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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. vol.31 no.1 Temuco mar. 2013

http://dx.doi.org/10.4067/S0717-95022013000100051 

Int. J. Morphol., 31(1):329-337, 2013.

 

Immunopathological Features Developing in the Mosquito Midgut after Feeding on Anopheles gambiae Mucin-1 / Interleukin-12 cDNA Immunized Mice

 

Desarrollo de Características Inmunopatológicas en el Intestino Medio del Mosquito Después de Alimentarse de Ratones Inmunizados con Mucina-1 / Interleucina-12 ADNc de Anopheles gambiae

 

Wilfred E. Injera*,**; Elphantus W. Kabiru**; Michael M. Gicheru**; John I. Githure* & John C. Beier*,***

* International Centre of Insect Physiology and Ecology, Nairobi, Kenya.

** Department of Zoological Sciences, Kenyatta University, Nairobi, Kenya.

*** Department of Epidemiology and Public Health, University of Miami, Miami, USA.

Correspondence to:


SUMMARY: The mosquito midgut is the organ into which the blood meal passes and in which, within a peritrophic membrane secreted by the epithelium, the blood is retained during digestion and absorption. The mosquito midgut is lined with an actin filled microvilli that are exposed to the harsh environment of the gut lumen such as food particle abrasion, digestive hydrolases and attack by pathogens and parasites that are taken in by the blood meal. These microvilli are protected them these effects by the peritrophic matrix, the glycocalyx and the mucin proteins that line their epithelial surfaces. Immunization of BALB/c mice with AgMUC1/IL-12 cDNA has been shown to kill mosquitoes when fed on these mice. Mucin is one of the proteins produced in the mosquito midgut after a blood meal. The fine structure of the mosquito midgut epithelium interacting with immune factors such as antibodies or immune cells is of special significance for interpreting early events in the interaction between the mosquito midgut lining and the specific immune components present in the blood of AgMUC1/IL-12 cDNA immunized BALB/c mice. Following bright light microscopy, scanning electron and transmission electron microscopic observations of the features seen in mosquito midgut sections from An. gambiae mosquitoes fed on BALB/c mice immunized with AgMUC1/IL-12 cDNA, the most likely immune mechanisms responsible for mosquito killing could be cell mediated, most likely antibody dependent cellular cytotoxicity. Both necrotic and apoptotic processes that could be the cause of mosquito death were seen to take place in the cells lining the midgut epithelium.

KEY WORDS: Midgut epithelium; Immunopathological features; Adherent cells; Activated hemocytes.


RESUMEN: El intestino medio es el órgano al cual pasa la sangre consumida por el mosquito y donde, mediante una membrana peritrófica secretada por el epitelio, esta sangre es mantenida durante la digestión y absorción. El intestino del mosquito está revestido por microvellosidades llenas de actina que son expuestas a las complejas condiciones en torno a la luz intestinal, tales como la abrasión producida por partículas de alimentos, hidrolasas digestivas y el ataque de patógenos y parásitos que son tomados en la sangre consumida. Estas microvellosidades se protegen de estos efectos mediante la matriz peritrófica, el glicocálix y las proteínas de mucina que revisten las superficies epiteliales. La inmunización con AgMUC1/IL-12 ADNc en ratones BALB/c ha demostrado ser útil para matar los mosquitos cuando se alimentan de estos ratones. La mucina es una de las proteínas producidas en el intestino medio del mosquito después de consumir sangre. La fina estructura del epitelio del intestino interactúa con factores inmunes tales como anticuerpos o células inmunes es de especial importancia para interpretar los eventos tempranos en la interacción entre el revestimiento del intestino medio y los componentes inmunológicos específicos presentes en la sangre de ratones BALB/c inmunizados con AgMUC1/IL-12 cDNA. Después de observar mediante microscopías de luz, electrónica de barrido y de transmisión las características de secciones del intestino medio del mosquito Anopheles gambiae alimentado de ratones BALB/c inmunizados con AgMUC1/IL-12 cDNA, mecanismos inmunes mediados por citotoxicidad celular dependiente de anticuerpos (ADCC) podrían ser los responsables de matar a los mosquitos. Los procesos necróticos y apoptóticos que pueden ser la causa de la muerte del mosquito tienen lugar en las células que recubren el epitelio del intestino medio.

PALABRAS CLAVE: Epitelio del intestino medio; Características inmunopatológicas; Células adherentes; Hemocitos activados.


