Gas exchange and dive behaviour in the diving beetle Platynectes decempunctatus (Coleoptera: Dytiscidae).

Many aquatic insects use bubbles on the body surface to store and supply O2 for their dives. There are two types of bubbles: air stores, which store O2 gained from air at the surface, and gas gills that allow passive extraction of O2 from water. Many insects using air stores and gas gills return to the surface to replenish their bubbles and, therefore, their requirement for O2 influences dive behaviour. In this study, we investigate gas exchange and dive behaviour in the diving beetle Platynectes decempunctatus that uses a sub-elytral air store and a small compressible gas gill. We measure the PO2 within the air store during tethered dives, as well as the amount of O2 exchanged during surfacing events. Buoyancy experiments monitor the volume of gas in the gas gill and how it changes during dives. We also directly link O2-consumption rate at three temperatures (10, 15 and 20 °C) with dive duration, surfacing frequency and movement activity. These data are incorporated in a gas exchange model, which shows that the small gas gill of P. decempunctatus contributes less than 10% of the total O2 used during the dive, while up to 10% is supplied by cutaneous uptake.

The critical thermal maximum of diving beetles (Coleoptera: Dytiscidae): a comparison of subterranean and surface-dwelling species.

Thermal tolerance limits in animals are often thought to be related to temperature and thermal variation in their environment. Recently, there has been a focus on studying upper thermal limits due to the likelihood for climate change to expose more animals to higher temperatures and potentially extinction. Organisms living in underground environments experience reduced temperatures and thermal variation in comparison to species living in surface habitats, but how these impact their thermal tolerance limits are unclear. In this study, we compare the thermal critical maximum (CTmax) of two subterranean diving beetles (Dytiscidae) to that of three related surface-dwelling species. Our results show that subterranean species have a lower CTmax (38.3-39.0°C) than surface species (42.0-44.5°C). The CTmax of subterranean species is ∼10°C higher than the highest temperature recorded within the aquifer. Groundwater temperature varied between 18.4°C and 28.8°C, and changes with time, depth and distance across the aquifer. Seasonal temperature fluctuations were 0.5°C at a single point, with the maximum heating rate being ∼1000x lower (0.008°C/hour) than that recorded in surface habitats (7.98°C/hour). For surface species, CTmax was 7-10°C higher than the maximum temperature in their habitats, with daily fluctuations from ∼1°C to 16°C and extremes of 6.9°C and 34.9°C. These findings suggest that subterranean dytiscid beetles are unlikely to reach their CTmax with a predicted warming of 1.3-5.1°C in the region by 2090. However, the impacts of long-term elevated temperatures on fitness, different life stages and other species in the beetle's trophic food web are unknown.

Comparative analysis of morabine grasshopper genomes reveals highly abundant transposable elements and rapidly proliferating satellite DNA repeats.

BACKGROUND: Repetitive DNA sequences, including transposable elements (TEs) and tandemly repeated satellite DNA (satDNAs), collectively called the "repeatome", are found in high proportion in organisms across the Tree of Life. Grasshoppers have large genomes, averaging 9 Gb, that contain a high proportion of repetitive DNA, which has hampered progress in assembling reference genomes. Here we combined linked-read genomics with transcriptomics to assemble, characterize, and compare the structure of repetitive DNA sequences in four chromosomal races of the morabine grasshopper Vandiemenella viatica species complex and determine their contribution to genome evolution. RESULTS: We obtained linked-read genome assemblies of 2.73-3.27 Gb from estimated genome sizes of 4.26-5.07 Gb DNA per haploid genome of the four chromosomal races of V. viatica. These constitute the third largest insect genomes assembled so far. Combining complementary annotation tools and manual curation, we found a large diversity of TEs and satDNAs, constituting 66 to 75% per genome assembly. A comparison of sequence divergence within the TE classes revealed massive accumulation of recent TEs in all four races (314-463 Mb per assembly), indicating that their large genome sizes are likely due to similar rates of TE accumulation. Transcriptome sequencing showed more biased TE expression in reproductive tissues than somatic tissues, implying permissive transcription in gametogenesis. Out of 129 satDNA families, 102 satDNA families were shared among the four chromosomal races, which likely represent a diversity of satDNA families in the ancestor of the V. viatica chromosomal races. Notably, 50 of these shared satDNA families underwent differential proliferation since the recent diversification of the V. viatica species complex. CONCLUSION: This in-depth annotation of the repeatome in morabine grasshoppers provided new insights into the genome evolution of Orthoptera. Our TEs analysis revealed a massive recent accumulation of TEs equivalent to the size of entire Drosophila genomes, which likely explains the large genome sizes in grasshoppers. Despite an overall high similarity of the TE and satDNA diversity between races, the patterns of TE expression and satDNA proliferation suggest rapid evolution of grasshopper genomes on recent timescales.

