Production of microsporidia pathogenic to the Colorado potato beetle (Leptinotarsa decemlineata) in alternate hosts

JOURNAL

OF INVERTEBRATE

PATHOLOGY

44,

166-171(1984)

Production of Microsporidia Pathogenic Beetle (Leptinotarsa decemlineata)

to the Colorado Potato in Alternate Hosts

ZDENGK HOSTOUNSK? Entomological

Institute,

Department 162 00 Praha

of Insect Pathology, Czechoslovak 6, Flemingovo nam. 2, Czechoslovakia

Academy

of Sciences,

Received July 8, 1982; accepted March 16, 1984 The microsporida Nosema gastroideae and N. equestris, which are highly pathogenic for Lephave been successfully produced in some other chrysomelid species, Gastrophysa poand G. viridula. As the principal target host, Leptinotarsa is very susceptible to these pathogens, and death occurs before massive sporulation by the microsporidia. By contrast, the infected larvae of G. polygoni or G. viridula are able to develop until the adult stage when most of the tissues become filled with spores. In addition, the larvae and adults of these species can be reared in the laboratory on Polygonum aviculare and Rumex obtusifolius. These plants have longer vegetative periods and are better sources of food than potato leaves. In both species of Gastrophysa the yields of spores related to unit weight were about five times higher than in Leptinotarsa. In the adults of G. viridula there was up to 4.8 x lo6 spores mggt body weight of N. gastroideae, or 9.1 x lo6 spores mg- ’ of N. equestris. The higher content of microsporidian spores facilitates their purification and isolation. KEY WORDS: Leptinotarsa decemlineata; Microsporidia; Nosema gastroideae; Nosema equestris; spore production; Gastrophysa polygoni; Gastrophysa viridula: Chrysomelidae. tinotarsa, lygoni

INTRODUCTION

Chemical control measures against the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera, Chrysomelidae) used so far have been unable to prevent increased spreading of this dangerous pest. Owing to a lack of information on biotic factors regulating the density of its population both in the area of its origin and new areas of its distribution, it can only be assumed that the natural enemies, mainly pathogens, eventually become adapted to the new host. Therefore, utmost attention should be given to every, even a solitary, finding of its pathogens (Lipa, 1968; Sidor, 1974). While searching for insect pathogens, we isolated several microsporidia which were capable of producing an acute infection in larvae of L. decemlineata (Hostounsky and Weiser, 1973, 1975, 1978, 1980). In contrast to McLaughlin and Bell (1970), who succeeded in producing spores of a microspo-

ridium, Glugea gasti, directly in Anthonemus grandis (both the original and the target host), our target host, L. decemlineata, was too susceptible to an infection with these microsporidia. Larvae died before the parasite could produce large number of spores. Therefore, we had to find alternate hosts for the production of these microsporidian spores. Satisfactory for this purpose were two economically unimportant European species of the genus Gastrophysa (Coleoptera, Chrysomelidae), G. polygoni and G. vivid&a. Infected larvae of these species developed almost normally into adults who survived until almost all tissues suitable for the parasite were filled with spores. The present paper describes methods for the breeding of hosts in the laboratory, infection methods, and measures to prevent contamination of the stock culture. Also being presented are data on the number of spores of Nosema gastroideae and N. equestris obtained from adults of Gastro166

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0 1984 by Academic

Press. Inc.

