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2012

Midgut and Fat Body Bacteriocytes in Neotropical Cerambycid Beetles (Coleoptera: Cerambycidae)

Olga Calderon CUNY La Guardia Community College

Amy Berkov CUNY City College

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This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] INSECT-SYMBIONT INTERACTIONS Midgut and Fat Body Bacteriocytes in Neotropical Cerambycid Beetles (Coleoptera: Cerambycidae)

1,2 2,3,4 OLGA CALDERON AND AMY BERKOV

Environ. Entomol. 41(1): 108Ð117 (2012); DOI: http://dx.doi.org/10.1603/EN11258 ABSTRACT Xylophagous insects derive nutrients from intractable substrates by producing or ingesting cellulolytic enzymes, or by maintaining associations with symbiotic microbes. Wood-boring cerambycid beetle larvae sometimes house maternally-transmitted endosymbiotic yeasts that are presumed to provide their hosts with nutritional beneÞts. These are thought to be absent from species in the large subfamily Lamiinae; nevertheless yeasts have been repeatedly isolated from the guts of neotropical lamiines. The objective of this study was to conduct transmission electron microscopy (TEM) studies of cerambycid larval midgut tissues to determine if gut yeasts were intracellular, or simply present in the gut lumen. Nine cerambycid larvae were harvested from two trees in the Brazil nut family (Lecythidaceae) in the rain forest of SE Peru; seven were identiÞed using mtDNA sequence data and processed for TEM. Yeasts cultured from larval frass or exuvia, and identiÞed with rDNA sequence data, were identical or similar to yeasts previously isolated from beetles. In TEM analyses yeast cells were found only in the gut lumens, sometimes associated with fragments of thick-walled xylem cells. Apparent bacteriocytes were found in either midgut or fat body tissue of three larval specimens, including two lamiines. This is the Þrst report of a potential fat body symbiosis in a cerambycid beetle. Future studies of cerambycid symbiosis should distinguish the identities and potential roles of free-living organisms in the gut lumen from those of organisms harbored within gut epithelial or fat body tissue.

KEY WORDS Barcode, mycetome, Periboeum pubescens, symbiont, Xylergates pulcher

Wood- and bark-boring beetles are surprisingly large to the challenge by feeding on wood already colonized and diverse guilds, including Ͼ70,000 described spe- by fungi and ingesting fungal enzymes (Martin 1987). cies (Lawrence 1982, Oberprieler et al. 2007). Mem- Others, such as the ambrosia beetles, transport a sym- bers of the families Anobiidae, Bostrichidae, Bupres- biotic fungal inoculum and feed directly on fungi, tidae, Cerambycidae, as well as some Brentidae and either exclusively or mixed with xylem fragments Curculionidae, successfully extract nutrients from the (Hulcr et al. 2007). There is a growing body of evi- least auspicious of substrates. Xylem and phloem dence that some xylophagous beetles (and numerous sapsÑand wood itselfÑare extremely low in nitrogen other insects) produce endogenous cellulases (Wa- (Mattson 1980). Even carbon is difÞcult to access tanabe and Tokuda 2010); these may act synergisti- because so much is incorporated into the recalcitrant cally with enzymes produced by symbiotic microbes structural polymers cellulose and lignin that, in con- or other sources. junction with hemicelluloses, compose plant second- Cerambycidae is the most species-rich lineage of ary cell walls. Complete cellulose metabolism requires wood-borers, including Ϸ35,000 described species a complex of three cellulolytic enzymes in the glyco- (Lawrence 1982). Some cerambycid species, like ano- side hydrolase (GH) family (Watanabe and Tokuda biids, host maternally-transmitted intracellular gut 2010), which typically are found in fungi and bacteria. yeasts. Although yeasts do not produce endogenous Digestion of cellulose also is impeded because it is cellulases, some are likely to metabolize hemicellulose cross-linked with lignin, which is even more resistant (Chararas et al. 1983, Schauer and Hanschke 1999, Suh and usually degraded by fungal peroxidases (Martin et al. 2003). Other possible functions include the syn- 1991, Brune et al. 1995). Some borers apparently rise thesis of vitamins or amino acids, or detoxiÞcation of plant secondary metabolites (Chararas et al. 1983, Dowd 1991, Shen and Dowd 1991). Schomann (1937) 1 Department of Natural Sciences, LaGuardia Community College, 31-10 Thomson Ave., Long Island City, NY 11101. conducted the most comprehensive early studies of 2 Department of Biology, City College and the Graduate Center, cerambycid symbiosis. The yeasts are smeared onto The City University of New York, Convent Ave. @ 138th St., New the exterior surfaces of eggs during oviposition, and York, NY 10031. ingested by Þrst-instar larvae when they consume the 3 Division of Invertebrate Zoology, American Museum of Natural History. Central Park West @ 81st St., New York, NY 10024. eggshell. Yeasts are housed within specialized myce- 4 Corresponding author, e-mail: [email protected]. tocytes in anterior midgut caeca, but the mycetocytes

