Macroevolution of venom apparatus innovations in auger snails (Gastropoda; Conoidea; Terebridae)

Cymatiidae are a family of marine gastropods that feed on a variety of other invertebrates. This family is generally not considered venomous, but there are indications that this family is capable of producing a variety of bioactive compounds, especially in the large paired foregut glands anterior to the proboscis. This thesis aims to make a comprehensive update to the knowledge on Cymatiidae, especially focusing on the foregut glands. These foregut glands were previously termed salivary glands; this thesis aims to determine whether these glands also have a role in toxin production. The family of Cymatiidae is investigated using a ‘top-down’ approach, starting with overall observations on biology in Chapter 2, moving to detailed observations on the foregut glands through magnetic resonance imaging (MRI), and detailed molecular investigation of the compounds produced in the foregut glands in Chapters 3 and 4. The protein expression in these glands are investigated through proteomics and transcriptomics to determine the presence of toxic compounds and the investment of these tissues in toxin production. Further, in Chapter 5, bioactivity-guided experiments are used to investigate the effects of the foregut gland extract on invertebrates and mammalian cell lines. Finally, in Chapter 6, the structure of a large pore-forming toxin (echotoxin) is investigated using transmission-electron microscopy. The paradigm-shifting conclusion of this thesis is that the large foregut glands are involved in toxin production. Transcriptomic analysis confirmed the presence of multiple venom homologues based upon consensus analysis of contiguous sequences (contigs) assembled from previously characterized venom glands. Proteomic analysis confirmed the presence of some of these at protein level, with the main product of the foregut glands being echotoxin. Transcriptomic comparison of triton foregut glands with other venom glands and multi-function salivary glands provides further indication that Cymatiidae indeed use their foregut glands in prey capture. Bioactivity assays indicated peptide-based compounds acting on ion channels were present in multiple Cymatiidae foregut glands. Difficulties in purification and sequencing prevented the structure of these compounds being determined. However, bioactivity assays confirmed the presence of at least one compound acting on nicotinic acetylcholine channels and produced by Monoplex pilearis and Monoplex parthenopeus, and one acting on L-type calcium channels is produced by Ranularia pyrum. Echotoxin is the main component of Cymatiidae foregut glands but its three-dimensional structure has not yet been determined. Transmission-electron microscopy confirmed that echotoxin monomers assemble into an octameric pore structure and hinted at a possible pre-pore state. In conclusion, the work in this thesis discusses the definition of venom in light of the findings herein that multiple members of Cymatiidae are capable of producing toxic compounds in their ii foregut glands. Cymatiidae foregut glands therefore should be considered as dual-function glands, filling the role of salivary and venom glands.

Fine structure of the stinger, histology and histochemistry of the venom gland in the scorpion Androctonus amoreuxi (Buthidae)

Heterotopic salivary tissue

Gross morphology and ultrastructure of salivary glands of the mute cicada Karenia caelatata Distant (Hemiptera: Cicadoidea)