PRODUCTION

physa in comparison with spores from L. decemIineata. MATERIALS

OF

MICROSPORIDIA

the yield

of

AND METHODS

G. polygoni feeds on Polygonum aviculure (Polygonaceae). It comes to the host

plant from its sites of hibernation when the plant has about 5-7 leaves. G. viridula feeds on Rumex, mainly R. obtusifolius (Polygonaceae). The first adults appear when the plant develops the flowering stem. The breeding stock was reared in plastic, aerated boxes (20 x 10 x 10 cm) on the leaves which were kept fresh in jars tilled with water. The food was changed about two to three times a week. Clusters of eggs for hatching were placed together with a small piece of the leaf in test tubes stoppered with cotton wool. Females of both species laid 30-40 eggs per day. At 2X, the larvae hatched in 4 days, at 15°C they hatched after 10 or more days. Hatched larvae were either transfered to breeding boxes or used in infection experiments. They started to feed in groups on the underside of the leaf while older larvae spread all over the leaf and fed separately on it. At 25°C and 12 hr photoperiodic illumination, the larva completed its development within 8-9 days. In the field, larvae pupate in the soil, but in the laboratory pupation occurred on the bottom of the breeding box. The pupal stage lasted 5-6 days. The adults started to feed immediately after emergence, and, at the age of 4 days, the adults started to mate. At this age new breeding pairs were selected for the stock because it is difficult to distinguish the sex of these young individuals on the basis of their externally visible characteristics. Under laboratory conditions, experiments with G. viridula could be started several weeks before these beetles appeared in the field because they can hibernate on potted R. obtusifolius. During the summer, vigorous plants were potted in larger pots (20 cm diameter) and their shoots were cut off. The plants started to grow and, in Sep-

IN

ALTERNATE

HOSTS

167

tember, healthy beetles were placed on them under a cloth. The beetles fed on the plants for a short period and then went into the soil in the pot for hibernation. The soil in the pots was kept moderately moist. During the winter the pots were stored in a sheltered place. In the spring, as soon as R. obtusifolius started to sprout in the field, the pots were transferred to a greenhouse and heavily watered. Several days later the beetles emerged and started to feed. As both G. polygoni and G. viridula collected in the field might be infected with microsporidia, the following measures were taken to prevent the introduction of undesired pathogens to the breeding stock: feces were inspected for intestinal parasites; an infection of other tissues was disclosed with a method suggested by Pasteur (1870) for silkworms. Breeding pairs were kept separately and eggs were collected with a small piece of the leaf and stored at lo-15°C. As soon as an appreciable number of eggs was available, fresh smears of both sexes of beetles were examined with a microscope for the presence of spores. Larvae of both species were exposed to the microsporidia immediately after hatching and consumption of their egg shells. For introducing the pathogen, a purified suspension of microsporidian spores (Hostounsky, 1981) was used. It was given to the larvae directly by employing a microbiological loop, or applied indirectly over the surface of the leaf. The suspension was left to dry and then the leaf was given to the larvae to feed on. Nearly 100% infection was obtained with the first method and 92-97% infection with the second method. Each dose had to be tested before use to avoid either too strong a dose, which may result in death of the larvae, or too weak a dose, which might prolong the life of the adult. As soon as the larvae stopped feeding on either the loop droplet or the treated food, they were placed on the host plant in the breeding box where they were maintained until adult emergence under conditions identical to those used for the

168

ZDENiiK

HOSTOUNSKP

breeding of the stock. After the death of the first individuals, the remaining beetles were starved for 1 or 2 days, and spores were recovered from them by employing the technique of Hostounskg (1981). Spore counts, both in the inoculum and in infected individuals, were made with a Biirker hemocytometer. Infected individuals were weighed and then ground in a glass homogenizer. RESULTS

The two coleopteran species, G. viridula and G. polygoni, can be easily reared in the laboratory. They are good hosts for various microsporidia isolated from different coleopteran species. With regard to the production of spores, the use of Gastrophysa shows several advantages in comparison with the target host of the mentioned Nosema species, which is L. decemlineata. Thus, for example, the infected larvae of Leptinotarsa die before the parasite fully develops and sporulates. The infected larvae of Gastrophysa, on the other hand, develop until the adult stage and the parasite can properly reproduce. Some further advantages for the use of Gastrophysa as a suitable host for the production of spores are revealed by the data in Table 1. It can be observed that the total amount of spores present in the consider-

