Applied Microbiology and Biotechnology (2021) 105:7505–7515

https://doi.org/10.1007/s00253-021-11541-2

ENVIRONMENTAL BIOTECHNOLOGY

Rhamnolipids and essential oils in the control of mosquito‑borne

tropical diseases
Ana Maria Salazar‑Bryam1 · Vinicius Luis Silva1 · Marina Rodrigues de Abreu1 · Renata Silva Matos1 ·
Mateus Aparecido Gonçalves da Rocha1 · Raphael Culim Neves1 · Maria Izabel Camargo‑Mathias1 ·
Claudio José Von Zuben2 · Roberta Barros Lovaglio3 · Jonas Contiero1,4

Received: 6 May 2021 / Revised: 13 August 2021 / Accepted: 22 August 2021 / Published online: 15 September 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract
The diseases transmitted by mosquito vectors are a great public health issue. Thus, effective vector control becomes the main
strategy to reduce their prevalence. However, insecticide resistance has become a huge concern for the mitigation of mosqui-
toes; here, we propose the use of rhamnolipids in emulsion with clove oil against Aedes aegypti and Culex quinquefasciatus.
The toxicity of rhamnolipids and clove oil to two species of mosquitoes transmitting tropical diseases was investigated. After
24 h, the ­LC50 was 140 mg/L when rhamnolipids were used and 154 mg/L when clove oil was used against Aedes aegypti
larvae. In the case of Culex quinquefasciatus, the ­LC50 was 130 mg/L for rhamnolipids and 19 mg/L for clove oil. When
the concentrations of the upper limits of one of the solutions (rhamnolipid or clove oil) were mixed, 100% mortality was
obtained after 24 h. The bioassay of insecticidal action for solutions of rhamnolipids and clove oil in the lower limit, upper
limit, and lethal concentration 50 to determine the effect on 50% of the population (KD50) achieved low results from KD50
to the upper limit compared to the other concentrations for both Aedes aegypti and Culex quinquefasciatus. The rhamnolipids
and clove oil at the upper limit concentration had the greatest repellent activity against the two mosquito species. Bioassays
using different concentrations of rhamnolipids revealed variations in the morphology of the intestinal epithelium (800 mg/L).
A concentration of 900 mg/L led to the most severe morphological changes in the organization of the epithelium and the
cells lining the intestines of these larvae. When larvae were exposed to a concentration of 1000 mg/L, the marginalization
of chromatin in the nucleus of epithelial cells was very severe, indicating the onset of cell death.
Key points
• The toxicity of rhamnolipids and clove oil has a larvicidal, insecticidal, and repellent effect.
• The combination of concentrations of these compounds enhances their action.
• Different concentrations of rhamnolipids led to severe morphological changes in the organization of the epithelium and
the cells and the intestines of larvae.

Keywords Rhamnolipids · Clove oil · Pseudomonas aeruginosa · Aedes aegypti · Culex quinquefasciatus

Introduction
* Jonas Contiero
jonas.contiero@unesp.br
Mosquito-borne diseases are concentrated mainly in tropi-
1
Department of General and Applied Biology, Institute cal countries. One-sixth of infections worldwide are asso-
of Biosciences, São Paulo State University-Unesp, ciated with vector-borne diseases and the highest incidence
Rio Claro, São Paulo, Brazil is related to mosquitoes (McGraw and O’Neill 2013; How-
2
Department of Biodiversity, Institute of Biosciences, São ard 2016). Dengue is a disease transmitted by the mos-
Paulo State University-Unesp, Rio Claro, São Paulo, Brazil quito Aedes aegypti and its incidence has increased over
3
Federal University of São Carlos (UFSCar), Natural the past 10 years, becoming a major public health problem,
Sciences Center - CCN - Campus Lagoa Do Sino, with approximately 2.4 million cases reported annually
Campina do Monte Alegre, São Paulo, Brazil
since 2012 (McGraw and O’Neill 2013; Howard 2016).
4
Institute for Research in Bioenergy, São Paulo State Aedes aegypti not only transmits this arboviral infection,
University-Unesp, Rio Claro, São Paulo, Brazil

