Evolution and functional morphology of ptychoidy in Ptyctima (Acari, Oribatida)

Oribatida (Acari, Arachnida) are diverse and abundant in temperate forest litter. As particle feeding saprophages or mycophages, their food is of relatively low quality, which supposedly results in slow movement, prolonged generation time and reduced reproductive potential. Hence, oribatid mites developed a number of different defensive mechanisms. The most complex mechanical defensive mechanism is ptychoidy, where the animals can retract their legs and mouthparts into a secondary cavity in the idiosoma and encapsulate by deflecting the prodorsum. In this state the animals do not exhibit soft membrane but only—through biomineralization—hardened, thick cuticle, and are therefore well protected against many predators. Certain prerequisites have to be met for the evolution of ptychoidy: the coxisternum must be free from solid exoskeletal connections, i.e. embedded in soft membrane; the cuticle of the opisthosoma has to be hardened; the coxisternum must be foldable; and a system able to accommodate huge volume changes is needed. Despite this complexity, ptychoidy evolved three times independently, once in the Ptyctima and twice in the Enarthronota (Mesoplophoridae and Protoplophoridae). We used synchrotron X-ray microtomography (SRµCT), scanning electron microscopy and high-speed videography to investigate the morphological and functional characteristics of ptychoidy in both superfamilies of the Ptyctima (Euphthiracaroidea and Phthiracaroidea). At first glance Euphthiracaroidea and Phthiracaroidea considerably differ in the arrangement of the ventral plates. Phthiracaroidea posses fused anal and adanal (anal valves) as well as fused genital and agenital plates (genital valves). The genital and anal valves are separated from each other and are embedded in soft membrane (anogenital membrane). Euphthiracaroidea posses either completely fused holoventral plates (complete fusion of anal and genital valves) or retain only a suture between anal and genital valves. In both cases the ventral plates are elongated and surrounded by the plicature plates, a sclerotization of the surrounding membrane. The musculature associated with the ventral plates differs accordingly. In Euphthiracaroidea the notogaster lateral compressor connects the notogaster and the junction between the ventral and plicature plates on the complete length and combined with the ventral plate adductor and ventral plate compressor bridges the ventral plates in the area of the preanal apodeme. A simultaneous contraction of these muscles leads to a transmission of forces via the preanal apodeme onto the notogaster resulting in a lateral compression. In Phthiracaroidea on the other hand the notogaster lateral compressor is restricted to the last third of the ventral plates, inserting on the anogenital membrane. A contraction of the notogaster lateral compressor—along with the postanal muscle—leads to a retraction of the temporarily unified ventral plates into the idiosoma. Other differences concern for example the manubrium (present in Euphthiracaroidea, absent in Phthiracaroidea), and the sensillus ridge allowing for an un-pinched stowage of the sensillus in encapsulated state (absent in Euphthiracaroidea, present in Phthiracaroidea). The coxisternal protractor, which is able to act as retractor as well as protractor of the legs, could only be found in the investigated species of Phthiracaroidea, but in none of the species of Euphthiracaroidea. In conclusion, the mode of pressure build-up associated with ptychoidy is functionally different in the two groups of Ptyctima, but morphologically based on the same characters. Molecular data and for example the location of the taenidiophore also suggest a common origin. A comparison to a potential sister group