Taxonomic features and comparisons of the gut microbiome from two edible fungus-farming termites (Macrotermes falciger; M. natalensis) harvested in the Vhembe district of Limpopo, South Africa.

BACKGROUND: Termites are an important food resource for many human populations around the world, and are a good supply of nutrients. The fungus-farming 'higher' termite members of Macrotermitinae are also consumed by modern great apes and are implicated as critical dietary resources for early hominins. While the chemical nutritional composition of edible termites is well known, their microbiomes are unexplored in the context of human health. Here we sequenced the V4 region of the 16S rRNA gene of gut microbiota extracted from the whole intestinal tract of two Macrotermes sp. soldiers collected from the Limpopo region of South Africa. RESULTS: Major and minor soldier subcastes of M. falciger exhibit consistent differences in taxonomic representation, and are variable in microbial presence and abundance patterns when compared to another edible but less preferred species, M. natalensis. Subcaste differences include alternate patterns in sulfate-reducing bacteria and methanogenic Euryarchaeota abundance, and differences in abundance between Alistipes and Ruminococcaceae. M. falciger minor soldiers and M. natalensis soldiers have similar microbial profiles, likely from close proximity to the termite worker castes, particularly during foraging and fungus garden cultivation. Compared with previously published termite and cockroach gut microbiome data, the taxonomic representation was generally split between termites that directly digest lignocellulose and humic substrates and those that consume a more distilled form of nutrition as with the omnivorous cockroaches and fungus-farming termites. Lastly, to determine if edible termites may point to a shared reservoir for rare bacterial taxa found in the gut microbiome of humans, we focused on the genus Treponema. The majority of Treponema sequences from edible termite gut microbiota most closely relate to species recovered from other termites or from environmental samples, except for one novel OTU strain, which clustered separately with Treponema found in hunter-gatherer human groups. CONCLUSIONS: Macrotermes consumed by humans display special gut microbial arrangements that are atypical for a lignocellulose digesting invertebrate, but are instead suited to the simplified nutrition in the fungus-farmer diet. Our work brings to light the particular termite microbiome features that should be explored further as avenues in human health, agricultural sustainability, and evolutionary research.

Respiratory Strategies in Relation to Ecology and Behaviour in Three Diurnal Namib Desert Tenebrionid Beetles.

The respiratory physiology of three diurnal ultraxerophilous tenebrionid beetles inhabiting either the dune slipface or gravel plain in the Namib Desert was investigated. The role of the mesothoracic spiracles and subelytral cavity in gas exchange was determined by flow-through respirometry. All three species exhibited the discontinuous gas exchange cycles with a distinct convection based flutter period and similar mass specific metabolic rates. There was variation in their respiration mechanics that related to the ecology of the species. The largest beetle species, Onymacris plana, living on the dune slipface, has a leaky subelytral cavity and used all its spiracles for gas exchange. Thus, it could use evaporative cooling from its respiratory surface. This species is a fog harvester as well as able to replenish water through metabolising fats while running rapidly. The two smaller species inhabiting the gravel plains, Metriopus depressus and Zophosis amabilis, used the mesothoracic spiracles almost exclusively for gas exchange as well as increasing the proportional length of the flutter period to reduce respiratory water loss. Neither species have been reported to drink water droplets, and thus conserving respiratory water would allow them to be active longer.

Metabolic rate does not vary with seasonal change in Anopheles arabiensis adults in South Africa.