Podisus nigrispinus Dallas (Hemiptera: Pentatomidae) is a zoophytophagous insect with a potential for use as a biological control agent in agriculture because nymphs and adults actively prey on various insects by inserting mouthparts and regurgitating the contents of the salivary glands inside the prey, causing rapid paralysis and death. However, the substances found in saliva of P. nigrispinus that causes the death of the prey are unknown. As a first step to identify the component of the saliva of P. nigrispinus, this study evaluated the ultrastructure and cytochemistry of the salivary glands of P. nigrispinus. The salivary system of P. nigrispinus has a pair of principal salivary glands, which are bilobed with a short anterior lobe and a long posterior lobe, and a pair of tubular accessory glands. The principal gland epithelium is composed of a single layer of cells enclosing a large lumen. Epithelial cells of the principal salivary gland vary from cubic to columnar shape, with one or two spherical and well-developed nuclei. Cells of the anterior lobe of the principal salivary gland have an apical surface with narrow, short, and irregular plasma membrane foldings; apical and perinuclear cytoplasm rich in rough endoplasmic reticulum; and mitochondria with tubular cristae. The basal portion of the secretory cells has mitochondria associated with many basal plasma membrane infoldings that are short but form large extracellular canals. Secretory granules with electron-dense core and electron-transparent peripheral are dispersed throughout the cytoplasm. Cells of the posterior lobe of the principal salivary gland are similar to those of the anterior lobe, except for the presence of mitochondria with transverse cristae. The accessory salivary gland cells are columnar with apical microvilli, have well-developed nucleus and cytoplasm rich in rough endoplasmic reticulum, and have secretory granules. Cytochemical tests showed positive reactions for carbohydrate, protein, and acid phosphatase in different regions of the glandular system. The principal salivary glands of P. nigrispinus do not have muscle cells attached to its wall, suggesting that saliva-releasing mechanism may occurs with the participation of some thorax muscles. The cytochemical and ultrastructural features suggest that the principal and accessory salivary glands play a role in protein synthesis of the saliva.

Phospholipases A and B activities of the oriental hornet ( Vespa Orientalis) venom and venom apparatus

Research into the reconstruction of ancient communities in terms of dietary regimes, habitat preferences and ecological interactions of species has focused predominantly on biogeochemistry or ecomorphology alone and not in combination. The Venta Micena site (Orce, Guadix–Baza basin, SE Spain) has an early Pleistocene vertebrate assemblage with exceptional biomolecular preservation. Collagen was successfully extracted from 77 bone samples of 18 species of large mammals, which allowed analyses of carbon- and nitrogen-isotopes. δ 13C, δ 15N and δ 18O ratios combined with ecomorphological indexes provide interesting clues on the autecology and palaeophysiology of extinct species, which help in deciphering aspects of community trophic structure and predator–prey interactions. Specifically, morphometric ratios (e.g., hypsodonty index and relative length of the lower premolar tooth row; [Palmqvist, P., Gröcke, D.R., Arribas, A., Fariña, R. 2003. Paleoecological reconstruction of a lower Pleistocene large mammals community using biogeochemical ( δ 13C, δ 15N, δ 18O, Sr:Zn) and ecomorphological approaches. Paleobiology 29, 204–228.]) allow classifying the ungulates among grazers ( Equus altidens, Bison sp., Praeovibos sp., Hemitragus albus, Hippopotamus antiquus, and Mammuthus meridionalis), mized-feeders ( Soergelia minor and Pseudodama sp.) and browsers ( Stephanorhinus sp. and Praemegaceros cf. verticornis). However, δ 13C values reveal that these ungulates consumed exclusively C 3 plants and significant differences in isotopic values between perissodactyls (monogastric, hindgut fermenters) and ruminants (foregut fermenters) must reflect physiological differences related to their rates of methane production and digestive efficiency. δ 18O ratios allow the interpretation of the dietary water source of these species, suggesting that fallow deer Pseudodama sp., goat H. albus and ovibovine S. minor obtained a significant fraction of their metabolic water from the vegetation consumed. Carnivore species have higher δ 15N values than herbivores, which records the isotopic enrichment expected with an increase in trophic level. However, the unexpectedly high δ 15N values of hippo H. antiquus and muskoxen Praeovibos sp. suggest that these ungulates predominantly consumed aquatic plants and lichens, respectively. Inferences on predator–prey relationships within this ancient community, derived from the dual linear mixing model, indicate resource partitioning among sympatric predators, suggesting that sabre-tooth Megantereon whitei and jaguar Panthera cf. gombaszoegensis were ambushers of forest environments while sabre-tooth Homotherium latidens and wild dog Lycaon lycaonoides were coursing predators in open habitat . The giant, short-faced hyena Pachycrocuta brevirostris scavenged the prey of these hypercarnivores.

