Anaerobic Bacteria Isolated from the Alimentary Canals of Alfalfa Leafcutting Bee Larvae G

Anaerobic bacteria isolated from the alimentary canals of alfalfa leafcutting bee larvae G. Douglas Inglis, L. Jay Yanke, Mark S. Goettel

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G. Douglas Inglis, L. Jay Yanke, Mark S. Goettel. Anaerobic bacteria isolated from the alimentary canals of alfalfa leafcutting bee larvae. Apidologie, Springer Verlag, 1998, 29 (4), pp.327-332. hal- 00891498

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Anaerobic bacteria isolated from the alimentary canals of alfalfa leafcutting bee larvae

G. Douglas Inglis* L. Jay Yanke, Mark S. Goettel

Agriculture and Agri-Food Canada Research Centre, P.O. Box 3000, Lethbridge, AB, T1J 4B1, Canada

(Received 2 January 1998; accepted 13 March 1998)

Abstract - Microorganisms were isolated from the alimentary canals of third-instar alfalfa leafcutting bee (Megachile rotundata) larvae under strict anaerobic conditions. In all 3 years of the study, small populations (8.0 colony-forming units per gut) of a limited number of bacterial taxa (primarily the facultatively anaerobic bacteria Paenibacillus macerans, Bacillus licheniformis, B. brevis, B. mycoides and P. polymyxa) were isolated. With the exception of three isolates of Clostridium longisporum recovered in 1993, no obligate anaerobic bacteria were isolated. The small size of populations and the inconsistency of recovery of bacteria in different years suggest that anaerobic microorganisms are not common inhabitants of leafcutting bee larval guts. Consequently they could not have a significant impact on the ecology of the alimentary canal nor influence the development of chalkbrood disease caused by the entompathogenic fungus, Ascosphaera aggregata. © Inra/DIB/- AGIB/Elsevier, Paris Megachile rotundata / larvae / alimentary canals / anaerobic microflora

1. INTRODUCTION disease of leafcutting bees throughout this area, and bee losses can exceed 50 %. Alfalfa leafcutting bees (Megachile Disease is initiated by ascospores in food rotundata Fabricius) are the principal pol- provisions that are ingested by early-instar linators used for production of seed alfalfa leafcutting bee larvae. The ascospores, puta- (Medicago sativa) in northwestern North tively stimulated by an elevated CO2 concen- America. Chalkbrood, caused by the asco- tration, germinate in the mid-gut, and hyphae mycetous fungus, Ascosphaera aggregata then penetrate into the hemocoel eventually has become an increasingly important causing death of the larvae [12, 15].

