Susceptibility of spores of the microsporidian Nosema algerae to sunlight and germicidal ultraviolet radiation

JOURNAL

OF

INVERTEBRATE

Susceptibility

PATHOLOGY

34, 164- 169 (1979)

of Spores of the Microsporidian Sunlight and Germicidal Ultraviolet

Nosema Radiation’

algerae

to

JAMES F. KELLY Department

of Entomology

and Nematology. Florida,

Institute Gainesville,

of Food Floridu

and Agricaltural 32611

Sciences.

University

oj’

AND DARRELL Insects

Affecting

Man

and Animals

W. ANTHONY

Research Laboratory. Gainesville, Florida

U.S. Department 32604

of Agriculture,

SEA,

AR,

Received November 10, 1978 Spores of the microsporidian Nosema algerue exposed to 1, 2. and 4 hr of sunlight and 121 @W/cm2 of germicidal ultraviolet light were fed to first-instar Anopheles albimanus. Twenty days after feeding, the incidence and intensity of infection (spores/mosquito) were recorded from adult mosquitoes. While sunlight-treated spores showed no significant decline in incidence of infection atIer 1-, 2-, and 4-hr exposures, intensity of infection decreased significantly after the 2- and 4-hr exposures. Incidence of infection of mosquitoes fed bactericidal ultraviolet light-treated spores declined 48.2, 76.2, and 99.9% after 1-, 2-, and 8-min exposures, respectively. Measured by intensity of infection, activity of bactericidal light-treated spores decreased 87.2% after 1 min. 91.7% after 2 min. and 99.9% after 8 min. Levels of radiation required to inactivate spores of N. algerae fell within the range reported for other Microsporidia. KEY WORDS: Microsporidia; Nosema algerae; sunlight; ultraviolet: spores, susceptibility to; Anopheles albimanus; biological control.

Laboratory studies (Anthony et al., 1972, INTRODUCTION 1978a, b) have shown that the microsporidThe bactericidal effect of ultraviolet radiian Nosema algerae may be potentially ation in sunlight, known for over a century control agent for (Downes and Blunt, 1877), has long pro- useful as a biological vided bacteriologists with a valuable means Anopheles albimanus. Also, field studies in Panama (Anthony et al., 1978b) demonof sterilization. This same effect has more recently hindered the development of en- strated that N. algerae can be introduced into breeding areas and produce infections tomopathogens for use in biological control programs (Ignoffo and Hostetter, 1977) by in natural populations of A. albimanus. The possibility of future field use of N. algerae causing rapid loss of infectivity. Investigators evaluating Microsporidia as and investigations on improved formulation and application methods have led us to potential microbial insecticides (Maddox, 1973, 1977; Nilova and Strelinkova, 1974; evaluate the susceptibility of this microsporidian to irradiation by sunlight and artifiWilson, 1974; Ignoffo et al., 1977; Kaya, cial ultraviolet light. 1977; Sikorowski and Lashomb, 1977; Teetor and Kramer, 1977) demonstrated in MATERIALS AND METHODS each case that microsDoridian snores share this susceptibility to natural or artificial UV Irradiation Procedures light. Spores of N. algerae (Walter Reed strain) were harvested from adult Heliothis zea. cleaned by a routine method (Kelly and I Institute of Food and Agricultural Sciences, UniKnell, 1979) and stored in pH 7, 0.17 M versity of Florida Agricultural Experiment Station Journal Series No. 1473. standard McIlvaine buffer at 4°C until use. 164 0022-201 l/79/050164-06$01.00/0 Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

IRRADIATION

OF

TABLE INCIDENCE

AND

INTENSITY

OF INFECTION

SUBJECTED

Exposure

TO SOLAR

IN Anopheles

RADIATION

(hr) control)

” Values

followed

165

al‘wwe

1 albimanus EXPOSED TO SPORES OF Nosema (MEANS OF Two REPLICATIONS)

1OOa 1OOa 1OOa 97.5a Ob

4.85 2 0.86a 5.03 2 1.12a 3.81 k 1.23b 2.65 + 0.99~ 0

control) by the same letter

in each column

are not significantly

algerae

Percentage infection” (n = 300)

Spores per mosquito (X 106)” (n = 50)

period

0 (Infected I 2 4 (Noninfected

Nosema

different

from

each other

at 0.05 level.

