A laboratory evaluation of the microsporidian Vavraia culicis as an agent for mosquito control

JOURNAL

OF

INVERTEBRATE

A Laboratory

PATHOLOGY

37, 117-122 (1981)

Evaluation of the Microsporidian Vavraia Agent for Mosquito Control

JAMES F. KELLY',~DARRELL

W. ANTHONY,ANDWHARLES

culicis

as an

R. DILLARD

Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, University Florida. Gainesville, Florida 32611, and tlnsects Affecting Man and Animals Research Laboratory, Agricultural Research. Science and Education Administration, U.S. Department of Agriculture, Gainesville, Florida 32604

of

Received March 11, 1980 The susceptibility

of the mosquitoes Aedes

aegypti, Aedes taeniorhynchus, Anopheles alquinquefasciatus. Culex salinarius, and Culex tarsalis to infection by the microsporidian culicis was determined. Using 1%hr exposures to 5 x 103, 1 x lo”, 5 x 10d’, and 1 x lo5 spores/ml, C. salinarius. C. tarsalis, and A. albimanus were found to be significantly more susceptible than A. aegypti. The most severe infections were observed in C. salinarius and C. tarsalis, although heavy infections of approximately 1 million spores per adult were recorded at the higher dosages in all species tested except A. aegypti. Production trials indicated that up to 5.4 x 10” spores could be routinely produced in individual corn earworms, Heliothis zea. Inactivation bimanus,

Culex

pipiens Vavraia

of the spores by sunlight was measured by observing the subsequent incidence of infection and spore production in A. albimanus. These two measurements provided an LT,,, of 5.5 and 3.3 hr. respectively. KEY WORDS: Vavraia culicis; Microsporida; Aedes aegypti; Aedes taeniorhynchus; Anopheles albimanus; Culex pipiens quinquefasciatus; Culex salinarius; Culex tarsalis; mosquito; biological control: in vivo production; Heliothis zea; sunlight inactivation.

INTRODUCTION On the basis of its broad host and geographic range, interference with the development of species of Plasmodium, and effects on host longevity and fecundity, the microsporidian Vavraia culicis was selected for further study as a potential agent for microbial control of mosquitoes. In their review of host records of Microsporida in Culicidae, Hazard and Chapman (1977) listed reports of V. culicis infections in 13 species of mosquitoes belonging to the genera Aedes, Anopheles, Culex, and Culiseta. V. culicis has been reported in mosquitoes collected in the Congo (Reynolds, 1966), Czechoslovakia (Weiser, 1946), Italy (Weiser and Colluzi, 1964), and Louisiana (Chapman, 1974). The potential value of V. culicis in malaria control was first proposed by Garnham (1956), who ’ Present address: Department of Zoology, Morrill Center, University of Massachusetts, Amherst, Mass. 01003. Science

suggested possible interference with Plasa phenomenon that was later confirmed (Bano, 1958) with

modium development,

Plasmodium cynomolgi.

In laboratory studies, Reynolds (1970) demonstrated that infections of V. culicis significantly lowered the longevity and fecundity of Culex pipiens fatigans. Reynolds (1972) later conducted field tests with V. culicis on the South Pacific island of Nauru. Although some persistence of infection was detectable 2 years after treatment, no significant reduction in mosquito populations was reported. The feasibility of producing V. culicis in a substitute lepidopteran host, recently demonstrated by Weiser (1978), represents a significant improvement over previous mass production methods, and would greatly facilitate future field tests. All spores of V. culicis produced by Reynolds (1972) for the test in Nauru (1.26 x lOlo spores) were produced in C. p. fatigans over a 4-month period. 117 0022-2011/81/020117-06$01.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

118

KELLY.

ANTHONY.