13
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7506
but is known to transmit other arboviruses, such as chi-
kungunya and Zika virus, the latter of which is related to
microcephaly and Guillain-Barré syndrome (McGraw and
O’Neill 2013; Lupi et al. 2013). Culex quinquefasciatus
is another vector of tropical diseases, such as lymphatic
filariasis caused by Wuchereria bancrofti; while Brugia
malayi and Brugia timori, which also cause the disease,
are vectors respectively Mansonia mosquitos and Anoph-
eles barbirostris. Despite its important incidence, this
tropical disease is in the group of neglected diseases and
its elimination remains a challenge (Hotez and Fujiwara
2014). Vector control strategies through mechanisms for
managing and eliminating vector-borne tropical diseases
generally involve the use of chemical insecticides, which
have potential negative effects and also lead to the occur-
rence of mosquito resistance (Chang et al. 2014). Bio-
logical pesticides constitute effective, environmentally
friendly alternatives and contribute little to the occurrence
of resistance on the part of vectors.
Essential oils have been studied for their effectiveness
in controlling different insects, including disease-trans-
mitting mosquitoes. Dias and Moraes (2014) report that
361 essential oils extracted from a wide variety of plants
have been tested against A. aegypti and 40% were consid-
ered to be active ­(LC50 < 100 mg/L). Saavedra et al. (2018)
analyzed molecules from 62 plants with activity against
the vector of chikungunya, dengue, and Zika virus using
linear QSAR models of bioactive molecules, which pro-
vide appropriate results for predicting insecticidal activ-
ity. Chandrasekaran et al. (2018) determined the larvicidal
efficacy of latex extracted from Carica papaya and synthe-
sized silver nanoparticles (CPAgNPs) against developing
immature juveniles of Aedes aegypti and Culex quinque-
fasciatus. Clove oil is one of the most widely studied due
to the active compound 4-allyl-2-methoxyphenol (euge-
nol), which has achieved positive results with regard to
vector control (Regnault-Roger et al. 2012). Rhamnolipids
are biosurfactants produced by Pseudomonas aeruginosa
and have characteristics that can be used when seeking
a green insecticide. These glycolipid biosurfactants have
been reported to be potential metabolites for biological
control. Recent studies have used rhamnolipids to control
larvae, adults (Da Silva et al. 2015), and pupae (Praba-
karana et al. 2015) of the mosquito A. aegypti, achieving
good larvicidal, insecticidal, and pupicidal activity. In
addition to having insecticidal properties, rhamnolipids
have physicochemical properties that enable the forma-
tion of emulsions, which allows these biosurfactants to
be mixed with other compounds that have insecticidal
activity, such as essential oils. Based on this principle,
we investigated the emulsifying properties of rhamnolip-
ids mixed with essential oils to assess the possibility of
13
Applied Microbiology and Biotechnology (2021) 105:7505–7515
synergistic activity between the action of the rhamnolipid
and that of the essential oil.
Materials and methods
Microorganism and culture media
The microorganism used in the experiments was Pseu-
domonas aeruginosa LBI 2A1 (Lovaglio 2011) from
the random mutant bank at the Industrial Microbiology
Laboratory, Department of General and Applied Biology,
UNESP, Rio Claro, state of São Paulo, Brazil. The mutant
was obtained from the parental strain P. aeruginosa LBI
isolated from an oil spill in soil (Benincasa 2001), using
the kit EZ-Tn5™ < KAN-2 > Tnp Transposome (Epi-
center), whose marker was the kanamycin resistance gene.
From previously published results, the mutant strain P.
aeruginosa LBI 2A1 was chosen for rhamnolipid produc-
tion for showing higher yields compared the its parental
strain (Lovaglio 2011; Salazar-Bryam et al. 2017). Puri-
fied glycerol without salt was used as a carbon source to
produce rhamnolipids. The experiments for the production
of rhamnolipids were carried out in 1-L Erlenmeyer flasks
containing 300 mL of culture medium (g/L): K ­ 2HPO4 0.3;
­MgSO4 ­7H2O 0.5; KCl 1.0; ­NaNO3 15; glycerol 100; and
1 mL/L of the trace element solution with the following
composition in g/L: sodium citrate dihydrate 2.0; ­FeCl3
­6 H 2O 0.28; Z­ nSO 4 ­7 H 2O 1.4; C
­ oCl 2 ­6 H 2O 1.2; C
­ uSO 4
­5H2O 1.2; ­MnSO4 ­H2O 0.8, maintained on a shaking table
at 200 rpm and 37 °C for 72 h.
Rhamnolipid extraction
The total volume of the flask was centrifuged after 72 h
of fermentation. The cell-free broth was acidified with
­H 3PO 4 85% 1:100 (v/v), leading to the precipitation of
the rhamnolipids. Extraction was performed with ethyl
