a consequence of fecal contamination of the integument following per os inoculation. Leslie L. Allee, 1 Mark S. Goettel, t'4 Alia Gol'berg, 2 H.S. Whitney 3 & D.W. ...
Mycopathologia ! 11 : 1 7 - 2 4 , 1990. 9 1990 Kluwer Academic Publishers. Printed in Belgium.
Infection by Beauveria bassiana of Leptinotarsa decemlineata larvae as a consequence of fecal contamination of the integument following per os inoculation Leslie L. Allee, 1 Mark S. Goettel, t'4 Alia Gol'berg, 2 H.S. Whitney 3 & D.W. Roberts I i Insect Pathology Resource Center, Boyce Thompson Institute, Tower Road, Cornell University, Ithaca, N Y 14853, USA; 2Agricultural Research Organization, Gilat Experiment Station, Mobile Post Negev 2, 85-280, Israel; 3Pacific Forestry Centre, Forestry Canada, 506 West Burnside Road, Victoria, British Columbia V8Z 1M5, Canada; 4present address: Crop Sciences Section, Research Station, Research Branch, Agriculture Canada, Lethbridge, Alberta, TIJ 4B1, Canada Received 31 May 1989; accepted 27 November 1989
Key words: Beauveria bassiana, entomopathogenic fungus, gut infection, histopathology, Leptinotarsa decemlineata, pathogenesis
Abstract
A study of per os inoculated larvae was undertaken to determine if the entomopathogenic fungus, Beauveria bassiana, is able to infect the Colorado potato beetle, Leptinotarsa decemlineata, via the alimentary tract. Surface or per os inoculated larvae which were immediately surface sterilized post inoculation did not succumb to infection, whereas those larvae not sterilized became infected. Histological studies of fed or starved, agnotobiotic (with microbial flora) and axenic larvae revealed that conidia can germinate in the gut regardless of the presence of gut microflora. However, infection via the alimentary tract was never observed in fed larvae and only noted in a single starved individual. It is concluded that infections of per os inoculated larvae occurred after surface contamination of the integument by viable conidia contained in the frass. The rate of food passage through the gut is probably important in preventing per os infections. Abbreviations: CFU: colony forming units, SDAY: Sabouraud dextrose agar plus 2~o yeast extract, SDAY + G: SDAY plus 0.2~o gentamycin sulphate, SWT: sterile water with 0.5~o Tween 80.
Introduction
Specific knowledge on the modes of infection of the entomopathogenic fungus Beauveria bassiana may assist in optimizing future insect control efforts. Although B. bassiana may infect insects via the respiratory system [6] and alimentary tract [2, 4, 14, 28], penetration through the external
integument is the most common route of invasion [1, 13, 21, 26]. Recent field trials with B. bassiana against the Colorado potato beetle, Leptinotarsa decemlineata, have produced mixed results [ 16] which may have been in part due to field conditions detrimental to conidial survival and host infection. Ultraviolet light and low levels of humidity
18 are factors damaging to conidia [15, 19, 22] that might be avoided if infection via the alimentary tract or gut occurred and could be enhanced. It is well established that B. bassiana penetrates the Colorado potato beetle via the external cuticle [ 12, 20]. Infection from within the gut has been assumed [7, 17, 24], but not confirmed experimentally. In the present study, the possibility of infection by B. bassiana of Colorado potato beetle larvae from within the gut was investigated. It is concluded that infection occurred via surface contamination of the integument by ingested conidia excreted in the frass.
Materials and methods Insects
Agnotobiotic (with microbial flora) Colorado potato beetles were reared on potato plants in cages at 27-30 ~ 4 0 ~ RH and 16 hr light: 8 hr dark. For axenic study, three-day-old eggs were washed in a mercuric chloride solution [27], rinsed with sterile water and aseptically transferred to sterile Colorado potato beetle diet (BioServe, Frenchtown, N J).
