Thermal resilience of Prostephanus truncatus (Horn): Can we derive optimum temperature-time combinations for commodity treatment?

Journal of Stored Products Research 86 (2020) 101568

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Thermal resilience of Prostephanus truncatus (Horn): Can we derive optimum temperature-time combinations for commodity treatment? Honest Machekano a, Reyard Mutamiswa a, b, Charles Singano c, Virgil Joseph a, Frank Chidawanyika b, Casper Nyamukondiwa a, * a

Department of Biological Sciences and Biotechnology, Botswana International University of Science and Technology (BIUST), Private Bag 16, Palapye, Botswana Department of Zoology and Entomology, University of the Free State, Bloemfontein, South Africa c Chitedze Agricultural Research Station, Department of Agricultural Research Services, P.O. Box, 158, Lilongwe, Malawi b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 August 2019 Received in revised form 13 November 2019 Accepted 16 January 2020 Available online xxx

Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae), is a wood-boring, destructive quarantine insect pest of stored cereal grains and tuber crops. Current disinfestation methods against this pest mainly include fumigants, whose usage in some countries has been contested and discontinued owing to increasing pesticide resistance, public health risks and environmental hazards. Grain temperature treatments thus, offer a sustainable non-chemical and near universally acceptable form of disinfestation for international commodity movement. Currently, blanket temperature treatments are applied regardless of as-yet-unknown P. truncatus developmental stage thermal mortality thresholds that simultaneously optimise grain quality. Here, we used established static and dynamic protocols to determine the low and high thermal profile of P. truncatus larvae and adults measured as critical thermal minima (CTmin), lower lethal temperatures (LLT0), chill coma recovery time (CCRT), supercooling points (SCPs), critical thermal maxima (CTmax), upper lethal temperatures (ULT0) and heat knock-down time (HKDT). We tested the adult ULT-time matrices on maize and sorghum grain quality (germination %) to determine the most effective temperature-time combination(s) retaining optimum grain germination quality. Our results showed adults had higher basal heat (CTmax and HKDT), cold (CTmin, CCRT and SCP) and potential thermal plasticity than larvae (P < 0.05). The LLTs and ULTs ranged
1 to
15  C and 41 e49  C respectively. Using LLT0 and ULT0, our results showed that for heat treatment, moderate temperature  long duration matrix; i.e. either 45.5  C  4 h or 47  C  2 h were more efficacious while retaining commodity quality. Similarly, for cold treatment;
9  C  4 h,
11  C  2 h,
13  C  1 h and
15  C  0.5 h were effective for complete mortality. These temperature-time combinations may be a sustainable alternative to fumigants in phytosanitary grain disinfestation against P. truncatus or related pests. Such pest- and commodity -specific thermal profiling is critical for development of effective standardised grain disinfestation protocols. © 2020 Elsevier Ltd. All rights reserved.

Keywords: Critical thermal limits Lethal temperatures Grain temperature treatment Commodity quality Postharvest pests

1. Introduction The larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrychidae), is a wood-boring, destructive quarantine insect pest of stored cereal grains and tuber crops in Africa, particularly maize and dried cassava (Hodges, 1986; Markham et al., 1991). Since its accidental arrival in East Africa from the meso-America in

* Corresponding author. E-mail address: [email protected] (C. Nyamukondiwa). https://doi.org/10.1016/j.jspr.2020.101568 0022-474X/© 2020 Elsevier Ltd. All rights reserved.

the late 1970s, it has rapidly spread and established in over 16 countries across the width and breadth of Africa (Dunstan and Magazine, 1981; Farrell, 2000; EPPO Global Database, 2019; Doganay et al., 2018) as well as parts of Europe (EPPO Global Database, 2019; Doganay et al., 2018). Damage on maize cobs or shelled grain can start in the field (pre-dispersal) (reviewed in Hodges, 1986; Food and Agriculture Organisation (FAO), 1997) and continue in storage (post dispersal) (Hodges, 1986; Tefera et al., 2011), together accounting for cumulative ~10e30% cob/grain/ seed loss in about three months (reviewed in Hodges, 1986; Boxall, 2002). Prostephanus truncatus highly sclerotized mandibles and

