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Review of the gross anatomy and microbiology of the Phasmatodea digestive tract.

Abstract

The sparse descriptions of the stick insect (Phasmatodea) digestive system as reported/provided in the literature are highly contradictory. This paper describes the digestive systems of several families of Phasmatodea (Timematidae, Heteropterygidae, Diapheromeridae, Pseudophasmatidae, and Phasmatidae) plus the gut microbiome of these and one other (Phylliidae) to both verify past findings and provide a general description of the Phasmatodea alimentary canal. The constrictions imposed by this anatomy on phasmid gut microbiology, its connections to recently released Phasmatodea transcriptomes, and how it differs from the anatomy of related orders in the Polyneoptera are discussed.

All Phasmatodea have ridged proventriculi lined or covered with small spines. Anterior projections of the midgut, sometimes described as gastric caeca, are only found in Euphasmatodea and often obscure the proventricuius. We define the cardia as the complex of foregut and midgut tissue where the type II peritrophic matrix is produced. Appendices of the midgut are an autapomorphy for Phasmatodea, but Timema have fewer and larger appendices relative to body size. We suggest caeca-like projections and the loss of large, proventricular teeth are apomorphies of Euphasmatodea. We identify a possible facultative symbiosis in Eucalyptus-feeding species that requires further study.

Key words

Alimentary canal, digestion, microbiome, phasmids, proventriculus, symbioses

Introduction

The order Phasmatodea (leaf and stick insects) consists of approximately 3000 known species often characterized by an elongated body cavity and procryptic camouflage (Azevedo et al. 2013). The relatively short and stout Timematidae are considered the sister group to the Euphasmatodea (Whiting et al. 2003, Terry & Whiting 2005, Friedemann et al. 2012, Gottardo et al. 2012, Tilgner et al. 1999). Recent works suggest Phasmatodea are a monophyletic group based on morphological (Tilgner et al. 1999, Friedemann et al. 2012, Beutel et al. 2013), mtDNA (Plazzi et al. 2011, Bradler et al. 2014), nuclear genomic (Buckley et al. 2009, Bradler et al. 2014), and transcriptomic (Misof et al. 2014) datasets. Autapomorphies include prothoracic defense glands and small, pyriform appendages on the posterior midgut (Tilgner et al. 1999, Beutel et al. 2013) recently demonstrated as having a unique excretory function (Shelomi & Kimsey 2014). The sister order to the Phasmatodea is most likely Embiidina (webspinners) forming a "Eukinolabia" clade, based on external morphology (Wipfler et al. 2011, Friedemann et al. 2012, Beutel et al. 2013), rDNA (Flook & Rowell 1998, Terry & Whiting 2005), expressed sequence tags (Letsch et al. 2012) and transcriptomics (Misof et al. 2014). Future phylogenetic analyses are still required to resolve the relationships of the Polyneopteran orders (Trautwein et al. 2012): in particular, comparisons of internal features are lacking due to the inconsistent anatomical studies.

Literature on phasmid digestive system anatomy is contradictory (Table 1). Common areas of contention are the existence of a proventriculus and gastric caeca. Some works claimed phasmids have one or the other (Heymons 1897, Cameron 1912, Caarels 2011), or neither (Chopard 1949), though never both, and some descriptions are incomplete (Bordas 1897, Clark 1976). Reports differ on whether or not the inner surface of the proventriculus is toothed, and on what counts as teeth: one study claimed that absence of proventricular dentition is an autapomorphy of the Euphasmatodea (Tilgner et al. 1999), based on a distinction between "spinules" and "teeth" ()udd 1948). Those claiming caeca exist all agree they are reduced in size, relative to those of Orthoptera for example, but it remains unclear whether they are differentiated from the midgut tissue. Descriptions and names of the "appendices of the midgut," the Phasmatodea-specific tubules arising from pear-shaped ampules on the posterior midgut, also vary, such as whether or not they are tracheated (Savage 1962). One author referred to them as "gastric caeca" (Azevedo et al. 2013), another as "Malpighian tubules" (Savage 1962). Unhelpful is the fact that these studies are often limited to one to three species each, and do not cover the diversity of Phasmatodea.

