The Immune System of Spiders

Spiders, as all other arthropods, have an open circulatory system, and their body fluid, the hemolymph, freely moves between lymphatic vessels and the body cavities (see Wirkner and Huckstorf 2013). The hemolymph can be considered as a multifunctional organ, central for locomotion (Kropf 2013), respiration (Burmester 2013) and nutrition, and it amounts to approximately 20 % of a spider’s body weight. Any injury includes not only immediate hemolymph loss but also pathogen attacks and subsequent infections. Therefore spiders have to react to injuries in a combined manner to stop fluid loss and to defend against microbial invaders. This is achieved by an innate immune system which involves several host defence systems such as hemolymph coagulation and the production of a variety of defensive substances (Fukuzawa et al.2008). In spiders, the immune system is localised in hemocytes which are derived from the myocardium cells of the heart wall where they are produced as prohemocytes and from where they are released as different cell types into the hemolymph (Seitz 1972). They contribute to the defence against pathogens by phagocytosis, nodulation and encapsulation of invaders. The humoral response includes mechanisms which induce melanin production to destroy pathogens, a clotting cascade to stop hemolymph loss and the constitutive production of several types of antimicrobial peptides, which are stored in hemocyte granules and released into the hemolymph (Fukuzawa et al.2008) (Fig.7.1). The immune system of spiders is an innate immune system. It is hemolymph-based and characterised by a broad but not very particular specificity. Its advantage is a fast response within minutes to a few hours. This is in contrast to the adaptive immune system of vertebrates which can react to very specific pathogens, thus resulting in much more specific responses. Moreover, it creates an immunological memory during the lifetime of the species. The disadvantage is that it needs more time to react with antibody production, usually many hours to a few days, and needs to be built up during early ontogenesis

Spider Venoms Potentially Lethal to Humans

Spiders have one pair of venom glands, and only a few families have reduced them completely (Uloboridae, Holarchaeidae) or modified them to another function (Symphytognathidae or Scytodidae, see Suter and Stratton 2013). All other 42,000 known spider species (99%) utilize their venom to inject it into prey items, which subsequently become paralysed or are killed. Spider venom is a complex mixture of hundreds of components, many of them interacting with cell membranes or receptors located mainly in the nervous or muscular system (Herzig and King 2013). Spider venom, as it is today, has a 300-million-yearlong history of evolution and adaptation and can be considered as an optimized tool to subdue prey. In Mesothelae, the oldest spider group with less than 100 species, the venom glands lie in the anterior part of the cheliceral basal segment. They are very small and do not support the predation process very effectively. In Mygalomorphae, the venom glands are well developed and fill the basal cheliceral segment more or less completely. Many of these 3,000 species are medium- to large-/very large-sized spiders, and they have created the image of being dangerous beasts, attacking and killing a variety of animals, including humans. Although this picture is completely wrong, it is persistent and contributes considerably to human arachnophobia. The third group of spiders, Araneomorphae or “modern spiders”, comprises 93% of all spider species. The venom glands are enlarged and extend to the prosoma; the openings of the venom ducts are moved from the convex to the concave side of the cheliceral fangs and enlarged as well. These changes save the chelicerae from the necessity of being large, and hence, on the average, araneomorph spiders are much smaller than mygalomorphs. Nevertheless, they possess relatively large venom glands, situated mainly in the prosoma, and may also have rather potent venom

Main Components of Spider Venoms

Venom glands are alreadypresent in theoldes t spider group, the Mesothelae. Theglands lie in the anterior portion of the cheliceral basal segment but are very small, and it is doubtful how much the venom contributes to the predatory success. In mygalomorph spiders, the well-developed venom glands are still in the basal segment of the chelicerae and produce powerful venom that is injected via the cheliceral fangs into a victim. In all other spiders (Araneomorphae), the venom glands have become much larger and reach into the prosoma where they can take up a considerable proportion of this body part. Only a few spiders have reduced their venom glands, either partially or completely (Uloboridae, Holarchaeidae and Symphytognathidae are usually mentioned) or modified them significantly (Scytodidae, see Suter and Stratton 2013). As well as using venom, spiders may also use their chelicerae to overwhelm an item of prey. It is primarily a question of size whether a spider chews up small arthropods without applying venom or if it injects venom first. Very small and/or defenceless arthropods are picked up and crashed with the chelicerae, while larger, dangerous or well-defended items are carefully approached and only attacked with venom injection. Some spiders specialize on prey groups, such as noctuid moths (several genera of bola spiders among Araneidae), web spiders (Mimetidae), ants (Zodarion species in Zodariidae, aphantochiline thomisids, several genera among Theridiidae, Salticidae, Clubionidae and Gnaphosidae) or termites (Ammoxenidae). However, these more or less monophagous species amount only to roughly 2 % of all known spider species, while 98 % are polyphagous. From these considerations, it follows that the majority of spider venoms are not tailored to any given invertebrate or insect group but are rather unspecialized to be effective over a broad spectrum of prey types that spiders naturally encounter

