# Comparative assessment of heat tolerance in weevils associated with a fire-prone ecosystem

Marion Javal, John S Terblanche, Chantelle Smit, Julien Haran

► To cite this version:

Marion Javal, John S Terblanche, Chantelle Smit, Julien Haran. Comparative assessment of heat tolerance in weevils associated with a fire-prone ecosystem. Ecological Entomology, 2023, 48 (2), pp.240-250. 10.1111/een.13218. hal-04007187v2

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ORIGINAL ARTICLE

Ecological
Entomology

<figure><img src="image_2.png" /></figure>

# Comparative assessment of heat tolerance in weevils associated with a fire-prone ecosystem

Marion Javal<fnref n="1" />,<fnref n="2" /> ID | John S. Terblanche<fnref n="1" /> ID | Chantelle Smit<fnref n="1" /> ID | Julien Haran<fnref n="3" /> ID

**Correspondence**

Marion Javal, Department of Conservation
Ecology & Entomology, Faculty of
AgriSciences, Stellenbosch University, South
Africa.

Email: marion.javal@cefe.cnrs.fr

**Present address**

Marion Javal, Centre d'Écologie Fonctionnelle et Évolutive (CEFE), Université de Montpellier, CNRS, EPHE, IRD, 34293, Montpellier, France.

**Associate Editor:** Krzysztof Miler

**Abstract**

1. Fire is an important cause of disturbance which directly shapes many ecosystems worldwide. While the effect of fire on the distribution and regeneration of plant species has been widely documented, little is known on how phytophagous insects are reacting to these disturbances.

2. This study explores the survival strategies of various weevil species, a highly diverse phytophagous beetle group in fire-prone ecosystems of the biodiversity hotspot of the Cape Floristic Region in South Africa. More specifically, we investigated how the lifestyle of species (location of larval development, phenology of adults, and flight ability) correlates with heat tolerance. We hypothesized that wingless species in particular will show better tolerance to heat as they have a limited ability to escape fire rapidly and usually remain hidden in the soil.

3. The thermal tolerance of a set of sixteen species with divergent lifestyles and geographic distribution was measured using a standard heat knockdown protocol at 48°C. Respirometry was then performed on the most resistant species using a ramping protocol in order to determine $CT_{max}$.

4. Our results show that the species tested exhibit high variation in thermal tolerance across taxonomic groups, clustering into three modalities: weak, intermediate, and high tolerance to heat stress. In addition, life history traits (diurnal vs. nocturnal adult activity and location of juvenile stages in plant tissues) likely better explain thermal tolerance at the species level than flight ability or the fire-proneness of ecosystems. Finally, results revealed that some non-flying weevil species are highly heat tolerant with $CT_{max}$ values reaching up to 50.2 and 51.9°C in species among the Ocladiinae and Brachycerinae subfamilies, respectively.

5. Climate change is leading to an increase in the impact and frequency of fires. In this context, this study highlights the diversity of strategies developed by arthropods to escape extreme heat in fire prone ecosystems. Further work is necessary to examine the generality of these patterns across other fire prone ecosystems to better understand behavioural compensation and evolutionary responses, especially given the forecast increases in fire driven by drying and warming associated with climate change.

<footnote n="1">¹Department of Conservation Ecology & Entomology, Faculty of AgriSciences, Stellenbosch University, South Africa</footnote>

<footnote n="2">²CEFE, University of Montpellier, CNRS, EPHE, IRD, Montpellier, France</footnote>

<footnote n="3">³CBGP, CIRAD, INRAE, IRD, Institut Agro, University of Montpellier, Montpellier, France</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2022 The Authors. *Ecological Entomology* published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.</footnote>
KEYWORDS

Bogert effect, fynbos, heat knockdown, thermolimit respirometry, insect, Coleoptera

INTRODUCTION

Fire is an important cause of disturbance which directly shapes many
ecosystems worldwide (Bond et al., 2005). Fire regime can be defined
by several parameters: the frequency and the intensity of the fire
events, but also their seasonality and the size of the surface burnt
(Gill & Allan, 2008). Fynbos is a fire-prone ecosystem characteristic of
the Cape Floristic Region (CFR, South Africa), in which fire has played
a major role in the evolutionary diversification of plants (Keeley
et al., 2012). It is associated with sandy and infertile soils and charac-
terised by the prominence of aphyllous graminoids and sclerophyllous
shrubs, most of the time including proteoid shrubs (Cowling
et al., 1997). The characteristics of this ecosystem such as the vegeta-
tion assemblage or the accumulation of dead material make it particu-
larly flammable (Kraaij & van Wilgen, 2014). In the context of climate
change, the average temperature as well as the frequency of extreme
events such as heat waves or fires are likely to increase on short time
scales (Harvey et al., 2022; IPCC, 2022). In the CFR in particular, the
total area burnt annually has increased significantly between 1980
and 2010 along with the occurrence of extremely warm and dry
weather conducive to fires, which can be directly correlated to climate
change (Kraaij et al., 2013). In addition, the increase in the frequency
of extreme weather following wildfires, together with the effects of
exotic plant species have reduced the regeneration of native plant
species, leading to a decrease in botanic biodiversity since 1966
(Slingsby et al., 2017). Understanding the responses of fynbos organ-
isms to these events is, therefore, crucial in estimating how global
changes will affect them (Swengel, 2001).

