134 P. von Dungern, H. Briegel Journal of Insect Physiology 47 2001 131–141
lithium carbonate were added to give a final volume of 1 ml, and uric acid was determined as before Van Handel,
1975. In certain experiments 5–10 mosquitoes were kept in cups 800
× 900 mm; 204 ml and their pooled
faeces treated similarly: they were eluted first in water, then in lithium carbonate, with the volumes adjusted to
the number of mosquitoes used, i.e. a final volume of 1 ml per mosquito.
Midguts were also dissected on ice, collected in a total volume of 250
µ l Tris buffer 0.05 M, pH 8.4 in cooled
centrifuge test-tubes Eppendorf, 1.5 ml, sonicated Branson B15, Microtip and subsequently stored at
220 °
C until analysis. Trypsin activity was measured at 25
° C against TAME tosyl-arginine methylester, Sigma
T 4626 following Graf and Briegel 1982. Aminopepti- dase activity was determined with LPNA leucine-p-
nitroanilideHCl; Sigma L 9125 as a substrate following the procedure of Graf and Briegel 1982. Proteolytic
activities are given as Umidgut, based on millimolar extinction coefficients of 0.409 for TAME and 9.62 for
LPNA Ho¨rler, 1995 and were also expressed as percent of their mean maximal activities after a standard blood
meal. Aliquots of 0.05–0.5 midguts were used through- out.
Protein was measured for individual mosquitoes as total nitrogen by the Kjeldahl procedure Minari and Zil-
versmit, 1963; von Dungern and Briegel, 2000. Total lipid was determined in chloroform–methanol extracts
by a vanillin–phosphoric acid reaction as described by Van Handel 1985 with soybean oil 0.1 in chloro-
form; Sigma S-7381 as standard.
Regressions and ANOVA analyses were performed using Statview 4.02. Sample differences were tested for
significance by t-test Sokal and Rohlf, 1981.
3. Results
Larval development is an intense phagoperiod accompanied by rapid growth, particularly during the
fourth instar Timmermann and Briegel 1998, 1999. Feeding and growth end with pupation 6–8 days after
hatching. To investigate the fate of surplus protein acquired through the incessant feeding period of larvae
III and IV we analyzed larval material at daily intervals for catabolic enzyme activities. Their body size was
measured and their total protein content determined.
Arginase activity was measured from the second lar- val instar to the pharate pupa. It increases linearly with
body size during the time of development, although with great variability Fig. 1. In Fig. 2 we have combined
all the mean arginase values for a complete activity pro- file which includes the imaginal life-span of Ae. aegypti.
At the time of the larvalpupal transition its activity doubled,
reaching the
maximal peak
of 23.3
nmolmininsect in newly molted pupae. Thereafter it
Fig. 1. Arginase activity in whole-body homogenates of larvae and
pharate pupae of Aedes aegypti. Single larvae were assayed except for the second instar where pools of 10–20 were used; data are from differ-
ent experiments. Symbols: LII, LIII I, LIV s and pharate pupa j. Activity follows a linear regression on body size: Y
= 3.17X20.39
N =
143, r
2
= 0.663, t
= 16.59, P,0.0001.
Fig. 2. Arginase activity in whole-body homogenates during the life-
cycle of Aedes aegypti. Data are means of nmolmininsect. Except for LII G where pools of 10–20 were used, individual mosquitoes were
assayed: LIII j, LIV h, P I, sugar-fed females s. For LII N =
5 and for all other instars N
= 6–39.
sharply decreased and in the teneral imago it fell to approximately 15 of the pupal maximum. Surprisingly,
activity persisted long into imaginal life; it remained constant for the first few days, and then gradually
decreased to 10–15 of the teneral value during 3–4 weeks. In teneral females arginase activity was equally
distributed between abdomen and headthorax 47.6 and 49.7 of whole-body activity respectively. After
centrifugation of a larval homogenate 16 000g, 15 min before incubation, the arginase activity in the supernatant
was only 10 of the total activity, indicating a mito- chondrial localization Tsuyama et al., 1980.