The Cytotoxic Mode of Action of the Venom of Cupiennius salei (Ctenidae)

The venom of the ctenid spider Cupiennius salei (Fig.16.1) is rich in components which belong to different functional groups. Besides low molecular mass compounds, the venom contains several disulphide-rich peptides, also called mini-proteins, which act as neurotoxins on ion channels or as enhancers of neurotoxins. Likewise, a variety of small cytolytic peptides, which destroy membranes very efficiently, and enzymes are present in the venom. Neurotoxins with cytolytic activity, cytolytic a-helical small cationic peptides and enzymes most probably attacking connective tissue and phospholipid membranes cause the overall cytotoxic effect of this venom. Synergistic and enhancing interactions between components enable the spider to achieve a maximum of toxicity with a minimum of venom quantity

Venom of Cupiennius salei (Ctenidae)

The venom of the ctenid spider Cupiennius salei contains a variety of low molecular mass compounds, many small cationic peptides, disulfiderich peptides, and several enzymes. Among the low molecular mass compounds are taurine, histamine, and citric acid. Some small cationic peptides act membranolytically and destroy prokaryotic as well as eukaryotic cells. More than 40 small cationic peptides (SCPs) have been identified and characterized so far, of which several exert strong cytolytic effects and enable other venom compounds to reach their targets. Most of the disulfiderich peptides follow the ICK motifand act as neurotoxins or support the activity of other neurotoxins as enhancers. More than a dozen different Cupiennius salei toxins (CsTx) can be distinguished so far. CsTx-1 is the most abundant and most insecticidal neurotoxin in the venom of C. salei. It is composed of two domains, an N-terminal part exhibiting the ICK motif, which inhibits L-type Ca2+ ion channels, and a highly cationic C-terminal tailadopting an a-helical conformation, which destroys cell membranes. Several CsTx peptides consisting of two chains, connected by two disulfide bonds, enhance in nontoxic concentrations the insecticidal activity of other toxins. Among the enzymes, a highly active hyaluronidase is crucial for destroying tissue and assisting other venom compounds to spread into the target organism. The combined effects of synergistic and enhancing interactions between these components enable C. salei to inject a minimum quantity of venom to achieve maximum toxicity, thus optimizing its venom investment

Defensin Levels in Spider Hemolymph

Spiders, like all arthropods, exclusively rely on an innate immune system localized in the hemocytes to protect against pathogen invasion. In the hemocytes of the wandering spider Cupiennius salei (C. salei), defensin expression was found to be constitutive. Defensins belong to the group of antimicrobial peptides, which appear in most taxonomic groups, and play an essential role in innate immunity. It has further been reported that during the primary immune answer of C. salei, the peptide content of hemocytes changes markedly, which may indicate the release of defensins from the hemocytes. However, no data on the peptide levels in C. salei hemolymph has so far been published. Formerly, the involvement in the primary immune answer was considered the only function of defensins. However, recent findings strongly suggest that the importance of defensins goes far beyond. There is evidence for defensins contributing to the adaptive immune response, to angiogenesis, and furthermore to tissue repair, i.e. to a variety of essential processes in living organisms. To date, only very little is known about the identity of C. salei defensins and their detailed mode of action. The goal of the work presented herein is the identification of hitherto unknown C. salei defensins in hemocytes and the hemolymph. Moreover, the levels of defensin expression under differential conditions are compared by the means of liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Functional differentiation of spider hemocytes by light and transmission electron microscopy, and MALDI-MS-imaging

The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70–80%) and granulocytes (20–30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionization mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

STRUCTURE OF CUPIENNIUS SALEI VENOM HYALURONIDASE Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. FUNCTION OF VENOM HYALURONIDASES Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques