Introduction
The housefly, Musca
domestica L., is a widespread insect intrinsic to humans, medically
important, and a mechanical transmitter of many pathogens such as viruses,
bacteria, protozoans, and worms, in addition to its rapid multiplication and
severe disturbance to humans (Khamesipour et al. 2018; Al-Khafagi and
Mohammed 2022). Many synthetic chemical pesticides, such as organophosphates,
pyrethroids, and organochlorines, have been used to control M. domestica
(Palacios et al. 2009). The excessive, repeated, and incorrect use of
these pesticides also led to the killing of parasites and predators (natural
enemies), weakening their role in the process of natural control and causing an
imbalance in the environment (Klakankhai 2022). Their incorrect use also led to
the emergence of resistance to pesticides, leading to the predominance of new
pests that did not exist previously (Fayyad et al. 2022). Therefore,
interest has begun in developing alternative control methods for manufactured
pesticides and devising new control methods. The scientists began using natural
organic pesticides such as plant extracts, vegetable oils, and solutions of
mineral metals, which do not have any harm to human health and the environment
and are environmentally friendly. Research is still ongoing that supports the
use of some algae by extracting some effective compounds. Preliminary
experiments were conducted to support the toxicological effects of algae (Mohammed 2018).
Algae are known as autotrophic primitive plants that convert
inorganic matter into organic matter through the process of photosynthesis (Lee
2019). These are simple in structure, lack vascular tissue, contain
chlorophyll, have simple reproductive structures, are not surrounded by a
sterile wall, and do not rise to the level in contrast to higher plants (Fayyad
et al. 2022). The effect of
algae extracts is due to the presence of numerous compounds such as alkaloids
and phenols that act as anti-insects leading to the destruction of insects and
affecting the hormones to decline the egg rate (Mohammed et al. 2018).
The present study aims to know the effect of the hot
alcoholic extract of E. ralfsii algae on some stages of houseflies as an
alternative to chemical insecticides.
Materials and
Methods
General
description
Rearing of housefly: The adult
insects (Musca domestica) were raised in the laboratory in wooden cages
(30 × 30 × 30 cm: L x W x H), covered with a muslin cloth, with one side in a
sleeve shape to deal with insects. A plastic container of 500 mL containing powdered sugar
and dried milk in a ratio of (1:1) (weight: weight) and glass bottles
containing cotton moistened with water were used to feed adults.
The
experiments were carried out at Mustansiriya University in Baghdad's animal
house, which is equipped with specialized insect incubators. For egg laying and larval growth, the cage
was also provided with another plastic container the same size as the previous
one containing the culture medium of 200 g of floating fish broth (protein 20%,
fat 4% and fiber 4%).
The culture
medium was grinded and sterilized in an autoclave at 121°C and 1 bar pressure
for 3 min. 10 g of dry yeast and sterile distilled water was added to get the
final volume of 100 mL (Hermize et al. 2016). The colony was perpetuated
for ten generations to avoid the presence of any residual effect of pesticides
to obtain a specific sensitive strain in constant conditions (28 ± 2°C and
relative humidity 65 ± 5%) before conducting life experiments (Martins 2013).
Collection of
algal sample: Algae samples were collected using plastic containers
and shard-edged tools on March 01, 2022, from the University of Baghdad, Al-Jadriya,
Iraq. The geographical coordinates of the location were longitude
33°01'.94"E and latitude 44°20'.41"N, from the bottom, with a depth
of 10–30 cm.
Preparation
of the algae powder: The isolated algae were washed well with tap water to
remove the mud and dirt and let dry at room temperature with continuous stirring. Later, these were ground with
an electric grinder, and the algae powder was kept in dry packages in the
refrigerator at 4°C until used. The method of Swain (1966) was followed to
prepare the Soxhlet alcoholic extracts of the green algae. The extract was
taken and filtered with (Whatman No.1) filter paper, and the remaining filtrate
was dried in an incubator at 37°C for 48 h.
To obtain the dry algae powder, it is stored in the
refrigerator until use. Different concentrations of algae powder (4, 8 and 16
mg/mL) were prepared by taking one gram of the extract and dissolving it into 5
mL of solvent (96% ethanol alcohol). The final volume of 10 mL was prepared to
obtain a concentration of 1 gm/10 mL which is equal to 100 mg/mL (stock
solution).
