Keywords:

Chitin

Entomopathogenic

Chitinolytic

Larvicidal

Eco-friendly

Entomopathogenic potential of thermotolerant microorganisms isolated from infected

Dermestes maculatus against Culex pipiens larvae

Potencial entomopatógeno de microorganismos termotolerantes aislados de Dermestes maculatus

infectados contra larvas de Culex pipiens

Potencial entomopatogênico de microrganismos termotolerantes isolados de Dermestes maculatus

infectados contra larvas de Culex pipiens

Ali Boulanouar*

Zineb Hamani

Benlarbi Larbi

Rev. Fac. Agron. (LUZ). 2026, 43(1): e264305

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v43.n1.V

Crop production

Associate editor: Dra. Lilia Urdaneta

University of Zulia, Faculty of Agronomy

Bolivarian Republic of Venezuela

Laboratory Development of Biological Resources and

Food Security/ TAHRI Mohamed University of Bechar -

ALGERIA.

Received: 28-08-2025

Accepted: 07-12-2025

Published: 28-12-2025

Abstract

During summer, when temperatures reach extreme records,

the inhabitants Sahara seek refuge in oases for shade and water.

These ecosystems are characterized by a unique microclimate.

Nevertheless, they face serious threats from two arthropod species:

venomous scorpions and mosquitoes, which act as vectors of

diseases. Chemicals impacting both biotic and abiotic components

of the ecosystem, and more critically human health. Chitinolytic

entomopathogenic microorganisms were isolated from dust

samples collected from the cadavers of the Dermestes maculatus.

Chitin extracted from shrimp shells (yield: 16.6 %) served as the

sole carbon source in the selective culture media employed for their

cultivation. Five strains were obtained: three fungi (Aspergillus

avus, A. fumigatus, Mucor sp.) and two bacteria (Bacillus sp.

and Actinomycete). Bioassays against third-instar Culex pipiens

larvae showed that Actinomycete (10⁶ CFU.mL

-1

) induced 90 % of

mortality, followed by A. fumigatus, Mucor sp., and Bacillus sp.

(80 %). Data were analyzed using one-way ANOVA and Duncan’s

test (p<0.05). Microscopic observations revealed severe larval

deformities. These ndings conrm the strong larvicidal potential

of microorganisms as eco-friendly alternatives to chemical

insecticides.

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Rev. Fac. Agron. (LUZ). 2026, 43(1): e264305 January-March. ISSN 2477-9409.

2-7 |

Resumen

Durante el verano, cuando las temperaturas alcanzan valores

extremos, los habitantes del Sahara buscan refugio en los oasis para

obtener sombra y agua. Estos ecosistemas se caracterizan por un

microclima único. Sin embargo, enfrentan graves amenazas de dos

especies de artrópodos: los escorpiones venenosos y los mosquitos,

que actúan como vectores de enfermedades. Los productos químicos

utilizados afectan tanto los componentes bióticos como abióticos del

ecosistema y, de manera más crítica, la salud humana. Se aislaron

microorganismos entomopatógenos quitinolíticos a partir de muestras

de polvo recolectadas de los cadáveres de Dermestes maculatus. La

quitina extraída de caparazones de camarón (rendimiento: 16,6 %) se

utilizó como única fuente de carbono en el medio selectivo para su

cultivo. Se obtuvieron cinco cepas: tres hongos (Aspergillus avus, A.

fumigatus, Mucor sp.) y dos bacterias (Bacillus sp. y Actinomiceto).

Los bioensayos contra larvas de tercer estadio de Culex pipiens

mostraron que Actinomiceto (10⁶ CFU.mL

-1

) provocó un 90 % de

mortalidad, seguido por A. fumigatus, Mucor sp. y Bacillus sp. (80

%). Los datos se analizaron mediante ANOVA de una vía y la prueba

de Duncan (p<0,05). Las observaciones microscópicas revelaron

deformidades larvales severas. Estos resultados conrman el fuerte

potencial larvicida de los microorganismos como alternativas

ecológicas a los insecticidas químicos.

Palabras clave: quitina, entomopatógenos, quitinolíticos, larvicida,

ecológico.

