The flesh fly, Parasarcophaga argyrostoma is abundant in Egypt and it
has some medical and veterinary importance since it is of particular interest
owing to its significant role in causing gastric, intestinal and nasal myiasis
and invading various tissues of man and animals (facultative parasites) and
often leading to series consequences. It normally lives in decomposing matter
and may be attracted to foul wounds for larvae position. Larvae have been found
in sore wounds and natural openings of man (Gardiner et
al., 1983). The efficient control of these insects has long been the
goal of workers in the field of medical entomology.
Chemical treatments create problems by leaving undesirable residues in food
and there are public fears about chemical residues on safety of users and the
environment, as well as the development of resistance to these insecticides.
Thus in 1996, the Food Quality Protection Act limited the use of many pesticides,
particularly those that share common toxicological mechanisms such as organophosphate,
carbamate insecticides and triazine herbicides (Lyon and
Newton, 1999). Thus, it is becoming clear that alternative pest management
tools are needed, which will be less hazardous to human, non target animals
and the environment, in the same time these alternative tools must be used in
the field application with minimum cost. In this context, sunlight-activated
photo-pesticides represent a possible alternative to traditional chemical compounds.
The use of photochemical processes as a tool to control the population of several
types of insects has been repeatedly examined in both laboratory experiments
and field studies (Ben Amor and Jori, 2000). At the
cellular level, most photosensitizers are able to induce apoptotic cell death
This work aims to use the HP as a non-toxic and nonmutagenic photopesticide to control P. argyrostoma and choose the optimum parameters that can be used in the field application to have high percentage of mortalities of flies with maximum safety level and minimum cost.
MATERIALS AND METHODS
This study was done as a part of Master thesis in the period of 2008-2010 in the national Institute of Laser Enhanced Sciences (NILES), Cairo University and Faculty of Sciences, Fayoum University, Egypt.
P. argyrostoma laboratory colonization: A large glass vessel
containing a few pieces of decaying meat was left in the open air for three
days and a few drops of water were added whenever the meat showed any signs
of dryness. In this condition the meat is a good larviposition site for wild
flesh flies. The vessel was then transferred to the laboratory with its top
covered with muslin. When the deposited larvae were fully developed and ready
to pupate, they were transferred into muslin cages where adults emerged. The
emerging adults were identified and kept separately. A stock culture was maintained
under laboratory conditions from which the different stages needed for the work
experiments were taken.
The breeding stock of adults was maintained in 50x50x80 cm muslin cages. On emergence, the adults were supplied with sugar, milk and water as food. Sugar and milk were supplied in the form of a concentrated solution in a ratio of 1:2 or dried in the form of granulated sugar mixed with milk powder in the proportion of 1:2. Water was supplied by dipping a piece of cotton, as a wick, in a bottle filled with water. The meat was introduced in a petri-dish, as a larviposition medium and was changed daily. Both fresh and previously frozen meat attracted gravid females for larviposition. The cages were examined once a day in the morning for the newly deposited larvae. The sugar milk and water were checked and the meat was renewed whenever necessary.
Preparation of hematoporphyrin stock solution (Treated bait): Each normal
bait was prepared by dissolving 100 g sugar in 70 mL distilled water. The treated
bait was prepared by dissolving 1.7 g of hematoporphyrin (from porphyrin products
(logan, utah) in 23 mL sugar solution and 2 mL of sodium hydroxide solution
(0.1 M). The stock solution was left on magnetic stirrer for 2 h. The final
solution was adjusted to pH = 6.5-7. The final HP concentration was measured
by spectrophotometer (Perkin Elmer, LAMBDA 40). The stock solution was used
within two weeks because it is stable for a few weeks when kept in dark at 4°C
(Thameur et al., 1998). According to Beer
s Lambert law the measured absorbance is being correlated to the concentration
of the solution. Diluted solutions were prepared by taking aliquots of the stock
solution and added to the normal bait.