Chemical detection of algae: Preliminary detection of the active compounds in the hot
alcoholic extract of E. ralfsii (as seen in Table 1) was based on the
method of Harbone (1984). The initial detection of the active compounds in the
hot alcoholic extract of algae, Enteromorpha ralfsii was done using
specific reactions and reagents according to previous protocols (Al-Khafagi and
Mohammed 2022; Palaniyappan
et al. 2023) as given in Table 1. GC-Mass
technology was used to detect the active compounds present in the hot alcoholic
extract of E. ralfsii by
following a special thermal system (Sekaran et al. 2010).
Detailed
description of laboratory experiments: The effect of different
concentrations of the hot alcoholic extract of E. ralfsii on the larval
and pupal stages of the housefly was investigated via the following laboratory
experiments.
First: Effect
of algal extract on third instar larvae of housefly
The effect of
concentrations (4, 8 and 16 mg/mL) of the hot alcoholic extract of E.
ralfsii algae on the third instar larvae of houseflies was investigated in
two ways.
Feeding of
algae extract through larval diet: The larval diet was treated with extract of E. ralfsii
according to the method of Klakankhai (2022). To prepare 60 g of diet, 6 mL of
4 mg/mL concentration was added and mixed for two minutes. The treated larval
food was divided into three plastic containers (diameter of 4.5 cm and height
of 3.5 cm). Ten larvae (third instar) from the breeding colony were transferred
to each container, which were then covered with muslin cloth to allow the
larval breathing and to block their escape from the container. The study was
repeated in a similar way using two other concentrations (8 and 16 mg/mL) of
algal extract.
For control treatment, only sterile distilled water was
added to the larval diet. The plastic containers were transferred to an
incubator at 28 ± 2°C, RH 65 ± 5%, and duration of illumination 12:12 (light:
dark). The experiment was monitored daily and the numbers of dead and distorted
larvae, dead pupae, and emerging adults of the surviving pupae from treated
larvae were recorded. The photographs of dead and distorted larvae were also
recorded.
Direct spray
on the housefly larvae: The larvae were treated with direct spraying of algal
extract following the method of Sharififard et al. (2011). The effect of
spraying different concentrations (4, 8 and 16 mg/mL) of algae extract E.
ralfsii on third instar larvae was evaluated. Thirty larvae (third instar)
were transferred from the culture colony to a plastic container and 4 mL of 4
mg/mL of the algal extract was sprayed from a distance of 5 cm using a hand
sprayer of 10 mL capacity. Afterwards, ten larvae were transferred to plastic
containers (diameter of 4.5 cm and height of 3.5 cm) containing 20 g of larval
culture medium. The experiment was also repeated using two other concentrations
(8 and 16 mg/mL) of algae extract. Three replicates were carried out for the
control treatment in which, the larvae were sprayed with 4 mL of sterile
distilled water. The replicates of the experiment were preserved after covering
them with a perforated muslin cloth (to prevent larvae from escaping) in the
incubator at 30 ± 1°C, RH 65 ± 5%, and illumination duration of 12:12 (light:
dark). The experiment was monitored daily and the numbers of dead and distorted
larvae, dead pupae and emerging adults of the surviving pupae were recorded for
two weeks. The dead and distorted housefly larvae were photographed.
Second:
Effect on housefly pupal mortality and emergence rates
The effect of
the alcoholic extract of algae at tested concentrations (4, 8 and 16 mg/mL) was
studied on housefly pupae (24 and 72 h old). Thirty pupae (24 h old) were taken
with three replications with the control treatment, 4 mL of each tested
concentration of the algal extract was sprayed using a hand sprayer of 10 mL
capacity from a distance of 5 cm to ensure good coverage of the algal extract
over all the pupae. Later, every 10 pupae were transferred to plastic
containers (diameter of base 4.5 cm and height of 3.5 cm), covered with
perforated tulle cloth, and transferred to the incubator (28 ± 2°C and 65 ± 5%
RH) and a duration of illumination 12:12 (light: dark). The experiment
monitored the dead and distorted pupae and recorded the insects emerging from
the treated pupae. Three replicates were carried out for the control treatment,
three concentrations (4, 8 and 16 mg/mL) of algal extract were sprayed on pupae
and larvae (24 and 72 h old), and photos of the experiment's deformities were
taken. The same procedures were carried out on larvae and pupae that were
sprayed with sterile distilled water as a control treatment.