Cutaneous respiration by diving beetles from underground aquifers of Western Australia (Coleoptera: Dytiscidae).

Insects have a gas-filled respiratory system, which provides a challenge for those that have become aquatic secondarily. Diving beetles (Dytiscidae) use bubbles on the surface of their bodies to supply O2 for their dives and passively gain O2 from the water. However, these bubbles usually require replenishment at the water's surface. A highly diverse assemblage of subterranean dytiscids has evolved in isolated calcrete aquifers of Western Australia with limited/no access to an air-water interface, raising the question of how they are able to respire. We explored the hypothesis that they use cutaneous respiration by studying the mode of respiration in three subterranean dytiscid species from two isolated aquifers. The three beetle species consume O2 directly from the water, but they lack structures on their bodies that could have respiratory function. They also have a lower metabolic rate than other insects. O2 boundary layers surrounding the beetles are present, indicating that O2 diffuses into the surface of their bodies via cutaneous respiration. Cuticle thickness measurements and other experimental results were incorporated into a mathematical model to understand whether cutaneous respiration limits beetle size. The model indicates that the cuticle contributes considerably to resistance in the O2 cascade. As the beetles become larger, their metabolic scope narrows, potentially limiting their ability to allocate energy to mating, foraging and development at sizes above approximately 5 mg. However, the ability of these beetles to utilise cutaneous respiration has enabled the evolution of the largest assemblage of subterranean dytiscids in the world.

The effects of temperature, activity and convection on the plastron PO2 of the aquatic bug Aphelocheirus aestivalis (Hemiptera; Aphelocheiridae).

The aquatic bug Aphelocheirus aestivalis (Fabricius 1794) utilises a plastron, a thin bubble layer on the surface of its body to extract O2 from the water. Millions of tiny hairs keep the bubble from collapsing, enabling the bug to remain submerged indefinitely. The development of fibre optic O2-probes has allowed measurements of O2 pressure (PO2) surrounding the plastron, and within the plastron although only for short periods. Here we developed methods to continuously measure plastron PO2, and investigate how it is affected by temperature (15, 20, 25°C), activity, and water circulation. We also made measurements of water PO2, temperature and velocity in the field and swimming velocity at the treatment temperatures. Results show that plastron PO2 is inversely related to temperature, associated with differences in metabolic demand, and that small bouts of activity or changes in water convection result in rapid changes in plastron PO2. A model was developed to calculate the conditions under which Aphelocheirus would exist without becoming O2-limited in relation to water temperature, PO2 and boundary layer thickness. This suggests that Aphelocheirus at one of two field sites may have a reduced metabolic scope even in well convected water in association with low PO2 and moderate temperature, and that in well convected, air-saturated water, bugs may have a reduced metabolic scope where water temperatures are between 20 and 25°C. If exposed to 5kPa PO2, Aphelocheirus cannot sustain resting metabolic rate even in well-convected water and would die at temperatures above approximately 25°C.

Flight metabolic rate of Locusta migratoria in relation to oxygen partial pressure in atmospheres of varying diffusivity and density.