acetate 1:1.25 (v/v). After stirring for 10 min, the mixture
was left to rest. The upper phase was then removed and the
solvent was evaporated in a rotary evaporator to obtain the
biosurfactant. The extraction procedure with ethyl acetate
was repeated with the lower phase.
Insect collection and maintenance
The insects used for the bioassays are described in Table 1.
Adults were fed 10% glucose and kept in plastic cages
at a controlled temperature of 27 ± 1 °C with a 12-h
photoperiod.
Applied Microbiology and Biotechnology (2021) 105:7505–7515
Table 1  Description of insects Insect
used in bioassays
Aedes aegypti
Culex quinquefasciatus
Egg laying and larval maintenance
To obtain eggs and larvae for the experiments, blood meals
were performed for 30 min using laboratory mice (approved
by the Ethics Committee on Animal Use [Protocol nº: 3698,
Decision No. 022/2011]). The cages in which the females
performed the blood meal received 600–mL flasks con-
taining 300 mL of mineral water. Each flask contained a
12 × 4 cm wooden palette for oviposition. After egg lay-
ing, the flasks were filled with mineral water until the eggs
were submerged and placed in new plastic boxes. The larvae
hatched 3 days after oviposition by the females and were fed
fish feed (Neto and Navarro-Silva 2004).
Essential oil
Oil extracted from leaves of Eugenia caryophyllata (clove)
was used in the bioassays of larvicidal, insecticidal, and
repellent activity. The oil was purchased from the Phy-
toterapica® company (São Paulo, Brazil). Chaieb et al.
(2007) show that the main component of clove oil is gen-
erally considered to be eugenol, with β-caryophyllene and
smaller amounts of other components, such as benzyl alco-
hol, but the proportions vary widely. They present a value
of 88.58% for eugenol, 5.62% for eugenyl acetate, 1.38%
for β-caryophyllene, 0.93% for 2-heptanone, 0.66% for ethyl
hexanoate, 0.19% for α-humulene, 0.10% for calamenene,
0.11% for calacorene, and 0.27% for humulenol, these ana-
lyzes being performed by GC–MS.
Studies on action of rhamnolipids produced
by Pseudomonas aeruginosa LBI 2A1
Twenty mosquitoes in the third larval instar were placed
in 60-mL flasks. Solutions of 100, 200, 400, 600, 800, and
1600 mg/L of the rhamnolipid, pH 7, were evaluated. The
­LC50 was calculated after 24 h of exposure to the rham-
nolipid (Modified from WHO, 2005). The experiments were
performed in triplicate. Mineral water was used as a control.
Bioassays of larvicidal action
Direct contact mortality tests were used to assess the toxic-
ity of the rhamnolipids and clove oil both separately and
7507
Transmitting disease Source
Dengue/chikungunya/Zika Virus Mérieux –
NutriSciences,
São Paulo, BR
Lymphatic filariasis Mérieux –
NutriSciences,
São Paulo, BR
together (with half the amount of each substance) to assess
the possibility of synergistic action between the substances.
Clove essential oil
Twenty mosquitoes in the third larval instar were placed in
60-mL flasks. Solutions of 5, 10, 20, 40, 80, 160, 320, 640,
and 1280 mg/L of clove oil were evaluated. The oil was
diluted in mineral water with ethanol (1% v/v). The ­LC50
was calculated after 24 h of exposure to the oil (Modified
from WHO, 1996). The experiments were performed in trip-
licate. Mineral water was used as a control.
Synergistic action of rhamnolipids in emulsion
with clove oil
To evaluate the larvicidal action of the rhamnolipid in emul-
sion with essential oil, the concentrations of rhamnolipid
and oil determined to be the ­LC50 as well as the lower and
upper limits were evaluated. Forty milliliters of the solution
to be evaluated were added to each flask. Sterile distilled
water was used as a negative control. To evaluate whether
a synergistic effect exists between rhamnolipid and clove
oil, the action of the compounds each separately versus the
compounds mixed in solution was compared. Sterile distilled
water was used as a negative control. The experiments were
performed in triplicate. The mortality rate was calculated
after 24 h of exposure to the emulsion.
Bioassays of insecticidal action
For the insecticidal action tests, the rhamnolipid and clove
oil solutions (both separately and together [emulsion])
that exhibited greater effectiveness in the larvicidal tests
were evaluated. Ten adults were placed in the cages and
3 mL were sprayed into each cage. The bottle was weighed
before and after spraying the solution to control the volume
released. Distilled water was used as a negative control. The
experiments were performed in triplicate (Modified from
Silva et al. 2015). The time required to kill half the popu-
lation with the solution (knock-down dosage [­ KD50]) was
calculated. For the survivors, water with 10% glucose was