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insatiate feeding (Vendl et al., 2018), has potential to reduce huge amounts of stored products to fine dust at low population densities (Osipitan et al., 2011; Tefera et al., 2011; Nhamucho et al., 2017). Moreover, it can also be a nuisance pest, attacking many other nonfood household and outdoor products e.g. shoes, wood, plastic utensils, curtains, sofas and others (Boxall, 2002; Nang’ayo et al., 2002; Muatinte and van den Berg, 2019). Apart from resistance to organophosphates and pyrethroids (Rumbos et al., 2013), the efficacious control of P. truncatus is difficult owing to its ability to thrive on wood in non-agricultural habitats (Muatinte et al., 2014; Arthur et al., 2019). Similarly, Muatinte and van den Berg (2019) recently reported the species can survive and successfully breed on many non-cultivated grass and indigenous tree species in Mozambique. In addition, P. truncatus can survive on dry overwintering field maize stalks and several unidentified dry and/or fresh indigenous wood and grass species in Africa (Nang’ayo et al., 2002; Muatinte and van den Berg, 2019). This host diversity likely facilitates its successful establishment and may likely broaden the magnitude of its biosecurity risk. This ecological threat therefore requires a reciprocal effort on sustainable management methods to safeguard livelihoods dependant on cereal agriculture. Stored product integrated pest management (IPM) involving multiple tactics (White, 1992), and incorporating the use of postharvest temperature treatments (Fields, 1992; Lurie, 1998; Tang et al., 2000; Stejskal et al., 2019) has recently regained renewed interest as a safe, effective and sustainable pest management option in stored product entomology (Hansen et al., 2011; Stejskal et al., 2019). Due to rising global concerns for public health risks and food safety issues associated with pesticides, the use of synthetic grain protectants on stored grain is increasingly being scrutinised. Given the quarantine nature of P. truncatus against the backdrop of the ‘ban’ on landmark fumigants such as methyl bromide (Fields and White, 2002; Byrns and Fuller, 2011) and the rising trend of resistance to current phytosanitary grain fumigants such as aluminium phosphide (Opit et al., 2012; Nayak et al., 2013; Holloway et al., 2016; Wakil et al., 2018), there is need for research to promptly fill the void for effective, non-chemical control of P. truncatus (Singano et al., 2020). Considering the increasing global trade in grain and humanitarian food aid, phytosanitary heat treatments against P. truncatus offer a non-chemical and near universally acceptable form of disinfestation for international grain movement across political and quarantine regulatory boundaries (Lurie, 1998; Hansen et al., 2011; Stejskal et al., 2019). Heat treatment is technically more applicable particularly in storage facilities because of their enclosed nature and relatively small sizes that enable effective temperature manipulation through the grain bin plenums (Hansen et al., 2011; Stejskal et al., 2019). Temperature is indiscriminate and kills all life stages of target pest insects including immobile juveniles and inconspicuous insects ‘hiding’ in grain kernels, cracks and crevices, where they would otherwise be inaccessible e.g. by dust pesticide formulations (Giga and Canhao, 1992). However, data on the most effective thermal lethal thresholds for most species are scarce, highly variable or scattered in literature (Mourier and Poulsen, 2000; Bruce et al., 2004; Stejskal et al., 2019). Thus, establishment of optimum species-specific time  temperature matrices that achieve maximum efficacy while minimising commodity damage for development of standardised temperature treatment protocols warrants investigation. The first key to the development of effective postharvest thermal disinfestation treatment protocols is the knowledge of lifestage specific thermal sensitivity of the target insect pest species (Tang et al., 2000; Izadi et al., 2019). Indeed, thermal tolerance varies across insect species, developmental stages, age, sex and other factors (reviewed in Bowler and Terblanche, 2008; see also Marais et al., 2009; Nyamukondiwa and Terblanche, 2009; Arias

et al., 2011; Li et al., 2019; Mutamiswa et al., 2019). Moreover, some species or developmental stages may be more vulnerable to lower than higher temperatures (Arias et al., 2011; Li et al., 2019) thus raising complexities in the development of pest management using universal thermal treatments. Therefore, for species and lifestage specific maximal efficacy, it is critical to determine whether low or high temperature treatments are optimal (Tang et al., 2000), and whether applied as acute or in a dynamic protocol (Tang et al., 2000; Izadi et al., 2019). Moreover, for species susceptible to high temperature treatments, the optimum time  temperature matrix retaining the best commodity quality at maximal pest efficacy needs to be established. To our knowledge, this information is missing for P. truncatus and other stored product pest insects (Stejskal et al., 2019 but see Bruce et al., 2004). Here, we investigated the basal thermal profile of P. truncatus through critical thermal activity thresholds and lethal temperatures for both larvae and adults and the likely costs and benefits of these on commodity quality. We link our thresholds to commodity quality using the most common cereal staples in southern Africa (i.e. maize and sorghum), aiming to provide a systematic approach to grain temperature treatment protocols as a safe grain disinfestation alternative to pesticides. 2. Materials and methods 2.1. Insect culture The initial colony of P. truncatus was obtained from Chitedze Research Station, Malawi (13 850 S; 33 380 E). This colony has been in the laboratory for more than 10 generations with regular augmentation with wild populations to maintain genetic diversity. The colony was maintained on maize grain with a moisture content of ~13% in climate chambers (HPP 260, Memmert GmbH þ Co.KG, Germany) at 32 ± 1  C and 80 ± 10% RH in 1000 ml glass jars with perforated metal screw-cap lids. Unsexed adult P. truncatus were placed in a series of these glass jars with each jar holding 500 g of maize grain. The jars were sealed with perforated screw cap lids for ventilation and to prevent the escape of insects. Sieving of grain and insects was done after three weeks assuming that oviposition had occurred so that offspring of a synchronized age would be obtained. The cultures were left undisturbed until adult emergence. Adult insects that emerged from the cultures within the first 7 days of emergence (0e7 days old) and larvae (third instar) were used in this study. Re-culturing of the insects was carried out at regular intervals to ensure availability of experimental organisms. 2.2. High temperature tolerance of P. truncatus 2.2.1. Ramping rate effects on critical thermal maxima (CTmax) To assess whether heating rate affects P. truncatus thermal tolerance, CTmax was measured using standardised protocols (see Nyamukondiwa and Terblanche, 2009; Nyamukondiwa and Terblanche, 2010; Mutamiswa et al., 2017; Machekano et al., 2018a). Ten P. truncatus larvae and adults were randomly placed in ‘organ pipes’ connected to a programmable water bath (Lauda Eco Gold, Lauda DR.R. Wobser GMBH and Co. KG, Germany) that was filled with 1:1 water: propylene glycol. Whilst in the organ pipes, insects were subjected to 10 min equilibration period at 32  C (optimal temperature) before ramping the temperature up at rates of 0.12, 0.25 and 0.5  C/min until their CTmax were recorded. A thermocouple (type K 36 SWG) connected to a digital thermometer (53/54IIB, Fluke Cooperation, USA) was inserted in a control chamber to record organ pipe temperature. This was repeated twice to yield sample sizes of n ¼ 20. Each insect body temperature was assumed to be in equilibrium with the chamber temperature as

