Oral infection of Hylobius pales by Metarrhizium anisopliae

JOURNAL

OF INVERTEBRATE

Oral Infection

PATHOLOGY

27, 377-383 (1976)

of Hylobius HANS

Department

pales by Metarrhizium

anisopliae

G. SCHABEL’

of Forestry, Duke University,

Durham, North Carolina 27706

Received June 19,1975 Microfeeding of Hylobiw pales with as few as 40 conidia of Metarrhizium anisopliae. under conditions that excluded any possibility of integumental contaminations, resulted in high mortality. When larger doses were employed, the insects succumbed faster. Histological sections revealed that the fungus invaded the host from within the buccal cavity. There was no evidence of germination and penetration inside the intestinal tract. Spores retained their viability after passing through the gut. In vitro, conidia mixed with the liquid contents of the midgut germinated within 20 hr. Germination occurred even though both yeasts and bacteria were present in the midgut contents. Fungus-killed Hylobius contained hyphae inside their digestive system, but the intima always remained intact.

INTRODUCTION Infection of insects by bacteria, protozoa, and viruses is generally initiated by ingestion of the pathogen. This route of infection is, however, unusual for most entomopathogenie nematodes and fungi. Only one fungus, the yeast Monosporella unicuspidata, is believed to invade its host, a dipterous larva, exclusively from within the intestinal tract (Keilin, 1920). The needle-shaped ascospores of this fungus supposedly assist in penetrating through the gut wall. With other fungi, there is little or questionable evidence as to what role, if any, oral and/or intestinal infections play. Histological proof is often lacking or unconvincing, because the experimental procedures used by early investigators did not exclude the possibility of integumental infections. Until the present time, no research with Metarrhizium anisopliae has irrefutably proven that invasion of the host can occur from within the digestive system (MtillerKogler, 1965). Evidence in favor of oral, as opposed to intestinal, infection does however exist. Notini and Mathlein (1944) as well as Veen (1966) noticed hyphae of Metarrhizium penetrating proximal portions of insect mouthparts. IPresent address: College of Natural Resources, University of Wisconsin, Stevens Point, Wisconsin 54481.

The present study was part of an overall investigation to determine how M. anisopliae infects the pales weevil, Hylobius pales, one of the most serious insect enemies of pine regenerations in Eastern North America. Peirson (1921) hypothesized that Beauveria bassiana, a hyphomycetous relative of Metarrhizium, probably infects pales weevils through feeding. Preliminary laboratory and field tests indicated a possibility that, provided the timing was right, biological control of this insect might be achieved with Metarrhizium by broadcasting large doses of inoculum over recently harvested pine stands (Walstad and Anderson, 1971). However, the doses found necessary for high mortality were so large as to make control economically prohibitive. Consequently, the question of oral and/or intestinal infection was of particular interest. This mode of infection would both obviate the need for high ambient humidity and permit a considerable reduction of inoculum. Spores would only have to be applied to the material on which the weevils feed. MATERIALS AND METHODS Insects. Freshly cut pine discs, as described by Ciesla and Franklin (1965), were used to trap pales weevils in the Duke Forest, Durham, North Carolina. Prior to use in experiments, the beetles were kept in the laboratory for at least 1 month and all dead in-

377 Copyright o 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

378

HANS

G.

dividuals were removed daily to prevent the spread of naturally developing mycosis. Pales weevil larvae were reared from the egg on meridic diet as formulated by Thomas (1969). Fungus. M. ani~~~~~~ewas isolated from a pales weevil which had died of mycosis shortly after being collected. Stock cultures were maintained on malt peptone yeast agar, prepared according to the specifications of Schaerffenberg (1964). Sporulating cultures were kept under refrigeration and used up to 6 months. New cultures were started subsequently by reisolating spores from diseased weevils. Conidia were suspended in 0.4% Tween 20 when used for the tests. Administration of i~ocu~um. In order to inject the inoculum directly into the foregut without contaminating the beetles externally, they were tied with fine copper wire in a way so they could not spread their wings or reach their mouthparts with either antennae or tarsi. A soft polyethylene tube (Intramedic PE 10, manufactured by Clay-Adams, Division of Becton, Dickinson and Co., Parsippany, New Jersey), was drawn out over a flame to a diameter of about 100 to 150 pm and connected to a micrometer syringe by a hypodermic needle of gauge 30. The tube was inserted into the buccal cavity and pharynx for about 1.5 mm. After the administration of approximately 20 ~1 of Tween (control) or spore suspension into each of 20 beetles, respectively, the tube was retained in place and its open end sealed with heated forceps. The beetles were suspended in moist chambers at room temperature and checked daily for mortality. Dead individuals were immediately removed and kept in moist Petri dishes for verification of the development of M. anisopliae. Some of the beetles regurgitated the injected suspension and most rejected the tubes after a while. Therefore, it was deemed impossible to inject suspension into the gut proper. In a subsequent test, droplets of suspension containing various spore amounts were applied directly to the mouthparts of 10 beetles with a micropipet, after these had been immobilized as previously described.

