietic progenitors. while the sub-types of hemocytes that circulate within the mosquito hemocoel are known, basic aspects of hemocyte biology, such as hemocyte numbers, proliferation and spatial distribution, remain poorly understood. for example, aside from a recent study on the interaction between hemocytes and the heart, the location, activity and number of sessile hemocytes remain unknown. likewise, the number of circulating hemocytes within individual mosquitoes continues to be debated, as similar techniques have sometimes led to markedly dissimilar counts. nevertheless, a strong consensus indicates that naïve adult mosquitoes contain between and circulating hemocytes, which is similar to the number of hemocytes present in the related dipteran, drosophila melanogaster. in adult mosquitoes and other insects the number of circulating hemocytes changes in response to infection, age and physiological state. it has often been hypothesized that this change is due to either a release of sessile hemocytes into circulation, or the replication or differentiation of prohemocytes. however, in adult insects little evidence supports these hypotheses, as no hematopoietic organ has been identified in the adult stage, and although the replication of circulating hemocytes has been visualized in insect larvae, there is a scarcity of direct evidence showing that this also occurs in adults. nevertheless, in mosquitoes the theory of hemocyte replication is strongly supported by bromodioxyuridine- and thymidine-based studies that have documented hemocyte proliferation somewhere in the body of adult aedes spp. in response to blood feeding or infection with filarial nematodes, respectively. in the present study we scrutinize the organization of hemocytes within the entire body of the malaria mosquito, anopheles gambiae, and present the first quantitative hemocyte map of any insect. this map describes the spatial distribution of all hemocytes and shows that approximately three quarters of mosquito hemocytes exist in circulation and one quarter exists as sessile cells. furthermore, we show that the number of hemocytes decreases with age but increases after an immune challenge, and that the increase in hemocyte numbers following infection is primarily due to mitosis by circulating hemocytes.  circulating and sessile hemocyte numbers: effect of age and infection examination of fluorescently labeled hemocytes revealed that at two days post-eclosion, naïve mosquitoes contain an average of circulating hemocytes and that this number decreases to and by six and sixteen days after emergence, respectively. at two days post-eclosion, naïve mosquitoes contain an average of sessile hemocytes, and this number decreases to and by six and sixteen days after emergence, respectively. thus, naïve mosquitoes contain an average of, and hemocytes at two, six and sixteen days post-eclosion. together, these data signify that the number of hemocytes in naïve mosquitoes drops with age analysis of variance p < ) and that, regardless of age, sessile hemocytes comprise approximately 25% of the total hemocyte population. at six and sixteen days post-eclosion, injury induces a 32% and 33% increase in the total number of hemocytes, respectively, and e. coli infection induces a 52% and 77% increase in the total number of hemocytes, respectively. at two days post-eclosion, however, injury and e. coli infection induce a more modest 5% reduction and 12% increase in the total number of hemocytes, respectively. statistical comparison of total hemocyte numbers in naïve, injured and e. coli infected mosquitoes by two-way anova showed that, across the three treatment groups, the total number of hemocytes drops with age, which is exemplified by the dramatic reduction that occurs between days two and six post-eclosion. two-way anova also revealed that treatment affects total hemocyte numbers, and post-hoc sidak’s multiple comparisons showed that this is due to a significant infection-induced increase in the total number of hemocytes. no significant interaction was detected between age and treatment group. independent analysis of circulating and sessile hemocyte numbers revealed that infection has a different effect on these two cell populations. shared between circulating and sessile hemocytes is that, across all treatment groups, there is an age-specific reduction in the number of hemocytes. however, although treatment does not have a significant effect on the number of sessile hemocytes, treatment induces a change in the number of circulating hemocytes. sidak’s post-hoc analysis revealed that this treatment-induced change in circulating hemocyte numbers is due to an increase in the number of circulating cells following infection, which is especially pronounced in older mosquitoes. finally, no significant interaction was detected between age and treatment group for either the circulating or sessile hemocyte populations. together, these data show that the number of circulating and sessile hemocytes dramatically drops several days after eclosion, and that the infection-induced increase in total hemocyte numbers is primarily due to an increase in the number of hemocytes that circulate throughout the hemocoel. the majority of sessile hemocytes exist