Studying the Phasmatodea digestive system is important not only for potential use in systematics, but also in the evolution of their diet: Unlike other Polyneoptera, phasmids are exclusively folivorous (Calderon-Cortes et al. 2012, Azevedo et al. 2013, Beutel et al. 2013). A long-standing question in phasmids is whether or not their digestion is symbiont-dependent. While cellulolytic gut symbionts would certainly facilitate digestion of the phasmids (1) leafy diets (Watanabe & Tokuda 2010), the acidic and moderately oxidizing phasmid digestive tract is not conducive to fermentative digestion (Caarels 2011), and its straight and simple shape suggests it is more likely to be inhabited by transient microbes passing with the diet (Douglas & Beard 1996, Lacey et al. 2007) than symbionts (Dillon & Dillon 2004, Watanabe & Tokuda 2010). Recent transcriptomic assays confirmed that Phasmatodea produce their own plant cell wall degrading enzymes (Shelomi et al. 2014a) in certain sections of the gut, theoretically enabling symbiont independent digestion of compounds like cellulose (Shelomi et al. 2013), but this finding is controversial (Moran et al. 2008, Caarels 2011) and does not address other potential functions of the microbiome, such as in defending against plant secondary toxins (Boone et al. 2013).

In this study we describe the digestive systems of species widely spread across the Phasmatodea phylogenetic tree (Fig. 1). Our goals were to resolve the confusion in the literature, determine whether or not a common digestive system ground pattern exists for the order (especially relative to other Polyneoptera), and identify species differences in the number and placement of the midgut appendices. In addition, we cultured and stored aerobic and anaerobic gut microbes of the phasmids, to identify possible phylogenetic or dietary patterns in microbiota composition, formulate functional hypotheses, and provide microbial strains for future assays testing these hypotheses.

Materials and methods

The following Phasmatodea species were used: Diapherodes gigantea Gmelin, Eurycantha calcarata Lucas, Extatosoma tiaratum Macleay, Medauroidea extradentata Brunner, and Ramulus nematodes De Haan (Phasmatidae), plus Sipyloideasipylus Westwood (Diapheromeridae), Aretaon asperrimus Redtenbacher (Heteropterygidae), Peruphasma schultei Conle & Hennemann and Anisomorpha buprestoides Stoll (Pseudophasmatidae), and Phyllium siccifolium L (Phylliidae). The latter two were only available for microbial assays. Phasmids were reared at room temperature in the Bohart Museum of Entomology, University of California, Davis, on an ad libitum diet of privet (Ligustrum sp.) for Pe. schultei, Eucalyptus citriodora for Ex. tiaratum and D. gigantea, live oak (Quercus sp.) for Eu. calcarata, and of rose (Rosa sp.) or blackberry (Rubus sp.) for all other species. Also examined were wild-caught Timema sp. Scudder (Timematidae) found on Ceanothus sp. from Gates Canyon, Vacaville, CA, USA (38.387811 -122.077398, 464m). All insects were maintained and used as per the University of California, Davis' Institutional Animal Care and Use Committee guidelines (http://safetyservices.ucdavis.edu/ps/a/ IACUC).

For anatomical studies, insects were preserved and dissected in 70% ethanol. Photos were taken from Nikon SMZ2B stereomicroscopes (Chiyoda, Japan), and drawings produced with a camera lucida and Adobe Photoshop[R] CS3 for image processing. For scanning electron microscopy, dissected tissues were preserved in Karnovsky's fixative at 4[degrees]C for at least 24 hours, mounted on stubs, sputter coated in gold with a Peleo Auto Sputter Coater SC-7 (Ted Pella Inc., Redding, CA), and viewed using a Philips XL30 TMP Scanning Electron Microscope (F.E.I. Company, Hillsboro, OR) with iTEM Software (Olympus Soft Imaging Solutions GmbH). The pH of the gut contents of fresh Ar. asperrimus specimens were measured using Hydrion[R]pH paper (Micro Essential Laboratory Inc., Brooklyn, NY) and with an AMANI-650 micro pH electrode (Warner Instruments, Hamden, CT), both of which gave comparable results.

To culture microbes, phasmids were anaesthetized on ice and surface sterilized in two changes of 70% ethanol anda rinse in sterile deionized water. Insects were dissected on parafilm with flame-sterilized forceps and scissors in a biosafety cabinet. Gut sections for large insects or whole guts for small ones were macerated in 1mL of sterile PBS (phosphate buffered saline, pH 7.4) with a micropestle, and samples streaked aseptically onto two plates each of 1/10 Tryptic Soy Agar (Remel, Lenexa, KS) with 1% cyclohexamide for bacteria or Rose Bengal Chloramphenicol Agar (BD, Franklin Lakes, N)) for fungi. One pair of plates per sample was incubated in an anaerobic chamber. All plates were incubated at room temperature. Plates were similarly prepared from samples of leaves collected in a sterile Whirl-Pak[R] bag directly from the plant and macerated in sterile PBS to isolate surface microbes, swabs from the insects' rearing cages to identify potential environmental contaminants, and phasmid eggs to check for vertical microbe transmission as seen in certain Hemiptera (Prado et al. 2009).