0046-225X/12/0108Ð0117$04.00/0 ᭧ 2012 Entomological Society of America February 2012 CALDERON AND BERKOV:CERAMBYCID BACTERIOCYTES 109 also are released into the gut lumen and excreted. In males, yeasts generally are eliminated before pupa- tion, whereas in females yeasts are Þrst freed into the intestinal lumen, and then eventually Þll tubular “in- tersegmental pouches” associated with the ovipositor (Schomann 1937). This early study documented con- siderable diversity among both microbes and the bee- tle structures associated with their maintenance and transmission. Gru¨ nwald et al. (2010) further explored this diversity by reexamining classic temperate exam- ples using ßuorescence in situ hybridization (FISH) techniques and sequence analysis. Their study re- vealed the presence of one cerambycid species with- out, and three species with, endosymbiotic yeasts; one of the latter also housed intracellular bacteria. Cerambycids in the two largest subfamilies (lami- ines and some cerambycines) lack the intersegmental pouches in which yeasts proliferate before oviposition (Schomann 1937; A. B., unpublished data). Although Ϸ79% of the temperate cerambycid genera that have been investigated appear to host symbiotic yeasts (Dowd 1991), beetles without specialized structures Fig. 1. Representative cerambycid larvae included in the for their transmission would not necessarily be ex- TEM analyses. (a) YPN-8 ϭ P. anceps, (b) YPN-32 ϭ P. pected to have obligate associations. Gru¨ nwald et al. crassimana (olivaceous form), (c) YPN-25 ϭ X. pulcher, (d) (2010) did not include any lamiines in their study, but YPN-35 ϭ P. pubescens, (e) the bracket indicates the region the cerambycine they investigated yielded yeast from of the proximal midgut prepared for TEM, the arrow shows the gut lumen but lacked endosymbionts. In our initial adherent fat body tissue (this individual was dissected for gut studies of yeasts associated with neotropical ceram- culture). bycids (Þve lamiine species and one cerambycine), we found that most larvae yielded gut yeasts, but there was no evidence of vertical transmission. ConspeciÞc the precise instars were not known at the time of beetles yielded diverse yeasts, whereas unrelated bee- collection, but the specimens selected for this project tles inhabiting the same branch sometimes yielded the ranged in length from Ϸ6 to 15 mm (Fig. 1). Each larva same yeast (Berkov et al. 2007). In this study our was placed on Þlter paper in an individual tight-Þt primary objective was to conduct transmission elec- petri dish to clear the gut of transient material before tron microscopy (TEM) studies of larval midgut tis- Þxation for microscopy; Þlter papers were moistened ϫ sues to determine if the gut yeasts were in fact intra- with sterile H2O and replaced daily 5Ð8 d. cellular. Alternatively the yeasts might be transients or Yeast Culture. Larval frass and exuvial samples were digested as a direct source of nutrients; a secondary cultured to determine if viable yeast cells were pres- objective was to determine if yeast cells survived pas- ent. As soon as larvae produced frass (1Ð2 d after sage through the larval gut. harvest), samples were collected from Þlter papers within the petri dishes, placed in individual 0.5-ml tubes with 300-␮l sterile saline solution, and macer- Materials and Methods ated with sterile probes. A sterile transfer pipette was Specimen Acquisition. We sampled neotropical ce- used to transfer 100 ␮l of each solution to an acidiÞed rambycids associated with the Brazil nut family, be- yeast medium (AYM) plate (with the pH adjusted to cause we had already created a DNA-sequence library 3.5 to inhibit bacterial growth). Plates were streaked to facilitate larval identiÞcation (Berkov et al. 2007). and incubated at ambient temperatures for 1Ð2 d. Specimens were collected in the lowland moist forest Single colonies, selected on the basis of colony mor- at Los Amigos Research Center, in the Department of phology, then were streaked for puriÞcation (twice Madre de Dios, Peru. The site is Ϸ268 m above sea for each isolate). level, with mean annual rainfall ranging from 2,700 to Genetic Identification of Cerambycid Larvae and 3,000 mm per year, and mean monthly temperatures Yeast Isolates. DNA sequence data were used to iden- ranging from 21 to 26ЊC (Pitman 2008). In September tify both the larvae and the yeast isolates. For DNA 2005, we severed bait branches from two trees in the preservation, we applied tissues (or isolates) to FTA Brazil nut family, Bertholletia excelsa Humboldt & Classic Cards (Whatman Inc., Florham Park, NJ). The Bonpland and Eschweilera coriacea (A. P. de Can- cards are impregnated with chemicals that lyse cells, dolle) S. A. Mori (at 12Њ 34.044Ј S, 70Њ 06.058Ј W and denature proteins, and bind DNA. When larvae were 12Њ 33.450Ј S, 70Њ 06.478Ј W, respectively). Branches Þxed for TEM (see TEM Analyses of Larval Guts), the were exposed to beetles on the forest ßoor for Ϸ6 wk. head capsules were placed on cards and covered with We then removed sections of bark, and harvested nine small pieces of paraÞlm, which were burnished to larvae (20Ð24 November 2005). Their identities and maximize the contact of cells with the DNA-binding 110 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1