Estimation of HP concentrations inside the insect body: After feeding on HP, the flies were frozen then homogenized with 5 mL dist. H2O using homogenizer. The suspension thus obtained was centrifuged for 25 min at room temperature and at 13000 rpm. The pellet was discarded, while known aliquots of supernatant were measured by reading the absorbance in sulfuric acid using spectrophotometer. The HP concentration was determined from Beers Lambert law using absorbency 423000 in sulfuric acid. The results were expressed as moles L-1 of HP recovered by fly.
Exposure studies: The flies were exposed in transparent cages (7x5x11 cm). The sunlight fluence rate was measured by Eldonet WinDose 2000 dosimeter (Real Time Co., Germany). The actual fluence rate was accounted through the average of the different intensities during the exposure time.
Dark experiment: In this experiment, the flies groups were left for one week in the dark and each group was supplied by one of HP treated bait (10¯2, 10¯3 and 10-4 ML-1). The behaviour of the flies and percentage of fly mortality were monitored.
Histological studies: Three groups of flies (10 flies/each) were separated from the rearing cage. All groups were supplied with 10¯2 M L-1 HP treated bait and left in the dark for 12 h as a feeding period. Each group was exposed to sunlight for one of the exposure times 2, 4 and 9 h. The died flies were dissected to get the mid gut. The alimentary canal of flies was removed and fixed using Bouins, then dehydrated by using ascending series of ethanol. The samples were cleared by xylol, after that infiltrated and imbedded in paraffin wax. Thin sections of midgut were prepared using the microtome, mounted on glass slides and treated with Haematoxylin and Eosin stains to investigate the extent of HP effect on the level of flies tissues.
Statistical analysis: The statistical package for social science (SPSS
version 17.0) program were used for statistical evaluation and analysis of variance
(frequently abbreviated ANOVA) (Miller and Miller, 1988).
RESULTS AND DISCUSSION
In this study, Hematoporphyrin IX was tested as a novel modality pesticide against one of the medical insects, Parasarcophaga argyrostoma. The results revealed the optimum parameters which induced the highest efficiency of HP in controlling HP treated Parasarcophaga argyrostoma using direct sunlight.
During the application of our experimental protocol, we found that there are many parameters, which can modulate the efficiency of HP as a photoinsecticide against P. argyrostoma. These parameters are HP concentrations, fluence rates, exposure times and incubation periods.
Figure 1 showed that P. argyrostoma was more affected
by the highest concentration (10¯2 M L-1) of HP giving
average mortality of 83% and the mortality of 10¯3 M L-1
of HP was 50%. This means that the most efficient HP concentration for controlling
of P. argyrostoma is 10¯2 M L-1 which is
different from the most efficient concentration used to control Musca domestica
(10¯3 M L-1) and Culex pipines (10-5
M L-1) studied in the previous work (El-Tayeb,1999).
|| Effect of different HP concentrations (10-2, 10-3
and 10-4 M L-1) on the % of mortality of P. argyrostoma
(20 flies/concentration/replicate) exposed to natural sunlight (fluence
rate: 236.50 Wm-2) for 9 h immediately after incubation with
HP for 12 h
||Effect of different sunlight doses (893.7, 1935 and 2645.1
Wm-2) on % mortality of P. argyrostoma (20 flies/dose/replicate)
which had been exposed to 10-2 M L-1 HP and then exposed
to sunlight for 3, 6 and 9 h
This effect can be ascribed to the role played by the integument and the size
of flesh fly. P. argyrostoma has most darkely pigmented body, heaviest
integument and greatest size (Khoobdel et al., 2008)
than M. domestica and C. pipiens. All these characters of the
body of P. argyrostoma allow to transmit light dose less than
lighter integument so the results of photodynamic reaction in case of P.
argyrostoma is slower than in case of M. domestica and C. pipines.
