Journal of the Ameican Mosquito Control Association, 10(3):451-455, 1994
Copyright @ 1994 by the American Mosquito Control Association, Inc.
PILOT SCALE PRODUCTION AND APPLICATION IN
WILDLIFE PONDS OF LAGENIDIUM GIGANTEUM
(OOMYCETES : LAGENIDIALES)
JAMES L. KERWIN,' DEBORAH A. DRITZ'.cND ROBERT K. WASHINO'
ABSTRACT. Lagenidium giganteum, a facultative parasite of mosquito larvae, has recently been
registeredby the U.S. Environmental Protection Agency for operational mosquito control. We report
here the first pilot scale production of the mycelium formulation. Scale-up from 10 to 650 liters was
accomplishedby a proportionate increaseof medium componentsand volume of water. Foaming of the
culture medium had not beenencounteredpreviously, but was a seriousproblem in pilot scaleproduction
due to the very rapid gowth of a large volume of L. giganteum. Addition of an antifoaming agent did
not adversely affect growth, but reduced the ability of the fungus to sporulate. Despite what was effectively
a 100-fold reduction in the desired application rate due to reduced sporulation, L. giganteum infected
sentinel mosquito larvae and reduced field populations for more than 2 months following application.
scalefermentation production ofthe fungus,and
demonstratesthat even suboptimal applications
can result in larval mortality in a wildlife habitat.
A preliminary test was conducted 2 wk before
the field application to insure that the shearforcesgeneratedby the circulation pump usedby the
aircraft to deliver the material to the spray booms
would not damage L. giganteum mycelia. Mycelia grown in 2 l0-liter fermentation batches
was filtered, concentrated in distilled water, and
mailed to the Sacramento-Yolo Mosquito
AbatementDistrict. Sacramento,California. This
material was diluted in water in the hopper of
the plane that was to be used for the field application. The pilot then flew the plane under conditions similar to those to be used during application. At 5-min intervals, for a total of 20 min,
the pilot landed and a small samplewas removed
from the hopper of the aircraft, with no signiflcant loss in viability observedeven after 20 min.
The Z. giganteum mycelium formulation
(USEPA Registration No. 56984-2, American
Type Culture Collection AccessionNo. 52675)
was usedfor thesefield trials. Inoculum for largescalefermentation was grown in l0 liters of medium in l4liter tanks in a 3-tank New Brunswick
ScientificCo. Labroferm FS-314unit using slight
modifications of the yeastextract-basedmedium
<iescribedby Kerwin and Washino (1988). The
inoculum was grown for 60 h, and lE liters was
then asepticallycollectedin a sterile 20liter carboy. This material was transportedto Bainbridge
Island, WA, and used to inoculate 650 liters of
the medium described above in a l,0OOJitercapacity custom-built fermentor.
The agitation rate was 200 rpm, which, due to
the length of the propellors and geometry of the
tank, was equivalent to ca. 350 rpm in our stan' Department of Botany AJ-30, University of Wash- dard lO-liter fermentation tanks. In the 1,000liter fermentor, the fungus was aerated initially
ington.Seattle,WA 98195.
2 Department of Entomology, University of Califor- at a rate of 3,000 cclmin, but this rate has to be
gadually cut back to ca. 2,OOOcclmin ca' 15 h
CA95616.
nia.Davis.
Lagenidium giganteum (Oomycetes:Lagenidiales), a facultative fungal parasite of mosquito
larvae, is the only biological control agent approaching operational use in mosquito control.
The infective stagesofthe fungusare biflagellate,
motile zoosporesthat selectivelyadhereto larval
cuticle. Infection is initiated by mechanical and
enzymatic activity of the encystedzoospore,allowing entry into the larva, with larval death
occurring within 24-72 h. IJpon maturation of
the mycelium, the fungus can reproduce either
sexually or asexually.Asexual reproduction amplifies infection, with a new round of zoospore
releaseoccurring every 24-72h, dependingupon
the mosquito host and environmental conditions. Sexualrgproduction culminatesin the production of oospores,which are dormant propagulescapableof surviving prolongeddesiccation,
environmental extremes, and mechanical abrasion. Thesesporesallow multiyear persistenceof
L. giganteumin somehabitats (Fetter-Laskoand
Washino 1983,Kerwin and Washino 1986).
Three formulations of L. gigantezrn, consisting of various combinations of the sexual and
asexualstages,have beenregisteredwith the U.S.
