Assessing the Morphological and Physiological Adaptations of the Parasitoid Wasp Echthrodesis Lamorali for Survival in an Intertidal Environment

Physiological Entomology (2017) 42, 173–180 DOI: 10.1111/phen.12187

Assessing the morphological and physiological adaptations of the parasitoid wasp Echthrodesis lamorali for survival in an intertidal environment

CANDICE A. OWEN1 , JULIE A. COETZEE2, SIMON VAN NOORT3,4 andANDREW D. AUSTIN5 1Department of Zoology and Entomology, Rhodes University, Grahamstown, South Africa, 2Department of Botany, Rhodes University, Grahamstown, South Africa, 3Department of Natural History, Iziko Museums of South Africa, Cape Town, South Africa, 4Department of Biological Sciences, University of Cape Town, Cape Town, South Africa and 5Department of Genetics and Evolution, School of Biological Sciences, Australian Centre for Evolutionary Biology and Biodiversity, The University of Adelaide, Adelaide, Australia

Abstract. As a result of a variety of chemical, environmental, mechanical and physiological difficulties, insects that spend their entire life spans in the marine orinter- tidal region are relatively rare. The present study assesses whether morphological and physiological adaptations have evolved in a maritime parasitoid wasp species Echthrode- sis lamorali Masner, 1968 (Hymenoptera: Platygastridae, Scelioninae), in response to environmental pressures on its respiratory functioning. Scanning electron and light microscopy of whole and sectioned specimens show the presence of structure-retaining taenidia in the tracheal tubes, although there is an absence of other major adaptations associated with the trachea or spiracles. Histological sectioning reveals the presence of unusual sacs in the female metasoma whose role is unknown, although they are hypothesized to most likely be linked to ovipositor control. Respirometry experiments illustrate the formation of a plastron when submerged, with the longevity of the wasps being increased by quiescence. The critical thermal range of E. lamorali is shown to be large: from −1.1 ∘C ± 0.16 to 45.7 ∘C ± 0.26 (mean ± SE). Behavioural and physiological adaptations in E. lamorali appear to have evolved in response to exposure to the heterogeneous environmental conditions experienced within the intertidal zone. Key words. Desis formidabilis, hydrostatic ovipositor control, plastron, respiration, scanning electron microscopy, spiracle, taenidia, thermal physiology, trachea.

Introduction despite a much wider host spider distribution from East London (Eastern Cape, South Africa), around the Peninsula to Namibia Echthrodesis lamorali Masner, 1968 (Hymenoptera: Platygastri- (Day, 1974; Dippenaar-Schoeman & Jocqué, 1997). The habi- dae, Scelioninae) is an endoparasitoid of the egg stage of the tat of this parasitoid sets the species apart as one of only three intertidal South African spider Desis formidabilis O.P. Cam- other such maritime wasps known worldwide (van Noort et al., bridge 1890 (Araneae: Desidae) (Lamoral, 1968; Masner, 1968; 2014). Females gain access to the multi-compartmentalized host Branch & Branch, 1981; van Noort, 2009; van Noort et al., spider egg sacs by chewing through their silken walls with strong 2014). This wasp displays high endemism, living and surviving mandibles, after which they oviposit in all eggs present (van only within the intertidal region along a small stretch of the Cape Peninsula (Western Cape, South Africa) (Owen et al., 2014), Noort, 2009; van Noort et al., 2014). Males eclose first and fight each other over access to their female kin, which emerge next Correspondence: Dr Candice A. Owen, Department of Zoology (van Noort et al., 2014). The mode of dispersal to other nests is and Entomology, Rhodes University, PO Box 94, Grahamstown, unknown, although it is hypothesized to occur post-copulation Eastern Cape, 6140, South Africa. Tel.: +27 84 716 4746; e-mail: through active searching at low tide by walking between rock [email protected] pools when the host nests are exposed (van Noort et al., 2014).