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previously outlined in similar insect taxa (Terblanche et al., 2007). Critical thermal maximum was defined as the highest temperature at which each individual insect lost coordinated muscle function which was regarded as a lack of coordinated response following stimuli e.g. mild prodding (e.g. Nyamukondiwa and Terblanche, 2010). 2.2.2. Heat knockdown time (HKDT) Heat knock down times (HKDTs) for P. truncatus larvae and adults were assayed as outlined by Weldon et al. (2011). Ten replicate adults and larvae were individually placed in numbered 0.65 ml microcentrifuge tubes and placed in a climate chamber connected to a camera (HD Covert Network Camera, DS2CD6412FWD-20, Hikvision Digital Technology Co., Ltd, China) that was linked to a computer. These tubes were later exposed to a knockdown temperature of 50 ± 0.3  C; 80% RH derived from preliminary critical thermal maxima investigations ranging 45.17 ± 0.95  C and 49.02 ± 0.79  C for larvae and adults respectively. This was repeated twice to yield sample sizes of n ¼ 20 per each treatment. All observations and data were recorded from the climate chamber video recording system. Heat knockdown time was defined as the time (in minutes) at which organisms lost activity following acute (50  C) heat stress in a climate chamber. 2.2.3. Upper lethal temperature assays (ULT) The upper lethal temperatures were assayed using established protocols (Chidawanyika and Terblanche, 2011; Mutamiswa et al., 2018), through a direct plunge technique in programmable water baths (Systronix, Scientific, South Africa), filled with a mixture of propylene glycol and water (1:1 ratio). Upper lethal temperature assays ranged from 41 to 49  C for 0.5e4 h durations until % mortality was recorded. Ten P. truncatus adults were placed in 60 ml polypropylene vials with perforated screw-cap lids, replicated five times (n ¼ 50). To avoid desiccation related mortalities, wet cotton wicks were placed in each vial to maintain optimum RH. Thereafter, the vials were loaded into a water-resistant zip-lock bag which was submerged in the water bath for each temperature/time treatment. Digital thermometers (Fluke 53/54IIB, Fluke Cooperation, USA) were used to monitor water bath temperatures for the whole duration of the experiment. Following treatment, propylene vials containing assayed P. truncatus adults were placed in a climate chamber set at 32  C; 80% RH and provided with maize grain (as food at 13% moisture content) during the entire recovery period. Survival was recorded 24 h after treatment. In this study, survival was defined as coordinated muscle response to stimuli such as gentle prodding, or normal behaviours such as walking, feeding or flying (24 h post treatment). 2.3. Low temperature tolerance in P. truncatus 2.3.1. Ramping rate effects on critical thermal minima (CTmin) To determine cooling rate effects on low temperature tolerance of P. truncatus, CTmin was assayed following standardised protocols (see Nyamukondiwa and Terblanche, 2010; Mutamiswa et al., 2017; Machekano et al., 2018a). As in heating rate experiments, ten P. truncatus larvae and adults were randomly placed in the ‘organ pipes’ connected to a programmable water bath filled with 1:1 water: propylene glycol to allow for sub-zero temperatures. Thereafter, temperature was ramped down at a rate of 0.12, 0.25 and 0.5  C/min until their CTmin were recorded. This was also repeated twice to yield sample sizes of n ¼ 20. Critical thermal minimum was defined as the lowest temperature at which each individual insect lost coordinated muscle function which was regarded as a lack of response to mild stimuli e.g. gentle prodding (e.g. Nyamukondiwa and Terblanche, 2010).