Environmental Protection Agency (JSEPA RegistrationNos. 56984-1,56984-2,and 56984-3),
with the Department of Health Services,State
ofCalifornia, acting as registrant.This biological
control agent can be produced with inexpensive
medium componentsusing standardstirred tank
fermentation (Kerwin and Washino I 986, I 987),
and, with some precautions, applied by ground
or air with techniquesand equipment currently
used by mosquito control agencies(Kerwin and
Washino 1988). This report summarizes pilot
451
452
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Moseurro CorcrRor Assoqenor
Vou 10,No. 3
after beginningthe fermentation, and then finally using a Transland spray systemwith an air-drivto 1,500 cclmin after 30 h becausethe fungus en pump. Air speed was 95-100 mph and the
was growing so rapidly that it was foaming out swath width was 10.5 m (40 ft.). The uniformity
the top of the fermentor between the agitator and density of mycelial application was checked
gaskets.This happeneddespitethe automatic in- in the following manner. Fungus sprayed from
jection of a liter of antifoam (Mazur, DF 6,000 the plane was collected from 3 passesin a 20K) over a period of 12 h as the fungus initiated cm-diam plastic bucket holding 200 ml water.
This material was returned to the laboratory,
rapid growth that causedexcessivefoaming.
Harvesting of the cells was initiated 60 h after concentrated by filtration through paper filter,
inoculation. The fungus was concentrated and and the collected cells examined microscopicalresuspendedin distilled water in 20Jiter carboys ly. Approximately 40 cells were examined, and
after about a l5-fold concentration. The follow- all appearedundamagedby the application proing day the carboys were surrounded with bags cess.These cells were subsequentlyresuspended
of ice, and transported to Davis, CA, where they in distilled water, and 30 colony-reared Cx. pipienslawae added to the pan. After 3 days there
were stored at l5'C until application.
was
l00o/oinfection of these larvae.
for
test
site
material
treatment
of
the
Sufhcient
Applications of the fungus were made in 3
at an approximate rate of 4.5 x l0ro cells/hawas
transported to an airstrip on Conaway Ranch, separatefields on the Conaway Ranch Conseradjacentto the field sitesto be treated. A sample vancy located several miles northwest of Sacraof this mycelia was kept in the laboratory, and mento, California. The most heavily monitored
germination and virulence to laboratory-reared field, 5l ha (127 acres)in size, was subdivided
in both distilled wa- into 12 plots. Plots I through 5 were treated with
Culex pipiens Linn. assessed
ter and in samplesof water drawn from each of L. giganteum:lplot 6 was a buffer; plots 7 through
5 plots treated with the fungus on the Conaway 9 were treated with the Vectobac@ formulation
Ranch. This was done using a concentration of of Bacillus thuringiensis var. israelensr.sat an effungus about l00x that ofthe field application fective rate of 1.25 liters/ha (l pinVacre); anc
to facilitate quantitative analysisof sporulation. plots I 0 through I 2 servedascontrols. Two other
When mycelium from the material used for fields, 55 ha and 65 ha, were treated with fungus
the field applications was diluted in distilled wa- alone.
Pretreatmentdipping was done in all fields 24ter, all 36 sentinel Cx. pipiens larvae added to
the pan were dead and infected by L. giganteum 48 h prior to application of the control agents,
in 15 h. Larval infection was confirmed by mi- using l0 dips per plot. Aquatic light traps were
croscopic examination. There was no larval in- placed in selectedlocations to monitor aquatic
fection in field-site water after 15 h, but after 3 invertebrates, and 2 sentinel buckets, each conin all of the water sam- taining 50 3rd-instar laboratory-reared Culex
days infection was 1000/o
ples from the field sites;however, when the per- tarsalis Coq.larvae, were placedin eachplot and
centageofcells that releasedzoosporeswas as- in the 2 outlying fields. Nontarget sampling of
sessedafter 72h,610/oof thosein distilled water invertebrates and monitoring of fungal activity
with sentinellarvae was repeatedapproximately
had releasedzoospores,but less than loloofthe
cells suspendedin water from the field sites had every2 wk for 2 months. Sampling ofindigenous
releasedzoospores.This compareswith the usual larvae by personnel from the Sacramento-Yolo
levels ofgreater than 980/oofcells releasingzoo- Mosquito Abatement District was done weekly
spores in distilled water and gxeater than 900/o from August 7 through October 6 using standard
germination in water from comparablefield sites 0.47-liter (l-pint) dippers. Ten dips were taken
when the fungus was grown in the same media in a transectacrossall plots of a given treatment.
in liquid shake culture or l0-liter fermentors. In past studies,we returned all larvae that were
These results suggestedthat the effective field collected from dip samples to the laboratory,
application rate of the fungus was less than Troo where they were held for several days to monitor
of that required for immediate high levels of infection. Becausethe mosquito abatement permosquito control. Subsequentlaboratory eval- sonnel were monitoring on an operational basis
uations using liquid shakecultures confirmed that and had limited time for this study, an alternate
the antifoam agent delayed zoosporogenesisand sampling strategy was used. Larvae sampled with
reduced the percentageof reproductively com- the dippers were pooled as the number of lstplus 2nd-instar, and 3rd- plus 4th-instar larvae.
petent cells.
