Effects of flow rate and temperature on cyclic gas exchange in tsetse flies (Diptera, Glossinidae)

Abstract

Air flow rates may confound the investigation and classification of insect gas exchange patterns. Here we report the effects of flow rates (50, 100, 200, 400 ml min⁻¹) on gas exchange patterns in wild-caught Glossina morsitans morsitans from Zambia. At rest, G. m. morsitans generally showed continuous or cyclic gas exchange (CGE) but no evidence of discontinuous gas exchange (DGE). Flow rates had little influence on the ability to detect CGE in tsetse, at least in the present experimental setup and under these laboratory conditions. Importantly, faster flow rates resulted in similar gas exchange patterns to those identified at lower flower rates suggesting that G. m. morsitans did not show DGE which had been incorrectly identified as CGE at lower flow rates. While CGE cycle frequency was significantly different among the four flow rates (p < 0.05), the direction of effects was inconsistent. Indeed, inter-individual variation in CGE cycle frequency exceeded flow rate treatment variation. Using a laboratory colony of closely related, similar-sized G. morsitans centralis we subsequently investigated the effects of temperature, gender and feeding status on CGE pattern variation since these factors can influence insect metabolic rates. At 100 ml min⁻¹ CGE was typical of G. m. centralis at rest, although it was significantly more common in females than in males (57% vs. 43% of 14 individuals tested per gender). In either sex, temperature (20, 24, 28 and 32 °C) had little influence on the number of individuals showing CGE. However, increases in metabolic rate with temperature were modulated largely by increases in burst volume and cycle frequency. This is unusual among insects showing CGE or DGE patterns because increases in metabolic rate are usually modulated by increases in frequency, but either no change or a decline in burst volume.

Introduction

Insects at rest are characterised by at least three distinct gas exchange patterns. Continuous gas exchange is a pattern in which spiracles appear to open and close continuously and asynchronously, often associated with fairly high metabolic rates (e.g. Gibbs and Johnson, 2004). Cyclic gas exchange (CGE) shows distinct ‘burst’ and ‘inter-burst’ periods, spiracle behaviour appears fairly well co-ordinated, and at least some CO₂ production is detectable even during the inter-burst period (e.g. Marais and Chown, 2003, Nespolo et al., 2007). Finally, discontinuous gas exchange (DGE) patterns, possibly the most curious and well characterised of insect gas exchange patterns (Chown et al., 2006), comprise three distinct periods when recording the insect's CO₂ emission in a flow-through, real-time system: a closed (C) spiracle phase, a flutter (F) phase and finally an open (O) phase (e.g. Hetz, 2007). Each of these three periods is defined by distinct rates of exchange of each respiratory gas and DGE results in a partial temporal decoupling of CO₂ release and O₂ uptake (Lighton, 1996). Discontinuous gas exchange cycles were first thoroughly characterized in lepidopteran pupae, and have since been reported in a wide variety of adult insects and non-insect arthropods, and may have evolved independently at least five times within the Insecta (reviewed in Lighton, 1996, Marais et al., 2005, Chown et al., 2006).

While continuous (or possibly cyclic) gas exchange is thought to be basal in insects at rest (Marais et al., 2005, Clusella-Trullas and Chown, 2008), considerable variation in gas exchange patterns has been documented both within and among species (Lighton, 1998, Chown, 2001, Marais and Chown, 2003, Gibbs and Johnson, 2004, Marais et al., 2005, Nespolo et al., 2007, Woodman et al., 2007). The modification of the strongly periodic (cyclic) pattern, possibly through adaptive change to avoid excess water loss or oxidative damage (Hetz and Bradley, 2005, White et al., 2007, Terblanche et al., 2008a, but see also Lighton and Turner, 2008), is thought to have resulted in the more complex form of discontinuous gas exchange. Alternatively, the discontinuous pattern may initially have characterized the system at rest (as a consequence of interacting CO₂ and O₂ setpoints (Chown and Holter, 2000)) and subsequently have been selected to reduce water loss or oxidative damage (Chown and Nicolson, 2004, Terblanche et al., 2008a).

By contrast with these arguments, that tend to view the three patterns as relatively discrete entities, recent studies have suggested that the patterns may rather represent a continuum (e.g. Bradley, 2007). Moreover, in at least some cases this continuum may be confounded in its interpretation by inappropriate experimental methods, and particularly, low gas flow rates relative to cuvette size or the detection chamber size of the CO₂ analyzer being used, which tend to reduce the resolution of the gas exchange pattern (Gray and Bradley, 2006, see also Bartholomew et al., 1981). Indeed, Gray and Bradley (2006) suggested that discontinuous gas exchange might be much more common (especially in small insects) than previously supposed because low flow rates favour an interpretation of the VCO₂ trace as cyclic gas exchange. In fact, they also cautioned that strong relationships among temperature and metabolic rate and, in turn, metabolic rate and gas exchange characteristics, might also confound interpretation of gas exchange patterns. Typically, DGE yields the lowest, and continuous gas exchange the highest metabolic rates when patterns are compared within a species, although metabolic rates during CGE and DGE are frequently statistically indistinguishable (Vogt and Appel, 2000, Marais and Chown, 2003, Gibbs and Johnson, 2004, Gray and Chown, 2008).

In the context of previous work suggesting that cyclic gas exchange is phylogenetically basal, and that discontinuous gas exchange is uncommon or indeed absent in several, more derived insect orders, such as the Diptera (Marais et al., 2005), it is clear that additional work is required to determine the extent to which experimental artefacts might have influenced these conclusions. Here, we therefore firstly examine the influence of rate of air flow on gas exchange patterns in Glossina morsitans morsitans using wild-caught individuals in Zambia. Subsequently, using a laboratory-reared colony we investigate the effects of temperature, feeding status and gender on gas exchange patterns in a closely related sub-species, G. morsitans centralis, under controlled laboratory conditions. These latter factors are investigated as they have relatively well documented influences on metabolic rate in insects and therefore may influence gas exchange patterns. In the case of temperature, metabolic rate is strongly affected for most insect species including tsetse (Terblanche et al., 2005, Terblanche and Chown, 2007) with an acute increase in temperature resulting in higher resting metabolic rate (see Terblanche et al., 2005, Terblanche et al., 2009 for detailed discussion of these effects in tsetse). Feeding can also influence metabolic rate through the specific costs of digestion of a particular meal, also known as the heat increment of feeding or specific dynamic action (Bradley et al., 2003, Nespolo et al., 2005). Finally, gender might influence insect metabolic rates either through sexual-dimorphism or costs associated with reproduction (Terblanche et al., 2005). Although gas exchange patterns have been reported from the Diptera (Lehmann, 2001, Marais et al., 2005, Gray and Bradley, 2006), the order is relatively poorly investigated in this context. Moreover, although metabolic rate variation has been investigated in the Glossinidae, gas exchange patterns in tsetse are not well known, at least partly due to the fact that most early investigations made use of closed-system respirometry or radioisotope excretion rates with relatively poor temporal resolution to estimate metabolic rates (Rajagopal and Bursell, 1966, Hargrove and Coates, 1990), and that metabolic rate variation was the subject of most, more-recent investigations (e.g. Terblanche et al., 2004, Terblanche et al., 2005, Terblanche et al., 2009, Terblanche and Chown, 2007).

Therefore, the specific aims of this study are (1) to investigate the effects of flow rates on the pattern of gas exchange, and (2) to examine the influence of temperature, feeding status and gender on metabolic rate and gas exchange pattern modulation.