The ovipositor actuation mechanism of a parasitic wasp and its functional implications.

Parasitic wasps use specialized needle-like structures, ovipositors, to drill into substrates to reach hidden hosts. The external ovipositor (terebra) consists of three interconnected, sliding elements (valvulae), which are moved reciprocally during insertion. This presumably reduces the required pushing force on the terebra and limits the risk of damage whilst probing. Although this is an important mechanism, it is still not completely understood how the actuation of the valvulae is achieved, and it has only been studied with the ovipositor in rest position. Additionally, very little is known about the magnitude of the forces generated during probing. We used synchrotron X-ray microtomography to reconstruct the actuation mechanism of the parasitic wasp Diachasmimorpha longicaudata (Braconidae) in four distinct phases of the probing cycle. We show that only the paired first valvulae of the terebra move independently, while the second valvula moves with the metasoma ('abdomen'). The first valvula movements are initiated by rotation of one chitin plate (first valvifer) with respect to another such plate (second valvifer). This is achieved indirectly by muscles connecting the non-rotating second valvifer and the abdominal ninth tergite. Contrary to previous reports, we found muscle fibres running inside the terebra, although their function remains unclear. The estimated maximal forces that can be exerted by the first valvulae are small (protraction 1.19 mN and retraction 0.874 mN), which reduces the risk of buckling, but are sufficient for successful probing. The small net forces of the valvulae on the substrate may still lead to buckling of the terebra; we show that the sheaths surrounding the valvulae prevent this by effectively increasing the diameter and second moment of area of the terebra. Our findings improve the comprehension of hymenopteran probing mechanisms, the function of the associated muscles, and the forces and damage-limiting mechanism that are involved in drilling a slender terebra into a substrate.

Intra-oropharyngeal food transport and swallowing in white-spotted bamboo sharks.

Despite the importance of intraoral food transport and swallowing, relatively few studies have examined the biomechanics of these behaviors in non-tetrapods, which lack a muscular tongue. Studies show that elasmobranch and teleost fishes generate water currents as a 'hydrodynamic tongue' that presumably transports food towards and into the esophagus. However, it remains largely unknown how specific musculoskeletal motions during transport correspond to food motion. Previous studies of white-spotted bamboo sharks (Chiloscyllium plagiosum) hypothesized that motions of the hyoid, branchial arches and pectoral girdle, generate caudal motion of the food through the long oropharynx of modern sharks. To test these hypotheses, we measured food and cartilage motion with XROMM during intra-oropharyngeal transport and swallowing (N=3 individuals, 2-3 trials per individual). After entering the mouth, food does not move smoothly toward the esophagus, but rather moves in distinct steps with relatively little retrograde motion. Caudal food motion coincides with hyoid elevation and a closed mouth, supporting earlier studies showing that hyoid motion contributes to intra-oropharyngeal food transport by creating caudally directed water currents. Little correspondence between pectoral girdle and food motion was found, indicating minimal contribution of pectoral girdle motion. Transport speed was fast as food entered the mouth, slower and step-wise through the pharyngeal region and then fast again as it entered the esophagus. The food's static periods in the step-wise motion and its high velocity during swallowing could not be explained by hyoid or girdle motion, suggesting these sharks may also use the branchial arches for intra-oropharyngeal transport and swallowing.

Effects of lighting schedule during incubation of broiler chicken embryos on leg bone development at hatch and related physiological characteristics.

Providing a broiler chicken embryo with a lighting schedule during incubation may stimulate leg bone development. Bone development may be stimulated through melatonin, a hormone released in darkness that stimulates bone development, or increased activity in embryos exposed to a light-dark rhythm. Aim was to investigate lighting conditions during incubation and leg bone development in broiler embryos, and to reveal the involved mechanisms. Embryos were incubated under continuous cool white 500 lux LED light (24L), continuous darkness (24D), or 16h of light, followed by 8h of darkness (16L:8D) from the start of incubation until hatching. Embryonic bone development largely takes place through cartilage formation (of which collagen is an important component) and ossification. Expression of genes involved in cartilage formation (col1α2, col2α1, and col10α1) and ossification (spp1, sparc, bglap, and alpl) in the tibia on embryonic day (ED)13, ED17, and at hatching were measured through qPCR. Femur and tibia dimensions were determined at hatch. Plasma growth hormone and corticosterone and pineal melatonin concentrations were determined every 4h between ED18.75 and ED19.5. Embryonic heart rate was measured twice daily from ED12 till ED19 as a reflection of activity. No difference between lighting treatments on gene expression was found. 24D resulted in higher femur length and higher femur and tibia weight, width, and depth at hatch than 16L:8D. 24D furthermore resulted in higher femur length and width and tibia depth than 24L. Embryonic heart rate was higher for 24D and 16L:8D in both its light and dark period than for 24L, suggesting that 24L embryos may have been less active. Melatonin and growth hormone showed different release patterns between treatments, but the biological significance was hard to interpret. To conclude, 24D resulted in larger leg bones at hatch than light during incubation, but the underlying pathways were not clear from present data.

Comparative and functional analysis of the digital mucus glands and secretions of tree frogs.

BACKGROUND: Mucus and mucus glands are important features of the amphibian cutis. In tree frogs, the mucus glands and their secretions are crucial components of the adhesive digital pads of these animals. Despite a variety of hypothesised functions of these components in tree frog attachment, the functional morphology of the digital mucus glands and the chemistry of the digital mucus are barely known. Here, we use an interdisciplinary comparative approach to analyse these components, and discuss their roles in tree frog attachment. RESULTS: Using synchrotron micro-computer-tomography, we discovered in the arboreal frog Hyla cinerea that the ventral digital mucus glands differ in their morphology from regular anuran mucus glands and form a subdermal gland cluster. We show the presence of this gland cluster also in several other-not exclusively arboreal-anuran families. Using cryo-histochemistry as well as infrared and sum frequency generation spectroscopy on the mucus of two arboreal (H. cinerea and Osteopilus septentrionalis) and of two terrestrial, non-climbing frog species (Pyxicephalus adspersus and Ceratophrys cranwelli), we find neutral and acidic polysaccharides, and indications for proteinaceous and lipid-like mucus components. The mucus chemistry varies only little between dorsal and ventral digital mucus in H. cinerea, ventral digital and abdominal mucus in H. cinerea and O. septentrionalis, and between the ventral abdominal mucus of all four studied species. CONCLUSIONS: The presence of a digital mucus gland cluster in various anuran families, as well as the absence of differences in the mucus chemistry between arboreal and non-arboreal frog species indicate an adaptation towards generic functional requirements as well as to attachment-related requirements. Overall, this study contributes to the understanding of the role of glands and their secretions in tree frog attachment and in bioadhesion in general, as well as the evolution of anurans.

Stiffness gradients facilitate ovipositor bending and spatial probing control in a parasitic wasp.

Many parasitic wasps use slender and steerable ovipositors to lay eggs in hosts hidden in substrates, but it is currently unknown how steering is achieved. The ovipositors generally consist of three longitudinally connected elements, one dorsal and two ventral valves that can slide along each other. For the parasitic wasp Diachasmimorpha longicaudata, it has been shown that protraction of the ventral valves causes incurving of the ventral valves towards the dorsal one, which results in a change in probing direction. We hypothesize that this shape change is due to differences in bending stiffness along the ovipositor. Alignment of the stiff tip of the dorsal valve with a more flexible ventral S-shaped region situated just behind the tip straightens this S-bend and results in upwards rotation of the ventral tip. We show that the S-shaped region of the ventral valves has a low bending stiffness because it contains soft materials such as resilin. In contrast, the large cross-sectional area of the dorsal valve tip area probably results in a high bending stiffness. Elsewhere, the dorsal valve is less stiff than the ventral valves. Our results support the hypothesis that the interaction between the stiff dorsal valve portion and the more flexible S-shaped region co-determines the configurational tip changes required for steering the ovipositor in any desired direction along curved paths in the substrate. This provides novel insights in the understanding of steering mechanisms of the hymenopteran ovipositor, and for application in man-made probes.