Several strategies to cope with fire have been described in plants
and animals (Pausas, 2019). Some species develop traits that allow them
to resist fire, by developing a protective structure for vital organs
(e.g., thick bark (Pausas, 2015), carapace (Sanz-Aguilar et al., 2011), shell
(Kiss & Magnin, 2006)), by resprouting from vegetative buds or by
reducing the intensity of fire (the "non-flammable strategy", mainly
described in plants (Pausas et al., 2017)). Dormancy is another strategy
to survive fire. For example, some species survive in the ground as egg,
pupae, or seed whose germination or emergence can be tightly linked to
fire (Keeley et al., 2012; Pausas et al., 2018a, 2018b). Finally, organisms
with sufficient dispersal abilities may flee the fire and/or retreat to areas
less subject to fire ("refugia", Keeley et al., 2012; Robinson et al., 2013),
to recolonise the burnt area exogenously (García et al., 2016). However,
fire survival strategies are relatively unstudied in animals compared to
plants, probably because of differences in the mobility capacity (Pausas
et al., 2018a, 2018b). In insects, in particular, few examples are docu-
mented. For these ectothermic organisms, temperature is a key environ-
mental driver of population dynamics since they directly depend on
environmental temperature to regulate their physiological functions
(Angilletta, 2009). Tolerance to extreme high temperatures associated
with fire may involve physiological adaptations (González-Tokman

et al., 2020; Ma et al., 2021) that lead to an increase in their maximum
temperature for coordinated locomotion, or critical thermal maximum
(CTmax, Cerdà et al., 1998). Behavioural adaptations are also known. For
example, some invertebrate species take refuge in protected parts of
plants, where the temperature is buffered (Brennan et al., 2011). Other
species survive by burrowing into the soil (Thom et al., 2015), and fire
can even be a cue for emergence (Jacobs et al., 2011), as it is for the
germination of some plants (Keeley et al., 2012). Finally, exogenous
recolonisation, a case in which individuals from unaffected areas relo-
cate in the burnt zone after the fire, is well described, particularly in
saproxylic insects (Evans, 2010).

Weevils (Coleoptera, Curculionoidea) constitute a hyper-diverse
lineage of exclusively phytophagous beetles (Oberprieler et al., 2007).
This insect group is especially well diversified in the CFR, with entire
new clades only described very recently (Meregalli et al., 2021; Haran
et al., 2022). More specifically, this region hosts radiating basal weevil
lineages adapted to xeric environments (e.g., Brachycerus and several
tribes among Brachycerini; Oberprieler, 2014) together with more
derived clades of various "true" weevil subfamilies among the Curcu-
lionidae (Alonso-Zarazaga & Lyal, 1999; Hernández-Vera et al., 2013;
Hansen et al. 2021). Due to their high diversity of species, but also of
lifestyles or host-plants found in this group, weevils constitute a pow-
erful model to investigate evolutionary dynamics, speciation pro-
cesses, and adaptation of insects to their environment (Toussaint
et al., 2014; Baird et al., 2021; Hévin et al., 2022). A particularly note-
worthy feature in the case of xeric and fire-prone ecosystems is the
high diversity of terricolous non-flying weevils, where the adults gen-
erally live in the leaf litter in the vicinity of their host-plant. This life-
style is usually associated with larval stages developing in or on the
root system or bulbs of the host plant encountered in several subfam-
ilies and tribes of weevils (e.g., Entiminae, Embritini, Oosomini, Tanyr-
hynchini; Cyclomiinae; Brachycerinae, Brachycerini, Cryptolaryngini;
Ocladiinae, Ocladiini; Oberprieler, 1995; Oberprieler, 2014; Haran
et al., 2020). The latter contrasts with the expectation that species
with good dispersal abilities would better cope with fire-prone eco-
systems since they can rapidly escape extreme heat conditions on the
soil surface and repeated cycles of fire. This apparent contradiction
was noticed during several field observations in the CFR, where spe-
cies of non-flying genera were observed on resprouting plants in the
middle of large, recently burnt areas (Cryptolarynx pyrophilus in Mon-
tagu, Haran et al., in press; Tanyrhynchus carinatus in Du Toitskloof
Pass, JH pers. obs.). Though many strategies may be at work in these
cases (quick recolonisation, larval stages protected deep in the soil),
the assumption that adults of non-flying insects occurring in fire-
prone ecosystems exhibit a higher heat tolerance than flying species
occurring in neighbouring regions with no fire cycles remains to be
explored. This is particularly relevant since other groups of non-flying
arthropods such as Collembola were previously found significantly
more heat tolerant in the CFR than in other regions (Liu et al., 2020).
In this study, we conducted a first investigation of the relative heat tolerance of adult weevils occurring both in fire-prone and non-burning ecosystems of South Africa. Using both acute constant and dynamic heat exposure protocols, we tested whether non-flying weevil species exhibit a higher tolerance to extreme heat than flying species as an adaptation to survive heat when hidden in the ground. We also produced preliminary data on the potential difference between weevil species occurring in fire-prone ecosystems and in non-fire-prone ecosystems of neighbouring regions in terms of heat tolerance. Patterns of heat tolerance were then contextualised with life history traits of the species tested.

# MATERIAL AND METHODS

## Species sampling

Weevils are inconspicuous organisms and sampling live specimens of a peculiar species in sufficient amounts can be challenging. In this context, the sampling was conducted opportunistically in 2018 and 2019 during spring, at the vicinity of Stellenbosch in the CFR and further in neighbouring regions. In all, adults of 16 species were obtained, with a number of individuals sampled ranking from 6 to 22 per population (Table 1). Twelve of these species are non-flying, as typical for their subfamilies (Brachycerinae, Cyclomiinae and a part of Entiminae among the Curculionidae (Bouchard et al. 2011; Marvaldi et al., 2014; Oberprieler, 2014; Figure 1)). Due to phylogenetic conservatism in flight abilities, no closely related species to the above groups with flight behaviour occurs in the CFR; therefore, the four flying species included were sampled among the Nanophyinae and Curculioninae subfamilies (Table 1). For two species (Tanyrhynchus carinatus and Cryptolarynx variabilis), two to three geographically distant populations (1–20 km apart) were obtained and retained for a preliminary assessment of the variability of thermal resistance at population level.