ably larger larva of Leptinotarsa is only about four times greater than in the relatively small adult Gastrophysa. When related to unit fresh weight, however, the yields of the spores appear to be considerably higher in Gastrophysa. It has been found that N. gastroideae can give, in G. viridula, up to 4.8 x lo6 spores mg-’ while in L. decemlineata it gives only 1.0 x lo6 spores mg - ‘. Similarly, N. equestris can give, in G. viridula, up to 9.1 x lo6 spores mg - ’ while it gives only 1.8 x lo6 spores n-4 -’ in L. decemlineata. This indicates an approximately fivefold better yield of the spores of Nosema in 1 mg of Gastrophysa. Furthermore, it has been determined that female adults of Gastrophysa contain more of the Nosema spores than do the infected males. This is mainly due to the relatively large size of the body, and especially due to considerably larger fat body (heavily infected tissue) in the females. However, a considerable part of the female’s body is occupied by the ovaries. Growth and development of the ovaries are faster in comparison with growth and sporulation of the parasite, although the parasite is occasionally able to invade the oocytes and enter the egg case (N. gastroideae). When related to milligrams of body weight, the content of spores is higher in the males. Thus, for example, N. gastroideae in G. viridula

TABLE PRODUCTIONOF SPORESOF Nosema

Microsporidium N. gastroideae

N.

equestris

Host species

1 AND N. cquestris

gastroideae

Spore yield per specimen (X 10’) Female

IN VARIOUS HOSTS

Number of spores per milligram fresh body weight (x 106)

Stage

Male

Male

Gastrophysa polygoni

Adults

1.7-1.8

2.3-3.1

0.7-0.8

0.5-0.7

Gastrophysa viridula

Adults

2.0-3.7

4.7-6.3

2.6-4.8

1.9-2.5

Leptinotarsa decemlineata

Larvae

Gastrophysa viridula

Adults

Leptinofarsa decemlineata

Larvae

0.6-l .O

6.0-9.0 5.0-7.0

9.0-13.0

10.0-20.0

Female

6.5-9.1

3.5-5.2 1.2-1.8

PRODUCTION

OF MICROSPORIDIA

produces 4.8 x IO6 spores mg - * in adult male, but only 2.5 x lo6 spores mg-’ occur in the body of a female. Similarly, N. equestris in G. viridula gives 9.1 x lo6 spores mg-’ in the male as compared to 5.2 x IO6 spores mg - * in the female. This shows that the number of spores is about twice as great in the male when related to equal size. The development of the parasite was examined microscopically when infected specimens of Gastrophysa were dissected periodically. It appeared that the main progress of the infection took place after adult emergence, during adult life. This explains the observation that, after dissection of old or dead adults, all suitable tissues for the parasite were packed with the spores, i.e., generalized infection. In contrast, dead larvae of L. decemlineata contained only localized foci of spores at the primary sites of infection, mainly at the distal terminations of the tracheae and in the fat body. Further development of the infection here is impossible because the larvae of Leptinotarsa die sooner, before the generalized infection and massive sporulation of the parasite could have occurred. Table 2 shows the developmental times in the host species concerned. In both species of Gastrophysa the development from egg to adult stage lasts approximately 18 days at 25°C. Leptinotarsa needs 37 days to reach the adult stage. This shows that, during the period in which Leptinotarsa is still developing, we can obtain adult Gastrophysa containing all suitable tissues filled with the spores.