of Ptyctima, the Collohmannioidea, indicates that the mode of pressure build-up of Euphthiracaroidea is ancestral (based on the elongated state of the ventral plates and the associated musculature). This is also affirmed by molecular studies showing that the Phthiracaroidea are a derived group originating from euphthiracaroid ancestors. Further investigations regarding ptychoidy should include the two remaining— unexamined—ptyctime groups, Synichotritiidae (Euphthiracaroidea) and Steganacaridae (Phthiracaroidea), and the two groups that have evolved ptychoidy independently within the Enarthronota, Mesoplophoridae and Protoplophoridae. Especially the two enarthronote groups are of particular interest since they developed ptychoidy on a different phylogenetic basis. External observation of the Mesoplophoridae for example leads to the assumption that their mode of pressure build-up might be similar to that of the Phthiracaroidea. However, anal and genital valves of Mesoplophoridae are embedded in a single ventral plate of partially notogastral origin. In Protoplophoridae the anal and genital valves are not observable externally in encapsulated state. They are covered by cuticular plates that were converted from static notogastral plates into moveable and retractable plates that potentially function as system for the build-up of pressure. These observations support the assumption of a convergent evolution of ptychoidy in the three groups.Hornmilben (Oribatida) sind in der Laubstreu gemäßigter Wälder arten- und zahlreich vertreten. Sie sind detritivor oder fungivor und verzehren diese Nahrung in Form von festen Partikeln. Diese relativ niederwertige Nahrung soll zu einer langsamen Bewegungsweise, einer verlängerten Generationsdauer und einer verminderten Reproduktionsrate führen. Deshalb haben Hornmilben zahlreiche verschiedene und sehr effektive Mechanismen zum Schutz vor Prädation entwickelt. Der wohl komplexeste mechanische Defensivmechanismus ist die Ptychoidie, welche es den Tieren erlaubt ihre Beine und das Gnathosoma in einen sekundären Hohlraum im Idiosoma einzuziehen und mit dem Prodorsum zu verschließen. In diesem eingekapselten Zustand präsentieren die Tiere nach außen hin nur noch feste, durch Sklerotisierung und Biomineralisierung gehärtete, Kutikula und keine weichhäutigen Membranen mehr. Dadurch erlangen sie einen effektiven Schutz vor zahlreichen Fressfeinden. Für die Evolution der Ptychoidie müssen bestimmte Vorbedingen erfüllt sein: das Coxisternum muss frei beweglich sein, darf also mit keiner anderen kutikulären Struktur fest verbunden sein; die Kutikula des Notogaster muss gehärtet sein; das Coxisternum muss zusammenklappbar sein; und es muss ein System geben, welches die mit der Ptychoidie assoziierte Volumenänderung kompensieren kann. Trotz dieser Komplexität ist die Ptychoide vermutlich dreifach unabhängig entstanden: einmal bei den Ptyctima und zweimal innerhalb der Enarthronota (bei den Mesoplophoridae und Protoplophoridae). Zur Untersuchung der morphologischen und funktionellen Merkmale der Ptychoidie in den beiden Überfamilien der Ptyctima (Euphthiracaroidea und Phthiracaroidea), verwendeten wir Synchrotron-Röntgen-Mikrocomputertomographie (SRµCT), Rasterelektronenmikroskopie und Hochgeschwindigkeitsaufnahmen. Schon auf den ersten Blick unterscheiden sich Euphthiracaroidea und Phthiracaroidea in der Ausprägung der Ventralplatten-Morphologie. Die Phthiracaroidea besitzen verschmolzene Anal- und Adanalplatten (Analklappen) sowie verschmolzene Genital- und Agenitalplatten (Genitalklappen). Die Anal- und Genitalklappen sind dabei voneinander abgegrenzt und sind in einer umlaufenden weichhäutigen Membran eingebettet (Anogenitalmembran). Die Euphthiracaroidea besitzen entweder Holoventralplatten (vollständig verschmolzene Anal- und Genitalklappen) oder behalten