135 P. von Dungern, H. Briegel Journal of Insect Physiology 47 2001 131–141
The same larval and pupal material also showed sub- stantial XDH activity during the developmental period. It
correlates linearly with larval body size: Y =
1.30X +
0.21 N
= 109, r
2
= 0.781, t
= 19.53, P,0.001. The maximal
values were close to 8 µ
molmininsect in late fourth instars. Larval urate nitrogen also showed a linear
increase: Y =
0.86X20.39 N =
124, r
2
= 0.824, t
= 23.91,
P,0.001, parallel
to total
protein nitrogen:
Y =
19.66X25.00 N =
116, r
2
= 0.947, t
= 44.47, P,0.001.
In absolute terms urate nitrogen increased from roughly 0.2
µ g per larva up to 4
µ g per larva; at the same time
protein nitrogen reached up to 80 µ
g per larva. From these data average daily syntheses of protein and urate
could be computed: 20 µ
g per larva for protein nitrogen and 0.05–0.1
µ g urate nitrogen. In the larval rearing
water, however, only traces of uric acid were detectable. To test the extent of uric acid excretion, single fourth
instar larvae were rinsed in distilled water and trans- ferred to 3 ml of distilled water. After 6 and 12 h, the
water was analyzed for urate but less than 10 ng of urate nitrogen were found, indicating negligible excretion. The
time interval of 6–12 h was short but for reasons of urate stability longer periods were found unsuitable. Thus lar-
vae synthesize urate which is not excreted.
To further characterize this larval uricotely, pupae of uniform size 2.05–2.15 mm cephalothorax length were
analyzed for XDH activity and urate at several time points. In Fig. 3 the means are combined with those of
the last two larval instars. During the fourth instar, both parameters rose steeply, parallel to the feeding activity.
As stated before, after pupation XDH declined to about half its activity but urate reached a stable plateau at
roughly 2.5
µ g nitrogen per female pupa. Fig. 3 also
indicates that roughly a third of this urate is excreted within 12 h of eclosion, together with the meconium.
Therefore, urate accumulates during the larval period; it is conserved in the pupa until eclosion when it is voided
within the first day of imaginal live.
Male pupae are considerably smaller than female pupae 1.8–1.9 mm but when normalized for size XDH
activity was similar to females. To localize the site of enzyme activities, early and late
LIV, female pupae, newly eclosed and 3-day-old females were divided into abdomen and headthorax. Expressed
as percent of the total XDH activity, the abdominal values of 50 in early LIV increased to 80 in 3-day-
old females. In contrast, in the headthorax segment the XDH activity decreased from 40 in early LIV to about
10 of the possible maxima in 3-day-old females. This indicates enzyme localization within the fatbody and a
massive proliferation of the fatbody tissue. The remain- ing activity was distributed among the Malpighian
tubules and midguts. In contrast, arginase activity of teneral females is equally distributed among the two
body segments.
Detailed XDH activity profiles for females have been
Fig. 3. Compilation of XDH activity and uric acid accumulation dur-
ing the development of Aedes aegypti. Whole-body preparations of larvae LIII j, LIV h, female pupae I and newly eclosed females
s, and white bar were assayed. Meconial urate at 12 h after eclosion sums up to the pupal level dotted segment. Enzyme activity is given
in µ
molmininsect and uric acid in µ
g nitrogeninsect, N =
10–39.
presented before von Dungern and Briegel, 2000, where it was shown to be expressed throughout imaginal
life and to rise steeply after a blood meal, proportional to the amount of protein ingested.
There was a rapid increase of arginase activity after blood-feeding, with a maximum around 18 h Fig. 4.
As in larvae, it showed a high variability despite constant blood meals of 3
µ l, indicating considerable individual
differences. Dry faecal samples were collected in a tem- poral sequence and quantified for urea and urate. A total
of 6.8 µ
g urea nitrogen and 16.7 µ
g urate nitrogen was recovered, both taken as 100 in Fig. 4b. Excretion of
urea preceded and terminated a few hours before the expulsion of urate was complete. The peak of arginase
activity correlates with the amount of dietary protein. With enemas of 1, 3, and 5
µ l of rat blood, arginase
activity increased with similar kinetics, but the maxima were gradually reached later, namely at 18, 24, and 32
h after the injection, with a corresponding enhancement from 60 to 100, and 120, respectively.