Fourteen of the species were sampled in fire-prone ecosystems of the CFR (Fynbos, and to a lesser extent Renosterveld, Table 1), including ten non-flying. Two species from non-fire-prone ecosystems of neighbouring regions were included: *Ocladius costiger* and *Sciobius pr. brevicollis* (both non-flying) specimen were collected in a desert biome of the Northern Cape province and in coastal tropical forests of KwaZulu-Natal, respectively. These ecosystems show no natural cycles of fires but can exhibit high temperatures, and temperatures in the Northern Cape region in particular are higher than in the CFR (van Wilgen et al., 2016). As these two species share life history traits with non-flying species from the CFR, they are expected to provide insights for a comparative assessment of fire tolerance of weevils in fire versus non-fire prone ecosystems.

While this study focuses on survival of adult weevils, we also recorded the location of larvae in or on the host plant since these stages are the most exposed to hot summers and fires and could unravel insights on survival strategies of the species sampled. All the flying species sampled develop in the aerial part of their host plant and cannot survive fire at larval stages. The adult stage on the other

hand, is expected to be able to escape the heat stress by flying. By contrast, the non-flying species sampled develop on or in underground tissues of the host plant and are thus possibly protected by buffering effect of the ground during fire event. Adult stages of these species are generally able to hide in the ground as well and have possibly been selected to resist high heat when staying a few centimetres below the surface of the ground. As a result, the lifestyle of both adults and larvae of non-flying species are relevant in a context of survival to fire events. Adult circadian activity of these weevils is also a relevant feature since they may emerge from the ground only at night and as such escape from extreme heats from solar radiation. Through detailed field observations, lifestyle of larval stages was recorded for all species and host plant taxa was documented for thirteen of them. The sampling further included eight nocturnal species and eight active during daytime at adult stage. The two species from non-fire prone ecosystems were both nocturnal (Table 1).

In order to reduce background thermal history effects (e.g., recent weather fronts; Terblanche & Hoffmann, 2020; Huey & Buckley, 2022), specimens were kept for two to four days after collection (Table 1) at room temperature (about 25°C) with foliage of their host as a food substrate. Species were identified taxonomically based on available literature and using the reference collections housed at Iziko Museum (Cape Town). At least one reference specimen per population was mounted, dried and deposited in the Continental Arthropod Collection at Centre de Biologie pour la Gestion des Populations, Montpellier, France (CBGP; https://doi.org/10.15454/D6XAKL) attached with a specific identification code (see Table 1).

## Thermal tolerance assessment

The relative heat resistance of species was first measured using a heat knockdown derived approach to rapidly sort ranking of tolerance (Huey et al., 1992). Specimens were individually set in an Eppendorf tube with ambient air. A thermocouple was used to track the air temperature of the tube, and set in order not to touch the tube walls. Eppendorf tubes were then placed in a water bath at 50°C in order to reach a stable air temperature of 48°C, a lethal temperature for most insects including beetles (Sunday et al., 2011). The duration between the introduction of the tube in the bath and the death of specimens was measured to the nearest second. Weevils exhibited a range of reactions to heat treatment, from rapid escape movement to resistance position without movement. In this last case, we observed that at time of death, weevils could not keep the position of resistance (legs and antennae relaxed) and regurgitate. At time of death, tubes were removed from the bath and left for 45 min at ambient temperature. Specimens that recovered from heat coma were not considered in the analyses. Specimens were dried and weighed individually after experiment microbalance (to 0.1 mg; AB104-S/Fact, Mettler Toledo International, Inc., Columbus, OH, United States).