nur eine feine Naht zwischen den beiden Klappen. In beiden Fällen sind die Platten stark verlängert und sind umrahmt von den Holoventralfalten, die eine Sklerotisierung der Anogenitalmembran darstellen. Die mit den Ventralplatten assoziierte Muskulatur unterscheidet sich dementsprechend ebenfalls. Der Lateralkompressor des Notogaster (nlc) verbindet bei den Euphthiracaroidea über die gesamte Länge den Notogaster mit der Gelenkstelle zwischen den (Holo-) Ventralplatten und Holoventralfalten. Die Gruppe bestehend aus nlc, Ventralplatten-kompressor (vpc) und Ventralplattenadduktor (vpa) überspannt dabei auf Höhe des präanalen Apodems wie eine Brücke die ventralen Platten. Die simultane Kontraktion dieser Muskeln bewirkt eine Übertragung der Kräfte via des präanalen Apodems auf den Notogaster und führt somit zu einer lateralen Kompression desselben. Bei den Phthiracaroidea hat der nlc seinen Ursprung zwar ebenfalls am Notogaster, jedoch nur im letzten Drittel des Körpers und inseriert, anders als bei den Euphthiracaroidea, auf der Anogenitalmembran. Die Kontraktion des nlc führt hier aber in Kombination mit der Kontraktion des postanalen Muskels (poam) zu einem Einziehen der Ventralplatten in das Idiosoma. Weitere Unterschiede betreffen beispielsweise das Manubrium (nur bei den Euphthiracaroidea vorhanden) und eine Nut, die ein Einklemmen des Sensillus’ zwischen Notogaster und Prodorsum im eingekapselten Zustand der Tiere verhindert (in dieser Ausprägung nur bei den Phthiracaroidea vorhanden). Der Protraktor des Coxisternum (csp) kommt nach dem bisherigen Stand unserer Forschung nur bei den Phthiracaroidea vor. Er ist in der Lage sowohl als Pro- wie auch als Retraktor der Beine zu fungieren. Das Druck-Kompensationssystem für die Volumenänderung unterscheidet sich funktionell deutlich zwischen den zwei Gruppen, obwohl zum großen Teil dieselben morphologischen Strukturen involviert sind. Molekulare Untersuchungen und zum Beispiel die Position der Taenidiophore sprechen ebenfalls für einen gemeinsamen Ursprung der beiden Überfamilien. Durch den Vergleich mit einer potentiellen Außengruppe der Ptyctima, den Collohmannioidea, kann angenommen werden, dass die laterale Kompression wie sie bei den Euphthiracaroidea zu finden ist den ursprünglichen Zustand darstellt (basierend zum Beispiel auf der in die Länge gezogenen Form der Ventralplatten und der damit assoziierten Muskulatur). Dies wird auch durch molekulare Studien belegt, die zeigen, dass die Phthiracaroidea eine abgeleitete Gruppe darstellen die ihren Ursprung in den Euphthiracaroidea haben. Weitere Forschungsvorhaben zur Ptychoidie sollten den Fokus auf die zwei verbleibleibenden (bisher nicht untersuchten) Gruppen der Ptyctima, Synichotritiidae und Steganacaridae, sowie die zwei Gruppen, die den Defensivmechanismus Ptychoidie innerhalb der Enarthronota entwickelt haben (Mesoplophoridae und Protoplophoridae), richten. Die beiden Gruppen der Enarthronota sind dabei von besonderem Interesse, da sie die Ptychoidie auf einer gänzlich anderen phylogenetischen Basis evolviert haben. Schon eine äußerliche Betrachtung der Mesoplophoridae lässt den Schluss zu, dass der Druckaufbau ähnlich wie bei den Phthiracaroidea funktioniert. Allerdings sind hier die Anal- und Genitalklappen anders als bei den Phthiracaroidea in eine unpaare ventrale Platte eingebettet, die zum Teil aus Platten des Notogaster aufgebaut ist. Bei den Protoplophoridae sind die Ventralplatten (Anal- und Genitalklappen) durch eine äußerliche Betrachtung eingekapselter Tiere nicht auszumachen. Sie sind durch ehemals statische Platten des Notogaster verdeckt, die zu beweglichen und einziehbaren Strukturen umgewandelt worden sind und jetzt potentiell dem Druckaufbau dienen. Diese äußerliche Betrachtung festigt die Annahme konvergenter Evolution der Ptychoidie bei den drei Gruppen