SCHABEL

In a third test, 35 beetles were allowed to ingest spores with their food, by offering them pine sections that had been immersed in suspensions of various spore concentrations. ~i~tologicuI technique. Beetles were fed pine twigs with conidia sandwiched between wood core and cambium. After 48, 70, 100, 120, and 144 hr, specimens were killed and prepared for sectioning. Other sections were made of larvae and beetles which had recently died of mycosis and also of a larva that had been inoculated by immersion in a spore suspension and suspended from a thread for 78 hr. The specimens were fixed in acetic formalin, dehydrated in an ethyl alcohol series, and cleared with succeeding intermediary solvents of methyl benzoate and benzene. Paraplast served as the embedding medium. The sections were cut at 8 pm and affixed to slides in a warm water bath (40°C) containing 1% Mayer’s albumin. The periodic acid staining procedure described by De Palma and Young (1963) served to differentiate the pathogen in the host tissues. Germination tests. The ability of conidia to germinate in vitro was tested by the following procedure. Digestive tracts of weevils were excised with both ends ligatured, then immersed in commercial strength (1:750) aqueous Zephiran chloride and four changes of distilled water, 5 min in each. With a finely drawn glass pipet, the promesenteron was punctured, its liquid contents withdrawn and placed on slides in Petri dishes. The droplets of this gut juice were then inoculated with lo-* ml of spore suspension, containing about 1000 conidia, stirred, and incubated at room temperature for 20 and 40 hr, respectively. The effect of gut transit on the viability of conidia was investigated by feeding weevils pine sections sandwiched with inoculum and then suspending them. About 24,48,67, and 86 hr after separation from the food, feces were collected and gut contents isolated from various parts of the alimentary tract. Isolations were made from this material by using streaking techniques on a selective medium

ORAL

INFECTION

described by Veen and Ferron (1966). Observations were made subsequently to determine growth of M. anisopliae.

OF

WEEVIL

379

remained low, with only 3% of the insects dead 50 days post-treatment. His tology

RESULTS Feeding Tests

In the first test, 100% mortality resulted from the injection of conidia into the foregut of the test weevils. The first casualties occurred 6 days after inoculation. All beetles were dead within 12 days and subsequently developed signs of M. anisopliae. Mortality among the control beetles, microfed with Tween, was 20% during the same period, and no mycosis was evident. In the second test, micropipetting approximately 450 spores directly to the mouthparts killed 90% of the beetles between the 8th and 16th day after treatment (Fig. 1). All the beetles were dead within 22 days and 80% of these developed signs of M. anisopliae. At doses of 40 and 5 conidia per beetle, the first mortalities occurred after more than 2 weeks, and at the lowest dose the mortality was almost identical with that in the control. In the third test, only 10% of the beetles feeding on pine twigs which had been immersed in spore suspensions containing 5, 10, and 20 million condida/ml died of Metarrhizium. For twigs immersed in a 50 million/ml suspension, the first deaths occurred on the ninth day and, within 31 days, 94% of the weevils were dead. Surviving beetles were still alive on the 50th day. Control mortality

Conidia were found in the intestinal tracts of all the beetles sectioned 48, 70, 100, and 120 hr after feeding. Most of the spores were found within gut contents (Fig. 2A), but a few were trapped in intimal folds of the crop (Fig. 2B). None showed signs of germination. In the larva, which had been inoculated by immersion 78 hr prior to fixation, hyphae were found to have penetrated through the nonsclerotized parts of the buccal cavity close to the pharyngeal opening (Fig. 2C) and also around the implantation of the mouthparts and the lingua. An infection cushion consisting of spores and hyphal elements and overlying melanized cuticle in the mouth was clearly visible as the point of origin for penetrating hyphae (Fig. 2D). In the same specimen, fungal growth was also evident in the interspace between foregut intima and neighboring tissues, with occasional breakthroughs into the hemocoel. No hyphae grew in the foregut lumen proper or anywhere else in the digestive system. Specimens fixed postmortem contained hyphae in all portions of the intestinal tract, but only sparingly so in the fore- and hindgut. Hyphae invaded the midgut lumen from within the hemocoel, but penetration of the intima of the fore- and hindgut could not be verified. Mycelium aggregated in dense masses under the intima of the hindgut. Germination

FIG. 1. Cumulative mortality after plication of Metarrhizium anisopliae mouthparts of adult Hylobius pales.

the controlled conidia to

apthe

Twenty hours after inoculation of contents from the midgut with M. anisopliae, about 95% of the conidia had germinated. Some germ tubes had extended to a length of 120 pm, and many conidia had two germ tubes. This growth took place in the presence of numerous yeast and bacterial cells. Colonies of M. anisopliae developed from many of the streaks made from feces and gut contents of spore-fed beetles, even as long as 86 hr after the insects had been separated from their food.