The sunlight dose (Fig. 2) plays an important role, in the
effect of photosensitizer (HP) on the survival of P. argyrostoma. It
is clear from this figure that the mortality percentage of the flies (13, 96,
96%) increases with increasing the fluence rate (893.7, 1935, 2645.1 W m-2,
respectively) of the light. This behavior is agree with the previous work (El-Tayeb,
1999; Luksiene et al., 2005) in which, they
found that the effect of HP on Liriomyza bryoniae, C. pipiens
larvae and M. domestica mortalities in the sunny seasons is more than
the other seasons because; with increasing sunlight intensity the number of
photons striking the target increases and so, the number of excited HP molecules
will be increased followed by a concomitant increase of the amount of singlet
oxygen produced by photochemical reaction.
|| Effect of different light exposure times (1, 3, 5 and 9 h)
on % of mortality of P. argyrostoma (20 flies/each exposure time/replicate)
which had been exposed to 10-2 M L-1 for 12 h. (fluence
rate of natural sunlight: 236.50 W m-2)
||Effect of different HP concentrations and incubation periods
on the accumulation rates of HP in P. argyrostoma body (20 flies/concentration/replicate).
The flies were exposed to the HP bait for 6,12 and 24 h
Also, this support Fondren et al. (1978) work
in which they studied the light intensity as a critical parameter in the photodynamic
toxicity of rose Bengal to the adult house fly, they showed that the accumulated
number of photons needed to kill 50% of a population decreased as the intensity
increased. This would interfere that; there is a regenerative capacity within
the insect that is more efficiently overcome by photodynamic action as the light
The different light exposure times have significant differences on the percentage
of survival of P. argyrostoma. It is clear from Fig. 3
that the percentage of mortality of flies (10, 26.66, 43.3 and 83%) increased
with increasing light exposure time (1, 3, 5 and 9 h, respectively) after treatment
with HP. The effective light exposure time in case of P. argyrostoma
control using HP is lesser than the light exposure time of the other photopesticides
because the half Lethal Time (LT50) for P. argyrostoma fed on 10¯3
M L-1 was 9 h. While the other photopesticides like Phloxin B, LT50
= 13.31h and tetrachloro- fluorescein, LT50 = 31.48 h when the flies fed on
10 ¯3 M L-1 (Fondren et al.,
Dark experiments revealed that the hematoporphyrin had no toxic effect in absence
of light hence there is no toxic effect on human and animals when it accumulated
inside their bodies. This is agreeing with (Kappus et
al., 1988) in which they tested the dark toxicity on human keratinocytes.
Previous study declared the dark toxicity in some of other photopesticides.
Creighton et al. (1980) reported the dark toxicity
of rose bengal to the cabbage looper, the corn earworm and the pickleworm. James
and Heitz (1987) showed that both phenylheptatriyne and alpha-terthienyl
displayed ovicidal activity against the eggs of the fruit fly in the dark. The
high carcinogenic potential of this class of compounds has kept them from being
exploited as much as would be expected if there was no carcinogenic risk.
The most attractive feature of HP was investigated in this study is its ability
to accumulate inside the biological tissues of flies by a rate which exceeds
the excretion rate of flies, especially when the concentration and incubation
time are long enough. The highest rate of accumulation was revealed in the flies
which were fed on the highest HP concentration (10-2 M L-1)
in the all HP ncubation periods. The HP concentration accumulated in the flies
bodies which were fed on 10¯2 M L-1 HP bait was 20x10-6
M L-1 HP accumulated in the flies bodies. The HP feeding concentration
of 10-3 and 10-4 M L-1 caused the same amounts
of HP accumulation (8 x10-6 M L-1 HP) inside the flies
bodies for the whole HP bait incubation periods (Fig. 4).
||Transverse sections of P. argyrostoma alimentary canal
(midgut) showing: (a) Control flies (b) Flies treated with HP and exposed
to sunlight for 2 h (c) Flies treated with HP and exposed to sunlight for
4 h and (d) Flies treated with HP and exposed to sunlight for 9 h
This may interprets the results of Fig. 1 in which the highest
mortality rate appeared with the highest HP concentration in the feeding medium.
This agree with Awad et al. (2008) which tested
the effect of HP as a photopestiside on Culex pipien larvae in a semi-field
Histological studies (Fig. 5a-d) confirmed
the results of Fig. 2 which support the previous interpretation
of which related to the cause of flies death due to starvation. The damaged
alimentary canal stopped to absorb the digested food to supply to the other
Results study of this work concluded that HP photosensitization process is a promising method for control of P. argyrostoma. Accordingly, it is possible to say that this study is available to wide field studies. The advantages of HP and the method of its application are enough to expect the success of this method in the field application.