An important component of South Africa's malaria elimination agenda is identifying the entomological drivers of residual transmission, especially those that present opportunities for enhanced vector control. Seasonal mosquito density correlates directly with malaria transmission in South Africa. Transmission is highest during the warm rainy season and lowest but not entirely absent during the cooler dry season. The factors that sustain dry-season mosquito survival remain unknown. The aim of this project was therefore to investigate seasonal change in metabolic rate to determine the presence or absence of winter dormancy in malaria vector mosquitoes. Metabolic rate, determined by CO2 production during closed-system respirometry, was measured from wild anophelines collected from KwaZulu-Natal Province, South Africa. Monthly sampling spanned all four seasons (summer, autumn, winter, and spring) in 2017. Anopheles arabiensis and An. parensis specimens formed the majority of the total 437 identified specimens (n = 216 and n = 162, respectively). Metabolic rate data from wild-caught mosquitoes showed no significant seasonal disparities for An. arabiensis and An. parensis males and females. Further laboratory experiments assessed the effect of manipulated photoperiod, representing seasonal day-length changes, on the metabolic rate of colonized An. arabiensis mosquitoes. Simulations of midwinter (10 h:14 h light dark) and midsummer (14 h:10 h) day-length showed no significant effect on the metabolic rate of these mosquitoes. Age (in days) had a significant effect on the metabolic rate of both male and female colonized adult An. arabiensis mosquitoes which may be linked to developmental factors during maturation of adults. These data suggest that the South African populations of the malaria vector species An. arabiensis and An. parensis do not curtail their breeding and foraging activities during the colder and drier winter months. Overwintering by diapause does not appear to be triggered in the adult mosquito stage in An. arabiensis. However, their respective population densities do decrease considerably during winter leading to reduced malaria transmission and the opportunity for control by winter larviciding of known breeding sites.

Respiratory patterns in field collected brown locust, Locustana pardalina, in the gregarious phase.

In this paper we report on the metabolic rates and respiratory patterns measured from gregarious brown locusts, Locustana pardalina, collected from the Nama Karoo region in South Africa. All five instar hopper stages and adults were collected over a three year period when significant numbers of locust swarms were seen. Flow-through respirometry was used to measure the CO2 emission from individual locusts from all the developmental stages and adults within a week of collection. Carbon dioxide emission scaled hypometrically with mass, 0.863 ±â€¯0.026. Except in the 1st and 5th instar stage there was no difference in the mass specific rate of CO2 emission (V̇CO2). These had significantly higher metabolic rates compared to the other stages which reflects their biology, with the 1st instar undergoing rapid growth and the 5th instar also undergoing rapid growth and development in preparation for becoming an adult. The 1st instars used a form of continuous gas exchange while all the other stages showed discontinuous gas exchange cycles. A clear burst phase and interburst periods could be seen. The 2nd and 3rd instars use mainly diffusion to expel CO2 and so exhibited an open form of the burst phase. There was an increase in CO2 volleys seen in the burst phase from the 4th instar stage onwards thus indicating an increased use of convection. There was no change in the duration or frequency of the discontinuous gas exchange cycles through the locust development or with body mass.

Gas exchange pattern transitions in the workers of the harvester termite.

The evolutionary genesis and the current adaptive significance of the use of the discontinuous gas exchange cycle (DGC) for respiration by insects is the subject of intense debate. Years of research have resulted in several leading hypotheses, one of which is the emergent-property hypothesis. This hypothesis states that DGC is an emergent property or consequence of interactions between the O2 and CO2 set points that regulate spiracular function, i.e. opening and closing. Workers of the harvester termite, Hodotermes mossambicus were selected as a model to test this hypothesis. The respiratory patterns of major workers, investigated using flow-through respirometry, were obtained at 100% relative humidity (RH) under varying temperature to evaluate the assumptions of the emergent-property hypothesis. Metabolic rate, measured as VCO2 increased significantly after 15 °C. As VCO2 increased in response to increasing temperature and activity, the gas exchange pattern displayed by workers transitioned to a continuous gas exchange. A true DGC, defined as showing all three phases and a coefficient of variation value close to 2, was not expressed under the experimental conditions. The conclusion drawn from this study of termite workers is that changes in respiratory patterns are most likely an emergent property of the insects' nervous and respiratory system.

Group hunting in a ponerine ant, Leptogenys nitida Smith.