380

HANS

G

SCHABEL

FIGURE

2

ORAL

INFECTION

DISCUSSION The hypothesis that Metarrhizium can invade pales weevils from within the intestinal tract could not be verified. However, both histological and indirect proof was obtained that oral invasion occurred. This emphasizes the need to differentiate clearly between oral and intestinal infections. The term “oral,” as commonly used in the literature of insect pathology, frequently connotes intestinal infection or seems to encompass both oral and intestinal. A thorough search for conidia that had pregerminated or germinated in the gut of weevils was futile. Radha et al. (1956) likewise only recovered ungerminated spores of M. anisopliae from the gut of Oryctes larvae and no one else has reported that conidia of this fungus germinated in the gut of any insect. The reasons for the failure of conidia to germinate inside the intestinal tract of the host are not readily evident. The pH range within the gut of pales weevils is 5.3 to 7.4 and thereby coincides with the range favored by Metarrhizium for growth (Schabel, 1973). Also, the digestive action apparently does not harm the conidia, since these retain their viability even after a prolonged stay in the intestinal tract. Also, in vitro germination in the midgut contents of H. pales was surprisingly rapid and complete. This test does not, of course, permit conclusions with respect to in vivo conditions in the fore- and hindgut. A similar study by Gabriel (1959) showed that conidial germination was inhibited in the presence of intestinal contents of several insect species in contrast to the results obtained in the present study. Numerous yeasts and bacteria developed concurrently with the growth of Metarrhi-

OF

WEEVIL

3x1

zium in midgut contents in vitro; therefore,

these organisms were not strong antibiotic inhibitors as are many soil microbiota (Huber, 1958). The speed at which food and spores move through the alimentary tract probably assures that most conidia pass through the host before they could respond to any germination stimulus. However, even conidia that had been retained in the gut as long as 3 days, time sufficient for germination under suitable conditions, showed no sign of pregermination. The moisture content within the intestinal tract of H. pales may also have a bearing on spore germination, since Metarrhizium. like most fungi, requires very moist conditions for germination (Walstad et al., 1970). According to Saini (1964), one of the functions of the cryptonephridial tubes of the hindgut of curculionids is to reabsorb water and salts from the rectal contents. It is possible, therefore, that spores that would have been preactivated in the anterior portions of the gut may be deactivated by the stringent conditions in the hindgut. Moisture may be plentiful in the promesenteron, but the scarcity of spores found in this portion of the intestine suggests that rapid passage occurs there. Oral invasion of insects with M. anisopliae, has been reported by Notini and Mathlein (1944) in Ephestia and by Veen (1966) in Schistocerca. This was also observed in sections of a larva of H. pales in the present study. The poor quality of sections obtained through the mouthparts of weevils prevented making statisfactory observations there. Lack of evidence for intestinal infection, however, combined with the high mortality in controlled tests, when the possibility for integumental contamination was excluded, indicates a strong probability that oral infection equally occurred in beetles.

FIG. 2. Photomicrographs of sections of the buccal cavity and intestinal tract of HylobiuF pales containing elements of Merarrhizium anisopliue. (A), Transverse section through the cryptonephridial hindgut of a beetle, 70 hr postinoculation. Note the ungerminated conidia enclosed by the gut contents. (B), Ungerminated conidium (arrow) trapped in intimal fold of the crop of a beetle. (C) Hypha in cuticle of the buccal cavity (bc) close to the pharyngeal opening (p). Larva inoculated 78 hr before. (D), Infection cushion (arrow) on cuticle within the buccal cavity of a larva, 78 hr after inoculation. The cuticle is heavily melanized and contains hyphal elements as clearly visible in sections with differential stain.

