UC Davis UC Davis Previously Published Works Title Vital staining of the stick insect digestive system identifies appendices of the midgut as novel system of excretion. Permalink https://escholarship.org/uc/item/3j91677h Journal Journal of morphology, 275(6) ISSN 0362-2525 Authors Shelomi, Matan Kimsey, Lynn S Publication Date 2014-06-01 DOI 10.1002/jmor.20243 Peer reviewed eScholarship.org Powered by the California Digital Library University of California JOURNAL OF MORPHOLOGY 275:623–633 (2014) Vital Staining of the Stick Insect Digestive System Identifies Appendices of the Midgut as Novel System of Excretion Matan Shelomi* and Lynn S. Kimsey Bohart Museum of Entomology, Department of Entomology and Nematology, University of California, Davis, California ABSTRACT The stick insects or phasmids (Phamsato- (grasshoppers and crickets), Embioptera (webspin- dea) have a series of pyriform ampulles with long, thin ners), or Notoptera (ice-crawlers and heel-walkers, filaments on the posterior end of their midgut referred formerly Grylloblattidae and Mantophasmatodea, to as the “appendices of the midgut.” Found only in respectively). The phasmid midgut is divided into phasmids, their function had never been determined until now. Their similarity to the Malpighian tubules, two sections based on morphology: the anterior which are ubiquitous insect organs of excretion, sug- third to half of the midgut is heavily pleated and gested a similar function. To differentiate between the folded, whereas the posterior third of the midgut appendices and the Malpighian tubules and compare is studded with a variable number of ampules that functional differences between the two tissue types, terminate with long, thin filaments that run vital staining (the injection of histological stains into toward the posterior of the insect, referred to as living organisms) was done in conjunction with light the “appendices of the midgut” and not found in and scanning electron microscopy in multiple phasmid any of the proposed Phasmid sister orders species. The results showed that the appendices origi- (Lacombe, 1971; Nation, 1983; Klass et al. 2002). nated in the basal phasmids (Timematidae) and grew more numerous in derived species. The appendices Although most authors describe the midgut as stain selectively, notably failing to pick up the indica- having two parts (Cameron, 1912; Chopard, 1949; tors of the two known systems of invertebrate excretory Clark, 1976), undifferentiated regions between the function, indigo carmine and ammonium carmine. pleated and appendices-containing sections and Appendices sequester stains in the ampule portion between the latter and the hindgut are occasion- before eliminating the compounds into the midgut. We ally counted as distinct midgut sections for four conclude by confirming that the appendices do have an sections total (Holtmann and Dorn, 2009). At the excretory function, but one unlike any other known in junction between the midgut and hindgut, as in invertebrates. Their function is likely cation excretion, playing a role in calcium regulation and/or organic most other insects, are over one hundred Malpigh- alkaloid sequestration. The appendices must thus be ian tubules, which are the primary organs of considered distinct organs from the Malpighian excretion. tubules. J. Morphol. 275:623–633, 2014. VC 2013 Wiley The functionality and anatomy of phasmid Mal- Periodicals, Inc. pighian tubules is well known, mostly due to the KEY WORDS: histochemistry; excretion; digestion; insects; phasmatodea Additional Supporting Information may be found in the online ver- sion of this article. INTRODUCTION Contract grant sponsor: National Science Foundation Graduate Research Fellowship; Grant number: 1148897; Contract grant The stick and leaf insects (Phasmatodea), or sponsor: UC Davis & Humanities Graduate Research Fellowship in phasmids, are an enigmatic order of leaf-chewing Entomology (2010–2014); Contract grant sponsor: The System- insects characterized by their long and thin body atics Research Fund [jointly administered by the Linnean Society plan, which places size and shape constraints on of London and the Systematics Association (2010)]. their digestive system. Past anatomical studies of *Correspondence to: Matan Shelomi; Department of Entomology the phasmids (Bordas, 1897; de Sinety, 1901; and Nematology, 1 Shields Ave, Davis, CA 95616. Cameron, 1912; Clark, 1976) reveal their gut is E-mail: [email protected] straight and narrow with no obvious enlargements or diverticulae. Although the foregut and hindgut Received 1 August 2013; Revised 28 October 2013; are typical, the midgut is modified in ways unlike Accepted 1 December 2013. those of the phasmids’ nearest relatives, hypothe- Published online 14 December 2013 in