For exposure to UV light or sunlight 5 x 10” shielded during irradiation were used as spores were added to 10 ml of distilled controls. All exposures to mosquitoes were water and placed in 60 x 15mm plastic made in 60 x 15mm plastic Petri dishes Petri dishes. The dishes containing the containing 5 x lo6 spores in 10 ml of disspores were positioned 4.5 cm from a stan- tilled water. After exposure to the spores the dard germicidal lamp (GE 30T8, maximum larvae were transferred to enamel pans and radiation 240-260 nm).’ Incident radiation reared normally at 28°C. In each of two at this distance was measured at 121 pW/ tests, squashes of three groups of 10 adult cm’ using a Hewlett-Packard radiant flux A. albimanus per treatment were examined detector and meter (Model Nos. 8330A, 20 days after exposure to spores to deter8334A). Exposures were made in the dark mine incidence of infection. The xp test was at 25°C for 1, 2, and 8 min. To avoid layer- used to determine levels of significance. ing and shielding of the spores the suspen- Intensity of infection (spores/mosquito) in sions were stirred on a magnetic stirrer each test was measured from five spore during the exposures. Control spores were suspensions per treatment, each suspension shielded from the UV light. containing 20 homogenized adults of A. alSunlight treatments were made in direct bimanus. Data were analyzed according to sunlight in September and October in the procedures of Dunnett (1955) and Gainesville, Florida, under cloudless skies. Tukey (unpublished manuscript cited by The spores were exposed in uncovered Steel and Torrie (1960)). plastic Petri dishes for periods of 1, 2, and 4 RESULTS AND DISCUSSION hr between 10:00 AM and 2:00 PM. Control Since low doses of N. algerae may cause spores were shielded from the sun in a light infections in A. albimanus, with little sealed box. Temperatures of spore suspen- or no decline in incidence, both incidence sions during sunlight exposures were and intensity of infection were evaluated in 31-32°C. all experiments. The importance of this Assay and Evaluation Procedures latter measurement is shown in Table 1. Newly hatched larvae of A. albimanus Based on incidence of infection alone, no were exposed for 18 hr to spores of N. significant change in infectivity was obalgerae that had been irradiated by artificial served for sunlight-irradiated spores after UV light or sunlight. Spores that were I-, 2-, and 4-hr exposures. However, the intensity of infection declined significantly after 2- and 4-hr exposures to sunlight. L’Mention of a commercial or proprietary product in Although, in this study, measurements of this paper does not constitute an endorsement of this solar radiation in the germicidal range were product by the U.S. Department of Agriculture.

166

KELLY

AND

TABLE INCIDENCE AND IRRADIATED

INTENSITY OF INFECTION IN Anopheles BY ARTIFICIAL ULTRAVIOLET LIGHT

Exposure period (min) 0 (Infected control) 1 2 8 (Noninfected control)

Spores per mosquito (x 106) (n = 50) 3.44 2 0.79 0.45 k 0.37 0.29 2 0.24
ANTHONY

2

albimanus AT 121 pW/cm*

EXPOSED TO SHARES (MEANS OF Two

OF Nosema REPLICATIONS)

algerae

Average percentage decline Spore production (n = 50) 87.2 91.7 99.9

Percentage infection” (n = 300) 96.6 2 2.9a 50.0 2 lO.Ob 23.0 k 7.6~ Od Od

(’ Values followed by the same letter are not significantly different from each other at 0.05 level.

