Acta Tropica 234 (2022) 106627

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Delivery and effectiveness of entomopathogenic fungi for mosquito and tick

control: Current knowledge and research challenges
Claudia Cafarchia a, *, Rebecca Pellegrino b, Valentina Romano a, Marco Friuli b,
Christian Demitri b, Marco Pombi c, Giovanni Benelli d, Domenico Otranto a, e
a
Department of Veterinary Medicine, University of Bari Aldo Moro, Str. prov. per Casamassima km 3, Valenzano, Bari 70010, Italy
b
Department of Engineering for Innovation, University of Salento, Lecce, Italy
c
Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy
d
Department of Agriculture, Food and Environment, University of Pisa, Pisa, Italy
e
Faculty of Veterinary Sciences, Bu-Ali Sina University, Hamedan, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Insects, ticks, and mites represent a threat to animal health globally, mainly due to their role as vectors of
Culicidae pathogens. Among the most important diseases, those transmitted by mosquitoes (e.g., malaria and arboviral
Ixodidae infections) and ticks (e.g., Lyme borreliosis, babesiosis, and viral haemorrhagic fever) have a huge impact on
Integrated vector management
human health. The principal methods available for reducing the public health burden of most vector-borne
Insecticide and acaricide formulation
Beauveria spp.
diseases are vector-based intervention relying to insecticides and acaricides. However, the use of these prod­
Metarhizium spp. ucts is challenged by the introduction of invasive species, the quick development of physiological insecticide and
acaricide resistance, and their non-target effects on human health and environment. In this scenario, insecticide/
acaricide-free control approaches based on the employment of entomopathogenic fungi (EPFs) are currently
considered a promising tool in Integrated Pest/Vector Management, even if their large-scale use is still limited. In
this article, we provide an overview on current knowledge about the role of EPFs for mosquito and tick man­
agement to assess solutions improving the delivery and efficacy of EPFs in the field. Laboratory research provided
solid evidence that EPFs represent a next-generation control tool to manage mosquito and tick populations.
However, the viability, infectivity, and persistence of fungal spores under field conditions are still inadequate.
Herein we also discuss the development and optimization of EPF-based lure and kill approaches through bio­
polymers to improve cost-competitive, safety and eco-friendly pest and vector control tools.

1. Introduction
A number of arthropod species, with special reference to insects and
ticks, represent a serious threat globally, mainly due to their role as pests
and/or vectors of pathogens of medical and veterinary importance.
Vector-borne diseases (VBDs) account for more than 17% of all infec­
tious diseases, causing more than 700,000 human deaths annually
(World Health Organization, 2020). Among the most important patho­
gens are mosquito-borne arboviruses (e.g., yellow fever, dengue, chi­
kungunya, West Nile, and Zika viruses), protozoans causing malaria, as
well as a wide range of tick-borne pathogens, such as those responsible
for Lyme borreliosis, babesiosis, and viral haemorrhagic fevers (Di
Giovanni et al., 2021). Due to the climate change and the continuous
movement of people and goods, several arthropod vectors can find
suitable habitats for their proliferation also in many uncolonized areas
(Wilke et al., 2019, 2020, 2021a, 2021b). For example, approximately
half of the world’s population is expected to be at risk of arbovirus
transmission by 2050 (Lessler et al., 2016; World Health Organization,
2020). The effect of such epidemics has not only an implication linked to
the disease, but also socioeconomic drawbacks linked both to the direct
costs of case management/hospitalization and cascading effects on the
economy (Quereshi, 2018).
Ticks are second to mosquitoes in their capacity to transmit zoonotic
diseases, acting as vectors of a wide number of pathogens mainly
causing anaplasmosis, babesiosis, borreliosis, Colorado tick fever, rocky
mountain spotted fever, tick-borne relapsing fever, southern tick-
associated rash illness, ehrlichiosis, heartland virus, lyme disease, Pos­
wassan disease, tularaemia, and rickettsiosis (Dantas-Torres et al., 2012;

* Corresponding author.
E-mail address: claudia.cafarchia@uniba.it (C. Cafarchia).

https://doi.org/10.1016/j.actatropica.2022.106627
Received 8 June 2022; Received in revised form 12 July 2022; Accepted 28 July 2022
Available online 29 July 2022
0001-706X/© 2022 Elsevier B.V. All rights reserved.
C. Cafarchia et al.
Guglielmone et al., 2014; Benelli, 2020). Moreover, ixodid ticks are final
hosts of Babesia spp. and Theileria spp. causing diseases in livestock
(Perveen et al., 2021). Additionally, ticks may harm animals directly by
inducing toxicosis (i.e., sweating sickness and tick paralysis caused by
salivary fluids containing toxins), skin wounds susceptible to secondary
bacterial/fungal infections, screw-worm infestations and anemia
(Perveen et al., 2021).
The principal methods available for reducing the public health
burden of most VBDs are vector-based interventions (Petersen et al.,
2019; Wilson et al., 2020). Until 1940, vector control relied on a thor­
ough understanding of vector ecology and epidemiology and imple­
mentation of environmental management tailored to the ecology and
behavior of local vector species. This complex interaction of factors has
been neglected and pesticide-based approaches still remain the most
common option to manage mosquitoes and ticks (Wilson et al., 2020).
However, these substances – mostly applied through environmental
spraying/nebulization - are not very effective in terms of species tar­
geting and require repeated applications over time with increasing
pesticide concentrations, thus resulting in resistance development,
presence of toxic molecules in the food chain, and negative effects on
non-target organisms (Desneux et al., 2007; Benelli, 2019; Benelli et al.,
2020). Therefore, pesticide-free approaches are increasingly being
investigated to deal with threats derived from exposure to anthropo­
genic stressors (Wilke et al., 2021a; Cutler et al., 2022), and the need to
incorporate more vector control interventions to eliminate these dis­
eases (World Health Organization, 2012; Benelli et al., 2021). Examples
of alternative control strategies are represented by vaccines against ar­
thropods or pathogens they transmit (de la Fuente et al., 2016; Nadeem
et al., 2022), releasing of genetically modified mosquitoes, Wolbachia-­
based approaches, sterile insect technique (SIT) (Wilke et al., 2018), the
employment of plant-isolated repellent, insecticides and acaricides
(Isman 2006, 2020; Benelli et al., 2016b; Benelli and Pavela, 2018;
Pavela et al., 2019), and the use of entomopathogens (virus, bacteria and
fungi) as biocontrol agents (Lacey et al., 2015; Shen et al., 2020; Ebani
and Mancianti, 2021).
In the present article, we reviewed current knowledge about the role
of entomopathogenic fungi (EPFs) formulations available for mosquito
and tick management, as well as of methods and devices employed for
their delivery, to optimize the efficacy and applicability in the field.
2. How entomopathogenic fungi infect mosquitoes and ticks?
EPFs are microorganisms having the ability to infect and kill ar­
thropods. The employment of these biological control agents (BCAs)
represents an effective alternative to manage arthropod populations in
an eco-friendly manner. The evolutionary history of EPFs (reviewed by
Vidhate et al., 2022) suggests that they were primarily associated with
plants, for which they were endosymbionts (endophytes). During their
evolution, these fungi diverged to a separate lineage, shifting from plant
endosymbionts to insect pathogenic organisms, bringing with them an
“arsenal” of enzymes which allowed to survive or cause infection in
different host species (Vidhate et al., 2022). Similarly, the diversifica­
tion of EPF host-specificity is demonstrated by genome sequencing of
generalist entomopathogenic fungi ranging from a wider (e.g., Meta­
rhizium anisopliae (Metschn.) Sorokin, 1883) to narrow (e.g., Meta­
rhizium acridum (Driver & Milner) J.F. Bisch) host-range. Thus, the
pathogenic mechanisms of these organisms are linked to their biology,
virulence factors and hosts receptivity (Shin et al., 2020; Islam et al.,
2021).
2.1. Biotic and abiotic factors affecting pathogenic mechanisms of EPFs
More than 700 fungal species from 100 genera have been isolated
from soil and insect cadavers (Shin et al., 2020). These fungi are
generally known to be harmless to humans and other non-target or­
ganisms and have been studied as BCAs of arthropods pests, as they do
2
Acta Tropica 234 (2022) 106627
not adversely affect the environment (Lacey et al., 2015). Today, EPFs
comprise different taxa belonging to Ascomata, and the most common
belongs to Hypocreales (Metarhizium, Beauveria, Hirsutella, Nomuraea
and Cordyceps) and Entomophthorales (Entomophthora, Pandora, Ento­
mophaga and Zoophthora). However, the EPFs most employed to manage
mosquitoes and ticks belong to the Hypocreales order, namely, Beauveria
bassiana (Bals.-Criv.) Vuill., 1912, for mosquitoes and Metarhizium spp.
for ticks (Tables 1–4). Species belonging to the above-mentioned genera
were largely studied among EPFs because of their double actions in
reducing natural arthropod populations and forming complex relation­
ships with plants, providing them with nitrogen assimilated during the
parasitisation of insects or making plants more resistant to insects (Lit­
win et al., 2020). In addition, the occurrence of EPFs in the environment
and their pathogenic mechanisms to target arthropod species have
contributed to focus research on their role as BCAs (see Tables 1–4). The
pathogenetic mechanisms of EPFs is linked to several factors, encom­
passing both biotic (i.e., host range, latency, spore density and dispersal)
and abiotic ones (i.e., physical and chemical soil properties, tempera­
ture, humidity, rainfall, and sunlight), which play a role, alone or in
combination, in selecting fungal species for biocontrol strategies. The
assessment of host range for a biocontrol agent is fundamental to reduce
potential risks for the environment or for non-target species (Rohrlich
et al., 2018). Indeed, the ecological host-range refers to the range of
species that an EPF infects in the field, whereas the physiological host
range is the species range that the BCA can infect under laboratory
conditions (Rohrlich et al., 2018). The assessment of the ecological host
range is a complex task, still not well-explored (Rohrlich et al., 2018),
thus knowledge about the potential virulence of EPFs usually rely on
studies conducted under laboratory conditions. To date, fungi belonging
to the Entomophthorales order tend to have a narrow ecological and
physiological host range limited to a small number of taxonomically
related species, whereas those belonging to Hypocreales order show a
broader host range (Rohrlich et al., 2018). However, among Hypo­
creales, differences in host range are often mentioned at the species or
strain level, most likely due to environmental conditions affecting their
virulence profiles (Rohrlich et al., 2018). Host immunity and biology
may also affect the pathogenic mechanisms of EPFs. Higher host density,
the dark of epidermal surface and the host immunity might affect the
EPF infections procedures (Abdul Qayyum et al., 2021). Young mosquito
larval stages are frequently more susceptible than adults (Tables 1 and
2). In addition, since mosquitoes and ticks develop a response against
fungal infections by upregulating antifungal compounds and/or acti­
vating an innate immune response through reactive oxygen species
(ROS), humoral melanisation and phagocytosis, EPFs suitable to be used
as BCAs need to carry resistance mechanisms to elude host immune
barrier. This is the case of B. bassiana, which upregulates superoxide
dismutase and accelerates the conversion of superoxide ions into mo­
lecular oxygen and hydrogen peroxide, enhancing its tolerance to
oxidative stress (Zibaee and Malagoli, 2014).
However, under natural conditions the whole infection process is
linked to abiotic factors that primarily affect the host–fungus in­
teractions. Generally, the EPFs belonging to Entomophthorales order are
major pathogens of insect pests in temperate habitats, while Hypo­
creales are prevalent in humid tropical habitats (Islam et al., 2021).
However, B. bassiana and M. anisopliae are found in all types of eco­
systems with B. bassiana dominating in natural habitats being affected
by soil tillage and agricultural practices (Islam et al., 2021). Addition­
ally, soil acidity, the heaviness of soil texture, and high organic matter
content led to the higher percentages of harbouring of all EPFs (Abdul
Qayyum et al., 2021). Beauveria bassiana and M. anisopliae isolates can
grow at a wide temperature range (8 to 37 ◦ C) but the optimal tem­
perature range for germination has consistently been found to be 20 ◦ C
to 30 ◦ C (Abdul Qayyum et al., 2021). In fact, temperature is a key factor
affecting viability and effectiveness of EPFs with strains from warmer
environments performing better at higher temperatures and those from
cooler climates at lower temperatures (Abdul Qayyum et al., 2021).
C. Cafarchia et al. Acta Tropica 234 (2022) 106627