* Correspondence and reprints ** Present address: Department of Entomology and Plant Pathology, Mississipi State, MS 39762- 9775, USA The gut microflorae of insects can be been shown to support the growth of a variety antagonistic to pathogens and affect the epi- of anaerobic bacteria (Yanke et al., unpublished). zootiology of disease (e.g. [3, 7]). Although In 1993, but not in 1994 or 1996, the media were L-1 a number of bacteria and fungi were isolated amended with 50 mg Nystatin (Sigma, to inhibit or 100 L-1 from leafcutting bee larval guts [10], these St. Louis, MI) fungi mg St. Louis, to inhibit did not inhibit chalkbrood Tetracycline (Sigma, MI) microorganisms bacteria, and all cultures were incubated in the bee larvae have a blind [11]. Leafcutting anaerobic chamber. Cultures were initially placed gut (no anus) up to the fourth-larval instar at 30 °C in 1993, but no fungal and only a few [17], and low redox potential of the mid- bacterial colonies were observed by 11 days. gut [16] indicates a low oxygen environ- Cultures were subsequently transferred to 37 °C for an additional 8 In 1994 and the ment. Although a number of facultative days. 1996, cultures were immediately placed at 37 °C, and anaerobic bacteria and fungi were previously colony-forming units (CFU) were counted and isolated from larval guts under aerobic colonies collected after 5 days. To isolate cellu- conditions the and diver- [10], prevalence lolytic bacteria, the homogenate was placed in sity of obligate anaerobic and facultative tubes of MD broth containing 1-cm2 pieces of aerobic microorganisms in the alimentary Whatman #1 filter paper as the carbohydrate canals of alfalfa leafcutting bee larvae have source [1]. From tubes displaying filter paper degradation, the medium was diluted in not been investigated. To ascertain if anae- serially a 10-fold dilution series, and 200 μL from each robic could have an microorganisms impact dilution was spread onto MD agar. Isolates were on the ecology of the larval guts, the objec- streaked for purity and placed in MD broth tive of this study was to isolate, under strict containing filter paper to confirm cellulolytic anaerobic conditions, microorganisms from activity. Collected bacteria were stored in 10 % the guts of third-instar larvae over a 3-year glycerol at -80 °C until characterized. period. Catalase, oxidase and gram stain reactions were determined for bacteria grown on MD agar for 24 h using standard methods, and all isolates were also tested for growth in ambient oxygen. 2. MATERIALS AND METHODS Bacteria were identified by gas-liquid chroma- tography of fatty acid methyl esters using the MIDI Microbial Identification bee cells were collected on 19 System (MIDI Leafcutting To bacteria August 1993, 18 July 1994, 3 August 1994 and Inc., Newark, DE). accomplish this, were on 25 July 1996 from nests in bee shelters situated grown aerobically trypticase soy agar Detroit, at 37 °C for 24 h and in an field of alfalfa at the (Difco, MI) fatty irrigated Agriculture acids extracted and to the and Agri-Food Canada Research Centre at Leth- methylated according MIDI [14]. Bacillus and Paenibacillus bridge. The cells were immediately transported to protocol were further characterized using the API the where ten arbitrarily selected pre- species laboratory CH50B St. Laurent, iden- defecation larvae were removed (Biomerieux, Quebec) (third instar) tification and surface-sterilized in 1 % sodium hypochlo- system [13]. rite with 0.1 % Tween 80 for 2 min, followed In 1996, alimentary canals from six arbitrarily by two rinses in sterile distilled water. Surface- selected third-instar larvae were excised, fixed sterilized larvae were stored for a maximum of in 2 % glutaraldehyde in 0.05 M phosphate buf- 12 h at 5 °C, warmed to room temperature, and fer, post-fixed with 1 % osmium tetroxide, dehy- placed in an anaerobic chamber (90: 10 % drated in ethanol, critical-point dried in liquid CO2:H; Forma-Scientific, Anaerobic System, CO2, sputter coated with gold, and examined with Model 1024). Alimentary canals were removed a Hitachi S-570 scanning electron microscope from each larva by making a longitudinal incision (SEM) at an accelerating voltage of 10 kV. and carefully extracting the intact gut in sterile saline (0.85 % sodium chloride). The digestive tracts were individually homogenized in 1 mL 3. RESULTS AND DISCUSSION of Bryant’s anaerobic dilution solution [2] using a Kontes micropestle, and 200 μL of the homo- of anaerobic bacteria genate were spread in duplicate on modified Small populations Dehority’s agar (MD agar) [1]; this medium has with a limited diversity (five taxa per year) were recovered from the alimentary canals lates recovered) and Bacillus licheniformis of pre-defecation leafcutting bee larvae in (24-60 %) were the most common taxa 1994 (= 8.0 CFU/gut) and 1996 (= 2.5 isolated. Bacillus brevis, B. mycoides and CFU/gut). Large numbers of pollen grains P. polymyxa were recovered at lower fre- were observed in guts of all six larvae exa- quencies (≥ 15 %). Only bacilli were obser- mined with the SEM. Alfalfa leafcutting bee ved in the alimentary canals of larvae exa- provisions are comprised of water, nectar mined with the SEM in 1996; two cell and pollen with M. sativa pollen being the morphologies were observed and included most prevalent taxon [8]. Although bacterial short robust rods (0.8-1.1 x 0.5-0.7 μ; cells were infrequently observed, in seve- figures I and 2) and longer bacilli (2.2-3.0 ral instances