 

INTRODUCTION

The mosquito midgut is the organ into which the blood meal passes and in which, within a peritrophic membrane secreted by the epithelium, the blood is retained during digestion and absorption. The monolayer of the mosquito midgut epithelial cells rests on a network of longitudinal and circular muscles that are entirely covered with an extra cellular basal membrane (lamina) (Chapman, 1999; Dow, 1986; Richards, 1975). As the midgut expands in the course of ingesting a blood meal, the distance between the longitudinal muscles increase and the single layered epithelium is distended to give a flat and thin epithelium. In addition to providing integrity to the gut, the muscles also confer the peristaltic actions of the gut allowing the movement of ingested food to the hindgut. There is no zonal segregation of different types of epithelial cells in the midgut of the female mosquito. This single layer of columnar epithelial cells with an apical brush border and a basal laminar is highly specialized to accomplish all the functions of secretion and absorption attributed to the midgut of the adult female mosquito (Bertram & Bird, 1961; Reinhardt & Hecker, 1973).

The mosquito midgut is lined with a single layered epithelium consisting of irregularly shaped cubical to columnar cells whose apical side is folded into numerous actin filled microvilli. It is these microvilli that are normally exposed to the harsh environment of the gut lumen and are subjected to damage caused by food particle abrasion, digestive hydrolases and attack by pathogens and parasites. These microvilli have however evolved mechanisms that protect them from these effects. These mechanisms include the peritrophic matrix, the glycocalyx and the mucin proteins that line their epithelial surfaces (Shen et al., 1999; Antonyraj et al., 1998; Willadsen & Billingsley, 1996).

The midgut morphology is drastically altered after blood feeding. There is need to have a very clear view of the normal midgut structure and compare to the midgut structures that have been altered through immune components interactions from the blood meal. There are no reports about the fine structure of the mosquito midgut epithelium interacting with immune factors such as antibodies or immune cells. Knowledge of such a midgut structure is of special significance for interpreting early events in the interaction between the mosquito midgut lining and the specific immune components present in the blood of BALB/c mice immunized with AgMUC1/IL-12 cDNA. The results of this examination will therefore be interpreted in the context of the fine structure of the midgut epithelial cells derived from the midgut of mosquitoes fed on mice immunized with empty vector cDNA compared to midguts of mosquitoes fed on mice immunized with AgMUC1/IL-12 cDNA. The aim of this study is therefore to define the immune pathologic features that lead to mosquito death after feeding on AgMUC1/IL-12 cDNA immunized mice with particular attention to the events taking place on the surface of the epithelial cells of the blood fed mosquito midgut.

MATERIAL AND METHOD

Preparation of blood fed mosquito midguts. Mosquitoes were fed on the experimental AgMUC1/IL-12 cDNA and the control empty vector/IL-12 cDNA immunized mice groups and their midguts dissected out at 8 hours post blood feeding.

Preparation of blood fed mosquito midgut sections for bright light microscopy. Dissected midguts for preparation of sections for bright light microscopy were frozen in OCT compound medium at ­20oC immediately they were dissected out. Cryostat sections (10 µm) were made and stained in 10% Giemsa stain in phosphate buffered saline for 15 minutes. The stained sections were examined under the bright light microscope using X10 and X40 objectives with the aim of identifying differential features appearing in the midgut sections from mosquitoes fed on the experimental AgMUC1/IL-12 cDNA immunized mice against those from the midgut sections of mosquitoes fed on the control empty vector/IL-12 cDNA immunized mice.

Preparation of sections for scanning electron microscopy (SEM). Dissected midguts for preparation of sections for scanning electron microscopy were collected into the electron microscopy (EM) fixative (0.05M Sodium cacodylate buffer Ph 7.4 containing 0.01 M Calcium chloride, 2.5% Glutaldehyde and 5% Sucrose) immediately they were dissected out and stored at 4oC until processing. The midguts were removed from the fixative during processing, cut longitudinally into two pieces and mounted onto tissue blocks. The pieces were impregnated with Gold and examined on the scanning electron microscope with the aim of identifying differential features appearing in the midgut of mosquitoes fed on the experimental AgMUC1/IL-12 cDNA immunized mice against those from mosquitoes fed on the control empty vector/IL-12 cDNA immunized mice.