Information about predator–prey relationships in aquatic systems can be helpful to improve the conservation management of endangered species. Restoration efforts for the endangered axolotl (Ambystoma mexicanum) involve the creation of refuges to increase the occurrence of suitable conditions for native species. Little is known about the predator–prey interaction between the axolotl and the native crayfish (Cambarellus montezumae) that also inhabits the refuges. To understand this interaction, we designed a set of experimental arenas and investigated both species at various life stages. Our findings suggest a switch in the predator–prey relationship between crayfish and axolotls at lengths between 2.6 and 3.5 cm. Individuals of both species below this size-range serve as prey, whereas larger individuals become predators. The 66.7% of crayfish that were preyed upon by axolotls were of intermediate size, whereas adult crayfish primarily consumed newly hatched axolotls (70%). We discuss the implications of this native species interaction with respect to their habitat conservation.

Podisus distinctus (Hemiptera: Pentatomidae) is a zoophytophagous insect with significant potential for use as a biological control agent in agriculture and forestry because their nymphs and adults actively prey on diverse insect species. The saliva of this insect possesses active substances that cause paralysis and death of the prey. As the first step in identifying compounds of P. distinctus saliva, this study describes the ultrastructure of the salivary glands of this predator. The salivary system of P. distinctus possesses a pair of main salivary glands with a short anterior lobe, a long posterior lobe, and a pair of tubular accessory glands. The main salivary gland of P. distinctus has no associated muscles, suggesting that the saliva-release mechanism occurs with the help of certain thorax muscles. The main salivary gland epithelium has a single layer of cells (varying from cubical to columnar) with cytoplasm rich in rough endoplasmic reticulum, spherical granules of different sizes, a nucleus with a predominance of decondensed chromatin, and nucleolus. The apical cell region has a few short microvilli and the basal region has plasma membrane infoldings. The epithelium of the accessory salivary glands possesses a single-layered epithelium of cubic cells delimiting a narrow lumen. The apical cell region has a high density of microvilli and pleomorphic mitochondria, whereas the central cell region is rich in rough endoplasmic reticulum with a well-developed nucleus and decondensed chromatin. The basal cell region is characterized by the presence of several basal plasma membrane infoldings associated with mitochondria and numerous openings to the hemocoel forming large channels. The ultrastructural characteristics suggest that the main salivary glands and accessory salivary glands play a vital role in protein synthesis for saliva production and that the accessory glands are involved in transport of materials of the hemolymph.

The morphology, assembly, and accumulation sites of rhabdovirus particles in Peregrinus maidis planthoppers infected with a Hawaiian isolate of maize mosaic virus (MMV) were studied. These particles were usually bullet-shaped, but were sometimes bacilliform, and averaged 234 and 247 nm, respectively, in length and 60 nm in width. They were found in most acini of the principal and accessory salivary glands and in brain, nerve ganglia, leg muscle, foregut, midgut, trachea, epidermis, and fat and connective tissues. In most tissues MMV particles accumulated mainly within intracytoplasmic, dilated cisternae that were connected to the perinuclear space. However, in the salivary glands virus particles accumulated mainly in intercellular and extracellular spaces and were found in secretion vesicles. MMV particles appeared to bud from three types of membranes: (i) inner, and rarely outer, nuclear membranes of cells in most tissues examined; (ii) intracytoplasmic membranes, e.g., endoplasmic reticulum in salivary glands; and (iii) plasma membranes of salivary gland cells and nerve axons. The plasma membrane has not been reported previously as a budding site for plant rhabdoviruses, although it is known as a major assembly site for animal rhabdoviruses.

