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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estimates of the numbers of sporozoites injected by individual mosquitoes into the skin of mice have been made by evaluating skin biopsy specimens post-feeding, using real-time polymerase chain reaction (PCR) assessment of parasite 18S rRNA.8 This study reported small numbers of infected mosquitoes that fed but did not leave behind detectable numbers of sporozoites within the skin. However, an inability of mosquitoes to infect or to leave behind residual sporozoites in the host’s skin does not necessarily imply an actual failure to deliver sporozoites. Such failure could also be caused by the re-ingestion of these sporozoites by the mosquito after their delivery into the skin. This possibility must be considered as a component of the quantitative dynamics of sporozoite delivery by mosquitoes.
Many years ago, Yorke and Macfie9 demonstrated the presence of sporozoites in the bloodmeal of an infected Anopheles maculipennis shortly after it had fed. Re-ingestion of sporozoites into the midgut of feeding mosquitoes was confirmed with An. gambiae and An. stephensi that had been experimentally infected with Plasmodium falciparum and then allowed to feed on a rat.10 Our long-term interest in the kinetics of mosquito delivery of sporozoites into the skin of rodents1,5,7 prompted us to re-examine the phenomenon of sporozoite re-ingestion by mosquitoes. Because previous studies9,10 may have failed to differentiate between sporozoites that had been re-ingested by mosquitoes after deposition into the host versus sporozoites released into the midgut lumen from ectopic oocysts emptying into the interior of the midgut,11 we took precautions to resolve this question. An. stephensi mosquitoes infected with P. berghei sporozoites that express enhanced green fluorescent protein (EGFP) were used for these studies because they are readily detectable with great sensitivity by fluorescence microscopy.5,12 The methodology described in the current article allows us to distinguish between sporozoites ingested into the midgut after their delivery into the mammalian host versus those released directly into the midgut from ectopic oocysts.
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MATERIALS AND METHODS |
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Microscopy. We used a Leica MZ16FA fluorescence stereoscopic microscope with a 2.0X stereoscopic objective lens (Leica Microsystems, Wetzlar, Germany). Illumination for fluorescence studies was with an EXFO X-Cite 120 F1 illumination system and with a GFP2 filter set (restricting excitation to 480 ± 20 nm and fluorescence signal emission to wavelengths longer than 510 nm). Images were acquired with a Leica DFC350 FX digital camera and saved as digital files for further analysis and processing. We used Leica FW4000 software for documentation and analysis.
Experimental studies. Mosquitoes were allowed to feed for 3–4 minutes on mice anesthetized by IP injection of ketamine (50 mg/kg) plus xylazine (10 mg/kg) and acepromazine (1.7 mg/kg). Midguts were then removed through the posterior segment of the abdomen, placed on a microscope slide, and examined by fluorescence microscopy to detect the presence of sporozoite-containing oocysts on the midgut wall. Midguts were then treated (see later in this article) to quench fluorescence from endogenous oocysts and sporozoites on the exterior of the midgut wall. The head and thorax of the mosquito were washed and moved to a clean slide for dissecting out the salivary glands and determination of their infection with sporozoites.
Quenching of enhanced green fluorescent protein fluorescence.
To distinguish newly ingested salivary gland sporozoites within the lumen of the midgut from endogenous sporozoites released from oocysts on the hemocoel side, midguts were dissected out and treated with 0.4 mg/ml Crystal Violet to quench fluorescence of oocysts and sporozoites at the exterior of the midgut.13 We used an aqueous dilution of a starting solution of 3 mg/ml of Crystal Violet (DIFCO—Gram Crystal Violet Primary Stain [Becton-Dickinson and Co., Franklin Lakes, NJ]). Midguts were treated for 2–3 minutes to quench EGFP emission, then washed with water, dried gently with bibulous paper, placed on a microscope slide, and pulled apart with fine forceps to release ingested blood. Each midgut was then covered with a 22-mm2 coverslip and observed by fluorescence microscopy. Total number of sporozoites within the bloodmeal of each midgut was determined by counts of sporozoites in randomly sampled fields under the coverslip. We found that after such treatment the fluorescence of endogenous sporozoites at the exterior of the midgut became undetectable (Figure 1A and 1B
), whereas newly re-ingested sporozoites released from the bloodmeal mass in the midgut lumen remained EGFP-positive (Figure 1C). As a control, cohorts of these mosquitoes that had not been fed on a mouse were treated in the same way to quench EGFP-emission by external oocysts and sporozoites. The midguts of these mosquitoes were then observed for the presence of fluorescence from sporozoites. In all cases (fed and non-fed mosquitoes), salivary glands were checked to establish that all mosquitoes had gland infections with sporozoites.