Preparation of sections for Transmission Electron Microscopy (TEM) . From the EM fixative the midguts were washed in distilled water then post fixed in 1% Osmium tetroxide in phosphate buffered saline. The midguts were then washed in distilled water, dehydrated through ascending concentrations of ethanol (50% - 70% - 80% - 90% - 96% and lastly absolute ethanol). The midguts were kept in each alcohol concentration for 10 minutes. From absolute ethanol, the tissues were transferred to a 50:50 mixture of ethanol and propylene oxide for 10 minutes then transferred to pure propylene oxide for another 10 minutes. They were then transferred to a 50:50 mixture of propylene oxide and epoxy (araldite) overnight. The tissues were then processed through two changes of araldite each lasting 24 hours then embedded in pure araldite in embedding molds and incubated at 60oC for 72 h to allow the araldite to harden. Ultra thin sections were cut, stained with 50% uranyl acetate in ethanol for 30 min then washed in distilled water. They were then transferred to lead citrate for 10 minutes then washed in distilled water. The sections were then dried and examined on Transmission Electron Microscope with the aim of identifying differential features appearing in the sections from mosquito midguts fed on the experimental AgMUC1/IL-12 cDNA immunized mice against those sections from mosquito midguts fed on the control empty vector/IL-12 cDNA immunized mice.

RESULTS

Bright light microscopy. Giemsa stained mosquito midgut sections from the An. gambiae mosquitoes fed on BALB/c mice immunized with AgMUC1/IL-12 cDNA and Empty vector/IL-12 cDNA were examined by bright light microscopy. Figures 1, 2 and 3 are midgut sections from mosquitoers fed on AgMUC1/IL-12 cDNA immunized mice. A common feature observed in these sections is the adherence of white blood cell-like cells on the epithelial surface of the mosquito midgut lining. This feature was not observed in the mosquito midgut sections made from mosquitoes fed on BALB/c mice immunized with empty vector/IL-12 cDNA (Figs. 4 and 5).

The other features observed in midgut sections from mosquitoes fed on AgMUC1/IL-12 cDNA immunized mice include the formation of nodules on the epithelial surface (Fig. 6), the appearance of some epithelial cells that appears to be undergoing apoptosis (Fig. 7), the appearance of some swollen epithelial cells probably filled up with midgut contents (Fig. 8), the appearance of some gaps through the epithelial cell lining (Figs. 9, 10 and 11) and the appearance of hemocyte-like cells along the basement membrane (Figs 12, 13 and 14).

Figs. 1, 2 and 3: Gemsa stained crystat sections prepared from blood fed An. gambiae mosquito midgut 8 hours post blood feeding on BALB/c mice immunized with AgMUC1/IL-12 cDNA. The arrows show the attachment of white blood cell-like cells along the epithelial lining.

Figs. 4 and 5. Gemsa stained crystat sections prepared from blood fed An. gambiae mosquito midgut 8 hours post blood feeding on empty vector/IL-12 cDNA immunized mice. There is no evidence of immunologic activities on either the epithilial lining or the basement membrane.

Fig. 6. Gemsa stained crystat sections prepared from blood fed An. gambiae mosquito midgut 8 hours post blood feeding on AgMUCl/IL-12 cDNA immunized mice. Arrow shows a nodule on the epithelium.

Fig. 7. Gemsa stained crystat sections prepared from blood fed An. gambiae mosquito midgut 8 hours post blood feeding on AgMUCl/IL-12 cDNA immunized mice. The arrows show epithelial cells whose shrinking nuclei appears to be undergoing fragmentation probably by apoptosis.

Fig. 8. Gemsa stained crystat sections prepared from blood fed An. gambiae mosquito midgut 8 hours post blood feeding on AgMUCl/IL-12 cDNA immunized mice. The arrow shows a swollen epithelial cell where midgut contents have probably sipped into the epithelial cell. This cell may eventually burst and lead to necrosis of the surrounding tissue.

Figs. 9, 10 and 11. Gemsa stained crystat sections prepared from blood fed An. gambiae mosquito midgut 8 hours post blood feeding on AgMUCl/IL-12 cDNA immunized mice. The arrows show some of the features that may turn pathologic, seen commonly in mosquitoes fed on the experimental mice. Gaps in the epithelial cell lining may allow the midgut contents to mix with the hemolymph and eventually lead to mosquito death.

Fig. 12, 13 and 14. Gemsa stained crystat sections prepared from blood fed An. gambiae mosquito midgut 8 hours post blood feeding on AgMUCl/IL-12 cDNA immunized mice. The arrows show hemocyte-like cells probably indicating hightened hemocyte activities.

Scanning Electron Microscopy (SEM). In the SEM midgut sections there appears to be the adherence of white blood cell-like cells onto the epithelial lining (Figs. 15 and 16). The adherence cell appears amoeboid in shape indicating that the attachment could be transient. There also appears to be cells entrapped into the epithelialium (Fig. 17).

Figs. 15, 16 and 17. Scannining Electron Microscopy micrographs prepared from blood fed An. gambiae mosquito midgut 8 hours post blood feeding on AgMUCl/IL-12 cDNA immunized mice. The arrows in figs. 15 (magn. X 5,000) and 16 (mag. X 7,500) show the attachement of cells, probably white blood cells, onto the epithelial lining. The arrow in fig. 17 (magn. X 3,500) show a group of cells, probably white blood cells, entrapped in the midgut epithelium.