In a second approach, we selected species that were the least affected by the HKD experiment (which suggests a high thermal tolerance) and could not fly (and, therefore, cannot escape thermal stress
<table><caption>TABLE 1 Ecology of weevil species used in the study</caption><thead><tr><th>Species</th><th>Family</th><th>Subfamily</th><th>Nocturnal/diurnal</th><th>Flight</th><th>Host plant</th><th>Localisation of larva in plant</th><th>Larval stages</th><th>Exposure to fire</th><th>HKD group (Figure 2)</th><th>Geographical coordinates</th><th>Localisation</th><th>Type of ecosystem</th><th>n (HKD)</th><th>Date of collection</th><th>Date of HKD test</th><th>Mean weight</th><th>Species code</th></tr></thead><tbody><tr><td>Cryptolorynx variabilis pop. 1</td><td>Brachyceridae</td><td>Brachycerinae</td><td>D</td><td>no</td><td>Oxalidaceae</td><td>Ground</td><td>enclosed (bulbs)</td><td>yes</td><td>C</td><td>33°57°01.4°S 18°52°30.6°E</td><td>Stellenbosch Mountain</td><td>fynbos</td><td>15</td><td>29/09/2018</td><td>1/10/2018</td><td>0.014 g</td><td>JHAR01185</td></tr><tr><td>Cryptolorynx variabilis pop. 2</td><td>Brachyceridae</td><td>Brachycerinae</td><td>D</td><td>no</td><td>Oxalidaceae</td><td>Ground</td><td>enclosed (bulbs)</td><td>yes</td><td>C</td><td>33°55°53.4°S 18°52°29.7°E</td><td>Jean Marais nature reserve</td><td>fynbos</td><td>22</td><td>30/09/2018</td><td>2/10/2018</td><td>0.017 g</td><td>JHAR01497</td></tr><tr><td>Cryptolorynx pyrophilus</td><td>Brachyceridae</td><td>Brachycerinae</td><td>D</td><td>no</td><td>Oxalidaceae</td><td>Ground</td><td>enclosed (bulbs)</td><td>yes</td><td>B</td><td>33°48°41.4°S 20°08°58.4°E</td><td>Montagu</td><td>fynbos</td><td>15</td><td>24/09/2018</td><td>26/09/2018</td><td>0.004 g</td><td>JHAR01528</td></tr><tr><td>Byrsops apicalis</td><td>Brachyceridae</td><td>Brachycerinae</td><td>D</td><td>no</td><td>unknown</td><td>Ground</td><td>free</td><td>yes</td><td>B</td><td>33°55°53.4°S 18°52°29.7°E</td><td>Jean Marais nature reserve</td><td>fynbos</td><td>14</td><td>30/09/2018</td><td>1/10/2018</td><td>0.021 g</td><td>JHAR01486</td></tr><tr><td>Brachycerus milleporus</td><td>Brachyceridae</td><td>Brachycerinae</td><td>N</td><td>no</td><td>Lilaceae</td><td>Ground</td><td>free</td><td>yes</td><td>A</td><td>34°14°28.5°S 18°51°12.8°E</td><td>Kogelbay</td><td>fynbos</td><td>10</td><td>23/09/2019</td><td>26/09/2019</td><td>0.055 g</td><td>JHAR01501</td></tr><tr><td>Ocladulus costiger</td><td>Brachyceridae</td><td>Ocladinae</td><td>N</td><td>no</td><td>Aizodaceae</td><td>Ground</td><td>enclosed (roots)</td><td>no</td><td>C</td><td>29°41°05.7°S 17°56°36.2°E</td><td>Springbok</td><td>other</td><td>9</td><td>29/09/2019</td><td>7/10/2019</td><td>0.023 g</td><td>JHAR04984</td></tr><tr><td>Hexatmetrus sp.</td><td>Brentidae</td><td>Nanophyinae</td><td>D</td><td>yes</td><td>Ericaceae</td><td>Aerial</td><td>enclosed (fruits)</td><td>yes</td><td>B</td><td>33°57°54.1°S 18°52°34.5°E</td><td>Stellenbosch Mountain</td><td>fynbos</td><td>17</td><td>29/09/2018</td><td>1/10/2018</td><td>0.009 g</td><td>JHAR04985</td></tr><tr><td>Eremus setifer</td><td>Curculionidae</td><td>Entiminae</td><td>N</td><td>no</td><td>Asteraceae</td><td>Ground</td><td>free</td><td>yes</td><td>B</td><td>33°57°01.4°S 18°52°30.6°E</td><td>Stellenbosch Mountain</td><td>fynbos</td><td>14</td><td>29/09/2018</td><td>2/10/2018</td><td>0.013 g</td><td>JHAR00991</td></tr><tr><td>Eremus ecatus</td><td>Curculionidae</td><td>Entiminae</td><td>N</td><td>no</td><td>Asteraceae</td><td>Ground</td><td>free</td><td>yes</td><td>A</td><td>34°03°46.6°S 18°52°25.5°E</td><td>Helderberg</td><td>Renosterveld</td><td>9</td><td>13/10/2018</td><td>15/10/2018</td><td>0.051 g</td><td>JHAR2187</td></tr><tr><td>Sciobius pr. brevicollis</td><td>Curculionidae</td><td>Entiminae</td><td>N</td><td>no</td><td>unknown</td><td>Ground</td><td>free</td><td>no</td><td>B</td><td>29°42°14.2°S 31°02°40.3°E</td><td>Mount Edgecombe</td><td>other</td><td>20</td><td>09/10/2018</td><td>11/10/2018</td><td>0.021 g</td><td>JHAR04986</td></tr><tr><td>Phlyctinus grootbosensis</td><td>Curculionidae</td><td>Entiminae</td><td>N</td><td>no</td><td>Asteraceae</td><td>Ground</td><td>free</td><td>yes</td><td>A</td><td>34°31°44.9°S 19°29°23.1°E</td><td>Grootbos nature reserve</td><td>fynbos</td><td>22</td><td>13/10/2018</td><td>15/10/2018</td><td>0.023 g</td><td>JHAR01300</td></tr><tr><td>Oosomus cf. varius</td><td>Curculionidae</td><td>Entiminae</td><td>N</td><td>no</td><td>Asteraceae</td><td>Ground</td><td>free</td><td>yes</td><td>A</td><td>34°31°44.9°S 19°29°23.1°E</td><td>Grootbos nature reserve</td><td>fynbos</td><td>19</td><td>13/10/2018</td><td>15/10/2018</td><td>0.003 g</td><td>JHAR01301</td></tr><tr><td>Tanyrhynchus carinatus pop. 1</td><td>Curculionidae</td><td>Entiminae</td><td>N</td><td>no</td><td>Poales</td><td>Ground</td><td>free</td><td>yes</td><td>A</td><td>33°41°52.0°S 19°04°26.3°E</td><td>DuToitskloof pass</td><td>fynbos</td><td>14</td><td>24/09/2018</td><td>26/09/2018</td><td>0.017 g</td><td>JHAR03044</td></tr><tr><td>Tanyrhynchus carinatus pop. 2</td><td>Curculionidae</td><td>Entiminae</td><td>N</td><td>no</td><td>Poales</td><td>Ground</td><td>free</td><td>yes</td><td>A</td><td>33°57°01.4°S 18°52°30.6°E</td><td>Stellenbosch Mountain</td><td>fynbos</td><td>22</td><td>29/09/2018</td><td>2/10/2018</td><td>0.015 g</td><td>JHAR00806</td></tr><tr><td>Tanyrhynchus carinatus pop. 3</td><td>Curculionidae</td><td>Entiminae</td><td>N</td><td>no</td><td>Poales</td><td>Ground</td><td>free</td><td>yes</td><td>A</td><td>33°57°54.1°S 18°52°34.5°E</td><td>Stellenbosch Mountain</td><td>fynbos</td><td>19</td><td>4/10/2018</td><td>5/10/2018</td><td>0.014 g</td><td>JHAR01487</td></tr><tr><td>Hypocellosus anaglypticus</td><td>Curculionidae</td><td>Cyclomiinae</td><td>D</td><td>no</td><td>unknown</td><td>Ground</td><td>free</td><td>yes</td><td>B</td><td>33°48°41.4°S 20°08°58.4°E</td><td>Montagu</td><td>fynbos</td><td>17</td><td>24/09/2018</td><td>26/09/2018</td><td>0.023 g</td><td>JHAR01534</td></tr><tr><td>Marius sp.</td><td>Curculionidae</td><td>Curculioninae</td><td>D</td><td>yes</td><td>Campanulaceae</td><td>Aerial</td><td>enclosed (fruits)</td><td>yes</td><td>B</td><td>33°57°01.4°S 18°52°30.6°E</td><td>Stellenbosch Mountain</td><td>fynbos</td><td>7</td><td>29/09/2018</td><td>4/10/2018</td><td>0.011 g</td><td>JHAR01646</td></tr><tr><td>Tychius placidus</td><td>Curculionidae</td><td>Curculioninae</td><td>D</td><td>yes</td><td>Fabaceae</td><td>Aerial</td><td>enclosed (fruits/stems)</td><td>yes</td><td>C</td><td>33°56°30.2°S 18°52°31.0°E</td><td>Stellenbosch Mountain</td><td>fynbos</td><td>6</td><td>29/09/2018</td><td>4/10/2018</td><td>0.011 g</td><td>JHAR01553</td></tr><tr><td>Acaliopistus guttatus</td><td>Curculionidae</td><td>Curculioninae</td><td>D</td><td>yes</td><td>Malvaceae</td><td>Aerial</td><td>enclosed (fruits)</td><td>yes</td><td>C</td><td>33°56°30.2°S 18°52°31.0°E</td><td>Stellenbosch Mountain</td><td>fynbos</td><td>11</td><td>29/09/2018</td><td>4/10/2018</td><td>0.010 g</td><td>JHAR01481</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Fire-prone vegetation types in the Western cape province of South Africa and habitats of several apterous weevils tested in this study. (a) Fynbos vegetation type in the Theewaterskloof nature reserve. (b) Resprouting vegetation in a burnt area near Montagu where *Cryptolarynx pyrophilus* was collected. (c) *Cryptolarynx variabilis*. (Brachycerinae; Cryptolaryngini). (d) *Byrsops apicalis* (Brachycerinae; Byrsopini). (e) *Brachycerus milleporus* (Brachycerinae; Brachycerini). (f) *Ocladius costiger* (Ocladiinae; Ocladiini). (g) *Eremnus setifer* (Entiminae; Tanyrhynchini). (h) *Oosomus cf. varius* (Entiminae; Oosomini). (i) *Tanyrhynchus carinatus* (Entiminae; Tanyrhynchini). (j) *Hypocolobus anaglypticus* (Cyclomiinae; Rhythirrinini).</figcaption></figure>