The present state of development of V. culicis as an agent for biological control, as described in the above studies, indicated to us the need for further research in the following areas: (1) determination of minimum dosages of V. culicis to produce heavy infections in some medically important mosquito hosts: (2) development of dependable, efficient method of mass production of spores; (3) ascertainment of the susceptibility of spores of V. cuEi& to irradiation by sunlight. MATERIALS

AND METHODS

Dosage -effect studies. All mosquitoes used in these tests were obtained from colonies of Aedes aegypti, A. taeniorhynchus, Anopheles albimanus, C. p. quinquefasciatus, C. tarsalis, and C. salinarius,

maintained at the Insects Affecting Man and Animals Research Laboratory in Gainesville. Spores of V. culicis originally obtained from Drs. E. U. Canning (Imperial College of Science and Technology, London) and J. Weiser (Czechoslovak Academy of Sciences, Prague) were propagated in corn earworms, Heliothis zea, and extracted according to the procedures of Kelly and Knell (1979). Spore suspensions were stored in standard McIlivaine buffer, pH 7.0, at 4°C for up to 2 months before use. Newly hatched, first-stage mosquito larvae were placed in lo-ml suspensions of 5 x 103, 1 x 104, 5 x 104, or 1 x lo5 spores/ml of distilled water. These exposures were made at 27°C for 18 hr in 60 x B-mm plastic Petri dishes, using 50 larvae per dish. Larvae were transferred to large plastic cups containing 250 ml of water and reared at 27°C. Four replicates were made of each of the four treated groups and the control group. Twenty days after exposure to spores, 20 adults were randomly selected in each replicate and examined in wet squash preparations to determine the incidence of infection. Spore production (spores/mosquito) was determined from 20 adults per replicate

AND

DILLARD

which were homogenized with a glass, hand-held homogenizer. Regression analysis was performed on data that had been transformed to the natural logarithmic scale. Differences between the regressions were measured using the t comparison, as described by Steel and Torrie (1960). Mass production trials. Eggs of H. zea were obtained from the Southern Grain Insects Research Laboratory, AR, SEA, U.S. Department of Agriculture, Tifton, Georgia. Larvae were removed from artificial media 5 days after hatch and starved for 18 hr in individual plastic cups. Groups of 20 larvae were allowed to feed on drops of spores containing the following dosages: 1 X lo6 spores/ml in 0.1 ml, 1 x lo6 spores/ml in 0.05 ml, 1 x lo5 spores/ml in 0.1 ml. A control group was also starved for 18 hr and then reared normally. After 18 hr, larvae were returned to the media and reared at 27°C. Spore counts were made of homogenates of individual H. zea adults immediately after death. Twenty adults per treatment were used. Data were transformed by dividing each value by 1 x 106. Means of transformed data were compared using Tukey’s w procedure. Susceptibility of spores to sunlight damage. Spore suspensions (1 X lo6 spores/ml) of V. culicis were placed in 60 x 15-mm

plastic Petri dishes to a depth of 1 cm, and exposed to direct sunlight for 1, 2, 3, and 4 hr. All exposures were made between 10 AM and 2 PM on July 11, 1978, in Gainesville, Florida, under cloudless skies. Spores shielded from direct sunlight in a sealed box were used as controls. The temperature of the spore suspensions during the sunlight exposure was 32” ? 1°C. The infectivity of the irradiated and control spores to A. albimanus was tested in the same manner as in the first experiment. Regression analysis was performed on the untransformed incidence of infection data. The means of spores produced per mosquito were divided by 1 x lo6 prior to regression analysis.

STUDIES

ON

RESULTS Dosage -Effect

Studies

At the higher dosages of V. cuficis incidences of infection of 95% or higher were recorded in all the species of mosquitoes tested (Table 1). The severity of infection, measured by the number of spores produced per mosquito, was significantly higher in C. salinarius, C. tarsalis, and A. albimanus than in the least susceptible species, A. aegypti (Table 2, Figs. 1, 2). Infections in A. taeniorhynchus and C. p. quinquefasciatus were at an intermediate level. At the 5 x lo4 and 1 x lo5 dosages, pupal mortality in C. salinarius averaged between 55 and 60%. The dosages, in spores per milliliter, required to produce an infection of 1 million spores in the mosquito species tested were calculated as follows: C. salinarius, 2.21 x 104; C. tarsalis, 2.22 x 104; A. albimanus, 6.12 X 104; C. p. quinquefasciatus, 8.09 X 104; A. taeniorhynchus, 9.01 x 104; A. aegypti, 1.24 x 106. In our experience, production of this number of microsporidian spores in mosquitoes is symptomatic of a heavy infection. In addition, Undeen and Maddox (1973) reported an average of 8.9 x IO5 spores of the microsporidian Nosema algerae in heavily infected adult Anopheles stephensi.