IN ALTERNATE

DISCUSSION

The results presented indicate that the use of Gastrophysa as a laboratory species suitable for production of microsporidian spores has certain advantages that merit practical considerations. There are several aspects which favor Gastrophysa over the target host, Leptinotarsa decemlineata. These are briefly discussed below. Some coleopteran species, which appear to be the original hosts of the microsporidia, are small or difficult to breed in the laboratory, have relatively long developmental periods, or show other difficulties which prevent production of large numbers of spores (cf. Otiorrhynchus). The two species of Gastrophysa can easily be reared in the laboratory on their natural food. The convenient food is R. obtusifolius, which is available naturally from the end of April until the first serious winter frost. The cut leaves remain useful for longer periods than potato leaves. By contrast, Leptinotarsa requires fresh potato leaves, which are available for a shorter period (Derridj, 1975). Further advantages of Gastrophysa are that they are very fertile. They complete their life cycle much sooner than Leptinotarsa and produce many generations (in laboratory; 7-8 generations per year) without the necessity of estivation. The successive batches of eggs can be stored for some time at lowered temperatures (lo1YC). In this way it is possible to obtain eggs with reduced or almost arrested em-

TABLE 2 COMPARISONOF DEVELOPMENTAL TIMING IN Gastrophysa polygoni, decimlineata (AT 25°C) Duration of developmental Species G. polygoni G. viridula L. decemlineatab

Egg 4 4

4-5

a Maximum life span in laboratory. b After Dirlbek (1971).

Larva 8

9 20

169

HOSTS

G. viridula,

stages (days)

Pupa

Total

Adult life

6 5 12

18 18 37

29-70 (135)” 2 1-42 (67)

AND Leptinotarsa

Number of eggs (female/day) 28-32 34-38 3.5

170

ZDENBK

HOSTOUNSKk

bryonic development. After transfer to 25°C the development is resumed and large amounts of the synchronized stages for massive infection can be obtained. Moreover, both species of Gastrophysa are quite susceptible to microsporidian infections, including the parasites of insect families other than Chrysomelidae. These, for example, include N. equestris parasitic in members of Curculionidae, and recently it has been found that G. viridula is also a very good host for N. algerae from anopheline mosquitoes (Hostounsky, 1982). The described results show that, at the final stages of infection, Gastrophysa contains a larger number of spores than does Leptinotarsa. These differences are important for the process of spore purification, which proceeds better when the ratio between spores and tissue debris is higher. The method described in this paper does not solve the problem of large-scale production of microsporidian spores for field use, which has been described elsewhere (Hostounsky, 1978). However, the method appears to be very suitable for isolation and reproduction of new microsporidia from the small coleopteran species. The literature includes only one example of reproduction of microsporidian parasites of coleopterans in the laboratory. This example rests with Glugea gasti, which has been reproduced in its original and target host Anthonomus grandis (see McLaughlin and Bell, 1970). Comparison of the infections caused by the three species, i.e., N. gastroideae, N. equestris, and G. gasti, revealed that they all invade essentially identical tissues of the hosts, and all three parasites cause generalized infections at later stages of the infection. In spite of the fact that Anthonomus is considerably larger than any of the Gastrophysa species, the yield of spores related to one specimen is almost the same. Thus, we can see that N. gastroideae in G. polygoni produces an average of 2.2 x 10’ spores (max. 3.2 x lOs), N. equestris in G. viridula produces 1.0 x IO8 (max. 2.0 x 108) spores, and G. gasti

in A. grandis produces 1.0 x lo8 (max. 2.5 x lo*) spores. The relatively rapid killing of L. decemlineata larvae by N. gastroideae and N. equestris is remarkable with respect to the general host-parasite relationships. Both these microsporidian species were originally isolated from living adults which did not differ from the healthy animals (a somewhat strange behavior was observed only in Otiorrhynchus equestris, the original host of N. equestris, which was found sitting on the wall of a house). A similar situation was found with all other microsporidia that have been hitherto isolated from coleopterans. The infection was noticed in the adult stage, eventually with transfer of the parasite to the progeny (Weiser, 1961). Also, a similar situation occurs in A. grandis where maximum numbers of spores of G. gasti have also been obtained from adults (McLaughlin and Bell, 1970). The infected specimens of G. polygoni and G. viriduia also developed and behaved quite normally in these experiments. The adults emerging from the infected larvae died only after their tissues were loaded with the spores. The different fates of the infected L. decemlineata in comparison to the infected Gastrophysa may be due to the different sensitivities of the hosts or, eventually, to special properties of the parasites. The populations of L. decemlineata develop in the newly occupied territory without selective pressure from other pathogens. This proceeds in spite of the fact that such pathogens do exist in the territory. For example, the original host of N. gastroideae, G. polygoni, normally lives on the weeds present in potato cultures and, in addition, it is taxonomically related to L. decemlineata. Exact reasons preventing spontaneous transmission of this and other such parasites to natural populations of L. decemlineata are not known. REFERENCES DERRIDJ,