An automated device for the digitization and 3D modelling of insects, combining extended-depth-of-field and all-side multi-view imaging

Digitization of natural history collections is a major challenge in archiving biodiversity. In recent years, several approaches have emerged, allowing either automated digitization, extended depth of field (EDOF) or multi-view imaging of insects. Here, we present DISC3D: a new digitization device for pinned insects and other small objects that combines all these aspects. A PC and a microcontroller board control the device. It features a sample holder on a motorized two-axis gimbal, allowing the specimens to be imaged from virtually any view. Ambient, mostly reflection-free illumination is ascertained by two LED-stripes circularly installed in two hemispherical white-coated domes (front-light and back-light). The device is equipped with an industrial camera and a compact macro lens, mounted on a motorized macro rail. EDOF images are calculated from an image stack using a novel calibrated scaling algorithm that meets the requirements of the pinhole camera model (a unique central perspective). The images can be used to generate a calibrated and real color texturized 3Dmodel by ‘structure from motion’ with a visibility consistent mesh generation. Such models are ideal for obtaining morphometric measurement data in 1D, 2D and 3D, thereby opening new opportunities for trait-based research in taxonomy, phylogeny, eco-physiology, and functional ecology

Neglected Very Long-Chain Hydrocarbons and the Incorporation of Body Surface Area Metrics Reveal Novel Perspectives for Cuticular Profile Analysis in Insects

Most of our knowledge on insect cuticular hydrocarbons (CHCs) stems from analytical techniques based on gas-chromatography coupled with mass spectrometry (GC-MS). However, this method has its limits under standard conditions, particularly in detecting compounds beyond a chain length of around C40. Here, we compare the CHC chain length range detectable by GC-MS with the range assessed by silver-assisted laser desorption/ionization mass spectrometry (Ag-LDI-MS), a novel and rarely applied technique on insect CHCs, in seven species of the order Blattodea. For all tested species, we unveiled a considerable range of very long-chain CHCs up to C58, which are not detectable by standard GC-MS technology. This indicates that general studies on insect CHCs may frequently miss compounds in this range, and we encourage future studies to implement analytical techniques extending the conventionally accessed chain length range. Furthermore, we incorporate 3D scanned insect body surface areas as an additional factor for the comparative quantification of extracted CHC amounts between our study species. CHC quantity distributions differed considerably when adjusted for body surface areas as opposed to directly assessing extracted CHC amounts, suggesting that a more accurate evaluation of relative CHC quantities can be achieved by taking body surface areas into account

Robotic-assisted pancreatic surgery in the elderly patient: experiences from a high-volume centre

Background: Robotic-assisted pancreatic surgery (RPS) has fundamentally developed over the past few years. For subgroups, e.g. elderly patients, applicability and safety of RPS still needs to be defined. Given prognosticated demographic developments, we aim to assess the role of RPS based on preoperative, operative and postoperative parameters. Methods: We included 129 patients undergoing RPS at our institution between 2017 and 2020. Eleven patients required conversion to open surgery and were excluded from further analysis. We divided patients into two groups; >= 70 years old (Group 1; n = 32) and < 70 years old (Group 2; n = 86) at time of resection. Results: Most preoperative characteristics were similar in both groups. However, number of patients with previous abdominal surgery was significantly higher in patients >= 70 years old (78% vs 37%, p = 70 years old stayed significantly longer at ICU (1.8 vs 0.9 days; p = 0.037), length of hospital stay and postoperative morbidity were equivalent between the groups. Conclusion: RPS is safe and feasible in elderly patients and shows non-inferiority when compared with younger patients. However, prospectively collected data is needed to define the role of RPS in elderly patients accurately. Trial registration Clinical Trial Register: Deutschen Register Klinischer Studien (DRKS; German Clinical Trials Register). Clinical Registration Number: DRKS00017229 (retrospectively registered, Date of Registration: 2019/07/19, Date of First Enrollment: 2017/10/18)

Life as a fortress structure, function, and adaptive values of morphological and chemical defense in the oribatid mite Euphthiracarus reticulatus (Actinotrichida)

