Comparison of Water Relation in Two Powderpost Beetles Relative to Body Size and Ontogenetic and Behavioral Traits

Comparison of Water Relation in Two Powderpost Beetles Relative to Body Size and Ontogenetic and... Abstract Heterobostrychus aequalis (Waterhouse) (Coleoptera: Bostrychidae) and Lyctus africanus Lesne (Coleoptera: Lyctidae) are distributed mainly in tropical regions. The primary mechanism allowing these beetles to survive in cold and arid habitats beyond the native tropical region is a reduced water loss rate. This study investigated the water relations of these two beetles in relation to their size, ontogenetic traits, and behavioral characteristics to determine how they can survive in desiccated wood. H. aequalis and L. africanus share similar water characteristic with beetles living in desert and woodlands. They have high percentage total body water (%TBW) content (58.38 ± 1.86% to 63.20 ± 1.38%), but low %TBW loss (4.28 ± 1.02% to 48.26 ± 8.28%) due to their impermeable cuticle (cuticular permeability [CP] value: 0–15.57 ± 4.90 µg cm–2 h–1 mmHg–1) at all life stages. Although the larvae of L. africanus exhibited relatively high %TBW loss, they had relatively shorter development times that minimized prolonged exposure to dry conditions inside the wood. The aggregative behavior of the adult could be responsible for maintaining a low water loss rate to compensate for their small body size. In contrast, the larvae of H. aequalis had larger body size and significantly lower CP values, allowing them to survive in the desiccated wood for a longer period of time. These results demonstrate the remarkably sophisticated strategies that insects employ as a trade-off between body size, ontogenetic development, and insect sociality (aggregative and non-aggregative behavior) to maintain their water balance in xeric environments. desiccation tolerance, water retention, hardwood, wood-attacking beetle, aggregative behavior Water conservation is a primary survival tactic for xeric-adapted arthropods, as they lack of the ability to replenish water loss by drinking, feeding, or absorbing water vapor from the environment. In addition, water loss during moulting is expectedly massive due to the drastic increase in cuticular permeability (CP) of newly formed cuticle. In contrast, for mesic-adapted arthropods, more water is available to diffuse passively into the body through the cuticle (Appel et al. 1986) to replace what is lost due to diffusion and respiration (Haverty and Nutting 1976). In general, insects improve their desiccation tolerance by limiting water loss rate via reduced CP; this can occur by enhancing the cuticular lipid and melanization process, increasing the amount of water that is permitted to be lost, increasing body water content, or various combinations of these traits (Bazinet et al. 2010). Increased desiccation resistance in larval insects can be achieved through various behavioral and physiological adaptations. For instance, larvae of the Antarctica midge Belgica antarctica Jacobs, which experience a prolonged cold and arid environment, can lose more than 70% of their water content at a rate of more than 10% per hour. In response the environmental conditions, the larvae formed dense clusters and increased the concentration of glycerol and trehalose two- to threefold to reduce the rate of water loss (Benoit et al. 2007). A similar observation was reported for the African chironomid Polypedium vanderplanki Hinton (Kikawada et al. 2005). B. antarctica also decreased its metabolic rate by reducing the oxygen consumption rate in response to dehydration (Benoit et al. 2007). For the meal worm Tenebrio molitor Linnaeus, the starved larvae survived by maintaining their water balance through atmospheric absorption when the relative humidity (RH) was greater than 88% (Machin 1975). Toolson (1982) reported that the shift from short chain epicuticular hydrocarbons to longer chain lengths reduced CP in the phorid fly Drosophila pseudoobscura Frolova. However, it is unclear how the powderpost beetles tolerate desiccation stress in the temperate regions, which the low temperature decelerates the development of these beetles and likely prolonged their exposure to dryness inside the woods. Bostrichidae, also known as powderpost beetles, are among the most destructive wood-attacking beetles, thus they are of economic importance in forestry and lumber industries (Peters et al. 2002). Although these beetles are predominantly distributed in tropical regions, they were introduced into temperate regions through imported timber or timber products. Of the 550 described species distributed worldwide (Ivie 2002), 16 species are currently found in Japan, and eight of them have become established in Japan: Heterobostrychus hamatipennis Lesne, Rhizopertha dominica (Fabricius), Dinoderus minutus Fabricius, Lyctus africanus Lesne (Coleoptera: Lyctidae), Minthea rugicollis (Walker), Lyctus sinensis Lesne, Lyctus linearis (Goeze), and Lyctus brunneus (Stephens) (Mito and Uesugi 2004). Infestations of these beetles are widespread, and they have become a major problem in Japan (Shigetaka 1980, Nobuchi 1986, Kawakami 1996, Furukawa et al. 2009, Bong 2015). Heterobostrychus aequalis (Waterhouse) (Coleoptera: Bostrychidae) was first found in seasoned imported lauan timber in Naha, Okinawa (Nobuchi 1986). In recent years, cases of damaged timber involving this beetle have been on the rise, but it is still unclear whether H. aequalis is established in Japan. On the other hand, L. africanus was found to be established in Japan in the 1980s (Iwata 1982, Mito and Uesugi 2004). Since then, the species has become a major pest in Japan because of its prevalent infestation of wooden structures throughout the country (Furukawa et al. 2009). Low temperatures in temperate regions decelerate development and likely prolong the beetles’ exposure to dryness inside wood. Water relations could be an important factor that allows these insects to thrive under high desiccation pressure across a wide range of geographical regions (Appel et al. 1983, Zachariassen 1996, Bong et al. 2013). The possible mechanism allowing powderpost beetles to survive beyond their native tropical region in cold and arid habitats is reduction of water loss rate as shown in other beetles (Bellés and Halstead 1985, Benoit et al. 2005). For example, the spider beetle Mezium affine Boieldieu survived for approximately 3 mo without food or water in the desiccated condition because of a remarkable ability to retain water inside their bodies (Benoit et al. 2005). For this reason, M. affine is one of the most persistent and widely distributed stored product pest (Bellés and Halstead 1985). Despite their similar habitat, H. aequalis and L. africanus differ conspicuously in morphology, ontogeny, and behavior. For instance, L. africanus is 3–4 times smaller than H. aequalis. Water balance is a challenge for small-bodied insects, as they have high surface area-to-volume ratios that may result in a significantly increased water loss rate (Hu et al. 2012). Thus, it was likely that L. africanus had a specific adaptive behavior to survive inside desiccated wood. Male L. africanus produce volatile esters to elicit aggregative behavior (Kartika et al. 2015). Despite of attracting conspecific to form a cluster for predation avoidance (Cornell et al. 1987, Stamp and Bowers 1988, Lawrence 1990, Sillén-Tullberg 1990) and growth (Tsubaki and Shiotsu 1982, Clark and Faeth 1997, Denno and Benrey 1997), the behavior can facilitate water conservation (Rasa 1997, Yoder and Grojean 1997, Glass et al. 1998). It was reasoned that aggregation formed a ‘superorganism’ which characterized the low ratios of surface area-to-volume to reduce water loss rate. In addition, the aggregation may create a humid microcosm between individuals when water is lost as shown in dust mite, Dermatophagoides farinae Hughes (Acari: Pyroglyphidae) (Glass et al. 1998). Aggregation behavior is absent in H. aequalis, which always appear as single individuals or in pairs. H. aequalis undergoes a lengthy immature developmental process that lasts approximately 24 wk, which is 12 times longer than that of L. africanus (Creffield and Howick 1979, Ho 1995, Bong et al. 2018). Thus, prolonged immature development for H. aequalis during winter may increase the risk of larval desiccation and eventually cause failure to develop. In all likelihood, H. aequalis and L. africanus have physiological and behavioral plasticity that enables them to survive prolonged exposure in dry wood. In this study, we examined the water relations of H. aequalis and L. africanus to determine how they