Eurydema spectabilis (Heteroptera: Pentatomidae) has a piercing‐sucking mouth type and feeds on plant sap. In this study, the morphological structure of the salivary glands, alimentary canal, and Malpighian tubules of E. spectabilis was examined using light and scanning electron microscopy. Salivary glands consist of the principal and accessory salivary glands. In E. spectabilis, digestion begins in the mouth and ends in the anus. Alimentary canal is divided into three parts: foregut, midgut, and hindgut. The foregut consists of pharynx, esophagus, and proventriculus. The esophagus connects to the proventriculus and resembles a narrow tube. The wall of the proventriculus has a recessed structure and is surrounded by a single cylindrical layer of epithelium and muscle. The midgut is divided into three regions: the first, second, and third ventricles (V1‐V3). V1 and V2 consist of single‐layered cylindrical epithelium. V3 contains a single layer of cuboidal epithelium. Gastric caeca were found in the midgut. The hindgut consisted of a pylorus followed by a well‐developed rectum. The wall of the rectum consists of a single‐layer cuboidal epithelium and muscle. Its lumen contains numerous bacteria and uric acid crystals. The pylorus consists of a single‐layered cylindrical epithelium. It is also the origin of Malpighian tubules. Malpighian tubules consist of two regions: proximal and distal. The morphological structure of the salivary glands, alimentary canal, and Malpighian tubules of E. spectabilis, which has not been studied before, was examined and discussed in comparison with other orders. It is also aimed to contribute to future studies.Research HighlightsIn E. spectabilis, the salivary glands are divided into principal and accessory salivary glands.Microvilli and numerous secretory granules were found in Malpighian tubules.Numerous uric acid crystals and bacteria were found in the rectum lumen.

We reported the histological and anatomical analyses of Legnotus limbosus's alimentary and excretory system using stereomicroscope, light and electron microscopy. As a result of the obtained data, the digestive tract of L. limbosus has three main parts: fore, mid, and hindgut. The salivary gland and gastric caeca are structures that assist digestion. The salivary gland is a pair consisting of the principal and accessory salivary glands. The foregut has the pharynx and esophagus. The pharynx structure is located immediately after the mouth and continues with the esophagus as a thin, long canal. The esophagus connects to the ventriculus 1 (V1). The midgut consists of three parts: V1, V2, and V3. The V1 and V2 have a single layer of cylindrical epithelium. However, the proximal and distal parts of V3 have cylindrical epithelium, while the lateral part exhibits a cuboidal form. The ileum and rectum make up the hindgut. The first has a cylindrical epithelium; the second has a squamous epithelium. The two pairs of Malpighian tubules, which are attached to the midgut-hindgut junction, are responsible for excretion and osmoregulation. Crystals with a deltoid shape are seen in the lumen of the Malpighian tubule and the rectum. This study is the first on the digestive and excretory system morphology of the Cydnidae family and will make significant contributions to studies on this subject in the Heteroptera, including this family.

Testing the Toxicofera: Comparative transcriptomics casts doubt on the single, early evolution of the reptile venom system

Denaturing high-performance liquid chromatography (DHPLC) is a relatively new method for separating amplicons in a mixture, and was recently developed for parasite detection in the blue crab Callinectes sapidus. That assay used a peptide nucleic acid (PNA) PCR hybridization blocking probe (PNA–PCR–DHPLC) to decrease the generic PCR bias of dominant templates (the host) in the mixture prior to separation on the DHPLC column, thus enhancing the less abundant parasite DNA. The same assay and rational can be used to investigate predator–prey interactions. However, in ecosystem studies with many predator–prey relationships, development of specific PNA-blocking probes for each predator would be too laborious. Here, we have developed a PCR–DHPLC assay excluding the dominant predator amplicons in a first DHPLC run, followed by re-amplification of the non-predator retention volumes and further separation and characterization in a second DHPLC run. This assay generated data on the specific trophic interactions between the calanoid copepod Limnocalanus macrurus and its prey from a seasonal sampling programme. The assay provides an efficient way for an unbiased screening of predator–prey relationships, and although developed for L. macrurus in this study, the approach has wide applicability for any predator–prey interaction.

Sessile lesion of the retromolar pad