in their natural environment). The upper thermal limits (CT<sub>max</sub>) of
these species were measured using thermolimit respirometry
(Lighton & Turner, 2004), following methods adapted from Boardman
and Terblanche (2015). All specimens were weighed before and after
respirometry on a microbalance. Airflow was regulated at

200 ml min⁻¹ (STPD) and controlled by a mass flow control valve (SideTrak, Sierra International, United States) into a Li-7000 infra-red CO₂/H₂O analyser fully calibrated for CO₂ at 386 ppm (balanced nitrogen) and water. The CO₂ production was recorded differentially (to ultimately produce an estimate of VCO₂) in ppm by the standard LiCor software (Li-Cor, Lincoln, NE, United States). Activity was monitored using an infra-red activity detector (AD-2, Stable Systems International, Las Vegas, NV, United States). Animals were given a 10-min equilibration period at 25°C, after which the temperature in the chamber was ramped up at a constant heating rate of 0.25°C min⁻¹ to 57°C using a programmable circulating and heating water bath (CC410wl, Huber, Berching, Germany). Baseline recordings were taken before and after each run to correct for potential drift, which was typically negligible. Expedia v. 1.9.10 (Sable Systems International) was used to transform recorded CO₂ values in ppm to VCO₂ in ml CO₂/h, to correct for potential baseline drift for all respirometry files, and to visualise activity data. CTₘₐₓ was defined as the point at which spiracles switched from high to low variability indicating loss of spiracular control (see Javal et al., 2019) and computed according to Boardman and Terblanche (2015).

