Impact of UV-radiation in entomopathogens in aquatic environments remains little investigated. The present study reports on the effect of UV-A on the larvicidal activity of Leptolegnia chapmanii zoospores in Aedes aegypti; on the production of zoospores in larvae killed by the pathogen and then exposed to UV-A; and on the activity of these zoospores against healthy larvae. Whereas the virulence of free zoospores in A. aegypti larvae was affected by a UV-A exposure time longer than 10. min, production of zoospores in larvae and their virulence were not hampered at a maximal 8. h exposure of dead larvae to UV-A. Findings suggest that dead larvae and zoosporangia provide a certain protection to zoospores against UV-A and emphasize the susceptibility of free encysted zoospores to such radiation.Centro de Estudios Parasitológicos y de Vectore

Fernandes, Éverton K. K.

García, Juan José

Luz, Christian

López Lastra, Claudia Cristina

Marreto, Ricardo N.

Rueda Páramo, Manuel Enrique

Journal of Invertebrate Pathology

English

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Journal of Invertebrate Pathology 130 (2015) 133–135Contents lists available at ScienceDirect
Journal of Invertebrate Pathology
journal homepage: www.elsevier .com/ locate/ j ipShort CommunicationEffect of ultraviolet-A radiation on the production of Leptolegnia
chapmanii (Saprolegniales: Saprolegniaceae) zoospores on dead
Aedes aegypti (Diptera: Culicidae) larvae and their larvicidal activityhttp://dx.doi.org/10.1016/j.jip.2015.08.002
0022-2011/ 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding author at: DMIPP, IPTSP, UFG, CP 131, 74001-970 Goiânia, GO,
Brazil.
E-mail address: wchrisluz@hotmail.com (C. Luz).Manuel E. Rueda Páramo a,b, Claudia C. López Lastra b, Juan J. García b, Éverton K.K. Fernandes a,
Ricardo N. Marreto c, Christian Luz a,⇑
a Instituto de Patologia Tropical e Saúde Pública (IPTSP), Universidade Federal de Goiás, Goiânia (UFG), GO, Brazil
bCentro de Estudios Parasitológicos y de Vectores (CEPAVE), Universidad Nacional de La Plata-CONICET, La Plata, Buenos Aires, Argentina
c Faculdade de Farmácia, UFG, Goiânia, GO, Brazila r t i c l e i n f o
Article history:
Received 14 January 2015
Revised 3 August 2015
Accepted 6 August 2015
Available online 7 August 2015
Keywords:
Water molds
Oomycetes
Entomopathogens
Mosquitoes
UV-A radiationa b s t r a c t
Impact of UV-radiation in entomopathogens in aquatic environments remains little investigated. The pre-
sent study reports on the effect of UV-A on the larvicidal activity of Leptolegnia chapmanii zoospores in
Aedes aegypti; on the production of zoospores in larvae killed by the pathogen and then exposed to
UV-A; and on the activity of these zoospores against healthy larvae. Whereas the virulence of free
zoospores in A. aegypti larvae was affected by a UV-A exposure time longer than 10 min, production of
zoospores in larvae and their virulence were not hampered at a maximal 8 h exposure of dead larvae
to UV-A. Findings suggest that dead larvae and zoosporangia provide a certain protection to zoospores
against UV-A and emphasize the susceptibility of free encysted zoospores to such radiation.
 2015 Elsevier Inc. All rights reserved.1. Introduction
Entomopathogens face challenging and complex biotic and abi-
otic stress scenarios in their natural environments. Sunlight, tem-
perature and humidity are among the most critical factors that
affect the persistence of these microorganisms (Benz, 1987).
Solar radiation consists of visible light, infrared and ultraviolet
(UV) radiation. UV radiation is divided into UV-A (315–400 nm),
UV-B (280–315 nm) and UV-C (100–280 nm), and its deadly and