Table 1. Cerambycid larvae collected for TEM analysis, with associated yeast isolates

Larva no. Host treea Larval ID (MM)b Subfamily Isolate no.e Similar sequence (MM)b YPN-8 BE-4 Palame anceps (1) Lamiinae 9 (F) C. endomychidarum (0) YPN-12 BE-4 Palame crassimanac (1) Lamiinae 14 (F) YP4 (0) YPN-32 EC-3 Palame crassimanac (0d) Lamiinae 49 (F) YP4 (0) YPN-25 EC-3 Xylergates pulcher (2) Lamiinae 38 (F) YP4 (0) YPN-97 EC-3 Xylergates pulcher (0) Lamiinae 97 (F) YP4 (0) YPN-35 EC-3 Periboeum pubescens (0) Cerambycinae 55 (E) C. endomychidarum (6) YPN-98 EC-3 Periboeum pubescens (0) Cerambycinae 98 (F) YP4 (0) YPN-9 BE-4 Genus sp. (excluded) NA 10 (F) YP1 (9) YPN-16 BE-4 Pupated (excluded) NA 19 (F) YP1 (0)

a BE-4, Bertholletia excelsa specimen 4; EC-3, Eschweilera coriacea specimen 3. b MM, the number of nucleotide substitutions between these sequences and reference sequences. c Palame crassimana olivaceousÕ form, which is genetically distinct and probably represents a cryptic species (Berkov 2002). d We were only able to amplify and sequence 386 bases from this specimen but they were identical to a reference sequence. e (F), isolated from larval frass; (E), isolated from larval exuvium. chemicals and then removed. For yeast preservation, products were puriÞed using a Geneclean II Kit (Qbio- a loop of each puriÞed isolate was mixed with a few gene, Carlsbad, CA). drops of sterile saline on a sterilized microscope slide, Cycle sequencing and sequencing of both strands and transferred to a card with a sterile transfer pipette. were performed in 12-␮l volumes on the cleaned PCR All cards were allowed to dry overnight, then stored products, either at CCNY (some yeasts) or at Genewiz at ambient temperatures in plastic resealable bags with Inc. (additional yeasts and all beetles; South PlainÞeld, silica gel, and transported to the City College of New NJ). At CCNY, cycle sequencing was with a Big Dye York (CCNY). Terminator Kit 1.1 (Applied Biosystems, Foster City, We processed the FTA Classic Cards according to CA), using the program 95Њϫ2 min /55Њϫ45 s /68Њϫ the manufacturerÕs instructions: 2-mm dots were 2 min, (95Њϫ2 min/55Њϫ45 s/68Њϫ5 min) ϫ 37 cycles, punched from the cards, washed three times with FTA 94Њϫ45 s/55Њϫ45 s /68Њϫ5 min, followed by sequenc- PuriÞcation Reagent, washed twice with TE buffer ing on an ABI Prism Genetic Analyzer. (pH 8.0) and dried for use in polymerase chain reac- Resulting sequences were submitted to GenBank tion (PCR) reactions. The target segment for the yeast (http://www.ncbi.nlm.nih.gov/Genbank/). We used Ϸ was 600 (or 850) base pairs (bp) of the D1/D2 the NIH BLAST database (http://www.ncbi.nlm.nih. domain of the large subunit ribosomal RNA gene (LSU gov/BLAST) to locate sequences similar to those of rDNA). The primers were LS1 (AGT ACC CGC TGA the yeasts, and to match larval sequences with our ACT TAA G) and either LR3 (CCG TGT TTC AAG previously acquired sequences from adult cerambyc- ACG GG) or LR5 (TCC TGA GGG AAA CTT CG). ids reared at the same site (Berkov et al. 2007; Each 50-␮l PCR reaction included 0.25-␮l GoTaq DNA GenBank accession numbers AF466934ÐAF466981, Polymerase (Promega, Madison, WI), 10-␮l reaction DQ346684ÐDQ346712). buffer, 6-␮l MgCl ,2␮l of each primer (10 ␮M), 4-␮l 2 Yeast SEM. To create reference images of yeast cells dNTP (10 mM), 26-␮l water, and a 2-mm dot from an for comparison with the TEM images, we prepared FTA Classic Card. Sequences were ampliÞed with an yeast isolates for scanning electron microscopy MJR DNA Engine Thermal Cycler (Bio-Rad Labora- tories Ltd., Mississauga, ON, Canada), using the pro- (SEM). Yeast isolates identiÞed previously as YP1 and Њϫ Њϫ Њϫ Њϫ YP4 (Table 1) were cultured for 48 h at 28ЊC, and 100 gram 95 5 min, (95 1 min/50 1 min/72 1 ␮ min) ϫ 35 cycles, 72Њϫ10 min. l of each Þxed in 4% glutaraldehyde (equal volumes The target segment for beetle larvae was Ϸ600 bp of of 8% glutaraldehyde and 0.2-M cacodylate buffer, pH ϫ the mitochondrial gene cytochrome oxidase I (COI). 7.2). Samples were iced 1 h, and 1 ml of each We used universal primers designed for a previous solution was added to a chilled vial containing 10 drops ϫ cerambycid study (Berkov 2002): L4 (GCT ACT ATA of 2% osmium tetraoxide. Vials were iced 5 min; cells ATT ATT GCT GTA CC) and H8 (CCA ATGCAC then were Þltered on a 25-mm polycarbonate Þlter TAA TCTGCC ATA TTA GA). When we were not (0.22-␮m pore size). Each Þlter was covered with a able to amplify the entire target segment, we used second, moist Þlter; the two Þlters were clamped be- various primers (L4Pan (GCA ACA ATA ATT ATT tween O-rings and rinsed in cold distilled water 3Ð5 GCA GTC CC), L5 ceram (GTT CTT TCT ATA GGA times ϫ 3 min. Cells then were dehydrated in an GCC GTA TTT GC), and L5, H1, and H6M from ethanol series (30Ð100% ϫ 5 min each). Filters were Berkov 2002) to amplify shorter, overlapping frag- rinsed in amyl acetate twice ϫ 5 min. For critical point ϫ ments. Each 40-␮l PCR reaction included 0.4-␮l drying, cells were rinsed seven times in liquid CO2 GoTaq DNA Polymerase, 4-␮l reaction buffer, 4-␮l 5 min. Filters were mounted on SEM pins by using ␮ ␮ ␮ Њ ϫ MgCl2,4 l of each primer (10 M), 4- l dNTP (10 doubled coated carbon tape, stored at 60 C 24 h, and mM), 20-␮l water, and a 2-mm dot from an FTA Classic coated with gold and palladium in a Denton Vacuum Card. The program was 95Њϫ5 min, (95Њϫ15 s/48Њϫ Desk II (Denton Vacuum, Moorestown, NJ). Yeast 10 s/72Њϫ30 s) ϫ 35 cycles, 72Њϫ10 min. All PCR cells were observed subsequently on a Zeiss DSM 940 February 2012 CALDERON AND BERKOV:CERAMBYCID BACTERIOCYTES 111 thermionic SEM (Carl Zeiss, Oberkochen, Germany) mens YPN-12, YPN-32); X. pulcher (YPN-25, YPN-97); with a resolution of 5 nm at 30 kV. and P. pubescens (YPN-98); were identical in sequence TEM Analyses of Larval Guts. We processed the to yeast YP4, previously