3

2.3.2. Chill coma recovery time (CCRT) Prostephanus truncatus larvae and adults CCRT were assayed following experimental modifications from Weldon et al. (2011). As in HKDT experiments, organisms (larvae and adults) were individually placed in 0.65 ml microcentrifuge tubes before being placed in ziplock bags and submerged into a water bath (Systronix, Scientific, South Africa) filled with 1:1 water: propylene glycol set at 0  C for 1 h. The temperature  time interaction used in this experiment has been reported to elicit chill-coma in other insect taxa (see Weldon et al., 2011; Sinclair et al., 2015). Following 1 h exposure to chill-coma temperature, micro-centrifuge tubes were immediately transferred from the water bath to the climate chamber set at 32  C, 80% RH and connected to a video recording camera linked to a computer. This was repeated twice to yield sample sizes of n ¼ 20 per each life stage. Observations were also recorded from the climate chamber video recording system. Chill coma recovery time was defined as the time (in minutes) required to regain coordinated movement (in larvae) and to stand upright on their legs (for adults) (Milton and Partridge, 2008) following chill coma. 2.3.3. Lower lethal temperature assays (LLT) As in ULT experiments, lower lethal temperatures were assayed using a direct plunge protocol in programmable water baths (see Chidawanyika and Terblanche, 2011; Mutamiswa et al., 2018). Lower lethal temperatures ranged from
1 to
15  C for 0.5e4 h durations, % mortality was recorded. Ten P. truncatus adults were placed in 60 ml polypropylene vials with perforated screw-cap lids, replicated five times (n ¼ 50) and then loaded into a zip-lock bag. The zip-lock bag with insects was submerged in the water bath filled with a mixture of propylene glycol and water (1:1 ratio to allow for sub-zero temperatures without freezing) for each temperature  time treatment. Following treatment, grain was added in each propylene vial with insects and placed in a climate chamber (32  C; 80% RH) before measuring survival after 24 h. Survival was defined as coordinated muscle response to stimuli such as gentle prodding, or normal behaviour such as flying (24 h post treatment). 2.3.4. Supercooling points (SCPs) Supercooling points of P. truncatus adults and larvae were assayed as outlined by Nyamukondiwa et al. (2013). A total of 20 insects were individually placed in 0.65 ml microcentrifuge tubes. A tip of a type-T copper-constantan thermocouple (762e1121, Cambridge, UK), inserted through the lid of the tube was attached to each insect with both thermocouple and individual insect being secured in place by a cotton wool. These thermocouples were connected to one of two 8-channel Picotech TC-08 (Pico Technology, Cambridge, UK) thermocouple interfaces and temperatures were recorded at 1s intervals using PicoLog software for Microsoft Windows® (Pico Technology, Cambridge, UK). Experiments commenced by briefly holding individual insects at 15  C for 10 min (to allow for insects’ body temperature equilibration) before ramping down at a rate of 0.5  C/min until their SCPs were recorded. SCP for each individual insect was determined as the lowest temperature recorded prior to a spike in temperature associated with latent heat of crystallization (Nyamukondiwa et al., 2013). 2.4. Maize and sorghum responses to high temperature treatment Maize and sorghum grain were procured from Botswana Agricultural Marketing Board (BAMB), Palapye, Botswana. Grain was exposed to the ULTs that showed 100% mortality on P. truncatus (Fig. 1C) to test for % germination. This germination experiment was not done for LLTs since low temperatures may not have negative

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effects on grain germination quality (Jayas and White, 2003; Braunbeck, 2014). One hundred seeds of each grain type were placed into separate Ziploc bags and dipped into a programmable waterbath (Systronix, Scientific, South Africa) set at different temperature-time combinations derived from P. truncatus adults ULT0; 45.5  C  4 h, 47  C  2 h, 48  C  1 h, 49  C  0.5 h and 50  C  0.5 h replicated three times. After treatment, seed germination tests were carried following standard protocols (International Seed Testing Association (ISTA), 2001). Cotton wool was used as a moisture retaining substrate at 28 ± 1  C. Germinated seeds were counted and expressed as a percentage of total seeds tested. In this study a seed was said to have germinated successfully when the radicle was visible indicating its ability to produce a satisfactory plant (International Seed Testing Association (ISTA), 2001). 2.5. Statistical analyses Statistical analyses were carried out in STATISTICA, version 13.0 (Statsoft Inc., Tulsa, Oklahoma) and R version 3.3.0 (R Development Core Team, 2016). The lethal temperature assay results did not meet assumptions of ANOVA; therefore, these were analysed using generalized linear models (GLM) assuming a binomial (for LLT and ULT), Gaussian (for CCRT, HKDT and SCP) and a logit link function in R statistical software. Germination % and critical thermal limits (CTLs) data met the linear model assumptions of constant variance and normal errors, therefore, the effects of ramping rate on CTmax and CTmin were analysed using one-way ANOVA in STATISTICA. Tukey-Kramer’s post-hoc tests were used to separate statistically heterogeneous groups. 3. Results 3.1. High temperature tolerance in P. truncatus 3.1.1. Ramping rate effects on CT max At an intermediate ramping rate of 0.25  C/min, the CTmax for P. truncatus adult was 49.03 ± 0.14  C and was significantly higher than that of the larvae (45.2 ± 0.20  C). Ramping rate significantly affected CTmax in larvae and adults of P. truncatus (Table 1; Fig. 1A). Faster heating rate showed high temperature tolerance (CTmax) in both larvae and adults, albeit more in adults than larvae (Fig. 1A). There was a significant difference in CTmax between larvae and adults exposed to the two slower ramping rates (0.12 and 0.25  C/ min). However, no significant developmental stage difference (P > 0.05) was recorded on CTmax following faster (0.5  C/min) ramping rate (Fig. 1A). 3.1.2. Heat knockdown time The mean HKDTs for P. truncatus larvae and adults were 2.74 ± 0.77 and 9.26 ± 1.5 min respectively (Fig. 1B). Adults took Table 1 Summary statistical results of the effects of ramping rates on Prostephanus truncatus larvae and adults critical thermal limits; recorded as critical thermal maxima (CTmax) and critical thermal minima (CTmin). DF ¼ Degrees of freedom, F ¼ FishereSnedecor test statistic (total df ¼ 114; error df ¼ 119). Fig. 1. Effects of high temperature on the larva and adult of P. truncatus. (A) Summary results showing mean ± 95% confidence limits heating rate effects on P. truncatus larvae and adults measured as critical thermal maxima. (B) Effect of life stage (larvae and adults) on heat knockdown time. (C) Survival of P. truncatus adults at high temperatures applied over four different durations. Error bars represent 95% CLs (N ¼ 20). Means with the same letter(s) are not significantly different from each other.