attached to the abdominal epidermis, trachea, and heart-associated periostial regions while a considerable amount of work has investigated the biology of mosquito circulating hemocytes, the location and activity of sessile hemocytes has received little or no attention. systemic analysis of sessile hemocytes in a. gambiae revealed that they are consistently attached to the thoracic and abdominal cuticular epidermis, the visceral organs, and inside all appendages, with the exception of the antennae and halteres. the anatomical location that contains the vast majority of sessile hemocytes is the abdominal wall, which in two-, six- and sixteen-day-old naïve mosquitoes contains an average of and hemocytes, indicating that aging results in a 50% reduction in the number of abdominal sessile hemocytes. in relation to the entire body, abdominal sessile hemocytes represent 78%, 71% and 65% of the total sessile hemocyte population at two, six and sixteen days post-eclosion, respectively. in naïve mosquitoes of all ages, approximately two thirds of the abdominal sessile hemocytes are located in the dorsal abdomen and one-third in the ventral abdomen. of the dorsal abdominal hemocytes, between 11% and 16% are present in the periostial regions and, in both the dorsal and ventral abdomen, a large proportion of abdominal sessile hemocytes exist attached to the respiratory trachea. while the ratio of dorsal versus ventral hemocytes is similar for all age groups, their spatial distribution changes with age. specifically, abdominal sessile hemocytes are more widely dispersed in younger insects when compared to older insects. in response to infection, the vast majority of abdominal sessile hemocytes phagocytose gfp-expressing e. coli, indicating that their ability to sequester pathogens is similar to that of circulating hemocytes. however, at all ages tested, injury or infection did not induce changes in abdominal sessile hemocyte numbers with one notable exception: the periostial regions. specifically, infection for hours led to a 251%, 377% and 204% increase in the number of periostial hemocytes in two-, six-, and sixteen-day-old mosquitoes, numbers that are in agreement with our recent report detailing the physiological interaction between the mosquito circulatory and immune systems. in two-day-old mosquitoes, the increase in periostial hemocyte numbers was commonly coupled with the presence of melanization products inside the pericardial cells that line the heart. sessile hemocytes exist attached to the indirect flight muscles and thoracic cuticular epidermis a large proportion of the thorax is composed of the indirect flight muscles, and sessile hemocytes were found distributed throughout them with no discernible pattern. light refraction by the myofibers prevented high resolution imaging of thoracic sessile hemocytes, but most of these cells had clearly phagocytosed bacteria following infection. while these cells were immunologically active, injury or infection did not impact thoracic hemocyte numbers. aging, on the other hand, impacted thoracic hemocyte numbers: two- and sixteen-day old mosquitoes contained more thoracic hemocytes than six-day-old mosquitoes. overall, the number of thoracic hemocytes averaged and in naïve and infected mosquitoes, respectively, or 14% and 13% of the total number of sessile hemocytes. sessile hemocytes exist inside the head, maxillary palps and legs sessile hemocytes are present in all major appendages, except for the antennae and halteres. hemocytes were present in the head, maxillary palps and legs of all individuals, and hemocytes in all of these locations were highly phagocytic. regardless of age or treatment, mosquitoes had approximately hemocytes in the head, and these were most commonly found near the neck and around the base of each antenna. the maxillary palps also contained approximately hemocytes, which in two-day-old mosquitoes were most commonly located near the base of these sensory appendages while in older mosquitoes were randomly distributed across their entire length. finally, all mosquitoes contained hemocytes in their legs, but the number of cells in these appendages dramatically changed with age: the legs of naïve mosquitoes at two, six and sixteen days post-eclosion contained an average of and hemocytes, respectively. this age-associated doubling of leg hemocytes is even more pronounced when considering that the total number of sessile and circulating hemocytes decreases with age. finally, injury or infection had no effect on the number of hemocytes inside the head, palps, and legs. few hemocytes were observed inside the wings: only 17%, 10% and 26% of two-, six- and sixteen-day-old mosquitoes contained hemocytes inside the wings, and when observed, the average number of cells was < per mosquito. also, while hemocytes were observed at the base of the antennae and halteres, they were not observed inside of these appendages. small numbers of hemocytes exist attached to the midgut and malpighian tubules the midgut and malpighian tubules had a small but variable number of sessile hemocytes, and no clear pattern was observed between any age or treatment groups. an average of hemocytes per mosquito were scattered along the basal surface of both