Masner (1968) postulates that this species must exhibit a vari- et al., 2008) because its distribution and physiological adap- ety of unique adaptations for coping with salt water inundation tations are all influenced by its thermal tolerance (Terblanche that may occur during dispersal. Loss of the wings (aptery), et al., 2007; Hazell et al., 2008). which may otherwise weigh an insect down when immersed, In light of the extreme habitat in which E. lamorali persists, the is an obvious adaptation typical in maritime species (Cheng, present study aims to determine whether there are any external or 1976) and occurs in both sexes of E. lamorali (Masner, 1968; internal morphological structures associated with the spiracles van Noort, 2009; van Noort et al., 2014). Wing reduction or and trachea that allow it to withstand saltwater inundation, loss also aids individuals in remaining in one area during the as well as to establish whether the insect forms a plastron strong wind activity typical of this habitat (Cheng, 1976; van in a similar manner to its host D. formidabilis. Furthermore, Noort, 2009) and, in the case of E. lamorali and other scelionine the study also aims to determine the critical thermal range of spider-egg parasitoids, a streamlined body eases physical issues E. lamorali to better understand how the species persists within associated with gaining access through the wall of the spider the intertidal zone. host’s silken egg sac (Austin, 1988; Austin et al., 2005; Stevens & Austin, 2007; van Noort et al., 2014). In maritime species, aptery is often not displayed in isolation, with other morpholog- Materials and methods ical adaptations also having evolved to cope with the extreme environment (Cheng, 1976; Foster & Treherne, 1976; Hinton, Specimen collection 1976). Therefore, it follows that E. lamorali is likely to exhibit additional adaptations for survival within the intertidal zone. All E. lamorali specimens used for morphological and thermal Lamoral (1968) illustrates how the wasp’s host, physiological examinations were collected in early 2012 within D. formidabilis, possesses dense, short, chitinous setae spaced the intertidal region on the stretch of rocky shore at ‘The Island’ at regular intervals on the edges of the openings to the book (Kommetjie, Cape Peninsula, South Africa; 34∘8′22.7034′′S, lungs. These are hypothesized to avert water entering and 18∘19′17.5794′′E) through active searching for spider nests preventing the ‘leaves’ of the book lungs from closing as a containing egg sacs. Egg sacs were placed in a mesh-lidded result of pressure when underwater, allowing for continued container in the laboratory and covered in paper towel moistened respiration (Lamoral, 1968). It is unknown whether E. lamorali with seawater every day to allow for emergence of the wasps. possesses similar structures in its trachea. Furthermore, the host spider develops a plastron when submerged (Lamoral, 1968; Masner, 1968; Branch & Branch, 1981). This mechanism is Specimen examination dependent on the presence of setae covering the spider’s body, which trap a film of air that is capable of exchanging gasses Both the external and internal features of the eclosed wasps with the surrounding environment without collapsing (Lamoral, were examined using scanning electron microscopy of entire 1968; Masner, 1968; Hinton, 1976; Branch & Branch, 1981). specimens and light microscopy of thin-sectioned individuals Evolving independently several times (Hebets & Chapman, mounted on glass slides. Independent dissections of E. lamorali 2000), a variety of insect species are demonstrated to pos- by O. Popovici (pers. comm.) revealed that the species lacks the sess this adaptation (Cheng, 1976; Foster & Treherne, 1976; posterior metasomal spiracles common in other Hymenoptera. Neumann & Woermann, 2009; Seymour & Matthews, 2013), By default, the mesosomal spiracles were the focus of investiga- which may take the form of either a compressible (termed a tion in the present study. ‘physical gill’) or an incompressible (termed a ‘plastron’) gill To examine the external spiracular structure, five wasps (Balmert et al., 2011; Seymour & Matthews, 2013). Physical were sputter-coated with gold as per standard methods, and gills are those that form as an unsupported layer of air over the images captured using a Vega LMU Scanning Electron Micro- insect’s body, whereas plastrons require some form of support, scope (Tescan, Kohoutovice, Czech Republic) (20 kV current; such as setae and microtrichia (Balmert et al., 2011; Seymour analysis photocapture and measurement software, Olympus & Matthews, 2013). Echthrodesis lamorali exhibits a densely Soft Imaging Solutions GmbH, Germany) housed in the Rhodes pilose body surface (Masner, 1968; van Noort, 2009; van Noort University Electron Microscopy Unit (Cross & Pinchuck, et al., 2014), suggesting the formation of an incompressible gill 1987). Captured images were visually assessed for the presence during submersion similar to that of its host spider. of any structures or adaptations that may assist with submerged In addition to likely morphological adaptations, the intertidal survival. zone often exhibits huge daily temperature fluctuations, and so For internal tracheal structure, specimens were prepared for species living in this environment need to have large critical ther- resin-immersion and subsequent sectioning by placing 10 live mal limits to maintain normal bodily functionality (Huey et al., insects into 2.5% glutaraldehyde [CH2(CH2CHO)2] in 0.1 m 1992; Hazell et al., 2008). Subsequent to exposure beyond the sodium phosphate (Na3PO4) buffer solution overnight to allow maximum and minimum threshold temperatures, the calcium for fixation of the tissues. Thereafter, the liquid was removed currents across cell membranes in an individual are disrupted, and the specimens rinsed twice in 0.1 m sodium phosphate buffer resulting in a loss of motor control and an inability to self-right for 10 min each time. The process of sealing in Avaldite/TAAB (Huey et al., 1992; Terblanche et al., 2007; Hazell et al., 2008). 812 resin (Cross & Pinchuck, 1987) is detailed in Table 1, where Knowing the thermal limits of a species is important for fully the wasps were exposed to a variety of dehydrating agents and understanding its ‘ecology, evolution and physiology’ (Hazell resins to ensure full fixation. Once left in pure resin overnight,