Transmission Electron Microscopy (TEM). In the transverse sections from the midgut of the mosquitoes fed on AgMUC1/IL-12 cDNA immunized mice, an epithelial cell nucleus shrinking probably as a result of the apoptotic processes was observed (Fig. 19). The nucleus of the affected cell was seen to have condensed considerably compared to that seen in the section from the mosquitoes fed on the control empty vector/IL-12 immunized group (Fig. 18). The basement membrane was also observed to have thickened (Fig. 19). The apoptotic cell (Fig. 19) was seen to have more prominent mitochondria compared to the normal epithelial cell (Fig. 18).

Fig. 18. Shows the general features of fine structure in the midgut epithelial cell. Midgut lumen (L), Microvilli (MV), Intercellular boundary (ICB), Golgi complex (G), Nucleus (N), Vesicles (VES). Mosquito fed on empty vector/IL-12 cDNA

Fig. 19. Shows fine structure in the epithelial cell from the midgut of An. gambiae mosquito fed on BALB/c mice immunized with AgMucl/IL-12 cDNA, 8 hrs post blood feeding. Note the shrinking of the nucleus, the thickening of the basement cell membrane (BCM) and more prominent Mitochomdeia (M). Hemocel (H).

DISCUSSION

The midgut epithelium, apart from being an immunologically competent organ, also serves as a structural barrier for microbes and parasites (Dimopoulos, 2003). The peritrophic membrane that is formed around the blood meal facilitates digestion and also protects the midgut epithelium from direct contact with the blood meal. Foreign pathogens that make it through the structural barriers encounters the innate immune system that comprises of a variety of effector mechanisms that ultimately can mount a successful defense mechanism (Medzhitov & Janeway, 2002). Anopheline mosquitoes have been reported to be strongly activating their immune system when the parasites are invading the midgut epithelial tissues (Dimopoulos et al., 1997).

The adherence of white blood cells onto the surface of the mosquito midgut epithelium (Figs 1, 2 and 3) could be due to the fact that the cells lining the midgut epithelium express mucin protein after a blood meal. The antibodies to this protein present in the blood meal from AgMUC1/IL-12 cDNA immunized mice will recognize and attach onto this protein forming antigen-antibody complexes on the epithelial surface. The antigen ­ antibody complexes may in turn attract either complement, Fc receptor or complement receptor bearing white blood cells to bind onto these surfaces. The adherent antibodies, complement and either Fc or complement bearing cellular immune components may react with their specific receptors turning their targets into a foci of immune activity.

The cells of the immune system that have the Fc and C3 receptors are known to be involved in mediating the phenomenon of antibody dependent cellular cytotoxicity (ADCC). The Fc receptor and C3 receptor bearing cells that are involved in ADCC include macrophages, NK cells and Neutrophils (Abbas et al., 1997). The pores made by ADCC have been reported to be larger than those made through complement action (Dourmashkin et al., 1980). Since the ADCC pores inflicted in the epithelial cells are large enough, the midgut contents would pass into the epithelial cell which would swell (Fig. 8) and probably go on to burst. Once the epithelial cell bursts, there could be mixing of the midgut contents and the hemocel, probably leading to the death of the affected mosquito.

In the mouse, the FcY receptors preferentially show specificity for IgG2a and IgG2b over IgG1 and IgG3 (Ravetch & Kinet, 1991). The FcY receptor is a high affinity receptor whose expression in monocytes, macrophages and neutrophils is enhanced by IFN-Y. In this study more IFN-Y is generated through activation of NK cells by IL-12. Thus the most likely white blood cell-like cells observed adhering on the epithelium of the An. gambiae midgut fed on AgMUC1/IL-12 cDNA immunized mice could be the NK cells (Figs. 1-3, 15 and 16). Although NK cells are also activated in the empty vector/IL-12 cDNA immunized mice, lack of specific anti-mucin antibodies in these mice blood meals makes them not attach onto the midgut epithelium (Figs. 4 and 5).

A genetically selected refractory mosquito strain, L3-5, has been shown to completely block plasmodium development through a melanotic encapsulation mechanism that appears to involve components of the innate immune system (Collins et al., 1986). Two types of melanotic encapsulation mechanisms have been described in insects; cellular encapsulation mediated by hemocytes (Gotz, 1986) and humoral encapsulation (Söderhäll & Cerenius, 1998). It appears that some white blood cells appear to be encapsulated within the epithelium (Figs. 6 and 17) where they appear like a nodule. This could be probably as a result of humoral encapsulation taking place on the surface of the epithelium. This encapsulation could as well be as a result of inflammatory response or through the process whereby the peritrophic membrane was formed and trapped the cells on the epithelial surface. If this nodule breaks there is chance that it might cause necrosis of the epithelial lining and through such injuries on the epithelial lining the midgut and hemocel contents could mix leading to a possible activation of the hemocytes and probably mosquito death.