Resumo

Durante o verão, quando as temperaturas atingem níveis extremos,

os habitantes do Saara buscam refúgio nos oásis em busca de sombra

e água. Esses ecossistemas são caracterizados por um microclima

único. No entanto, enfrentam sérias ameaças de duas espécies de

artrópodes: escorpiões venenosos e mosquitos, que atuam como

vetores de doenças. Os produtos químicos utilizados afetam tanto os

componentes bióticos quanto abióticos do ecossistema e, de forma

mais crítica, a saúde humana. Microrganismos entomopatogênicos

quitinolíticos foram isolados a partir de amostras de poeira coletadas

de cadáveres de Dermestes maculatus. A quitina extraída de cascas

de camarão (rendimento: 16,6 %) foi utilizada como única fonte de

carbono no meio seletivo para seu cultivo. Cinco cepas foram obtidas:

três fungos (Aspergillus avus, A. fumigatus, Mucor sp.) e duas

bactérias (Bacillus sp. e Actinomiceto). Os bioensaios realizados com

larvas de terceiro instar de Culex pipiens mostraram que Actinomiceto

(10⁶ CFU.mL

-1

) causou 90 % de mortalidade, seguido por A. fumigatus,

Mucor sp. e Bacillus sp. (80 %). Os dados foram analisados por meio

de ANOVA de uma via e teste de Duncan (p<0,05). As observações

microscópicas revelaram deformidades larvais severas. Esses

resultados conrmam o forte potencial larvicida dos microrganismos

como alternativas ecológicas aos inseticidas químicos.

Palavras-chave: quitina, entomopatogênico, quitinolítico, larvicida,

ecológico.

Introduction

Insects are the most diverse organisms on Earth, with about one

million described and millions more yet undiscovered (Li and Wiens,

2023). While many play vital ecological roles, others seriously

damage crops and aect human health (Bharadwaj et al., 2025).

Mosquitoes (Aedes, Culex, Anopheles) remain major vectors of

malaria, dengue, and other arboviral diseases. Although still widely

used, chemical insecticides cause resistance, harm non-target species,

and pollute fragile ecosystems like oases. In contrast, biological

control using entomopathogenic microorganisms oers a safer and

more sustainable alternative. Several biocontrol agents, notably

Bacillus thuringiensis israelensis (Bti) and Bacillus sphaericus, are

currently applied worldwide against mosquito larvae. However, their

ecacy decreases markedly under extreme desert conditions, where

elevated temperatures and low humidity reduce spore viability and

larvicidal performance. This limitation emphasizes the importance

of identifying new thermotolerant entomopathogenic strains capable

of maintaining activity in such environments. The main objective of

the present study is to isolate and characterize new extremophilic

entomopathogenic microorganisms from necrophagous insects,

Dermestes maculatus beetles, inhabiting extreme desert environments

of southwestern Algeria, using selective media prepared with extracted

chitin. These poorly investigated regions of the Grand Sahara (some

still unexplored by humans) oer a unique opportunity to discover

microbial resources adapted to thermal and arid stress. The present

study explores their potential for replacing synthetic insecticides,

focusing on: (i) Eco-friendly approaches to preserve ecosystem

balance. (ii) High specicity against target mosquito species. (iii)

Human safety with no toxic residues or long-term risks. (iv) Cost-

eective tools to overcome insecticide resistance (Bharadwaj et al.,

2025). (v) Integration within sustainable Vector Management (IVM).

(vi) Validation by scientic evidence for large-scale application.

This study aims to isolate and identify chitinolytic microorganisms

associated with necrophagous insects and to evaluate their pathogenic

potential against Culex pipiens. The ultimate goal is to contribute

to the development of sustainable biological control strategies for

mosquito population management in vulnerable oasis ecosystems.

Materials and methods

This study focused on isolating entomopathogenic microorganisms

with chitinolytic activity for sustainable mosquito control. Strains

were recovered from necrophagous insect cadavers (Dermestes

maculatus De Geer), cultured on chitin-based selective media, then

puried and preserved. The most active isolates were characterized

morphologically and tested for pathogenicity against Culex pipiens

larvae (gure 1).

Figure 1. Experimental methodology.

Screening of potential entomopathogenic microorganisms

Chemical extraction of chitin

Chitin of marine origin (crustaceans), is commonly used to isolate

chitinolytic microorganisms due to its structural similarity to natural

chitin Izadi et al. (2025). Using chitin as the sole carbon source

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Boulanouar et al. Rev. Fac. Agron. (LUZ). 2026, 43(1): e264305

3-7 |

reliable morphological identication. (iii) Absence of metamorphosis

changes seen in L4, reducing bias and misidentication. (iv) Lower

mortality and pupation risk during handling and transport. (v) Optimal

size and clear species-specic traits for laboratory experiments.

Selection and sorting of L3: Final sorting of L3 larvae was

performed carefully in the laboratory under a stereomicroscope,

following the morphological characteristics.

Standardization of experimental conditions: To replicate

natural conditions inside laboratory, and ensure that mortality results

from the tested microorganism, height controlled environmental

parameters must be respected (table 1).