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Control: interference effects of blood. To see if ingested blood from the midgut had an effect on reliability of counts of sporozoites within the bloodmeal mass, we prepared a suspension of sporozoites from salivary gland dissections and did counts to determine the concentration of sporozoites per 1 µL in the suspension. We compared this with counts made when aliquots from the same sporozoite suspension were mixed with the midgut contents of non-infected mosquitoes that had just taken bloodmeals on mice not infected with malaria.
Control: distinguishing between endogenous oocyst versus re-ingested salivary gland sporozoites. To further confirm that the luminal sporozoites that we observed were salivary gland sporozoites re-ingested by the mosquito rather than endogenous oocyst sporozoites already residing within the midgut, we used two procedures previously shown to differentiate between these developmental stages of sporozoites. These were (1) assessment of gliding motility of sporozoites on microscope slides in the presence of 2% bovine serum albumin (BSA),14 which is characteristic of sporozoites of salivary gland but not oocyst origin and (2) assessment of the circumsporozoite precipitation (CSP) reaction,15 which also is characteristic of sporozoites of salivary gland but not oocyst origin.16 The CSP reaction was done by adding 6 µg of monoclonal antibody 3D11 to each slide preparation of sporozoites in the presence of 2% BSA. For each experiment, a suspension of dissected-out salivary gland sporozoites and a suspension of dissected-out midgut sporozoites from the same cohorts of mosquitoes were assessed in parallel for motility and CSP-reactivity as baseline controls. Slides were coded and read blindly.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. There were no detectable, ingested sporozoites in the midgut lumens of ~10% of the mosquitoes. Approximately half of the mosquitoes ingested 100 or fewer sporozoites; there was a progressive decrease in the numbers of mosquitoes found to ingest larger numbers of sporozoites. No sporozoites were ever detected in the midgut lumens of the 30 infected, control mosquitoes that had not been allowed to feed on mice. These data include only mosquitoes that were shown to possess both midgut infections with oocysts and salivary gland infections with sporozoites. To investigate whether significant numbers of re-ingested sporozoites might have been omitted from our totals because of loss through the anus, we used capillary heparinized, hematocrit tubes to collect drops of blood from the posterior of mosquitoes during and after bloodmeals. No sporozoites were ever seen within drops of blood emerging from the anus during and post-bloodmeal.
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). Evaluation of motility of these sporozoites in the presence of 2% BSA (Figure 3A) indicated that 74.5% of these sporozoites recovered from the midgut lumen after a bloodmeal exhibited classic gliding motility. As baseline controls, 83.5% of sporozoites from dissected-out salivary glands obtained from a cohort of these mosquitoes exhibited gliding motility, whereas 0% of sporozoites from midguts of non-fed mosquitoes exhibited gliding (N = 25 mosquitoes for each group). It was important to examine these sporozoites as soon as possible after the blood-meal because sporozoite motility was lost after extended residence in the midgut lumen.
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) indicated that 67.7% of these sporozoites recovered from the midgut lumen after a bloodmeal exhibited CSP reactivity. As baseline controls, 69.4% of sporozoites from dissected-out salivary glands obtained from a cohort of these mosquitoes had positive CSP reactions whereas only 0.8% of sporozoites from midguts of non-fed mosquitoes exhibited CSP reactivity.
In an attempt to determine whether the site of the blood-meal influenced the numbers of sporozoites re-ingested, we allowed infected mosquitoes to feed on either the ear pinna or the ventral abdomen of mice for our standard 3–4-minute feeding time (N = 26 mosquitoes for each group). Those fed on the ear ingested a mean of 83.8 ± 15.8 (SE) sporozoites, whereas those fed on the abdomen ingested a mean of 114.1 ± 30.4 (SE) sporozoites but the differences between the means were not significant as determined by Student paired t test (P 
0.32).
We tested the possible role that ingested blood cells might have on interfering with our ability to visualize and count sporozoites from within the midgut lumen. We found virtually no differences between counts of sporozoites in aliquots of a suspension of sporozoites from dissected-out salivary glands versus counts taken when the corresponding aliquots of sporozoites were added to appropriate amounts of the blood-meal contents of non-infected mosquitoes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another of our technical concerns related to a report that some oocysts may develop in ectopic sites within the midgut, resulting in the release of some oocyst sporozoites directly into the midgut lumen,11 a concern not previously dealt with by others10 who assumed that all sporozoites found in the midgut lumen had been re-ingested after release from the mosquito proboscis. Not all workers have been able to demonstrate ectopic development of oocysts,17 so we felt obliged to do controlled studies to establish whether this was occurring within our own system. Our studies failed to demonstrate sporozoites associated with midguts of the non-fed mosquitoes studied after treatment of their midguts to eliminate fluorescence from external oocysts. Because this treatment does not eliminate fluorescence from sporozoites within the mid-gut lumen, we would have observed such sporozoites from ectopic oocysts, if they had been present in any of the 30 infected but non-fed mosquitoes so examined.