aggregations of bacilli were x 0.5-0.7 μ; figures 3 and 4). In 1993, 23 observed on the surfaces of pollen grains or additional bacterial isolates grew after the on the peritrophic membrane (figures 1-4). incubation temperature was raised from 30 In both 1994 and 1996, Paenibacillus mace- to 37 °C (32 total bacteria); 29 were Bacil- rans (comprising 24-45 % of the total iso- lus spp. with three cellulolytic isolates identified as Clostridium longisporum. No cel- grew at ≥ 37 °C but not at 30 °C. Nest tem- lulolytic bacteria were isolated from larval peratures are substantially less than 37 °C for guts in 1994 or 1996. much of the day and night period, and microorganisms resident in the alimentary All of the Bacillus and Paenibacillus canal would be expected to grow at 30 °C. we isolated from larval are cos- species guts The results of this study indicate that anae- and are as faculta- mopolitan categorized robic microorganisms are not abundant in tive anaerobes Three of the B. bre- [6]. taxa, alfalfa leafcutting bee larval guts, and there- B. and P. have vis, licheniformis macerans, fore would not be expected to have a signi- been isolated from previously leafcutting ficant impact on the ecology of the alimen- and bee under aerobic condi- honey guts tary canal or on the development of tions [5, 10]. An actinomycete-like bacte- chalkbrood disease. rium was also consistently recovered from larval guts in 1994 (6-24 %, n = 3-12). Acti- the aerobe nomycetes, primarily obligate ACKNOWLEDGMENTS Streptomyces, have also been reported from the canals of and alimentary leafcutting We thank the following people at the Agri- honey bees under aerobic conditions [4, 10]. culture and Agri-Food Canada Research Centre The actinomycete-like bacterium that we at Lethbridge: Tim Myers and Grant Duke for isolated grew very poorly in both anaero- collecting leafcutting bees in 1994; Byron Lee bic and aerobic environments, produced for conducting the SEM; and Dr Denis Gaudet white filamentous colonies, thin hyaline and Dr Colin D’Silva for their comments on the This is 2 in but it did not manuscript. manuscript Lethbridge hyphae (< μm diameter), Research Centre contribution #3879670. sporulate in culture. We were unable to isolate or observe fungi, even though Tricho- sporonoidies megachiliensis, a facultative anaerobe, is commonly associated with leaf- Résumé - Bactéries anaérobies isolées du tube des larves de la cutting bee larval guts [9]. digestif mégachile de la luzerne. La mégachile de la luzerne Despite having blind guts with redox (Megachile rotundata Fabricius) est le prin- potentials indicative of a low oxygen envi- cipal pollinisateur utilisé dans le nord-ouest ronment, we recovered only small popula- de l’Amérique du Nord pour la production tions of anaerobic bacteria with low taxo- de luzernes porte-graines (Medicago sativa). nomic diversity from leafcutting bee larvae Ascosphaera aggregata Skou, l’agent du in three different years. The majority of bac- couvain plâtré, a acquis une importance de teria isolated from larval guts under aero- plus en plus grande en tant que parasite des bic conditions are probably transients having mégachiles. Les ascospores sont ingérées been ingested during consumption of pro- par les larves, germent dans l’intestin moyen visions and subsequently excreted in frass et le champignon pénètre dans l’hémocèle, [ 10, 11]. Our observations indicate that anae- pouvant provoquer la mort de la larve. robic bacteria isolated from alfalfa leafcut- Jusqu’au quatrième stade larvaire, la larve ting bee larval guts are also transient. With possède un tube digestif fermé et les poten- the exception of Clostridium, the bacteria tiels redox montrent que l’environnement we isolated are facultative anaerobes, and est pauvre en oxygène. Afin de déterminer si many have been isolated previously from le tube digestif de la larve de troisième stade the alimentary canals of leafcutting bees possède une microflore anaérobie, on a isolé, under aerobic conditions [10]; Clostridium en conditions strictement anaérobies, des spores are cosmopolitan and can survive microorganismes à partir d’intestins larvaires prolonged exposures to oxygen. Further- excisés. Au cours des trois années qu’a duré more, many of the bacteria isolated in 1993 l’expérience, on a isolé de petites populations ( < 8,0 unités formant une colonie/- schließen, daß es nur eine geringe Verbrei- intestin) d’un nombre restreint de taxons de tung anaerober Mikroorganismen im bactéries (principalement les bactéries anaé- Magendarmkanal von M. rotundata gibt. robies facultatives Paenibacillus macerans, Entsprechend können sie weder eine große Bacillus licheniformis, B. brevis, B. mycoides Auswirkung auf die Bedingungen im Darm- et P. polymyxa). À l’exception de trois isolats kanal ausüben noch Einfluß auf die Ent- de Clostridium longisporum trouvés en 1993, wicklung der Kalkbrut haben, die durch den aucune bactérie anaérobie obligée n’a été insektenpathogenen Pilz Ascosphaera isolée. De rares bacilles ont été aussi obser- aggregata hervorgerufen wird. © Inra/DIB/- vés sur le pollen et les membranes péritro- AGIB/Elsevier, Paris phiques. La taille réduite des populations et la présence irrégulière des bactéries d’une Megachile rotundata / Larven / Magen- année sur l’autre laisseraient penser que les darmkanal / anaerobe microorganismes anaérobies ne sont pas des hôtes habituels des intestins de larves de mégachiles. 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