Parameter Conditions

Water Quality

Dechlorinated freshwater (pH 6.5-8, low conductivity,

low-moderate hardness, low turbidity); replace

periodically.

Temperature

Optimal range for the species studied (25 ± 2 °C),

sudden uctuations were avoided.

Photoperiod

Timeframe 12:12 h (light/dark).

Intensity

Soft light intensity, were

avoided direct strong light on

containers.

RH (%) 15-25 % (close to the seasonal value)

Dissolved Oxygen

Mechanical aeration was not required, but low-

oxygen water was avoided and adequate surface area

was ensured for larvae.

Feeding Regime

Standard larval diet (sh food, yeast) in controlled

amounts, 0.1-0.2 mg dry food per larva/day.

Container Size and

Density

Larval density was maintained within recommended

limits to avoid competition and cannibalism.

Handling Practices

To replicate natural abiotic conditions, mechanical

disturbance was minimized, reducing stress and

ensuring that mortality was due to the microbial

treatment.

Predators and

Contaminants

No other organisms in the water (aquatic worms,

predatory insect larvae, parasitic fungi, etc.). Handle

with clean, disinfected tools.

Microbial load measurement: Fragments from isolated colonies

were aseptically transferred into 60 mL of sterile water. Microbial

suspensions were homogenized in sterile distilled water with 0.05 %

Tween 80, preheated to 40 °C for full dissolution (Goettel et al.,

2010). Fungal and Actinomycete suspensions were adjusted to 10⁶

CFU.mL

-1

using a hemocytometer, and Bacillus suspensions to 10⁶

cells.mL

-1

via spectrophotometry. Two serial dilutions (10⁵ and 10⁴

CFU.mL

-1

) were prepared for bioassays.

Data analysis: Twenty larvae were placed in each container and

exposed to dierent concentrations of each entomopathogenic strain,

with three replicates per treatment. Larval survival was monitored

daily for seven days, and microscopic examinations were conducted to

assess pathogenic eects. Control mortality (ranging from 5 to 20 %)

was corrected using Abbott’s formula (Abbott, 1925). Daily mortality

data collected over seven consecutive days (N = 7 observation times)

provided sucient information to perform regression analyses and

estimate LT₅₀ values for each applied dose.

Evaluation of biological eects

The entomopathogenic activity of each isolate was evaluated

through three parameters: (i) larval mortality over seven days

allows specic selection of chitinase-producing bacteria and fungi.

However, commercial chitin may contain impurities or mixed origins,

potentially aecting microbial proles. Locally puried chitin of

known arthropod origin, properly treated to remove proteins, lipids,

and minerals, can therefore provide greater specicity.

Shrimp shells were obtained from Mostaganem, an Algerian

coastal city. The shells were peeled, thoroughly washed, dried at 45

°C, and then ground into a ne powder. Three grams of the powdered

material were treated with 30 mL of 1 M HCl to remove mineral

content (demineralization). The resulting residue was subsequently

treated with 30 mL of 1.25 M NaOH at 90 °C for 2 hours to eliminate

proteins (deproteinization). For optional depigmentation, hydrogen

peroxide (H₂O₂) was applied, followed by rinsing with acetone

to remove pigments and lipids, as described by Izadi et al. (2025).

Finally, the puried chitin was dried at 80 °C for 10 hours.

Preparation of chitin-based culture media

We replaced the carbon source in CDA, GN culture media with

a similar amount of puried chitin where it becomes the only carbon

source available in the prepared culture media.

Isolation and purication of microbial strains

Dermestes maculatus cadavers were collected from IGLI

oasis, which are likely serve as reservoirs of entomopathogenic

microorganisms. Samples were collected under sterile conditions

and transported to the laboratory within 24 h to preserve microbial

viability.

Necrophagous insect carcasses were ground into powder,

5 g suspended in 45 mL sterile saline as stock, followed by serial

dilutions (10

-1

-10

-4

). Aliquots were inoculated onto selective chitin-

based media and incubated (bacteria 37 °C/24-48 h; fungi 3 days;

Actinomycetes 28 °C/21 days). CDA

chitin

, chloramphenicol for fungal

isolation, GN

chitin

solid medium for Bacillus and Chitin-based solid

medium B vitamin for Actinomycetes isolation. Chitinolytic strains

were isolated and identied via morphological, microscopic, and

staining methods. All tests were performed in triplicate.

Bioassays on Culex pipiens larvae

Larval sampling

Sampling site: Larval sampling was carried out in the IGLI

palm grove, Algeria (30.4440799 ° N, 2.2995554 ° W), at a stagnant

drainage channel partially shaded by palm fronds and enriched with

moderate organic matter.

