Carrera, M.V.

Léry, Xavier

Pollet, André

Zeddam, Jean-Louis

Horizon / Pleins textes

Insect Pathogens and Insect Parasitic Nematodes
[OBClwprs Bulletin Vol. 31. 2008
pp.40-43
Evaluation of the per os insecticidal activity of baculoviruses by a
nebulization method
Maria Victoria Carrera I.Z, Jean-Louis Zeddam I, Andre Pollet I, Xavier Lery 2,
Miguel Lopez-Ferber 3
J lRD (UR072), Pontificia Universidad Catolica del Ecuador, Laboratorio de Bioquimicay
Microbiologia molecular, Quito, Ecuador; 2 l RD (UR072), Centre de Recherche, 30380
Saint-Christol-les-Ales, France; J Ecole des Mines, LGEl, 30319, Ales, France
Abstract: Precise evaluation of the biological activity of Baculoviruses by the usual droplet method is
not appropriate for boring insect larvae that remain on plant surfaces for a limited time before
penetrating the substrate. Alternative systems using surface contamination of the substrate (Potter
tower, immersion) are often expensive and/or difficult to implement and are not always sufficiently
reliable because they produce non homogeneous and non reproducible results. A new system was
developed for evaluating precisely the biological activity of several granulovirus isolates on potato
tuber moths. In this system. the spraying of viral suspensions was carried out by a Pulmo-Aide
Sunrise® Compressor/Nebulizer usually used for the treatment of pulmonary infections. This system
delivered small drops by the Venturi effect. PVC tubes directed the aerosol to the potato surface where
it is deposited homogeneously. The system worked well in different locations where the atmospheric
pressures were significantly different. To provide an internal control, we used samples of purified viral
suspensions at appropriate dilutions mixed with Coomassie blue. Spectophotometer measurements of
the optical density of the colorant deposited on the surface after nebulization showed that the
reproducibility of the method was always greater than 95 %. This method is reliable, inexpensive and
easy to use.
Key words: aerosol. nebulization, surface contamination. granulovirus, insect borer
Introduction
The droplet method usually used for the evaluation of the biological activity of baculoviruses
is not appropriate in the case of borers like the potato tuber moths (Lepidoptera; Gelechiidae)
whose mining larvae remain on the infected surface only for a few minutes. So, the biological
activity of the Phthorimaea operculella granulovirus (PhopGV) was generally evaluated by
immersing the eggs (Sporleder et al., 2005) or the potato tubers (Zeddam et al .. 1999) in viral
solutions which contain a certain number of macerated PhopGV-infected larvae (larva-
equivalents). This protocol has since been improved by the use of purified virus granules
instead of larva-equivalents. However, these methods do not allow the precise determination
of the granule concentration present on the potato surface. which could lead to a high
variability between experiments. A more precise technique is the pulverization of purified
granules using a Potter tower. but its use as a routine methodology is expensive and thus not
adapted for emerging countries. A cheap. precise and repeatable method of the evaluation of
biological activity of a virus isolate is required to allow proper comparisons and quality
control. Small droplet generators are widely used in the treatment of pulmonary diseases and
the technology to produce droplets of reproducible size is well developed. We have examined
the possibility of using such devices to deliver precise and repeatable virus doses onto a given
surface.
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Material and methods
Contamination device
Th e co ntami nation device is com posed of a medical co mp resso r/ne bulizer (DeVilbiss Pulmo-
Aide Sunr ise® Compressor/Nebu lize r, model 5650. Sunrise Medi cal. USA ) cou pled to a
co mb ination of 125 mm-diameter PV C tubes co nstituting the tower and the cham ber for
pulv erisation of the vira l suspensions (Fig I). Different constructions were tested by
mod ifying the horizontal and vert ical lengths as well as the pos ition of the de compress ion
hole. The total cos t o f the whole device was less than 200 US$.
Figure 1. Th e nebulizat ion devi ce. A: Nebu lizer . B: Nebulization cham ber. C: Pulv eri zati on