Source of variation Critical thermal maxima Developmental stage Ramping rate Developmental stage * Ramping rate Critical thermal minima Developmental stage Ramping rate Developmental stage * Ramping rate

DF

F

p

1 2 2

97.05 159.01 25.93

˂ 0.001 ˂ 0.001 ˂ 0.001

1 2 2

1502.13 16.21 22.17

˂ 0.001 ˂ 0.001 ˂ 0.001

H. Machekano et al. / Journal of Stored Products Research 86 (2020) 101568

5

significantly more time to be knocked down by acute heat stress (50 ± 0.3  C) compared to larvae (c2 ¼ 157.98, d.f ¼ 1, P ˂ 0.05; Fig. 1B), indicating high temperature tolerance.

was a significant difference in SCPs between larvae and adults (c2 ¼ 41.31, d.f ¼ 1, P ˂ 0.05) (Fig. 2D), with larva recording lower (more negative) SCPs than adults.

3.1.3. Upper lethal temperature assays High temperature and duration of exposure significantly affected survival of P. truncatus adults (P ˂ 0.001) (Table 2; Fig. 1C). An increase in temperature above 43  C and duration of exposure significantly increased mortality in adults of P. truncatus (Fig. 1C). In addition, high temperature  duration of exposure interaction effect was not significant (P > 0.05) (Table 2). For durations of 0.5, 1, 2 and 4 h, upper lethal temperatures for 100% mortality (ULT0) ranged from 45.3 to 49  C (Fig. 1C).

3.3. Maize and sorghum responses to high temperature treatment

3.2. Low temperature tolerance in P. truncatus 3.2.1. Ramping rate effects on CT min At intermediate rate of 0.25  C/min, the CTmin for P. truncatus adults (4.2 ± 0.17  C) was significantly lower than that of the larvae (10.9 ± 0.08  C; Fig. 2A). Ramping rate significantly influenced CTmin in P. truncatus larvae and adults (P ˂ 0.001) (Table 1; Fig. 2A). Slower cooling rates of 0.12  C/min were associated with significantly higher adult CTmin than the faster cooling rates of 0.25  C and 0.5  C/min. There were no significant differences between the two latter rates (Fig. 2A). Furthermore, there was no significant effect of cooling rates on larval CTmin (Fig. 2A). 3.2.2. Chill coma recovery time There was a significant difference in CCRT between P. truncatus larvae and adults (c2 ¼ 30.41, d.f ¼ 1, P ˂ 0.05) (Fig. 2B). Chill coma recovery times averaged 7.15 ± 1.6 and 3.78 ± 0.97 min for larvae and adults respectively (Fig. 2B), with adults recovering faster than larvae (P ˂ 0.05) following an exposure to chill-coma temperature of 0  C for 1 h (Fig. 2B). 3.2.3. Lower lethal temperature assays Low temperature decrease and duration of exposure significantly influenced P. truncatus adults’ survival (P ˂ 0.001) (Table 2; Fig. 2C). Consistent with ULT assays, an increase in duration and magnitude of low temperature exposure resulted in significant increase in mortality of P. truncatus adults (Fig. 2C). Similarly, duration of exposure  low temperature interaction was significant (P ˂ 0.01) (Table 2). Lower lethal temperatures showing 100% mortality (LLT0) ranged from
8 to
15  C for durations of 0.5e4 h (Fig. 2C) while ULT0 ranged from 45.3 to 49  C for durations of 0.5e4 h. 3.2.4. Supercooling points The average SCPs for P. truncatus larvae and adults were
19.45 ± 1.18 and
16.65 ± 1.16  C respectively (Fig. 2D). There Table 2 Summary statistical results of the effects of temperature, duration of exposure and their interactions on survival of Prostephanus truncatus adults following upper lethal temperature (ULT) and lower lethal temperature (LLT) treatments. Analysis were done using generalized linear models (GLZ) assuming binomial distribution with a logit link function in R version 3.3.0. DF ¼ Degrees of freedom. Parameter Upper lethal temperature Duration Temperature Temperature * Duration Lower lethal temperature Duration Temperature Temperature * Duration