of Spores of V. culicis Spore production (spores/adult H. zea) attained by feeding spores of V. culicis to

Mass

Vavraia

119

culicis

H. zea was as follows (values followed by the same letter are not significantly different at the 0.05 level, n-80): 1 x lo6 (0.05 ml), 5.37 x lo8 + 2.51 x 10s, a; 1 x lo6 (0.1 ml), 4.28 x lo8 2 2.68 x 108, a; 1 x 10” (0.1 ml), 0.831 x lo* + 1.30 x 10s, b; control, Oc. Thus the maximum spore production was observed at the high dosage. Spore production did not change significantly when spores were fed at concentrations of 1 X IO7 spores/ml (1 X lo6 spores in 0.1 ml) or 5 x lo6 spores/ml (1 x lo6 spores in 0.05 ml). Though a slightly better conversion rate was achieved by feeding H. zea 1 x lo5 spores (approximately 2.8 log increase versus approximately 2.5 logs at the 1 x IO6 dosages), infection levels were significantly lower at the 1 x lo5 dosage. Susceptibility of Spores Sunlight Damage

As measured by both intensity (spores/ mosquito) and incidence of infection in A. albimanus, the spores of V. culicis showed a linear decline in activity after exposure to sunlight (Table 3, Fig. 3). Using incidence of infection as a measure of spore viability, the LTso of sunlight-exposed spores was 3.1 hr, and the LT9,, was 6.1 hr. In comparison, on the basis of declines in intensity of infection, the LT,, of sunlight for spores of V. culicis was 1.6 hr and the LT,, was 3.8 hr. DISCUSSION

Production

TABLE INCIDENCE

OF INFECTION

IN MOSQUITOES

Percentage Dosage (X 10’) 0.5 1 5 10 a Results

c. salinarirrs

C. tarsalis

42 2 14 80 + 9 100 100 are means

of four

492 14 44 2 7 100 100 replications.

A. albimanus 61 t 24 IO k 15 98 ” 1 100

of V. culicis to

The microsporidian V. culicis appears to have significant potential for control of 1 EXPOSED

infection

TO SPORES

(N

OF Vavraia

cdicis”

= 1920) of

A. taeniorhynchys 93 It 8 94 -e 8 99t2 100

c. p. quinquefasciatus 562 79 rt 96 k 99 2

11 9 4 2

A. uegypti 83 93 96 99

“_ 2 + 5

12 6 4 2

120

KELLY,

ANTHONY,

AND

TABLE INTENSITY

OF INFECTION

IN MOSQUITOES Spores

DOS&$.? (X

259 746 1970 2630

1 5 10

k ? f -t

165 218 520 1090

403 475 2120 2420

Note. Results

are means of four of mosquitoes followed

k + ? f

175 133 470 1150

358 231 886 1370

replications. by the same letter

+ -t i k

aegypti, infected

1. Comparison of spore Aedes taeniorhynchus, with Vuvraia culicis.

production and Culex

103) (N

OF Vu~~ruiu

199 67 348 230

were

440 360 828 860

not significantly

in Aedes salinarius

k k k 2

culicis

= 96)

A.

c. p.

tueniorhynchus””

some mosquito hosts. Using dosages of 5 x lo4 spores/ml, heavy infections were produced in both C. salinarius and C. tarsalis, with 50-60% mortality in the former species. Using slightly higher dosages (1 x lo5 spores/ml) severe infections were recorded in A. albimanus, C. p. quinquefasciatus, and A. taeniorhynchus. Despite the high incidences of infection recorded for A. aegypti, low spore production indicated only limited susceptibility to development of large numbers of V. culicis. In their studies of the microsporidian parasite of mosquitoes, Nosema algerae, Hazard and Lofgren (1971) reported that, even at high dosages, infections in A. aegypti were always limited to nerve tissue. Light infection levels of V. culicis in A. aegypti may also be related to restricted tissue specificity. Studies of tissue specificity of V. culicis in mosquitoes (Canning, 1957; Weiser and Coluzzi, 1972) have shown that gonadal tis-

FIG.