Sampling period: Sampling was carried out on June 2020,

corresponding to the peak seasonal abundance of Culex pipiens larvae

in the study area, warm temperatures and the presence of stagnant

water bodies ensure high population densities.

Sampling time: To minimize heat stress and reduce larval

movement toward the bottom of the water column, which typically

occurs at higher temperatures, collection was conducted during the

early morning hours (06h00-08h00).

Sampling method: Larvae were collected using a 350 mL white

plastic dipper at a 45 °C angle, with ten consecutive dips taken along

a 15 m transect to account for spatial variability. Specimens were

separated from debris in white enamel trays, then transferred to 500

mL containers (≤30 larvae/container) to avoid stress and cannibalism.

Criteria for choosing the larval stage for sampling: Sampling

specically targeted third-instar (L3) Culex pipiens larvae for the

following reasons: (i) High susceptibility to entomopathogens with a

suciently long larval period for post-treatment monitoring. (ii) Larger

body size than L1-L2, allowing easier handling, sorting, dosing, and

Table 1. Steps followed during the standardization of experimental

conditions.

% Corrected mortality =

% Observed mortality – Control mortality

100 - % Control mortality

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Rev. Fac. Agron. (LUZ). 2026, 43(1): e264305 January-March. ISSN 2477-9409.

4-7 |

to estimate cumulative death and LT₅₀; (ii) pupal inhibition and

post-emergence mortality, reecting developmental blockage or

early adult death; and (iii) adult deformities and ight incapacity,

indicating sublethal physiological eects. This approach allowed a

comprehensive assessment of both lethal and sublethal impacts on

Culex pipiens development.

Statistical analysis: All statistical analyses were performed using

SPSS software. Data were expressed as mean ± standard deviation

(SD). A one-way analysis of variance (ANOVA) was conducted to

evaluate the eect of microbial treatments on larval mortality. When

signicant dierences were detected (p<0.05), means were separated

using Duncan’s multiple range test.

Results and discussion

The chitin yield was 16.6 %, which aligns with recent studies

reporting that crustacean shells generally contain 15 to 40 % chitin,

depending on species and extraction method used (Izadi et al., 2025).

Microbial cultures and puried strains: Following the

inoculation of the stock solution of the necrophagous insects in the

chitinous media CDA

chitin

, GN

chitin

and Vitamin B

Chitin

, ve distinct

microbial isolates were puried. Three fungal (Aspergillus avus,

Aspergillus fumigatus, and Mucor sp.) and two bacterial (Bacillus sp.

and Actinomycete).

Larval bioassay results

Control group: Control groups consistently showed 0 %

mortality, full ight capability, and no post-emergence mortality, and

are therefore presented as a common baseline for all treatments.

Eect of Aspergillus avus on mosquito larvae and adult

emergence

At 10⁶ CFU.mL

-1

, A. avus caused the highest mortality, reaching

70 % by day 7, with only 10 % of adults retaining ight ability,

indicating marked sublethal eects. At 10⁵ CFU.mL

-1

, mortality

was moderate (50 %), and 20 % of adults remained ight-capable,

suggesting reduced impairment. At 10⁴ CFU.mL

-1

, mortality declined

to 40 %, but ightlessness (30 %) and post-emergence mortality

(20 %) were more evident, reecting severe physiological stress

among survivors (gure 2). These ndings conrm the dual action

of entomopathogenic fungi, combining lethality with functional

impairment. Similar results were reported by Lamy et al. (2025)

and Valdez et al. (2025), highlighting their strong potential against

mosquitoes and other insect pests.

Eect of Aspergillus fumigatus on mosquito larvae and adult

emergence

For A. fumigatus, the concentration of 10⁶ CFU.mL

-1

induced

rapid mortality, rising from 10 % on day 1 to 70 % by day 5, with no

increase thereafter. Flight ability was strongly impaired, with only

10 % of adults able to y, while 10 % exhibited ightlessness and

10 % died post-emergence. At 10⁵ CFU.mL

-1

, mortality plateaued

at 40 % by day 5, but functional impairments were higher (30 %

ightlessness, 20 % post-emergence mortality). At 10⁴ CFU.mL

, mortality was lower (30 %), but 30 % of adults remained ight-

capable, suggesting less damage among survivors (gure 3). These

ndings align with recent reports showing that entomopathogenic

fungi both reduce adult survival and compromise adult tness in

mosquito hosts (Aguilar-Durán et al., 2023; Renuka et al., 2023).

