IRON ACCUMULATION IN AEDES AEGYPTI LARVAE, FOOD BOLUS AND
FECAL PELLETS
Paulo
Pedrosa*1, Desiely Silva Gusmão2, Carlos Eduardo Rezende1
& Francisco José Alves Lemos2. Universidade Estadual do Norte
Fluminense, 1Laboratório de Ciências Ambientais; 2Laboratório
de Biotecnologia, Av. Alberto Lamego, 2000, Campos dos Goytacazes, 28015-620,
Rio de Janeiro, Brasil.
E-mail:
pedrosa@cbb.uenf.br
Abstract
The
mosquito larval stage is always aquatic and shuttles from the subsurface where
it feeds on microorganisms to the surface to obtain oxygen through a
snorkel-like breathing apparatus. Mosquito larvae can be found in numerous
habitats and the distribution of some mosquito species depends on their ability
to cope with extreme situations such as polluted water and water with a high
saline. The biological responses (e.g. accumulation, toxicity) of A. aegypti larvae face to heavy metals
is, however, poorly known. Our goal is to investigate iron accumulation in A. aegypti larvae, food bolus and fecal
pellets and the role of peritrophic matrix as a barrier to the iron passage
through it.
Introduction
The distribution of mosquitoes is largely based on the
availability of suitable sites for larval growth. Because larval mosquitoes are
aquatic, water is essential. However, natural waters vary considerably in
composition, from the nearly distilled resulting from rain to salt marshes and
alkaline lakes (Hagedorn, 1996). On the other hand, mosquitoes larvae have
developed physiological mechanisms that allow them to invade niches that are
extreme in terms of salt composition. Also, aquatic systems are threatened more
and more by many types of pollutants including heavy metals (Peters, 1992). The
biological responses (e.g. accumulation, toxicity) of A. aegypti larvae to heavy metals is, however, poorly known. The
gut of most of the insects contains an extra cellular layer separating the food
of the epithelial cells. This layer is denominated of peritrophic matrix (PM)
(Lehane,1997). Our knowledge concerning this structure is still very
incomplete. However, the study of this structure has been intensifying due to
its probable function as a barrier against pathogens and toxic agents present
in the food.
Also, being detritivorous and being preyed upon by
insectivorous vertebrates and arthropods, they can play a significant role in
accumulating and further transferring toxic metals to higher trophic levels
(Devkota and Schmidt, 2000). Jamil and Hussain (1992) showed that the transfer
of heavy metal in an aquatic ecosystem could be very high. Our goal was to
investigate the role of peritrophic matrix as a barrier to high levels of iron
by measuring the accumulation of this heavy metal in larvae,
food bolus and fecal pellets of A.
aegypti.
Methods
We used A. aegypti mosquitoes larvae from the colony maintained in the
Laboratory of Biotecnology of Bioscience and Biotechnology Center at North
Fluminense State University.
150 larvae were maintained in petri dishes containing
20 mL of distilled water (control) and FeSO4 solutions (0.5mM, 5mM
and 50mM in distilled water). After 16h the larvae were dissected in
saline solution (0.15M NaCl) and their food bolus, carcass (larva without food
bolus) and feces were dried at 90º
C for 36h. The samples were weighed and
decomposed with 5 ml of concentrated nitric acid on a hot plate. After initial
digestion was added 150 ml of hydrogen peroxide to the
samples. After evaporation to dryness the residue was dissolved in 10 mL of 0.5
M nitric acid and analyzed by ICP-AES.
The iron concentrations in the blank samples were also
determined and these values were subtracted from samples values to avoid any
unwanted contamination during sample preparation.
Mean and standard deviation of all determinations of
the heavy metal was calculated and the data are given in mmol Fe g -1 dry weight of the sample.
Results and Discussion
Control larvae maintained in distilled water (control)
presented a concentration of 3,9 mmol Fe g-1 while in the iron enriched medium (0.5 mM, 5 mM,
50 mM) the concentrations were higher (66 mmol Fe g-1, 132 mmol Fe g-1, 290 mmol Fe g-1, respectively). We observed that iron
concentration in the larva, carcass, food bolus and feces is increased when the
larvae are maintained in the medium containing different Fe concentration
(0.5mM, 5mM and 50mM, respectively) (figure 1). At the highest concentration
(50 mM) we don’t observe the iron saturation of the tissues except to the feces
(figure 1). When we compare iron concentration in the carcass (figure 1B) and
food bolus (figure 1C) we observe that most of the ingested iron is retained in
the gut lumen.
Larvae maintained in absence of iron didn’t produce
feces. Differently, iron-treated larvae excrete a huge amount of feces (data
not shown). The enhanced production of feces enveloped by peritrophic matrix (a
chitin-protein extra cellular gut matrix) appears to be a physiological
response of the larvae for the iron elimination.
The feces of larvae submitted to 5 mM FeSO4
were washed to separate the iron from peritrophic matrix. Only 13% of iron was
solubilized indicating that most of the iron
is associate to the PM and/or is in an insoluble form (e.g. iron oxide).
Based on the bioconcentration factors corresponding 0.5 mM FeSO4
(Larva = 1.102, carcass = 1.101, food bolus = 8.102
and feces = 2.103) we can
conclude that iron is concentrated in the food bolus suggesting the possibility
of the peritrophic matrix as an effective barrier against iron incorporation
(figure 2). Besides to block iron penetration, PM also can bind this heavy
metal. Preliminary measurements (data not shown) demonstrate that isolated PM
can sequestrate approximately 700 mmol Fe g-1.
In order to verify whether PM could function as a
barrier to heavy metals we did a preliminary study to see the iron
incorporation into the larvae tissues. First, we observed that A. aegypti
larvae when maintained under three different iron concentrations solutions
excreted great amount of brownish feces (feces are composed of PM and food
residue) while control larvae didn’t excrete at all. As the larvae was kept in
a FeSO4 solutions without
any kind of food we can conclude that
iron was responsible for the color and production of the feces. The
figures 1 and 2 show that the iron concentration observed in food bolus and
feces are much higher than in larvae and carcass. These data indicate that most
of iron is retained in the larvae midgut lumen and excreted in an insoluble
form as a feces component. As fecal pellets proved to serve in the transport of
organic matter from surface waters into deeper layers, they might be involved
in the transport of pollutants far from the source of pollution (Turner and
Ferrante, 1979). It has been shown that fecal pellets may contain elevated
concentrations of heavy metals (Boothe
and Knaeur, 1972). The PM is a probable responsible for the
bioaccumulation of iron in the larvae midgut. It binds and retains most of the
ingested iron in the food bolus. Dissected PM from 50 mM FeSO4 treated
larva appear quite distinct to the control larvae PM with a magnify
fluorescence.
ACKNOWLEDGEMENTS
We acknowledge the FAPERJ, CNPq, UENF and FENORTE for
their financial support.
References
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Jamil, K., Hussain, S. (1992). Arch. Environ. Contam. Toxicol. 22(4), 459-463.
Lehane, M.J. (1997) Annu. Rev. Entomol. 42, 525-550.
Peters, W. (1992) Peritrophic membranes. In: Bradshaw S.D., Burggren W.,
Heller, H.C., Ishii, S., Langer, H., Neuweiler, G. e Randall D.J.
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D
CA
B
A
Figure 1.
Measurements of iron in larva (A), carcass (B), food bolus (C) and feces (D)
according to increasing medium iron concentration. Each point represents the
mean of three experiments.
B
D
CA
A
Figure 2.
Relationship between medium iron concentration and bioconcentration factor in
larva (A), carcass (B), food bolus (C) and feces (D).