For field applications, the concentratedfungus This allowed rough estimatesof eggoviposition
was diluted in sufficient water firreviously loaded over the previous 24 days,and survival ofyounger instars to older larvae.
into the hopper ofthe plane) to allow an appli
The laboratory evaluations of sporulation decation rate of l0 liters/ha (ca. I gallon/acre). A
SchweizerAgCat wasusedto apply the mycelium scribed above suggestedthat initial fungal activ-
453
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7-Aug
24-Aug
31-Aug
7'SeP
14-Seo
21-SeP
27-SeP
6-Oct
plots, buffer plot, and plots treated with either
Fig. l. Number oflarvae collected in l0 dips from control
2nd) instars and number of (3rd + 4th)
;;;Gr;(1st'+
"r
reporied
B.t.i. or l-agenidium giganteui.Oit"are
instars. Aulust 7 correiponds to day 22 postapplication'
and 4th instars) larval populations beginning 3
ity in the experimental plots would be minimal'
wk after applications (August 7 sampling date),
senof
2
wk
Tiris was confirmed during the first
with varying degreesof success
tinel monitoringwhen no infection occurreddur- and continuing
3 wk (Fig. l). Culex tarsalls-was
following
the
for
ing the initial monitoring period, "$ 94{.o.""
speciesduring the first part ofthe
dominant
the
(l
inl9o
infection
sentin€l
had
Ligenidiumplot
peri od, andAnophelesfreeborni Aitken
feciion ofsentinel larvae) 2 wk postapplication' sampling
during the second half' Reduction
It was decided to continue monitoringthe treated *us "Uottduttt
of late larval instars by the
abundance
the
of
inof
fields to see if the initial very low levels
pronounced during Sepfection would be amplified as the larval breeding fungus was especially
stressedthat the larval numbe
It
should
tember.
seasoncontinued.
estimate of abundance,
This strategy was successful beginning 4. wk bers are an instantaneous
larvae that were
account
into
take
not
did
and
after application, wh€n L. giganteum infections
would be infected beor
alive,
still
but
infected
plots
treated
test
5
the
of
*ere dbtumented in 3
pupation. Larval abundance figures for the
with the fungus' Sentinel larvae were also in- fore
plots, therefore' are an underesti
gigaiteum
L.
plot,
desfectedin a secondLagenidium'treated
true impact of the fungus on the
the
of
-atiott
ignated the levee field, and in one of the sites
mosquito
plots
away
PoPulation.
4
located
was
which
treated with B./.1.,
A very rough estimate of oviposition vs' surfrom the nearestsite treated with the fungus' The
was obtained by separatingthe abundance
vival
i'
check
8'l'
the
to
possibly
transferred
funguswas
2 younger instars from the 2 older stages'
ofthe
Uy waterfowl, either by adherenc€to feathers and
were up to 8 times more young (lst- and
There
larvae
passage
fungus-infected
of
feet, or by
phe2nd-instar) larvae compared to old instars in the
through the gut following ingestion. This
plots
treated with l. Siganteum d.urile 4 of the
last
the
during
again
-8
nomenon was observed
periods. Given the limitations of this
sampling
sampling period, which was 2 months after funwe suggestthat recycling of
ie"httiqne,
sampling
with
were
infected
when
sentinels
gal application,
probably
impacting the larval
was
lnttgns
ttre
L. giganteumin 2 of the B./.i. plots.
populations.
5
adthe
in
levels
^
infection
Feicent sentinel
Aquatic light trap collections and microscopic
jacent
-days plots treated with l,. giganteum were:-5
of nontarget invertebrates, which
examination
!
5o/o;29
16
days-2
posttreatment-0o/o;
Diptera, Hemiptera, MolColeoptera,
+
included
l0o/o;63 days43 days-5
days-47 + 380/o;
showed no adverse efects
Annelida
and
lusca,
gqb
5).