Light-dark rhythms during incubation of broiler chicken embryos and their effects on embryonic and post hatch leg bone development.

There are indications that lighting schedules applied during incubation can affect leg health at hatching and during rearing. The current experiment studied effects of lighting schedule: continuous light (24L), 12 hours of light, followed by 12 hours of darkness (12L:12D), or continuous darkness (24D) throughout incubation of broiler chicken eggs on the development and strength of leg bones, and the role of selected hormones in bone development. In the tibiatarsus and femur, growth and ossification during incubation and size and microstructure at day (D)0, D21, and D35 post hatching were measured. Plasma melatonin, growth hormone, and IGF-I were determined perinatally. Incidence of tibial dyschondroplasia, a leg pathology resulting from poor ossification at the bone's epiphyseal plates, was determined at slaughter on D35. 24L resulted in lower embryonic ossification at embryonic day (E)13 and E14, and lower femur length, and lower tibiatarsus weight, length, cortical area, second moment of area around the minor axis, and mean cortical thickness at hatching on D0 compared to 12L:12D especially. Results were long term, with lower femur weight and tibiatarsus length, cortical and medullary area of the tibiatarsus, and second moment of area around the minor axis, and a higher incidence of tibial dyschondroplasia for 24L. Growth hormone at D0 was higher for 24D than for 12L:12D, with 24L intermediate, but plasma melatonin and IGF-I did not differ between treatments, and the role of plasma melatonin, IGF-I, and growth hormone in this process was therefore not clear. To conclude, in the current experiment, 24L during incubation of chicken eggs had a detrimental effect on embryonic leg bone development and later life leg bone strength compared to 24D and 12L:12D, while the light-dark rhythm of 12L:12D may have a stimulating effect on leg health.

Functional principles of steerable multi-element probes in insects.

Hemipterans, mosquitoes, and parasitic wasps probe in a variety of substrates to find hosts for their larvae or food sources. Probes capable of sensing and precise steering enable insects to navigate through solid substrates without visual information and to reach targets that are hidden deep inside the substrate. The probes belong to non-related taxa and originate from abdominal structures (wasps) or mouthparts (hemipterans and mosquitoes), but nevertheless share several morphological characteristics. Although the transport function clearly differs (egg laying and acquisition of liquid food), the functional demands on the mechanical behaviour of the probe within the substrate tend to be similar. The probe needs to be thin to limit substrate deformation, and long, in order to attain substantial path lengths or depths. We linked the morphology across taxa to the different functional requirements, to provide insights into the biology of probing insects and the evolution of their probes. Current knowledge of insect probes is spread over many taxa, which offers the possibility to derive general characteristics of insect probing. Buckling during initial puncturing is limited by external support mechanisms. The probe itself consist of multiple (3-6) parts capable of sliding along one another. This multi-part construction presumably enables advancement and precise three-dimensional steering of the probe through the substrate with very low net external pushing forces, preventing buckling during substrate penetration. From a mechanical viewpoint, a minimum of three elements is required for 3D steering and volumetric exploration, as realised in the ovipositors of wasps. More elements, such as in six-element probes of mosquitoes, may enhance friction in soft substrates. Alternatively, additional elements can have functions other than 'drilling', such as saliva injection in mosquitoes. Despite the gross similarities, probes show differences in their cross sections, tip morphologies, relative lengths of their elements, and the shape of their interconnections. The hypothesis is that the probe morphology is influenced by the substrate properties, which are mostly unknown. Correlating the observed diversity to substrate-specific functional demands is therefore currently impossible. We conclude that a multipart probe with sliding elements is highly effective for volumetric substrate probing. Shared functional demands have led to an evolutionary convergence of slender multi-element probes in disparate insect taxa. To fully understand 3D probing, it is necessary to study the sensory and material properties, as well as the detailed kinematics and dynamics of the various probes in relation to the nature of the selective pressure originating from the species-specific substrates. Such knowledge will deepen our understanding of probing mechanisms and may support the development of slender, bio-inspired probes.