not available, more rapid rates of inactiva-, exposures. Among the factors listed by tion of spores exposed to 121 pW/cm2 of Barker (1968) that control incident solar artificial UV light indicated that this radiaradiation in this range are: natural fluxes in tion was at a higher level than that occurthe thickness of the ozone layer, latitude of ring in sunlight. Incidence of infection di- exposure site (higher radiation levels nearer minished 48.2% after 1 min of UV expothe equator), time of day (maximum penesure, 76.2% after 2 min, and 99.9% after 8 tration between 10:00 AM and 2:00 PM), and min (Table 2). Spore production per mos- time of year (greatest damage possible bequito (intensity of infection) decreased an tween April and September). average of 87.2% after 1 min of bactericidal Comparisons of N. afgerae susceptibility to UV light with that reported for other UV radiation, 91.7% after 2 min, and 99.9% Microsporidia are complicated by the variafter 8 min. ety of media used for exposing spores and a Sunlight resistance of N. algerae rewide range of UV sources, as listed in Table ported here falls within the range reported for other Microsporidia (Table 3): Octo4. Vairimorpha necatrix spores in aqueous sporea muscaedomesticae, 3 hr (Teetor and suspensions were inactivated in 4- 10 min, Kramer, 1977); Vairimorpha necatrix. while spores exposed on bean leaves re3-28+ hr (Maddox, 1977), 24 hr (Kaya, tained some activity after 6 hr (Kaya, 1977). Ignoffo et al. (1977), also studying V. neca1977); Nosema trichoplusiae, 18 hr (Madtrix, predicted a half-life of 2.1 hr when dox, 1977); Nosema upis, 15-51 hr (White, 1919). spores attached to H. zea eggs were exThis range of sunlight susceptibility posed to UV light. Reduction in activity of shown for Microsporidia may not be due spores of Nosema fumiferanae was demonsolely to species or strain differences. As strated after 1 hr of bactericidal UV expo1974). Spores of 0. muswas demonstrated by Maddox (1977), pur- sure (Wilson, ity of spores and the medium used during caedomesticae in aqueous suspension were sunlight exposures greatly affect inactivainactivated in 30 min, while dried spores tion rates. Clean, dried spores of V. neca- lost infectivity after 1.5 min (Teetor and trix exposed on glass slides were the most Kramer, 1977). Nilova and Strelinkova susceptible to sunlight while those mixed (1974) estimated the LD,,, of Pleistophora schubergi and Nosema agrotidis to be with soil were the most resistant (Table 3). Variable inactivation times reported in 60,000 and 100,000 pW/cm2. the cited studies might also reflect differWhile comparisons with prior laboratory ences in incident radiation in the 220- to studies (Tables 3,4) show that N. algerae is 290-nm range during the indicated sunlight similar to other Microsporidia in its sus-

Oct. -Mar.

Maryland

N. apis

N. fumiferanae

Wilson (1974)

Oct. -Mar.

Oct. -Mar.

New York

0. muscaedomesticae

Ontario

Oct.-Mar.

May

Illinois Mississippi

May May

Time of year

Connecticut Illinois

Location bean leaves glass slides corn leaves artificial

mixed with soil on glass slides mixed with soil on cotton leaves

on on on on

Crude aqueous suspension Spores in Petri dishes Spores on cherry leaves

Aqueous suspension

Spores Spores Spores Spores diet Spores Spores Spores Spores

Medium

MICROSP~RIDIA TO SOLARRADIATION

N. heliothis

N. trichoplusiae

V. necatrix V. necatrix

Pathogen

OF

3

Sikorowski and Lashomb (1977) Teetor and Kramer (1977) White (1919)

Kaya (1977) Maddox (1977)

Reference

SUSCEPTIBILITIES

TABLE

in in in in

78 hr 3 hr 4.5 hr 9 hr

100% in 37-51 hr 100% in 15-32 hr 21% in 4 hr, 90% in shr

Some activity after 28 hr 100% in 3 hr 100% in 18 hr Significant decline after 4 hr 100% in 3 hr

100% 100% 100% 100%

Results (% decline in infectivity)

? ii h 2 8

$

E F 2 z

E

N. fumijeranae

6E 30-W germicidal lamp

5500

13.7-1026 5500

N. agrotidis 0. muscaedomesticae

Wilson (1974)

13.7- 1026

P. schubergi

Nilova and Strelinkova (1974) Teetor and Kramer (1977)

Chromato-Vue cc-20 (ultraviolet producers)

V. necatrix

140 (250-260 nm) 1800 (290-400 nm)

Measured radiation (pWlcm2)

IRRADIATION

Kaya (1977)

UV source with peak radiation at 254 nm

Light source

TO ULTRAVIOLET

V. necatrix

Pathogen

OF MICROSPORIDIA

4

Ignoffo et al. (1977)

Reference

SUSCEPTIBILITY

TABLE LAMPS

Aqueous suspension Spores in Petri dishes Aqueous suspension Spores on cherry leaves

Aqueous suspension Spores on Bean leaves Aqueous suspension

H. zea eggs

Medium

BY GERMICIDAL

100% in 30 min 100% in 4 hr

LD,, = 1 pW/cm2 100% in 15 min

100% in 4- 10 min Some activity after 360 min LDs, = 600 pW/cm*

96.3% in 4 hr

Results (% decline in infectivity)

4

; 2 8

k

z F -c

IRRADIATION

ceptibility artificial

to inactivation by sunlight and UV light, the resistance of N. algerue may increase disproportionately in actual field use. Unlike the terrestial habitats of other .Microsporidia discussed here, the natural aquatic environment of spores of N. algerue could afford additional protection against harmful UV radiation. Dissolved organic material, normally associated with A. albimanus breeding sites, would absorb large quantities of solar radiation in the ultraviolet range (Wetzel, 1975) and offer some protection for the spores. ACKNOWLEDGMENTS Technical assistance for this study provided by C. R. Dillard and C. L. Sheffield, Department of Entomology and Nematology, University of Florida, is gratefully acknowledged. Measurements of bactericidal ultraviolet radiation were made possible by J. Stanley of the Insect Attractants, Behavior and Basic Biology Research Laboratory, USDA, Gainesville, Florida.