isolated from P. mimetica at seven larval specimens that we were able to identify the same locality (Yeast 4 in Berkov et al. 2007), and (Table 1) for TEM analysis. Before returning to NY, similar in sequence to Candida cerambycidarum (Suh larval head capsules were removed and applied to et al. 2005). Isolates from Palame anceps (YPN-8) and Whatman cards for DNA preservation, and the rest of Periboeum pubescens (YPN-35) were identical or sim- each specimen was preserved in 4% glutaraldehyde/ ilar in sequence to C. endomychidarum, which, like C. paraformaldehyde solution ϫ 24 h. At CCNY larval cerambycidarum, is a member of the C. membranifa- guts were removed and cut into sections Ϸ2 mm long, ciens clade (Suh et al. 2005). Yeast isolates 10 and 19 which were rinsed in PBS ϫ 5 min, then placed in were identical or similar to in sequence to YP1, a yeast chilled 2% osmium tetraoxide solution ϫ 40 min. Sam- previously isolated from P. mimetica, X. pulcher, and ples were rinsed three times with distilled water, then the weevil Piazurus incommodus (Yeast 1 in Berkov et twice in PBS. The specimens then were dehydrated at al. 2007), and similar to C. mycetangii, isolated from the room temperature with ethanol (70, 80, 90, ϫ 1 and mycangia of ambrosia beetles (Kurtzman 2000). Yeast 100% ϫ 2), for 10 min each step). Samples were rinsed isolates 10 and 55 differ from the closest LSU rDNA ϫ twice in propylene oxide 10 min, then placed in the sequence by Ͼ1% and may represent novel species. following propylene oxide: Embed-812 resin (Electron Fig. 2a shows an SEM image of yeast cells from a Microscopy Sciences, HatÞeld, PA) solutions: 2:1, 1:1, previous isolate of YP4; in this study yeasts with iden- ϫ and 1:2, 30 min each. Samples were placed in cap- tical sequences were isolated from Þve frass samples sules containing a mixture of 100% Embed-812 resin (Table 1). The maximum cell length in both YP4 and ϫ with 1-mm DMP-30 accelerator, desiccated 1h,and YP1 was Ϸ2 ␮m, with YP4 slightly smaller than YP1. In Њ Ϸ dried at 60 C for 48 h. Resin blocks were sectioned TEM images yeast cells easily were identiÞed and using a Sorvall Porter-Blum MT-2B Ultra-Microtome distinguished from bacteria by their thick walls and (Ivan Sorvall Inc., Newton, CT). Ribbons were col- nuclei (Fig. 2b). Yeast cells were not located in the gut lected on metal grids and stained for no Ͻ24hin ϫ lumens of the three small specimens of Palame, but uranyl acetate, then in lead citrate 10 min. Samples remained in the gut lumens of all four larger speci- from the proximal midgut (Fig. 1e) were observed mens. In specimens of Periboeum pubescens (YPN-35, under the Zeiss EM 902 thermionic TEM (Carl Zeiss) YPN-98), yeast cells were associated with fragments of at magniÞcations ranging from 3000X to 85,000X, and xylem cells (not shown), and were sometimes budding imaged with an SIS MegaView III camera (Olympus, (Fig. 2c). Bacterial cells also were present in the gut Mu¨ nster, Germany). lumens of three of the four larger specimens (Fig. 2d); they were also sometimes associated with fragments of Results thick-walled xylem cells (Fig. 2e). Abundant dividing Genetic Identification of Neotropical Larvae and cocci were present in the gut lumen of Xylergates Yeasts. Sequence data enabled us to identify seven pulcher (YPN-25; Fig. 2d), a specimen with intracel- cerambycid larvae (Table 1, GenBank Accession num- lular cocci (see Bacteriocytes in Midgut and Fat Body bers JN204238ÐJN204244). All larval sequences were Tissue). identical to, or differed by Ͻ0.4% from, reference COI Larval Midgut Anatomy. Like other insects sequences acquired from sympatric individuals; this is (Billingsley and Lehane 1996), the cerambycid consistent with our expectations for intraspeciÞc di- midgut epithelium terminates in a brush border of vergences in these beetles (Berkov 2002, Berkov et al. microvilli lining the gut lumen (Mv, Figs. 3a, 3c). 2007). The larvae included Palame anceps (Bates) The epithelium, consisting of a single layer of epi- (N ϭ 1, Fig. 1a); Palame crassimana Bates “olivaceous” thelial cells, rests upon a continuous basement form (N ϭ 2, Fig. 1b); Xylergates pulcher Lane (N ϭ membrane (BM). The epithelium includes colum- 2, Fig. 1c); and Periboeum pubescens (Olivier) (N ϭ 2, nar (Figs. 3aÐc), regenerative (R, Fig. 3a), and Fig. 1d). The latter two species typically are larger endocrine cells (not shown). The epithelial cells than species of Palame (Fig. 1). Species of Palame and appear to be similar to those of a noctuid caterpillar Xylergates belong to the tribe Acanthocinini in the (Levy et al. 2004): the apical portions are rich in subfamily Lamiinae, not thought to host endosymbi- mitochondria (Fig. 3c), the plasma membrane of the otic yeasts (Martin 1987). Periboeum pubescens be- basal portion forms a convoluted basal labyrinth longs to the tribe Elaphidiini in the Cerambycinae, a (BL, Fig. 3b), and the nucleus and numerous vac- subfamily including some species with endosymbi- uoles are found in the middle portion (Fig. 3a). onts. Exterior to the basement membrane is a layer of Yeast Isolated from Frass; Yeast and Bacteria within spongy connective tissue (CT, Figs. 3aÐb) with col- the Gut Lumen. All larval frass and the exuvium lagen Þbrils that serves as a nutrient conduit be- yielded yeast (Table 1). Sequence analysis revealed tween the absorptive midgut cells and fat body cells the presence of Þve distinct strains, all of which were (Smith 1968). This layer includes circular and lon- similar in sequence to yeasts previously isolated from gitudinal muscle Þbers (MF), and associated tra- beetles (GenBank Accession numbers JN204245Ð cheoles (Tr, Figs. 3aÐb). Metabolically active fat JN204253). Isolates from P. crassimana (larval speci- body tissue surrounds the gut (not shown). 112 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1