TO SPORES

(x

A.

0.5

” Species

2 EXPOSED

per mosquito”

olbimanus”

IO’)

DILLARD

150 172 175 177

different

A.

yuinquefuscirrtus’L” 218 354 1020 920

from

each

-t -t k t

other

530 209 100 135

at the 0.05

uepypti” 5 115 268 213

i t 2 L

14 320 150 83

level.

sue is not affected. However, Canning (1957) has pointed out that the destruction of the Malpighian tubules, which is common in infections of V. culicis, may cause toxic excretions to accumulate, and be the source of any harmful effects to the host. Such harmful effects may include lessening the adult longevity and the fecundity of susceptible species of mosquitoes. Using dosages that produced only light infections in our studies (0.6-1.2 x lo4 spores/ml), Reynolds (1972) demonstrated that the net reproduction rate of C. p. fatigans infected with V. culicis declined up to 24%. Reynolds (1972) further noted that reduction in fecundity was directly related to spore dosage, the latter being limited experimentally by the lack of an efficient mass production technique. Further studies, using dosages higher than those available previously, are required to adequately determine the potential of V. culicis in les-

FIG. 2. Comparison of spore production in Anopheles albimanus. Culex pipiens quinquefusciatus, and Culex tarsalis infected with Vavraia culicis.

STUDIES

INCIDENCE

AND

INTENSITY

OF INFECTION

Vuvruiu

culicis

Exposure (hrl 0 (Infected 1 2 3 4 (Noninfected

ON

Vavraia

TABLE

3

IN Anopheles SUBJECTED

control)

1060 610 325 190 18

control) LT,,, = 1.6 hr LT!,,, = 3.3 hr

a Results

ulbimnnus TO SOLAR

Spores/mosquito (x (A’ = 24)

are means

of four

k k ? k k 0

121

culicis

EXPOSED

TO I x IO5 SpoaEsimI

OF

RADIATION”

Percentage infection (N = 480)

loY1

404 174 222 91 13

90 78 60 51 29 LT,,, LT,,,

zk + k + k 0

10 7 15 7 11

= 3.1 hr = 5.5 hr

replications.

sening the longevity

and fecundity of C. p. and of other susceptible hosts. Reduction of longevity and fecundity in insects infected with microsporidia has long been known (Zimmack et al., 1954), and has been demonstrated in studies of N. algerae infecting C. p. fatigans (Reynolds, 1971), A. stephensi (Undeen and Alger, 1974), and A. albimanus (Anthony et al., 1978a). Since there often was no meaningful effect on the immatures in the laboratory studies discussed here, the efficacy of this pathogen seems to depend largely on its effect on the resulting adults. Further tests are necessary, however, to determine the effect of V. culicis on target species in the natural environment where the combined effect of the pathogen and natural stresses may increase larval pathogenicity. quinquefusciatus,

FIG. 3. Incidence of infection and spore production in Anopheles ulbimanus exposed to sunlight-treated spores of Vavraia culicis.

The fe.asibility of using Lepidoptera for mass culturing spores of a microsporidian parasite of mosquitoes was first demonstrated in studies of N. algerae (Undeen and Maddox, 1973; Anthony et al., 1978b). Recently, Weiser (1978) reported that 3 x lOa mature spores of V. culicis were produced in a single specimen of Barathra (= Mamestra) brussicae. While the mean level of spore production in our studies with H. zea was only slightly higher (5.37 x lo8 spores/specimen at the 1 x lo6 spores/ 0. l-ml dosage), single specimens produced up to 1.15 x lo9 spores. Further studies of the susceptibility of other lepidopterans to V. culicis are necessary to determine if more suitable hosts than H. zea and B. brassicae exist. While variable experimental conditions may greatly hinder precise comparisons of the effect of sunlight on microsporidian spores (Kelly and Anthony, 1979), V. culicis appears to be similar to other Microsporida in its susceptibility to solar radiation (White, 1919; Wilson, 1974; Kaya, 1977; Maddox, 1977; Sikorowsky and Lashomb, 1977; Teetor and Kramer, 1977; Kelly and Anthony, 1979). Available evidence (Kelly and Anthony, 1979) indicates that V. culicis may be more susceptible to sunlight damage than N. algerae. This vulnerability to solar radiation must be considered in preparing formulations of V. culicis for future field use. More rapid rates of spore damage re-