Statistical
analysis
The data was
statistically analyzed using SPSS (2012) and the comparison of multiple means was
done using least significant difference (LSD).
Results
Enteromorpha
ralfsii is a species of macroalgae in the group green algae Chlorophyceae of
the family Ulvaceae (Fig. 1). The preliminary detection of the active compounds
from the hot alcoholic extract of algae showed the presence of alkaloids,
tannins, flavonoids, and saponins, while glycosides, terpenes and phenols were
absent (Table 2).
The data of gas chromatography
(GC-Mass) showed the presence of seven major
compounds in the hot alcoholic extract of E. ralfsii (Table 3 and Fig. 2). The comprehensive spectroscopic analysis of the
compounds representing 86.32% of the total mass forming and the remaining 14%
was not verified due to their low plenty from the hot alcoholic extract of E.
ralfsii. The percentage of area was pentadecane (12.67%), nonadecane
(4.60%), tetradecane (3.02%), octadecane (11.20%), hexadecane (6.7%) and salicylic acid (6.10%).
First: Effect
on housefly larval mortality
Table 4 shows
the effect of tested concentrations of the hot alcoholic extract of E.
ralfsii alga fed to third-stage larvae of housefly. The mean larval mortality
was significantly different with values of 2.67 ± 0.33, 3.00 ± 0.57, and 5.33 ±
0.33 after feeding the algal extract at concentrations of 4, 8 and 16 mg/mL,
respectively. Morphological abnormalities observed in the treated and dead
larvae were represented as bursting of the digestive tract and excretion of
digestive fluid with the blackening of the dead larvae (Fig. 3). A decrease in the mean natural larval emergence was observed
with an increase in the concentration of algal extract in the larval diet, and
natural emergence was least (2.67 ± 0.88) at higher algal concentration of 16
mg/mL (Table 4).
Table 5 showed that after a direct spray of algal
extract at different concentrations, a decrease in the mean natural emergence
was observed with an increase in the concentration of hot alcoholic extract.
The mean larval mortality (3.33 ± 0.67) of the third instar larvae treated with
the concentration of 16 mg/mL was significantly different compared with the
control (0.67 ± 0.33).
Second:
Effect on housefly pupal mortality and emergence rates
The efficacy
of the hot alcoholic extract of E. ralfsii on the treated housefly pupa
(24 h old) can be seen in Table 6. The pupae mortality at concentrations of 8
and 16 mg/mL were the highest with values of 4.33 ± 0.67 and 5.00 ± 0.57,
respectively. These values were substantially higher than the pupae mortality
in the control (0.67 ± 0.33). All concentrations gave equal partial emergence
(2.00) of twisted pupae.
The natural emergence was significantly reduced with all
tested concentrations compared to the control (9.33 ± 0.33). Among tested
concentrations, least natural emergence (3.00 ± 0.57) was observed with highest
concentration (16 mg/mL) followed by 8 mg/mL (3.67 ± 1.85).
Table 7 indicates a significant difference (P < 0.00) in the mortality for the
treated pupae (72 h old) with algal extract concentrations The pupal mortality
was increased with an increase in concentrations (4, 8 and 16 mg/mL) of algal
extract with values of 1.00 ± 0.57, 4.33 ± 0.33 and 6.67 ± 0.33, respectively. The
natural emergence from the pupa was highest in the control treatment (10.00 ±
0.00), which differed significantly from the natural emergence of other tested
concentrations. The natural emergence was decreased
with an increase in the concentration of algal extract. The normal emergence of
adults decreased from 9.00 ± 0.57 to 5.67 ± 0.33 and 3.33 ± 0.00 at concentrations
of 4, 8 and 16 mg/mL, respectively (Table 7).