formed regenerative cells during both digestion and ecdysis (Levy et al. 2004); these may represent epi- thelial cells being shed into the gut lumen. Bacteriocytes in Midgut and Fat Body Tissue. We did not Þnd evidence of intracellular yeast cells in any of the samples studied. Three of the four larger larvae, including both specimens of the lamiine Xylergates pulcher (YPN-25, YPN-97; Table 1) and the ceramby- cine Periboeum pubescens (YPN-98), appeared to have bacteriocytes with intracellular bacteria. In X. pulcher (YPN-25, Fig. 5) and P. pubescens (YPN-98, Fig. 6), intracellular bacteria were dividing within host tis- sues, whereas in X. pulcher (YPN-97, Fig. 6), a putative bacteriocyte with partially degraded bacteria ap- peared to have been released into the gut lumen. In X. pulcher (YPN-25), apparent cocci-type bac- teria (lacking nuclei, with diameter Ϸ0.5 ␮m, dividing by Þssion), were located within single cells amid lobes of fat body tissue (Fig. 5) that adhered to the gut. The bacteriocytes were found consistently in the vicinity of large lipid droplets. Cocci also were dividing within the gut lumen of this specimen (Fig. 2d). In the gut lumen of the second specimen of X. pulcher (YPN-97), we found what appears to be a bacteriocyte shed from midgut epithelial tissue (Fig. 6b), a phenomenon dis- cussed by Schomann (1937) and shown in Gru¨ nwald et al. (2010). It may have been shed along with epi- thelial cells like those shown in Fig. 4. The rod-shaped bacteria appear to be degenerating, with their mem- branes pulling away from the cell walls (Fig. 6d). Periboeum pubescens (YPN-98) had inclusions that we interpret to be bacteria (lacking nuclei, with di- ameters Ͻ1.0 ␮m), in what appear to be the basal portions of midgut epithelial cells (Fig. 6a). We are not positive about the location because of the fragmentary nature of tissues prepared for TEM, but the bacteria appear close to the basement membrane and associ- ated connective tissue, bordering adherent fat body tissue. Some of the putative bacteria appear to be dividing (Figs. 6a, 6c).