Fungus Beauveria bassiana strain ARSEF 252 (USDA-
ARS Collection of Entomopathogenic Fungal Cultures, Ithaca, NY) was used for per os and topical treatments. This strain was originally isolated in 1978 from a Colorado potato beetle in Maine. Conidia were produced on half-strength Sabouraud dextrose agar and stored at 4 ~ Colony forming units (CFU) per gram of conidial preparation were estimated by homogenizing 10 mg of conidia in 10 ml sterile water with 0.57o Tween 80 (SWT), preparing 10-fold dilutions, and spreading 0.1 ml of each dilution with a sterile glass rod on each of five 100-ram petri plates of Sabouraud dextrose agar plus 2~o yeast extract (SDAY). Colonies were counted after incubation at 26 ~ for three days.
Topical and per os inoculation
For tropical treatment, 6-10 one-day-old starved larvae were fed on potato leaves for 3-4 hr, placed on filter paper in 35-mm Petri dishes and sprayed with a suspension of conidia (1 mg/ml) in SWT. The spraying was done with a 1/8 JJ Air Atomizer nozzle (Spraying Systems Co., Wheaton, IL), at a height of 267 mm and an air flow rate of 15 liter/min. A small hole was drilled into the nozzle chamber from the side opposite the orifice. The nozzle was positioned at the end of the air flow system with the orifice pointing down. An 18-gauge needle attached to a barrel from a 5-ml syringe was inserted into the top hole. This served as a funnel into which conidiai suspension was introduced while the air flow was on. The suspension was pulled into the chamber by the venturi effect and sprayed in totality in a few seconds. Dose was estimated by spraying 35-mm Petri dishes containing SDAY plus 0.2~ gentamycin sulphate (SDAY + G) and counting CFUs. Assuming a 2 mm 2 surface area per insect, the topical dose was approximately 8.2 x 103 CFU/larva. This test was repeated nine times. For pet" os inoculation, 21 one-day-old starved larvae were fed on a fresh potato leaf extract mixed with 1, 4, or 8 mg conidia/ml. The leaf extract was prepared by macerating potato leaves using a mortar and pestle and filtering through cheese cloth. To reduce surface contamination, larvae were fed for 5-10 minutes through 0.5-1.0mm holes in stretched Parafilm M (American Can Company, Greenwich, CT). Larvae were observed at 60 x to verify that the gut contained green potato extract. Dosage was estimated by determining the weight difference between seven groups of 10 fed and unfed larvae. The mean difference was 0.6 + 0.29 mg per 10 larvae or approximately 0.06 ~1 per larva. Assuming this rate of ingestion, the per os doses were approximately 5.1 x 103, 2.2 x 104, and 4.5 x 104 CFU/larva. This was verified by homogenizing nine larvae in SWT immediately after feeding and surface sterilizing in 0.5~ sodium hypochlorite; insect homogenates, plated on SDAY + G, produced virtually identical CFU
19 counts per #1 to those estimated above. This determination was repeated four times. In a separate treatment, fecal pellets were collected 5, 9, 21, 32 and 46 hr after peros inoculation, mixed with SWT and plated on SDAY + G. In addition, 4-25 larvae were sacrificed every 6-12 hr for up to 72 hr post inoculation by dissecting on the surface of SDAY + G in 35-mm diameter petri dishes, one larva per dish. Dissected tissues and organs were allocated to separate portions of the agar surface. Gut tissues were triturated in sterile water and spread locally on the surface of the medium. In other cases the gut, with head and tail attached, was left intact. Eight larvae were also inoculated per os (4.5 • l04 conidia/larva); 4 were homogenized and plated immediately while 4 were held on leaves and homogenized after 24 hr. All culture sites were examined for growth of B. bassiana following incubation at 27 ~ for 3-7 days.
Histology
Axenic and agnotobiotic one-day-old starved larvae were fed droplets of leaf extract containing 1 x 10 7 conidia/ml. For axenic insects, the extract was first filter sterilized and the larvae were held on sterile Colorado potato beetle diet. Agnotobiotic larvae were fed either potato leaves or the Colorado potato beetle diet, or were starved for 48 hr and then fed potato leaves. Controls, fed leaf extract only, were kept for each feeding regime. Five larvae from each treatment were removed at 24, 48, 60, and 72 hr post inoculation, fixed in Bouin's, and embedded in paraffin. Entire larvae were sectioned at 7 #m, stained by oxidation in periodic acid, basic fuchsin and light green [8], and examined for the presence of conidia and hyphae. A further five larvae from each treatment were held until death and verified for beauveriosis. These treatments were repeated three times.