particularly mutagenic power in terrestrial and aquatic organisms
is attenuated during its passage through the atmosphere.
Penetration of solar radiation into aquatic ecosystems depends
on the concentration of dissolved or particulate material, and any
harmful impact on organisms is related to the total dose (Häder
et al., 2007).
Aedes aegypti is the main vector of virus infections in the tropics.
Aquatic larval stages of this species develop in smaller water col-
lections eventually exposed to sunlight. A successful control of lar-
vae is often hampered by the difficulty to contact all larvae with a
control agent, and by the increasing resistance to syntheticinsecticides (Morrison et al., 2008). Specific microbial insecticides
present a powerful additional option for integrate control strate-
gies. The aquatic oomycete Leptolegnia chapmanii (Saprolegniales)
has recently gained considerable attention for mosquito control
due to its high virulence against larval stages (Scholte et al.,
2004; Pelizza et al., 2011, 2013; Leles et al., 2013). Interest about
the impact of abiotic factors on L. chapmanii has focused on the
influence of temperature, pH and salinity on the infectivity of zoos-
pores (Pelizza et al., 2007), and there is initial evidence about the
impact of solar radiation on the activity of this pathogen against
A. aegypti larvae (Rueda Páramo et al., 2015). A better understand-
ing of the influence of solar radiation on L. chapmanii is highly
important for the development of an innovative biolarvicide based
on this pathogen. The present study reports on the impact of UV-A
radiation on L. chapmanii and its activity against A. aegypti larvae
under laboratory conditions.2. Materials and methods
2.1. Origin, rearing and preparation of A. aegypti
The A. aegypti colony tested here derived from larvae collected
in 2012 in Goiânia, Brazil. The mosquitoes were reared under
134 M.E. Rueda Páramo et al. / Journal of Invertebrate Pathology 130 (2015) 133–135laboratory conditions at the IPTSP-UFG as described by Lima et al.
(2009). Second (L2) and third instar larvae (L3), about 72 h after
molting and fed with small amounts of cat food pellets (Black
Jack; Alisul Alimentos S.A., São Leopoldo, Rio Grande do Sul,
Brazil), were used for the tests.
2.2. Origin of L. chapmanii and preparation of zoospores
L. chapmanii CEP 010 (Collection of Entomopathogenic Fungi of
Insects and other Arthropods, CEPAVE) also deposited as ARSEF
5499 used in the present study was isolated from an Ochlerotatus
albifasciatus larva collected in La Plata, Buenos Aires, Argentina,
in 1996 (López Lastra et al., 1999).
For each repetition, zoospores were produced with 300 L2/L3
previously exposed for 48 h to five dead larvae killed by ARSEF
5499. Zoospores were suspended in 1000 ml of distilled water at
a final 103 zoospores/ml and immediately used in the assays.
2.3. UV-A radiation assays
White plastic containers (25 ml) with 10 ml of zoospore sus-
pension (103 zoospores/ml) at a 1 cm water depth each were
arrayed in eight groups. Each group contained six containers (five
replicates and one control). Containers were then exposed to UV-
A in a photostability Chamber CARONTM 6545-2 (Marietta, OH,
USA) with total irradiance of 31.514 mWm2 at 25 C at increasing
exposure time periods: 0 (control), 0.17, 0.5, 1, 2, 4, 6 and 8 h, that
corresponded to a total dose of 0 (control), 19.29, 56.72, 113.45,
226.90, 453.80, 680.70, 907.60 kJ m2, respectively. The spectral
distribution of the lamps in the Caron photostability chamber is
given in Fig. 1. Spectral irradiance was measured with an Ocean
Optics USB 2000 Spectroradiometer (Dunedin, FL, USA). After irra-
diation, containers were covered completely with aluminum foil