Discussion Our results indicate that the cerambycine P. pubes- cens and the lamiine X. pulcher, a member of a sub- Fig. 2. Extracellular gut microbes. (a) SEM image of YP-4, the yeast strain isolated from Þve frass samples (the family thought to lack endosymbiotic gut yeasts, po- arrows show scars from previous budding), (b) TEM image tentially house intracellular microbes. It is ironic that, of a yeast cell from the gut lumen of X. pulcher (YPN-97) whereas previous investigations of cerambycid sym- showing the thick cell wall (CW) and nucleus (N), (c) TEM biosis have focused on midgut yeasts, these microbes image of a budding yeast cell from the gut lumen of P. appear to be bacteria, and in one case they were found pubescens (YPN-98), the arrow indicates budding, (d) TEM within fat body rather than midgut cells. All speci- image of dividing cocci from the gut lumen of X. pulcher mensÑeven those representing conspeciÞc larvaeÑ (YPN-25), (e) TEM image of bacteria and degraded xylem seem to be telling different stories: reiterating the ϭ ϭ cells from P. pubescens (YPN-98), X xylem cells; bars 1.0 variability that led Grunwald et al. (2010) to caution ␮m, (a, b, c, e) and 0.2 ␮m (d). ¨ about the “possible limited signiÞcance when trans- ferring Þndings from one cerambycid to another.” In one specimen of X. pulcher we observed several In this study of four neotropical cerambycid species, elongated strands of epithelial cells that terminate in we recovered evidence of intracellular inclusions that microvilli and appear to extend into the gut lumen we interpret to be bacteria in three of the four spec- (Fig. 4). The cells are not bound by a basement mem- imens representing the two larger species (X. pulcher brane, and intracellular components are not clearly and P. pubescens), but not in the three specimens visible. Epithelial cells are replaced with newly representing the smaller species. Xylergates pulcher February 2012 CALDERON AND BERKOV:CERAMBYCID BACTERIOCYTES 113

Fig. 3. Midgut anatomy of the cerambycid beetle P. pubescens (YPN-35), (a) overview from microvilli to connective tissue, bar ϭ 2.0 ␮m, (b) basal portion of an epithelial cell with convoluted basal labyrinth, bar ϭ 1.0 ␮m, (c) apical portion of an epithelial cell with dense mitochondria, and microvilli with vesicles, bar ϭ 1.0 ␮m; BL ϭ basal labyrinth, BM ϭ basement membrane, CT ϭ connective tissue, Lu ϭ lumen, M ϭ mitochondrion, MF ϭ muscle Þbers, Mv ϭ microvilli, N ϭ nucleus, R ϭ regenerative cell, Tr ϭ tracheole, V ϭ vacuole, Ve ϭ vesicle. was represented by two larval specimens that attempted to screen the proximal midgut samples housed putative bacteria with different morpholo- comprehensively (depending on the size of the gies, embedded within different tissues and, pre- larva, and the quality of sectioning and staining, sumably, with different roles in the insectÕs life 1Ð10 h per specimen). Conventional histology cycles. It is possible that this apparent heterogeneity would be a more appropriate method for section by is a result of our sampling or processing, although we section viewing of the gut. Transient microbes do not normally colonize gut tissues (Dillon and Dillon 2004), but intracellular bac- teria need not be obligate mutualists. Intracellular bacteria can have a facultative, rather than obligate, association with the host (Zientz et al. 2004), and even mutualistic bacteria can be acquired from the envi- ronment, rather than from the parent (Kikuchi et al. 2007). Furthermore the bacteria might be parasitic, in which case the intracellular phase is often transient (Zientz et al. 2004), or commensalistic, or Þtness con- sequences can fall along a continuum with outcome inßuenced by context (Dillon and Dillon 2004). The intracellular organisms documented in this study are not likely to represent virulent pathogens. The larvae didnÕt show obvious symptoms of disease, and Fig. 4. Lobed epithelial tissue in X. pulcher (YPN-97), we would not expect severely compromised larvae to bar ϭ 5.0 ␮m; Mv ϭ microvilli, N ϭ nucleus. survive the stress of 5Ð8 d starvation on sterile, moist- 114 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1