DF

c2

p

3 5 15

276.27 1073.51 19.73

˂ 0.001 ˂ 0.001 0.182

3 7 21

238.65 1176.19 70.15

˂0.001 ˂0.001 ˂0.001

Temperature-time treatments significantly influenced % germination of maize and sorghum (P ˂ 0.001) (Table 3; Fig. 3A; B). Combinations of moderate temperature to long durations i.e. 45.5  C  4 h and 47  C  2 h did not significantly reduce the germination rate of maize and sorghum grain compared to the untreated control grain (Fig. 3A; B). However, high temperature to short durations (49  C  0.5 h, 50  C  0.5 h, 48  C  1 h) reduced germination rate of maize compared to untreated control grain (Fig. 3A). Similarly, 49  C  0.5 h and 48  C  1 h exposure resulted in significant decrease in sorghum germination rate relative to control (Fig. 3B). Unlike in maize, a high temperature  short duration (i.e. 50  C  0.5 h) did not significantly reduce sorghum grain germination compared to control. 4. Discussion This work presents the thermal profile of P. truncatus in response to the renewed interest in exploring postharvest heat treatments (Stejskal et al., 2019) as a sustainable alternative to synthetic pesticides. The latter, applied mainly as fumigants has come with many challenges, ranging from public health hazards, pesticide resistance, environmental contamination and others (reviewed in Chaundhry, 1997; see also Nayak et al., 2013; Holloway et al., 2016; Wakil et al., 2018). The sustainable use of temperature treatment on stored products is hinged on the knowledge of lethal thermal profiles for the target species and developmental stages (Tang et al., 2000). Temperature affects many insect biochemical processes and physiological functions which underlie survival (Neven, 2000; Hochachka and Somero, 2002). As temperature goes sub-optimal, prolonged low or high temperature stress may manifest as cumulative complex physiological damage that can cause mortality (Neven, 2000; Chown and Nicolson, 2004). Development of species’ life-stage temperature x time matrix lethal thresholds is essential for pest management using temperature treatments. Defining the temperature x timing also optimises on energy costs, improves storage designs and informs appropriate choice of construction materials for efficient energy delivery kinetics in grain storage facilities (Fields, 1992; White, 1992; Dosland et al., 2006; reviewed in Stejskal et al., 2019). To our knowledge, information on insect species and life-stage specific thermal thresholds for the development of commodity specific treatment protocols in stored product protection is scarce (though see Stejskal et al., 2019). The current study unravels interesting differences in thermal resilience between P. truncatus developmental stages (larvae and adults), separated as heat and cold tolerance to effectively juxtapose thermal tolerance to grain thermal sensitivity responses. Based on recommendations by Golob (2002) and our results; we used the ULT0 thresholds of the more thermally resilient P. truncatus life stage; the adult, to derive optimum temperature x time matrices that ensure complete insect mortality at minimum grain germination quality losses. Our results show that moderate temperature x long duration matrix treatments (45.5  C x 4 h or 47  C x 2 h) were efficacious on insect mortality while retaining optimum grain germination rate (%) than high temperature x short durations. Similarly, for cold treatment;
9  C x 4 h,
11  C x 2 h,
13  C x 1 h and
15  C x 0.5 h showed high insect mortality (LLT0). However, we did not test these low temperature combinations on grain germination %

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Fig. 2. Effects of low temperature on the larva and adult of P. truncatus. (A) Summary results showing cooling rate effects on P. truncatus larvae and adults measured as critical thermal minima. (B) Effect of life stage (larvae and adults) on P. truncatus chill coma recovery time. (C) Survival of P. truncatus adults at different low temperatures applied over four different durations. (D) Effect of life stage (larvae and adults) on Prostephanus truncatus supercooling points. Error bars represent 95% CLs (N ¼ 20). Means with the same letter(s) are not significantly different from each other.

Table 3 Effect of temperature-time treatments on the germination % of maize and sorghum. DF ¼ Degrees of freedom, F ¼ FishereSnedecor test statistic (error df ¼ 12).

Maize Sorghum

Effect

DF

F

p

Treatment Treatment

5 5

17.4603 22.6172

˂ 0.001 ˂ 0.001

since previous studies suggest that they also retain good grain germination quality (Jayas and White, 2003; Braunbeck, 2014). These protocols may be useful in industry to tease apart and refine the existing ‘blanket’ temperature treatments on grain disinfestation (Fields, 1992; Dosland et al., 2006; Hansen et al., 2011; Mubayiwa et al., 2018). At intermediate/control ramping rate (0.25  C/min) the CTmax for P. truncatus adult was significantly higher than that of the larvae, signifying higher basal fitness advantage than the larva. Similarly, at all other tested ramping rates, adults consistently maintained higher basal heat tolerance (CTmax) compared to larvae. Adult HKDT was over three times higher than that of the larvae, indicating that upon faced with acute heat stress, adults are likely better resilient compared to larval P. trancatus. This result is inconsistent with