Table 1
Effectiveness of different formulations of Beauveria spp. for mosquito control: in vitro studies over the last ten years.
Fungi Mosquito Infected EPF formulations Mode of Mortality%/time Notes References
species stage somministration or lethal time (LT)

Beauveria Aedes aegypti Larvae Oil-in-water emulsion CT x 24 h CT: Blastospore showed better de Oliveira Barbosa
bassiana of conidia and IT x 10 days Blastospores activity than conidia. Water Bitencourt et al., 2021;
blastospores: 100%/7 days; emulsions yielded better results Deng et al., 2019;
water conidial Conidia than the water suspensions. Rodrigues et al., 2021
suspension: 90–92.6%/7–15
3.3 × 103–1 × days.
108conidia/ml. IT: WT 95%/10
Water blastospore days;
suspension: 1 × 108 Cyt2Ba 100%/9
blastospores/ml. days
Adults Water or PBS with CT x 3 h-30 days CT: 90–100%/ Bb-Cyt2Ba is more effective Deng et al., 2019;
Tween 20 or 80 IT x 1 min 10–18 days than Wt. Dong et al., 2012;
conidial suspension of IT: <85%/18 days CT yielded better results than Ramirez et al., 2019,
wild type or IT. 2018
genetically modified
strains:
1 − 2.3 × 108 conidia/
ml
Aedes Larvae Water with Tween 80 IT x 10 days WT 70–100%/10 Bb-Cyt2Ba was more effective. Deng et al., 2019, 2017
albopictus conidial suspension of days; LT50 was lower for the strain
wild type or Cyt2Ba 100%/9 Bb-AaIT.
genetically modified days;
strains: Bb- AaIT 90%/10
1 × 107 conidia/ml days;
LT50: Wt=
7.7–6.7 days;
Bb-AaIT= 5.5–4.4
days
Adults Soybean oil conidial Spray Spray: 100%/10 Bb-Cyt2Ba was more effective. Deng et al., 2019,
suspension; CT or Ingestion days LT50 was lower if conidia were 2017; Lee et al., 2019a;
Water conidial CT x 3 h CT: Wt 80–100%/ ingested and in genetically Ramirez et al., 2021;
suspension with 10–11 days; modified strains. B. brogniartii Shoukat et al., 2020b
Tween 80; Cyt2Ba 100%/8 was less effective.
Water conidial days;
ingestion with 10% Bb-AaIT 100%/8.5
glucose; days;
Water conidial Ingestion:
suspension of wild Bb-AaIT 100%/10
type or genetically days; Wt 100%/10
modified strains: days.
1–3 × 108 conidia/ml
Anopheles Adults Water suspension with CT x 3h 80–100%/8–14 Valero-Jiménez et al.,
coluzzii Tween 80: days. 2017, 2014
mineral oil conidial
suspension:
1 × 109 spores/ml
Anopheles Adults Mineral oil conidial Spray 80%/2–5 months Blanford et al., 2012a
gambiae suspension: 1 × 109
spores/ml
Anopheles Adults Water conidial CT x 1–6 h CT:80–100%/4–7 Blanford et al., 2012b,
stephensi suspension: 1 × 108 Spray days; 2012a; Ishii et al.,
conidia/ml; Spray: 80%/5 2017; Lai et al., 2017
Water mineral oils months.
conidial suspension:
1 × 109 conidia/ml
Culex pipiens Larvae Conidial suspension in CT x 3 days 73.33% /3 days Hamama et al., 2022
distilled water with
Tween 80:
LC50 1.24×106 spore/
ml
Adults Spray The mortality of C. pipiens Lee et al., 2019a;
; varied according Bb strains.
Water conidial Spray: LT50: from
suspension with 4.3 to 6.3 days.
Tween 80 of different
Bb strains:
1 × 108 conidia/ml.
Culex Larvae Water conidial IT x 24–92 h 34.7–52.94%/ B. bassiana had synergistic Koodalingam and
qinquefasciatus (L2) suspension with 48h-92 h larvicidal effects with Dayanidhi, 2021;
Tween 80: Bb-28: imidacloprid. Vivekanandhan et al.,
1 × 108 conidia/ml; 83.33–100%/1 Bb-28 ethyl acetate mycelial 2018
water suspension with day extracts was comparable to that
of the commercial insecticide
(continued on next page)