[FIGURE 1 OMITTED]

To identify isolated microbe colonies to species, PCR was used to amplify the 16S rRNA gene with the 27F and 1492R primers for bacteria and the D1/D2 domain sequence of 26S rRNA gene using the NL1 forward and NL4 reverse primers for fungi (Golomb et al. 2013). PCR products were purified using a QIAquick PCR Purification kit (Qiagen, Valencia, CA) and products sequenced using the forward primers at the UC Davis DNA Sequencing Facility (http:// dnaseq.ucdavis.edu/, Davis, CA) on a 3730 Capillary Electrophoresis Genetic Analyzer with BigDye Terminator v3.1 Cycle Sequencing chemistry (Applied Biosystems, Foster City, CA). Gene sequences were identified by comparing them to the NCB1 database using BLAST (Altschul et al. 1990). As cultured microbes can be used for later bioprospecting assays of strains for industrial applications, such as biodegradation, biofuels, or pharmaceuticals (Lacey et al. 2007, Poulsen et al. 2011, Sitepu et al. 2013), isolated bacterial and fungal colonies were preserved in 20% glycerol at -80[degrees]C and are publicly available from the Phaff Yeast Culture Collection (phaffcollection. ucdavis.edu).

[FIGURE 2 OMITTED]

Results

1. Generality of phasmid gut morphology.--Of the eleven species available, anatomical studies were performed on nine, all but two (R. nematodes and M. extradentata) from different subfamilies sensu Bradler et al. (2014) (Fig. 1). Though insufficient numbers of all species were available for quantitative comparative analyses, we were still able to address the contradictions in the Phasmatodea literature, as they are primarily qualitative. All species examined (Figs 2a-j) had a similar digestive tract. The oesophagus continues without demarcation into an expandable, transparent crop lined with cuticle and filled with large, green fragments of shredded leaves. The midgut starts within or just before or after the 3rd thoracic segment. Within the midgut lumen, particulate matter is much smaller and oxidized brown. The midgut can be clearly divided into four parts. The "anterior midgut" is pleated externally and internally except for along a single dorso-longitudinal furrow. The number of pleats varies with species but does not correlate with body size: female Eu. calcarata had the most with more than fifty, while female D. gigantea had the least with fewer than ten. The pleating gradually decreases then disappears, becoming a smooth-surfaced "middle midgut," whose length can be greater or less than that of the anterior midgut depending on the species or gender. The" posterior midgut," defined as the region studded irregularly with the appendices (elaborated on later), is followed by a smooth "post posterior midgut." The undifferentiated hindgut starts within the 4th or 5th abdominal segment, marked by the origin of the Malpighian tubules. These number in the dozens in all species, with excretory and calciferous tubules as described in Savage (1962).

[FIGURE 3 OMITTED]

Comprehensive measurements of pH (Table 2) were only done on Ar. asperrimus, however other species showed the same patterns both in our preliminary work and in other literature (Caarels 2011, Monteiro et al. 2014). No significant differences were found between male and female insects. The foregut lumen pH was acidic (3-5) and increased along the midgut, becoming basic (7.5-9) at the appearance of the appendices and returning to acidic (5-7) at the start of the hindgut.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

2. Proventriculus and gastric caeca.--In all species, the crop terminates in a muscular proventriculus, originating at the base of the second or third thoracic segment. Externally along the gut, the junction between crop and proventriculus is unmarked. In most cases, the proventriculus is also partially concealed by part of the midgut, as visualized in Fig. 2a, opening into the midgut lumen with a small valve. Internally, the proventriculus is differentiated from the crop by thirty to sixty longitudinal ridges covered in tiny, chitinous spinules (Fig. 3). This dentition varies across species (Fig. 4): some have a single row of spines along the apex of each ridge, some have several irregular "rows" of spines along the apex, and some have spines in the valleys between the ridges as well. Note that Timema, as reported in Tilgner et al. (1999), also have six large, sclerotized "teeth" in addition to the spined ridges. In nearly all cases, determining the proventriculus' existence requires dissecting away the midgut obscuring it and checking the inside wall for these ridges.

In all phasmids except Timema, small pouches of anterior midgut tissue, sometimes stil I pleated, mark the start of the anterior midgut, obscuring part of the foregut. These pouches are the "gastric caeca" occasionally mentioned in the literature: however, their tissue lacks a differentiated luminal epithelium relative to the anterior midgut, nor do they harbor large quantities of symbiotic microbes, and thus may not be considered "true" caeca (Beadle 1972, Shelomi et al. 2013). They are greatly reduced in size when compared to the caeca of Acrididae (Hodge 1943), never longer than 2 mm except in gigantic species such as Eu. calcarata or D. gigantea. They number in three, six, eight or twelve. A thin membrane connects the midgut to the proventriculus (Fig. 5), with the "gastric caeca" comprising most of the midgut tissue anterior to this membrane. The food bolus is enclosed within a peritrophic matrix (PM), which persists throughout the rest of the gut and into the frass (Caarels 2011). It is produced at the membrane between the proventriculus and the midgut, meaning the complex is a "cardia": a structure comprised of foregut and midgut tissue producing a type II PM as seen in some Diptera and Lepidoptera, as well as the Polyneoptera orders Dermaptera and Embiidina (Boonsriwong et al. 2007, Lehane 1997).