Table 1. Embedding protocol followed to fix specimens in resin. mean for the Cape Peninsula (Owen, 2016), at a rate of 0.25 ∘Cmin−1. Individuals were kept at this temperature for Solution specimens exposed to: Period of time exposed to solution (min) 5 min before being transferred into the individual test chambers that either contained atmospheric air or filtered, autoclaved 1% osmidium tetroxide (OsO4)90 0.1 m sodium phosphate 10 seawater. Specimens were then randomly placed into 200-μL 0.1 m sodium phosphate 10 chambers (CH10478; Loligo) filled with either seawater (sealed

30% ethanol (CH3CH2OH) 10 to prevent air-bubble formation) or air, allowing for both control 50% ethanol 10 and experimental chambers of both treatments in each trial 70% ethanol 10 for comparison of same-trial consumption rates. The chamber 80% ethanol 10 tray was then submerged in the water bath flow-through unit 90% ethanol 10 attached to the water bath and placed on top of a microplate Absolute ethanol 10 reader (OX11900; SDR-436; Loligo). Absolute ethanol 10 Propylene oxide (CH CHCH 0) 20 Each trial was run for a total of 24 h with oxygen readings 3 2 –1 Propylene oxide 20 (cO2:mgL ) in each chamber recorded and assimilated using 75/25 pro-resin 60 sdr, version 38 (PreSens GmbH) every 1 min. Data collected in 50/50 pro-resin 60 chambers containing wasps that did not gain full motor control 25/75 pro-resin 60 within 24 h after the end of the study were removed from the Pure resin Overnight final analysis. Those wasps that were still alive and active after 24 h were euthanized in 96% ethanol for further assessment of their volume. These were determined by treating the head the solution was poured into capsule moulds with one specimen and body of each individual as two ellipsoids, the volumes at the tip of each and baked at 60 ∘C for 36 h in an oven. of which were calculated using a standard ellipsoid equation All sectioning was performed using a glass blade [prepared from measurements of each specimen using a SZX16 stacker using a LKB Knife Maker Type 780 1B at 80∘ (Sweden)] microscope and SDF Plapo 1XPF camera (Olympus) with the in an RMC MT-7 Microtome (Tucson, Arizona), set to cut associated steam motion, version 1.9 software (Olympus). The 120-μm thick sections. Both longitudinal and transverse sections volumes of the head and body were summed to determine the of a variety of specimens were prepared. These were then total volume for the entire organism. placed on glass slides; stained with 1% toluidine blue stain in The first 2 h of data were removed from the dataset to allow for a sodium carbonate solution; rinsed with 30% 70% and then oxygen drift common at the start of such experiments (Köster 100% ethanol in sequence; and images captured using an BX50 et al., 2008). Thereafter, the remaining data were analyzed Microscope (Olympus, Japan) and the associated analysis using regression analysis in statistica, version 10 (Statsoft, software. These images were visually examined for the presence Inc., 2011). Because experimental error is frequent in such of any adaptations. investigations as a result of the small scale of readings (P. Patrick, pers. comm.), the slopes of these lines were compared for all trials within each treatment using a Thompson Tau test, Plastron development which removes statistical outliers. Oxygen consumption in each chamber was then calculated, where after values for wasps in To determine whether E. lamorali is capable of forming a air could be compared with those submerged in water using a plastron, the respiratory rate of 131 adult E. lamorali individu- Student’s t-test for independent samples in statistica, version als, collected from field populations at Kommetjie in mid-2015, 10. The equation for oxygen consumption is: was determined in 12 separate trials using a 25 ∘C calibrated [( ) ] mw − mc ( ) respirometer (PreSens GmbH, Germany) in both air (62 wasps) VO2 = × Vc − Vw ×βO2 and seawater (69 wasps). All trials were conducted at the 100