Statistical analyses

Data were analysed using R 4.0.4 (R Core Team, 2021) with appropri-
ate packages including MuMln (Barton, 2022) and DoBy (Højsgaard &
Halekoh, 2021). For the HKD experiment, data were expressed as
proportions of specimens able to survive at 48°C over a period of
45 min. Survival data were modelled by specifying a full factorial gen-
eralised linear model (GLM) with logistic link function for binary out-
come (i.e., dead/alive). Probability and estimated S.E. for survival were
obtained from fitted GLMs and were used to draw survival lines that
are presented in figures. Visual inspection of HKD data allowed the
graphic categorisation of the species/populations in three groups
(Figure 2a-c) according to their reaction pattern. The less heat-
tolerant group (group A) was represented by species for which all
specimens were dead in less than 500 s, the more heat tolerant group
(group C) included species for which survival probability was higher
than 60% by the end of the trial, and the intermediate group (group B)
included all species in between these two extremes. We ran a one-
way ANOVA followed by a Tukey post hoc test to explore every pos-
sible pairwise comparison within the three groups. The same combina-
tion of tests was used to compare flying species (n = 4, Table 1) and
non-flying populations (n = 15, Table 1), respectively.

RESULTS

Survival probabilities of all species (HKD) followed three main types
of responses (weak, intermediate, and high heat tolerance, Figure 2a).
The less heat-tolerant group (group A) gathered five flightless,
nocturnal species at adult stage, one of which was represented by its
three populations (*Tanyrhynchus carinatus*). All these species have one
<figure><img src="image_4.png" /><figcaption>**FIGURE 2** Survival probabilities of weevil species during heat knockdown (HKD) experiments at 48°C. top left: Overview of survival probabilities of all species tested clustered in three groups: Weak (a), intermediate (b), and high heat-tolerant (c). Bottom left and right: Details for the species included in the three groups.</figcaption></figure>

thing in common: they all develop in the soil in the larval stage, and feed ectophytically. The remaining flightless species clustered in the intermediate (group B, 5 species) and highly tolerant groups (group C, 2 species). The intermediate group gathered a majority of diurnal species except for *Eremnus setifer* and *Sciobus pr. brevicollis*. Species with flight abilities clustered only in the intermediate and highly tolerant groups (2 species each). Finally, the most heat tolerant species (group C) included mainly diurnal species except for *Ocladius costiger* (Table 1 and Figure 2). The two species from non-fire prone ecosystems fell in the intermediate and high tolerant groups (*Sciobus pr. brevicollis* and *Ocladius costiger* respectively).

Overall, results showed that the three groups we defined were quite homogeneous in terms of survival probabilities: 14 pairwise comparisons out of 21 were non-significant in group A, 15 out of 21 in group B and group C was the only one for which species survival probabilities were all statistically similar (Table S1). In group A, survival probability of *Brachycerus milleporus* was significantly higher than that of *Eremnus occatus* (*p* < 0.001) and *Oosomus* sp. (*p* < 0.001). *Eremnus occatus* survival probability was significantly lower than that of *Phlyctinus grootbosensis* (*p* = 0.020) and *Tanyrhynchus carinatus* pop 2 (*p* = 0.023). Finally, *Oosomus* sp. had a significantly lower survival probability than *Phlyctinus grootbosensis* (*p* < 0.001) and *Tanyrhynchus carinatus* 2 and 3 (*p* < 0.001 and *p* < 0.05, respectively). In group B, *Cryptolarynx pyrophilus* has a significantly higher survival probability

<table><caption>**TABLE 2** Summary of data of $CT_{max}$ measurements for *Ocladius costiger* and *Cryptolarynx variabilis*</caption><thead><tr><th rowspan="2">Specimen number</th><th colspan="2">Ocladius costiger</th><th colspan="2">Cryptolarynx variabilis</th></tr><tr><th>CT<sub>max</sub> (°C)</th><th>Mass (g)</th><th>CT<sub>max</sub> (°C)</th><th>Mass (g)</th></tr></thead><tbody><tr><td>1</td><td>47.6</td><td>0.0072</td><td>53.4</td><td>0.0023</td></tr><tr><td>2</td><td>51.6</td><td>0.0088</td><td>53.3</td><td>0.0105</td></tr><tr><td>3</td><td>49.8</td><td>0.0072</td><td>48.9</td><td>0.0031</td></tr><tr><td>4</td><td>49.4</td><td>0.0085</td><td></td><td></td></tr><tr><td>5</td><td>52.6</td><td>0.0092</td><td></td><td></td></tr><tr><td>Mean</td><td>50.2</td><td>0.0082</td><td>51.9</td><td>0.0053</td></tr><tr><td>SEM</td><td>1.95</td><td>0.0004</td><td>2.5</td><td>0.0026</td></tr></tbody></table>

than any other species (*p* < 0.001 in all cases but *Miarus* sp. for which *p* < 0.05). The comparison of flying species revealed that only *Tychius placidus* and *Acallopistus guttatus* on the one hand and *Miarus* sp. and *Hexatmetus* sp. on the other hand had a similar resistance to HKD. Among the 15 non-flying species/population, 47 of the 105 pairwise comparisons revealed significant differences (*p* < 0.001; Table S1). Details of the pairwise comparisons are given in Table S1.

$CT_{max}$ was estimated from thermolimit respirometry for *Ocladius costiger* and *Cryptolarynx variabilis* since they were the only species
from group C that could not fly and were little or unaffected by the
HKD experiment. CTmax values ranged from 47.6 to 52.6°C for Ocla-
dius costiger (n = 5, mean = 50.2°C, SEM = 1.95) and from 48.9 to
53.4°C for Cryptolarynx variabilis (n = 3, mean = 51.9°C, SEM = 2.51),
and the mean values were not significantly different between the two
species (p = 0.161, Table 2). These two species exhibit a small (about
0.015 g for Cryptolarynx variabilis) to average (about 0.023 g for Ocla-
dius costiger) body weight when compared to all species tested
(Table 1).