provided and monitoring for mortality was performed after
24 h.
13
7508
Repellent action bioassays
For the repellent action tests, both the rhamnolipid and clove
oil solutions were evaluated, along with the emulsion with
the greatest effectiveness in the larvicidal action tests. The
solutions were sprayed on the mice that served as the blood
meal; each animal received 3 mL of solution. The bottle was
weighed before and after spraying the solution to control the
volume released. The mice were anesthetized with ketamine.
Each cage contained one mouse and 10 adult mosquitoes.
Exposure time of the mouse to the mosquitoes was 40 min.
The amount of mosquitoes perched on the mouse was evalu-
ated. Distilled water was used as a negative control. The
experiments were performed in triplicate (Modified from
Silva et al. 2015).
Larval histology studies
Fixing the material
After the exposure of insects to different concentrations of
rhamnolipid, the material (kept on ice and packed in micro-
tubes containing 100 µL of 0.1 M sodium phosphate buffer
[PBS], pH 7.4) was sent to the Histology Laboratory of the
Department of General and Applied Biology, UNESP, Rio
Claro, SP, Brazil. The material was immediately fixed in 4%
paraformaldehyde for 48 h at room temperature.
Dehydration and packaging
After the time required for fixation, the material was washed
three times in 0.1 M sodium phosphate buffer, pH 7.4, dehy-
drated in an increasing series of ethyl alcohol (70%, 80%,
90%, and 95% [30-min baths each]), and soaked in LEICA
resin for 7 days. The material was then placed in plastic
molds that were filled with resin plus polymerizer and placed
into an oven at 37 °C until the complete polymerization of
the blocks containing the material.
Block sectioning/hematoxylin and eosin (HE) staining
After polymerization, the blocks were sectioned with a
thickness of 3 µm in a LEICA microtome. The sections were
collected on glass slides and with six sections/slide. The
slides were then dried at room temperature to be stained first
by hematoxylin for 7 min and subsequent washing for 4 min
in running water. Aqueous eosin was then used for 5 min,
followed by rinsing under running water for 5 min.
Statistical analysis
Mortality data were corrected using Abbott’s formula (1925)
when 10% mortality was found in the negative control. The
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Applied Microbiology and Biotechnology (2021) 105:7505–7515
Table 2  Toxicity of rhamnolipid and clove oil to mosquito larvae dur-
ing direct contact bioassay. The experiments were conducted in trip-
licate
Aedes aegypti Culex quinquefasciatus
Substance assessed LC50 mg/L (95% CI) LC50 mg/L (95% CI)
Rhamnolipid 140 (93.64–185.8) 130 (81.86–178.8)
Clove oil 154 (98.25–203.71) 19 (7.04–31.06)
CI, confidence interval
­LC50 was calculated using Probit Analysis (R software,
version 3.2.0) with the function described by Pacheco and
Rebelo (2013).
The normality of the data was analyzed using the Shap-
iro–Wilk test (α = 0.05). A parametric test (ANOVA) was
applied to determine significant differences between treat-
ments (α = 0.05), for which the R software (version 3.2.0)
was also used.
Results
Larvicidal action bioassays
The toxicity of the rhamnolipids and clove essential oil was
compared for two species of mosquitoes that transmit tropi-
cal diseases (Table 2). After 24 h, the L
­ C50 was 140 mg/L
when rhamnolipids were used and 154 mg/L when clove
oil was used against A. aegypti larvae. In the case of C.
quinquefasciatus, the L­ C50 was 130 mg/L for rhamnolipids
and 19 mg/L for clove oil.
When mixing the L ­ C50 and UL of each solution, mortality
increased from 50 to 100% in 24 h, indicating synergistic
action between the compounds (Table 3), likely due to the
emulsifying activity of rhamnolipid, which makes the oil
bioavailable in water to come into contact with the larvae.
Bioassays of insecticidal action
The concentrations of the rhamnolipid and clove oil solu-
tions at the lower limit, upper limit, and L­ C50 were evalu-
ated. The time for the knock-down effect for 50% of the
population ­(KD50) is displayed in Table 4. For both the
rhamnolipid and clove oil solutions, the concentration
of the solution at the upper limit had a lower ­KD50 com-
pared to the other concentrations for both A. aegypti and C.
quinquefasciatus.
Figure 1 shows that related to adult insect mortality, cuti-
cle rupture/degradation occurs, also causing dehydration of
the cell membranes and leading to the death of the insect.
Based on the previous results, an evaluation of adult
mortality was also performed (Table 5). Concentrations at
Applied Microbiology and Biotechnology (2021) 105:7505–7515 7509