3
C. Cafarchia et al.
Table 1 (continued )
Fungi Mosquito Infected EPF formulations Mode of
species stage somministration
concentration 250 mg/
l extract showed strong
Adults Water conidial Spray
suspension 1.3 × 1011
conidia/m2
Beauveria Aedes aegypti Adults Soy bean oil conidial CT
bassiana suspension
and 1 × 108 conidia/ml
Beauveria
brongniartii Aedes Adults
albopictus
Culex pipiens Adults
IT: Immersion test; CT: Contact test; Wt: Wilde type; Bb-AaIT: Beauveria bassiana genetically modified to express an insect-specific scorpion neurotoxin (AaIT); Bb-
Cyt2Ba: B. bassiana, genetically modified to express the Bacillus thuringiensis toxin Cyt2Ba; Bb-28: B. bassiana-28.
Spore germination and conidia viability of all EPFs are usually high
when the relative humidity (RH) is near saturation (>95.5%), but
B. bassiana and M. anisopliae can infect their respective hosts under low
atmospheric humidity conditions, possibly due to adequate humidity
within the microhabitats (Abdul Qayyum et al., 2021). In addition,
humidity and precipitation play a key role in the dispersion of conidia
from substrates, and large quantities of spores and conidia may be
removed from the arthropod cuticle during precipitation. Finally, the
solar UVA (320–400 nm) and UVB (290–320 nm) negatively affect the
conidia viability and germination due to the degradation of cytoplasmic
membranes and organelles and to the direct and indirect damage of
DNA, which, in turn, depends on the intensity, duration of conidia’s
exposure, or EPF isolates. Usually, there is a considerable reduction of
relative germination and total inactivation of Beauveria spp. or Meta­
rhizium spp. conidia after only a few hours exposure to UV-B irradiation.
Metarhizium anisopliae seems to be more tolerant to UV-B than
B. bassiana. However, it has been demonstrated that the native isolates
of Beauveria spp. and Metarhizium spp. are more tolerant to solar expo­
sition being also more virulent than not-native strains, due to their
ability to survive in the environment from which they have been isolated
(Cafarchia et al., 2015; Sutanto et al., 2021). As for the pH, the best
growing condition occurs between pH= 3–9, but pH = 4.4 and pH=6 are
optimal for the growth of mycelium and spores production, respectively
(Teng, 1962). Beauveria bassiana and Metarhizium spp. do not require a
specific nitrogen source, but urea is very effective for sporulation,
especially at high concentrations. The amount of EPF spore production is
also proportionally linked to the concentration of sugars (e.g., maltose,
sucrose, raffinose, and glucose) in the cultures. Additionally, the min­
erals are essential compounds for spore production and the addition of
0.2 mg of manganese or iron in the culture is useful for an increase in
spore production of 15–20%, respectively (Teng, 1962). The spores of
EPFs have been shown to be very resistant to insecticides and fungicides
commonly employed in agronomic practices as gasses are produced by
plant tissues after treatment with a harmful effect to B. bassiana spores
and an increasing of its germination up to 30–90% (Teng, 1962).
2.2. A closer look to pathogenic mechanisms of EPFs
The common route for EPF infections is cuticle penetration, though
several alternative routes of infections such as ingestion of EPF spores
have been also suggested (Mannino et al., 2019). EPFs recognize hosts
by the G protein–coupled receptors (GPCR) (which facilitate nutrient
sensing, stress response, and fungal development) and by the
4
Acta Tropica 234 (2022) 106627
Mortality%/time Notes References
or lethal time (LT)
B. bassiana-22 extract. Bb-28
insecticidal activity against 1st
to 4th instar larvae.
40%/21 days Popko et al., 2018
B.bassiana: Ae. albopictus and Ae aegyptiwere Ramirez et al., 2018
94.9%/15 days susceptible to the both
B. brongniartii: Beauveria spp. while Cx. pipiens
88%/15 days appear to be resistant to B.
B.bassiana: 100%/ brongniartii Ramirez et al., 2021
11 days most likely due to a different
B. brongniartii: phenoloxidase cascade genes
95%/15 days expression.
B.bassiana: 90%/ Ramirez et al., 2021
11 days
B. brongniartii:
10%/15 days
transmembrane protein, activating the mitogen-activated protein kinase
(MAPK) and cAMP-dependent protein kinase (PKA) downstream path­
ways. In addition, EPF spores can passively reach the host surface
through the movement of air and water and attach to exocuticle by
hydrophobic forces. Indeed, dry conidia are covered by hydrophobins,
which are typical proteins of external layer of conidia. Different genes
drive the adhesion process in EPFs and, in particular, two adhesion
proteins are described: Hyd1 and Hyd2 for B. bassiana and Mad1 and
Mad2 for M. anisopliae (Islam et al., 2021; Vidhate et al., 2022). On the
surface of host, conidia germinate producing hypha and/or appresso­
rium, which are useful to reach the hemocoel by penetration of host
cuticle which is driven by extracellular activity of enzymes (i.e., lipases,
esterases, proteases and chitinases) produced by fungi.
In addition to hydrolysing enzymes, the appressorium secretes oxalic
acid which softens the cuticle and facilitates the penetration of the
fungus. Reaching the host hemocoel, fungus secretes various secondary
metabolites (toxins) and effector proteins to circumvent host immunity.
To accomplish the infection, fungal cells are differentiated into yeast-
like cells called blastospores that use resources from hemocoel causing
host mortality. After EPFs kill the host, they convert back to mycelial
growth and cuticle penetration increases again. After conquering the
host, conidia regenerated on the host surface are mainly dispersed by
non-biological factors, such as water and wind (Shin et al., 2020; Islam
et al., 2021; Vidhate et al., 2022). The hydrophobic property of conidia
is not only necessary to attach to the host cuticle and achieve successful
infection, but also to facilitate dispersion with water (Shin et al., 2020).
Currently, studies performed using gene knockout mutants of EPFs
and transcriptome analysis suggested that different genes are involved
in each phase of EPF infections of arthropods (i.e., attachment of con­
idia, germination, penetration, growth, and generation of secondary
infectious conidia, reviewed by Shin et al., 2020). For example, the
mutant Bbmpk1 of B. bassiana, which is involved in the formation of
appressorium, could not penetrate the cuticle from inside to outside,
thus resulting in a fungus lacking regenerating properties. Therefore,
EPFs infecting mosquitoes and ticks are also able to control the vectorial
competence of vectors since the number of bloodsucking arthropods that
can bite and get infected are reduced. Interestingly, it has also been
shown that the mortality caused by EPFs on mosquito adult females
infected by dengue virus serotype 2 (DENV-2) is five times higher than
mortality registered for uninfected adult females (Blanford et al., 2005;
Dong et al., 2012). In addition, the ability of EPFs in inducing an
immunological response, Toll and JAK/STAT pathways, in infected
mosquitoes seem to play a role in making mosquitoes resistant to
C. Cafarchia et al. Acta Tropica 234 (2022) 106627

Table 2
Effectiveness of different formulations of Metarhizium spp. for the mosquito control: in vitro studies over the last ten years.
Entomopathogenic Mosquito Infected EPF formulation Mode of Mortality/time; Notes References
fungi species Stage somministration lethal time (LT)

Metarhizium Aedes aegypti Larvae Water conidial with or CTx24 h or 7 55–100%/6–15 Survival time was Alkhaibari et al., 2018,
anisopliae; without Tween; days; days; related to the fungus 2017; de Oliveira
Metarhizium Water conidial IT LT50 = 1.27–3 days and spore Barbosa Bitencourt
brunneum; suspension plus concentration. The et al., 2021; Gomes
Metarhizium avermectins (Phytoverm highest concentration et al., 2015; Noskov
humberi; 0.2%; SPC of conidia and et al., 2019; Paula
Metarhizium Pharmbiomed’, Russia); M. anisopliae et al., 2019; Prado
robertsii; Water or mineral oil represented the more et al., 2020; Rodrigues
blastospore suspension: active EPF et al., 2021;
from 1 × 104 spores/ml combination; Vivekanandhan et al.,
to 1 × 109 spores/ml Blastospore in water 2020a
or oils suspension
were more effective
than water conidial
suspension; the
survival rate was
lower with the
combinations of
neem + fungus or
avermectins + fungus.
IT was the more
frequent test
performed.
Adults M. anisopliae CT x 1 min Blastospores100%/ Mineral oil added to Carolino et al., 2014;
Water or oil conidial − 72–120 h. 4 days; conidia showed better de Paula et al., 2021;
suspension with Tween Spray Conidia 80–100%/ results than testing Falvo et al., 2020;
20–80: 1 × 106–2 × 109 CT with black 5–15 days; conidia alone. The Mehmood et al., 2022;
conidia/ml; cotton clothes x CT black cloth additions of Paula et al., 2013a,
Oil blastospores 32–120 h Imi+Fungi imidacloprid increase 2013b; Paula et al.,
suspension with Tween 56.7–61.7%/7 days mortality. The use of 2018; Rodrigues et al.,
20: 1 × 108 synthetic lures 2019
blastospores/ml; increased the
Water conidial mortality of 16%.
suspension with Tween Blastospores showed
80 and imidacloprid: better results than
1 × 109 conidia/ml conidia.
1 × 108 conidia/cm2
Metarhizium Aedes Larvae Water conidial CT x 3 days LC50 1.09×105 Bilal et al., 2012
anisopliae albopictus suspension with Tween LC90 1.90×1013
80: 1 × 104 − 1013
conidia/ml
Adults Water conidial Spray 92.5%/7 days Shoukat et al.,
suspension with Tween 2020a
80: 3 × 108 spores/ml
Metarhizium Anopheles Adults Water conidial CT x 6 h CT:100%/7 days Mnyone et al.,
anisopliae gambiae suspension: about 1.0 2012; Ondiaka
× 1011 conidia/m2. et al., 2015
Oil conidial suspension
in 1:1 Enerpar oil
(Enerpar M002®, BP
Southern Africa Ltd)
and Shellsol oil
(Shellsol T®, South
Africa Chemicals).
Metarhizium Anopheles Larvae Water conidial IT 97.3–100%/1–3 Similar mortality rates at Alkhaibari et al.,
anisopliae stephensi suspension: 75 μg/ml; days all conidia concentrations. 2017;
Metarhizium Water conidial The effectiveness of Vivekanandhan
brunneum suspension with Tween blastospore suspension et al., 2020a
80 or dry conidia; was higher than conidia
Water blastospores suspension at
suspension: concentration 1 × 108
1 × 108 propagules/ml propagules/ml
Adults Water mineral oil CT x 6 h 70–100%/10–14 Mortality varies according Blanford et al.,
conidial suspension: days Metarhizium spp. Reduced 2012b;
1 × 109 conidia/ml feeding propensity in Vivekanandhan
mosquito following fungal et al., 2020a
infection was observed.
Metarhizium Culex Larvae Water conidial IT x 7 days 37.94–100%/1–10 The conidial suspension in Alkhaibari et al.,
anisopliae qinquefasciatus suspension: 75 μg/ml; days water was more effective 2017;
Metarhizium Water conidial than the blastospores Koodalingam and
brunneum suspension with Tween suspension and dry Dayanidhi, 2021;
80: conidia. Vivekanandhan
1 × 108–1 × 109
(continued on next page)