REFERENCES ANTHONY, D. W., LOTZKAR, M. D., AND AVERY, S. W. 1978a. Fecundity and longevity of Anopheles albimanus exposed at each larval instar to spores of Nosema algerae. Mosquito News, 38, 116- 121. ANTHONY, D. W., SAVAGE, K. E., HAZARD, E. I., AVERY, S. W., BOSTON, M. D., AND OLDACRE, S. W. 1978b. Field tests with Nosema algerae Vavra and Undeen (Microsporidia, Nosematidae) against Anopheles albimanus Wiedemann in Panama. Misc. pub.

Enromol.

Sot.

Amer.,

11, 17-28.

ANTHONY, D. W., SAVAGE, K. E., AND WEIDHAAS, D. E. 1972. Nosematosis: Its effect on Anopheles albimanus Wiedemann and a population model of its relation to malaria transmission. Proc. Helminthol. SOC. Wash., Special Issue 39, 428-433. BARKER, R. E. 1%8. The availability of solar radiation b&w 290 nm and its importance in photomodification of polymers. Photochem. Photobiol., 7, 275 -295.

DOWNES, A., AND BLUNT, T. P. 1877. Researches on the effect of light upon bacteria and other organisms. Proc.

Roy.

Sot.,

26,488-500.

DUNNETT, C. W. 1955. A multiple comparisons pro-

169

OF Nosema olgerae

cedure for comparing several treatments with a control. J. Amer. Statist. Assoc., 50, 1096-1121. IGNOFFO, C. M., AND HOSTETTER, D. L. 1977. Summary statement. In “Environmental Stability of Microbial Insecticides” (B. F. Eldridge, ed.), Vol. 10, pp. 117-119. Misc. Pub. Entomol. Sot. Amer., College Park, Md. IGNOFFO, C. M., HOSTETTER, D. L., SIKOROWSKI, P. P., SUTTER, G., AND BROOKS, W. M. 1977:Inactivation of representative species of entomopathogenic viruses, a bacterium, fungus, and protozoan by an ultraviolet light source. Environ. Entomol., 6, 411-415. KAYA, H. K. 1977. Survival of spores of Vairimorpha ( =Nosema) necatrix (Microsporidae: Nosematidae) exposed to sunlight, ultraviolet radiation and high temperature. J. Znvertebr. Pathol., 30, 192-198. KELLY, J. F., AND KNELL, J. D. 1979. A simple method of cleaning microsporidian spores. J. Znvertebr.’

Pathol.,

33, 352.

MADDOX, J. V. 1973. The persistence of the Microsporida in the environment. Misc. Pub. Entomol. Sot. Amer., 9, 99-104. MADDOX, J. V. 1977. Stability of entomopathogenic Protozoa. Misc. Pub. Entomol. Sot. Amer., 10, 3-19. NILOVA, G. N., AND STRELINKOVA, L. V. 1974. The effect of ultraviolet irradiation on the viability of spores of Pleistophora schubergi and Nosema agrotidis. Parazitologiia, 5, 463-468 (in Russian, English summary). SIKOROWSKI, P. P., AND LASHOMB, J. H. 1977. Effect of sunlight on the infectivity of Nosema heliothidis spores isolated from Heliothis zea. J. Znvertehr. ,Pathol.,

30, 95-96.

STEEL, R. G. D., AND TORRIE, J. H. 1960. “Principles and Procedures of Statistics.” McGraw-Hill, New York. TEETOR, G. E., AND KRAMER, J. P. 1977. Effect of ultraviolet radiation on the microsporidian Octosporea muscaedomesticae with reference to protectants provided by the host Phormia regina. J. Znvertebr.

Pathol.,

30, 348-353.

WETZEL, R. G. 1975. “Limonology.” Saunders, Philadelphia. WHITE, G. F. 1919. Nosema-disease. U.S. Dep. Agr. Bull.,

780,

l-59.

WILSON, G. G. 1974. The effects of temperature and ultraviolet radiation on the infection of Choristoneura fimiferana and Malacosoma pluviale by a microsporidan parasite, Nosema (Perezia) fumiferunae (Thorn.). Canad. J. Zoo/., 52, 59-63.