Table 3  Toxicity of rhamnolipid-oil emulsion to mosquito larvae after 24 h of direct contact bioassay (% of mortality). The experiments were
conducted in triplicate
Emulsion (rhamnolipid:clove oil)
LL:LL LC50:LL UL::LL LL:LC50 LC50:LC50 UL:LC50 LL:UL LC50:UL UL:UL

A. aegypti 30 63 100 56 100 100 100 100 100

C. quinquefasciatus 41 57 100 59 100 100 100 100 100

LL, lower limit; UL, upper limit; LC50, lethal concentration

Table 4  Toxicity of rhamnolipid and clove oil to adult mosquitoes the lower limit, upper limit, and L
­ C50 were mixed, which
using direct contact bioassay. The experiments were conducted in were established in the direct contact bioassays with adult
triplicate
mosquitoes.
Substance Concen- Aedes aegypti, Culex quinque-
assessed tration ­KD50 min (95% fasciatus, ­KD50 Repellent action bioassays
used CI) min (95% CI)

Rhamnolipid LL 31 (19.45–40.12) 43 (37.56–50.31) The repellent action of rhamnolipids and clove oil was evalu-
LC50 18 (9.45–27.24) 24 (11.85–31.8) ated within 40 min after application of the product (Fig. 2).
UL 11 (8.25–17.13) 16 (10.70–22.11) In both cases, the solution at the upper limit concentration
Clove oil LL 15 (10.11–22.63) 10 (6.01–14.2) achieved the greatest repellent activity against the two mos-
LC50 9 (5.10–18.24) 7 (5.04–11.06) quito species.
UL 25 (12.01–33.8) 5 (3.51–8.19) Evaluating the repellent action of the emulsions (Fig. 3),
those with one of the solutions at the upper limit concentra-
CI, confidence interval; LL, lower limit; UL, upper limit; KD50,
knock-down time 5
tion achieved greater repellency, with significant differences
compared to the control (p value < 0.05), indicating possible

Fig. 1  Optical microscopy of

adult mosquitoes after applica-
tion of rhamnolipid
Negative control
Mosquitoes after rhamnolipid
application

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Table 5  Toxicity of rhamnolipid-oil emulsion to adult mosquitoes after 24 h during direct contact bioassay (% of mortality). The experiments
were conducted in triplicate
Emulsion (rhamnolipid:clove oil)
LL:LL LC50:LL UL::LL LL:LC50 LC50:LC50 UL:LC50 LL:UL LC50:UL UL:UL

A. aegypti 25 51 100 52 100 100 100 100 100

C. quinquefasciatus 31 51 100 50 100 100 100 100 100

LL, lower limit; UL, upper limit; LC50, lethal concentration 50

Fig. 2  Percentage of mosquitoes a) b)

perched on mouse serving as
blood meal after using rham-
nolipids and clove oil. a Aedes
Percentage of mosquitoes that landed

Rhamnolipid Clove oil

Rhamnolipid Clove oil
aegypti. b Culex quinquefas-
ciatus. The experiments were
conducted in triplicate
in the mouse (%)