Fig. 5. aÐe. Intracellar cocci in fat body tissue from X. pulcher (YPN-25), 5b is an enlargement of the inset from 5a; bars ϭ 1.0 ␮m; arrows indicate dividing cells, BM ϭ basement membrane, L ϭ lipid droplet. ened Þlter paper (although many of them chewed, the Þrst observation of a potential fat body symbiosis probably ingested, and may have even derived nutri- in a cerambycid beetle. This is the only case in which ents from Þlter paper fragments). we can speculate about a possible function (in lipid Because of the heterogeneity of our results, our metabolism); although fat body cells are involved in working hypothesis is that the bacteria dividing within the synthesis of fat, glycogen, and proteins (Arrese host cells represent facultative mutualistsÑwith the and Soulages 2010), the cocci were consistently lo- caveat that in some cases presumed mutualists turn out cated in the vicinity of lipid droplets (Figs. 5aÐe). to have negative impacts on host Þtness (Gibson and We are not able to identify the cocci or other in- Hunter 2009). The most interesting result of this study tracellular bacteria, but we did isolate bacteria and was the documentation of intracellular, dividing cocci acquire 16S ribosomal RNA (rRNA) sequences from within fat body cells (Fig. 5). Fat body symbioses are the guts or surfaces of larvae collected concurrently well-documented in most cockroaches, a planthopper, with those included in this TEM study (Fig. 1e; A. B., and a bostrichid (Noda 1977, Kleespies et al. 2003, Lo unpublished data). These bacterial isolates, which et al. 2007), but this is, to the best of our knowledge, grew on a medium that had been acidiÞed to optimize February 2012 CALDERON AND BERKOV:CERAMBYCID BACTERIOCYTES 115