Eect of Mucor sp. on mosquito larvae and adult emergence

At 10⁶ CFU.mL

-1

, Mucor sp. caused a sharp mortality increase from

10 % on day 1 to 60 % by day 7, with complete loss of ight ability

among survivors (0 % ight-capable). At 10⁵ CFU.mL

-1

, mortality

reached 80 % by day 7, with 10 % of adults retaining ight capacity

but showing marked physiological impairment. At 10⁴ CFU.mL

-1

mortality was also high (80 %) but with a slower progression, while

20 % of survivors were ight-capable and no post-emergence mortality

was observed (gure 4). This suggests lower virulence at reduced doses

but persistence of functional eects. Similar outcomes were reported

by Zhu et al. (2023), who demonstrated strong pathogenic eects of

Mucor hiemalis on insect hosts. Eect of Bacillus sp. on mosquito

larvae and adult emergence

Figure 2. (a) Daily evolution of mortality rates following exposure

to varying doses of Aspergillus avus. (b) Post-exposure

outcome of larvae surviving beyond 7 days.

Figure 3. (a) Daily evolution of mortality rates following exposure

to varying doses of A. fumigatus. (b) Post-exposure

outcome of larvae surviving beyond 7 days.

100%

10%

20%

10%

20%

30%

10% 10%

20%

-20%

20%

40%

60%

80%

100%

120%

140%

Control 10

^6 10

^5 10^4

(b) Aspergillus flavus

Flight-capable Flightlessness Post-emergence mortality

10%

20%

30%

40% 40%

50%

70%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 3 4 5 6 7

Mortality

Days

(a) Aspergillus flavus

Control 10^6 10^5 10^4

10%

20% 20%

30%

70% 70% 70%

10%

20%

30%

40%

50%

60%

70%

80%

0 1 2 3 4 5 6 7 8

Mortality

Days

(a) Aspergillus fumigatus

Control 10^6 10^5 10^4

100%

10% 10%

30%

10%

30%

20%

10%

20% 20%

-20%

20%

40%

60%

80%

100%

120%

140%

Control 10^6 10^5 10^4

(b) Aspergillus fumigatus

Flight-capable Flightlessness Post-emergence mortality

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Boulanouar et al. Rev. Fac. Agron. (LUZ). 2026, 43(1): e264304

5-7 |

At 10⁶ CFU.mL

-1

, Bacillus sp. caused 60 % larval mortality by day

7 with high post-emergence death and complete ight loss, whereas

at 10⁵ CFU.mL

-1

mortality was lower (40 %) but sublethal eects

remained severe (gure 5). Interestingly, the highest mortality (80 %)

occurred at 10⁴ CFU.mL

-1

, suggesting a non-linear dose-response. The

lower virulence observed at 10⁶ CFU.mL

-1

compared to 10⁵ CFU.mL

-1

may result from bacterial aggregation and quorum-sensing inhibition

at high densities, which reduce toxin production and trigger stronger

host immune defenses (Lavine and Strand, 2002; Natrah et al., 2011;

Hossain, et al., 2025). These results highlight both lethal and sublethal

impacts of Bacillus sp. across concentrations, supporting its potential as

an eective biocontrol agent.

Figure 5. (a) Daily evolution of mortality rates following exposure

to varying doses of Bacillus sp. (b) Post-exposure

outcome of larvae surviving beyond 7 days.

Eect of Actinomycete on mosquito larvae and adult

emergence

At 10⁶ CFU.mL

-1

Actinomycete caused mortality up to 90 % by day

6, with no ight-capable adults and 10 % post-emergence mortality,

conrming strong virulence (gure 6). At 10⁵ CFU.mL

-1

, mortality

stabilized at 40 %, with 40 % of adults ight-capable, though 10 %

exhibited ightlessness and 10 % died after emergence. At 10⁴,

mortality was low (20 %), but 50 % of adults retained ight, while

20 % showed ightlessness and 10 % post-emergence mortality.

These results suggest dose-dependent eects, where higher

concentrations strongly reduce survival, while lower ones primarily

impair physiology. Comparable studies by Al-Nagar et al. (2024) and

Singh and Dubey (2015) support the entomopathogenic potential of

Actinomycetes through both lethal and functional impacts.

ANOVA revealed a signicant eect of microbial treatment on

larval mortality (F = 5.87, p<0.001). Mucor sp. and Actinomycete

caused the highest mortality, while Bacillus sp. showed weaker eects;

A. avus and A. fumigatus produced moderate, similar responses.

Fungal isolates, particularly Mucor sp., were the most virulent, likely

due to rapid growth and chitinolytic activity. These results conrm the

stronger biocontrol potential of fungi, with bacterial strains showing

complementary, dose-dependent eects.