of these two organs, and a high proportion of these hemocytes were phagocytic. hemocytes may be involved in developmental processes mosquito hemocytes have largely been studied because of their role in immunity. however, a series of labeling experiments suggest that they may also be involved in development. first, in vivo hemocyte staining showed that abdominal sessile hemocytes are more prevalent in newly emerged mosquitoes when compared to older mosquitoes, and that in young mosquitoes they are evenly dispersed throughout the abdominal wall. second, muscle staining revealed that at one to two days post-emergence, the histolysis of larval swimming muscles has not been completed. finally, co-staining of hemocytes and muscle revealed that at one to two days post-emergence some of the abdominal sessile hemocytes had internalized muscle fibers and pyknotic nuclear materials that were likely remnants of the larval swimming muscles. muscle degradation by sessile hemocytes was not observed at days six and sixteen post-emergence, a time when all larval swimming muscles had been completely broken down. thus, hemocytes may be involved in shaping the internal architecture of adult mosquitoes during the first few days after eclosion. infection induces mitosis in circulating hemocytes during this study we observed that infection induces an increase in hemocyte numbers. the increase in hemocyte numbers is due to an increase in circulating cells, and visual examination of cm-dii/hoechst stained hemocytes suggested that a small proportion of circulating cells were undergoing cell division. for example, some cells contained two nuclei, and the two nuclei at times appeared to be interconnected. to test whether hemocytes divide in circulation, we exposed hemocytes to compounds that delay or halt mitosis and directly measured cell replication using tubulin and nuclear staining. as an initial assay, mosquitoes were treated with colchicine, and hemocytes were then perfused, fixed, and labeled with anti-tubulin antibody and hoechst because colchicine binds tubulin and interferes with microtubule polymerization, thus slowing down mitosis and enriching the number of mitotic cells, we reasoned that if hemocyte replication were occurring then treatment with this chemical would allow for the direct visualization of mitotic events. indeed, a small percentage of hemocytes were observed undergoing mitosis, with the mitotic stages of prophase, metaphase, anaphase and telophase, as well as cytokinesis, all represented. most mitotic events led to two daughter cells of similar size. however, grossly asymmetric cytokinesis was also observed. the smaller cells resulting from these asymmetric divisions were similar in size to the small hemocytes commonly referred to as prohemocytes, and together with the observation that these smaller cells are phagocytic, these data suggest that what has been previously called a prohemocyte could represent a small form of a granulocyte. finally, the vast majority of hemocytes that were undergoing mitosis had phagocytosed bacteria, indicating that replication is induced in immunologically active cells. while the detection of any mitotic cell in a static tissue is often considered as a sign of considerable cellular proliferation, we set out to measure more accurately the rate of hemocyte mitosis. to quantify mitotic events, hemocytes were treated with taxol, a chemical that stabilizes microtubules, leading to the arrest of mitosis and the production of highly condensed microtubule asters. as expected, taxol treatment confirmed that circulating hemocytes undergo replication and showed that the rate of replication increases in response to infection. specifically, % of circulating hemocytes from five-day-old naïve mosquitoes were observed undergoing mitosis. this mitotic index increased to % by nine hours post e. coli infection and then dropped to around % by and hours post-infection. at least one mitotic cell was observed in half of the naïve mosquitoes examined, 100% of the infected mosquitoes assayed at six and nine hours post-infection, and 60% to 70% of infected mosquitoes assayed at the other time points. the average mitotic index of all samples collected after infection was %. assuming that the process of hemocyte mitosis in mosquitoes occurs at roughly the same speed as mitosis in hemocyte-like drosophila s cells, autonomous cell division in mosquitoes accounts for an approximately 18% increase in hemocyte numbers by hours of infection. considering that our method only allowed for the reliable identification of mitotic cells from pro-metaphase onward, we estimate that the actual increase in hemocyte numbers approaches 25% per day. cytoskeleton rearrangement occurs during hemocyte-mediated immune responses while examining hemocytes for mitotic events, we noticed that arrangement of microtubules was different in a small proportion of the larger phagocytic cells when compared to the smaller phagocytic cells, suggesting that cytoskeleton dynamics are important during the phagocytosis response. specifically, some large and heavily phagocytic hemocytes were more rounded and contained a dense ring of tubulin around their margins, whereas most hemocytes from naïve and infected mosquitoes