Locust gut epithelia do not become more permeable to fluorescent dextran and bacteria in the cold.

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown whether the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts (Locusta migratoria) with fluorescent bacteria before exposing them to -2°C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.

The histology of sutures in chicken skulls: Types, conservation, and ontogeny.

Sutures are fibrous joints that occur between bone elements in vertebrate skulls, where they play a variety of roles including facilitating skull growth and function. In addition, a variety of studies examining sutures from diverse perspectives in many taxa have enabled the determination of anatomical homologs. Surprisingly, one important aspect of sutures-histology-remains unknown in the key model organism of the chicken. To fill this gap in our knowledge, we generated histological sections of six different cranial sutures across a range of developmental stages in embryonic chicken. Despite having a skull that is largely co-ossified or fused as an adult, we found that the types, components, and ontogeny of sutures in chicken skulls are very similar to sutures in other vertebrates. We did, however, find a new transient stage in the ontogeny of sutures between endochondral bone elements, in which one element has ossified and one was still cartilaginous. Moreover, to better understand the morphogenetic events at the onset of suture formation, we compared the developmental histology of six sutures with that of the space between the two ossification centers of the frontal-a location expected to be void of suture structures. We found that the mesenchymal cells in sutures condense and form a middle vascular layer. This was not found to be the case in the space between the two ossifications of the frontal, where instead only osteoid occurs.

Hyperkalaemia, not apoptosis, accurately predicts insect chilling injury.

There is a growing appreciation that insect distribution and abundance are associated with the limits of thermal tolerance, but the physiology underlying thermal tolerance remains poorly understood. Many insects, like the migratory locust (Locusta migratoria), suffer a loss of ion and water balance leading to hyperkalaemia (high extracellular [K+]) in the cold that indirectly causes cell death. Cells can die in several ways under stress, and how they die is of critical importance to identifying and understanding the nature of thermal adaptation. Whether apoptotic or necrotic cell death pathways are responsible for low-temperature injury is unclear. Here, we use a caspase-3 specific assay to indirectly quantify apoptotic cell death in three locust tissues (muscle, nerves and midgut) following prolonged chilling and recovery from an injury-inducing cold exposure. Furthermore, we obtain matching measurements of injury, extracellular [K+] and muscle caspase-3 activity in individual locusts to gain further insight into the mechanistic nature of chilling injury. We found a significant increase in muscle caspase-3 activity, but no such increase was observed in either nervous or gut tissue from the same animals, suggesting that chill injury primarily relates to muscle cell death. Levels of chilling injury measured at the whole animal level, however, were strongly correlated with the degree of haemolymph hyperkalaemia, and not apoptosis. These results support the notion that cold-induced ion balance disruption triggers cell death but also that apoptosis is not the main form of cell damage driving low-temperature injury.

Chilling induces unidirectional solute leak through the locust gut epithelia.

Chill-susceptible insects, like the migratory locust, often die when exposed to low temperatures from an accumulation of tissue damage that is unrelated to freezing (chilling injury). Chilling injury is often associated with a loss of ion balance across the gut epithelia. It has recently been suggested that this imbalance is at least partly caused by a cold-induced disruption of epithelial barrier function. Here, we aimed to test this hypothesis in the migratory locust (Locustamigratoria). First, chill tolerance was quantified by exposing locusts to -2°C and recording chill coma recovery time and survival 24 h post-cold exposure. Longer exposure times significantly increased recovery time and caused injury and death. Ion-selective microelectrodes were also used to test for a loss of ion balance in the cold. We found a significant increase of haemolymph K+ and decrease of haemolymph Na+ concentration over time. Next, barrier failure along the gut was tested by monitoring the movement of an epithelial barrier marker (FITC-dextran) across the gut epithelia during exposure to -2°C. We found a significant increase in haemolymph FITC-dextran concentration over time in the cold when assayed in the mucosal to serosal direction. However, when tested in the serosal to mucosal direction, we saw minimal marker movement across the gut epithelia. This suggests that while cold-induced barrier disruption is present, it is apparently unidirectional. It is important to note that these data reveal only the phenomenon itself. The location of this leak as well as the underlying mechanisms remain unclear and require further investigation.

Experimental evolution of response to anoxia in Drosophilamelanogaster: recovery of locomotion following CO2 or N2 exposure.

Many insects enter coma upon exposure to anoxia, a feature routinely exploited by experimentalists to handle them. But the genetic and physiological bases of anoxic coma induction and recovery are only partially understood, as are the long-term consequences for the animal's performance. We examined three populations of Drosophila melanogaster (designated B) that have been inadvertently under selection for rapid recovery from CO2 exposure for nearly 40 years (around 1000 generations) resulting from routine maintenance practices. We contrasted CO2 and N2 (presumed a less reactive gas) knockdown and recovery times of these B flies with six populations of common ancestry (A and C populations) that were not exposed to CO2 over the same period. We found that B populations showed faster and more consistent locomotor recovery than A or C populations after CO2 knockdown, a result also observed with N2 knockdown. A and C populations showed much higher variance in recovery time after CO2 exposure than after N2 exposure, suggesting gas-specific effects on pathways associated with locomotor recovery. Although these selection treatments result in considerable variation in life history attributes and body size, with the characteristic intermediacy of B populations, their superiority in resistance to gas exposure and locomotor recovery suggests that this is a direct consequence of prior repeated exposure to anoxia, broadly, and CO2, specifically. Hence we describe a powerful new evolutionary model for the genetic and physiological investigation of anoxic coma in insects.