KELLY,

122

ANTHONY,

corded by intensity of infection data indicate that this measurement is a more sensitive assay of spore infectivity than is incidence of infection information. This fact was noted earlier in studies with N. ulgerue (Kelly and Anthony, 1979). ACKNOWLEDGMENTS We thank Drs. Elizabeth Canning, Imperial College. London, and Jaroslav Weiser, Czechoslovakian Academy of Science, Prague, for providing us with spores of Vavraia calicis. We also extend our appreciation to Susan Avery, Insects Affecting Man and Animals Research Laboratory. U.S. Department of Agriculture, SEA, AR, Gainesville, Florida. for her assistance in preparing the figures.

REFERENCES ANTHONY, D. W.. LO~ZKAR, M. D.. AND AVERY, S. W. 1978a. Fecundity and longevity of Anopheles albimanus exposed at each larval instar to spores of Nosema algerae. Mosqaito 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 (Microsporida, Nosematidae) against Anopheles albimanas Wiedemann in Panama. Misc. Publ. Entomol. Sot. Amer.. 11, 17-28. BANO, L. 1958. Partial inhibitory effect of Plistophora culicis on the sporogonic cycle of Plasmodium cynomolgi in Anopheles stephensi. Nature (London), 181, 430. CANNING. E. U. 1957. On the occurrence of Plistophora culicis Weiser in Anopheles gambiae. Riv. Malario. 36, 39-50. CHAPMAN, H. C. 1974. Biological control of mosquito larvae. Anna. Rev. Entomol. 19, 33-59. GARNHAM, P. C. C. 1956. Microsporidia in laboratory colonies of Anopheles. Bull. WHO, 15, 845-847. HAZARD, E. I., AND CHAPMAN, H. C. 1977. Microsporidan pathogens of Culicidae (mosquitoes). In “Pathogens of Medically Important Arthropods” (D. W. Roberts and M. A. Strand, eds.). Vol. 55, pp. 63- 107. Bull. WHO (Suppl.). HAZARD, E. I., AND LOFGREN, C. S. 1971. Tissue specificity and systematics of a Nosema in some species of Aedes, Anopheles. and Culex. J. Invertebr. Pathol.. 18, 16-24. KELLY, J. F., AND ANTHONY. D. W. 1979. Susceptibility of spores of the microsporidian Nosema algerae to sunlight and germicidal ultraviolet radiation. J. Invertebr. Pathol. 34, 16& 169. KELLY, J. F., AND KNELL, J. D. 1979. A simple method of cleaning microsporidian spores. J. fnvertebr. Pathol.. 33, 252. KAYA, H. K. 1977. Survival of spores of Variamorpha (= Nosema) necatrix (Microsporidae: Nosematidae) exposed to sunlight, ultraviolet radiation and high temperature. J. Invertebr. Pathol., 30, 192- 198.

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DILLARD

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. Parasitologiia, 5, 463-468. (in Russian, English summary). REYNOLDS. D. G. 1966. Infection ofC.fLltigurzs with a microsporidian. Nature (London), 210, 967. REYNOLDS. D. G. 1970. Laboratory studies of the microsporidian Plistophora calicis (Weiser) infecting Calex pip/ens fatigans Wied. Bull. Entomol. Res.. 6, 339-349. REYNOI.DS. D. G. 1971. Parasitism of Calex jutigans by Nosema stegomyiae. .I. Invrrtebr. Pathol.. 18, 429. REYNOLDS, D. G. 1972. Experimental introduction of a microsporidian into a wild population of Culex pipiens jktigans Wied. Ball. WHO, 46, 807-812. SIKOROWSKI, P. P., and LASHOMB. J. H. 1977. Effect of sunlight on the infectivity of Nosema heliothidis spores isolated from Heliothis