Comparative analysis of TL₅₀ values

TL₅₀ results (table 2) revealed clear dierences in virulence among

the tested microorganisms. Fungal isolates acted faster than bacterial

ones, while bacterial eects were slower but cumulative. Mucor sp.

was the most virulent (TL₅₀ = 2 days at 10⁶ CFU.mL

-1

), consistent

with reports of high pathogenicity of Mucor species against insect

larvae (Zhu et al., 2023).

A. fumigatus showed moderate virulence (4.5 days), consistent

with previous reports of its larvicidal activity against mosquito larvae

(Balumahendhiran et al., 2019), and A. avus was slower (6 days),

likely due to delayed toxin production (Arreguín-Pérez et al., 2023).

Among bacteria, Actinomycete was most eective (TL₅₀ ≈ 4.7 days;

10%

50% 50% 50% 50%

60% 60%

20%

40%

60%

80%

100%

0 1 2 3 4 5 6 7 8

Mortality

Days

(a) Mucor sp.

Control 10^6 10^5 10^4

100%

10%

20%

10%

0%0%

20%

0% 0%

-50%

50%

100%

150%

Control 10^6 10^5 10^4

(b) Mucor sp.

Flight-capable Flightlessness Post-emergence mortality

Figure 4. (a) Daily evolution of mortality rates following exposure

to varying doses of Mucor sp. (b) Post-exposure outcome

of larvae surviving beyond 7 days.

10%

20%

40%

60%

80% 80%

10%

20%

30%

40%

50%

60%

70%

80%

90%

0 1 2 3 4 5 6 7 8

Mortality

Days

(a) Bacillus sp.

Control 10^6 10^5 10^4

100%

0% 0% 0%0% 0%

10% 10%

40%

30%

10%

-40%

-20%

20%

40%

60%

80%

100%

120%

140%

Control 10^6 10^5 10^4

(b) Bacillus sp.

Flight-capable Flightlessness Post-emergence mortality

0% 0%

20%

30%

60%

80%

90%

-20%

20%

40%

60%

80%

100%

0 1 2 3 4 5 6 7 8

Mortality

Days

(a) Actinomycete

Control 10^6 10^5 10^4

100%

40%

50%

0% 0%

10%

20%

10% 10% 10%

-40%

-20%

20%

40%

60%

80%

100%

120%

140%

Control 10^6 10^5 10^4

(b) Actinomycete

Flight-capable Flightlessness Post-emergence mortality

Figure 6. (a) Daily evolution of mortality rates following exposure

to varying doses of Actinomycete. (b) Post-exposure

outcome of larvae surviving beyond 7 days.

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Rev. Fac. Agron. (LUZ). 2026, 43(1): e264305 January-March. ISSN 2477-9409.

6-7 |

90 % mortality), in line with studies showing strong larvicidal activity

of actinomycete metabolites against mosquito larvae (Seratnahaei et

al., 2023). Bacillus sp. acted more slowly (6.5 days), consistent with

the short residual activity and environmental sensitivity reported for

Bacillus-based biolarvicides (Zogo et al., 2019). Nonlinear dose-

response trends suggested spore aggregation or host stress at high

inoculum levels (Parco et al., 2023).

Table 2. Comparative TL₅₀ values of tested entomopathogenic

strains.

Entomopathogenics

Doses

A. avus A. fumigatus Mucor

sp.

Bacillus

sp.

Actinomycete

10⁴ CFU.mL

-1

- - 4.5 4.5 -

CFU.mL

-1

7.0 - 5.0 -

CFU.mL

-1

6.0 4.5 2.0 6.5 4.7

Treated Culex pipiens larvae showed strong pathological eects,

including reduced vitality, impaired motility, and rapid decomposition,

especially in the head, abdomen, and siphon. Microscopic analysis

revealed severe cuticle degradation in chitin-rich areas (gure 7),

conrming integument weakening as a main mortality mechanism.

Pigmentation changes, such as whitening and darkening with reddish

hues, indicated melanization and microbial metabolite activity

(Vega and Kaya, 2012; Arreguín-Pérez et al., 2023). Development

was also disrupted, with delayed or premature pupation, prolonged

metamorphosis, and undersized non-viable pupae. Surviving adults

exhibited malformations, including wing deformities and ight

incapacity, consistent with entomopathogen exposure (Lacey, 2017).

These results, in line with previous studies (Lacey, 2017), highlighted

both lethal and sublethal impacts. Overall, the ndings conrmed the

strong pathogenic activity of tested microorganisms and their promise

as eco-friendly agents in mosquito control.