can survive in desiccated wood. The goals of this study were to address the following questions: Are there differences in water relations between adults of the two test beetle species, considering that L. africanus exhibits aggregative behavior? Does size matter for water retention of small-bodied L. africanus? How do the larvae of H. aequalis conserve the water needed for development over an extended period of 24 wk? Materials and Methods Rearing Method Adult H. aequalis and L. africanus were, respectively, reared in a closed container (20.0 cm length × 16.0 cm width × 9.0 cm height) and a closed jar (8.5 cm diameter × 13.0 cm height) in a walk-in environmental chamber (at 26 ± 2°C and 65 ± 10% RH) in the Deterioration Organisms Laboratory (DOL) at the Research Institute for Sustainable Humanosphere, Kyoto, Gokasho, Uji, Japan. The beetles were fed an artificial diet consisting of dried yeast (24% Asahi Food and Health Care, Tokyo, Japan), starch (50%, Nacalai Tesque, Kyoto, Japan), and lauan (Shorea spp.) wood sawdust (26%) in a 16.0 cm length × 8.0 cm width × 2.0 cm depth block for H. aequalis, and 8.0 cm length × 4.0 cm width × 2.0 cm depth block for L. africanus (Kartika and Yoshimura 2013). CP Study Percentage total body water (%TBW) content, %TBW loss over desiccation time, and CP of H. aequalis and L. africanus were examined gravimetrically (Appel and Tanley 1999, Shelton and Grace 2003). For H. aequalis, 10 late instar larvae weighing between 40 and 60 mg, 12 pupae weighing between 30 and 60 mg, and five adult males weighing between 25 and 40 mg and five adult females weighing between 30 and 50 mg aged 1-wk old were used. The insects were individually placed in the specimen tubes (15 mm diameter × 50 mm height), and examined gravimetrically. The study was replicated with one insect each time for each stage. For L. africanus, the study was replicated four to five times for each stage, with each replicate consisting of 10 insects (10 late instar larvae weighing between 2.4 and 3.9 mg, 10 pupae weighing between 2.2 and 2.5 mg, 10 adult males weighing between 1.2 and 1.5 mg and 10 females weighing between 1.3 and 1.5 mg) to simulate their aggregative nature in environment. Only newly emerged adults from the food source were subjected to analysis. The insects were weighed to the nearest 0.01 mg using a digital analytical balance (Sartorius Extended ED2245, Sartorius AG, Göttingen, Germany). The test insects were placed in an 11 L glass desiccator containing 1 kg of anhydrous CaSO4 (Fisons Scientific Apparatus, Leicestershire, United Kingdom). Before testing, the desiccant was dried at 100°C for 48 h. The desiccator was maintained at 6% RH with a saturation deficit of 27.56 mmHg at 28.6 ± 0.2°C. The test insects were weighed for mass loss at 2, 4, 6, 8, 10, and 24 h (Appel and Tanley 1999, Shelton and Grace 2003). After 24 h, the test insects were dried at 60°C for 72 h and then weighed to obtain the dry weight. Insect water loss during the first 2 h of desiccation was used to calculate the CP of an insect because water loss that occurs during this time interval represents cuticular water loss (Sponsler and Appel 1990, Shelton and Grace 2003). The CP value was calculated as water loss using the following equation: [initial weight − weight loss at 2 h] (µg) per surface area (cm2) per time (h) per saturation deficit (mmHg) (Edney 1977). Surface area of the specimen was calculated using Meeh’s formula (Meeh 1897): S = 12M2/3, where S = body surface area (cm2) and M = initial mass (g). Edney (1977) reported that insects living in xeric habitats exhibited CP values of 0–30 µg cm–2 h–1 mmHg–1, whereas those found in mesic and hygric habitats exhibited CP values of 31–60 µg cm–2 h–1 mmHg–1 and > 60 µg cm–2 h–1 mmHg–1, respectively. %TBW content and %TBW loss of an insect were calculated as follows: %TBW content=[(initial mass–dry mass)/initial mass]×100% %TBW loss=[(initial weight–weight at each hour)/(initial weight–dry weight)]×100% Statistical Analysis % TBW content and TBW loss were arcsine square root transformed. Body mass, CP value, and the transformed values were checked for normality at the 0.05 significance level using the Kolmogorov-Smirnov test. When the criteria of normality were not met, a log10 transformation was performed, and the data were retested for normality. The data were then analyzed using a one-way followed by Tukey’s Honest Significant Difference test. Body water content was analysed using one-way analysis of covariance (ANCOVA) and separated by least significant difference with initial weight as a covariate. ANCOVA was used because it eliminates the influence of the variation in body size on the physiological response, generating a more reliable result (Packard and Boardman 1999, Hu et al. 2012). All analyses were performed using SPSS analysis version 11.0 (SPSS Inc., Chicago, IL). Comparison With Other Beetles in Relation to Habitats, Environmental Variables, and Water Relations Water relation data for other beetles were collected from previous studies (Table 1). The association between water relation (CP and %TBW loss) of beetles and habitats and environmental variables were examined using CANOCO 5.0 (ter Braak and Ŝmilauer 2012). The unconstrained ordination method (detrended correspondence analysis) was used to examine the gradient length. Principal component analysis was used because the water relation of beetles along the environmental variables in their habitat was found to be homogenous. Table 1. Habitat types and environmental variables in relation to water relation of beetles Beetle species Habitat types Environmental variables Water relation References Temperature (°C) Relative humidity (%) Precipitation (mm/month) CP (µg cm–2 h–1 mmHg–1) TBW loss (%) Charidotella bicolor Plantation 41 50 80.05 9.99 50.36 Hull-Sanders et al. (2003) Deloyala guttata Plantation 41 50 40.6 8.41 70.05 Hull-Sanders et al. (2003) Eleodes armata Desert 37 22 24.13 2.5 4 Ahearn (1970), Hadley (1978) Cryptoglossa verrucosa Desert 37 22 24.13 1.6 2 Ahearn (1970), Hadley (1978) Centrioptera municata Desert 39 11 12.7 2.2 4 Ahearn (1970), Hadley (1978) Onymaeris plana Desert 35 47 3 1.53 1.25 Edney (1971) Onymaeris laeviceps Desert 35 47 3 3.41 3 Edney (1971) Lepidochora argentogrisea Desert 35 47 3 1.91 3 Edney (1971) Onymacris rugatipennis Desert 35 47 3 1.87 1.5 Edney (1971) Lepidochora porti Desert 35 47 3 3.49 3 Edney (1971) Calosis amabilis Desert 35 47 3 1.09 1.5 Edney (1971) Gyrosis moralesi Desert 35 47 3 2.24 4 Edney (1971) Ctenolepisma terebrans Desert 35 47 3 0.68 5 Edney (1971) Trigonopus sp. Woodland 26.5 71 24 4.13 6 Edney (1971) Paederus fuscipes Plantation 28 80 500 15.3 79.2 Bong et al. (2013) Heterobostrychus aequalis Hardwood 26 12 (m.c) 0 15.3 11.77 Present study Lyctus africanus Hardwood 26 12 (m.c) 0 4.52 17.27 Present study Beetle species Habitat types Environmental variables Water relation References Temperature (°C) Relative humidity (%) Precipitation (mm/month) CP (µg cm–2 h–1 mmHg–1) TBW loss (%) Charidotella bicolor Plantation 41 50 80.05 9.99 50.36 Hull-Sanders et al. (2003) Deloyala guttata Plantation 41 50 40.6 8.41 70.05 Hull-Sanders et al. (2003) Eleodes armata Desert 37 22 24.13 2.5 4 Ahearn (1970), Hadley (1978) Cryptoglossa verrucosa Desert 37 22 24.13 1.6 2 Ahearn (1970), Hadley (1978) Centrioptera municata Desert 39 11 12.7 2.2 4 Ahearn (1970), Hadley (1978) Onymaeris plana Desert 35 47 3 1.53 1.25 Edney (1971) Onymaeris laeviceps Desert 35 47 3 3.41 3 Edney (1971) Lepidochora argentogrisea Desert 35 47 3 1.91 3 Edney (1971) Onymacris rugatipennis Desert 35 47 3 1.87 1.5 Edney (1971) Lepidochora porti Desert 35 47 3 3.49 3 Edney (1971) Calosis amabilis Desert 35 47 3 1.09 1.5 Edney (1971) Gyrosis moralesi Desert 35 47 3 2.24 4 Edney (1971) Ctenolepisma terebrans Desert 35 47 3 0.68 5 Edney (1971) Trigonopus sp. Woodland 26.5 71 24 4.13 6 Edney (1971) Paederus fuscipes Plantation 28 80 500 15.3 79.2 Bong et al. (2013) Heterobostrychus aequalis Hardwood 26 12 (m.c) 0 15.3 11.77 Present study Lyctus africanus Hardwood 26 12 (m.c) 0 4.52 17.27 Present study m.c., moisture content. View Large Table 1. Habitat types and environmental variables in relation to water relation of beetles Beetle species Habitat types Environmental variables Water relation References Temperature (°C) Relative humidity (%) Precipitation (mm/month) CP (µg cm–2 h–1 mmHg–1) TBW loss (%) Charidotella bicolor Plantation 41 50 80.05 9.99 50.36 Hull-Sanders et al. (2003) Deloyala guttata Plantation 41 50 40.6 8.41 70.05 Hull-Sanders et al. (2003) Eleodes armata Desert 37 22 24.13 2.5 4 Ahearn (1970), Hadley (1978) Cryptoglossa verrucosa