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First published online November 30, 2007
Journal of Experimental Biology 210, 4359-4367 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.010694
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Strategies for regulation of hemolymph pH in acidic and alkaline water by the larval mosquito Aedes aegypti (L.) (Diptera; Culicidae)

Thomas M. Clark*, Marcus A. L. Vieira, Kara L. Huegel, Dawn Flury and Melissa Carper

Indiana University South Bend, South Bend, IN 46615, USA


Figure 1
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Fig. 1. Effects of acid–base exposure on drinking rates and total body water. Animals were reared and assayed in media containing NaCl (RS; 59.9 mmol l–1), or in media in which the only added NaCl was that present in the food (low-NaCl RS). (A) Effects of pH on drinking rates in the presence and absence of NaCl. Drinking rates of acclimated larvae were determined using FITC-dextran ingestion rates (average molecular mass 4.3 kDa). Drinking rates were highest in acidic rearing solutions (column effects; P<1x10–8, F=50.8, d.f.=47). The presence or absence of NaCl did not influence the effect of pH on drinking rates (column effects; F=1.37, P>0.22). Rearing solutions containing NaCl are indicated by •, while those without added NaCl are indicated by {circ}. (B) Effects of ambient pH on percentage body water. Body water was reduced in larvae chronically exposed to acidic media, and was elevated when larvae were acutely exposed to more acidic media (single factor ANOVA of arcsine-transformed body water ratio). The presence or absence of NaCl did not influence the effects of acute pH challenges on body water. RS 7 represents larvae reared and assayed in RS 7, RS 7–3 represents larvae reared in RS 7 and assayed in RS 3, RS 4 represents larvae reared and assayed in RS 4, and RS 4–3 represents larvae reared in RS 4 and transferred to RS 3. For larvae reared and assayed in low NaCl conditions, low-NaCl RS 7 represents larvae reared and assayed in low-NaCl RS 7, while low-NaCl RS 7–3 represents larvae reared in low-NaCl RS 7 and assayed in low-NaCl RS 3. Data are presented as means ± s.e.m.

 

Figure 2
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Fig. 2. Mitochondrial luminosity within the Malpighian tubules of animals reared in acidic (pH 4), neutral or alkaline (pH 11) media. Mitochondrial densities were influenced by environmental pH within the proximal tubule (P<0.05, F=4.24, d.f.=17) and within the distal tubule (P<0.003, F=8.92, d.f.=17, single factor ANOVA). N=6/pH value. All measurements were made with an exposure time of 1/8 s and at the same magnification (x100). Data are presented as means ± s.e.m.

 

Figure 3
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Fig. 3. Mitochondrial luminosity of anal papillae (AP) of animals reared in acidic (pH 4), neutral or alkaline (pH 11) media. All measurements were made with an exposure time of 1/8 s and at the same magnification. Luminosity was determined for the base, midsection and tip of one AP from each animal. Luminosity was always greatest in proximal and distal regions, and lowest in the central region. Larvae reared in acidic media (RS 4) showed reduced mitochondrial densities in the midsection and tip, and mitochondrial densities averaged across regions were also reduced in RS 4 (P<0.05).

 

Figure 4
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Fig. 4. The rectum of a larva that had ingested azolitmin in vivo, reared and assayed in alkaline water (pH 11). Azolitmin is blue in alkaline water (pH >6.2), and red in acidic water (pH <6.2). A total of 36 larvae were assayed (12 larvae each in RS 4, RS 7 and RS 11). The rectal contents were red (pH <6.2) in all larvae. Top, the original image. Bottom, the same image adjusted for brightness (+39) and contrast (+35) using Adobe Photoshop.

 

Figure 5
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Fig. 5. The pH within the AP changes in response to changes in ambient pH in vivo. Top, larva exposed to bromothymol blue in vivo in pH 4 media (RS 4). Under these conditions bromothymol blue enters the AP. Bromothymol blue is yellow at pH <6.8 and blue at pH >6.8. In acid media, the interior of the AP is thus acidic (pH <6.8). Bottom, AP that have been `loaded' by exposure of larvae to bromothymol blue in acidic media followed by transfer to pH 11 media (RS 11) for at least 24 h. Note that the rectal lumen is acidic under these conditions.

 

Figure 6
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Fig. 6. Length (A) and mass-specific length (B) of the AP as a function of salinity. Larvae were reared in different concentrations of artificial seawater. (A) Length was significantly influenced by salinity (P<0.05, F=3.97, d.f.=25, single factor ANOVA). (B) Mass-specific length of the AP (relative to dry mass) of larvae reared at different salinities. No significant mass-specific effects of salinity were observed (P>0.27, F=1.37, d.f.=25).

 

Figure 7
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Fig. 7. Length (A) and mass-specific length (B) of the AP as a function of pH. The influence of pH was determined in the absence ({circ}) and presence (•) of added NaCl. (A) Length was significantly influenced by pH in the presence (P<0.005, F=8.24, d.f.=29) but not the absence (P>0.19, F=1.75, d.f.=29) of NaCl. (B) In the presence of NaCl, pH had a highly significant influence on mass-specific length (P<0.0001, F=13.23, d.f.=29). In the absence of NaCl, no influence of pH on mass-specific length was observed (P>0.86, F=0.15, d.f.=29).

 





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