Flying insects have the highest mass-specific metabolic rate of all animals. Oxygen is supplied to the flight muscles by a combination of diffusion and convection along the internal air-filled tubes of the tracheal system. This study measured maximum flight metabolic rate (FMR) during tethered flight in the migratory locust Locusta migratoria under varying oxygen partial pressure (PO2 ) in background gas mixtures of nitrogen (N2), sulfur hexafluoride (SF6) and helium (He), to vary O2 diffusivity and gas mixture density independently. With N2 as the sole background gas (normodiffusive-normodense), mass-independent FMR averaged 132±19 mW g-0.75 at normoxia (PO2 =21 kPa), and was not limited by tracheal system conductance, because FMR did not increase in hyperoxia. However, FMR declined immediately with hypoxia, oxy-conforming nearly completely. Thus, the locust respiratory system is matched to maximum functional requirements, with little reserve capacity. With SF6 as the sole background gas (hypodiffusive-hyperdense), the shape of the relationship between FMR and PO2  was similar to that in N2, except that FMR was generally lower (e.g. 24% lower at normoxia). This appeared to be due to increased density of the gas mixture rather than decreased O2 diffusivity, because hyperoxia did not reverse it. Normoxic FMR was not significantly different in He-SF6 (hyperdiffusive-normodense) compared with the N2 background gas, and likewise there was no significant difference between FMR in SF6-He (normodiffusive-hyperdense) compared with the SF6 background gas. The results indicate that convection, not diffusion, is the main mechanism of O2 delivery to the flight muscle of the locust when demand is high.

Gas exchange and dive characteristics of the free-swimming backswimmer Anisops deanei.

Many aquatic insects utilise air bubbles on the surface of their bodies to supply O2 while they dive. The bubbles can simply store O2, as in the case of an 'air store', or they can act as a physical 'gas gill', extracting O2 from the water. Backswimmers of the genus Anisops augment their air store with O2 from haemoglobin cells located in the abdomen. The O2 release from the haemoglobin helps stabilise bubble volume, enabling backswimmers to remain near neutrally buoyant for a period of the dive. It is generally assumed that the backswimmer air store does not act as a gas gill and that gas exchange with the water is negligible. This study combines measurements of dive characteristics under different exotic gases (N2, He, SF6, CO) with mathematical modelling, to show that the air store of the backswimmer Anisops deanei does exchange gases with the water. Our results indicate that approximately 20% of O2 consumed during a dive is obtained directly from the water. Oxygen from the water complements that released from the haemoglobin, extending the period of near-neutral buoyancy and increasing dive duration.

Respiratory function of the plastron in the aquatic bug Aphelocheirus aestivalis (Hemiptera, Aphelocheiridae).

The river bug Aphelocheirus aestivalis is a 40 mg aquatic insect that, as an adult, relies totally on an incompressible physical gill to exchange respiratory gases with the water. The gill (called a 'plastron') consists of a stationary layer of air held in place on the body surface by millions of tiny hairs that support a permanent air-water interface, so that the insect never has to renew the gas at the water's surface. The volume of air in the plastron is extremely small (0.14 mm(3)), under slightly negative pressure and connected to the gas-filled tracheal system through spiracles on the cuticle. Here, we measure PO2 of the water and within the plastron gas with O2-sensing fibre optics to understand the effectiveness and limitations of the gas exchanger. The difference in PO2 is highest in stagnant water and decreases with increasing convection over the surface. Respiration of bugs in water-filled vials varies between 33 and 296 pmol O2 s(-1), depending on swimming activity. The effective thickness of the boundary layer around the plastron was calculated from respiration rate, PO2 difference and plastron surface area, according to the Fick diffusion equation and verified by direct measurements with the fibre-optic probes. In stagnant water, the boundary layer is approximately 500 µm thick, which nevertheless can satisfy the demands of resting bugs, even if the PO2 of the free water decreases to half that of air saturation. Active bugs require thinner boundary layers (∼ 100 µm), which are achieved by living in moving water or by swimming.