Force-transmitting structures in the digital pads of the tree frog Hyla cinerea: a functional interpretation.

The morphology of the digital pads of tree frogs is adapted towards attachment, allowing these animals to attach to various substrates and to explore their arboreal habitat. Previous descriptions and functional interpretations of the pad morphology mostly focussed on the surface of the ventral epidermis, and little is known about the internal pad morphology and its functional relevance in attachment. In this study, we combine histology and synchrotron micro-computer-tomography to obtain a comprehensive 3-D morphological characterisation of the digital pads (in particular of the internal structures involved in the transmission of attachment forces from the ventral pad surface towards the phalanges) of the tree frog Hyla cinerea. A collagenous septum runs from the distal tip of the distal phalanx to the ventral cutis and compartmentalises the subcutaneous pad volume into a distal lymph space and a proximal space, which contains mucus glands opening via long ducts to the ventral pad surface. A collagen layer connects the ventral basement membrane via interphalangeal ligaments with the middle phalanx. The collagen fibres forming this layer curve around the transverse pad-axis and form laterally separated ridges below the gland space. The topological optimisation of a shear-loaded pad model using finite element analysis (FEA) shows that the curved collagen fibres are oriented along the trajectories of the maximum principal stresses, and the optimisation also results in ridge-formation, suggesting that the collagen layer is adapted towards a high stiffness during shear loading. We also show that the collagen layer is strong, with an estimated tensile strength of 2.0-6.5 N. Together with longitudinally skewed tonofibrils in the superficial epidermis, these features support our hypothesis that the digital pads of tree frogs are primarily adapted towards the generation and transmission of friction rather than adhesion forces. Moreover, we generate (based on a simplified FEA model and predictions from analytical models) the hypothesis that dorsodistal pulling on the collagen septum facilitates proximal peeling of the pad and that the septum is an adaptation towards detachment rather than attachment. Lastly, by using immunohistochemistry, we (re-)discovered bundles of smooth muscle fibres in the digital pads of tree frogs. We hypothesise that these fibres allow the control of (i) contact stresses at the pad-substrate interface and peeling, (ii) mucus secretion, (iii) shock-absorbing properties of the pad, and (iv) the macroscopic contact geometry of the ventral pad surface. Further work is needed to conclude on the role of the muscular structures in tree frog attachment. Overall, our study contributes to the functional understanding of tree frog attachment, hence offering novel perspectives on the ecology, phylogeny and evolution of anurans, as well as the design of tree-frog-inspired adhesives for technological applications.

Mechanisms of ovipositor insertion and steering of a parasitic wasp.

Drilling into solid substrates with slender beam-like structures is a mechanical challenge, but is regularly done by female parasitic wasps. The wasp inserts her ovipositor into solid substrates to deposit eggs in hosts, and even seems capable of steering the ovipositor while drilling. The ovipositor generally consists of three longitudinally connected valves that can slide along each other. Alternative valve movements have been hypothesized to be involved in ovipositor damage avoidance and steering during drilling. However, none of the hypotheses have been tested in vivo. We used 3D and 2D motion analysis to quantify the probing behavior of the fruit-fly parasitoid Diachasmimorpha longicaudata (Braconidae) at the levels of the ovipositor and its individual valves. We show that the wasps can steer and curve their ovipositors in any direction relative to their body axis. In a soft substrate, the ovipositors can be inserted without reciprocal motion of the valves. In a stiff substrate, such motions were always observed. This is in agreement with the damage avoidance hypothesis of insertion, as they presumably limit the overall net pushing force. Steering can be achieved by varying the asymmetry of the distal part of the ovipositor by protracting one valve set with respect to the other. Tip asymmetry is enhanced by curving of ventral elements in the absence of an opposing force, possibly due to pretension. Our findings deepen the knowledge of the functioning and evolution of the ovipositor in hymenopterans and may help to improve man-made steerable probes.

Functional and evolutionary consequences of cranial fenestration in birds.