Desert 37 22 24.13 1.6 2 Ahearn (1970), Hadley (1978) Centrioptera municata Desert 39 11 12.7 2.2 4 Ahearn (1970), Hadley (1978) Onymaeris plana Desert 35 47 3 1.53 1.25 Edney (1971) Onymaeris laeviceps Desert 35 47 3 3.41 3 Edney (1971) Lepidochora argentogrisea Desert 35 47 3 1.91 3 Edney (1971) Onymacris rugatipennis Desert 35 47 3 1.87 1.5 Edney (1971) Lepidochora porti Desert 35 47 3 3.49 3 Edney (1971) Calosis amabilis Desert 35 47 3 1.09 1.5 Edney (1971) Gyrosis moralesi Desert 35 47 3 2.24 4 Edney (1971) Ctenolepisma terebrans Desert 35 47 3 0.68 5 Edney (1971) Trigonopus sp. Woodland 26.5 71 24 4.13 6 Edney (1971) Paederus fuscipes Plantation 28 80 500 15.3 79.2 Bong et al. (2013) Heterobostrychus aequalis Hardwood 26 12 (m.c) 0 15.3 11.77 Present study Lyctus africanus Hardwood 26 12 (m.c) 0 4.52 17.27 Present study Beetle species Habitat types Environmental variables Water relation References Temperature (°C) Relative humidity (%) Precipitation (mm/month) CP (µg cm–2 h–1 mmHg–1) TBW loss (%) Charidotella bicolor Plantation 41 50 80.05 9.99 50.36 Hull-Sanders et al. (2003) Deloyala guttata Plantation 41 50 40.6 8.41 70.05 Hull-Sanders et al. (2003) Eleodes armata Desert 37 22 24.13 2.5 4 Ahearn (1970), Hadley (1978) Cryptoglossa verrucosa Desert 37 22 24.13 1.6 2 Ahearn (1970), Hadley (1978) Centrioptera municata Desert 39 11 12.7 2.2 4 Ahearn (1970), Hadley (1978) Onymaeris plana Desert 35 47 3 1.53 1.25 Edney (1971) Onymaeris laeviceps Desert 35 47 3 3.41 3 Edney (1971) Lepidochora argentogrisea Desert 35 47 3 1.91 3 Edney (1971) Onymacris rugatipennis Desert 35 47 3 1.87 1.5 Edney (1971) Lepidochora porti Desert 35 47 3 3.49 3 Edney (1971) Calosis amabilis Desert 35 47 3 1.09 1.5 Edney (1971) Gyrosis moralesi Desert 35 47 3 2.24 4 Edney (1971) Ctenolepisma terebrans Desert 35 47 3 0.68 5 Edney (1971) Trigonopus sp. Woodland 26.5 71 24 4.13 6 Edney (1971) Paederus fuscipes Plantation 28 80 500 15.3 79.2 Bong et al. (2013) Heterobostrychus aequalis Hardwood 26 12 (m.c) 0 15.3 11.77 Present study Lyctus africanus Hardwood 26 12 (m.c) 0 4.52 17.27 Present study m.c., moisture content. View Large Results Water Relations In general, the fresh body mass of H. aequalis was 17- to 24-fold higher (32.00 ± 2.55 mg to 52.90 ± 2.85 mg) than that of L. africanus (1.36 ± 0.05 mg to 3.10 ± 0.29 mg) (Table 2). The fresh body mass of both species decreased significantly as larva developed to the adult stage (H. aequalis, F = 7.463; df = 3, 28; P = 0.001; L. africanus, F = 55.222; df = 3, 14; P < 0.001). Despite the difference in body mass between the two beetles, both species showed a comparably high %TBW content, ranging from 58.38 ± 1.86% to 63.20 ± 1.38%. Table 2. Physiological parameters of preadult and adult powderpost beetles (mean ± SD) Species Stage n Initial mass (mg) Body water content (mg)a TBW content (%) TBW loss (%)b CP (µg cm–2 h–1 mmHg–1) Ratios of surface area to volume Water loss rate (mg g–1 h–1)a Heterobostrychus aequalis L 10 52.90 ± 2.85a* 32.40 ± 1.85a* 61.35 ± 1.40a 5.93 ± 1.70ab* 2.52 ± 1.68a 32.14 ± 1.75a* 2.09 ± 1.40 a P 12 48.50 ± 2.78a* 29.50 ± 1.90a* 60.53 ± 0.92a 4.28 ± 1.02a* 5.89 ± 3.45a 33.21 ± 2.47a* 4.82 ± 2.66 a F 5 40.60 ± 3.26ab* 24.20 ± 2.08a* 59.50 ± 0.64a 11.55 ± 2.30b 13.76 ± 4.78a 35.12 ± 2.13ab* 13.77 ± 5.02 a M 5 32.00 ± 2.55b* 20.00 ± 1.76a* 62.36 ± 0.83a 11.99 ± 0.51b 15.57 ± 4.90a* 38.03 ± 2.38b* 16.58 ± 5.25 a* Lyctus africanus L 40 3.10 ± 0.29x* 1.93 ± 0.12x* 62.04 ± 0.70x 48.26 ± 8.28x* 5.96 ± 3.99x 82.79 ± 2.64x* 14.32 ± 9.83 x P 40 2.38 ± 0.06y* 1.50 ± 0.04x* 63.20 ± 1.38x 14.81 ± 2.94y* 8.43 ± 4.87x 89.98 ± 0.81y* 20.83 ± 12.03 x F 50 1.46 ± 0.05z* 0.85 ± 0.03x* 58.38 ± 1.86x 21.15 ± 6.22y 4.52 ± 2.77x 105.90 ± 1.25z* 12.92 ± 7.92 x M 50 1.36 ± 0.05z* 0.82 ± 0.02x* 60.49 ± 1.91x 13.38 ± 4.61y 0.00 ± 0.00y* 108.45 ± 1.38z* 0.00 ± 0.00 x* Species Stage n Initial mass (mg) Body water content (mg)a TBW content (%) TBW loss (%)b CP (µg cm–2 h–1 mmHg–1) Ratios of surface area to volume Water loss rate (mg g–1 h–1)a Heterobostrychus aequalis L 10 52.90 ± 2.85a* 32.40 ± 1.85a* 61.35 ± 1.40a 5.93 ± 1.70ab* 2.52 ± 1.68a 32.14 ± 1.75a* 2.09 ± 1.40 a P 12 48.50 ± 2.78a* 29.50 ± 1.90a* 60.53 ± 0.92a 4.28 ± 1.02a* 5.89 ± 3.45a 33.21 ± 2.47a* 4.82 ± 2.66 a F 5 40.60 ± 3.26ab* 24.20 ± 2.08a* 59.50 ± 0.64a 11.55 ± 2.30b 13.76 ± 4.78a 35.12 ± 2.13ab* 13.77 ± 5.02 a M 5 32.00 ± 2.55b* 20.00 ± 1.76a* 62.36 ± 0.83a 11.99 ± 0.51b 15.57 ± 4.90a* 38.03 ± 2.38b* 16.58 ± 5.25 a* Lyctus africanus L 40 3.10 ± 0.29x* 1.93 ± 0.12x* 62.04 ± 0.70x 48.26 ± 8.28x* 5.96 ± 3.99x 82.79 ± 2.64x* 14.32 ± 9.83 x P 40 2.38 ± 0.06y* 1.50 ± 0.04x* 63.20 ± 1.38x 14.81 ± 2.94y* 8.43 ± 4.87x 89.98 ± 0.81y* 20.83 ± 12.03 x F 50 1.46 ± 0.05z* 0.85 ± 0.03x* 58.38 ± 1.86x 21.15 ± 6.22y 4.52 ± 2.77x 105.90 ± 1.25z* 12.92 ± 7.92 x M 50 1.36 ± 0.05z* 0.82 ± 0.02x* 60.49 ± 1.91x 13.38 ± 4.61y 0.00 ± 0.00y* 108.45 ± 1.38z* 0.00 ± 0.00 x* Stage: L, larva; P, pupa; F, female adult; M, male adult. Mean values followed by the same letter within the species are not significantly different (Tukey’s HSD; α = 0.05). Mean values followed by an asterisk between species of the same instar are significantly different (Student’s t-test; α = 0.05). aParameter analyzed using ANCOVA and separated by least significant difference with initial mass as a covariate. bPercentage of TBW loss at 24 h. View Large Table 2. Physiological parameters of preadult and adult powderpost beetles (mean ± SD) Species Stage n Initial mass (mg) Body water content (mg)a TBW content (%) TBW loss (%)b CP (µg cm–2 h–1 mmHg–1) Ratios of surface area to volume Water loss rate (mg g–1 h–1)a Heterobostrychus aequalis L 10 52.90 ± 2.85a* 32.40 ± 1.85a* 61.35 ± 1.40a 5.93 ± 1.70ab* 2.52 ± 1.68a 32.14 ± 1.75a* 2.09 ± 1.40 a P 12 48.50 ± 2.78a* 29.50 ± 1.90a* 60.53 ± 0.92a 4.28 ± 1.02a* 5.89 ± 3.45a 33.21 ± 2.47a* 4.82 ± 2.66 a F 5 40.60 ± 3.26ab* 24.20 ± 2.08a* 59.50 ± 0.64a 11.55 ± 2.30b 13.76 ± 4.78a 35.12 ± 2.13ab* 13.77 ± 5.02 a M 5 32.00 ± 2.55b* 20.00 ± 1.76a* 62.36 ± 0.83a 11.99 ± 0.51b 15.57 ± 4.90a* 38.03 ± 2.38b* 16.58 ± 5.25 a* Lyctus africanus L 40 3.10 ± 0.29x* 1.93 ± 0.12x* 62.04 ± 0.70x 48.26 ± 8.28x* 5.96 ± 3.99x 82.79 ± 2.64x* 14.32 ± 9.83 x P 40 2.38 ± 0.06y* 1.50 ± 0.04x* 63.20 ± 1.38x 14.81 ± 2.94y* 8.43 ± 4.87x 89.98 ± 0.81y* 20.83 ± 12.03 x F 50 1.46 ± 0.05z* 0.85 ± 0.03x* 58.38 ± 1.86x 21.15 ± 6.22y 4.52 ± 2.77x 105.90 ± 1.25z* 12.92 ± 7.92 x M 50 1.36 ± 0.05z* 0.82 ± 0.02x* 60.49 ± 1.91x 13.38 ± 4.61y 0.00 ± 0.00y* 108.45 ± 1.38z* 0.00 ± 0.00 x* Species Stage n Initial mass (mg) Body water content (mg)a TBW content (%) TBW loss (%)b CP (µg cm–2 h–1 mmHg–1) Ratios of surface area to volume Water loss rate (mg g–1 h–1)a Heterobostrychus aequalis L 10 52.90 ± 2.85a* 32.40 ± 1.85a* 61.35 ± 1.40a 5.93 ± 1.70ab* 2.52 ± 1.68a 32.14 ± 1.75a* 2.09 ± 1.40 a P 12 48.50 ± 2.78a* 29.50 ± 1.90a* 60.53 ± 0.92a 4.28 ± 1.02a* 5.89 ± 3.45a 33.21 ± 2.47a* 4.82 ± 2.66 a F 5 40.60 ± 3.26ab* 24.20 ± 2.08a* 59.50 ± 0.64a 11.55 ± 2.30b 13.76 ± 4.78a 35.12 ± 2.13ab* 13.77 ± 5.02 a M 5 32.00 ± 2.55b* 20.00 ± 1.76a* 62.36 ± 0.83a 11.99 ± 0.51b 15.57 ± 4.90a* 38.03 ± 2.38b* 16.58 ± 5.25 a* Lyctus africanus L 40 3.10 ± 0.29x* 1.93 ± 0.12x* 62.04 ± 0.70x 48.26 ± 8.28x* 5.96 ± 3.99x 82.79 ± 2.64x* 14.32 ± 9.83 x P 40 2.38 ± 0.06y* 1.50 ± 0.04x* 63.20 ± 1.38x 14.81 ± 2.94y* 8.43 ± 4.87x 89.98 ± 0.81y* 20.83 ± 12.03 x F 50 1.46 ± 0.05z* 0.85 ± 0.03x* 58.38 ± 1.86x 21.15 ± 6.22y 4.52 ± 2.77x 105.90 ± 1.25z* 12.92 ± 7.92 x M 50 1.36 ± 0.05z* 0.82 ± 0.02x* 60.49 ± 1.91x 13.38 ± 4.61y 0.00 ± 0.00y* 108.45 ± 1.38z* 0.00 ± 0.00 x* Stage: L, larva; P, pupa; F, female adult; M, male adult. Mean values followed by the same letter within the species are not significantly different (Tukey’s HSD; α = 0.05). Mean values followed by an asterisk between species of the same instar are significantly different (Student’s t-test; α = 0.05). aParameter analyzed using ANCOVA and separated by least significant difference with initial mass as a covariate. bPercentage of TBW loss at 24 h. View Large The %TBW loss at all stages of H. aequalis and L. africanus increased curvilinearly with desiccation time (Fig. 1, Table 3). However, H. aequalis showed low %TBW loss, which was less than 12% at all stages (Table 2, Fig. 1a). The %TBW loss of immature individuals was significantly lower than that of the adults (F = 6.012; df = 3, 28; P = 0.003), resulting in higher CP values (13.76 ± 4.78 µg cm–2 h–1 mmHg–1 to 15.57 ± 4.90 µg cm–2 h–1 mmHg–1) in the adults compared to the immature stages (larval, 2.52 ± 1.68 µg cm–2 h–1 mmHg–1; pupal, 5.89 ± 3.45 µg cm–2 h–1 mmHg–1). The pupae and adults of L. africanus showed significantly lower %TBW