382

HANS G. SCHABEL

Unlike the conditions in a peristaltically active gut, conidia are more likely to become anchored in the numerous folds of the buccal cavity. Infection cushions, as observed in this study, may then enhance the invasion of the pathogen through the mouth wail. Robinson (1966) attributed mycoses occurring at low relative humidity to intestinal infection, thereby implying, like Veen (1966), that the microclimatic conditions inside a host may be more suitable for fungal germination and growth than the external ones. This is supported by the fact that, in the pales weevil larva in which oral infection could be ascertained, fungal growth was decidedly more advanced in the proximity of the mouthparts than anywhere else in the host body, where spores penetrated percutaneously. Oral infection of pales weevils suggests that field trials should be conducted in which the ino~ulum be directed at the feeding activity of the beetles. This could be achieved by dipping or spraying young trees with spore suspension, a modification of the technique formerly employed in the application of DDT to pine transplants. If this approach were used, the amount of inoculum needed may be considerably less than that required for broadcast types of application. Also, this work could be synchronized with planting and would, therefore, obviate extra labor costs. Ecological implications of utilizing a nonspecific pathogen, i.e., one also pathogenic to many nontarget insects, would be minimized. Application of spores to the mouthparts resulted in high mortalities at doses as low as 450 conidia per beetle, although these results were obtained with stressed specimens. Feeding the weevils with spore-contaminated pine twigs was a closer approximation to natural conditions. Mortality in this case was only appreciable at the highest dose applied, but some of the beetles survived and the mortality was protracted. For fast and reliable mortality, a field dose would, therefore, have to be in excess of 50 million spores/ml of immersion fluid. Reasonable survival of inoculum in the field would be prerequisite

and could possibly be assured by the use of suitable adjuvants. ACKNOWLEDGMENTS The author wishes to thank Dr. R. F. Anderson, Department of Forestry, Duke University, and Dr. W. M. Brooks, Department of Entomology, North Carolina State University, for helpful suggestions during the course of this study and advice in preparation of this manuscript.

REFERENCES CIESLA, W. M., AND FRANKLIN, R. T. 1965. A method of collecting adults of the pales weevil, Hylobius pales and the pitch-eating weevil, Pachylobius picivorus (Coleoptera: Curculionidae). J. Kans. Enromof. Sot.. 38, 2055206. DE PALMA, P. A., AND YOUNG, G. G. 1963. Rapid staining of Candida ufbicans in tissue by periodic acid oxidation, basic fuchsine and light green. Srain Technol.. 38,251-259. GABRIEL, B. P. 19.59.Fungus infections of insects via the alimentary tract. J. lnsecr Pathol., 1,3 19-330. HUBER, J. 1958. Untersuchungen zur Physiologie insektentotender Pilze Arch. Mikrobio~., 29,2X-276. KEILIN, D. 1920. On a new saccharomycete Monosporella unicuspidata gennnom, nsp.. parasitic in the body cavity of a dipterous larva (Dasyhelea obscura Winnertz). Parasitology, 12,83-9 I. MULLER-K&LER, E. 1965. “Pilzkrankheiten bei Insekten,” 444 pp. Paul Parey, Berlin and Hamburg. NOTINI, G., AND MATHLEIN, R. 1944. Groenmykos foerorsakad av Metarrhizium anisopliae (Metsch.) Sorok. I. Groenmykosen som biologiskt insektbekaempningsmedel. Medd. Vaextskyddanst., 43. l-58.

PEIRSON, H. B. 1921. The life history and control of the paies weevil (~ylobi~pales). Harvard Forest Bufl.. 3, 33 PP. RADHA, K., NIRULA, K. K., ASD MENOK, K. P. V. 1956. The green muscardine disease of Oryctes rhinoceros L. II. The causal organism. Rev. Appl. Entomol., A 45,336-337. ROBINSOIX,R. K. 1966. Studies on penetration of insect integument by fungi. Pesf Articles and News Summaries.Sect. 3, 12, 131-142. SAINI, R. S. 1964. Histology and physiology of cryptonephridial system of insects. Trans. Roy. Entomol. Sot. London, 116,341-392. SCHABEL, H. G. 1973. “The Mode of Infection of Hylobias pales (Herbst) with ~eiarrhiziam an~opl~ae (Metsch.) Sorok. Ph.D. Thesis, Department of Forestry, Duke University, Durham, N.C. SCHAERFFENBERG, B. 1964. Biological and environmental conditions for the development of mycoses caused by Beauveria and Metarrhizium. J. lnsecr Parhol..

6.8-20.

ORAL INFECTION

383

OF WEEVIL

H. A. 1969. A meridic diet and rearing technique for the pales weevil larva. J. Econ. Entomol., 62, 1491-1494. VEEX., K. H. 1966. Oral infection of second-instar nymphs of Schistocerca gregatia by Metarrhizium

WALSTAD,

anisopliae. J. Invertebr. Pathol., 8,254-256. VEEN, K. H., AND FERRON, P. 1966. A selective medium for the isolation of Beauveria tenella and Metarrhizium anisopliae. J. Invertebr. Pathol., 8,268-269.

W. J., 1970. Effects of environmental conditions on two species of muscardine fungi (Beauveria bassiana and

THOMAS,

J. D., AND ANDERSON, R. F. 1971. Effectiveness of Beauveria bassiana and Metarrhizium anisopliae as control agents for the pales weevil. J.

Econ. Entomol.. WALSTAD, J. D.,

Metarrhizium 22 1-226.

64,322-323. ANDERSON,

anisopliae).

R.

F.,AND

J. Invertebr.

STAMBAUGH,

Pathol.,

16,