One of the strongest arguments that had been offered on behalf of the release of sporozoites into the midgut interior from ectopic oocysts was that "feces" collected from mosquitoes fed on mice were able to induce parasitemia after injection into uninfected mice.11 However, an alternate explanation, as demonstrated by our own study, is that the sporozoites collected in the feces were from salivary glands and had been re-ingested by the feeding mosquitoes after delivery into the mouse. Mosquitoes sometimes concentrate the cellular component of their bloodmeal by releasing tiny drops of plasma-rich fluid from their anus during feeding. However, it required collection of such fluid from hundreds of mosquitoes to obtain infective sporozoites in this prior study,11 so passage of sporozoites via the feces must have been a relatively rare event. We examined the fluid released from the posterior of individual mosquitoes post-feeding and never found any sporozoites by microscopy.
In further confirmation that the intra-luminal sporozoites we observed had been released from salivary glands and re-ingested by the feeding mosquitoes, we found that these in-traluminal sporozoites were fully motile and exhibited full CSP reactivity, whereas sporozoites from the midguts of non-fed mosquitoes exhibited neither motility nor CSP reactivity. The acquisition of motility14 and CSP reactivity15 by salivary gland sporozoites is an unambiguous way of differentiating them from oocyst sporozoites. We conclude that sporozoites from ectopic oocysts could not account for the sporozoites that we observed within the bloodmeal mass in the lumen of the midgut after feeding.
The re-ingestion of some sporozoites by mosquitoes during feeding is not a surprising phenomenon. Some saliva injected into the host during mosquito engorgement is likely to be re-ingested with the blood back into the midgut. The food canal within the proboscis, through which blood is sucked into the mosquito, has more than 100 times the cross-sectional area of the parallel channel for saliva down through which saliva flows into the tip of the food canal. It has been estimated that the rate of blood flow into Aedes aegypti is of the order of 104–105 times greater than the opposing flow rate of saliva down the salivary channel and into the bite site.18 One should thus expect some sporozoites within secreted saliva to be re-ingested during mosquito engorgement.
Re-ingestion of sporozoites is likely enhanced when the mosquito is actively imbibing blood rather than injecting sporozoites into avascular tissue while probing for a blood source. Accordingly, one might expect more sporozoites to remain in the skin during extended, non-productive probing by the mosquito than after rapid localization and ingestion of a blood source. Thus, if mosquitoes tend to probe for longer periods of time in less vascularized tissue before they are able to encounter a blood source, one might expect greater likelihood of sporozoite transmission into such less vascularized areas of skin. To see if there were differences in numbers of sporozoites re-ingested after feeding on different sites, we compared infected mosquitoes that had fed on the ear pinna versus the ventral abdomen. Greater numbers of sporozoites were found in the midguts of mosquitoes that had fed on the abdomen but we were unable to show that these differences were statistically significant. Further work is needed in this area.
As previously noted, it has been reported that some individual, infected mosquitoes fed but did not leave behind detectable numbers of sporozoites within the skin8 or were unable to induce a blood infection1,2 and it was concluded that there was no transmission of sporozoites in these cases. However, the possibility of re-ingestion of all or most of the injected sporozoites cannot be ruled out in cases of transmission failure. The numbers of sporozoites re-ingested by mosquitoes appear to be of the same order of magnitude as the numbers that remain in the mammalian host and initiate the infection.5,6,8,9,19,20 Thus, this phenomenon of sporozoite re-ingestion likely plays a significant role in transmission dynamics of malaria by mosquitoes. The technique that we have described to measure sporozoite re-ingestion will be a useful addition to other quantitative measures of the kinetics of sporozoite transmission during the infection of the mammalian host by mosquito-injected sporozoites.
Received April 20, 2006. Accepted for publication August 13, 2006.
Acknowledgments: We are grateful to Allen Clarkson and Ute Frevert for helpful comments on the manuscript.
Financial support: This study was supported by NIH Grant #AI63530 to JV.
* Address correspondence to Jerome P. Vanderberg, Department of Medical Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY, 10010. E-mail: Jerome.Vanderberg{at}med.nyu.edu 
Authors’ addresses: Chahnaz Kebaier and Jerome Vanderberg, Department of Medical Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY 10010.
Reprint requests: Jerome Vanderberg, Department of Medical Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY 10010, E-mail: Jerome.Vanderberg{at}med.nyu.edu.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  Y. Jin, C. Kebaier, and J. Vanderberg Direct Microscopic Quantification of Dynamics of Plasmodium berghei Sporozoite Transmission from Mosquitoes to Mice Infect. Immun., November 1, 2007; 75(11): 5532 - 5539. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