loss, which was less than 20% (Table 2, Fig. 1b), compared to the value for larvae, which was approximately 50% (F = 7.048; df = 3, 14; P = 0.004). The CP value of L. africanus was low at all stages. Table 3. Power function regression coefficient (mean ± SE) for percentage of total body water lost over time for preadult and adult powderpost beetles, y = axb Species Stage a b F P r2 Heterobostrychus aequalis Larva 0.32 ± 0.08 0.91 ± 0.09 180.43 <0.0001 0.973 Pupa 0.99 ± 0.12 0.45 ± 0.05 182.43 <0.0001 0.973 Female 3.21 ± 0.56 0.38 ± 0.07 74.25 0.0003 0.937 Male 2.76 ± 0.53 0.44 ± 0.08 73.02 0.0004 0.936 Lyctus africanus Larva 5.78 ± 1.29 0.68 ± 0.08 123.91 0.0001 0.961 Pupa 3.82 ± 0.84 0.40 ± 0.09 49.50 0.0009 0.908 Female 2.36 ± 0.62 0.69 ± 0.10 88.07 0.0002 0.946 Male 1.18 ± 0.47 0.78 ± 0.14 54.85 0.0007 0.917 Species Stage a b F P r2 Heterobostrychus aequalis Larva 0.32 ± 0.08 0.91 ± 0.09 180.43 <0.0001 0.973 Pupa 0.99 ± 0.12 0.45 ± 0.05 182.43 <0.0001 0.973 Female 3.21 ± 0.56 0.38 ± 0.07 74.25 0.0003 0.937 Male 2.76 ± 0.53 0.44 ± 0.08 73.02 0.0004 0.936 Lyctus africanus Larva 5.78 ± 1.29 0.68 ± 0.08 123.91 0.0001 0.961 Pupa 3.82 ± 0.84 0.40 ± 0.09 49.50 0.0009 0.908 Female 2.36 ± 0.62 0.69 ± 0.10 88.07 0.0002 0.946 Male 1.18 ± 0.47 0.78 ± 0.14 54.85 0.0007 0.917 y is %TBW lost and x is desiccation time (h). View Large Table 3. Power function regression coefficient (mean ± SE) for percentage of total body water lost over time for preadult and adult powderpost beetles, y = axb Species Stage a b F P r2 Heterobostrychus aequalis Larva 0.32 ± 0.08 0.91 ± 0.09 180.43 <0.0001 0.973 Pupa 0.99 ± 0.12 0.45 ± 0.05 182.43 <0.0001 0.973 Female 3.21 ± 0.56 0.38 ± 0.07 74.25 0.0003 0.937 Male 2.76 ± 0.53 0.44 ± 0.08 73.02 0.0004 0.936 Lyctus africanus Larva 5.78 ± 1.29 0.68 ± 0.08 123.91 0.0001 0.961 Pupa 3.82 ± 0.84 0.40 ± 0.09 49.50 0.0009 0.908 Female 2.36 ± 0.62 0.69 ± 0.10 88.07 0.0002 0.946 Male 1.18 ± 0.47 0.78 ± 0.14 54.85 0.0007 0.917 Species Stage a b F P r2 Heterobostrychus aequalis Larva 0.32 ± 0.08 0.91 ± 0.09 180.43 <0.0001 0.973 Pupa 0.99 ± 0.12 0.45 ± 0.05 182.43 <0.0001 0.973 Female 3.21 ± 0.56 0.38 ± 0.07 74.25 0.0003 0.937 Male 2.76 ± 0.53 0.44 ± 0.08 73.02 0.0004 0.936 Lyctus africanus Larva 5.78 ± 1.29 0.68 ± 0.08 123.91 0.0001 0.961 Pupa 3.82 ± 0.84 0.40 ± 0.09 49.50 0.0009 0.908 Female 2.36 ± 0.62 0.69 ± 0.10 88.07 0.0002 0.946 Male 1.18 ± 0.47 0.78 ± 0.14 54.85 0.0007 0.917 y is %TBW lost and x is desiccation time (h). View Large Fig. 1. View largeDownload slide Percentage of total body water lost over time in (a) H. aequalis and (b) L. africanus. Fig. 1. View largeDownload slide Percentage of total body water lost over time in (a) H. aequalis and (b) L. africanus. Water Relation of Beetles in Relation to Habitats and Environmental Variables The eigenvalues for the first two axes of the ordinations were 0.8781 and 0.1219. Desert, woodland, and hardwood habitats are associated with low precipitation and low RH. Beetles inhabiting these environments exhibited low CP values (0.68–4.52 µg cm–2 h–1 mmHg–1) and low %TBW loss (1.25–17.27%) (Table 1, Fig. 2). Beetles inhabiting plantations where crops or vegetation were grown (Pf, Cb, and Dg) exhibited relatively higher CP values of 8.41–15.3 µg cm–2 h–1 mmHg–1 compared to beetles living in the desert environment. These beetles also lost an excessive amount of water as a consequence of dry conditions, illustrating their adaptiveness to habitats with high precipitation and high RH. Of the two wood-attacking beetles examined in our study, adult L. africanus showed similar environment adaptiveness with tenebrionid beetles that inhabit desert environments (Fig. 2). Fig. 2. View largeDownload slide Principal component analysis ordination diagram showing the water relation (CP and %TBW loss) of the beetles (circles) living in xeric to mesic habitats (triangles) in relation to environmental variables (arrow). First axis is horizontal, second axis is vertical. The beetle species are: Ca = Calosis amabilis, Cb = Charidotella bicolor, Ct = Ctenolepisma terebrans, Cm = Centrioptera municata, Cv = Cryptoglossa verrucosa, Dg = Deloyala guttata, Ea = Eleodes armata, Gm = Gyrosis moralesi, Ha = Heterobostrychus aequalis, Lya = Lyctus africanus, La = Lepidochora argentogrisea, Lp = Lepidochora porti, Ol = Onymaeris laeviceps, Or = Onymacris rugatipennis, Op = Onymaeris plana, Pf = Paederus fuscipes, Tsp = Trigonopus sp. Fig. 2. View largeDownload slide Principal component analysis ordination diagram showing the water relation (CP and %TBW loss) of the beetles (circles) living in xeric to mesic habitats (triangles) in relation to environmental variables (arrow). First axis is horizontal, second axis is vertical. The beetle species are: Ca = Calosis amabilis, Cb = Charidotella bicolor, Ct = Ctenolepisma terebrans, Cm = Centrioptera municata, Cv = Cryptoglossa verrucosa, Dg = Deloyala guttata, Ea = Eleodes armata, Gm = Gyrosis moralesi, Ha = Heterobostrychus aequalis, Lya = Lyctus africanus, La = Lepidochora argentogrisea, Lp = Lepidochora porti, Ol = Onymaeris laeviceps, Or = Onymacris rugatipennis, Op = Onymaeris plana, Pf = Paederus fuscipes, Tsp = Trigonopus sp. Discussion The powderpost beetles H. aequalis and L. africanus, which distributed predominantly in warm and moist regions, may be more widespread than is recognized, as they reportedly are able to establish themselves in temperate regions (Iwata 1982, Nobuchi 1986, Kawakami 1996, Mito and Uesugi 2004, Azmi et al. 2011). Creffield and Howick (1979) hypothesized that prolonged immature development during winter may increase the risk of desiccation in larvae and eventually results in development failure. However, the present study demonstrated that both H. aequalis and L. africanus larvae exhibited low CP. In particular, the CP value of H. aequalis larvae was twofold lower than that of adults. This feature allows the beetles to continue develop in desiccated environments throughout their relatively lengthy larval development process. These findings are another reminder of the many evolutionary solutions that allow insects to survive under adverse conditions. In general, our study showed that H. aequalis and L. africanus confined to hardwood shared similar water relation characteristics with beetles living in desert and woodland habitats (Fig. 2). When confined in wood with 12% moisture content (Ho 1995, Desch and Dinwoodie 1996), H. aequalis and L. africanus might obtain water solely from the wood they consume. However, the water gain may be insufficient to replace the amount of water lost if the beetles tend to lose excessive amounts of water. The beetles exhibited high %TBW content as well as an impressive ability to conserve water (low %TBW loss, except for the immature instar of L. africanus) due to their impermeable cuticle (CP value: 0–15.57 ± 4.90 µg cm–2 h–1 mmHg–1) at all life stages. The %TBW content and CP of these powderpost beetles within current result were consistent with those of the cowpea weevil, Callosobruchus maculatus (Fabricius) (Coleoptera: Chrysomelidae), and the bean weevil, Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae) (Prasantha et al. 2015). These weevils are host-specific and feed on legumes for development. Similar to the powderpost beetles, their high %TBW content (~50%) and low CP (4.5–7.4 µg cm–2 h–1 mmHg–1) enable them to maintain the body water level necessary to function properly and survive the dry conditions in the legumes. It is also possible that these confined microhabitats (cracks and excavated spaces in woods or regumes) create an ideal microclimate for the beetles or serve as insulator against low ambient temperatures. Thus, water loss through diffusion is decreased. In open macrohabitats with less or without vegetation like arid tropical areas, tenebrionid beetles possess extraordinary physiological features, particularly in terms of water conservation, as a way to survive in these extreme conditions where water and food are absent (Zachariassen 1996). This phenomenon explains the water conservation differential in beetles occupying macrohabitats with moisture- and vegetation-rich regions (e.g., Cb, Dg, and Pf in plantations) (Fig. 2), where more water is available to diffuse passively into the body through the cuticle (Appel et al. 1986) to replace what is lost due to diffusion and respiration (Haverty and Nutting 1976). Physiologically, Lyctus species are desiccation sensitive. Parkin (1943) and Bootle (1983) proved that the sympatric species L. brunneus only survived in wood with 10−50% moisture content, and larvae developed optimally in wood with 15% moisture content. Wood moisture contents below 