Discussion
Enteromorpha
ralfsii is known to be a dominant species in saline coastal wetlands with high
nitrogen levels (Gibson et al. 2001; Hayden et al. 2003). It
floats or moves between the water's surface and the bottom, or it can be
associated with rocks and plants (Fish and Fish 1989). The thallus (body) algae
is characterized by its smooth surface "silky", medium green,
filamentous branching, extending to a length of 2–15 cm, consisting of several
rows of cells (multi-seriate) (Lee 2019). Numerous active compounds were found
in the algae extract mentioned by Sekaran et al. (2010) and Alghazeer et
al. (2017). The secondary metabolites such as alkaloids, tannins,
and saponins showed considerable effect against insects (Adesina and Rajashekar 2018). Alkaloids are organic
substances that contain nitrogen in their composition and have strong
physiological effects, even in very low concentrations (Zandavar and Babazad
2023). Tannins are polyphenolic compounds that are nitrogen-free,
antiseptic, antimicrobial, and anti-inflammatory, tannins are antiseptic
substances that protect against fungal and insect diseases. Saponins are
natural compounds with chemical properties similar to glycosides, but they are
distinguished by the fact that they produce soapy foam when shaken with water
(Harbone 1984). Studies have shown that saponins have a physiological activity
that is toxic to humans and animals, some of them are decomposing red blood
cells Swain (1966).
Table 1: Methods for detecting active compounds (Harbone 1984; Sekaran et al. 2010; Palaniyappan et al. 2023)
|
Positive result |
Method |
Test/reagent |
Active compounds |
|
Forming a red precipitate |
Added 1 % Hydrochloric acid to the extract, then drops
of Dragendorff reagent (potassium bismuth iodide solution) |
Dragendorff’s test |
Alkaloids |
|
Purple-green color is formed |
Two mL of chloroform and a concentration acetic acid
are added to the extract in an ice bath, then added two drops of concentrated
sulfuric acid |
Liebermann’s test |
Glycosides |
|
Forming a white precipitate |
Added 1% domain
solution containing sodium chloride to the extract |
Gelatin test |
Tannins |
|
a - Golden yellow color appears b - A yellow ring forms between the two layers that
turn reddish-brown |
a- Added a few drops of concentrated sulfuric acid and
2 mL of chloroform to the extract, shake and leave until set. b- 2 mL of chloroform and 3 mL of concentrated
sulfuric acid are added to the extract. |
Salkowski test |
Terpenoid |
|
Radiates green |
One mL of the extract is placed in a watch glass and
heated in a water bath to dryness, then mixed with Wilson's reagent (5 mg
boric acid + 5 mg acidic acid + 3 mL acetone) and dried again, then applied
under U.V 365 nm. |
Wilson-tauboc |
Flavonoids |
|
Forming a dark blue or violet color |
Added 3-4 drops of iron chloride solution to the
extract |
Ferric chloride test |
Phenols |
|
The foam stays for 10 minutes |
Add 1 mL of the extract in a test tube containing 5 mL
of distilled water, and shake the tube well to form foam. |
Foam test |
Saponins |
Table
2: Presence and absence of active compound in a hot
alcoholic extract of E. ralfsii
|
No. |
Active
compounds |
Hot
alcoholic extract of E. ralfsii |
|
1 |
Alkaloids |
+ |
|
2 |
Glycosides |
- |
|
3 |
Tannins |
+ |
|
4 |
Terpenoid |
- |
|
5 |
Flavonoids |
+ |
|
6 |
Phenols |
- |
|
7 |
Saponins |
+ |
+ = Present Active compound
- = Absence
Active compound
Table 3: The main
compounds detected by the gas chromatography (GC-Mass) technique of the hot
alcoholic extract of E. ralfsii
|
Compound |
Retention time (min) |
Area (%) |
|
12.72 |
12.67 |
|
|
Nonadecane |
13.76 |
4.60 |
|
14.11 |
42.03 |
|
|
Tetradecane-8-Methyl |
16.77 |
3.02 |
|
Octadecane-8-Methyl |
17.07 |
11.20 |
|
Hexadecane |
21.8 |
6.70 |
|
Salicylic acid |
24.07 |
6.10 |
|
Total |
86.32 |
|
In the present study, GC-mass analysis shows groups of
hydrocarbons such as pentadecanone, octadecane-8-methyl, and hexadecane-tetra
methyl that were the main and common compounds in the crude alcoholic extracts
of E. ralfsii. These compounds are considered to have strong
antimicrobial activity and reduce the effect of free radicals. This includes
delaying the maturation of the eggs and increasing the thickness of the pupa to
prevent the insect from emerging (Shaker 2019). Salicylic acid is a phenolic
compound that was found in the algal extract in our study. It is widely used as
a crystalline organic acid, colorless in organic synthesis and plant hormone
functions derived from the metabolism of salicin present in the region (6.1%)
from the analysis of GC Mass. One keratolytic is salicylic acid. It is a member
of the same drug class (salicylates) as aspirin, which plays an important role in improving the plant's ability to withstand insects
through acquired systemic resistance (ASR) and making their pesticides more
effective (Hayat et al. 2010; Mohammed et al. 2018).