findings in Tribolium castaneum (Herbst) (Mahroof et al., 2003), where larvae had higher heat tolerance than the adults. This suggests a species- and life-stage- differences in thermal biology and calls for the development of context specific protocols for the management of storage pests using temperature treatments, as opposed to the current blanket temperature treatments (Dosland et al., 2006; Hansen et al., 2011). Higher ramping rate (0.5  C/min) increased heat tolerance (CTmax) for both developmental stages (Fig. 1A) in consonance with previous studies (Mutamiswa et al., 2017). This response may give P. trancutus a fitness advantage under global change, where mean temperatures are expected to increase, heat waves becoming more intense, frequent, longer lasting and the rate of temperature increase becoming faster (Meehl and Tebaldi, 2004; Thuiller, 2007; Walther et al., 2009). Conversely, slower ramping rates reduced CTmax for both P. truncatus developmental stages. This reduced heat tolerance could be attributed to energy depletion (costs) at lower ramping due to prolonged time of exposure (Rezende et al., 2011; Santos et al., 2011; Sørensen et al., 2013). This result suggests that rate of heating grain during disinfestation treatment has implications more on the adult’s ability to tolerate high temperatures than the larvae. This implies that slower ramping rates (0.12  C/min)

H. Machekano et al. / Journal of Stored Products Research 86 (2020) 101568

Fig. 3. Summary results of the effects of lethal temperature  time combinations on germination % of (A) maize, and (B) sorghum grain. Germination was defined by the visible radicle indicating the seed’s ability to successfully develop into a plant (International Seed Testing Association (ISTA), 2001).

may thus be more effective than higher ones, for two reasons; first the target lethal temperatures may be lower (due to reduced CTmax) and second higher mortality was achieved at more moderate than higher temperatures (see Fig. 1A). We used the more heat-tolerant life stage (adults), to determine effective time  temperature combination matrices through testing ULT0 thresholds on P. truncatus survival. As in Mahroof et al. (2003), we hypothesised that the resultant ULT0 would also apply for larvae since their lethal thresholds were significantly lower than that of adults. For all temperature x time combinations, P. truncatus adults showed ULT100 (100% survival) at 41  C. ULT0 (0% survival) was achieved at 45.5  C x 4 h, 47  C x 2 h, 48  C x 1 h and 49  C x 0.5 h. Similarly, at moderate temperature x long durations, ULT0 was achieved using the longer durations vis 45.5  C x 4 h and 47  C x 2 h. Maize and sorghum grain exposed to these moderate temperature x long durations (45.5  C x 4 h and 47  C x 2 h) retained germination qualities, consistent with untreated grain, whereas higher temperature  short durations i.e. 48  C x 1 h, 49  C x 0.5 h and 50  C x 0.5 h, significantly reduced germination compared to the untreated grain except for sorghum grain at 50  C x 0.5 h. This differential response between maize and sorghum grain at 50  C x 0.5 h implies that the type of grain plays a significant role in developing grain treatment protocols. Our results implied that

7

where P. truncatus is the target pest for pre-shipment heat disinfestation for maize or sorghum grain, moderate temperature x long duration matrices are more optimal as phytosanitary treatments as they optimise both efficacy and grain germination %. However, field trials may be needed to further validate these proposed treatments cognisant of variations in heat conductivity and kinetics across storage structure plenums and grain types (Bruce et al., 2004). Developmental stage differences in P. truncatus responses to low temperatures followed a similar trend with high temperature results. At intermediate/control ramping rate (0.25  C/min), adults were significantly more tolerant to lower temperatures than larvae (Fig. 2A). At all the tested ramping rates, adults consistently maintained a lower CTmin than larvae. In addition, adults had a significantly shorter CCRT than the larvae, in consonance with CTmin results. Low temperature exposure also showed more cold tolerance (i.e. reduced CTmin) following faster cooling rates, although this was evident for adults only. This result pointed to some degree of short-term plasticity for low temperature in the adult, and not the larva. Lack of plasticity at slower ramping rates may also be attributed to cumulative tissue damage caused by more prolonged stress exposure (Rezende et al., 2011; Santos et al., 2011; Sørensen et al., 2013). This may also be associated with alteration of membranes, loss of metabolic homeostasis, accumulation of toxic compounds, DNA damage and others (see discussions in Colinet et al., 2018). Our results are in keeping with previous reports that temperature thresholds are life-stage dependant (Klok and Chown, 2001; Marais et al., 2009; Mutamiswa et al., 2019; reviewed in Bowler and Terblanche, 2008) and that plasticity is complex, and varies with species, life stage, environmental history and the metrics tested (Mutamiswa et al., 2019). We thus suggest, with caveats that superior thermal resilience of adults over larvae at both thermal extremes may be related to their mobility (Bowler and Terblanche, 2008) and environmental history e.g. microhabitats (Li et al., 2019), consistent with similar reports e.g. for Sirex noctilio F. (Hymenoptera: Siricidae) (Li et al., 2019). From an evolutionary perspective, P. truncatus larva is less mobile and resides almost entirely inside grain kernels, a microenvironment with a degree of cushioning from outside heterogeneous ambient environments. Adults, on the other hand, are highly mobile with higher exposure to variable environmental ambient conditions. In consequence, observed higher adult basal thermal fitness over larva may be accounted for by environmental history (see discussions in Nyamukondiwa and Terblanche, 2010; Arias et al., 2011). Similarly, adults in some species, e.g. Tenebrio molitor L. (Coleoptera: Tenebrionidae) were shown to improve thermal tolerance more than the larvae following differential environmental histories (Arias et al., 2011). As in heat, we used the more cold tolerant life stage (adult) to test the LLT0 of P. trancatus. For all time x temperature combinations, P. truncatus adults showed LLT100 (100% survival) at
1  C and did not attain LLT0 (0% survival) at temperatures above
9  C for all tested temperature x time combinations. LLT0 (0% survival associated with cold treatment) was achieved at
9  C x 4 h,
11  C x 2 h,
13  C x 1 h and
15  C x 0.5 h. These temperatures were consistent with the SCPs recorded for adults where the internal body contents froze, likely resulting in death. This may mean that P. trancatus larvae is likely chill susceptible, and may not survive freezing of bodily fluids (reviewed Zachariassen, 1985; see also Block, 1990). Low temperature treatments are known to reduce respiration of stored grain and extend its storage time while simultaneously preventing the development of insects and moulds (Jayas and White, 2003; Ning et al., 2012; Braunbeck, 2014; Luo et al., 2014). Interestingly, larvae had more negative SCPs (more cold tolerance) compared to the adults. The reasons behind this are unknown and mechanisms facilitating this developmental stage differences are the focus for future studies.