Surface sterilization
Results
To prevent infections from occurring due to surface contamination of the integument during feeding, all larvae were submerged in 0.5~o sodium hypochlorite for 6 minutes [ 18] immediately afterper os inoculation and then rinsed three times in SWT. An additional group of topically inoculated larvae was surface sterilized in order to confirm the effectiveness of the wash. Uninoculated larvae, with and without surface sterilization, were used as controls. Following inoculation and surface sterilization, all larvae were incubated individually in 35-mm Petri dishes lined with filter paper. A potato leaf, replaced every 24 hrs, was provided as food. Petri dishes were held at an average 90~o humidity, 26 ~C without light. Mortality was recorded daily for 6 days. Dead larvae were held on wet filter paper or water agar. Those cadavers which produced an external sporulating layer orB. bass&na were considered as having died as a result of beauveriosis.
Mortality of topically and per os inoculated larvae
Larvae topically inoculated with 8.2 X 10 3 CFU/insect followed by surface sterilization developed no symptoms or signs of beauveriosis for up to six days after inoculation. Topical inoculation without surface sterilization resulted in 9 6 ~ beauveriosis mortality at six days. Mortality of uninoculated control larvae was 6?/0 and no fungal infection was observed. Larval mortalities six days after per os inoculation and surface sterilization were as follows: 58.8~ for larvae inoculated with 5.1 x 10 3 CFU/insect, 67.2~o with 2.2 x 10 4 C F U , and 73.0~o with 4.5 x 10 4 CFU. Larvae inoculated per os and homogenized after 24 hrs contained 99.9~o fewer conidia than larvae homogenized immediately following treatment (24 vs 4.5 X 104). Fecal pellets contained viable conidia up to 32 hr post-inoculation with peak exit at 12-24 hr. Larvae were frequently seen with fecal pellets trailing behind or adhering to their pos-
20 there was abundant growth of B. bassiana from gut contents of the same larvae.
Histology
Fig. 1. A tendril offecal matter adhering to the posterior (arrow) of a first instar larva ofLeptinotarsa decemlineata. Bar represents 1 mm.
terior integument (Fig. 1). Gut tissues, dissected fromper os inoculated larvae and plated, were not penetrated before 52 hr. During the same period,
Conidia were observed within the alimentary tract in allper os inoculated treatments at 24 hr. Germinated conidia were found throughout the gut lumen in conidia-fed, axenic and agnotobiotic larvae held on artificial diet (Fig. 2). Fecal matter within the lumen also contained hyphal growth. In contrast, germinated conidia were rarely seen in guts of conidia-fed larvae held on potato leaves. Also, hyphal growth was never observed in these larvae. Penetration of the gut wall was never observed in any conidia-fed larvae, axenic or agnotobiotic, or in most starved larvae (Fig. 3). However, penetration of the foregut at the entrance to the midgut or proventricular region was noted in a single starved individual 48 hr
Fig. 2. Beauveria bassiana conidia germination (g) in the gut lumen of Leptinotarsa decemlineata 24 hr after per os inoculation. Bar represents 20/lm.
21
Fig. 3. Hyphae of Beauveria bassiana growing in the gut lumen of fed larvae of Leptinotarsa decemlineata 48 hr after per os
inoculation. Note that the hyphae are not penetrating the gut wall Bar represents 20 ~m.
Fig. 4. Hyphae of Beauveria bassiana penetrating the foregut wall (arrow) between the esophogus (e) and the midgut (m) of a starved larva of Leptinotarsa decemlineata, 48 hr after per os inoculation. Bar represents 20/~m.
22
Fig. 5. Hyphae of Beauveria bassiana penetrating the foreleg of a Lepthzotarsa decemlineata larva (arrows) from a clump on the external integument, 60 hrs after per os inoculation. Bar represents 20 ltm.
post-inoculation (Fig. 4). Penetration of the external integument was observed in most larvae 60 hr post inoculation (Fig. 5). Most penetrations occurred from clumps which were thought to be fecal matter adhering to the integument (Fig. 1). Conidia-fed starved larvae died in 2-3 days while inoculated fed larvae died in 3-4 days. All mortality was attributed to beauveriosis.