5
C. Cafarchia et al. Acta Tropica 234 (2022) 106627

Table 2 (continued )
Entomopathogenic Mosquito Infected EPF formulation Mode of Mortality/time; Notes References
fungi species Stage somministration lethal time (LT)

conidia/ml; et al., 2020a,

Water conidial 2020b
suspension with Tween
80 or dry conidia;
Water blastospores
suspension:
1 × 108 propagules/ml
Adults M. anisopliaewater Spray 50%/21 days Popko et al., 2018
conidial suspension 1.3
× 1011 conidia/m2
Metarhizium Culex pipiens Larvae Water conidial CT x 48 h 88%/22.6 h Hamama et al.,
anisopliae suspension with Tween 2022
80: LC50 1.85×106
spore/ml

IT: Immersion test; CT: contact test; EPFs: Entomopathogenic fungi.

arboviral infections (Xi et al., 2008; Dong et al., 2012; Garza-Hernández
et al., 2013). However, despite based on different pathogen-vector
models, studies focusing on tsetse flies or triatomine vectors of the
protozoan T. congolense or T. cruzi, respectively, showed contrasting
results. The survival of triatomine (Rhodnius prolixus Stål) infected with
EPFs (Metarhizium spp. and B. bassiana) was greater when they were
infected with T. cruzi if compared to pathogen-free insects (Garcia et al.,
2016). On the contrary, the survival of Glossina fuscipes Newstead
infected with the M. anisopliae was lower when infected with
T. congolense then pathogen-free ones (Wamiti et al., 2018). Therefore,
the ability of EPFs to control the vector competence of mosquitoes or
ticks needs to be carefully evaluated before proposing the EPF as a BCA.
Indeed, if the parasite (e.g., T. cruzi) confers resistance to the insect
against the EPFs, the use of these fungi as a BCA could spur toward the
selection for vector competence (Garcia et al., 2016). For the reasons
above, future studies are needed to shed light on the EPFs potential for
reducing VBD transmission. In addition, it should be also determined
whether the selection of resistance to EPFs could have side consequences
on mosquito and tick competence for selected pathogens as well as on
arthropod vector behavior (see also Benelli, 2020).
2.3. EPF formulations: in vitro and in vivo studies against mosquitoes and
ticks
The use of EPFs as BCAs usually requires the isolation of the
microorganism strain in pure culture and the evaluation of its efficacy
through in vitro bioassays, followed by pilot tests under field conditions.
Most studies on EPFs against mosquitoes and ticks focused on in vitro
laboratory bioassays; most of them were carried out testing B. bassiana
and Metarhizium spp. as BCAs (Tables 1–3). The most frequent formu­
lation for in vitro studies is a water conidial suspension, and the modes of
application are by using contact and immersion tests. Few studies were
done in field and/or semi-field conditions, where EPF conidia were
usually formulated as water solutions or oil-based formulations and
applied with spraying methods (Table 4).
Immersion tests were employed for studying the EPF efficacy on
mosquito larvae, while contact, inoculation, or spray methods were used
when tested against adults (Tables 1 and 2). Regarding ticks, an im­
mersion test for 3 min led to good results on adults, while the spray
method was more useful to test the EPF efficacy on larval instars
(Table 3). The spray method on animals or in the environment was the
most commonly method used in field trials (Table 4). Conidia concen­
tration higher than 1–5 × 106 were useful to kill mosquitoes and ticks
and the lethal concentration (LC) and the lethal time (LT) varied ac­
cording to the mosquito or tick life stages (Tables 1–3). Mosquito larval
instars are more susceptible, and the LT was generally <7 days regard­
less the tested mosquito species. On the contrary, the LT for adult
mosquitoes was >10 days. For ticks, the LT was generally higher (>10
days) than those estimated for mosquitoes and unfed tick larvae
(LT>7days) were more susceptible (Tables 1–3). Despite the encour­
aging results obtained in vitro, the commercial availability of EPF-based
products is still lagging due to low virulence and inconsistent field
performances (Table 4). This is mainly due to the slow mortality rate of
infected pests, high EPFs doses required in the field and the environ­
mental factors influencing the vitality of EPFs (Abdul Qayyum et al.,
2021). To overcome these limits, different EPF formulations were pro­
posed to obtain BCA formulations with the high insecticidal or acaricidal
efficacy, resistance to environmental stressors, and persistence in the
environment.
With the advances in genetic recombination and transformation
techniques, transgenic mosquito-killing pathogenic fungi were con­
structed and tested in laboratory. In particular, the expression of
endogenous (e.g., cuticle-degrading enzymes) or exogenous genes (e.g.,
toxic peptides from arthropod predators/other arthropod pathogens) in
EPFs could provide excellent opportunities to enhance the field efficacy
and cost-effectiveness of EPF-based products (Shen et al., 2020; Vidhate
et al., 2022). For example, a cytolytic protein from Bacillus thuringiensis
(Cyt2Ba) with specificity to Culex spp., Aedes spp. and Anopheles spp.
larvae was expressed in B. bassiana to improve its insecticidal efficacy
against mosquitoes (Table 1). A neurotoxin targeting voltage gated so­
dium (Nav) channel was inserted into the genome of M. anisopliae using
the Metarhizium collagen-like protein (MCL1) promoter, resulting in
nine-fold increased toxicity against Aedes aegypti L. adults (Table 2).
Finally, it has been demonstrated that the virulence of EPFs might be
enhanced by the addition of sub-lethal doses of insecticides in the
formulation (Table 2). In particular, the synergism of EPFs with the
insecticides is because EPFs increase the susceptibility of
insecticide-resistant mosquitoes versus the active ingredients to which
they had developed resistance, most likely due to the reduction of the
insecticide-detoxifying enzymes following EPF infections (Farenhorst
et al., 2009; Knols et al., 2010). These findings might open the way to
propose a further integrated approach to successfully manage arthropod
vector populations.
3. Conventional entomopathogenic fungi formulations and
delivering: strengths and weaknesses
Nowadays, more than 170 commercial products based on EPFs are
available; most of them are formulated with B. bassiana and M. anisopliae
(Islam et al., 2021). Despite more than 700 species established as
insect-pathogenic fungi (Khachatourians and Qazi, 2008), the
commercialization of these products still requires greater improvements
to make them competitive against chemical pesticides (Rangel et al.,
2008). In particular, the EPFs slow killing activities (i.e., usually 2–3
weeks vs. 2–3 h for most chemical pesticides) reduces their commer­
cialization. To boost viability and virulence of EPFs, different

6
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Table 3
Effectiveness of different entomopathogenic fungi (EPF) formulations for the control of ticks: in vitro studies in literature of the last 10 years.
Fungi Ticks Stage EPF formulations Mode of Mortality Notes References
somministration (%)/time;Lethal
Time (LT)