Fig. 6. Possible bacteriocytes from P. pubescens (YPN-98) epithelial cells (a, c), and the X. pulcher (YPN-97) gut lumen (b, d). In (a) and (c), arrows indicate dividing cells in the basal portions of the epithelial cells; bar ϭ 1.0 ␮m, BM ϭ basement membrane. In (b) and (d), both the bacteriocyte (b) and enclosed bacteria (d) appear to be disintegrating; arrows show bacterial membranes pulling away from the cell walls; bars ϭ 1.0 ␮m. yeast growth and inhibit bacterial growth, yielded indicated that our protocol favored the growth of a sequences similar or identical to those of bacteria particular yeast, but the results were fully consistent identiÞed from temperate cerambycid pest species with our previous experiments, in which gut isolates (Schloss et al. 2006, Bahar and Demirbag 2007, Geib et from under-bark feeders were very homogenous al. 2009); we do not suggest that these isolates repre- (Berkov et al. 2007). Although the gut yeasts identiÞed sent intracellular bacteria. Thus far the temperate ce- in this study were extracellular, they were not random rambycid Tetropeum castaneum presents the only ev- subsets of yeasts present in the environment; species idence of a consistent association with intracellular of Candida do not appear to be well-represented in bacteria; those bacteria fall within the Sodalis clade of either phylloplane or soil communities (Botha 2006, endosymbionts associated with hosts including tsetse Fonseca and Ina´cio 2006). In a Chilean temperate rain ßies and grain weevils (Gru¨ nwald et al. 2010). forest, Gonza´lez et al. (1989) isolated yeasts from Although we did not locate intracellular yeasts, they decaying wood, and found that species of Candida were surprisingly persistent in the gut lumens of larger were especially common during the initial and Þnal specimens. Occasional budding, observed even 5Ð8 d stages of decay. Concurrent with this study we isolated after the beetle larvae had been collected (Fig. 2c), yeasts YP1 and YP4 from both wood samples and suggests that the cerambycid midgut is a hospitable numerous additional larval guts (A. B., unpublished environment for at least some yeasts. Even free-living data). This is not inconsistent with our suggestion that organisms, if consistently present, may be contributing cerambycids, which represent early visitors to felled to host Þtness (Dillon and Dillon 2004, Kikuchi et al. branches and trees, might disperse yeasts as the larvae 2007); it is certainly possible that yeast contribute feed at the cambium layer (Berkov et al. 2007). xylanases (Suh et al. 2003) that interact synergistically Wood-boring cerambycids appear to have quite with cellulolytic enzymes from other sources. Their variable interactions with yeasts and bacteria; this is consistent isolation from larval frass (Table 1) indi- not really surprising in such a large and diverse beetle cates that some yeast cells survive gut passage and family. The microbes appear to be part of an inter- potentially are dispersed by cerambycids; their ab- acting guild and there may be a gradient of associa- sence from the gut lumens of smaller specimens prob- tions, from intracellular to extracellular, from obligate ably was because of prolonged starvation. The isola- to facultative, and from mutualistic to commensalistic tion of a single strain from each frass sample may have or parasitic. Mann and Crowson (1983) suggest that, 116 ENVIRONMENTAL ENTOMOLOGY Vol. 41, no. 1 in both chrysomelids and cerambycids, ancestral lin- Brune, A., E. Miambi, and J. A. Breznak. 1995. Roles of eages have specialized structures associated with sym- oxygen and the intestinal microßora in the metabolism of biont transmission and the loss of symbionts may be lignin-derived phenylpropanoids and other monoaro- related to changes in feeding habits. matic compounds by termites. Appl. Environ. Microbiol. Future comparative studies of cerambycid symbi- 61: 2688Ð2695. osis should be designed from a phylogenetic perspec- Chararas, C., M.-C. Pignal, G. Vodjdani, and M. Bourgeay- tive. Although we lack a resolved phylogeny, Napp Causse. 1983. Glycosidases and B group vitamins pro- duced by six yeast strains from the digestive tract of (1994) showed that (with the exception of the Asemi- Phoracantha semipunctata larvae and their role in insect nae) cerambycid subfamilies are monophyletic and development. Mycopathologia 83: 9Ð15. fall within two monophyletic clades; this provides Dillon, R. J., and V. M. Dillon. 2004. The gut bacteria of some sampling framework. Optimally, future studies insects: nonpathogenic interactions. Annu. Rev. Entomol. should incorporate larger sample sizes and protocols 49: 71Ð92. for rapid screening. Next generation DNA sequencing Dowd, P. F. 1991. Symbiont-mediated detoxiÞcation in in- promises to be an increasingly time- and cost-effective sect herbivores, pp. 411Ð440. In P. Barbosa, V. A. Krischik, technology for culture-free exploration of the com- and C. G. Jones (eds.), Microbial mediation of plant- plete gut microbiome (Snyder et al. 2009). The sig- herbivore interactions. Wiley, New York. niÞcance of this work justiÞes this investment. Inter- Fonseca, A´ ., and J. Ina´cio. 2006. Phylloplane yeasts, pp. 263Ð actions of xylophagous insects and their gut 301. Biodiversity and ecophysiology of yeasts. Springer, microbiota inform our understanding of their evolu- Berlin Heidelberg, Germany. Geib, S. M., M.D.M. Jimenez-Gasco, J. E. Carlson, M. Tien, tionary ecology, the host ranges of invasive wood- and K. Hoover. 2009. Effect of host tree species on cel- borers and, because the fate of the carbon sequestered lulase activity and bacterial community composition in in senescing trees often depends on colonization by the gut of larval Asian longhorned beetle. Environ. En- heterotrophs, carbon-cycling in forests. tomol. 38: 686Ð699. Gibson, C. M., and M. S. Hunter. 2009. Negative Þtness consequences and transmission dynamics of a heritable Acknowledgments fungal symbiont of a parasitid wasp. Appl. Environ. Mi- We gratefully acknowledge Meredith Blackwell, Sung-Oui crobiol. 75: 3115Ð3119. Suh, and Nhu H. Nguyen (Louisiana State University) for Gonza´lez, A. E., A. T. Martı´nez, G. Almendros, and J. Grin- providing a crash course in beetle gut yeast, Pedro Centeno bergs. 1989. A study of yeasts during the deligniÞcation (Amazon Conservation Association) for cutting the bait and fungal transformation of wood into cattle feed in the branches, helping to harvest larvae in Peru, and the photo- Chilean rain forest. A. van Leeuw. J. Microbiol. 55: 221Ð graph in Fig. 1e, Karina Ramirez (INRENA) for assisting with 236. permits, Jorge Morales (Electron Microscopy Facility, Gru¨ nwald, S., M. Pilhofer, and W. Ho¨ll. 2010. Microbial CCNY) for training O.C. in EM techniques and manuscript associations in gut systems of wood- and bark-inhabiting review, Indira Paddibhatla (CCNY) for helping to interpret longhorned beetles (Coleoptera: Cerambycidae). Syst. the EM results and manuscript review, and Malikqua Lan- Appl. Microbiol. 33: 25Ð34. caster (RCMI Sequencing Facility, CCNY). Thanks also to Hulcr, J., M. Mogia, B. Isua, and V. Novotny. 2007. Host INRENA for permitting Þeld research in Peru. Funding for speciÞcity of ambrosia and bark beetles (Col., Curculion- this project was provided by National Science Foundation idae: Scolytinae and Platypodinae) in a New Guinea rain- RIG 0542276 and PSC-CUNY PSCREG-67538-00 36 (to A.B.) forest. Ecol. Entomol. 32: 762Ð772. and PSC-CUNY CLT Professional Development Incentive Kikuchi, Y., T. Hoskawa, and T. Fukatsu. 2007. Insect-mi- Grants (to O.C.). crobe mutualism without vertical transmission: a stinkbug acquires a beneÞcial gut symbiont from the environment every generation. Appl. Environ. Microbiol. 73: 4308Ð References Cited 4316. Kleespies, R. G., C. Nansen, T. Adouhoun, and A. M. Huger. Arrese, E. L., and J. L. Soulages. 2010. Insect fat body: en- 2003. Ultrastructure and temperature sensitivity of bac- ergy, metabolism, and regulation. Annu. Rev. Entomol. teriomes in Prostephanus truncatus (Horn). IOBC/WPRS 55: 207Ð225. Bahar, A. A., and Z. Demirbag. 2007. 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