Pathological symptoms in Culex pipiens larvae following

treatments

Figure 7. Dierent symptoms observed during treatments.

study, chitin was successfully extracted from shrimp shells (yield:

16.6 %), highlighting their potential as a valuable resource.

Five chitinolytic microorganisms were isolated and identied:

Aspergillus avus, Mucor sp., Bacillus sp., Aspergillus fumigatus,

and Actinomycete. Bioassays against Culex pipiens larvae conrmed

their pathogenicity, with mortality increasing proportionally to

concentration. Notably, strong larvicidal and sublethal eects were

observed, indicating their potential use in mosquito biocontrol.

Although preliminary, these results support the use of chitinolytic

microorganisms as eco-friendly alternatives to synthetic insecticides.

Further molecular, biochemical, and ecological studies are needed to

validate their eciency under natural conditions.

Literature cited

Abbott, W. S. (1925). A method of computing the eectiveness of an insecticide.

Journal of Economic Entomology, 18(2), 265-267. https://doi.org/10.1093/

jee/18.2.265

Aguilar-Durán, J. A., Villarreal-Treviño, C., Fernández-Santos, N. A.,

Hamer, Gabriel L., & Rodríguez-Pérez, M. A. (2023). Virulence

of entomopathogenic fungi isolated from wild mosquitoes against

Aedes aegypti. Entomological Research, 53(2), 158-166. https://doi.

org/10.1111/1748-5967.12640

Al-Nagar, N. M. A., Shawir, M. S., & Abdelgaleil, S. A. M. (2024). Mosquitocidal

activity of extracts derived from soil Actinomycete isolates. Alexandria

Science Exchange Journal, 45(4), 719-729. https://doi.org/10.21608/

asejaiqjsae.2024.395086

Arreguín-Pérez, C. A., Miranda-Miranda, E., Folch-Mallol, J. L., & Cossío-

Bayúgar, R. (2023). Identication of virulence factors in entomopathogenic

Aspergillus isolates. Microorganisms, 1(8), 2107. Jhttps://www.ncbi.nlm.

nih.gov/pmc/articles/PMC10457961/

Bharadwaj, N., Sharma, R., Subramanian, M., Ragini, G., Nagarajan, S. A., & Rahi,

M. (2025). Omics approaches in understanding insecticide resistance in

mosquito vectors. International Journal of Molecular Sciences, 26(5),

1854. https://doi.org/10.3390/ijms26051854

Goettel, M. S., Eilenberg, J., & Glare, T. R. (2010). Entomopathogenic fungi and

their role in regulation of insect populations. In L. A. Lacey (Ed.), Manual

of techniques in invertebrate pathology (2nd ed., pp. 387-432). Academic

Press. https://doi.org/10.1016/C2010-0-66784-8

Izadi, H., Asadi, H., & Bemani, M. (2025). Chitin: A comparison between its main

sources. Frontiers in Materials, 12: 1537067. https://doi.org/10.3389/

fmats.2025.1537067

Balumahendhiran, K., Vivekanandhan, P., & Shivakumar, M. S. (2019). Mosquito

control potential of secondary metabolites isolated from Aspergillus avus

and Aspergillus fumigatus. Biocatalysis and Agricultural Biotechnology,

21, 101334. https://doi.org/10.1016/j.bcab.2019.101334

Lacey, L. A. (2017). Entomopathogens used as microbial control agents. In

Microbial control of insect and mite pests (pp. 3-12). Academic Press.

https://doi.org/10.1016/B978-0-12-803527-6.00001-9

Lamy, D. L., Gnambani, E. J., Sare, I., Millogo, S. A., Sodré, F. A., Namountougou,

M., Viana, M., Baldini, F., Diabaté, A., & Bilgo, E. (2025). Metarhizium

pingshaense increases susceptibility to insecticides in highly resistant

malaria mosquitoes Anopheles coluzzii. Wellcome Open Research, 9, 290.

https://doi.org/10.12688/wellcomeopenres.21238.2

Lavine, M. D., & Strand, M. R. (2002). Insect hemocytes and their role in

immunity. Insect Biochemistry and Molecular Biology, 32(10), 1295-

1309. https://doi.org/10.1016/S0965-1748(02)00092-9.