[FIGURE 6 OMITTED]

3. Appendices of the Midgut.--For Euphasmatodea, the posterior midgut always starts between the 3rd and 5th abdominal segments. The posterior midgut is usually shorter than the anterior midgut and about 46mm in length, except in longer species such as females of D. gigantea and R. nematodes. The appendix tubules are long, extending from the midgut towards the posterior end of the insect where they end blindly in the hemolymph. The ampules are less than 1 mm in length and open into the exoperitrophic space of the midgut via a small pore (Shelomi & Kimsey 2014). The number of appendices varies among individuals, but correlates to some extent with gut circumference: The thin males of R. nematodes have about twenty, S. sipylus and the males of P. schultei and D. gigantea have less than fifty, and the large females of D. gigantea and Eu. calcarata have nearly 100 appendices. Our record was a female Eu. calcarata with over 160 appendices. The exception to these patterns is Timema, which have only two pairs of appendices on opposite sides of the body, as reported before (Tilgner et al. 1999, Shelomi & Kimsey 2014). Timema ampules and their openings into the midgut were twice the size of those from Euphasmatodea (Fig. 6). In all cases the appendix tubules are one-third the diameter of the Malpighian tubules and motile: in Euphasmatodea they are coiled like springs, and contract and expand throughout the body cavity (Shelomi & Kimsey 2014). Ampules originate either between oronthe longitudinal muscles of the midgut, which may affect the way they move within the body cavity (de Sinety 1901). In all species, the appendices are heavily tracheated: Branches from midgut tracheal trunks surround the appendix bulbs, and multiple different tracheoles feed each appendix tubule along its length (Fig. 7). These tracheoles supply oxygen both for the muscles surrounding the tubules and responsible for their motion, and for the active excretory and other functions of the appendices themselves.

[FIGURE 7 OMITTED]

4. Culturable Microbes.--Table 3 shows a selection of microbe strains cultured from stick insects, all currently available at the Phaff collection. They are mostly aerobes and facultative anaerobes, primarily in the bacterial orders Actinomycetales, Enterobacteriales, Lactobacillales, Pseudomonadales, and Xanthomonodales. Some of the species found in the gut were also successfully cultured from the leaves the insects fed on or in their environment. No significant differences in microbial composition were found among the gut sections of single species or among different species: variations were likely idiosyncratic to the individual and/or due to variations in pH and plant allelochemical presence (Dillon & Dillon 2004).

Discussion

The span of subfamilies examined in this study, including Timematidae, suggests our results can serve as a generalization for the Phasmatodea, and are a reliable tool to address contradictions and identify and correct errors in previous literature. However, it is possible some of the species in Table 1 not included in our study were correctly described, having lost features otherwise common to the Phasmatodea. Overall gut morphologies were similar to those described in Bordas (1897) and de Sinety (1901), and all our phasmids had proventriculi, in contrast to earlier works. These earlier authors may have overlooked the phasmid proventriculus because it is usually wholly or partially hidden by the midgut, and the border between crop and proventriculus is not clearly visible on the exterior side of the foregut. Proventriculi always contained spiny ridges, including in Ex. tiaratum in contrast to the findings of Caarels (2011). Similar ridges and spines were noted in Mantophasmatodea (Klass et al. 2002) and Embiidina (Lacombe 1971). The robust proventriculi of Phasmatodea shred the large, green leaf sections from the crop into smaller, oxidized particles, whose increased surface area would facilitate enzymatic digestion (Shelomi et al. 2014a,b). Dissection and analysis of the luminal side of the digestive system is necessary to view the macro- and micro-structure of the phasmid proventriculus. Different species showed different patterns of proventricular dentition (Fig. 4), which may be a morphological character of taxonomical significance for Phasmatodea as in other insects (Szinwelski et al. 2009).