up to 8 h. Subsequently, ten L2/L3 were set in each container and
maintained at 25 C and 12 h photophase. Mortality of larvae was
registered 48 h afterwards. Larvae were not fed during the assays.
Each treatment consisted of three independent repetitions.
In another test series, a single dead L2/L3, that had been
exposed to L. chapmanii zoospores 24 h earlier, was set in a plastic
container (2 ml) with 1.5 ml of distilled water. It was then exposed
to UV-A as mentioned and subsequently transferred to a container
(20 ml) with 10 ml of distilled water and maintained at 25 C and
12 h photophase for 24 h. The concentration of zoospores in each
container was then determined with a Neubauer chamber. Five
healthy L2/L3 were set in each container, and mortality recorded
48 h later.0
200
400
600
800
1000
1200
1400
1600
310 320 330 340 350 360 370 380 390 399
Sp
ec
tra
l i
rr
ad
ia
nc
e 
(m
W
 m
-2
 n
m
-1
)
Wavelength (nm)
Fig. 1. Spectral irradiances of lamps of the photostability chamber CARON 6545-2.
The chamber provided UV-A radiation at a total irradiance of 31.514 mWm2.Mortality data were transformed to arsine square-roots and
analyzed as numbers of zoospores by analysis of variance and
the Student–Newman–Keuls test for multiple comparison (IBM
SPSS 12.0).3. Results
The highest cumulated mortality (93.3%) at a 48 h incubation
time of larvae was obtained with either zoospores that were previ-
ously not exposed to UV-A (positive control) or exposed at a short-
est 10 min to UV-A. Survival of larvae increased significantly
within the next hours of exposure of zoospores to UV-A (0 h and
10 min > 30 min > 1 h up to 8 h; F7,64 = 19.9; P < 0.001), and mor-
tality dropped to a minimal 18.9% at an 8 h exposure (Fig. 2).
There was no reduction at all of larvicidal activity of zoospores in
the first 10 min of exposure to UV-A. Activity declined then to
highest (0.8%) rate of loss of larval mortality per minute of expo-
sure (related to maximal 93.3% attained at 0 and 10 min of expo-
sure) between a 10 and 60 min exposure to UV-A, and dropped
finally to a mean loss of 0.08% per min in the following 7 h (Fig. 2).
In the second test series, the total number of zoospores pro-
duced by L. chapmanii on larvae, 48 h after death and subsequent
exposure to UV-A up to 8 h, varied between 104 zoospores/larva
(control and 30 min of exposure to UV-A) and 5  104 zoospores/
larva (10 min and 8 h of exposure to UV-A). There was no signifi-
cant effect of the time of exposure on the number of zoospores/
larva (F7,16 = 0.6; P = 0.75; Table). Cumulative mortality of new
healthy L3 applied with the zoospores that derived from the previ-
ous tests and incubated for 48 h varied between 49.3% (2 h expo-
sure to UV-A) and 94.7% (10 min exposure to UV-A), and likewise
there was no significant effect of the time of exposure of dead lar-
vae to UV-A (0 up to 8 h) on the mortality caused by zoospores on
new larvae (F7,16 = 1; P = 0.5; Table 1).
In both test series, all control larvae had survived for the same
length of time.4. Discussion
Results clearly showed that free, encysted L. chapmanii zoos-
pores suspended in distilled water are susceptible to UV-A
radiation at 25 C, and that their susceptibility was related to the
time of exposure and corresponding dose. Virulence of zoospores
obviously was not affected by a unique short exposure time upExposure to UV-A (hours)
0 2 4 6 8
C
um
ul
at
ed
 m
or
ta
lit
y 
(%
)
0
20
40
60
80
100
Fig. 2. Cumulative mortality (% ± standard error) of Aedes aegypti third instar larvae
after a 48 h incubation with Leptolegnia chapmanii zoospores previously exposed to
UV-A radiation up to 8 h.
Table 1
Mean number of zoospores (± standard error, SE) produced by Leptolegnia chapmanii