Li, X., & Wiens, J. J. (2023). Estimating global biodiversity: The role of

cryptic insect species. Systematic Biology, 72(2), 391-403. https://doi.

org/10.1093/sysbio/syac069

Natrah, F. M. I., Ruwandeepika, H. A. D., Pawar, S., Karunasagar, I., Sorgeloos,

P., Bossier, P., & Defoirdt, T. (2011). Regulation of virulence factors by

quorum sensing in Vibrio harveyi. Veterinary Microbiology, 154(1-2),

124-129. https://doi.org/10.1016/j.vetmic.2011.06.024

Parco, J., Valverde-Rodríguez, A., Cornejo, A., Briceño, H., Barrionuevo, L.,

& Romero, J. (2023). Eciency of entomopathogenic bacteria and

fungi on Oligonychus yothersi in vitro and on Persea americana Mill.

plants. Revista de la Facultad de Agronomía (LUZ), 40(4), e234033.

https://produccioncientificaluz.org/index.php/agronomia/article/

view/40991/47103

Hossain, M. S., Anjum, M. F., Rabbani, M. A., Hasan, M. R., Sohag, M. M.

H., Tasnim, N., Karmakar, D., Akter, S., Rahman, M. M., & Karim,

M. R. (2025). Bacillus altitudinis: a sustainable mosquito controlling

biopesticide isolated from the rhizospheric soil of Nypa fruticans in

mangrove forest. BMC Microbiology, 25, 733. https://doi.org/10.1186/

s12866-025-04168-0

Seratnahaei, M., Eshraghi, S. S., Pakzad, P., Zahraei-Ramazani, A., & Yaseri, M.

(2023). Larvicidal eects of metabolites extracted from Nocardia and

-Significant reduction in vitality and motility

of treated larvae.

-Rapid decomposition of dead larvae, mainly

in the head, abdomen, and respiratory siphon.

-Pronounced body whitening in treated

larvae, especially in head and abdominal

regions (absent in controls).

-Darkening or reddish discoloration of the

body (melanization response).

-Gut emptying or epithelial degeneration.

-Osmotic imbalance causing body swelling or

desiccation.

-Microscopy revealed severe cuticle damage

in high-chitin areas (respiratory siphon,

tracheal branches).

-Delayed or abnormal molting (larvae trapped

between stages).

-Cephalic outgrowths appeared, leading to

impaired nervous system function.

-Deformation of wing musculature and

halteres were observed, resulting in inhibited

flight ability.

- Early emergence of malformed adults.

Conclusion

Protecting human health from mosquito-borne nuisances remains

essential, while conventional chemical insecticides face growing

limitations due to resistance and environmental concerns. In this

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Boulanouar et al. Rev. Fac. Agron. (LUZ). 2026, 43(1): e264304

7-7 |

Streptomyces species against the forth larval stage of Anopheles stephensi

(Diptera: Culicidae). Journal of Arthropod-Borne Diseases, 17(2), 187-

196. https://doi.org/10.18502/jad.v17i2.13623

Renuka, S., Vani, H. C., & Alex, E. (2023). Entomopathogenic fungi as a potential

management tool for the control of urban malaria vector Anopheles

stephensi (Diptera: Culicidae). Journal of Fungi, 9(2), 223. https://doi.

org/10.3390/jof9020223

Singh, R., & Dubey, A. K. (2015). Endophytic Actinomycetes as emerging source

for therapeutic compounds. Indo Global Journal of Pharmaceutical

Sciences, 5(2), 106-116. https://doi.org/10.35652/igjps.2015.11

Valdez, D., Farah, S., Espinoza, W., Veliz, F., Villon, H., & Herrera, L. (2025).

Biocontrol of Cosmopolites sordidus using entomopathogenic fungi under

laboratory conditions, Ecuador. Revista de la Facultad de Agronomía (LUZ),

42(2), e254217. https://doi.org/10.47280/RevFacAgron(LUZ).v42.2

Vega, F. E., & Kaya, H. K. (Eds.). (2012). Insect pathology (2nd ed.). Academic

Press. https://doi.org/10.1016/B978-0-12-384984-7.00001-4

Zogo, B., Tchiekoi, B. N., Ko, A. A., Dahounto, A., Ahoua Alou, L. P., Dabiré,

R. K., Baba-Moussa, L., Moiroux, N., & Pennetier, C. (2019). Impact

of sunlight exposure on the residual ecacy of biolarvicides Bacillus

thuringiensis israelensis and Bacillus sphaericus against the main

malaria vector Anopheles gambiae. Malaria Journal, 18, 55. https://doi.

org/10.1186/s12936-019-2687-0

Zhu, G., Ding, W., Zhao, H., Xue, M., Chu, P., & Jiang, L. (2023). Eects of the

entomopathogenic fungus Mucor hiemalis BO

-1

on the physical functions

and transcriptional signatures of Bradysia odoriphaga larvae. Insects,

14(2), 162. https://doi.org/10.3390/insects14020162