136 P. von Dungern, H. Briegel Journal of Insect Physiology 47 2001 131–141
Fig. 4. A Arginase activity in the female fatbody h of blood-fed
Aedes aegypti . B Faecal urea s and urate I during the course of
a gonotrophic cycle. All females were of uniform size and received 3 µ
l of blood. Both catabolites were measured from the same faecal samples. Data are in percent of their respective maxima: for arginase
3.45 nmolminfatbody; for cumulative urea 6.79 µ
g nitrogen and for uric acid 16.67
µ g nitrogen; N
= 4–12.
Since blood-fed females were reported to respond to the presence of drinking water by increased ureotely at
the expense of uricotely Briegel, 1986a, this effect was reinvestigated at the enzymatic level. Females with
identical blood meals of 3 µ
l either were kept completely dry during the digestive period or had access to drinking
water ad libitum from water-soaked cotton wicks. XDH and arginase activities were determined as well as faecal
urea and urate. XDH activity did not differ among the two groups. Arginase activity, for unknown reasons,
remained at 20–40 of the maximal values reported in Fig. 4, despite the presence of drinking water. The cumu-
lative output, however, showed more drastic results. With water available, urea excretion increased by 30,
whereas urate output dropped by 40 Fig. 5.
Males never feed on blood, they rely exclusively on carbohydrates throughout their life and consequently are
assumed to refrain from protein catabolism. As men- tioned before, in pupae no sex-related differences were
found for XDH activity. Within 2 days after eclosion, however, males excrete more urate than their sisters and
similar cumulative amounts until death 3 to 8 days after eclosion. Taking body size into account, starving males
Fig. 5. Effect of dessiccation on protein catabolism in blood-fed
Aedes aegypti . A XDH activity, B and C cumulative excretion of
uric acid and urea when females had access to water h, N =
6–11 or were without water I, N
= 3–7. Data are percent of maximal values
observed without water: XDH 4.42 µ
molminfatbody; urate 18.96 µ
g nitrogenfemale; urea 6.79
µ g nitrogenfemale. The dotted line in A is
a plot of the standard curve for XDH.
excreted twice the amount of urate per body volume or protein until death than did their sisters. This is reflected
in a
higher size-specific
XDH activity:
13.0 µ
molminmm
3
in males and 9.6 µ
molminmm
3
in females; the corresponding values for abdominal fatbody
were 8.0
µ molminmm
3
in males
versus 6.6
µ molminmm
3
in females. The functional role of XDH is pleiotropic. Since its
involvement in pigment synthesis has been established Ziegler and Harmsen, 1969, we carried out a prelimi-
nary comparison between two strains of extreme body coloration. XDH activity was followed during develop-
137 P. von Dungern, H. Briegel Journal of Insect Physiology 47 2001 131–141
ment and after a blood meal of a very pale queensland- ensis
strain QUWI and a very dark formosus type UGFO of Ae. aegypti. In both strains, XDH increased
in linear fashion until pupation, although slightly steeper in the larvae of the pale QUWI genotype. This strain
took half a day longer until pupation, hence grew slightly larger and reached a 10 higher enzyme activity at the
onset of pupation. Consequently, in teneral pupae the XDH was 1.3 times higher than in the dark UGFO types.
But pupal urate synthesis was only 0.15
± 0.02
µ g of size-
normalized nitrogen versus 0.27 ±
0.03 µ
g nitrogen in the dark form, or 0.25
± 0.02
µ g urate nitrogen in the UGAL
strain. Teneral females of both strains had equal protein contents when normalized for size, and after a blood
meal there were no significant differences in XDH activities and its temporal pattern. The dark form had a
1.3-fold higher lipid content at eclosion.
4. Discussion