+
n:
I
SD,
(mean
a
B
on theseorganismsfollowing fungal application'
Dip sampling of the l2 plots suggestedthat-I'
There is substantial precedentfor fermenting
(3rd
instar
late
indigenous
gigaiteum reduced
454
Joumar or rxr AusruceN Mosqurro Covrnor AssocrerroN
filamentous fungi such as penicillium and lspergillus spp. for commercial production of enzymes and pharmaceuticals,and Beauveria bassiana and Metarhizium anisopliae for insect
control. This is also true for various strains of
bacteria including the toxin-producing Bacillus
thuringiensis,which have been grown commercially in large-scalefermentors for several decades.By comparison, oomyceteousfungi have
rarely been cultivated in batcheslarger than one
liter, and there is no information on industrialscaleproduction of this group of organisms.It is
fortunate, therefore, that media developed for
liquid shake and small-scalefermentation production of L. giganteuz (Jaronski et al. 19g3.
Kerwin et al. 1986) appear to be suitable for
larger scaleproduction. The complication in production of this fungus for mosquito control is
that mycelia can readily be produced that are
incapable of either asexual or sexual reproduction. Motile zoosporesmust be produced by one
of these 2 modes of reproduction for larvae to
be infected, so mycelial production p er se cannot
be used to assessthe usefulnessof a given set of
culture protocols.
Current fermentaticn yields for the asexual
stageof L. giganteum are comparable to those
for the microbial insecticide-8.t.i. This stage,then,
would appearto be ideal for large-scaleindustrial
production; however, the problems with the
asexualstageinclude its short shelflife, the need
to keep the mycelium completely hydrated, its
susceptibility to being overwhelmed by contaminating microorganisms following formulation,
Iack of stability under extremesof temperature,
and special handling required to keep the formulated product from becoming anaerobic
(Domnaset al. 1977, 1982;Jaronskiet al. 1983:
Lord and Roberts1985;Su et al. 1986).
There have been attempts to mitigate these
problems by manipulating storageconditions of
agar cultures (Su et al. 1986) or encapsulating
mycelium in calcium alginate (Axtell and Guzman 1987). Although these attempts have had.
some degreeof success,mycelial stability is still
limited to less than ca. 3 months. More importantly, the cost and effort involved with these
formulations are prohibitive in the current mosquito control market.
We were successfulin our first attempt at pilot
scale production of I. giganteum, which is not
expectedexcept for the very simplest fermentations. The one complication we encounteredwas
the adverse effects of the antifoam agent used.
An alternative to using antifoaming agentsis to
increasethe fermentor tank head pressure,and
it is possible that this strategy alone will solve
the foaming problem during future fermentations.
Vou 10,No. 3
The oosporeor sexualstage,unlike the asexual
stage,is a thick-walled spore, resistant to abrasion and desiccation,which can be stored for at
least 7 yearsand still remain viable in either the
laboratory or the field (Fetter-Laskoand Washino 1983, Brey and Remaudiere 1985, Kerwin
and Washino 1988). Low fermentation yields,
which are currently 2 orders of magnitude below
what is necessaryfor commercial viabilitv. are
the major impediment to use of oorpo.ii fo,
operational mosquito control. Optimization of
oospore yields is a major focus of our current
researchefforts.
The major advantageof using this agentrather
than other control methods hasbeenamply demonstratedin this paper.If B.t.i. or any chemical
agent had been applied at less than lolooftheir
recommended rates, mosquito control would
have beennegligible,and the breedingsite would
have had to be immediately retreated. By treating with a microbial agent capable of recycling
and amplifuing the applied material, mistakesin
application due to operator error or equipment
calibration can be compensatedfor by this versatile and adaptable fungus.
This study was coordinated by M. Ferguson
and J. Lyons of the Technology Development
Center,Sacramento,CA, in conjunction with the:
Botany Department, University of Washington,
Seattle;Entomology Department, University of
California, Davis; California Mosquito and Vector Control Association; California Waterfowl
Association;N. Romeo, Bainbridge Island, WA;
ConawayRanch Conservancy;Sacramento-yolo
Mosquito and Vector Control District California Department of Pesticide Registration; California Department of Health Services(the current registrant for this product); and U.S. Fish
and Wildlife Service. This research was supported in part by a grant to J.L.K. from the National Insritutes of Health (Rol AI22gg3); Special Mosquito Augmentation Fund, University
of California; and the Technology Development
Center, Davis, California.
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'situ
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aqutlc
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i
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-lfi"tittg
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..iJs
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n"
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-fe-.rni-t-,
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2'.350-354.
Lagenidium
of
stage
ntal
aeriJ apptication of the ase
associatedwith
[iga"teumto, "ontrol of mosquitoes