Discussion
Fungal infection via the alimentary tract is relatively uncommon in insects. Although gut infection by B. bassiana has been shown to occur in some species other than L. decemlineata, few studies have excluded the possibility of infection by surface contamination of the integument [5]. Also, in many studies micro-injection devices were used for inoculation; puncture or damage to the gut wall often occurs as evidenced by a high
incidence of septicemia [ 14]. We used the natural feeding of neonate larvae to introduce the fungal inoculum p e r os. Feeding was immediately followed by surface sterilization which effectively prevented external contamination. As plated homogenates of p e r os inoculated surface sterilized larvae showed no loss of dose, the lower infection rate in p e r os as compared to topically inoculated larvae was not due to ingestion of the sterilization wash. Several factors are likely to influence p e r os infection: spores must remain viable and germinate in the conditions prevailing in the gut (i.e. unfavorable pH, presence of digestive enzymes and microbial flora), have sufficient contact time with the gut wall to allow germination and penetration, and must be capable of penetrating the gut wall [5, 20]. In the present study, viability and germination of conidia in the gut seemed unaffected by the conditions in the gut. Unlike the high gut pH found to inhibit B. bassiana germi-
23 nation in B o m b y x mort [ 14] and Heliothis zea [21 ], the gut pH of Colorado potato beetle was found to be 6.4 [3]. Such a pH would be condusive to germination ofB. bassiana [23, 25]. Furthermore, the glycoalkaloid solanine, contained in potato leaves, is not significantly toxic to B. bassiana [7]. Dillon & Charnley [9] found that the gut pH of Schistocerca gregaria was optimal for germination of Metarhizium anisopliae, yet germination was inhibited in normally fed locusts. Since germination and penetration did occur in the guts of axenic locusts, they attributed the inhibition to a fungitoxic compound produced by bacteria in the hindgut [10, 11]. In the present study, conidia germinated in the guts of both axenic and agnotobiotic Colorado potato beetle larvae, indicating that the presence of the gut microflora had little, if any, effect on germination. In per os inoculated B. mori larvae, infection from the gut apparently occurred after fungal metabolites disintegrated midgut cells, not by direct hyphal penetration [29]. In contrast, midgut ceils of per os inoculated Colorado potato beetle larvae remained intact until hyphae penetrated from the outside. These hyphae grew through the hemolymph from conidia germinating on the integument. The per os dose was lost rapidly from the guts of leaf-fed larvae. Most germination and hyphal growth occurred in the guts of axenic artificial diet-fed larvae. These larvae consumed much less food and grew more slowly than leaf-fed individuals. In addition, gut penetration occurred only in a starved individual. This suggests that the rate of food passage through the gut may be the most important factor in preventing per os infections. Live conidia were found in the frass, and infection of the integument occurred by 60 hr after per os inoculation. Infections occurred from clumps of frass on the integument surface. We conclude that infection of per os inoculated larvae resulted from contamination of the integument by conidia contained in the frass. Indeed, the frass of L. decemlineata is very sticky before it dries and it was often seen attached to the integument. Although the results of this study indicate that
field application of B. bassiana targeted for per os inoculation (i.e. in bait) would not necessarily result in infections through the gut, such a method may be beneficial by extending the inoculum availability in the field. Ingested conidia would be shielded from the detrimental effects of sunlight and low humidity for a period of up to 24 hr inside the insect gut. The embedding of conidia in frass may afford additional protection. Furthermore, gut wall penetration in a starved larva suggests that feeding habits may be important. If a larva that had ingested B. bassiana spores stopped feeding, as in cool weather or in preparation for molting, gut infection might occur. Timely addition of an antifeedant may thus enhance infection.
Acknowledgements The authors thank Clay Weeks for excellent technical assistance. This work was supported by the Natural Sciences and Engineering Research Council of Canada, the USAID Bean/Cowpea Collaborative Research Support Program, Forestry Canada, and the Jesse Smith Noyes Foundation.
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Corresponding author: Leslie L. Alice Boyce Thompson Institute Tower Rd. Ithaca, NY 14853, USA