had a cytoskeleton that was crosshatched or radial in appearance. perhaps hemocytes with dense tubulin rings are those that are engaging in the ‘pooled’ phagocytosis process previously described in aedes aegypti. discussion insects lack adaptive immunity, as classically defined. however, with an innate immune system they have filled virtually every imaginable terrestrial and freshwater niche, and by many measures have become the most successful group of multicellular organisms. the insect immune system is composed of both cellular and humoral factors and relies upon the actions of several types of immune cells called hemocytes. these cells exist within an open circulatory cavity, where they phagocytose and encapsulate foreign elements and help coordinate the humoral response to infection. because insects are of paramount economic and medical importance, it is surprising that major gaps still exist in our basic knowledge of hemocyte biology. among these gaps are the numbers of hemocytes present in mosquitoes, as well as their spatial distribution. specifically, the number of circulating hemocytes in mosquitoes continues to be debated, and until this study, the spatial distribution of sessile hemocytes within the hemocoel had not been addressed. aging and immune stimuli are known to impact circulating hemocyte numbers in multiple insect species, including mosquitoes. it has often been assumed that the release of hemocytes from a discrete hematopoietic organ, or the replication of a progenitor cell type known as the prohemocyte, leads to increases in circulating hemocyte numbers. however, no hematopoietic organ has been found in any adult insect, the release of sessile hemocytes into circulation has not been reported in this life stage and there is a scarcity of data on the replication of circulating hemocytes in adult insects. using novel techniques, we herein present the first quantitative map of hemocyte distribution in any insect. along with describing the spatial increases in hemocyte numbers following infection and the decreases in hemocyte numbers associated with aging, we also report direct evidence of mitosis by mosquito circulating hemocytes. a variety of techniques have been employed to study hemocyte biology. hemocytes are usually collected from circulating populations by some variation of hemolymph perfusion or extraction, and most prior studies have relied on ex vivo examination of live or fixed cells, although flow cytometry has also been used. in vivo studies have been conducted in larval drosophila, but with one exception, no direct in vivo examination of hemocytes has been conducted in non-drosophilids. here, using cm-dii to stain hemocytes in vivo we quantified the number of circulating hemocytes and the distribution and numbers of sessile hemocytes within the entire mosquito. we found that sessile hemocytes form a substantial proportion of the total hemocyte population and are heavily phagocytic. many of the sessile hemocytes in the abdomen, the compartment of the hemocoel where they are most abundant, are found attached to the trachea or near the ostia of the heart. we hypothesize that aggregation near the ostia and trachea represents an adaptation for increased immune surveillance, as this places hemocytes in areas of high hemolymph flow and in areas of potential pathogen entry, respectively. indeed, immune factors are transcribed in heart-associated tissues, and we hypothesize that these factors are produced by immunologically active periostial hemocytes. finally, in addition to being a strategic site for pathogen capture, hemocyte positioning at tracheal sites could also enhance their oxygen supply, as has been hypothesized in lepidopteran larvae. total hemocyte numbers decline over the first six days of an adult mosquito’s life, as had been previously shown in the circulating hemocytes of aedes aegypti and anopheles gambiae. the reason for this decline is not clear, but it may be related to development. specifically, in d. melanogaster hemocytes are involved in the digestion of apoptotic cells during ecdysis-associated tissue remodeling, a finding that is consistent with our observation that hemocytes degrade larval swimming muscles following eclosion. in d. melanogaster hemocytes are known to originate from two distinct cell lineages. in larvae, embryonic hemocytes occur as immunologically active sentinel cells, while lymph gland derived hemocytes are deployed following an immune challenge and, presumably, during the process of lymph gland degeneration that occurs during eclosion. we hypothesize that the dramatic age-specific decline in hemocyte numbers seen in this study could be due to lineage-specific hemocyte apoptosis following the completion of ecdysis-associated tissue remodeling. circulating hemocyte counts in a. gambiae indirectly support this hypothesis, as pupae contain considerably more hemocytes than three-day-old adults. multiple studies have reported that circulating hemocytes increase in number following immune stimulation. our circulating hemocyte data are in agreement with these findings, but our data on sessile hemocytes show that the only locations where infection induces a consistent and significant increase in sessile hemocytes are the