Tested dose Tested dose

synergistic action. When using the solutions at the ­LC50,
an increase in larvicidal action was found, with significant
differences compared to the control (p value < 0.05). This
increase also indicates possible synergistic action between
the compounds.
Larval histology
The results of the evaluation of the histopathological effects
on the midgut (mesenteron) of Aedes aegypti larvae exposed
to rhamnolipids at concentrations of 0.8 to 1.0 g/L are dis-
played in Fig. 4. This evaluation was performed to determine
what modifications occur due to exposure to the substance.
This is an important compartment of the larval body that
coordinates feeding/digestion/absorption processes. Moreo-
ver, the gut has a histological organization that compartmen-
talizes it morphophysiologically, subdividing it into other
three distinct regions: (a) anterior region (closest to the head
of the larva, here called R1); (b) median region (located in
the middle of the larval body, R2), and (c) posterior region
(final region of the intestine, R3) (Fig. 4).
Control group
The evaluation of the larvae allocated to the control group
confirmed that, histologically, these three regions of the
intestine were covered by a simple epithelium (with only
one layer of cells) that changed as it traveled the length of
the organ due to the different physiologies of each region
(Fig. 4A, D). The cells of this lining epithelium have mor-
phology compatible with the physiology of each region.
Thus, when the changes caused due to exposure to the toxic
substance tested herein reached the histological organization
of the epithelium and cell morphology, the entire physiology
of the organ was altered. Since the objective of this study
was to perform a comparative analysis of the results, the
description of the larvae in the control group (not exposed to
the toxic substance) was used to confirm the presence of an
intact lining epithelium composed in the R1 region (Fig. 4A,
B) of mononucleated cubic cells with uniform cytoplasmic
and nuclear marking by the stains (Fig. 4B). In the R2 region
(Figs. 4A and 4C), the cells were higher, with a tendency to
be more cylindrical than cubic, depending on their physiol-
ogy, since the digestion/absorption of nutrients begins in this
region. Thus, the cubic shape of the cells changes naturally
to cylindrical, but the cells continue to be mononucleated,
presenting more strongly positive marking by the stains
(Fig. 4C). In the posterior region (R3) (Figs. 4A and 4D), the
cells acquire a completely cylindrical morphology, typical of
cells that perform absorption and secretion, and were there-
fore higher, remaining mononucleated, strongly positive to
the dyes and with the apical portion in most cells releasing
secretion vesicles to the lumen of the intestine (Fig. 4D),

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Fig. 3  Percentage of mosquitoes A

perched on mouse serving as
100%
blood meal using rhamnolipid
emulsion:clove oil. LL, lower
limit; UL, upper limit; L
­ C50,

Percentage of mosquitoes resting on the

lethal concentration 50. A Aedes
aegypti. B Culex quinquefas-
ciatus. The experiments were
62%
conducted in triplicate

mouse (%)
42%

21% 20%
12% 11%
5% 3% 2%
0%
LI:LI LS:LI LC50:LC50 LI:LS LS:LS
B Rhamnolipid Emulsion: Clove Oil
100%
Percentage of mosquitoes resting on the

64% 63%
mouse (%)

45%

24%

15% 15%
12% 12% 10%

0%
LI:LI LS:LI LC50:LC50 LI:LS LS:LS
Rhamnolipid Emulsion: Clove oil

which assists in the digestive process as well as the elimina-
tion of residual products not used by the body.
Discussion
When analyzing Table 2, it is possible to observe the toxicity
results for the rhamnolipids were virtually the same for both
species. In contrast, there were differences in the toxicity of
the concentrations of clove oil evaluated. At the ­LC50, rham-
nolipids reduce the surface tension of the water to 30 mN/m,
which can cause larvae of both genders to experience diffi-
culty breathing because they cannot maintain themselves on
the surface. Thus, the siphon position is affected, causing the
death of the insect due to deficient gas exchange (Da Silva
et al. 2015). For the studies involving rhamnolipid emulsions
with clove oil, concentrations obtained as the lower limit,
upper limit, and lethal concentration 50 (LL; UL; L­ C50) were
used and significant differences (p value < 0.05) were found
between the combinations (rhamnolipid:clove oil) that did
not achieve 100% mortality (LL:LL, L ­ C50:LL, LL:LC50,
LL:UL). When concentrations of the upper limits of one of
the solutions (rhamnolipid or clove oil) were mixed, 100%
mortality was obtained after 24 h.
In the mixture of the lower limits of the rhamnolipid
and clove oil concentrations, no difference in mortality was
found compared to the control of each solution alone. How-
ever, when mixing the lower limit with any other concentra-
tion (LL:LC50, LL:UL, for example), an increase in mortality
was found in comparison to the controls in the case of both
the rhamnolipid and clove oil (Table 3). The surface tension
of the water with clove oil at concentrations of the lower
limit, upper limit, and L
­ C50 was 75 mN/m. When mixed with
the rhamnolipid, the surface tension of the final solution was