Beauveria Dermacentor Larvae Water conidial Spray 41–64%/21days Beauveria ERL836 Sullivan et al., 2020
bassiana albopictus suspension with 0.02% (granular) was more
organosilicone virulent than B. bassiana
surfactant: 108 conidia/ GHA BotaniGard®
ml
Haemaphysalis Eggs Water conidial CT x 3 min Hatchability 0% Zhendong et al., 2019
longicornis suspension with 0.1%
Tween 80: 1 × 107
conidia/ml
Larvae IT x 3 min 100%/15 days
(U)
Nymphs 100%/20 days
(U)
Adults
(EF)
Haemaphysalis Nymphs Water conidial IT x 30 s 92%/21 days Ren et al., 2016
quinghaiensis suspension with 0.05%
Tween 80:
109conidia/ml
Rhipicephalus Larvae Water conidial Spray 100%/7 days* ICIPE 279 strain Murigu et al., 2016
decoloratus (U) suspension with 0.05% LT50 3.0–4.1 originated from soil.
Triton X-100: 1 × 109 days Amitraz-susceptible
conidia/ml larvae were more
Oil conidial emulsion sensitive to treatment
with 15% canola oil than amitraz-resistant
larvae.
Rhipicephalus Eggs Water conidial IT x 3 min Hatchability: Perinotto et al., 2012
microplus suspension with 0.1% 49.5%
Tween 80: 108 conidia/
ml
Larvae Water conidial IT x 1–3 min IT: <40–99% Conidia showed a better Bernardo et al., 2018;
suspension with Spray /14–20 days activity than blastospore. Fernández-Salas et al., 2019;
0.01–0.1% Tween 80: Spray: 91–93%/ Spray method yielded Perinotto et al., 2012; Zeina
2.0 × 107–108 conidia/ 12 days better results than IT and Laing, 2022
ml; Acaricidal susceptible
Oil conidial emulsion larvae were more EPF
with 15% avocado oil: susceptible than resistant
108 conidia/ml one.
Adults Water conidial IT x 30 s-10 min IT: 63–100%/ Blastospore showed a Bernardo et al., 2018;
(EF) suspension with 7–14 days better activity than Monteiro et al., 2013;
0.01–0.1% Tween 80: Hatchability: conidia. Perinotto et al., 2012; Sun
2.0 × 107–109 conidia/ 38% et al., 2013; Tunholi-Alves
ml Eggs production et al., 2016; Zeina and
Ballista® o Break-thru® index: 12.6% Laing, 2022; Zeina et al.,
Baubassil® 2022
Rhipicephalus Larvae Water conidial IT x 3 min E 98.7%/15 days On unfed larvae, the Cafarchia et al., 2015
sanguineous suspension with 0.1% U 100%/15 days water suspension was
Tween 80: 1–5 × more effective than
107conidia/ml against engorged ones.
Nymphs E 85%/20 days On unfed nymphs, the Cafarchia et al., 2015
U 87.5%/20 days water suspension was
more effective than in
engorged ones, but the
time of activity is less on
engorged nymphs (85%/
10 days)
Adults E 100%/15 days On engorged adults, the Cafarchia et al., 2015
U 85%/20 days water suspension was
more effective and faster
in explicating its
bioactivity than on unfed
ones
Metarhizium Amblyomma Adults Water conidia IT x 5 min 100%/7 days Garcia et al., 2018
anisopliae parvum suspension with 0.1% (E9)
Tween 80
1 × 109 conidia/ml
Dermacentor Larvae Water conidial Spray 100%/6 days Sullivan et al., 2020
albipictus suspension 0.02%
organosilicone
surfactant: 108 conidia/
ml
Nymphs IT x 30 s Lee et al., 2021
(continued on next page)

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Table 3 (continued )
Fungi Ticks Stage EPF formulations Mode of Mortality Notes References
somministration (%)/time;Lethal
Time (LT)

Haemaphysalis Water conidial IT: >90%/30

longicornis suspension with 0.03% days
siloxane solution: 1 ×
108 conidia/ml
Ixodes ricinus Larvae Water conidial CT x 7 weeks 96.8%/7 weeks Wassermann et al., 2016
suspension with liquid
medium: 1 × 107
conidia/cm2
Rhipicephalus Larvae Water with 0.05% Spray 69.2% With ICIPE7 strain. Murigu et al., 2016
decoloratus Triton X-100 conidial Amitraz-resistant larvae
suspension were more sensitive to
treatment than amitraz-
susceptible larvae with all
except one strain. Testing
two strains, the mortality
was 100% in both
Rhipicephalus Larvae Water conidial IT x 1–3–5 min IT: 77–100%/ Blastospore showed a Bernardo et al., 2018;
microplus suspension with 0.1%− CT x 21 days 14–21 days. better activity than Fernández-Salas et al., 2019;
0.01% Tween 80 or CT 42.9%/21 conidia. Immersion test Quinelato et al., 2012;
Triton X-100 at 0.02% days. yielded better results than Webster et al., 2018
2.0 × 107- 1.2 × 108 the contact test.
conidia/ml
Adults Water conidial IT x 1–3 min IT:71–100%/14 Blastospore showed a Bernardo et al., 2018;
suspension with 0.01%− Spray (on the soil days better activity than Fernández-Salas et al., 2019;
0.1%Tween 80; 10% surface) Spray: conidia. Mineral oil Marciano et al., 2020, 2021
mineral or 10% soybean 75.78–100%/21 emulsions yielded better
oil conidial suspension days. results than vegetable oil
1.0 × 107–1.3 × 108 emulsions.
conidia/ml
Rhipicephalus Eggs Water conidial Spray Spray: 30.22%/ Contact test achieved Luz et al., 2016; Nur Izzati
sanguineous suspension with CT 30 days better mortality rates over et al., 2021
0.1–0.2% Tween 80: CT: the spray test. Vegetable
1.7 × 106–1.0 × 107 Water 90%/14.9 oil emulsions were more
conidia/ml. days; effective than water
Vegetable oil 3.8 × 104 Oil 90%/14.6 suspension.
conidia/ml days.
Larvae Oil solution 1% D- IT x 5 s Water Oil emulsions yielded Prado-Rebolledo et al., 2017
limonene suspension suspension: better results than water
1 × 107 conidia/ml 98.8%/7 days. suspension.
Oil emulsion:
100%/7 days
Adults Water 0.01% Tween 80 CT Water Oil emulsions yielded Alves et al., 2017; Barreto
or mineral oil suspension: better results than water et al., 2016
suspension 33.4%/5 days- suspension.
1 × 107–1 × 108 84.2%/NR
conidia/ml Oil emulsion:
Commercial strains: 3.6%/5 days-
Metarril® SP Organic 98.9%/NR
and Metarril® WP Egg hatchability:
Water: 59.09%
Oil: 13.3%
Metarhizium Dermacentor Larvae Water suspension with Spray 100%/6 days Strain F52 (Met52®EC) Sullivan et al., 2020
brunneum albipictus 0.02% organosilicone
surfactant:
108 conidia/ml
Ixodes scapularis Nymphs Water suspension with CT x 30 s 100%/7 days Spray method achieved Bharadwaj and Stafford,
0.02% Tween 80: from CT x 3/30/300 better toxicity rates over 2012
7.4 × 103 to 7.4 × 109 min other methods.
conidia/ml Spray x 3/30/
300 min
Rhipicephalus Eggs Water suspension with CT Hatchability of M. brunneum activity was Samish et al., 2014
annulatus 0.01% Triton X-100: 1 × eggs: better in summer over the
108 conidia/ml Summer 6.1% winter.
Winter 0.0%
Metarhizium Ixodes ricinus Nymphs 106blastospores/g CT 97.3–100%/10 Lorenz et al., 2020
pemphigi in beads with starch or days
chitin as additive
Metarhizium Rhipicephalus Larvae Water conidia or Spray Conidia Conidia showed a better Bernardo et al., 2018;
robertsii microplus blastospore suspensions IT x 1 min 81.8–99%/13–14 activity than blastospores. Iwanicki et al., 2018
with 0.01–0.02% Tween days
80: Blastospores
1.0–5.0 × 107 conidia/ <40–87.2%/
ml 13–14 days
(continued on next page)

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Table 3 (continued )
Fungi Ticks Stage EPF formulations Mode of Mortality Notes References
somministration (%)/time;Lethal
Time (LT)

1.0 × 106 − 1.0 × 107

blastospores/ml
Adults Water suspension with IT x 1 min IT: Blastospore Blastospores were more Bernardo et al., 2018
(EF) 0.01 Tween 80: 93%/14 days; effective than conidia.
1.0 × 107conidia/ml conidia
1.0 × 107blastospores/ 63–64.8%/8–14
ml days

EF: Engorged female; U: Unfed; IT: Immersion test; CT: Contact test.