Besides Timema, all phasmids had "gastric caeca" at the anterior midgut, although whether this term is appropriate is debatable as the caeca are histologically undifferentiated from the rest of the anterior midgut (Beadle 1972, Shelomi et al. 2013, Shelomi & Kimsey 2014). These midgut projections are connected to the cardia (Fig. 5): a region at the intersection of the foregut and anterior midgut that produces the type II peritrophic membrane. The term "cardia" has also been used to describe the proventriculus itself (King 1988), and may also be confused with the cardial / stomodeal / oesophageal valve between foregut and midgut. Lack of standardized terminology for or clear demarcation between these different regions of the alimentary canal may explain why so much literature on Phasmatodea anatomy appears contradictory (Table 1), and complicates efforts to compare digestive tracts across orders.

In all species the appendices of the midgut were tracheated and extended deep into the posterior end of the insect, terminating blindly in the hemolymph similarly to the primary Malpighian tubules, in contrast to Savage (1962). These organs are not known in any other Polyneopteran (Hodge 1943, Bartel 1947, Walker 1949, Lacombe 1971, Klass et al. 2002) and are likely an autapomorphy of Phasmatodea (Beutel et al. 2013). Timema have only a few, large appendices while (considerably longer) Euphasmatodea species have dozens of smaller bulbs, suggesting the increases in the numbers of appendices are an apomorphy of Euphasmatodea and/or due to allometric differences. Evidence suggests appendices have a unique excretory role (Shelomi & Kimsey 2014). They clearly alkalinize the posterior midgut lumen (Table 2), perhaps via secretion of bicarbonate ions into the lumen by the appendices' cells, which express high levels of carbonic anhydrase as found by chemical tests (Monteiro et al. 2014) and transcriptomics assays (Shelomi et al. 2014a), or by excretion of a yet unidentified compound (Shelomi & Kimsey 2014). The prior increase in pi I in the anterior midgut may be explained by compartmentalized enzymatic digestion of polysaccharides such as cellulose (Monteiro et al. 2014, Shelomi et al. 2014a) or by the presence and handling of plant phenolics and tannins. The increased acidity at the midgut/hindgut junction is due to the influx of uric acid from the Malpighian tubules.

None of the hypothesized sister orders to the Phasmatodea have conclusively" Phasmid like" digestive tracts, especially due to absence of midgut appendices. The detritivorous Embiidina have a nearly straight gut with a single bend after the ileum, an undifferentiated midgut, and a proventriculus larger than the crop covered internally with sclerotized spines (Bartel 1947, Lacombe 1971). The carnivorous Mantophasmatodea also have a large proventriculus "armed with weak, papillose sclerites that terminate in three successive whorls of weakly sclerotized lobes" and an undifferentiated midgut (Klass et al. 2002). The Grylloblattodea have a large proventriculus lined with twelve" longitudinal divisions characterized by rows of flexible (unsclerotized), backwardly directed lamellae" (Walker 1949). The heavily spined and multi-ridged Phasmatodea proventriculus is thus an apomorphic trait that evolved with their obligatorily folivorous diet: breaking down and oxidizing the relatively large leaf fragments swallowed to a small enough size that the endogenous phasmid digestive enzymes (Shelomi et al. 2014a) can act on them. Revised, comparative internal anatomical analyses of several species each of Embiidina, Grylloblattodea, and Mantophasmatodea using modern tools such as electron microscopy or computer tomography would facilitate comparative and systematic work in Polyneoptera.

Insects that depend on microbial fermentation for digestion have diverticulae or enlarged and alkaline fermentation chambers to house the symbionts (Dillon & Dillon 2004). The demonstrated absence of such modifications or of known lignocellulolytic microbes in the phasmid gut supports symbiont independence for phasmid plant cell wall digestion, as previous works hypothesized (Watanabe & Tokuda 2010, Caarels 2011) or suggested using culturing-independent methods (Shelomi et al. 2013). Most phasmid gut and fecal microbes with known insect associations lack the host specificity or phylogenetic congruence seen with true symbionts (Kikuchi et al. 2009). For example the yeast Candida carpophila has been found in Blastophaga psenes (fig wasp), Xestobium plumbeum (bark beetle), Batrocera oleae (olive fly), and ants, as well as general habitats such as fruits, soil, and tree surfaces (Lachance et al. 2011). Lactococcus lactis is commonly found on plant surfaces and dairy environments (Salama et al. 1995). Other ubiquitous or cosmopolitan species found are the bacteria Kluyvera sp., Lactococcus lactis ssp. lactis, Serrada marcescens, and Stenotrophomonas maltophilia; and the yeasts Ctyptococcus ramirezgomezianus, Pichia guilliermondii, and Rhodotonda mucilanginosa (Li et al. 2005, Zacchi & Vaughan-Martini 2002). The known ecology of these microbes suggests they were likely acquired through feeding on plant materials rather than maintained in the gut.