Background Oribatid mites are among the primordial decomposer faunal elements and potential prey organisms in soil. Among their myriad morphological defenses are strong sclerotization and mineralization, cuticular tecta, and the “ptychoid” body-form, which allows to attain an encapsulated, seed-like appearance. Most oribatid mites possess a pair of exocrine glands that produce blends of hydrocarbons, terpenes, aromatics, alkaloids and cyanogenic compounds. Many species evolved “holistic” defensive strategies by combining several morphological and chemical traits. Methods We describe the morphological and chemical bases of defense in the ptychoid oribatid Euphthiracarus reticulatus. The functional morphology was investigated with synchrotron X-ray microtomography (SRCT) and high-speed life-radiography. Gland secretions were collected from 20,000 adult specimens, purified and fractionated by preparative capillary gas chromatography (pcGC) and analyzed by gas chromatography / mass spectrometry (GC/MS), high-resolution mass spectrometry (HRMS), and nuclear magnetic resonance spectroscopy (NMR). The adaptive values of morphological and chemical defenses were estimated in bioassays against three predators: a similar-sized gamasid mite (Stratiolaelaps miles, ca. 0.8 mm, with slender chelicera for piercing membranous cuticular regions), and two larger staphylinid beetles, Stenus juno (ca. 7 mm, bearing a harpoon-like sticky labium and sickle-shaped mandibles) and Othius punctulatus (ca. 14 mm, bearing plesiomorphic chewing mandibles). Results The secretions comprised two components: the diterpene -springene and a novel compound with a mass of 276 g/mol eventually elucidated as 2-(but-1-en-1-yl)-4-butylidene-3-(pent-2-en-1-yl)-pentanedial, to which we assign the trivial name -acaridial. Upon attacks by S. juno, E. reticulatus reacted quickly: within 150 ms from the first contact the encapsulation was almost completed less time than the beetle needed to retract the labium and transfer the mite to the mandibles. Chemically-defended specimens of E. reticulatus effectively repelled all predators. After depletion of oil-gland reservoirs, however, O. punctulatus easily fed on the mites while S. miles and S. juno were not able to overcome the morphological barrier of strong cuticle and ptychoid body form. Conclusion Such an effective, holistic defense strategy, involving both morphological and chemical traits, probably carries high resource-costs, but it allows adult euphthiracaroid mites to occupy an almost “enemy-free space” despite the high diversity of predators in soil.(VLID)286348

Life as a fortress – structure, function, and adaptive values of morphological and chemical defense in the oribatid mite Euphthiracarus reticulatus (Actinotrichida)

Background: Oribatid mites are among the primordial decomposer faunal elements and potential prey organisms insoil. Among their myriad morphological defenses are strong sclerotization and mineralization, cuticular tecta, and the “ptychoid” body-form, which allows to attain an encapsulated, seed-like appearance. Most oribatid mites possess a pair of exocrine glands that produce blends of hydrocarbons, terpenes, aromatics, alkaloids and cyanogenic compounds.Many species evolved “holistic” defensive strategies by combining several morphological and chemical traits. Methods: We describe the morphological and chemical bases of defense in the ptychoid oribatid Euphthiracarus reticulatus. The functional morphology was investigated with synchrotron X-ray microtomography (SRμCT) and highspeed life-radiography. Gland secretions were collected from 20,000 adult specimens, purified and fractionated by preparative capillary gas chromatography (pcGC) and analyzed by gas chromatography / mass spectrometry (GC/MS), high-resolution mass spectrometry (HRMS), and nuclear magnetic resonance spectroscopy (NMR). The adaptive values of morphological and chemical defenses were estimated in bioassays against three predators: a similar-sized gamasid mite (Stratiolaelaps miles, ca. 0.8 mm, with slender chelicera for piercing membranous cuticular regions), and two larger staphylinid beetles, Stenus juno (ca. 7 mm, bearing a harpoon-like sticky labium and sickle-shaped mandibles) and Othius punctulatus (ca. 14 mm, bearing plesiomorphic chewing mandibles). Results: The secretions comprised two components: the diterpene β-springene and a novel compound with a mass of 276 g/mol – eventually elucidated as 2-(but-1-en-1-yl)-4-butylidene-3-(pent-2-en-1-yl)-pentanedial, to which we assign the trivial name δ-acaridial. Upon attacks by S. juno, E. reticulatus reacted quickly: within 150 ms from the first contact the encapsulation was almost completed – less time than the beetle needed to retract the labium and transfer the mite to the mandibles. Chemically-defended specimens of E. reticulatus effectively repelled all predators. After depletion of oil-gland reservoirs, however, O. punctulatus easily fed on the mites while S. miles and S. juno were not able to overcome the morphological barrier of strong cuticle and ptychoid body form. Conclusion: Such an effective, holistic defense strategy, involving both morphological and chemical traits, probably carries high resource-costs, but it allows adult euphthiracaroid mites to occupy an almost “enemy-free space” despite the high diversity of predators in soil

Life as a fortress – structure, function, and adaptive values of morphological and chemical defense in the oribatid mite Euphthiracarus reticulatus (Actinotrichida)