DISCUSSION

This study provides the first investigation of heat tolerance of multiple
species of a phytophagous insect group in fire-prone ecosystems of the
CFR. Within the weevil superfamily (Curculionoidea), the species we
assessed displayed a remarkable range of reactions to a standardised
heat stress at the adult stage. The general trend observed in this study
indicates an ability to tolerate heat stress that is mostly species-specific,
which is notable for phylogenetically related insects occurring in the
same ecosystem. This trend, however, echoes the patterns observed in
dung beetles (Scarabaeidae), for example, where substantial variation in
heat tolerance exists across species (Gotcha et al., 2021). Within the
weevils analysed, some species showed a high sensitivity to the HKD
assay while others exhibited a very high heat tolerance, similar to high-
est records among beetles (i.e., Roberts et al., 1991). It should be noted
that these previous records were measured on tenebrionid beetles from
the Namib desert with putative morphological adaptations to heat such
as long legs, larger size, and thick cuticle (Marden, 1987; Naidu, 2001).
Since CTmax often correlates positively with body size both within and
across species (O'Donnell et al., 2020; Wendt & Verble-Pearson, 2016),
high heat resistance of the minute weevils tested here (a few milligrams)
is unexpected and calls for further investigations. These results show
that in addition to ants and tenebrionid beetles, weevils should be con-
sidered among the handful of arthropod lineages whose CTmax values
exceed 50°C (Bennett et al., 2018). However, a range of intrinsic and
extrinsic factors can influence heat tolerance estimates (Mitchell et al.
2017; Clusella-Trullas et al. 2021) and we recommend future investiga-
tions to consider larger sample sizes, high resolution microclimatic data,
and complementary approaches to refine this finding. We also do not
explore thermal acclimation effects and phenotypic plasticity more
broadly. In some taxa, high levels of plasticity can be expressed rapidly
under specific environmental conditions and may alter conclusions of
resistance or heat susceptibility (González-Tokman et al., 2020), espe-
cially if fire-prone weevils possess a pronounced ability to mount heat
hardening (protective) responses triggered by fire-related cues.

Heat tolerance in flying versus non-flying species

With regard to the first hypothesis we proposed, we found no clear
pattern allowing conclusion about a correlation between flight abilities
and heat tolerance. Contrary to expectations, all species with flight

abilities clustered in the groups of moderate and highly tolerant spe-
cies. In other words, species with a physical ability to rapidly escape
peaks of heat and fire were also the ones showing a strong tolerance
to heat stress. A first possible explanation is the positive correlation
between flight abilities and heat tolerance due to the heat induced by
flight muscles during insects' activity (Stevenson, 1985). A second
explanation relates to the specific life-cycle and micro-environment
experienced by the immature and adult stages of the flying species
tested (Acallopistus, Hexatmetus, Miarus and Tychius; all from the CFR).
The Bogert effect proposes that the less mobile life stages of ecto-
therms show greater thermal tolerance than mobile life stages to com-
pensate for their inability to behaviourally escape thermal stress
(Marais & Chown, 2008; Mitchell et al., 2013). One could expect
thermo-tolerance to decrease with ontogeny and the transition from
the larval to the adult stage, since it is associated with the acquisition
of an increased mobility and capacity to avoid heat stress (Bowler &
Terblanche, 2008). The species studied here follow the phenology of
their hosts: they oviposit in tissues of the aerial part of their host
plants (seeds or stems) during spring while temperatures are mild, and
the development lasts until seed set, during the summer (JH pers.
obs.). As such, the last larval instars and the pupae but also teneral
adults may experience sustained or extreme heat with little possibility
to escape until they emerge as sclerotised, flying adults.

In this context, high heat tolerance might have been selected as it is
a requirement for the colonisation and survival of all stages on aerial
plant tissues exposed to summer heat typical of the region. Interestingly,
the non-flying species showing the highest heat tolerance are also the
ones for which immature stages cannot readily escape the plant struc-
tures used for development. In the two species of Cryptolarynx, larval
development takes place enclosed in the bulbs of Oxalis species
(Oxalidaceae) and teneral adults may stay in this structure for weeks
(Haran et al., in press). As bulbs of Oxalis are not always standing deep
in the ground, the immature may experience extreme heat (due to fire
or not) without the possibility to escape. Similarly, the larvae of Ocladius
costiger occupy the base of the woody root system of its host and
remain enclosed in it near to the surface of the ground. Such physical
constraints have probably selected resistance to heat stress and the high
tolerance observed in these species. Inversely, a common denominator
for the least tolerant species (group A) is that the larval stage develops
in the soil, but not in a particular plant structure. Consequently, these
individuals are potentially able to burrow deeper into the soil in case of
severe heat stress, which could be considered as a case of behavioural
thermoregulation (Marvaldi et al., 2014; Oberprieler, 2014; Pringle
et al., 2015). Thus, these species are not confronted with high thermal
stresses and their tolerance is, therefore, low, which highlight the impor-
tance of microenvironment in stress resistance.