10% disrupted embryo and larval development (Beeson and Bhatia 1937, Smith 1955). Although both test beetles in the present study share a similar habitat (i.e., residing in desiccated wood), the larvae and pupae of L. africanus lost approximately 50 and 15% of TBW, respectively. However, their CP value was comparably low. The high water loss can be explained by the small body size of Lyctus, which results in a high surface area to volume ratio, resulting in more water vaporizing from the larval cuticle relative to total water mass. A series of previous studies reported a significant association between body size and desiccation resistance (Hadley 1994). For instance, adults of C. maculatus from South India are larger than those of Brazilian strains and lose water slowly (Yoder et al. 2010). The fungus-growing termite Microtermes gilvus (Hagen), which has a high surface area-to-volume ratio (40.28–69.75) showed a higher rate of water loss and %TBW loss compared to the larger Macrotermes carbonarius (Hagen), which has a lower surface area to volume ratio (29.26–53.66) (Hu et al. 2012). The ability to reduce water loss rates in association with a reduced body size has often been implicated in the evolution of behavioral adaptation (Cohen and Pinto 1977, Hood and Tschinkel 1990, Hu et al. 2012), e.g., M. gilvus foraged only below ground or concealed within foraging mud tubes as a way to reduce water loss rates, while the larger termite, M. carbonarius that can tolerate desiccation stress was found foraging above-ground in the open air (Hu et al. 2012). Although the surface area to volume ratio of adult Lyctus in the present study was threefold higher than that of H. aequalis, the water loss rate remained comparable or even lower than that of the larger adult H. aequalis. Desiccation tolerance tests of 10 individuals per group for each L. africanus instar, suggest that the main function of evolved aggregative behavior in Lyctus is to reduce water loss rates in ways similar to those known for other arthropods. For instance, the interindividual distance of German cockroaches (Blattella germanica Linnaeus) decreased to form grouping when RH decreased (Dambach and Goehlen 1999). Adult giant Madagascar hissing cockroaches (Gromphadorhina portentosa (Schaum)) in a group of six retained more water content compared to isolated individuals (Yoder and Grojean 1997). Broly et al. (2014) studied the terrestrial isopod Porcellio scaber Latreille and reported that the correlation between the water loss rate of an individual and group size was best fitted by a power function. Any addition of an individual to a small group (1–40 individuals) significantly reduces the individual water losses compared to a large group of more than 50 individuals (Broly et al. 2014). The aggregation pheromone of L. africanus may be responsible for attracting both females and males to promote courtship behavior and act as a signal to aggregate all members to a suitable resource to initiate boring activity, as shown in lesser and larger grain borer (Fadamiro and Wyatt 1996, Edde 2012). The evolved aggregative behavior in adult L. africanus could help maintain relatively low %TBW loss and water loss rates in adult stages to compensate for their small body size, which may contribute to a better fitness performance and ecological success, as has been shown in caterpillars (Klok and Chown 1999). In general, immature arthropods have been reported to lose water more rapidly than adults (Yoder et al. 1997, Bong et al. 2013). As a result of these observations, it is currently believed that significant high cuticular water loss in Lyctus beetles is compensated for by their relatively short immature developmental time (e.g., ~2 wk from egg eclosion to adult; Kartika and Yoshimura 2013), which minimizes prolonged exposure to dry conditions. In contrast, larval H. aequalis undergo an approximately 24-wk development period inside seasoned wood before turning into adults (Creffield and Howick 1979, Ho 1995, Bong et al. 2018). Under desiccated conditions, the larvae are exposed for months to variable temperatures, and, in general, CP and water loss of insects increase with increasing temperature (Ramlov and Lee 2000, Renault et al. 2005, Lyons et al. 2014). Thus, water loss in larval H. aequalis could be intense during summer months. However, there was no apparent increase water loss rate in the larval instar stage, as low water loss rate and %TBW loss were documented. The low surface area to volume ratio (large body size) of this species undoubtedly contributes to the low water loss in larval H. aequalis. In addition, the CP value of larval H. aequalis was two- to fivefold lower than that of the other development stages within the species as well as between test species in the study. The low CP value for immature H. aequalis is equivalent to that of the very drought-resistant larvae of the gall fly, Eurosta solidaginis (Cocquillet), which also have prolonged development time over extended periods during severely low humidity conditions (Ramlov and Lee 2000), and to that of tenebrionid beetle larvae (Mead-Briggs 1956). In summary, the results show the impressive abilities of H. aequalis and L. africanus to survive for months in desiccated wood due to their ability to enhance water conservation by resisting water loss and having an impermeable cuticle. The results demonstrate strategies that insects and other arthropods employ as a trade-off between body size, ontogenetic development, and insect sociality (aggregative and non-aggregative behavior) to maintain their water balance in xeric environments. 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Keeney . 2010 . Enhanced tolerance to water stress in adults of the South India strain of the seed beetle, Callosobruchus maculatus (Coleoptera: Bruchidae), as a product of large body size . Eur. J. Entomol . 107 : 271 – 275 . Google Scholar CrossRef Search ADS Zachariassen , K. E . 1996 . The water conserving physiological compromise of desert insects . Eur. J. Entomol . 93 : 359 – 367 . © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Entomology Oxford University Press

Comparison of Water Relation in Two Powderpost Beetles Relative to Body Size and Ontogenetic and Behavioral Traits

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Entomological Society of America
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© The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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0046-225X
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10.1093/ee/nvy062
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Abstract

Abstract Heterobostrychus aequalis (Waterhouse) (Coleoptera: Bostrychidae) and Lyctus africanus Lesne (Coleoptera: Lyctidae) are distributed mainly in tropical regions. The primary mechanism allowing these beetles to survive in cold and arid habitats beyond the native tropical region is a reduced water loss rate. This study investigated the water relations of these two beetles in relation to their size, ontogenetic traits, and behavioral characteristics to determine how they can survive in desiccated wood. H. aequalis and L. africanus share similar water characteristic with beetles living in desert and woodlands. They have high percentage total body water (%TBW) content (58.38 ± 1.86% to 63.20 ± 1.38%), but low %TBW loss (4.28 ± 1.02% to 48.26 ± 8.28%) due to their impermeable cuticle (cuticular permeability [CP] value: 0–15.57 ± 4.90 µg cm–2 h–1 mmHg–1) at all life stages. Although the larvae of L. africanus exhibited relatively high %TBW loss, they had relatively shorter development times that minimized prolonged exposure to dry conditions inside the wood. The aggregative behavior of the adult could be responsible for maintaining a low water loss rate to compensate for their small body size. In contrast, the larvae of H. aequalis had larger body size and significantly lower CP values, allowing them to survive in the desiccated wood for a longer period of time. These results demonstrate the remarkably sophisticated strategies that insects employ as a trade-off between body size, ontogenetic development, and insect sociality (aggregative and non-aggregative behavior) to maintain their water balance in xeric environments. desiccation tolerance, water retention, hardwood, wood-attacking beetle, aggregative behavior Water conservation is a primary survival tactic for xeric-adapted arthropods, as they lack of the ability to replenish water loss by drinking, feeding, or absorbing water vapor from the environment. In addition, water loss during moulting is expectedly massive due to the drastic