Background: Oribatid mites are among the primordial decomposer faunal elements and potential prey organisms in soil. Among their myriad morphological defenses are strong sclerotization and mineralization, cuticular tecta, and the “ptychoid” body-form, which allows to attain an encapsulated, seed-like appearance. Most oribatid mites possess a pair of exocrine glands that produce blends of hydrocarbons, terpenes, aromatics, alkaloids and cyanogenic compounds. Many species evolved “holistic” defensive strategies by combining several morphological and chemical traits. Methods: We describe the morphological and chemical bases of defense in the ptychoid oribatid Euphthiracarus reticulatus. The functional morphology was investigated with synchrotron X-ray microtomography (SRμCT) and high-speed life-radiography. Gland secretions were collected from 20,000 adult specimens, purified and fractionated by preparative capillary gas chromatography (pcGC) and analyzed by gas chromatography / mass spectrometry (GC/MS), high-resolution mass spectrometry (HRMS), and nuclear magnetic resonance spectroscopy (NMR). The adaptive values of morphological and chemical defenses were estimated in bioassays against three predators: a similar-sized gamasid mite (Stratiolaelaps miles, ca. 0.8 mm, with slender chelicera for piercing membranous cuticular regions), and two larger staphylinid beetles, Stenus juno (ca. 7 mm, bearing a harpoon-like sticky labium and sickle-shaped mandibles) and Othius punctulatus (ca. 14 mm, bearing plesiomorphic chewing mandibles). Results: The secretions comprised two components: the diterpene β-springene and a novel compound with a mass of 276 g/mol – eventually elucidated as 2-(but-1-en-1-yl)-4-butylidene-3-(pent-2-en-1-yl)-pentanedial, to which we assign the trivial name δ-acaridial. Upon attacks by S. juno, E. reticulatus reacted quickly: within 150 ms from the first contact the encapsulation was almost completed – less time than the beetle needed to retract the labium and transfer the mite to the mandibles. Chemically-defended specimens of E. reticulatus effectively repelled all predators. After depletion of oil-gland reservoirs, however, O. punctulatus easily fed on the mites while S. miles and S. juno were not able to overcome the morphological barrier of strong cuticle and ptychoid body form. Conclusion: Such an effective, holistic defense strategy, involving both morphological and chemical traits, probably carries high resource-costs, but it allows adult euphthiracaroid mites to occupy an almost “enemy-free space” despite the high diversity of predators in soil

Under pressure: force resistance measurements in box mites (Actinotrichida, Oribatida).

Background Mechanical defenses are very common and diverse in prey species, for example in oribatid mites. Here, the probably most complex form of morphological defense is known as ptychoidy, that enables the animals to completely retract the appendages into a secondary cavity and encapsulate themselves. The two groups of ptychoid mites constituting the Ptyctima, i.e. Euphthiracaroidea and Phthiracaroidea, have a hardened cuticle and are well protected against similar sized predators. Euphthiracaroidea additionally feature predator-repelling secretions. Since both taxa evolved within the glandulate group of Oribatida, the question remains why Phthiracaroidea lost this additional protection. In earlier predation bioassays, chemically disarmed specimens of Euphthiracaroidea were cracked by the staphylinid beetle , whereas equally sized specimens of Phthiracaroidea survived. We thus hypothesized that Phthiracaroidea can withstand significantly more force than Euphthiracaroidea and that the specific body form in each group is key in understanding the loss of chemical defense in Phthiracaroidea. To measure force resistance, we adapted the principle of machines applying compressive forces for very small animals and tested the two ptyctimous taxa as well as the soft-bodied mite . Results Some Phthiracaroidea individuals sustained about 560,000 times their body weight. Their mean resistance was about three times higher, and their mean breaking point in relation to body weight nearly two times higher than Euphthiracaroidea individuals. The breaking point increased with body weight and differed significantly between the two taxa. Across taxa, the absolute force resistance increased sublinearly (with a 0.781 power term) with the animal's body weight. Force resistance of was inferior in all tests (about half that of Euphthiracaroidea after accounting for body weight). As an important determinant of mechanical resistance in ptychoid mites, the individuals' cuticle thickness increased sublinearly with body diameter and body mass as well and did not differ significantly between the taxa. Conclusion We showed the feasibility of the force resistance measurement method, and our results were consistent with the hypothesis that Phthiracaroidea compensated its lack of chemical secretions by a heavier mechanical resistance based on a different body form and associated build-up of hemolymph pressure (defensive trade-off)