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7512 Applied Microbiology and Biotechnology (2021) 105:7505–7515

Fig. 4  Histological sections of

intestine of Aedes aegypti larvae
exposed to rhamnolipids and
stained with hematoxylin–eosin
(HE)

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reduced to 37 mN/m. By reducing the surface tension of
the water, rhamnolipids prevent the larvae from feeding. In
addition, clove oil has the active phenolic compound euge-
nol, which interferes with GABA receptors in the nervous
system of the insect (Regnault-Roger et al. 2012). Thus, in
addition to the interaction between the hydrophobic siphon
region and the rhamnolipid, the movement of the larvae is
also affected. The mixture of rhamnolipid and clove oil may
have a synergistic effect because the rhamnolipid ensures the
bioavailability of the oil, allowing it to have faster or greater
effect at a lower concentration, as occurred when mixing the
solutions at the ­LC50.
The insecticidal action of adult mosquitoes shows, in
addition to mortality in the adults, the rupture/degradation
of the insect cuticle also occurred (Fig. 1). According to Kim
et al. (2011), rhamnolipids weaken the cuticles of aphids,
also causing dehydration of the cell membranes and leading
to the death of the insect. Da Silva et al. (2015) reported
this effect on the cuticle of Aedes aegypti. One hundred per-
cent mortality was achieved when using a solution at the
upper limit concentration mixed with any other concentra-
tion (Table 5). When using solutions at the L ­ C50 mixed with
the lower limit of the other solutions, mortality was greater
than 50%, but this increase did not achieve statistical sig-
nificance (p value > 0.05) compared to the control of the
solution (either rhamnolipid or clove oil at the ­LC50). Using
the solution of rhamnolipid and clove oil at the ­LC50, mortal-
ity was 100%, with significant differences (p value < 0.05)
when compared to the control of each solution at the ­LC50.
This demonstrates possible synergistic activity between the
two solutions. In addition to the rupturing of the cuticle by
the rhamnolipid, it is likely that the clove affects with the
flight behavior, metabolic regulation, or heart pressure of
the insect (Pflüger and Stevenson 2005). Rhamnolipids and
clove oil were used as larvicidal agents, mosquito insec-
ticides and repellents against Aedes aegypti and Culex
quinquefasciatus. These compounds exhibited high efficacy
at low concentrations (140 and 130 mg/L of rhamnolipids;
154 and 19 mg/L of clove oil, respectively). When tested in
the form of an emulsion, the same good larvicidal, insec-
ticidal, and repellent activity was found against both spe-
cies, with 100% insect mortality in most cases. Da Silva
et al. (2015) put forth the hypothesis that the mosquito may
recognize the smell of rhamnolipids as an unfavorable char-
acteristic and is therefore repelled. The present results lend
strength to this hypothesis. In addition, clove oil is a vola-
tile aromatic compound that is easily recognized by insects,
which are repelled (Dias and Moraes 2014; Regnault-Roger
et al. 2012). In an emulsion, clove oil likely enhances the
smell of rhamnolipids, thereby increasing the repellent activ-
ity of both solutions. These results indicate that a possible
synergistic interaction between the compounds can increase
their effectiveness and applicability.
7513
Regarding the effect caused by the exposure of larvae
to rhamnolipids, it is observed in the bioassays with larvae
exposed to rhamnolipids (treatment group 1—exposure to
concentration of 800 mg/L) that changes were found in the
morphology of the intestinal epithelium (Fig. 4E–P) follow-
ing exposure to a concentration 800 mg/L (Fig. 4E–H). The
epithelial cells of the R1 region (Figs. 4E and 4F) began
to undergo a morphological change from cubic to almost
cylindrical. The same change was found in the other two
regions (R2 and R3) (Figs. 4E and 4G, H), with more evi-
dent changes in the latter (Fig. 4H). Specifically in the R3
region, the apical portion of the lining cells (portion facing
the lumen of the intestine) exhibited intense vacuolization,
with a strong indication of the occurrence of cell damage
(Fig. 4H). Exposure to the concentration 900 mg/L (treat-