increase in cuticular permeability (CP) of newly formed cuticle. In contrast, for mesic-adapted arthropods, more water is available to diffuse passively into the body through the cuticle (Appel et al. 1986) to replace what is lost due to diffusion and respiration (Haverty and Nutting 1976). In general, insects improve their desiccation tolerance by limiting water loss rate via reduced CP; this can occur by enhancing the cuticular lipid and melanization process, increasing the amount of water that is permitted to be lost, increasing body water content, or various combinations of these traits (Bazinet et al. 2010). Increased desiccation resistance in larval insects can be achieved through various behavioral and physiological adaptations. For instance, larvae of the Antarctica midge Belgica antarctica Jacobs, which experience a prolonged cold and arid environment, can lose more than 70% of their water content at a rate of more than 10% per hour. In response the environmental conditions, the larvae formed dense clusters and increased the concentration of glycerol and trehalose two- to threefold to reduce the rate of water loss (Benoit et al. 2007). A similar observation was reported for the African chironomid Polypedium vanderplanki Hinton (Kikawada et al. 2005). B. antarctica also decreased its metabolic rate by reducing the oxygen consumption rate in response to dehydration (Benoit et al. 2007). For the meal worm Tenebrio molitor Linnaeus, the starved larvae survived by maintaining their water balance through atmospheric absorption when the relative humidity (RH) was greater than 88% (Machin 1975). Toolson (1982) reported that the shift from short chain epicuticular hydrocarbons to longer chain lengths reduced CP in the phorid fly Drosophila pseudoobscura Frolova. However, it is unclear how the powderpost beetles tolerate desiccation stress in the temperate regions, which the low temperature decelerates the development of these beetles and likely prolonged their exposure to dryness inside the woods. Bostrichidae, also known as powderpost beetles, are among the most destructive wood-attacking beetles, thus they are of economic importance in forestry and lumber industries (Peters et al. 2002). Although these beetles are predominantly distributed in tropical regions, they were introduced into temperate regions through imported timber or timber products. Of the 550 described species distributed worldwide (Ivie 2002), 16 species are currently found in Japan, and eight of them have become established in Japan: Heterobostrychus hamatipennis Lesne, Rhizopertha dominica (Fabricius), Dinoderus minutus Fabricius, Lyctus africanus Lesne (Coleoptera: Lyctidae), Minthea rugicollis (Walker), Lyctus sinensis Lesne, Lyctus linearis (Goeze), and Lyctus brunneus (Stephens) (Mito and Uesugi 2004). Infestations of these beetles are widespread, and they have become a major problem in Japan (Shigetaka 1980, Nobuchi 1986, Kawakami 1996, Furukawa et al. 2009, Bong 2015). Heterobostrychus aequalis (Waterhouse) (Coleoptera: Bostrychidae) was first found in seasoned imported lauan timber in Naha, Okinawa (Nobuchi 1986). In recent years, cases of damaged timber involving this beetle have been on the rise, but it is still unclear whether H. aequalis is established in Japan. On the other hand, L. africanus was found to be established in Japan in the 1980s (Iwata 1982, Mito and Uesugi 2004). Since then, the species has become a major pest in Japan because of its prevalent infestation of wooden structures throughout the country (Furukawa et al. 2009). Low temperatures in temperate regions decelerate development and likely prolong the beetles’ exposure to dryness inside wood. Water relations could be an important factor that allows these insects to thrive under high desiccation pressure across a wide range of geographical regions (Appel et al. 1983, Zachariassen 1996, Bong et al. 2013). The possible mechanism allowing powderpost beetles to survive beyond their native tropical region in cold and arid habitats is reduction of water loss rate as shown in other beetles (Bellés and Halstead 1985, Benoit et al. 2005). For example, the spider beetle Mezium affine Boieldieu survived for approximately 3 mo without food or water in the desiccated condition because of a remarkable ability to retain water inside their bodies (Benoit et al. 2005). For this reason, M. affine is one of the most persistent and widely distributed stored product pest (Bellés and Halstead 1985). Despite their similar habitat, H. aequalis and L. africanus differ conspicuously in morphology, ontogeny, and behavior. For instance, L. africanus is 3–4 times smaller than H. aequalis. Water balance is a challenge for small-bodied insects, as they have high surface area-to-volume ratios that may result in a significantly increased water loss rate (Hu et al. 2012). Thus, it was likely that L. africanus had a specific adaptive behavior to survive inside desiccated wood. Male L. africanus produce volatile esters to elicit aggregative behavior (Kartika et al. 2015). Despite of attracting conspecific to form a cluster for predation avoidance (Cornell et al. 1987, Stamp and Bowers 1988, Lawrence 1990, Sillén-Tullberg 1990) and growth (Tsubaki and Shiotsu 1982, Clark and Faeth 1997, Denno and Benrey 1997), the behavior can facilitate water conservation (Rasa 1997, Yoder and Grojean 1997, Glass et al. 1998). It was reasoned that aggregation formed a ‘superorganism’ which characterized the low ratios of surface area-to-volume to reduce water loss rate. In addition, the aggregation may create a humid microcosm between individuals when water is lost as shown in dust mite, Dermatophagoides farinae Hughes (Acari: Pyroglyphidae) (Glass et al. 1998). Aggregation behavior is absent in H. aequalis, which always appear as single individuals or in pairs. H. aequalis undergoes a lengthy immature developmental process that lasts approximately 24 wk, which is 12 times longer than that of L. africanus (Creffield and Howick 1979, Ho 1995, Bong et al. 2018). Thus, prolonged immature development for H. aequalis during winter may increase the risk of larval desiccation and eventually cause failure to develop. In all likelihood, H. aequalis and L. africanus have physiological and behavioral plasticity that enables them to survive prolonged exposure in dry wood. In this study, we examined the water relations of H. aequalis and L. africanus to determine how they can survive in desiccated wood. The goals of this study were to address the following questions: Are there differences in water relations between adults of the two test beetle species, considering that L. africanus exhibits aggregative behavior? Does size matter for water retention of small-bodied L. africanus? How do the larvae of H. aequalis conserve the water needed for development over an extended period of 24 wk? Materials and Methods Rearing Method Adult H. aequalis and L. africanus were, respectively, reared in a closed container (20.0 cm length × 16.0 cm width × 9.0 cm height) and a closed jar (8.5 cm diameter × 13.0 cm height) in a walk-in environmental chamber (at 26 ± 2°C and 65 ± 10% RH) in the Deterioration Organisms Laboratory (DOL) at the Research Institute for Sustainable Humanosphere, Kyoto, Gokasho, Uji, Japan. The beetles were fed an artificial diet consisting of dried yeast (24% Asahi Food and Health Care, Tokyo, Japan), starch (50%, Nacalai Tesque, Kyoto, Japan), and lauan (Shorea spp.) wood sawdust (26%) in a 16.0 cm length × 8.0 cm width × 2.0 cm depth block for H. aequalis, and 8.0 cm length × 4.0 cm width × 2.0 cm depth block for L. africanus (Kartika and Yoshimura 2013). CP Study Percentage total body water (%TBW) content, %TBW loss over desiccation time, and CP of H. aequalis and L. africanus were examined gravimetrically (Appel and Tanley 1999, Shelton and Grace 2003). For H. aequalis, 10 late instar larvae weighing between 40 and 60 mg, 12 pupae weighing between 30 and 60 mg, and five adult males weighing between 25 and 40 mg and five adult females weighing between 30 and 50 mg aged 1-wk old were used. The insects were individually placed in the specimen tubes (15 mm diameter × 50 mm height), and examined gravimetrically. The study was replicated with one insect each time for each stage. For L. africanus, the study was replicated four to five times for each stage, with each replicate consisting of 10 insects (10 late instar larvae weighing between 2.4 and 3.9 mg, 10 pupae weighing between 2.2 and 2.5 mg, 10 adult males weighing between 1.2 and 1.5 mg and 10 females weighing between 1.3 and 1.5 mg) to simulate their aggregative nature in environment. Only newly emerged