Field observations on the emigration and foraging behaviours of the southern African ponerine ant, Leptogenys nitida, were undertaken at Mtunzini, Natal, South Africa. These colonies have a single ergatoid queen and 200-1000 workers. The nest sites are found in the leaf litter and these nests are moved frequently over distances ranging from 0.5 to 5 m. Leptogenys nitida is a diurnal predator of arthropods dwelling in the leaf litter. Up to 500 workers participate in each foraging trail, and are not led by definite scouts. Ants form clear trunk trails and fan out at various intervals to search for prey. The prey is searched for and retrieved cooperatively. From laboratory tests it was determined that ants will follow pygidial gland extracts, with the poison gland extract eliciting a limited response. The type of army ant behaviour observed in L. nitida seems to be different to that observed in other ponerine ants.

Embryonic development and rates of metabolic activity in early and late hatching eggs of the major malaria vector Anopheles gambiae.

Anopheles gambiae eggs generally hatch at the completion of embryo development; two-three days post oviposition. However, staggered or delayed hatching has been observed whereby a single batch of eggs shows marked variation in time-to-hatch, with some eggs hatching 18 days post oviposition or later. The mechanism enabling delayed hatch has not been clearly elucidated but is likely mediated by environmental and genetic factors that either induce diapause or slow embryo development. This study aimed to compare metabolic activity and embryonic development between eggs collected from sub-colonies of the baseline Anopheles gambiae GAH colony previously selected for early or late time-to-hatch. Egg batches from early and late hatch sub-colonies as well as from the baseline colony were monitored for hatching. For both time-to-hatch selected sub-colonies and the baseline colony the majority of eggs hatched on day two post oviposition. Nevertheless, eggs produced by the late hatch sub-colony showed a significantly longer mean time to hatch than those produced by the early hatch sub-colony. The overall proportions that hatched were similar for all egg batches. CO2 output between eggs from early and late hatch sub-colonies showed significant differences only at 3 and 7 days post oviposition where eggs from the early hatch and the late hatch sub-colony were more metabolically active, respectively. No qualitative differences were observed in embryo development between the sub-colonies. It is concluded that all viable embryos develop to maturity at the same rate and that a small proportion then enter a state of diapause enabling them to hatch later. As it has previously been shown that it is possible to at least partially select for late hatch, this characteristic is likely to involve genetic as well as environmental factors. Delayed hatching in An. gambiae is likely an adaptation to maximise reproductive output despite the increased risk of desiccation in an unstable aquatic environment.

Pump out the volume--The effect of tracheal and subelytral pressure pulses on convective gas exchange in a dung beetle, Circellium bacchus (Fabricus).

Many flightless beetles like the large apterous dung beetle Circellium bacchus, possess a subelytral cavity (SEC) providing an extra air space below the elytra which connects to the tracheal system (TS) via metathoracic and abdominal spiracles. By measuring subelytral and intratracheal pressure as well as body movements and gas exchange simultaneously in a flow-through setup, we investigated the contribution of convection on Circellium respiratory gas exchange. No constriction phase was observed. TS and SEC pressures were always around atmospheric values. During interburst phase open abdominal spiracles and a leaky SEC led to small CO(2)-peaks on a continuous CO(2) baseline, driven by intermittent positive tracheal pressure peaks in anti-phase with small negative subelytral pressure peaks caused by dorso-ventral tergite action. Spiracle opening was accompanied by two types of body movements. Higher frequency telescoping body movements at the beginning of opening resulted in high amplitude SEC and TS pressure peaks. High frequency tergite movements caused subelytral pressure peaks and led to a saw tooth like CO(2) release pattern in a burst. We propose that during the burst open mesothoracic spiracles increase the compliance of the subelytral cavity allowing big volumes of tracheal air being pulled out by convection.

Helicoverpa armigera (Lepidoptera: Noctuidae) larvae that survive sublethal doses of nucleopolyhedrovirus exhibit high metabolic rates.