Even if their digestion is non-microbial, however, phasmid gut microfauna can play other, facultative roles such as preventing infection by pathogenic microbes (Mead et al. 1988) or reducing heat shock (Oliver et al. 2010). This could have impacts on conservation: the failure to rear the endangered New Zealand walking stick Dryococelus australis Montrouzier (Buckley et al. 2009, Carlile et al. 2009) in the San Diego and Bucharest Zoos due to development of gastrointestinal disease could be attributed to a change in gut flora (Benyo Andras, pers. com.). In particular, the finding of Serrada marcescens, a monoterpene-degrading bacteria (Boone et al. 2013), in all phasmids fed monoterpene-rich Eucalyptus suggests the microbe may function in these species to reduce the toxicity of the diet: a hypothesis that would explain how such phasmids can consume as many leaves as they do during outbreak years in Australia, where they are a significant cause of defoliation and Eucalypt dieback (Jurskis & Turner 2002). However, this bacteria species is cosmopolitan and was also found in the guts of blackberry-feeding Phyllium, so it is not exclusive to Eucalyptus feeders. Future work comparing the monoterpene-degrading ability of these different strains [Phaff Collection 09-1566, 10 203, 10-255] could show if differences exist between the same species of bacteria found in phasmids with diets varying in monoterpene content. These mutualisms are likely facultative (Moran et al. 2008) given that they are more strongly associated with diet or habitat than species (Thakuria et al. 2010, Ferrari & Vavre 2011) and that we found no evidence of vertical transmission of microbes via eggs, itself unlikely given the hatching behavior of phasmids (Severin & Severin 1911). One can theoretically test if microbes are obligate symbionts via antibiotics, however this is best done with a target microbe in mind (Visotto el al. 2009), and it can be difficult to determine whether reduced fitness in such experiments is due to the absence of microbes or the toxicity of the antibiotic to the insect directly (de Vries et al. 2004). Though these phasmids were lab-reared, and rearing conditions cause rapid changes in gut microbiota (Husseneder et al. 2009), we do not expect digestion in wild phasmids to be any less symbiont independent.

In summary, our analysis spanning multiple subfamilies and using scanning electron microscopy to examine finer details has resolved several inconsistencies in the Phasmatodea literature. In particular, we presume that spiny proventriculi--derived from an omnivorous ancestor and modified for herbivory--are ubiquitous or at least plesiomorphic in Phasmatodea, and "gastric caeca" plus an oesophageal valve are apomorphic for Euphasmatodea. Whether the "gastric caeca" are true caeca or merely small, undifferentiated extensions of the anterior midgut is a semantic matter, but to avoid confusion we propose referring to the entire system as the cardia (Fig. 5). We also propose "spinules" as the name for the smaller proventricular projections in Phasmatodea after Tilgner et al. (1999), to avoid confusion with larger, macroscopic "teeth" as in Timerna and Blattodea (Wigglesworth 1966).

We also hypothesize that the anatomy and microbiology of the Phasmatodea gut reflect the evolutionary pressure exerted by their mimetic camouflage and diet (Azevedo et al. 2013). Having a body cavity so much longer than it is wide precludes the development of the large diverticulae or paunches needed for microbial activity as in Blattodea (Watanabe & Tokuda 2010) or countercurrent flow to increase enzymatic activity time as in Acrididae (Terra 1990). Instead, a spiny proventriculus physically digests the leaves prior to enzymatic digestion in the anterior midgut, whose pleats increase available surface area. Further compensating for the lack of microbes is the unusually large complement of Phasmatodea digestive enzymes, including multiple copies of cellulase and pectinase genes (Shelomi et al. 2014a). This synergism between physical and chemical digestion allows phasmids to survive on a purely herbivorous diet, and even thrive to the levels that cause economic or ecological damage (Jurskis & Turner 2002). Lastly, while we found no evidence for symbiont-mediated plant cell wall digestion, the role of the gut microbiota in Phasmatodea may still play a role in their survival, with potential impacts on the establishment of new species or on conservation efforts.

Acknowledgements

The authors would like to thank Dr. Steve Heydon and the staff of the Bohart Museum of Entomology for insect care, the staff of the Phaff Yeast Culture Collection for maintaining microbe collections, and the Department of Pathology and Laboratory Medicine at UC Davis for scanning electron microscopy. This research was supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1148897, the 11C Davis & Humanities Graduate Research Fellowship in Entomology, and a 2010 grant by the Systematics Research Fund, jointly administered by the Linnaean Society of London and the Systematics Association. These sources had no role in study design or execution, or the writing and submitting of this article.