sized by various authors (Flook and Rowell, 1998; Wiley Online Library (wileyonlinelibrary.com). Plazzi et al. 2011) to be either the Orthoptera DOI 10.1002/jmor.20243 VC 2013 WILEY PERIODICALS, INC. 624 M. SHELOMI AND L.S. KIMSEY work of Ramsay (1954, 1955), who developed the always eliminate indigo carmine (Maddrell et al. most commonly used assays for Malpighian tubule 1974), whereas ammonium carmine is sequestered research on a walking stick, Carausius morosus. in the pericardial tissue (Palm, 1952). If indigo Phasmid Malpighian tubules are differentiated carmine stains the appendices of the midgut, their into two types: the superior tubules that function status as “true” Malpighian tubules would be in excretion of nitrogenous wastes plus ion and confirmed. water homeostasis as in other insects, and the In this study, we examined the anatomy of the inferior or “calciferous” tubules that store calcium digestive system of several phasmids to determine (Savage, 1962), possibly for production of the cal- the consistency of their morphologies, in particular cium oxalate layer unique to phasmid eggs (Clark, the number and form of their appendices of the 1958). These inferior tubules only develop in late- midgut to resolve the confusion over these organs instar phasmids, whereas the primary tubules from the literature. We also used vital staining to appear in the embryonic stage (Savage, 1962). identify stains that color the appendices differ- Superior and inferior tubules are grouped into ently than the Malpighian tubules and midgut tis- clusters at a 1:5 ratio, respectively, and open in sue. Such stains would not only facilitate future conical ampullae exactly at the junction between anatomical work on phasmids (as the appendix the midgut and hindgut. tubules are invisible to the naked eye and difficult The literature on the appendices of the midgut, to discern from among the Malpighian tubules), by contrast, is sparse and occasionally contradic- but would also serve as a functional test of the tory. The appendices are the first tubules to appendices’ excretory ability. In particular, vital appear in the embryonic phasmid, developing staining would demonstrate whether the appendi- before the third cuticulogenesis event with supe- ces are part of the indigo carmine (Malpighian rior tubules developing afterwards (Savage, 1962; tubule-like) or ammonium carmine (pericardial tis- Louvet, 1974). To date, no author has determined sue-like) excretory systems. the function of the tubules: even Ramsay never applied his own methods to the appendices, possi- MATERIALS AND METHODS bly due to their small size at one third the diame- ter of Malpighian tubules (Ramsay, 1955). Several Phasmids used were of the following species: ten Ramulus nematodes,15R. artemis, and twenty Medauroidea extraden- authors have referred to the appendices of the tata (Phasmatidae), plus over 100 Aretaon asperrimus (Hetero- midgut as a third type of Malpighian tubule, with pterygidae), and over 50 Peruphasma schultei Savage (1962) claiming they are “true Malpighian (Pseudophasmatidae). Phasmids were reared in cultures main- tubules” because their cellular construction is tained at room temperature in the Bohart Museum of Entomol- identical to the latters’. Savage also claimed the ogy, University of California, Davis, on an ad libitum diet of privet (Ligustrum sp.) for P. schultei only and of roses (Rosa tubules are untracheated and short, ending “a few sp.) for all other species. We also examined ten wild-caught millimeters behind the midgut hindgut junction,” Timema sp. (Timematidae) found on Ceanothus sp. from Mix which contradicts Ramsay’s findings. Their shape Canyon, Vacaville CA. All insects were maintained and used as and similarity to Malpighian tubules has lead per the University of California, Davis’ Institutional Animal many to hypothesize that the appendices are Care and Use Committee guidelines. For simple light microscopy, phasmids were preserved and excretory in nature (de Sinety, 1901), though other dissected in 70% ethanol or in phasmid saline (0.932% NaCl, possibilities include secretion of digestive enzymes, 0.077% KCl, 0.05% CaCl2). Most photos were taken from Nikon storage of symbiotic microbes, and sequestration of SMZ2B stereomicroscopes. Images were also taken using a toxic plant secondary chemicals. Nikon TE2000U fluorescence inverted microscope. For tissue slices, whole insects with longitudinal slits across the body wall One test for excretory function has been vital were fixed in Bouin’s solution for 3 days at room temperature staining: a histological technique where stains are and stored in 70% ethanol. Tissue samples were dissected out, injected into a living insect prior to dissection dehydrated at room temperature in a graded