periostial regions. we have previously shown that this increase in periostial hemocytes is due to the adhesive capture and subsequent migration of circulating hemocytes, which suggests that hemocyte replication in adult mosquitoes occurs in circulation. in the present study, tubulin staining of hemocytes showed that a small proportion of circulating hemocytes continuously undergo mitosis, and that the proportion of mitotic hemocytes increases following infection. as stated by wieder, tubulin-based mitotic assays represent the ‘one true direct measure of cellular proliferation’, although this procedure often underestimates the rate of mitosis. therefore, the mitotic indices reported here should be viewed as a conservative measure of mitosis. regardless, our results suggest that hemocytes are capable of proliferating by autonomous division. furthermore, because all phases of mitosis are observed in circulation, the data strongly suggest that the increases in circulating hemocyte numbers following infection are primarily due to mitosis by already circulating cells. finally, based on the likely division time of hemocytes and our measured mitotic indices, proliferation of circulating hemocytes in infected mosquitoes explains a substantial proportion of the proliferation seen in our systemic measures. whether replication also occurs within the sessile hemocyte population remains unknown, but no hematopoietic organ was identified during the course of this study. in adult insects it has been hypothesized that new hemocytes arise from the replication and differentiation of a hemocyte morphotype known as the prohemocyte. hemocytes matching this physical description were commonly seen during this study. however, our observation of these prohemocytes ‘budding’ from larger ones suggests that this hypothesis is incorrect and, furthermore, that the opposite might be true: we hypothesize that small granulocytes that are immunologically active are produced from mature granulocytes. the phagocytic nature of the small hemocytes observed in this study is in conflict with the classical definition of prohemocytes, but our finding is loosely in agreement with the view that prohemocytes are not multipotent stem cells but are instead fate restricted. ultrastructural evidence supports our hypothesis by showing that the cell types are very similar in subcellular structure. furthermore, hemocytes are bathed in a nutrient rich medium and are known to increase in size upon immune stimulus. therefore, it would not be surprising if these smaller daughter cells are produced with a minimum of sacrifice to the mother cell but are able to mature into capable immune cells in a relatively short period of time. recent data from mammalian systems show that asymmetric partitioning of cellular components between daughter cells is common and purposeful. the authors propose that certain types of asymmetric mitosis might represent a physiological mechanism for producing a ‘pristine’ daughter cell from a mother cell that contains abundant waste. we speculate that asymmetric mitosis could play a role in maintaining a healthy hemocyte population following a phagocytic response to infection.  in conclusion, the data presented herein represent three advancements in our understanding of insect hemocyte biology. first, by creating the first insect quantitative hemocyte map, we describe the anatomical distribution of hemocytes and show that sessile hemocytes form a major component of the mosquito immune system. second, by performing qualitative and quantitative analyses in different physiological states, we show that the number of hemocytes changes significantly with age and in response to infection. lastly, we present direct evidence of mitosis in the circulating hemocytes of an adult insect, showing that hemocyte proliferation in adult mosquitoes does not require a discrete organ or progenitor cell type. 
mosquito hemocytes are circulated throughout the hemocoel by the swift flow of hemolymph, and data show that some hemocytes also exist as sessile cells that are attached to tissues. within the abdominal wall, the areas of highest hemocyte density are the periostial regions, which are ideal locations for pathogen capture as these are areas of high hemolymph flow. 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. 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. these data describe the spatial and temporal distribution of mosquito hemocytes, and map the cellular response to infection throughout the hemocoel. 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. using 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. mosquitoes 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.
 mosquitoes 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 by the swift flow of hemolymph, 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.  using 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, which are ideal locations for pathogen capture as these are areas of high hemolymph flow.  these data describe the spatial and temporal distribution of mosquito hemocytes, and map the cellular response to infection throughout the hemocoel. hemocytehemocoelcirculatingsessilehemolymphphagocytosisimmunitymosquitoanopheles gambiae 