Aquatic Ecophysiology Research Platform (Rhodes University where VO2 is the consumption rate per volume of wasp per −1 and the South African Institute for Aquatic Biodiversity, Gra- unit of time (O2 mL min ); mw is the slope of the line of the hamstown) in a temperature-controlled laboratory set to the change in oxygen during the experiment for each test wasp −1 working temperature of the experiment and with a standardized chamber (cO2 min ); mc is the mean slope of the lines of the 12 : 12 h light/dark photocycle. Because seawater filtered using change in oxygen during the experiment for all controls in the −1 a 0.7-μm pore size filter paper still exhibited a high amount same trial (cO2 min ); Vc is the volume of the chamber; Vw of consumption in pre-experimental test control chambers, all the volume of each wasp; and βO2 is the oxygen capacitance of water used was filtered and autoclaved in a SA300-VL autoclave each medium (Alton et al., 2012). Calculations to determine the (Sturdy, Taiwan) to remove all biological organisms that may oxygen capacitance at the working temperature of each medium consume oxygen other than the test wasps. are given in the Supporting information (Appendix S1). Wasps were first acclimatized in a single, large, sealed chamber in a waterbath flow-through unit (CH10495; Loligo, Thermal physiology Denmark) attached to a PP20R water bath (PolyScience, Niles, Illinois) at 18 ∘C for 10 min. Because temperature can have To determine the critical thermal minima and maxima of significant effects on respiration (Verberk et al., 2015), the E. lamorali, 10 wasps of mixed sexes were placed individ- temperature was then ramped down to 17 ∘C, the environmental ually into Eppendorf tubes containing filter paper moistened

© 2017 The Royal Entomological Society, Physiological Entomology, 42, 173–180 176 C. A. Owen et al. with seawater to maintain the moisture content inside the enclo- adaptations of the species. Once all outliers had been removed by sure, and then submerged in a GP200 R4 programmable water the Thompson Tau test, a total of 27 wasps in air, 53 air controls, bath (Grant Instruments, U.K.) that was filled with 70% ethanol 23 wasps in water and 15 water controls remained for analy- for ramp-down trials and distilled water in ramp-up trials. An sis. Freshly submerged wasps were observed rapidly expanding operative thermometer placed inside an empty Eppendorf con- and contracting their limbs for a brief period, followed by the firmed close correlation between internal chamber conditions cessation of movement. Although the contents of the chambers and water temperatures. The wasps were allowed to acclima- were largely obscured during trials, the wasps in air-filled cham- tize at 18 ∘C for 10 min, after which the temperature was ramped bers appeared to be active throughout the entire period of the up or down at a rate of 0.25 ∘Cmin–1. Specimens were checked experiment, and remained so when the chambers were opened for motor function every 4 min (every change of 1 ∘C) by tap- at the end of the trials. On removal from the seawater chambers, ping the Eppendorf and checking for falling and self-righting no individuals were immediately active, with movement being behaviour. Each trial was replicated three times, resulting in a regained within 24 h after removal. total of 30 individuals in each experiment. The temperatures Echthrodesis lamorali individuals sealed in air filled cham- at which insects lost motor control were recorded and a mean bers exhibited significantly higher oxygen consumption rates was calculated for all 30 individuals as the upper (ramp-up) and than those isolated in seawater (t = 17.422, P < 0.0001), −1 lower (ramp-down) critical thermal limits. When specimens lost with a mean ± SE of 14 111 346 ± 646 061 O2 mL min and −1 their self-righting response, they were removed from the water 1206 190 ± 259 581 O2 mL min , respectively (Fig. 4). Because bath and maintained at room temperature. Recovery to full motor oxygen was consumed from the water, it can be concluded that function was determined 1 and 24 h after falling. the wasp formed a plastron when submerged, although their metabolic requirements were lowered by entering a quiescent state indicated by a lack of movement and greatly reduced Results oxygen consumption in this treatment.