S. 1975. etude

au champ de I’effet de la

PRODUCTION

OF MICROSPORIDIA

succession des variCtts de pomme de terre: Ackersegen, Bintje et Kennebec (So/unum tuberosum L.) sur la biologie du Doryphore (Leptinotarsa decemlineutu Say.) (Coltopt&re, Chrysomelidae). Ann. Zool. I&ol. Anim., 7, 227-246. DIRLBEK, J. 1971. New findings concerning Colorado potato beetle and the possibilities of protection (In Czech). SI-OVTI, Studijni informace, iada Ochrana rostlin, c. 2, pp. I-51. HO.STOUNSK*, Z. 1978. Successful transmission and multiplication of microsporidians from Coleoptera on a substitute lepidopteran host. J. Protozool.. 25, 37A, No. 110. HOSTOUNSK~, Z. 1981. The utilization of a natural sedimentation and Brownian movement in a concentration and separation of microsporidian spores from insect tissue. J. Znvertebr. Pathol., 38, 431-433. HOSTOUNSK~, Z. 1982. Formation of various morphological types in the spores of Nosema algerue after their oral transfer to Gastrophysa viriduln (Coleoptera). J. Protozool., 29, 504A. No. 123. HOSTOUNSKP, Z., AND WEISER, J. 1973. Nosema gastroideae sp. n. (Nosematidae, Microsporidia) infecting Gustroidea polygoni and Leptinotursa decemlineuta (Coleoptera, Chrysomelidae). Acta Entomol.

Bohemoslov.,

70, 345-350.

HOSTOUNSK~, Z., AND WEISER, J. 1975. Nosema polygrammae sp. n. and PlistophoraJdelis sp. n. (Mi-

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Nosematidae) infecting Polygrammu (Coleoptera, Chrysomelidae) in 0. Spol. Zoo/., 39, 104-l 10. HOSTOUNSK*, Z., AND WEISER, J. 1978. Pleistophora grossa sp. n. (Pleistophoridae, Microsporidia), parasite of Chrysomelid beetles in Yugoslavia. Vest. 6s. Spol. Zool., 42, 112-l 14. HOSTOUNSKL, Z., AND WEISER, J. 1980. A microsporidian infection in Otiorrhynchus equestris (Coleoptera, Curculionidae). V&t. 6. Spol. Zool., 44, 160165. LIPA, J. J. 1968. Nosema leptinotursae sp. n. a microsporidian parasite of the Colorado Potato Beetle, Leptinotarsu decemlineata Say. J. Znvertebr. Puthoi., 10, 111-115. MCLAUGHLIN, R. E., AND BELL, M. R. 1970. Mass production in vivo of two protozoan pathogens, Mattesia grandis and Glugea gasti, of the bollweevil, Anthonomus grandis. J. Znvertebr. Puthol., 16, 84-88. PASTEUR, L. 1870. l&udes sur la maladie des vers g soie. I. 322 pp. Gauthier, Paris. SIDOR, c. 1974. Survey of some results obtained in investigations of diseases of Colorado potato beetle Leptinotarsa decemlineata Say. (In Serbo-Croatian). Agronomski Glusnik, br. 9-12, 537-546. WEISER, J. 1961. Die Mikrosporidien als Parasiten der Insekten. Monogr. Angew. Entomol.. 17, l-149. undecimlineata Cuba. V&t.