formulations and delivery systems have been proposed. In the following studies showed that it may not be durable over time because of oil ab­
paragraphs, the most common EPF formulations and their delivery sorption by plant tissues with consequent loss of EPF protection (Mas­
methods are presented, discussing their strengths and weaknesses. carin and Jaronski, 2016).
Among other proposed EPF formulations, granules have drawn
attention mainly to control pests populating soil and plants. In this case,
3.1. Conventional formulations of EPFs
EPFs are produced on solid substrates, (e.g., grains such as rice, corn and
millet), then they are dried and mixed with inert materials (e.g., clay
The term “formulation” of biopesticides, including microbial BCAs,
minerals and ground plant residues) to form granules of controlled and
defines their processing with inert materials to induce advantageous
uniform size and with the active ingredient strongly adhered to or
properties (Kala et al., 2020). The aim is to obtain qualitatively high,
incorporated into them (Faria and Wraight, 2007). This kind of formu­
safe, and effective products, improving storage, delivery, and virulence
lation is usually dispersed in the soil in a dry form. It is relatively easy to
of the active ingredient or BCA (Luz et al., 2012; Lacey et al., 2015). In
produce and to apply, but biotic and abiotic factors play a crucial role on
general, EPF formulations contain fungal spores and propagules, inert
their effectiveness. In fact, when dispersed in the soil, granules can be
material to deliver them, and additives to protect and to improve the
moved by wind and rain, so it is not possible to achieve a uniform
EPF persistence and functionality (Skinner et al., 2014). Wettable
application.
powder formulations (i.e., a powder dispersed in water and applied as a
Despite numerous commercial EPF-based products are already
suspension) are the most common and ready to apply. These formula­
available on the market (e.g. Supplementary Data Table S1), they are
tions usually contain 50–80% of active components (i.e., technical
still more expensive than chemicals (e.g., for mosquitoes: 45 € /Kg for
powder), 15–45% filler, 1–10% dispersant, and 3–5% surfactant
B. bassiana-based pesticide vs. 24 €/Kg for cypermethrin- and
(Table 5). These kinds of formulations require several additives for being
tetramethrin-based products). This difference in costs is justified both
effective without any negative impact on the production and the
for the higher production costs of EPF-based bioinsecticide and for the
persistence of EPFs. For example, the aerial conidia of B. bassiana and
high registration costs of new products, eventually limiting the re­
M. anisopliae are highly hydrophobic, so their suspension in water is not
searches and commercialization of new products at competitive prices.
possible without a wetting agent (Bidochka et al., 1995). Therefore,
In addition, EPF formulations usually require a higher frequency of
surfactants are usually added in the EPF formulations in order to reduce
application to improve EPF persistence in field conditions. The mass
the surface tension of water and enhance the contact with target surfaces
production of EPFs remains critical to obtain EPF conidia or propagules
(i.e., arthropod exoskeleton, plant surfaces, etc.), which are usually
resistant or tolerant to the environmental stresses. However, technical
hydrophobic. However, care must be taken when selecting surfactants
solutions have been recently proposed to increase EPF survival in the
because they tend to be lethal for fungi, due to their lipophilicity
field and to reduce the number of applications and the volume of bio­
(Jackson et al., 2010). In addition, dispersants are added to disperse the
insecticide for arthropod control. Conidia encapsulation is one of the
finely ground particles in water (Zimdahl, 1993).
strategies explored to reduce the environmental effects of UV light,
The above formulations are usually applied using a standard insec­
temperature, and related desiccation of EPFs (John et al., 2011). Cap­
ticidal/acaricidal spray or hydraulic applicators or by drenching the soil.
sules are produced through the microencapsulation process by adding
Recently, oil miscible concentrate formulations are becoming popular.
polymers to conventional EPF formulations. There are different conidial
Oils (e.g., mineral, paraffin, vegetable, corn and cottonseed oils) favor
encapsulation methods (Vemmer and Patel, 2013), which can be carried
wetting the waxy lipophilic or hydrophobic surface of most arthropods,
out mainly using natural polymers. Up to their preparation, capsules are
thus assisting in the penetration and disruption of a waxy layer of host
dehydrated for storage and then rehydrated for spraying application.
cuticle by spore adhesion and stimulation of spore germination. In
Even if different studies have proven the efficacy of encapsulation
addition, oils are added to formulations to improve the shelf life of EPF-
techniques in protecting conidia and enhancing EPF pathogenicity, their
based insecticides and acaricides, even at low humidity, and to increase
field efficacy still needs to be investigated (Rodrigues et al., 2016).
their efficiency in field because they improve resistance of fungi to solar
Additionally, since the delivery method of capsules is inundative, high
radiation most likely due to UV absorption (Mascarin and Jaronski,
conidia concentration (i.e., 5 × 1012–5 × 1013 ha− 1) are usually
2016). Overall, the oil EPF formulations (EPF-oil) display a greater
necessary for pest/vector control, thus making encapsulated EPFs less
infectivity than conventional water-based suspensions of dry conidia
cost-effective than synthetic pesticides (Mascarin and Jaronski, 2016).
(Bateman and Alves, 2000). The EPF-oil can be emulsified with water
This finding suggests a role of the delivery methods in influencing the
and may contain other additives to improve EPF properties.
cost of EPF-based products. It has been demonstrated that the infective
The additive has no toxic effect on plants and related products
period of fungal BCAs was several days or weeks when randomly
because they are mainly composed of natural substances. The most
sprayed in agriculture (Pilz et al., 2011) or months or even years (Milner
prominent oil formulation is the invert emulsion (water-in-oil type)
et al., 2010) if they were delivered in the environments using soil
where water is homogenized with oil at elevated speed so that water
inoculation systems. In addition, the persistence and efficacy of fungal
droplets will be surrounded by oil droplets in the final emulsion. This
BCAs is also affected by substrates (Acharya et al., 2015). Beauveria
formulation protects water from evaporation in dry storage condition,
bassiana formulations applied on soil in laboratory conditions present a
which is useful for conidial germination (Batta, 2016). Although the
higher viability (up to 4–5 months), if compared to that registered when
above-mentioned formulation can provide several advantages, recent

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Table 4
Effectiveness of entomopathogenic fungi (EPF) Beauveria bassiana and Metarhizium spp. for mosquito and tick control: field and semi-field trials over the last 10 years.
Arthropod Mosquito or Fungi Infected Semi-field/ EPF formulation Mode of EPF activity/ Notes References
target tick species stage field somministration time
conditions

Mosquitoes Aedes aegypti Metarhizium Adults Semi-field Water Black clothes in 48–64%/5 Paula et al.,
anisopliae condition: suspension with pet traps was days 2018
Beauveria black clothes Tween 80: 1 × placed in each
bassiana were 108 conidia/ml room with fifty
suspended in female
PET traps and mosquitoes.
female
mosquitoes
were released
into each room
after 5 days.
Metarhizium Adults Semi-field Oil suspension: Five black cloths A reduction Better results Paula et al.,
anisopliae conditions: 1 × 109 conidia/ impregnated with in survival of can be achieved 2013a
(ESALQ 818) rooms ml the suspension female adding
simulating (final A. aegypti of imidacloprid
intra-domicile concentration 1 42% over the (IMI) to the
environment × 108 conidia/ control. impregnation,
and containing cm2). with a maximum
3 wick feeders reduction in
with 30 ml survival of
sucrose A. aegypti female
of 50% over the
control using
ESALQ 818 + 10
ppm IMI
Anopheles Metarhizium Larvae Semi-field Oil formulation: Sprayable Reduction in Tests were Seye et al.,
gambiae anisopliae conditions: 5.3 × 106 method: using a adult repeated in rainy 2013
trials pools spores/ml hand sprayer. emergence of and dry seasons,
with larger about 97% adult emergence
containers with respect rates (%) did not
covered with to the significantly
netting cage control/8 differ.
days
Anopheles Metarhizium Adults Semi-field Water mineral In huts with two Survival was Mnyone
arabiensis anisopliae condition: oils conidial volunteers. reduced by et al., 2012
Anopheles IP46 experimental suspension: 39–57%
stephensi Beauveria huts 1 × 109 conidia/ A. stephensi
bassiana designated to ml Ma: 100%/34
193–825 represent local days
housing Bb: 100%/37
Trial 1: eave days
netting A. arabiensis
Trial 2: eave Bb: 100%/26
curtain days
Trial 3: eave
curtains and
panels
Trial 4: eave
baffles
Trial 5: bed
net strips
Culex Beauveria Adults Field Water Sprayed on the 100%/ Bb and Ma Popko et al.,
qinquefasciatus bassiana conditions: in suspension: vertical walls of 17.3–20.4 activity was 2018
(BotaniGard underground 1.3 × 1011 each USDS site days better in the
22WP dry storm drain conidia/m2 and filter paper in spring over the
powder); systems a plastic vial autumn
Metarhizium (USDS) wall housed in plastic
anisopliae and filter trays suspended
(Met52 EC paper from USDS
suspension) ladders.
Ticks Haemaphysalis Metarhizium Nymphs Semi-field: Suspension Sprayed on the Ma Lee et al.,
longicornis anisopliae application on with 0.03% soil. 80%/7 days; 2019b
the soil siloxane 100%/14
solution: 1 × days.
108 conidia/ml
Hyalomma Beauveria Adults Field Water Sprayable In spring: A diluted González
lusitanicum bassiana conditions: suspension of method: products reduction in solution of B. b et al., 2016
rabbits commercial sprayed into tick achieved better
burrows products: burrow entrances abundance of results rather
(Spain) 2.43×108 using a mist 78.63% on than a
conidia/ml blower sprayer. day +30 and concentrated
63.28% on one.
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Table 4 (continued )
Arthropod Mosquito or Fungi Infected Semi-field/ EPF formulation Mode of EPF activity/ Notes References
target tick species stage field somministration time
conditions