External spiracular structures Thermal physiology No unique adaptations were evident around the external surface of the mesosomal spiracles (Fig. 1). These presented as A wide thermal tolerance range was demonstrated for the simple elongated non-valved openings with no indication of the species, with minimum and maximum temperatures at which presence of an atrium near the opening, and were similar to other individuals lost motor control as low as −3 ∘C and as high as members of the family. 48 ∘C, respectively. The mean ± SE minimum and maximum Longitudinal sections through E. lamorali specimens also temperatures at which individuals lost motor control were showed no hair-like or other structures are present in the trachea −1.1 ∘C ± 0.16 and 45.7 ∘C ± 0.26, respectively. In ramp-down (Fig. 2A). Furthermore, transverse sections through the mesoso- experiments, all specimens recovered full motor functionality mas confirmed no structures further into the trachea (Fig. 2B). within 1 h, whereas it took 24 h for them to recover in the The trachea did, however, exhibit taenidia, or thickened rings of ramp-up. cuticle along the length of the tube (Fig. 2B).

Discussion Other morphological structures Although E. lamorali clearly exhibits an ability to cope with All female specimens exhibited bifurcation of the ventral water inundation possibly assisted by tracheal taenidia that may metasoma into an upper fat- and egg-filled region, and a lower retain the integrity of the trachea when under pressure from cuticle-lined sac-like section filled with an unknown substance submersion (Lamoral, 1968), the present study demonstrates (Fig. 3). Internal sclerotized tubules (possibly tracheae) joining that the species lacks tracheal and spiracular modifications the two sections and, within the lower area, were clearly visible that might assist with long periods of submersion. From a (Fig. 3B). This division disappeared closer to the centre of the morphological perspective, the respiratory system of this wasp insect, where the dorsal fatty area assumes the full extent of the is apparently not specifically adapted to water inundation. metasoma. Unfortunately, all attempts to cut transverse sections Two sacs filled with either air or an unknown substance run through female metasomas led to collapse of the tissues as a along the entire length of the ventrolateral metasoma of female result of structural weakness because of the two sac-like spaces E. lamorali, tapering towards the centre of the tagma. These are on either side of the tagma. Male specimens did not have this linked to the dorsal, fat- and egg-filled portion of the metasoma bifurcation. by small tubes, suggesting the need for exchange of contents between the two regions. The function of the sclerotized sac-like areas and their contents is unknown. However, the most likely Physiological adaptations explanation for the presence of these sacs is provided by Austin (1985), Field & Austin (1994), Austin et al. (2005) and Murphy Investigations into the physiology of E. lamorali exhibited et al. (2007), as discussed below. The Platygastroidea can be more adaptation to inundation than those into the morphological divided into two groups, each with a differing ovipositor control

Fig. 1. Lateral view of the external features of the spiracle on the mesosoma of Echthrodesis lamorali.

Fig. 2. (A) Longitudinal sections through the entire length of Echthrodesis lamorali specimens revealed no internal structure in the trachea. (B) Transverse section through the mesosoma of Echthrodesis lamorali revealed taenidia (in red box) but no other internal structure in trachea. mechanism (Austin et al., 2005). These are known as the ‘Scelio Austin et al., 2005). Based on the muscle structure and the type’ and the ‘Ceratobaeus type’ (Austin et al., 2005). Field presence of the large sacs in the metasoma, E. lamorali appears & Austin (1994) detail the oviposition mechanics in the genus to exhibit the Ceratobaeus type ovipositor system. Therefore, Scelio and demonstrated that ovipositor control is achieved these sacs may act as fluid- or air-filled spaces that are involved solely through hydrostatic pressure changes in the metasoma in ovipositor extension and retraction. (Field & Austin, 1994; Austin et al., 2005; Murphy et al., 2007). It is also possible that, as a result of the loss of metasomal In the Ceratobaeus type, this control is achieved through partial spiracles, the wasps use these structures as air sacs that are muscle contraction and hydrostatic pressure (Austin, 1985; filled via the propodeal spiracles and tracheae. Oxygen could be

Fig. 3. (A) The dorsal fat- and egg-filled segment along the ventral region of the metasoma of afemale Echthrodesis lamorali specimen. (B) The ventral fluid-filled section in the metasoma (red arrows) of a female E. lamorali specimen is connected to the dorsal region with sclerotized tubes (black arrows).