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(MS, LSK) Dept of Entomology and Nematology, University of California-Davis, 1 Shields Ave, Davis CA 95616, USA. Email: lskimsey@ucdavls.edu

(MS) Department of Entomology, Max-Planck-Institute for Chemical Ecology, Hans- Knoll-Str. 8, 07745 Jena, Germany. Email: mshelomi@ucdavis.edu

(IRS, KLB-M) Dept of Food Science and Technology, University of California- Davis, 1 Shields Ave, Davis CA 95616, USA. Email: klbmills@ucdavis.edu; irsitepu@ucdavis.edu
Table 1. Review of previous Phasmatodea anatomical studies,
including terms given for the midgut appendices. Arranged by
first author. Species listed are given their currently accepted
names. A dash (--) indicates the article does not mention said
organ. PMG = posterior midgut. Prov. = proventriculus. Valve =
oesophageal valve at end of foregut.

Author           Species                          Caeca   Prov.

Azevedo et       Cladomorphus phyllinus Gray       --      Yes
al. 2013

Beadle 1972      Carausius morosus Sinety          --      --

Bordas 1897      Acanthoderus spinosus Gray,       --      Yes
                 Hermarchus pythonius Westwood,
                 Sipyloidea erechtheus Westwood

Caarels 2011     Extatosoma tiaratum Macleay       No      Yes

Cameron 1912     Bacillus rossi Rossi              Yes     No

Chopard 1949     Order Phasmatodea                 No      No

Clark 1976       Extatosoma tiaratum Macleay       --      Yes

de Sinety 1901   Carausius morosus Sinety          Yes     --

Heymons 1897     Bacillus rossi Rossi              Yes     No

Judd 1948        Order Phasmatodea                 --      Yes

Marshall &       Diapheromera femorata Say         No      No
Severin 1906

Monteiro et      Cladomorphus phyllinus Gray       No      Yes
al. 2014

Ramsay 1955      Carausius morosus Sinety          --      --

Rutschke et      Carausius morosus Sinety          No      --
al. 1976

Savage 1962      Carausius morosus Sinety          --      --

Tilgner et       Timema cristinae Vickery          No      Yes
al. 1999

Author           Toothed?    Valve       PMG Tubules + Name Used

Azevedo et          Yes       --        Yes, "gastric caeca-like"
al. 2013

Beadle 1972         --        Yes     Yes, "conical tubular organs"

Bordas 1897         Yes       Yes      Yes, "glandes coniques ...
                                          appendice filiforme"

Caarels 2011        No        --                   --

Cameron 1912        --        Yes     Yes, "conical tubular organs"

Chopard 1949        --        Yes          Yes, "small glands"

Clark 1976          --        --             Yes, "papillae"

de Sinety 1901      --        Yes    Yes, "Appendices de l'intestin
                                                 moyen"

Heymons 1897        --        Yes    Yes, "Anhange des Mitteldarmes"

Judd 1948           Yes       Yes                  --

Marshall &          --        Yes    Yes, "Anhange des Mitteldarmes"
Severin 1906

Monteiro et         --        --            Yes, "appendices"
al. 2014

Ramsay 1955         --        --            Yes, "appendices"

Rutschke et         --        --       Yes, "Glandulae piriformes"
al. 1976

Savage 1962         --        --       Yes, "appendices" are "true
                                            Malpighian tubes"

Tilgner et          Yes       No        Yes, "pyriform, filament
al. 1999         (Spinules                 bearing processes"
                 plus six
                  teeth)

Table 2. pH of the Aretaon asperrimus digestive tract. See
methods. Values given are for mean pH [+ or -] standard
deviation, with ranges in parentheses, based on readings taken at
the exact middle of each segment. AMG=anterior midgut.
HG=hindgut. MMG=middle midgut. PMG=posterior midgut. PPMG=post
posterior midgut.

Sex   n          Crop                 AMG                 MMG

M     10   3.97 [+ or -] 0.5   4.74 [+ or -] 1.2   5.31 [+ or -] 0.9
                (3.5-5)            (3.75-7)              (4-7)
F     13   3.86 [+ or -] 0.6   4.36 [+ or -] 1     5.18 [+ or -] 1.2
                 (3-5)              (3.5-7)           (3.5-7.25)

Sex          PMG                PPMG                 IIG

M     8.61 [+ or -] 0.3   8.06 [+ or -] 0.1   6.84 [+ or -] 1.1
          (8-9.25)            (8-8.13)            (5-8.88)
F     8.57 [+ or -] 0.4   8.65 [+ or -] 0.5    6.5 [+ or -] 0.8
         (7.5-9.13)          (7.63-9.2)           (5-7.25)

Sex       Hemolymph

M     7.18 [+ or -] 0.3
         (6.88-7.75)
F     6.91 [+ or -] 0.1
           (6.6-7)

Table 3. Microbes isolated from the Phasmatodea digestive tract
and deposited in the Phaff Yeast Culture Collection (UCDFST).
Microbes with a [dagger] are of the same species as a strain (listed in
the parenthesis and marked with a [dagger]) isolated from environmental
samples such as the diet. Microbes with a * are likewise but of
the same genus: a longer RNA sequence is needed to determine
whether or not they are the same species. B=Bacteria,
FF=Filamentous Fungi, Y=Yeast.