Ostrich-like birds (Palaeognathae) show very little taxonomic diversity while their sister taxon (Neognathae) contains roughly 10,000 species. The main anatomical differences between the two taxa are in the crania. Palaeognaths lack an element in the bill called the lateral bar that is present in both ancestral theropods and modern neognaths, and have thin zones in the bones of the bill, and robust bony elements on the ventral surface of their crania. Here we use a combination of modeling and developmental experiments to investigate the processes that might have led to these differences. Engineering-based finite element analyses indicate that removing the lateral bars from a neognath increases mechanical stress in the upper bill and the ventral elements of the skull, regions that are either more robust or more flexible in palaeognaths. Surgically removing the lateral bar from neognath hatchlings led to similar changes. These results indicate that the lateral bar is load-bearing and suggest that this function was transferred to other bony elements when it was lost in palaeognaths. It is possible that the loss of the load-bearing lateral bar might have constrained diversification of skull morphology in palaeognaths and thus limited taxonomic diversity within the group.

A Brief Review of Cephalopod Behavioral Responses to Sound.

Sound is a widely available cue in aquatic environments and is used by many marine animals for vital behaviors. Most research has focused on marine vertebrates. Relatively little is known about sound detection in marine invertebrates despite their abundance and importance in marine environments. Cephalopods are a key taxon in many ecosystems, but their behavioral interactions relative to acoustic stimuli have seldom been studied. Here we review current knowledge regarding (1) the frequency ranges and sound levels that generate behavioral responses and (2) the types of behavioral responses and their biological relevance.

Graded behavioral responses and habituation to sound in the common cuttlefish Sepia officinalis.

Sound is a widely available and vital cue in aquatic environments, yet most bioacoustic research has focused on marine vertebrates, leaving sound detection in invertebrates poorly understood. Cephalopods are an ecologically key taxon that likely use sound and may be impacted by increasing anthropogenic ocean noise, but little is known regarding their behavioral responses or adaptations to sound stimuli. These experiments identify the acoustic range and levels that elicit a wide range of secondary defense behaviors such as inking, jetting and rapid coloration change. Secondarily, it was found that cuttlefish habituate to certain sound stimuli. The present study examined the behavioral responses of 22 cuttlefish (Sepia officinalis) to pure-tone pips ranging from 80 to 1000 Hz with sound pressure levels of 85-188 dB re. 1 µPa rms and particle accelerations of 0-17.1 m s(-2). Cuttlefish escape responses (inking, jetting) were observed between frequencies of 80 and 300 Hz and at sound levels above 140 dB re. 1 µPa rms and 0.01 m s(-2) (0.74 m s(-2) for inking responses). Body patterning changes and fin movements were observed at all frequencies and sound levels. Response intensity was dependent upon stimulus amplitude and frequency, suggesting that cuttlefish also possess loudness perception with a maximum sensitivity around 150 Hz. Cuttlefish habituated to repeated 200 Hz tone pips, at two sound intensities. Total response inhibition was not reached, however, and a basal response remained present in most animals. The graded responses provide a loudness sensitivity curve and suggest an ecological function for sound use in cephalopods.

Flightlessness affects cranial morphology in birds.

Flightless birds belonging to phylogenetically distant clades share several morphological features in the pectoral and pelvic apparatus. There are indications that skull morphology is also influenced by flightlessness. In this study we used a large number of flightless species to test whether flightlessness in modern birds does indeed affect cranial morphology. Discriminant analyses and variation partitioning show evidence for a relationship between skull morphology and the flightless condition in birds. A possible explanation for the change in cranial morphology can be linked to the reduced selective force for light-weight skulls in flightless birds. This makes an increase in muscle mass, and therefore an enlargement of muscle insertion areas on the skull, possible. We also compared the ontogenetic trajectory of Gallus with the adult morphology of a sample of flightless species to see whether the apomorphic features characterizing the skull of flightless birds share the same developmental basis, which would indicate convergent evolution by parallelism. Skull morphology (expressed as principal component scores) of palaeognathous flightless birds (ratites) is dissimilar (higher scores) to juvenile stages of the chicken and therefore seem peramorphic (overdeveloped). Principal component scores of adult neognathous flightless birds fall within the range of chicken development, so no clear conclusions about the ontogenetic trajectories leading to their sturdier skull morphology could be drawn.