Echthrodesis lamorali females may store air to aid them in maintaining fertilized eggs. The respirometry experiments lead to two indisputable con- clusions concerning respiration by E. lamorali. First, the presence of oxygen consumption when submerged in seawater, albeit at a low rate, indicates that a plastron capable of exchanging gasses with the surrounding water is formed around the wasp when submerged, as occurs in its host, D. formidabilis (Lamoral, 1968). This result is supported by the dense pilosity that covers the whole body of both sexes (Masner, 1968; van Noort et al., 2014), and their silvery appearance when submerged. Among insects, true plastrons are usually exhibited by species that spend the majority of their time underwater, such as the notonec- Fig. 4. Echthrodesis lamorali individuals sealed in air filled chambers tid hemipterans and dytiscid beetles, although they are been exhibited significantly higher oxygen consumption rates than those recorded in some arachnid species that are not adapted for a fully isolated in seawater. aquatic lifestyle (Balmert et al., 2011; Seymour & Matthews, 2013). It is important to note that the formation of a plastron channelled via this network to supply the metasoma, suggesting around the bodies of submerged wasps may primarily provide that the connecting tubes visible in the thin sections are part of the alternative function of buoyancy (Balmert et al., 2011; Sey- the tracheal system. These sacs may also serve a physiological mour & Matthews, 2013). As discussed above, E. lamorali is function, providing the wasps with antifreeze necessary to not fully adapted for permanent, or long periods of submersion, maintain body functioning at sub-zero temperatures (Sinclair although it appears to exhibit adaptations to assist with acciden- et al., 2003), or assisting with salt- and water-balance as a result tal submersion. A small volume of air attached to an insect will of frequent salt water inundation. The sacs are only observed generally serve as a buoyancy method to propel a submerged in female specimens and therefore may be associated with an insect to the water surface, and therefore back in contact with increasedsex-linkedneedfordispersaltolocatefreshspider atmospheric air (Balmert et al., 2011; Seymour & Matthews, eggs for oviposition. Males mate with eclosed females within 2013). Furthermore, it may also prevent entrapment within a individual egg sacs containing trapped air (van Noort et al., small amount of water (Balmert et al., 2011), which may eas- 2014) and may not need to be as adapted for dispersal and ily occur as insect bodies are slightly denser than water and thus associated survival under submerged conditions in sea water, tend to sink or become trapped in water bodies if they break the although it is unclear whether females will ever purposefully surface tension of a liquid (Seymour & Matthews, 2013). submerge themselves and for how long they can survive water Second, the statistically significant difference in oxygen con- inundation. Finally, another possible explanation for the large sumption between wasps under normal air exposure and those sacs may be that they are filled with oxygen-rich air. This may immersed in seawater illustrates a further inundation-coping aid the females during dispersal in wet conditions from the mechanism in this organism. This difference, along with the nest they eclosed in to the one in which they will oviposit. observations that wasps quickly stop moving when immersed