day +60 The treatment

In summer: had less impact
reduction in against
tick Rhipicephalus
abundance of pusillus,
35.7% on day Haemaphysalis
+30 and hispanica, Ixodes
29.01% on ventalloi,
day +60 Dermacentor
marginatus.
Ixodes ricinus Metarhizium Larvae and Semi-field Blastospore Foliar treatment 92% larval Metarhizium Wassermann
anisopliae nymphs conditions: suspension:107 with the mortality strain used et al., 2016
plastic blastospores/ suspension. over 57% originates from a
containers cm2 mortality of dead tick from
half-filled with contro l /3 the field,
earth and months. resulting in
embedded 94% higher
half-way into mortality of infectiousness
the ground nymphs over
30% of
control /3
months.
Ixodes Metarhizium NR Field, home Conidial Sprayed on home Bharadwaj
scapularis brunneum sites suspension sites. 50%/1 week et al., 2012
oil-based 0%/2 weeks
formulation
+0.05%
nootkatone: 2.8
× 109 conidia/
m2
Rhipicephalus Metarhizium Adults Semi-field Conidial Spayed on soil Mortality:s Samish et al.,
(Boophilus) brunneum (engorged condition: soil suspension: surface. ummer: 93%/ 2014
annulatus females), surface in distillated 1 week;w
Eggs water 0.01% inter 62.2%/
with Triton X- 6 weeks.
100: 1 × 108 Hatchability
conidia/ml of eggs:s
ummer 6.1%;
w
inter 0.0%
Rhipicephalus Metarhizium Larvae Field: on cattle Water Applied on Ma 100%/7 Amitraz- Murigu et al.,
decoloratus anisopliae (unfed) suspension with cattle/once a days LT50 resistant larvae 2016
Beauveria 0.05% Triton X- week x 4 weeks 2.6–4.2 days were more
bassiana 100 and 15% Bb 100%/7 sensitive to
canola oil: 1 × days LT50 treatment than
109 conidia/ml 3.0–4.1 days amitraz-
susceptible
larvae, except
for a strain.
Rhipicephalus Beauveria Adults Field Water Sprayable Mortality of Tofiño Rivera
(Boophilus) bassiana (EF) conditions: suspension: 1 × method: directly treated ticks et al., 2018
microplus (Baubassil ®) Cebuine cows 106 conidia/ml sprayed on tick- 84.8%/5
infested animals. weeks
Rhipicephalus Beauveria Adults Field Oil formulation: Sprayable Mortality of Egg mass, Zeina et al.,
microplus bassiana conditions, 108 conidia/ml method: directly treated ticks hatching 2022
Farm sprayed on tick- ranged from percentage, egg
infested cows. 13 to 38%/4 production
days index,
nutritional index
and
reproductive
index were
reduced
Rhipicephalus Metarhizium Larvae Semi-field Two granular Manual An efficacy of Without Marciano
microplus robertsii (IP conditions: formulations: spreading of 38.4% for 25 reapplying the et al., 2021
146) soil in grass Blastospores granules over the kg/ha BLS fungal
pots (BLS)- granular surface of granules / 8 formulations to
formulation: previously days; grass pots, the
2.5 × 106 BLS/g irrigated soil. An efficacy of relative efficacy
Microsclerotia 64.6% for 50 was reduced on
(MS)-granular kg/ha MS the 2nd
formulation: granules / 8 evaluation
1.25×104 MS/g days. period (176
days)
(continued on next page)

11
C. Cafarchia et al. Acta Tropica 234 (2022) 106627

Table 4 (continued )
Arthropod Mosquito or Fungi Infected Semi-field/ EPF formulation Mode of EPF activity/ Notes References
target tick species stage field somministration time
conditions

After a 2nd
application of
fungal granules
(218 days), the
efficacy
increased again
but it was lower
than the 1st
application due
to the climate
change (summer
vs winter)
Rhipicephalus Metarhizium Larvae, Field: on Conidial Sprayed onto Mortality: Rot et al.,
sanguineus brunneum Nymphs, vertebrate suspension: vertebrates host. Larvae 49%; 2013
Adults host in sterile Nymphs 79%;
distilled water Adults
with 0.01% 25.3%;
Triton X-100 Triton 51.5%;
:1 × 108 Mineral oil
conidia/ml; 92.7%;
Spray Starch-
formulation: sucrose 85%;
-Triton X-100 Skim milk
− 10% mineral 38.3%.
oil
-mixture of 2%
starch and 2%
sucrose
− 1%skim milk

NR: not reported; Bb: Beauveria bassiana; Ma: Metarhizium anisopliae.

Table 5
Strengths and weaknesses of conventional entomopathogenic fungi (EPF) formulations.
EPF formulations Preparation Advantages Drawbacks

Dry Granules EPF grown on solid substrates, dried and mixed with Easy to produce and apply Very susceptible to biotic and abiotic factors.
formulations inert materials to form granules No uniform application achieved
Baits Similar to granules, however the employed substrate No repellent effect on arthropods Selected additives variable for each target
is attractive to target pest/vector species.
Detailed knowledge about the biology, ecology
and behavior of the arthropod target is needed
Liquid Wettable Usually 50–80% technical powder, 15–45% filler, Possibility to protect conidia and Not easy to prepare.
formulations powders 1–10% dispersant and 3–5% surfactant, then mixed improve their effectiveness. Dispersants and surfactants may negatively
with water Use of standard application affect EPF performances.
methods In water suspension, conidia can germinate
and lose infectivity after only 24 h
Oil Mineral oil or methylated seed oil, but also vegetable Improve shelf life and viability In some cases, the protection provided by oils is
formulations oils added to the formulation under conditions of low humidity. not durable over time
Improve resistance to solar
radiation and temperature.

Greater infectivity
No toxic effects

applied on wood or concrete (i.e., from 1 week to 2–3 months) (Acharya
et al., 2015).
3.2. Conventional delivery methods of EPFs
The selection of a proper application method depends on the kind of
formulation used on the arthropod targets (Montesinos and Bonaterra,
2019). Furthermore, the application method itself influences the treat­
ment effectiveness because it is responsible of spore distribution in
agricultural and urban/peri‑urban settings. Weather and environmental
conditions must be properly controlled to ensure survival and germi­
nation of conidia (Skinner et al., 2014). To date, foliar sprays, treatment
of soils through granular formulations, dipping of plant roots and the
EPF transmission by indirect vectors, are the most proposed delivery
methods to control arthropod pests (Baverstock et al., 2009). Foliar
spraying is a simple and well-known technique largely used to apply
chemical pesticides (Preininger et al., 2018). Correct droplet size,
coverage, and the uniformity of the application are essential to guar­
antee the effectiveness of this type of delivery method for the arthropod
control (Dorr et al., 2015).
The soil treatment through granular formulations is particularly
useful for controlling ticks inhabiting the soil (95% of their life cycle)
when not actively feeding on hosts (Mascarin and Jaronski, 2016).
Although this method can be used only with pests and vectors popu­
lating soils or arthropods spending a long part of their life cycle in the
soil, it presents several advantages. In this case, abiotic factors have a
minor impact on EPFs. For example, at the soil level there is higher
humidity, which enhances conidia germination and mycelia growth.