Table
4: Effect of different concentrations of the hot alcoholic
extract of E. ralfsii on housefly larvae (treatment as larval diet)
|
Concentration
mg/mL |
|
||
|
Dead larvae
(Mean ± SD) |
Partial
emergence (Mean ± SD) |
Natural
emergence (Mean ± SD) |
|
|
4 |
2.67 ±
0.33b |
0.667 ±
0.33a |
6.67 ±
0.33b |
|
8 |
3.00 ±
0.57b |
1.00 ± 0.50a |
6.0 ± 0.57b |
|
16 |
5.33 ±
0.33a |
2.00 ±
1.00a |
2.67 ± 0.88 |
|
Control |
0.667 ±
0.33c |
0.00 ±
0.00a |
9.33 ± 0.33 |
|
L.S.D. |
1.331** |
NS 2.369 |
1.882** |
|
P- value |
0.0003 |
0.335 |
0.0003 |
NS
(Non-Significant), ** (P < 0.01)
The mean
bearing different letters within the same column differ significantly among
themselves
%20417-424_files/image002.png)
Fig. 1: Phenotypic
structure of E. ralfsii in nature and under a microscope at 40X
magnification
The mortality of larvae could be due to the toxic effect
of compounds present in the hot alcoholic extract of E. ralfsii, such as
alkaloid compounds, which are known to be highly toxic to many insects and that
may act as inhibitors of feeding and thus starvation to death of larvae (Zandavar and Babazad
2023).
Table
5: Effect of different concentrations of hot alcoholic
extract of E. ralfsii on third instar larvae of house flies (treatment
as direct spray)
|
Concentration
(mg/mL) |
Larval
mortality (Mean ± SD) |
Natural
emergence (Mean ± SD) |
|
4 |
1.33
± 0.88ab |
6.67
± 0.88ab |
|
8 |
2.33
± 0.88ab |
6.0
± 0.88ab |
|
16 |
3.33
± 0.67a |
6.67
± 0.67b |
|
Control |
0.67
± 0.33b |
9.33
± 0.33 |
|
LSD |
2.369* |
2.369* |
|
P- value |
0.0426 |
0.0426 |
* (P <
0.01)
The means
bearing different letters within the same column differ significantly among
themselves
Table
6: Effect of different concentrations of the hot alcoholic
extract of E. ralfsii on the pupae (24 h old) of houseflies
|
Concentration
mg/mL |
Pupa (24 h old) |
||
|
Pupal
mortality (Mean ± SD) |
Partial
emergence (Mean ± SD) |
Natural
emergence (Mean ± SD) |
|
|
4 |
2.67 ±
0.33b |
2.00 ±
0.57a |
5.33 ±
0.88b |
|
8 |
4.33 ±
0.67a |
2.00 ±
1.52a |
3.67 ±
1.85b |
|
16 |
5.00 ±
0.57a |
2.00 ±
0.57a |
3.00 ±
0.57b |
|
Control |
0.67 ±
0.33c |
0.00 ±
0.00a |
9.33 ±
0.33a |
|
LSD |
1.630** |
NS 2.824 |
3.522** |
|
P- value |
0.0012 |
0.330 |
0.010 |
NS
(Non-Significant), ** (P < 0.01)
The mean
bearing different letters within the same column differ significantly among
themselves
%20417-424_files/image004.png)
Fig. 2: The main Compounds detected by
the gas chromatography (GC-Mass) technique of a hot alcoholic extract of E.
ralfsii algae
The high potency of the green algal extracts could be
attributed to the presence of toxic compounds, specifically Nonadecane and
Tetradecane, which inhibit the AChE enzyme. Furthermore, green algae are rich
in saponin and flavonoid compounds (Adesina and Rajashekar 2018). These
flavonoids inhibit the protein responsible for cholesterol transportation
during larval development resulting in larval mortality. In addition to the
presence of Salicylic acid that prevents the natural emergence of insects, it
is an exfoliating material that causes changes to the pupal shell through
surface scratches that reduce larval performance by preventing the insect from
exiting the pupal (Lortzing et al. 2019).