To determine the effect of sublethal doses of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) on the metabolic rate of H. armigera, the respiration rates of third instar H. armigera larvae inoculated with sublethal doses of HearSNPV were evaluated. Respiration rates, measured as the rate of CO(2) production (VCO(2)), were recorded daily using closed-system respirometry. By 4 days post-inoculation (dpi), the metabolic rates of LD(25) or LD(75) survivors were significantly higher than that of uninoculated controls. When dose data were pooled, the VCO(2) values of larvae that survived inoculation (0.0288mlh(-1)), the uninoculated controls (0.0250mlh(-1)), and the larvae that did not survive inoculation (0.0199mlh(-1)) differed significantly from one another. At 4dpi, the VCO(2) of the uninoculated controls were significantly lower than the VCO(2) of inoculation survivors, but significantly higher than the VCO(2) of inoculation non-survivors. Inoculation survivors may have had high metabolic rates due to a combination of viral replication, organ damage, and an energy-intensive induced cellular immune response. The high 4dpi metabolic rate of inoculation survivors may reflect an effective immune response and may be seen as the metabolic signature of larvae that are in the process of surviving inoculation with HearSNPV.

The role of the mesothoracic spiracles in respiration in flighted and flightless dung beetles.

The relative role of the mesothoracic and abdominal spiracles in respiration was examined using flow-through respirometry in four dung beetle species from different habitats. Two species of flightless beetles, Scarabaeus (Pachysoma) gariepinus and Scarabaeus (Pachysoma) striatum, from the arid western region of southern Africa and a large flighted species, Pachylomerus femoralis, from a more mesic habitat were compared with Circellium bacchus, a flightless beetle from a low rainfall eastern area. All species showed a form of the discontinuous gas exchange pattern at rest. The mesic flighted species used a closed, flutter, open, cycle (CFO) while those species from more arid habitats used a closed, ventilation, cycle (CV) or a closed, burst cycle (CB). The relative importance of the mesothoracic spiracles in CO(2) emission varied between the species, even between those from the same genus and habitat. C. bacchus and P. femoralis represent extremes of CO(2) emission from the mesothoracic spiracles; from almost total to almost none, respectively. Overall, mesothoracic CO(2) emission and convection were more pronounced in the dry habitat species, supporting the hypothesis that both strategies aid in the reduction of water loss.

Shaken, not stirred: a serendipitous study of ants and earthquakes.

There is anecdotal evidence for profound behavioral changes prior to and during earthquakes in many organisms, including arthropods such as ants. Behavioral or physiological analysis has often, in light of these reports, been proposed as a means of earthquake prediction. We report here a serendipitous study of the effect of the powerful Landers earthquake in the Mojave Desert, USA (Richter magnitude 7.4) on ant trail dynamics and aerobic catabolism in the desert harvester ant Messor pergandei. We monitored trail traffic rates to and from the colony, trail speed, worker mass distributions, rates of aerobic catabolism and temperature at ant height before and during the earthquake, and for 3 days after the earthquake. Contrary to anecdotal reports of earthquake effects on ant behavior, the Landers earthquake had no effect on any measured aspect of the physiology or behavior of M. pergandei. We conclude that anecdotal accounts of the effects of earthquakes or their precursors on insect behavior should be interpreted with caution.

The role of the subelytral spiracles in respiration in the flightless dung beetle Circellium bacchus.

The role of the subelytral cavity in flightless beetle species as an adaptation to water saving in arid habitats is still in dispute. We found that relatively little CO(2) was released from the subelytral cavity of a large apterous beetle Circellium bacchus during simultaneous measurements of CO(2) emission from the anterior mesothoracic spiracles and posterior body, which included the subelytral spiracles. However, when we sampled air directly from inside the subelytral cavity, we discovered that this pattern was reversed. A discontinuous gas exchange cycle (DGC) was recorded from the posterior body half, revealing a flutter phase that had been absent from the anterior mesothoracic DGC. The anterior mesothoracic and posterior subelytral spiracles act in synchrony to maintain high CO(2) and water vapour levels inside the subelytral cavity. In addition, the O(2) concentration of the air within the subelytral cavity is lower than the air around the elytral case, irrespective of the time of sampling. These findings lead us to conclude that the subelytral spiracles work in a coordinated fashion with the anterior spiracles to create a DGC, which allows us to extend the hypothesis of the function of the subelytral cavity as a respiratory water-saving device.

Respiratory airflow in a wingless dung beetle.