to wer. D: De co mpression hol e
Repeatability and homogeneity ofthe spray
A Coomassie blu e so lutio n (500 mglL) was used to calibrate o ur system. T he neb ul izat ion
cham ber was filled with 1 to 5 ml of this co lorant solut ion. Different volumes were tested to
ass ess whether the system provided cons istent results o ver this range. Fiv e 16 mm -di am eter
plast ic covers lips were randoml y pla ced on the treated surface located at the base of the
ve rtica l P VC tube . T he covers lips were used during the tests to co llec t the co lorant so lutio n
deposited onto a known area and , therefore eva luate the homogen e ity (i. e. the variability of
the quantity of co lo rant amo ng the 5 slipcovers corres po nd ing to a si ng le ap pli cation) and the
amo unt of so lutio n recovered . Afte r 10 to 20 m in o f pulve risation, the coversl ips we re
recovered and dri ed . Fi rs t. the hom ogen eit y o f the drop dis tributio n was eva luated by light
m icroscopy. Th en. the d ried cov ersli ps were was hed with 0,2 ml of water. The optical densi ty
(OD) of thi s so lutio n was measured at 556 nm wav elen gth (corresponding to the maximum
absorp tion of the Coomassie blue) usin g a spec tropho tome ter. Th e OD values we re co mpared
to a standa rd curve . Te n ex pe rime nts we re carried out for each device tested. T he
ho mogeneity of the experiments was estimated by the va riation coeffic ient (VC = Sx!x) of the
OD ob tained for each cov e rslip. The rep eatability was estima ted " by co mpariso n of the
I
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42
different VC obtained in the 10 experiments. A non parametric test of Kruskal-Wallis was
used to compare the deposit of colorant onto the different coversJips for each construction.
These results were compared with those obtained using a Potter tower.
Granulovirus application andyields estimation
Viral bio-assays were performed using purified supensions of the Tunisian isolate of PhopGV
(Taha et al.. 2000). Different purified granule dilutions (corresponding to final deposits of 0,6.
6. 60. 600 and 6000 viral inclusion bodies/mrrr', respectively) were used with or without
colorant to control the quantity of virus sprayed onto the treated surface by the system. For
these controls. coverslips were laid and treated as previously described and the virus
concentration was measured at 00 450 nrn, corresponding to the wavelength at which the
absorbance of the granulovirus solution is the greatest. The quantity of virus was evaluated
using the formula derived from Tchang and Tanada (I 978). modified by Zeddam et al,
(2003): 6.8 x 108 x 00450 x dilution == Number of granules/mf.
Results and discussion
Nebulization device
Various configurations of the device were evaluated by nebulizing a Coomassie solution as a
control of the quality of the deposits. The optimum homogeneity and repeatability were
obtained using the PVC pulverisation device (Fig.l). At Saint-Christol-les-Ales (France, 140
m.a.s.l.). its dimensions were the following: Horizontal length: 385 rnm, Vertical length: 345
mm. Decompression hole: 28 mm in diameter placed at 260 mm above the basis of the device.
The system developed at Ales had to be adapted for use under different atmospheric pressure
conditions. i. e. in Bogota (Colombia. 2600 m.a.s.l.) and Quito (Ecuador, 2800 rn.a.s.l.).
Namely. the place of the decompression hole and the length of the horizontal chamber were
modified for these two latter locations. The hole was enlarged to 28 mm in diameter and
displaced 175 mm above the device base and the length of the horizontal chamber was
reduced to 290 mm.
Repeatability and homogeneity ofthe deposits
In Ales, a 94.0 % homogeneity and a repeatability of 97.5 % G. e. the variability of the
quantity of colorant deposited onto the 5 discs among the 10 repetitions) with variation
coefficients of 0.0599 and 0,0252. respectively were obtained. No difference was observed
between repetitions (Kruskal-Wallis test. Kc == 8.08; P < 0,00. One mg of Coomassie blue
spray resulted in 8.065 ng of blue deposited/mrn', representing a 9 % recovery (for an intemal
diameter of the pulverisation tower of 120 rnrnl.The same protocol applied in Quito. with a