Insect         Organ         Microbes (UCDFST Strain ID)

Anisomorpha    Whole gut     Y: Candida palmioleophila (10-220),
buprestoides                 Cryptococcus flavescens (10-219),
                             Rhodotorula mucilaginosa (10-221)

Aretaon        Whole gut     B: Clostridium celerecrescens (10-228),
asperrimus                   Clostridium sphenoides (10-230)

                             FF: Acremonium alternatum (10-245)

Diapherodes    Crop          B: Serratia marcescens (10-259)
gigantea
                             Y: Cryptococcus ramirezgomezianus (10-258)

               Midgut        B: Enterobacter cloacea [dagger] (10-257,
                             11-270 [dagger])

               Hindgut       B: Enterobacter cloacea [dagger] (10-263,
                             11-270 [dagger]), Erwinia persiana (10-
                             256), Serratia marcescens (10-255)

Eurycantha     Gastric       B: Brevibacterium oceani like (09-1475),
calcarata      caeca         Kocuriapalustris [dagger] (09-1473, 09-
                             1392 [dagger], 09-1394 [dagger]),
                             Lactococcus lactis lactis (09-1471),
                             Ochrobactrum tritici (09-1469),
                             Pseudomonas putida [dagger] (09-1468, 09-
                             1554 [dagger], 11-253 [dagger]),
                             Stenotrophomonas maltophila (09-1472)

               Midgut        B: Lactococcus lactis lactis (09-1479),
               (anterior)    Stenotrophomonas maltophila (09-1481)

               Midgut        B: Acinetobacter guillouiae * (09-1482,
               (posterior)   09-1390 *, 09-1418 *), Corynebacterium
                             variabile (09-1485), Kluyvera cryocrescens
                             (09-1467), Kocuria palustris [dagger] (09-
                             1487, 09-1392 [dagger], 09-1394 [dagger]),
                             Lactococcus lactis lactis (09-1484)

               Midgut wall   B: Enterobacter asburiae * (11-281,
                             11-276, 11-285, 11-287, 11-277, 11-270 *),
                             Staphylococcus epidermidis (11-286),
                             Stenotrophomonas maltophilia (11-278),
                             Yokenella regensburgei (11-288)

                             Y: Kazachstania unispora (11-274),
                             Kazachstania servazzii (11-273, 11-268,
                             11-272, 11-275)

               Midgut        B: Acinetobacter guillouiae * (09-1462,
               contents      09-1390 *, 09-1418 *), Kluyvera
                             cryocrescens (09-1453, 09-1455),
                             Lactococcus lactis lactis (09-1448, 09-
                             1449, 09-1461), Staphylococcus hominis
                             like (09-1464)

                             FF: Emericella heterothalic (09-1439)

                             Y: Rhodotorula sloofiae (09-1445)

               Hindgut       B: Kluyvera cryocrescens (09-1453,
               contents      09-1455), Kluyvera intermedia (09-1517),
                             Pectobacterium cypripedii (09-1454)

                             FF: Aspergillus sp. (09-1451), Penicillium
                             sp. [dagger] (09-1450, 09-1413 [dagger],
                             ll-250 [dagger])

               Hemolymph     B: Dietzia sp. (09-1541), Kocuria
                             palustris [dagger] (09-1458, 09-1392
                             [dagger], 09-1394 [dagger]),
                             Methylobacterium extroquens (09-1542),
                             Microbacterium testaceum [dagger] (09-
                             1457, 09-1421 [dagger]), Stenotrophomonas
                             maltophila (09-1459, 09-1460),
                             Tsukamurella tyrosinosolvens (09-1456)

Extatosoma     Crop          B: Serratia marcescens (09-1369, 09-1370)
tiaratum
                             Y: Candida carpophila (09-1360, 09-1362)

               Midgut        B: Serratia marcescens (09-1365)

                             Y: Cryptococcus ramirezgomezianus (11-260,
                             11-264, 11-284), Candida carpophila (09-
                             1363)

               Hindgut       B: Serratia marcescens (09-1366)

                             Y: Pichia guillermondii (09-1361)

Medauroidea    Whole gut     Y: Cryptococcus saitoi (12-344)
extradentata

Phyllium       Midgut        B: Serratia marcescens (10-208)
siccifolium
               Hindgut       B: Serratia marcescens (10-203)
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