on dead Aedes aegypti third instar larvae after exposure to increasing UV-A radiation
(31.514 mWm2) and cumulative mortality (± SE) of new larvae exposed to radiated
cadavers with zoospores.
Time of exposure (h) UV-A Zoospores/larva Cumulative
Dose (kJ m2) (104)a Mortality (%)b
0 0 1 ± 0.3 86.3 ± 7.3
0.17 19.3 1.5 ± 0.9 94.7 ± 3.5
0.5 56.7 1 ± 0.4 68.7 ± 7.7
1 113.5 1.3 ± 0.5 69.3 ± 16.4
2 226.9 1.1 ± 0.2 49.3 ± 16.2
4 453.8 1.1 ± 0.4 71.3 ± 16.2
6 680.7 1.3 ± 0.6 73.3 ± 13.3
8 907.6 1.5 ± 0.3 84 ± 14.1
F7,16 = 0.6 F7,16 = 1
P = 0.75 P = 0.5
a Values based on individual tests (3 repetitions with 5 replicates each), larvae
were exposed to UV-A, 12 h after death, and the numbers of zoospores were
quantified 36 h after initiating radiation.
b Each dead larva was tested with 5 new L3; mortality presented at a 48 h
exposure at 25 C.
M.E. Rueda Páramo et al. / Journal of Invertebrate Pathology 130 (2015) 133–135 135to 10 min but a longer exposure of zoospores to UV-A instantly
affected their larvicidal activity in A. aegypti.
Further findings suggested that dead larvae in which the oomy-
cete is actively producing zoosporangia and zoospores, provided, to
a certain extent, protection against UV-A radiation and maintained
virulence against the larvae during the test period. This suggests
that a direct application of dead larvae previously infected with
L. chapmanii to disseminate zoospores in breeding sites seems to
be strategy worth being evaluated for biological control of mosqui-
toes with a longer residual effect than zoospores alone.
The outcomes of the present study together with previous
results (Rueda Páramo et al., 2015) corroborate presumptions
about the susceptibility of L. chapmanii zoospores to UV radiation.
However, at this stage nothing is known about any adaptation or
recovery capacities of L. chapmanii after exposure to UV-radiation
or of specific photo-repair mechanisms of DNA damages or oxida-
tive stress caused as was reported for other entomopathogens
(Chelico and Khachatourians, 2008; Rangel et al., 2011; Fang and
St. Leger, 2012).
Females of A. aegypti preferably oviposit in breeding sites pro-
tected from continuous exposure to sunlight (Reiter, 2007).
Depending on the diurnal course of the sun and properties of the
breeding site and its immediate surroundings, the exposure time
to solar radiation and its intensity on the aquatic microbiota will
vary (Häder et al., 2007).
The highest activity of L. chapmanii against A. aegypti can be
expected in breeding sites that are exposed to little or no sunlight
(e.g., tree holes and other shaded habitats). Interestingly, the first
mosquito larva (A. triseriatus) infected with this entomopathogen
was detected in a tree hole (Seymour, 1984). In conclusion, we
emphasize a need to strengthen investigations about the general
impacts of solar radiation and its more specific impacts on the
effectiveness of L. chapmanii in A. aegypti.Acknowledgments
We thank Juscelino Rodrigues, IPTSP, Goiânia, Brazil and Maria
Ines Urrutia, Centro Superior para el Procesamiento de la
Información, La Plata, Argentina, for their technical assistance
and Richard A. Humber for reviewing the manuscript. This study
was funded by the Coordination for the Improvement of Higher
Level -or Education- Personnel (CAPES, MERCOSUR, grant number
PPCP-016/2011), the Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET to support a doctoral fellowship)
and the National Agency for Technology and Science, ANPCyT,
grant number PICT 0622).
References
Benz, G., 1987. Environment. In: Fuxa, J.R., Tanada, Y. (Eds.), Epizootiology of Insect
Diseases. Wiley, New York, pp. 177–203.