ment group 2—exposure to concentration of 900 mg/L) led
to the most severe morphological changes to the organization
of the epithelium and the cells lining the intestines of the lar-
vae (Fig. 4I–L). The first change appeared in the R1 region,
with stratification of the epithelium; that is, the cell layer that
covered this region was no longer singular, undergoing an
increase in cell division processes and consequently chang-
ing the epithelial organization, with a greater number of cell
layers (stratification) (Fig. 4I–L). Such a change indicates
that the substance was highly toxic to the individual and
epithelial stratification may occur in an attempt to preserve
the internal environment of the organism from the action of
the toxic substance. In the R2 region (Fig. 4K), in addition
to stratification, the epithelium exposed to this concentration
also presented cells with an intense cytoplasmic vacuoliza-
tion and marginalization of the nuclear chromatin (Fig. 4K),
which strongly indicated the onset of the cell death process.
In the bioassay involving exposure of the larvae to a con-
centration of 1000 mg/L ( treatment group 3—exposure to
concentration of 1000 mg/L) (Fig. 4M–P), the changes found
in the different regions of the intestinal epithelium were very
similar to those seen in the bioassays involving exposure to
the concentration of 800 mg/L. It should be noted, however,
that the marginalization of chromatin in the nucleus of the
epithelial cells was very severe (Figs. 4N and 4P), indicating
the onset of the cell death process, which will determine the
biological success of the individual.
Due to their amphiphilic nature, glycolipids can interact
directly with plasma membranes (Otzen 2017). It is sug-
gested that the mode of action of rhamnolipids in the case
of zoospore-producing plant pathogens occurs through the
direct lysis of zoospores with intercalation of glycolipids
within plasma membranes, which are not protected by a cell
wall (Stanghellini and Miller 1997). Rhamnolipids can also
cause destabilization or lysis in mycelial cells. The parti-
tion of rhamnolipids into membranes depends heavily on
the composition of lipids (Aranda et al. 2007). It has been
shown that purified mono- and di-rhamnolipids are capable
13
7514
of interleaving in phosphatidylcholine and phosphatidyletha-
nolamine bilayers, notably changing their morphology (Ortiz
et al. 2006; Sanchez et al. 2006, 2009; Abbasi et al. 2012,
2013). These inserts, therefore, produce structural distur-
bances that can affect the function of the membranes. These
compounds also alter the physicochemical properties of the
bilayer and disturb the hydration status of the water/lipid
interface. Depending on the lipid composition of the mem-
brane and its concentration, rhamnolipids are also capable
of permeabilizing membranes (Sánchez et al. 2010), which
could result in their lysis. Amphiphilic compounds, such
as glycolipids, form aggregates in solutions, depending on
their concentration. Above their critical micellar concentra-
tion (CMC), they appear in the form of both aggregates and
monomers.
Author contribution AMS-B and VLS were responsible for conduct-
ing the rhamnolipid production experiments and bioassays. RCN and
MAGR collaborated on the bioassays carried out. MRA and RSM per-
formed the histological analyzes. CJVZ was responsible for providing
and preparing bioassays with Aedes aegypti and Culex quinquefascia-
tus, as well as discussing and correcting the text. MIC-M was respon-
sible for the histological design, analysis, discussion, and correction
of the text. RBL participated in the discussion of the results and cor-
rection of the text. JC wrote the manuscript, guided the students, and
supported the development of the project through assistance with the
Development Agencies.
Funding Financial support was provided by the São Paulo Research
Foundation (Fapesp) and the Coordination for the Improvement of
Higher Education Personnel (CAPES).
Data availability The authors declare that the data supporting the find-
ings of this study are available within the article.
Declarations
Ethics approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Conflict of interest The authors declare no competing interests.
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