Nadeem et al. (2022) mentioned that larvae die of
starvation was due to the presence of terpenoid compounds, including saponins,
which are considered feeding inhibitors (Mazid et al. 2011; Kachhwaha
2017). It was also found that these compounds affect the gastrointestinal
tract, especially the epithelial cells, and the occurrence of a state of
poisoning and death after a period of feeding and the death of larvae may be
due to the presence of flavonoids because of their ability to bind with
digestive enzymes in the insect’s body and thus lead to reduced metabolism
(Palaniyappan et al. 2023), or these compounds may interfere with the
work of the endocrine system, which leads to a defect in the process of growth
and increases in the destruction of the insect (Kreem and Annon 2018 ).
Through the union of these compounds with the fatty substances present in the
gastrointestinal tract, these fatty substances are expelled without the benefit
(Shaker 2019; Gao et al. 2022). The effect of compounds
including Phenolic, including Flavonoids, increases the rate of larval
mortality, which may be due to a decrease in the rate of food digestion, which
leads to the killing of larvae due to lack of nutrition (Kazim 2013; Salman and
Ahmed 2017; Nadeem et al. 2022).
Table
7: Effect of different concentrations of the hot alcoholic
extract of E. ralfsii on the pupae (72 h old) of houseflies
|
Concentration
mg/mL |
Pupal
mortality (Mean ± SD) |
Natural
emergence (Mean ± SD) |
|
4 |
1.00 ± 0.57c |
9.00
± 0.57a |
|
8 |
4.33 ± 0.33b |
5.67
± 0.33b |
|
16 |
6.67 ± 0.33a |
3.33
± 0.33c |
|
Control |
0.00 ± 0.00c |
10.00
± 0.00a |
|
LSD |
1.215* |
1.215* |
|
P- value |
0.0001 |
0.0001 |
*(P < 0.01)
The means
bearing different letters within the same column differ significantly among
themselves
%20417-424_files/image006.png)
Fig. 3: Housefly larvae after treatment
with different concentrations of hot alcoholic extract of E. ralfsii.
The blackening of larva and exit of digestive juices at high concentrations is
clearly visible
The reason for the mortality rates recorded for the
pupae treated with alcoholic extract at the ages of 24 and 72 h may be
attributed due to the effective properties of terpenes compounds including
saponins. The mortality rates of pupae treated with the tested concentrations
may be due to the interaction that occurs between the chemical compounds and
the alienation hormone in the pupal body (Wink 1988).
Rodrigue et al. (2023) showed that the toxic
compounds present in the extract such as saponins, and tannins, may affect the
cells that are in a state of continuous division, especially in the early
stages of the puparium, to complete the growth of the organs and prepare for
the exit of the adults, which leads to the death of the pupae while they are
inside the puparium. Hayat et al. (2010) mentioned the presence of salicylic acid, which is one
of the types of phenols that have a physiological effect through changing
osmosis and could cause changes in the pupal shells, which could not be opened.
Conclusion
It was
concluded that the hot alcoholic extract of E. ralfsii is biologically
effective in controlling Musca domestica L. The hot alcoholic extract
contains numerous active compounds and toxic substances such as alkaloids,
tannins, flavonoids, and saponins, which interfere with the physiological
processes and increase the mortality of larvae and pupae of houseflies.
Therefore, it is necessary to think about increasing the production of algae
for its potential use in the control of houseflies at the time of their peak
spread and reproduction. Ultimately, the availability of E. ralfsii in
large quantities is one of the effective sources to eliminate the housefly by
spraying it alcoholic extract on the waste, piles of garbage, animal manure
waste, and housefly breeding sites.
Acknowledgments
The authors
would like to thank Mustansiriyah University of Baghdad, Iraq for providing
support in the current work.
Author
Contributions
All authors
wrote reviews, edited, and contributed equally to the work. All authors have
read reviewed and agree to publish the version.
Conflicts of
Interest
Authors are
responsible for the correctness of the statements provided in the manuscript. The
authors declare that they have no competing interests. We hereby confirm that
all the Figures and Tables in the manuscript are us.
Data
Availability
All data and
materials are available if requested.
Ethics
Approval
This is an
observational study. Mustansiriyah University Research Ethics Committee has
confirmed that no ethical approval is required. The study was conducted on the
natural occurrence of algae extracts and usage of house flies that are
available in the environment and ethical approval is not demanded.
Funding Source
The authors
did not receive support from any organization for the submitted work. The
authors have no relevant financial or non-financial interests to disclose.
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