12
C. Cafarchia et al.
Moreover, EPFs in the soil are less affected from solar radiation and
temperature since soil can moderate temperature, avoiding fluctuation.
Nevertheless, the presence of other fungi in the soil can negatively affect
the EPFs persistence and effectiveness (Sinha et al., 2016).
Other kinds of application methods, such as dipping of plant roots
and the transmission by indirect vectors, are not commonly employed
since they can be not properly controlled in term of amount of conidia
transported and of specificity for the targeted arthropods (Bamisope
et al., 2018).
Despite the efficacy of different formulations and delivery methods
of EPFs was demonstrated, the selectivity of the treatment was not
achieved until now.
To overcome the above discussed limitations, EPFs can be delivered
by using lure and kill approach, relying to pheromones, kairomones,
and/or food baits to attract arthropod pests and vectors (Bextine and
Thorvilson, 2002). For many insect species, this approach is valid and
can be used to deliver both synthetic and eco-friendly insecticides
(Benelli et al., 2022). However, even lure and kill approaches have
demonstrated effectiveness against bloodsucking arthropods as
mosquitoes or ticks (Traore et al., 2020; Fraser et al., 2021; Gow­
rishankar et al., 2021), their long last efficiency in the environment
needs to be better explored. Furthermore, several of these baits are
formulated by using chemical polymers (e.g., polyacrylamide gels),
which are not biodegradable (Buczkowski et al., 2014).
Currently, controlled-release systems (CRS) are another option to
protect conidia and release them in a controlled and more effective way
without recurring to the feeding cues, so without the need to ingest the
microbial BCA thus saving its action by contact. CRS have been already
employed to deliver chemical pesticides such as organophosphates
(Kolaczinski and Curtis, 2004; Ahmad and Mahdi, 2018; Fouet and
Kamdem, 2019; Tay et al., 2020). They are based on absorbing materials
such as synthetic polymers-based hydrogels shaped in capsules. How­
ever, these methods are very effective for small synthetic molecules but
they are not useful for conidia having bigger size and cannot be released
in a controlled way (Neri-Badang and Chakraborty, 2019).
3.3. Innovative EPF formulations and delivery methods
Despite numerous commercial EPF-based products are already
available on the market, they are still more expensive than chemicals.
Apart from the cost related to the choice of inert material and additives,
the mass production of EPFs remains a critical step to obtain conidia/
propagules resistant or tolerant to the environmental stresses, and to
favor new delivery approaches (Baker et al., 2020). Recently new
eco-friendly formultions and delivery methods have been proposed, as
well as more cost-effective strategies for IPM/IVM. Additionally, the
integration of EPFs into a precision control methods further reduces
their impact on non-target insects/organisms.
Among them, “assisted autodissemination” is an innovative way of
EPF delivery that can be considered as part of a “push-pull” strategy
(Gaugler et al., 2012). Target arthropods are contaminated with EPFs in
infected sites, then infected pests can disperse EPFs directly to other
pests through contact and indirectly via conidia deposited on their
breeding site (Scholte et al., 2004). Since EPFs can self-replicate, they
can infect entire arthropod populations when contaminating individuals
returning to their breeding sites, thus leading to a cost reduction,
because a wide infection can occur even if the rate of fungal inoculum to
hosts is low (Baverstock et al., 2009). Of note, assisted autodissemina­
tion is usually more efficient if the infection of arthropods is facilitated
by using particular EPF formulations or by using “traps”.
Among EPF formulations, the employment of “electrostatically
charged powders” for EPF delivery is considered an efficient system to
improve conidia uptake by arthropods visiting contaminated baits and
the transmission within individuals of the same species, because
arthropod cuticle can accumulate electrical charges (McGonigle and
Jackson, 2002). To date, the most studied carrier used to formulate
13
Acta Tropica 234 (2022) 106627
charged powders is carnauba wax, a vegetable wax obtained from the
leaves of a palm tree native to Brazil, because of its dielectric charac­
teristic (Rumbos and Athanassiou, 2017; Muniz et al., 2020).
Although the use of the above formulation has reported many ad­
vantages in term of efficacy of EPF infections and dissemination, the
search of the more suitable site for EPF contamination limits its
employment. In recent years, studies are focused on the use of “traps”
that can recall target arthropods to the site of EPF infection (Guimapi
et al., 2020). Although research and the market are increasingly
focusing on low environmental impact solutions, to date, most of the
traps used in insect pest management are based on sex pheromones and
are mainly produced using synthetic and not-biodegradable/renewable
polymers for their structure (e.g. polyethylene) and for the preparation
of the pesticide release system (e.g., polyacrylamide gels, acrylate
resins) (Lucchi et al., 2018; Benelli et al., 2019; Carlos et al., 2022; Friuli
et al., 2022; Ricciardi et al., 2022).
To make this strategy eco-friendlier, the use of biomaterials which
can mimic natural characteristics of target insect habitats or breeding
site was proposed as strategy to delivery EPFs (Friuli et al., 2022). This
system is useful for extending the active period of the microbial BCA, but
also for reducing the starting EPF conidia concentration to obtain a
cost-competitive system to chemical control. Recently, a precision pest
management approach called biomimetic lure and kill (BLK) has been
proposed (Friuli et al., 2022). This system is based on biocompatible
biopolymers and biomimetics principles, already widely employed in
the medical field (Langer and Vacanti, 1993; Friuli et al., 2021b). In BLK,
a biocompatible biomaterial is exploited to host a natural bioinsecticide
(Benelli et al., 2014; Batista et al., 2017; Friuli et al., 2021a) and, at the
same time, engineered to reproduce a comfortable environment for a
particular arthropod behavior (e.g., feeding, and resting sites selection)
by imitating the natural conditions (biomimetic lure) to attract arthro­
pods. Consequently, it has a lure and kill effect without necessarily using
chemical pesticides and attractants. Biomaterial-based hydrogels are
biocompatible, and their parameters such as water content, rheological
and mechanical properties can be easily adjusted and adequate to the
target arthropod species (Demitri et al., 2008; Ropek and Kulikowski,
2009; Sannino et al., 2009, 2010; Chang and Zhang, 2011). Such
approach may be further considered for field use of EPFs to obtain a
cost-effective control target system to manage bloodsucking arthropods
with special references to mosquitoes and ticks.
4. Conclusions and future challenges
Our literature analysis outlined that many studies conducted over
the last decade had provided solid evidence that EPFs can represent a
next-generation tool to manage mosquito and tick populations as sig­
nificant complement/substitute of conventional chemical pesticides.
Nevertheless, useful guidelines and evaluation schemes are still needed
to grant clearance for wider use and application of novel technologies
based on these biological agents. From a technical point of view, several
hurdles need to be cleared. At present, EPFs such as B. bassiana and
M. anisopliae cover an important position for their easy delivery, as well
as their ability to infect both soft- and hard-bodied arthropods by direct
penetration of the host cuticle (Shahid et al., 2012). Thus, unlike other
microbial BCAs, such as bacteria and viruses, they can be used to
manage not only pests and vectors with chewing mouthparts, but also
blood-sucking arthropods, such as mosquitoes, without the need to
penetrate in the target species by ingestion (Scholte et al., 2007; Shahid
et al., 2012; Wang and Feng, 2014). The properties of EPFs under lab­
oratory conditions showed excellent and encouraging results with high
level of mortality on tested mosquitoes or ticks, even if the LT was higher
than those registered by using conventional pesticides. The ability of
EPF to self-regenerate and disseminate could result in exceeding the
limits due to the time required to cause mortality in infected arthropods.
In addition, their role in controlling VBDs also support their usefulness
as BCAs. However, since studies on the ability of EPFs to control vector
C. Cafarchia et al.
competence are scant, in our opinion it is imperative that their role in
controlling vector borne diseases needs to be clearly explored before
proposing the EPFs as a bio-controller of a specific species, to exclude
collateral consequences for mosquito/ticks’ competence. Additionally,
since EPFs do not trigger resistance in targeted arthropods (Benelli et al.,
2016a), they can be considered to manage arthropod populations
resistant to pesticides or might be employed in association to pesticide
sublethal doses in integrated control systems, thus resulting in a
reduction of pesticides in the environment as well as on animal and
human health. In this framework, further research on EPF-based nano­
formulations is also required Amerasan et al., 2016; Athanassiou et al.,
2018; Santos et al., 2021a, 2021b).
Based on the literature herein discussed, the viability, infectivity,
and persistence of fungal spores under field conditions are still inade­
quate. It will be useful to develop long-lasting formulations that can
remain viable and infect/kill target species for at least 6 months after
application. For this reason, the delivery methods need to be optimized
to maximize exposure of potential hosts both in- and outdoors with
minimal usage of spores. To date, the most used application methods are
sprayable techniques using wettable powders or oil formulations, which
currently suffer from many problems related to the survival of spores in
the environment and loss of their virulence due to abiotic factors. Even if
numerous strategies have been studied to improve the persistence of
EPFs in the field, and their efficacy on target hosts, the employment of
biopolymers seems to be a good option to match delivery and persistent
of EPFs at the same time. The employment of new lure and kill ap­
proaches through biopolymers seems to be useful to improve the de­
livery of EPFs but it will be better optimized for each target pest species.
Thus, the employment of better-optimized lure and kill approaches
through biopolymers will be useful to obtain cost-competitive, safety
and eco-friendly pest control tools in the future.
Funding
This research received no external funding.
CRediT authorship contribution statement
Claudia Cafarchia: Conceptualization, Writing – original draft, Data
curation, Supervision, Writing – review & editing. Rebecca Pellegrino:
Investigation, Writing – original draft. Valentina Romano: Writing –
original draft. Marco Friuli: Writing – original draft, Investigation, Data
curation. Christian Demitri: Supervision, Data curation. Marco Pombi:
Writing – review & editing. Giovanni Benelli: Conceptualization, Su­
pervision, Writing – review & editing. Domenico Otranto: Supervision,
Writing – review & editing.
Declaration of Competing Interest
The authors declare no competing interests.
Data availability
No data was used for the research described in the article.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.actatropica.2022.106627.
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