Effect of differences in tendon properties on functionality of the passive stay apparatus in horses.

OBJECTIVE: To determine the effect of differences in structural and mechanical tendon properties on functionality of the passive stay apparatus in horses. SAMPLE: 5 forelimbs each from nondwarf Friesians, dwarf Friesians, and ponies. PROCEDURES: Harvested forelimbs were loaded to test the passive stay apparatus. Tendons that stabilize the distal portion of the limb (superficial digital flexor tendon, deep digital flexor tendon, and tendo interosseus [suspensory ligament]) were isolated, and force-elongation data were obtained. Bone lengths, initial tendon lengths, and initial tendon cross-sectional areas were measured, and Young moduli were calculated. A model was used to determine whether joint angles could be explained by these 4 factors only. RESULTS: Dwarf limbs were unable to stand passively under loading because tendons that prevent overextension of the distal limb joints were too long and compliant to prevent over-extension. Tendon properties of limbs of nondwarf Friesians appeared to be intermediate between those of ponies and dwarf Friesians. CONCLUSIONS AND CLINICAL RELEVANCE: Dysfunction of the passive stay apparatus in dwarf Friesians could be related to differences in structural and material properties of the tendons that result in hyperextension of the joints under loading. Nondwarf Friesians had intermediate tendon properties, which might be a breed-specific variation. Results indicated that certain tendon properties were associated with load failure of the stay apparatus and provided additional information about the functionality and requirements of the passive stay apparatus.

The kinematics of feeding and drinking in palaeognathous birds in relation to cranial morphology.

Cranial kinesis is an important feature in avian feeding behaviour and involves the transmission of quadrate movement to the upper bill by the Pterygoid-Palatinum Complex (PPC). The PPC in Palaeognathae is remarkably different from that found in Neognathae. In this study we analyse whether the special morphology of the PPC is an adaptation to the feeding behaviour of the Palaeognathae. Behavioural analyses of the rhea Rhea americana showed that the feeding behaviour of the rhea is typical ;Catch and Throw' behaviour, independent of the size of the food item. Drinking is achieved by a scooping movement followed by a low-amplitude tip-up phase. During feeding rhynchokinetic movements of the upper bill were observed. However, cranial kinesis was limited and may differ from rhynchokinesis in neognathes as a clear bending zone seemed absent. Since the movement patterns are considered very similar to the basic feeding behaviour in neognathous birds it is concluded that the specific morphology of the PPC is not the result of specific functional demands from palaeognathous feeding behaviour.

Cranial kinesis in palaeognathous birds.

Cranial kinesis in birds is induced by muscles located caudal on the cranium. These forces are transferred onto the moveable parts of the skull via the Pterygoid-Palatinum Complex (PPC). This bony structure therefore plays an essential role in cranial kinesis. In palaeognathous birds the morphology of the PPC is remarkably different from that of neognathous birds and is thought to be related to the specific type of cranial kinesis in palaeognaths known as central rhynchokinesis. We determined whether clear bending zones as found in neognaths are present in the upper bill of paleognaths, and measured bending forces opposing elevation of the upper bill. A static force model was used to calculate the opening forces that can be produced by some of the palaeognathous species. We found that no clear bending zones are present in the upper bill, and bending is expected to occur over the whole length of the upper bill. Muscle forces are more than sufficient to overcome bending forces and to elevate the upper bill. The resistance against bending by the bony elements alone is very low, which might indicate that bending of bony elements can occur during food handling when muscles are not used to stabilise the upper bill. Model calculations suggest that the large processi basipterygoidei play a role in stabilizing the skull elements, when birds have to resist external opening forces on the upper bill as might occur during tearing leafs from plants. We conclude that the specific morphology of the palaeognathous upper bill and PPC are not designed for active cranial kinesis, but are adapted to resist external forces that might cause unwanted elevation of the upper bill during feeding.