The other mechanism that could lead to mosquito death is where the NK cells could attach onto the epithelial cells lining the midgut and they delivers a lethal hit that results in the dissolution of the affected epithelial cell. Two such epithelial cells whose nuclei appear to be undergoing such dissolution process were observed (Figs. 7 and 19). The cell nucleus defragmentation can be seen in the two cells. As the target cell undergoes lysis, the cells may shrink (Fig. 7 and 19), creating intercellular spaces (Figs. 9-11). There could also be sippage of the midgut contents into the hemocell through these intercellular spaces especially considering that the epithelial lining is greatly distended with the presence of the blood meal in the mosquito midgut. The mixing of the midgut contents and the hemocel through these intercellular spaces could be the other mechanism that could lead to the death of the affected mosquito. Where the apoptotic cells do not give way to intercellular spaces, there would most likely be repair whereby the adjacent non-affected epithelial cells come together closing the space left behind by the damaged cell, a mechanism that would be a kin to that observed with epithelial cells invaded by plasmodium ookinetes (Gupta et al., 2005). The documented innate mosquito immune responses to malaria infection may partly be as a result of the injury that is caused by the parasite invasion of the epithelial tissue as well as the microbial components of the midgut that may be present at the site of invasion (Dimopoulos). Such could be similarly occurring following injury to the epithelial cell resulting from the pores made by the NK cell lytic activity. It is not known what would be the consequence of the activation of the mosquito innate immune mechanism in the case of immune mediated injury to the mosquito midgut.

The midgut epithelium has been reported to be an immunocompetent organ that participates in the mosquito defense mechanisms (Dimopoulos). Any immune reactions occurring on the epithelium will trigger off the serine protease cascade that in turn will activate defense reactions in the mosquito hemocel (Hoffman & Reichhart, 2002). Such immune defense mechanisms will involve the activation of hemocytes. The basement membrane was observed to have thickened (Fig. 19) and since it is an immunologically active organ this thickening could be the one causing the immune like responses from the hemocyte-like cells that were observed under bright light microscopy (Figs. 12-14). The hemolymph contains large quantities of immune components and hemocytes that circulate freely or are attached to different organs (Hoffman et al., 2002). Hemocytes produce immune components and are also capable of phagocytosis and encapsulation of foreign particles such as apoptotic cells (Hoffman et al.). Hemocytes have been reported to be defense mechanisms of the mosquito immune system (Dimopoulos). In this study it appears that the appearance of these hemocyte-like cells could be as a result of the immune activities taking place at the epithelium (Fig. 6) and the resultant enlargement of the basement membrane (Fig. 19). In mosquitoes fed on AgMUC1/IL-12 cDNA immunized mice, activated hemocyte like cells were seen in amoeboid forms along the basement membrane (Figs. 12-14) probably as a result of the heightened immune activity along the enlarged basement membrane (Fig.19).

Following microscopic observations of the features seen in mosquito midgut sections from Anopheles gambiae mosquitoes fed on BALB/c mice immunized with AgMUC1/IL-12 cDNA, the most likely immune mechanisms responsible for mosquito killing could be cell mediated. In this case the cells responsible for mosquito killing could be the Fc receptor bearing NK cells which effect their killing mechanisms through ADCC. Both necrotic and apoptotic processes that could lead to mosquito killing were observed in the cells lining the midgut epithelium.

ACKNOWLEDGEMENTS. We are grateful to ICIPE and ILRI for provision of research facilities and the excellent technical support of Chimtawi, Milka and Jeremiah of ICIPE and Chuma of ILRI; Prof. Kiama and Mr. Njoroge of the Department of Veterinary Science, University of Nairobi for their invaluable assistance with Electron Microscopy.

This work was funded by the NIH grant number NIH ICIDR U19A145511 and NIH Forgarty ABC grant D43TWO1142.

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Correspondence to:

Wilfred Emonyi Injera

School of Medicine
College of Health Sciences
Moi University
P.O Box 4606-30100 Eldoret,
KENYA
Tel: +254-733-593 099
+254-724-152 908

Email address: weinjera@yahoo.com
weinjera@gmail.com

Received: 12-06-2012
Accepted: 20-08-2012

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