slightly modified device. gave similar results with 94 % homogeneity and 95.8 % repetitivity
with 0.0620 and 0.0419 VC. respectively, for a 10,4 % recovery. We obtained similar results
for nebulized volumes in the range 1 to 5 ml. We carried out a comparison between
pulverisations with a Potter tower and our system. before and after its calibration. Using a
Potter tower a lumpy aspect was observed. indicating a bad dispersal due to a drop size too
large. Light microscopical observations detected a great homogeneity in the sizes of the drops
generated by the nebulization system.
Granulovirus application yields obtained with the device
After calibration with the Coomassie blue solution. the device was tested for its suitability for
uniform application of the granulovirus onto the treated surface. The results were as good as
those obtained with the colorant solution (data not shown). However. the yield was different
a
43
in the case of the PhopGV application. 2.5 ml of viral suspension containing 10'0 granules
nebulized with our protocol allowed us to deposit 10.000 granules/mm", representing a 1.13
% recovery. The difference between the quantities of blue and virus recovered may be due to
a superior loss of virus in the horizontal part of the device. Using the immersion method. we
estimated that only 1 of 106 granules were finally adsorbed onto the potato surface (Carrera et
al .. 2002). Thus, with the same initial quantity of material. the yield of the virus deposited
using the new device would be superior by more than 104 times to the yield given by the
immersion method. which means that a much smaller amount of virus is required to perform
the assays.
The method presented here appears very useful for the realization of standardized per as
infection of potato tuber moth larvae. It is particularly suitable for the precise evaluation of
the biological activity of granuloviruses and was successfully used for this purpose (Carrera et
al .. 2002). This method presents three main advantages: it is cheap. repeatable. and easy to
perform.
This device is presently used in laboratories located in several countries (France. Ecuador.
Colombia and Costa Rica). It gave reliable results as the same lethal concentration values
were obtained for the same viral isolate tested in each of these laboratories. One of the great
advantages is the possibility of using a same calibrated method to compare the virulence of
distinct PhopGV (or other granulovirus) isolates against laboratory colonies of potato moths
maintained in different laboratories.
Acknowledgements
The authors gratefully acknowledge Dr. Darrell R. Stokes (Emory University. USA). Dr.
Stephane Oupas (IRD. France) and Dr. Olivier Dangles (IRD. France) for their comments and
help with the translation of this article.
References
Carrera. M. V.• Zeddam. J-L.. Lery, X.. Pollet. A.. Lopez-Ferber. M. 2002. Evaluation of the
biological activity of Phthorimaea operculella granulovirus. Proc. Assoc. Fran. de Protec.
des Plantes (AFPP). 6th Int. Conf. on Pests in Agricult.• 4th_6th Dec .• Montpellier, France:
447-452.
Chang. P.M. & Tanada, Y. 1978. Serological study on the transmission of a granulosis virus
of the armyworm. Pseudaletia unipuncta (Lepidoptera: Noctuidae), to other lepidopterous
species. J. Invertebr. PathoI. 31: 106-117.
Sporleder. M.. Kroschel, J .. Huber. J.• & Lagnaoui, A. 2005: The granulovirus PoGV in its
host Phthorimaea operculella. Entomol. Exp. Appl. 116: 191-197.
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Zeddam, J.L., Pollet. A; Mangoendiharjo. S.. Ramadham. T.H. & Lopez-Ferber. M. 1999:
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Zeddarn, J.L.. Vasquez Soberon, R.M .• Vargas Ramos, Z. & Lagnaoui, A. 2003: Produce ion
viral y tasas de aplicacion del granulovirus usado para el control biologico de las polillas
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Ra.lf-Udo Ehlers, JOrg Enkerli, Itamar Glazer,
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lOBe wprs BuJletin
Bulletin OILB~srop Vol. 31, 2008
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Organisation Intemationale de Lutte Biologique et Integree contre les Animaux et les Plantes Nuisibles
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