Non-flying lifestyle comes with a range of strategies
to escape or cope with heat

The ten non-flying species of weevil from the CFR exhibited a wide
range of reactions to heat stress. Noticeably, several congeneric
species clustered in distinct groups based on heat resistance (Eremnus
spp. in groups A and B and Cryptolarynx spp. in groups B and C) and
representatives of a single subfamily were found in all three groups (Bra-
chycerus, Byrsops, and Cryptolarynx among the Brachycerinae). A promi-
nent parameter associated with heat tolerance in these non-flying
species is the phenology of adult activity, corresponding to active move-
ment for feeding and moving to search for a partner or for an oviposi-
tion site. All the less tolerant species (group A) are active at night
whereas all species clustering in group B and C (except E. setifer) are
diurnal. This was also observed in other insects groups such as ants
(Garcia-Robledo et al., 2018). It corroborates the thermal adaptation
hypothesis, which not only states that more thermally variable environ-
ments host eurytherm organisms (Janzen, 1967) but also that ecto-
therms' thermal limits positively correlate with the temperature
extremes of their environment (Addo-Bediako et al., 2000, even though
some exceptions are known, especially for high temperatures: Bujan
et al., 2020). Diurnal weevil species are exposed to the high summer
temperature experienced in the CFR (Altwegg et al., 2014; van Wilgen
et al., 2016), which may explain why they evolved high heat resistance.
Conversely, the non-flying species active during the night generally hide
in the leaf litter at the base of their plant during the day. As they have
apparently not developed a physiological adaptation to heat stress, they
seem to survive this constraint by escaping it physically burrowed
underground and thus protected to some extent from heat extremes.

Burning versus non-burning ecosystems

This study included only a few weevils from neighbouring non-
burning ecosystems to the CFR (*Sciobius pr. brevicollis* and *Ocladius
costiger*). These two species are non-flying and are active at night, but
showed a moderate and high tolerance to HKD, respectively. *Ocladius*
exhibited CTmax values as high as those observed in the highly heat
tolerant *Cryptolarynx variabilis*. This species is occurring in the western
part of the Northern Cape region, where temperatures are higher than
in the CFR but shows no natural cycles of fires (van Wilgen
et al., 2016). As previously reported, the larval stages of this species
dig galleries in the woody root system of the host plant, with limited
possibility to move deeper to escape heat at the surface of the
ground. These first insights suggest that heat tolerances of non-flying
weevil species are associated with general climate conditions experi-
enced in an ecosystem rather than by the fire regime itself.

CONCLUSION

This study provides preliminary trends on the tolerance to heat of sev-
eral species of weevils associated with a fire-prone ecosystem. We
show that there is no general phylogenetic conservatism for heat toler-
ance of adults, but rather that lifestyle plays a major role in physiological
adaptation to heat stress. Day versus night activity of adults and
enclosed versus free lifestyle of larvae were associated with a higher tol-
erance to heat stress in adults. As fire generally occurs during summer

months and mostly affects immature stages, the higher tolerance of spe-
cies developing as endophagous in the ground with no possibility to
escape may represent an adaptation to this disturbance. That said, spe-
cies with similar lifestyles from neighbouring non-burning regions
showed similar heat tolerance. These observations suggest that there is
a general adaptation to heat stress due to lifestyle constraints. However,
whether the heat is caused by solar radiation or fire does not seem to
be a prominent parameter. Among the species tested, *Cryptolarynx* spe-
cies are probably the best candidates to conduct further investigations
on resistance to extreme heat of arthropods. These very small and slow-
moving species showed a remarkable tolerance to heat stress in the
adult stage while immature stages spend the hottest months enclosed in
the sealed bulb of their host plants only a few centimetres below the
surface. The physiological adaptations of these species may provide rel-
evant insights on how phytophagous insects cope with extreme temper-
ature conditions and how they will react to a warming climate.

AUTHOR CONTRIBUTIONS

Marion Javal and Julien Haran designed the research; John S. Terblanche
provided the equipment; Julien Haran conducted the insect sampling
and collected insect data; Julien Haran, Marion Javal, and Chantelle Smit
produced the experimental work; Marion Javal analysed the data; Mar-
ion Javal and Julien Haran drafted the manuscript. All authors read and
approved the final version.

ACKNOWLEDGEMENTS

The authors thank the Western Cape Nature Conservation Board
(Permit No. CN44-30-4229) and the Cape Research Centre,
South African National Parks (Permit No. CRC/2019-2020/012-2012/
V1) for permission to collect specimens in areas under their control. The
authors acknowledge Paula Strauss, Gary Beukman, and Michael Lut-
zeyer for access to the Grootbos Private Nature Reserve and their
friendly assistance during sampling. The authors thank Laurène Dubreuil
for introducing Julien Haran and Marion Javal to the Montagu region.
Henrika Bosua (Stellenbosch University) is warmly thanked for her help
with the respirometry measurements. This study was supported by
recurring funding from Montpellier University (Marion Javal) and Cirad
(Julien Haran).

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed
as a potential conflict of interest.

DATA AVAILABILITY STATEMENT

Data for the HKD experiment is available here: https://doi.org/10.
6084/m9.fishshare.21647015

ORCID

Marion Javal ID https://orcid.org/0000-0001-7878-2936

John S. Terblanche https://orcid.org/0000-0001-9665-9405

Chantelle Smit https://orcid.org/0000-0001-7568-7992

Julien Haran https://orcid.org/0000-0001-9458-3785
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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

### Table S1.

**How to cite this article:** Javal, M., Terblanche, J.S., Smit, C. & Haran, J. (2023) Comparative assessment of heat tolerance in weevils associated with a fire-prone ecosystem. *Ecological Entomology*, 48(2), 240–250. Available from: https://doi.org/10.1111/een.13218