8

H. Machekano et al. / Journal of Stored Products Research 86 (2020) 101568

Nevertheless, increased supercooling may be caused by increased concentration of insect carbohydrate cryoprotectants in the haemolymph or evacuation of the gut contents to reduce or mask ice nucleating agents (Storey and Storey, 1988; Lee, 1989; Toxopeus and Sinclar, 2018). Our LLT results suggest that, where heat treatment is not available as an option, and cognisant of the larval reduced SCPs (see Fig. 2D), temperatures 
20  C may be an option for grain low temperature (cold) treatment against P. truncatus. However, exact exposure duration at this temperature for optimal efficacy was not tested here and ought to be investigated. High basal heat and more cold tolerance exhibited by the adults, and some degree of plasticity may improve P. truncatus fitness as a grain pest, and successful establishment in novel environments (Webster et al., 2017). Moreover, its successful establishment in Africa (Dunstan and Magazini, 1981; Farrell, 2000; though see Gunderson and Stillman, 2015) may also have come from its basal thermal fitness advantage and plasticity, as has been reported for other invasive pest species, e.g. Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (Nyamukondiwa et al., 2010), Chilo partellus Swinhoe (Lepidoptera: Crambidae) Mutamiswa et al. (2017) and Tuta absoluta Meyrick (Machekano et al., 2018b). This high thermal fitness, coupled with a wide range of wild and domestic alternative plant and non-plant food sources (hosts) (Nang’ayo et al., 2002; Muatinte and van den Berg, 2019), makes P. truncatus likely highly adaptable to diverse habitats. This is indeed testimony of its current wide geographical range extensions (Arthur et al., 2019). Stored grain environments are usually typified by high temperature, low RH and low oxygen. As such, stored product insects should be able to tolerate these stressors, individually or using shared physiological mechanisms (see discussions in Sinclair et al., 2013; Gotcha et al., 2018). It is however unclear whether high thermal fitness is linked to desiccation (Sinclair et al., 2013; Gotcha et al., 2018) or € rtner, 2001; Boardman and Terblanche, hypoxia stress tolerance (Po 2015). Further research may be needed to investigate the likely underlying mechanisms for these combined stressors, and implications on the combination of modified atmospheres and heat as grain disinfestation treatments (Donahaye et al., 1996). Our results point to (1) high thermal resilience and (2) different thermal profiles for two developmental stages of P. truncatus, key information needed for the development of efficacious postharvest grain thermal disinfestation treatment protocols. We conclude that where P. truncatus is the target pest for pre-shipment or grain disinfestation for maize or sorghum, moderate temperature x long duration matrix (either 45.5  Cx 4 h or 47  C x 2 h) is recommended for maximum efficacy and optimum commodity quality. Where low temperature treatment is the available option, 
20  C may be an option. These time temperature combinations may be useful as long term sustainable alternatives to synthetic fumigants in phytosanitary grain disinfestation. Further field research data may be needed to validate these results and test cooking quality characteristics for maize and sorghum flour after exposure to our temperatures treatments before the protocols may be implemented. Declaration of competing interest The authors declare no conflict of interest whatsoever in publishing this research. Acknowledgements We acknowledge Botswana International University of Science and Technology for support to HM, JV and CN; University of Free State for support to RM and FC and University of Zimbabwe for support to CS. We also appreciate several anonymous reviewers for the valuable comments on an earlier version of this manuscript.

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