© 2017 The Royal Entomological Society, Physiological Entomology, 42, 173–180 Morphology and physiology of E. lamorali 179 in water, and are subsequently immobile for a period of less saltwater inundation, there are likely to be further adaptations, than 1 h when removed from seawater, suggests that E. lamorali particularly from a behavioural perspective, that remain to be becomes quiescent (Foster & Treherne, 1976). When in such a determined. state, most bodily functions are reduced to such an extent that the entire insect’s metabolism slows to a significantly lower rate compared with its usual active state. This allows an individual Supporting Information to survive adverse conditions where the demand for oxygen or other resources that it would usually need to survive are greatly Additional Supporting Information may be found in the reduced (Foster & Treherne, 1976). A reduced metabolic state online version of this article under the DOI reference: hasbeenrecordedinavarietyofotherinsectsthatrelyonaplas- DOI: 10.1111/phen.12187 tron when submerged (Seymour & Matthews, 2013). Seymour Appendix S1. Calculations used to determine the insect vol- & Matthews (2013) report an assessment of the metabolic rates umes, as well as the oxygen capacitance of air and seawater used of five plastron breathing insects and show that they exhibit an in respirometry experiments. average of only 38% of the predicted metabolic rates of other similarly-sized air-breathing insects. Echthrodesis lamorali is found to exhibit a wide thermal tol- Acknowledgements erance. Based on the environment in which it lives, however, where turbulent wave action reduces the temperature at regular We thank Ms Shirley Pinchuck and Mr Marvin Randall (Rhodes intervals during both high and low tides, and the rocks compris- University Microscopy unit, South Africa) for their assistance ing the habitat are heated by the sun at low tide (Teal, 1962; with all specimen preparation, sectioning and microscopy; Dr Foster & Treherne, 1976; Lubke, 1998; Sanford, 2002), a wide Ovidiu Popovici (Facultatea de Biologie, Universitatea Alexan- thermal tolerance would be expected. Although the upper criti- dru Ioan Cuza IASI, Romania) for e-mail correspondence cal thermal limit is generally accepted as being more controlling regarding the unique ovipositor control in the Scelioninae; Pro- of a species’ life history (Chown et al., 2015), a lower criti- fessor Alan Hodgson (Rhodes University, South Africa) for cal thermal sub-zero tolerance threshold is significant. Sinclair assistance in interpreting the images produced; and Justin Kemp et al. (2003) state that a tolerance of temperatures around zero (Aquatic Ecophysiology Research Platform, Rhodes Univer- is particularly important because this is the freezing point of sity, South Africa) and Paula Patrick (South African Insti- water. For maritime species, their aqueous environment freezes tute for Aquatic Biodiversity, South Africa) for assistance with ∘ at approximately −2 C, depending on the salinity (National experimental set-up and data analysis. This work was funded Ocean Service, 2016). Solidification of liquid within an insect by the Rhodes University Prestigious Scholarship Programme can be fatal, with cellular dehydration and disruption of chem- (Henderson Award), Rhodes University Research Council, NRF ical reactions across the cellular membranes inhibiting normal Incentive Funding for rated researchers, an NRF research grant functioning (Sinclair et al., 2003). GUN 79004, the NRF Innovation Award and the Ada and Bertie The mean ± SE temperature in 2015 at Kommetjie is reported Levenstein Award. Collection permits were granted by Cape ∘ as 15.53 ± 0.06 C (Owen, 2016) as recorded underneath limpet Nature, SanParks and Eastern Cape Nature Conservation. The shells to capture the accurate environment to which host nests authors declare that they have no conflicts of interest to report. and thus E. lamorali individuals are exposed. No temperatures below 7.5 ∘C are reported (Owen, 2016). Pörtner (2001) notes that physiological acclimation to temperature can drive genetic References differentiation and thus evolution. With this in mind, the very cold lower critical thermal tolerance limit may be indicative of Alton, L.A., White, C.R., Wilson, R.S. & Franklin, C.E. (2012) The the evolutionary origin of E. lamorali, although this is difficult energetic cost of exposure to UV radiation for tadpoles is greater when to determine. There are two plausible options for the evolution they live with predators. Functional Ecology, 26, 94–103. Austin, A.D. (1985) The function of spider egg sacs in relation to of the species, namely an ancestor based on the colder southern parasitoids and predators, with special reference to the Australian Ocean Islands [although D. formidabilis is not present there, fauna. Journal of Natural History, 19, 359–376. some relatives are (Pugh, 2004)] or a mainland African ancestor, Austin, A.D. (1988) A new genus of baeine wasp (Hymenoptera: which could have evolved during a historically colder period Scelionidae) from New Zealand associated with moss. New Zealand (Pörtner, 2001). Journal of Zoology, 15, 173–183. Very few insects are able to colonize an intertidal environment Austin, A.D., Johnson, N.F. & Dowton, M. (2005) Systematics, evolu- (Cheng, 1976). In comparison, a large proportion of all recorded tion, and biology of Scelionid and Platygastrid wasps. 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