The sealed subelytral cavity of many flightless beetle species is widely acknowledged to be an adaptation to water saving in arid-habitat species. However, this hypothesis relies on the acceptance of two largely untested assumptions: (i) that the movement of respiratory gases is unidirectional from anterior to posterior and (ii) that the coordinated action of the spiracles directs this flow. We tested these assumptions by simultaneously measuring CO(2) and O(2) exchange at the anterior mesothorax, independently of gas exchange in the posterior body, which included the subelytral cavity, of a large apterous beetle, Circellium bacchus. Flow-through respirometry revealed a marked discontinuous gas-exchange cycle (DGC) pattern from the anterior half of the body. Very little CO(2) was released from the posterior body, where the DGC was not apparent. Labelled air was shown to flow forwards from the posterior to the anterior body. Individual sampling from the mesothoracic spiracles revealed that the right mesothoracic spiracle, lying outside the elytral cavity, is the primary route for respiratory gas exchange in C. bacchus at rest. This discovery necessitates a reassessment of the currently assumed role of the subelytral cavity in water conservation and is, to our knowledge, the first demonstration of forward airflow associated with the unilateral use of a single thoracic spiracle for respiration in an insect.

Respiratory patterns and metabolism in tenebrionid and carabid beetles from the Simpson Desert, Australia.

The respiratory physiology of four species of Australian desert-dwelling beetle was examined using a flow-through respirometry system over the temperature range of 20-40°C. The two species of tenebrionid beetles (Heleus waitei and Pterohelaeus sp.) did not exhibit the discontinuous gas exchange cycles (DGC), a mechanism to possibly reduce respiratory water loss rates, observed in other arid-dwelling beetles. There were small increases in metabolic rate with temperature resulting in a Q 10 of 1.84 for H. waitei and 1.99 for Pterohelaeus sp. Furthermore, H. waitei has no increase in metabolic rate over the temperature range 25-35°C (Q 10=1). The two species of carabid beetles (Cerotalis sp. and Carenum sp.) displayed the DGC respiratory pattern, having long flutter and burst periods. Both species also exhibited spiracular movement or muscular pumping in the burst period. Relatively low Q 10 values of 1.31 and 1.64 were measured for Cerotalis sp. and Carenum sp., respectively. Cerotalis sp. had no increase in metabolic rate over the temperature range 20-35°C (Q 10=1). In both species the temperature-associated increases in metabolic rate were modulated by increases in DGC frequency. All the four beetle species studied have fused elytra, a closed subelytral cavity and are nocturnally active, which should assist in reducing respiratory water loss rates, and occupy similar microhabitats. Thus we propose that the difference in respiratory patterns found between the tenebrionid and carabid beetles is related to their thorax morphology, food type and food availability.

Metabolic rate and respiratory gas-exchange patterns in tenebrionid beetles from the Negev Highlands, Israel.

This study correlates the pattern of external gas exchange with the diel activity of nine species of tenebrionid beetle from the Negev Desert, Israel. The study species are active throughout the summer months when daytime temperatures are high and no rain falls. There were no differences in standard metabolic rate, determined by flow-through respirometry, among the nine species. All the nocturnally active beetles exhibited a form of continuous respiration, whereas the two diurnally active and one crepuscular species exhibited a cyclic form of respiration referred to as the discontinuous gas-exchange cycle (DGC). The DGCs recorded have a long flutter period consisting of miniature ventilations, and 29-48 % of the total CO(2) output occurred during this period. In this study, the flutter period played an important role in the modulation of metabolic rate, in contrast to other studies in which the burst period has been shown to be important. We suggest that the long flutter period is important in reducing respiratory water loss in arid-dwelling arthropods. This study lends support to the hypothesis that discontinuous gas exchange is important in reducing respiratory water loss from beetles that need to minimise dessication because of the high water vapour pressure gradient they experience. If the use of underground burrows were responsible for the evolution of discontinuous gas exchange, then we would expect all nine tenebrionid species to use DGCs since both the nocturnally and diurnally active species bury in the sand during periods of inactivity. We conclude that the activity patterns of the beetles are more important than their habitat associations in designating the type of respiration used.