Chelico, L., Khachatourians, G.G., 2008. Isolation and characterization of nucleotide
excision repair deficient mutants of the entomopathogenic fungus, Beauveria
bassiana. J. Invertebr. Pathol. 98, 93–100.
Fang, W., St. Leger, R.J., 2012. Enhanced UV resistance and improved killing of
malaria mosquitoes by photolyase transgenic entomopathogenic fungi. PLoS
ONE 7, e43069.
Häder, D.P., Kumar, H.D., Smith, R.C., Worrest, R.C., 2007. Effects of solar UV
radiation on aquatic ecosystems and interactions with climate change.
Photochem. Photobiol. Sci. 6, 267–285.
Leles, R., López Lastra, C.C., Garcia, J.J., Fernandes, É.K.K., Luz, C., 2013. A simple
method for the detection of Leptolegnia chapmanii from infected Aedes aegypti
larvae. Can. J. Microbiol. 59, 425–429.
Lima, W.P., Neto, F.C., Macoris, M.L.G., Zuccari, D.A.P.C., Dibo, M.R., 2009.
Estabelecimento de metodologia para alimentação de Aedes aegypti (Diptera-
Culicidae) em camundongos swiss e avaliação da toxicidade e do efeito residual
do óleo essencial de Tagetes minuta L. (Asteraceae) em populações de Aedes
aegypti. Rev. Soc. Bras. Med. Trop. 42, 638–641.
López Lastra, C.C., Steciow, M.C., García, J.J., 1999. Registro más austral del hongo
Leptolegnia chapmanii (Oomycetes: Saprolegniales) como patógeno de larvas de
mosquitos (Diptera: Culicidae). Rev. Iberoam. Micol. 16, 143–145.
Morrison, A.C., Zielinski-Gutierrez, E., Scott, T.W., Rosenberg, R., 2008. Defining
challenges and proposing solutions for control of the virus vector Aedes aegypti.
PLoS Med. 5, e68.
Pelizza, S.A., López-Lastra, C.C., Becnel, J.J., Bisaro, V., Garcia, J.J., 2007. Effects of
temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour
(Peronosporomycetes) in mosquito larvae. J. Invertebr. Pathol. 96, 133–137.
Pelizza, A.S., Cabello, M.N., Tranchida, M.C., Scorsetti, A.C., Bisaro, V., 2011.
Screening for a culture medium yielding optimal colony growth, zoospore
yield and infectivity of different isolates of Leptolegnia chapmanii (Straminipila:
Peronosporomycetes). Ann. Microbiol. 61, 991–997.
Pelizza, S.A., Scorsetti, A.C., Tranchida, M.C., 2013. The sublethal effects of the
entomopathic fungus Leptolegnia chapmanii on some biological parameters of
the Dengue vector Aedes aegypti. J. Insect Sci. 13, 1–8.
Rangel, D.E.N., Fernandes, É.K.K., Braga, G.U.L., Roberts, D.W., 2011. Visible light
during mycelial growth and conidiation of Metarhizium robertsii produces
conidia with increased stress tolerance. FEMS Microbiol. Lett. 315, 81–86.
Reiter, P., 2007. Oviposition, dispersal, and survival in Aedes aegypti: implications
for the efficacy of control strategies. Vector Borne Zoonotic Dis. 7, 261–273.
Rueda Páramo, M.E.R., López Lastra, C.C., García, J.J., 2015. Persistence and
pathogenicity of a native isolate of Leptolegnia chapmanii against Aedes aegypti
larvae in different anthropic environments. Biocontrol Sci. Technol. 25,
238–243.
Scholte, E.J., Knols, B.G.J., Samson, R.A., Takken, W., 2004. Entomopathogenic fungi
for mosquito control: a review. J. Insect Sci. 4, 19.
Seymour, R.L., 1984. Leptolegnia chapmanii, an oomycete pathogen of mosquito
larvae. Mycologia 76, 670–674.


Effect of ultraviolet-A radiation on the production of <i>Leptolegnia chapmanii</i> (Saprolegniales: Saprolegniaceae) zoospores on dead <i>Aedes aegypti</i> (Diptera: Culicidae) larvae and their larvicidal activity

http://sedici.unlp.edu.ar/bitstream/handle/10915/86413/Documento_completo.pdf?sequence=1

Effect of ultraviolet-A radiation on the production of <i>Leptolegnia chapmanii</i> (Saprolegniales: Saprolegniaceae) zoospores on dead <i>Aedes aegypti</i> (Diptera: Culicidae) larvae and their larvicidal activity

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