adults from the food source were subjected to analysis. The insects were weighed to the nearest 0.01 mg using a digital analytical balance (Sartorius Extended ED2245, Sartorius AG, Göttingen, Germany). The test insects were placed in an 11 L glass desiccator containing 1 kg of anhydrous CaSO4 (Fisons Scientific Apparatus, Leicestershire, United Kingdom). Before testing, the desiccant was dried at 100°C for 48 h. The desiccator was maintained at 6% RH with a saturation deficit of 27.56 mmHg at 28.6 ± 0.2°C. The test insects were weighed for mass loss at 2, 4, 6, 8, 10, and 24 h (Appel and Tanley 1999, Shelton and Grace 2003). After 24 h, the test insects were dried at 60°C for 72 h and then weighed to obtain the dry weight. Insect water loss during the first 2 h of desiccation was used to calculate the CP of an insect because water loss that occurs during this time interval represents cuticular water loss (Sponsler and Appel 1990, Shelton and Grace 2003). The CP value was calculated as water loss using the following equation: [initial weight − weight loss at 2 h] (µg) per surface area (cm2) per time (h) per saturation deficit (mmHg) (Edney 1977). Surface area of the specimen was calculated using Meeh’s formula (Meeh 1897): S = 12M2/3, where S = body surface area (cm2) and M = initial mass (g). Edney (1977) reported that insects living in xeric habitats exhibited CP values of 0–30 µg cm–2 h–1 mmHg–1, whereas those found in mesic and hygric habitats exhibited CP values of 31–60 µg cm–2 h–1 mmHg–1 and > 60 µg cm–2 h–1 mmHg–1, respectively. %TBW content and %TBW loss of an insect were calculated as follows: %TBW content=[(initial mass–dry mass)/initial mass]×100% %TBW loss=[(initial weight–weight at each hour)/(initial weight–dry weight)]×100% Statistical Analysis % TBW content and TBW loss were arcsine square root transformed. Body mass, CP value, and the transformed values were checked for normality at the 0.05 significance level using the Kolmogorov-Smirnov test. When the criteria of normality were not met, a log10 transformation was performed, and the data were retested for normality. The data were then analyzed using a one-way followed by Tukey’s Honest Significant Difference test. Body water content was analysed using one-way analysis of covariance (ANCOVA) and separated by least significant difference with initial weight as a covariate. ANCOVA was used because it eliminates the influence of the variation in body size on the physiological response, generating a more reliable result (Packard and Boardman 1999, Hu et al. 2012). All analyses were performed using SPSS analysis version 11.0 (SPSS Inc., Chicago, IL). Comparison With Other Beetles in Relation to Habitats, Environmental Variables, and Water Relations Water relation data for other beetles were collected from previous studies (Table 1). The association between water relation (CP and %TBW loss) of beetles and habitats and environmental variables were examined using CANOCO 5.0 (ter Braak and Ŝmilauer 2012). The unconstrained ordination method (detrended correspondence analysis) was used to examine the gradient length. Principal component analysis was used because the water relation of beetles along the environmental variables in their habitat was found to be homogenous. Table 1. Habitat types and environmental variables in relation to water relation of beetles Beetle species Habitat types Environmental variables Water relation References Temperature (°C) Relative humidity (%) Precipitation (mm/month) CP (µg cm–2 h–1 mmHg–1) TBW loss (%) Charidotella bicolor Plantation 41 50 80.05 9.99 50.36 Hull-Sanders et al. (2003) Deloyala guttata Plantation 41 50 40.6 8.41 70.05 Hull-Sanders et al. (2003) Eleodes armata Desert 37 22 24.13 2.5 4 Ahearn (1970), Hadley (1978) Cryptoglossa verrucosa Desert 37 22 24.13 1.6 2 Ahearn (1970), Hadley (1978) Centrioptera municata Desert 39 11 12.7 2.2 4 Ahearn (1970), Hadley (1978) Onymaeris plana Desert 35 47 3 1.53 1.25 Edney (1971) Onymaeris laeviceps Desert 35 47 3 3.41 3 Edney (1971) Lepidochora argentogrisea Desert 35 47 3 1.91 3 Edney (1971) Onymacris rugatipennis Desert 35 47 3 1.87 1.5 Edney (1971) Lepidochora porti Desert 35 47 3 3.49 3 Edney (1971) Calosis amabilis Desert 35 47 3 1.09 1.5 Edney (1971) Gyrosis moralesi Desert 35 47 3 2.24 4 Edney (1971) Ctenolepisma terebrans Desert 35 47 3 0.68 5 Edney (1971) Trigonopus sp. Woodland 26.5 71 24 4.13 6 Edney (1971) Paederus fuscipes Plantation 28 80 500 15.3 79.2 Bong et al. (2013) Heterobostrychus aequalis Hardwood 26 12 (m.c) 0 15.3 11.77 Present study Lyctus africanus Hardwood 26 12 (m.c) 0 4.52 17.27 Present study Beetle species Habitat types Environmental variables Water relation References Temperature (°C) Relative humidity (%) Precipitation (mm/month) CP (µg cm–2 h–1 mmHg–1) TBW loss (%) Charidotella bicolor Plantation 41 50 80.05 9.99 50.36 Hull-Sanders et al. (2003) Deloyala guttata Plantation 41 50 40.6 8.41 70.05 Hull-Sanders et al. (2003) Eleodes armata Desert 37 22 24.13 2.5 4 Ahearn (1970), Hadley (1978) Cryptoglossa verrucosa Desert 37 22 24.13 1.6 2 Ahearn (1970), Hadley (1978) Centrioptera municata Desert 39 11 12.7 2.2 4 Ahearn (1970), Hadley (1978) Onymaeris plana Desert 35 47 3 1.53 1.25 Edney (1971) Onymaeris laeviceps Desert 35 47 3 3.41 3 Edney (1971) Lepidochora argentogrisea Desert 35 47 3 1.91 3 Edney (1971) Onymacris rugatipennis Desert 35 47 3 1.87 1.5 Edney (1971) Lepidochora porti Desert 35 47 3 3.49 3 Edney (1971) Calosis amabilis Desert 35 47 3 1.09 1.5 Edney (1971) Gyrosis moralesi Desert 35 47 3 2.24 4 Edney (1971) Ctenolepisma terebrans Desert 35 47 3 0.68 5 Edney (1971) Trigonopus sp. Woodland 26.5 71 24 4.13 6 Edney (1971) Paederus fuscipes Plantation 28 80 500 15.3 79.2 Bong et al. (2013) Heterobostrychus aequalis Hardwood 26 12 (m.c) 0 15.3 11.77 Present study Lyctus africanus Hardwood 26 12 (m.c) 0 4.52 17.27 Present study m.c., moisture content. View Large Table 1. Habitat types and environmental variables in relation to water relation of beetles Beetle species Habitat types Environmental variables Water relation References Temperature (°C) Relative humidity (%) Precipitation (mm/month) CP (µg cm–2 h–1 mmHg–1) TBW loss (%) Charidotella bicolor Plantation 41 50 80.05 9.99 50.36 Hull-Sanders et al. (2003) Deloyala guttata Plantation 41 50 40.6 8.41 70.05 Hull-Sanders et al. (2003) Eleodes armata Desert 37 22 24.13 2.5 4 Ahearn (1970), Hadley (1978) Cryptoglossa verrucosa Desert 37 22 24.13 1.6 2 Ahearn (1970), Hadley (1978) Centrioptera municata Desert 39 11 12.7 2.2 4 Ahearn (1970), Hadley (1978) Onymaeris plana Desert 35 47 3 1.53 1.25 Edney (1971) Onymaeris laeviceps Desert 35 47 3 3.41 3 Edney (1971) Lepidochora argentogrisea Desert 35 47 3 1.91 3 Edney (1971) Onymacris rugatipennis Desert 35 47 3 1.87 1.5 Edney (1971) Lepidochora porti Desert 35 47 3 3.49 3 Edney (1971) Calosis amabilis Desert 35 47 3 1.09 1.5 Edney (1971) Gyrosis moralesi Desert 35 47 3 2.24 4 Edney (1971) Ctenolepisma terebrans Desert 35 47 3 0.68 5 Edney (1971) Trigonopus sp. Woodland 26.5 71 24 4.13 6 Edney (1971) Paederus fuscipes Plantation 28 80 500 15.3 79.2 Bong et al. (2013) Heterobostrychus aequalis Hardwood 26 12 (m.c) 0 15.3 11.77 Present study Lyctus africanus Hardwood 26 12 (m.c) 0 4.52 17.27 Present study Beetle species Habitat types Environmental variables Water relation References Temperature (°C) Relative humidity (%) Precipitation (mm/month) CP (µg cm–2 h–1 mmHg–1) TBW loss (%) Charidotella bicolor Plantation 41 50 80.05 9.99 50.36 Hull-Sanders et al. (2003) Deloyala guttata Plantation 41 50 40.6 8.41 70.05 Hull-Sanders et al. (2003) Eleodes armata Desert 37 22 24.13 2.5 4 Ahearn (1970), Hadley (1978) Cryptoglossa verrucosa Desert 37 22 24.13 1.6 2 Ahearn (1970), Hadley (1978) Centrioptera municata Desert 39 11 12.7 2.2 4 Ahearn (1970), Hadley (1978) Onymaeris plana Desert 35 47 3 1.53 1.25 Edney (1971) Onymaeris laeviceps Desert 35 47 3 3.41 3 Edney (1971) Lepidochora argentogrisea Desert 35 47 3 1.91 3 Edney (1971) Onymacris rugatipennis Desert 35 47 3 1.87 1.5 Edney (1971) Lepidochora porti Desert 35 47 3 3.49 3 Edney (1971) Calosis amabilis Desert 35 47 3 1.09 1.5 Edney (1971) Gyrosis moralesi Desert 35 47 3 2.24 4 Edney (1971) Ctenolepisma terebrans Desert 35 47 3 0.68 5 Edney (1971) Trigonopus sp. Woodland 26.5 71 24 4.13 6 Edney (1971) Paederus fuscipes Plantation 28 80 500 15.3 79.2 Bong et al. (2013) Heterobostrychus aequalis Hardwood 26 12 (m.c) 0 15.3 11.77 Present study Lyctus africanus Hardwood 26 12 (m.c) 0 4.52 17.27 Present study m.c., moisture content. View Large Results Water Relations In general, the fresh body mass of H. aequalis was 17- to 24-fold higher (32.00 ± 2.55 mg to 52.90 ± 2.85 mg) than that of L. africanus (1.36 ± 0.05 mg to 3.10 ± 0.29 mg) (Table 2). The fresh body mass of both species decreased significantly as larva developed to the adult stage (H. aequalis, F = 7.463; df = 3, 28; P = 0.001; L. africanus, F = 55.222; df = 3, 14; P < 0.001). Despite the difference in body mass between the two beetles, both species showed a comparably high %TBW content, ranging from 58.38 ± 1.86% to 63.20 ± 1.38%. Table 2. Physiological parameters of preadult and adult powderpost beetles (mean ± SD) Species Stage n Initial mass (mg) Body water content (mg)a TBW content (%) TBW loss (%)b CP (µg cm–2 h–1 mmHg–1) Ratios of surface area to volume Water loss rate (mg g–1 h–1)a Heterobostrychus aequalis L 10 52.90 ± 2.85a* 32.40 ± 1.85a* 61.35 ± 1.40a 5.93 ± 1.70ab* 2.52 ± 1.68a 32.14 ± 1.75a* 2.09 ± 1.40 a P 12 48.50 ± 2.78a* 29.50 ± 1.90a* 60.53 ± 0.92a 4.28 ± 1.02a* 5.89 ± 3.45a 33.21 ± 2.47a* 4.82 ± 2.66 a F 5 40.60 ± 3.26ab* 24.20 ± 2.08a* 59.50 ± 0.64a 11.55 ± 2.30b 13.76 ± 4.78a 35.12 ± 2.13ab* 13.77 ± 5.02 a M 5 32.00 ± 2.55b* 20.00 ± 1.76a* 62.36 ± 0.83a 11.99 ± 0.51b 15.57 ± 4.90a* 38.03 ± 2.38b* 16.58 ± 5.25 a* Lyctus africanus L 40 3.10 ± 0.29x* 1.93 ± 0.12x* 62.04 ± 0.70x 48.26 ± 8.28x* 5.96 ± 3.99x 82.79 ± 2.64x* 14.32 ± 9.83 x P 40 2.38 ± 0.06y* 1.50 ± 0.04x* 63.20 ± 1.38x 14.81 ± 2.94y* 8.43 ± 4.87x 89.98 ± 0.81y* 20.83 ± 12.03 x F 50 1.46 ± 0.05z* 0.85 ± 0.03x* 58.38 ± 1.86x 21.15 ± 6.22y 4.52 ± 2.77x 105.90 ± 1.25z* 12.92 ± 7.92 x M 50 1.36 ± 0.05z* 0.82 ± 0.02x* 60.49 ± 1.91x 13.38 ± 4.61y 0.00 ± 0.00y* 108.45 ± 1.38z* 0.00 ± 0.00 x* Species Stage n Initial mass (mg) Body water content (mg)a TBW content (%) TBW loss (%)b CP (µg cm–2 h–1 mmHg–1) Ratios of surface area to volume Water loss rate (mg g–1 h–1)a Heterobostrychus aequalis L 10 52.90 ± 2.85a* 32.40 ± 1.85a* 61.35 ± 1.40a 5.93 ± 1.70ab* 2.52 ± 1.68a 32.14 ± 1.75a* 2.09 ± 1.40 a P 12 48.50 ± 2.78a* 29.50 ± 1.90a* 60.53 ± 0.92a 4.28 ± 1.02a* 5.89 ± 3.45a 33.21 ± 2.47a* 4.82 ± 2.66 a F 5 40.60 ± 3.26ab* 24.20 ± 2.08a* 59.50 ± 0.64a 11.55 ± 2.30b 13.76 ± 4.78a 35.12 ± 2.13ab* 13.77 ± 5.02 a M 5 32.00 ± 2.55b* 20.00 ± 1.76a* 62.36 ± 0.83a 11.99 ± 0.51b 15.57 ± 4.90a* 38.03 ± 2.38b* 16.58 ± 5.25 a* Lyctus africanus L 40 3.10 ± 0.29x* 1.93 ± 0.12x* 62.04 ± 0.70x 48.26 ± 8.28x* 5.96 ± 3.99x 82.79 ± 2.64x* 14.32 ± 9.83 x P 40 2.38 ± 0.06y* 1.50 ± 0.04x* 63.20 ± 1.38x 14.81 ± 2.94y* 8.43 ± 4.87x 89.98 ± 0.81y* 20.83 ± 12.03 x F 50 1.46 ± 0.05z* 0.85 ± 0.03x* 58.38 ± 1.86x 21.15 ± 6.22y 4.52 ± 2.77x 105.90 ± 1.25z* 12.92 ± 7.92 x M 50 1.36 ± 0.05z* 0.82 ± 0.02x* 60.49 ± 1.91x 13.38 ± 4.61y 0.00 ± 0.00y* 108.45 ± 1.38z* 0.00 ± 0.00 x* Stage: L, larva; P, pupa; F, female adult; M, male adult. Mean values followed by the same letter within the species are not significantly different (Tukey’s HSD; α = 0.05). Mean values followed by an asterisk between species of the same instar are significantly different (Student’s t-test; α = 0.05). aParameter analyzed using ANCOVA and separated by least significant difference with initial mass as a covariate. bPercentage of TBW loss at 24 h. View Large Table 2. Physiological parameters of preadult and adult powderpost beetles (mean ± SD) Species Stage n Initial mass (mg) Body water content (mg)a TBW content (%) TBW loss (%)b CP (µg cm–2 h–1 mmHg–1) Ratios of surface area to volume Water loss rate (mg g–1 h–1)a Heterobostrychus aequalis L 10 52.90 ± 2.85a* 32.40 ± 1.85a* 61.35 ± 1.40a 5.93 ± 1.70ab* 2.52 ± 1.68a 32.14 ± 1.75a* 2.09 ± 1.40 a P 12 48.50 ± 2.78a* 29.50 ± 1.90a* 60.53 ± 0.92a 4.28 ± 1.02a* 5.89 ± 3.45a 33.21 ± 2.47a* 4.82 ± 2.66 a F 5 40.60 ± 3.26ab* 24.20 ± 2.08a* 59.50 ± 0.64a 11.55 ± 2.30b 13.76 ± 4.78a 35.12 ± 2.13ab* 13.77 ± 5.02 a M 5 32.00 ± 2.55b* 20.00 ± 1.76a* 62.36 ± 0.83a 11.99 ± 0.51b 15.57 ± 4.90a* 38.03 ± 2.38b* 16.58 ± 5.25 a* Lyctus africanus L 40 3.10 ± 0.29x* 1.93 ± 0.12x* 62.04 ± 0.70x 48.26 ± 8.28x* 5.96 ± 3.99x 82.79 ± 2.64x* 14.32 ± 9.83 x P 40 2.38 ± 0.06y* 1.50 ± 0.04x* 63.20 ± 1.38x 14.81 ± 2.94y* 8.43 ± 4.87x 89.98 ± 0.81y* 20.83 ± 12.03 x F 50 1.46 ± 0.05z* 0.85 ± 0.03x* 58.38 ± 1.86x 21.15 ± 6.22y 4.52 ± 2.77x 105.90 ± 1.25z* 12.92 ± 7.92 x M 50 1.36 ± 0.05z* 0.82 ± 0.02x* 60.49 ± 1.91x 13.38 ± 4.61y 0.00 ± 0.00y* 108.45 ± 1.38z* 0.00 ± 0.00 x* Species Stage n Initial mass (mg) Body water content (mg)a TBW content (%) TBW loss (%)b CP (µg cm–2 h–1 mmHg–1) Ratios of surface area to volume Water loss rate (mg g–1 h–1)a Heterobostrychus aequalis L 10 52.90 ± 2.85a* 32.40 ± 1.85a* 61.35 ± 1.40a 5.93 ± 1.70ab* 2.52 ± 1.68a 32.14 ± 1.75a* 2.09 ± 1.40 a P 12 48.50 ± 2.78a* 29.50 ± 1.90a* 60.53 ± 0.92a 4.28 ± 1.02a* 5.89 ± 3.45a 33.21 ± 2.47a* 4.82 ± 2.66 a F 5 40.60 ± 3.26ab* 24.20 ± 2.08a* 59.50 ± 0.64a 11.55 ± 2.30b 13.76 ± 4.78a 35.12 ± 2.13ab* 13.77 ± 5.02 a M 5 32.00 ± 2.55b* 20.00 ± 1.76a* 62.36 ± 0.83a 11.99 ± 0.51b 15.57 ± 4.90a* 38.03 ± 2.38b* 16.58 ± 5.25 a* Lyctus africanus L 40 3.10 ± 0.29x* 1.93 ± 0.12x* 62.04 ± 0.70x 48.26 ± 8.28x* 5.96 ± 3.99x 82.79 ± 2.64x* 14.32 ± 9.83 x P 40 2.38 ± 0.06y* 1.50 ± 0.04x* 63.20 ± 1.38x 14.81 ± 2.94y* 8.43 ± 4.87x 89.98 ± 0.81y* 20.83 ± 12.03 x F 50 1.46 ± 0.05z* 0.85 ± 0.03x* 58.38 ± 1.86x 21.15 ± 6.22y 4.52 ± 2.77x 105.90 ± 1.25z* 12.92 ± 7.92 x M 50 1.36 ± 0.05z* 0.82 ± 0.02x* 60.49 ± 1.91x 13.38 ± 4.61y 0.00 ± 0.00y* 108.45 ± 1.38z* 0.00 ± 0.00 x* Stage: L, larva; P, pupa; F, female adult; M, male adult. Mean values followed by the same letter within the species are not significantly different (Tukey’s HSD; α = 0.05). Mean values followed by an asterisk between species of the same instar are significantly different (Student’s t-test; α = 0.05). aParameter analyzed using ANCOVA and separated by least significant difference with initial mass as a covariate. bPercentage of TBW loss at 24 h. View Large The %TBW loss at all stages of H. aequalis and L. africanus increased curvilinearly with desiccation time (Fig. 1, Table 3). However, H. aequalis showed low %TBW loss, which was less than 12% at all stages (Table 2, Fig. 1a). The %TBW loss of immature individuals was significantly lower than that of the adults (F = 6.012; df = 3, 28; P = 0.003), resulting in higher CP values (13.76 ± 4.78 µg cm–2 h–1 mmHg–1 to 15.57 ± 4.90 µg cm–2 h–1 mmHg–1) in the adults compared to the immature stages (larval, 2.52 ± 1.68 µg cm–2 h–1 mmHg–1; pupal, 5.89 ± 3.45 µg cm–2 h–1 mmHg–1). The pupae and adults of L. africanus showed significantly lower %TBW loss, which was less than 20% (Table 2, Fig. 1b), compared to the value for larvae, which was approximately 50% (F = 7.048; df = 3, 14; P = 0.004). The CP value of L. africanus was low at all stages. Table 3. Power function regression coefficient (mean ± SE) for percentage of total body water lost over time for preadult and adult powderpost beetles, y = axb Species Stage a b F P r2 Heterobostrychus aequalis Larva 0.32 ± 0.08 0.91 ± 0.09 180.43 <0.0001 0.973 Pupa 0.99 ± 0.12 0.45 ± 0.05 182.43 <0.0001 0.973 Female 3.21 ± 0.56 0.38 ± 0.07 74.25 0.0003 0.937 Male 2.76 ± 0.53 0.44 ± 0.08 73.02 0.0004 0.936 Lyctus africanus Larva 5.78 ± 1.29 0.68 ± 0.08 123.91 0.0001 0.961 Pupa 3.82 ± 0.84 0.40 ± 0.09 49.50 0.0009 0.908 Female 2.36 ± 0.62 0.69 ± 0.10 88.07 0.0002 0.946 Male 1.18 ± 0.47 0.78 ± 0.14 54.85 0.0007 0.917 Species Stage a b F P r2 Heterobostrychus aequalis Larva 0.32 ± 0.08 0.91 ± 0.09 180.43 <0.0001 0.973 Pupa 0.99 ± 0.12 0.45 ± 0.05 182.43 <0.0001 0.973 Female 3.21 ± 0.56 0.38 ± 0.07 74.25 0.0003 0.937 Male 2.76 ± 0.53 0.44 ± 0.08 73.02 0.0004 0.936 Lyctus africanus Larva 5.78 ± 1.29 0.68 ± 0.08 123.91 0.0001 0.961 Pupa 3.82 ± 0.84 0.40 ± 0.09 49.50 0.0009 0.908 Female 2.36 ± 0.62 0.69 ± 0.10 88.07 0.0002 0.946 Male 1.18 ± 0.47 0.78 ± 0.14 54.85 0.0007 0.917 y is %TBW lost and x is desiccation time (h). View Large Table 3. Power function regression coefficient (mean ± SE) for percentage of total body water lost over time for preadult and adult powderpost beetles, y = axb Species Stage a b F P r2 Heterobostrychus aequalis Larva 0.32 ± 0.08 0.91 ± 0.09 180.43 <0.0001 0.973 Pupa 0.99 ± 0.12 0.45 ± 0.05 182.43 <0.0001 0.973 Female 3.21 ± 0.56 0.38 ± 0.07 74.25 0.0003 0.937 Male 2.76 ± 0.53 0.44 ± 0.08 73.02 0.0004 0.936 Lyctus africanus Larva 5.78 ± 1.29 0.68 ± 0.08 123.91 0.0001 0.961 Pupa 3.82 ± 0.84 0.40 ± 0.09 49.50 0.0009 0.908 Female 2.36 ± 0.62 0.69 ± 0.10 88.07 0.0002 0.946 Male 1.18 ± 0.47 0.78 ± 0.14 54.85 0.0007 0.917 Species Stage a b F P r2 Heterobostrychus aequalis Larva 0.32 ± 0.08 0.91 ± 0.09 180.43 <0.0001 0.973 Pupa 0.99 ± 0.12 0.45 ± 0.05 182.43 <0.0001 0.973 Female 3.21 ± 0.56 0.38 ± 0.07 74.25 0.0003 0.937 Male 2.76 ± 0.53 0.44 ± 0.08 73.02 0.0004 0.936 Lyctus africanus Larva 5.78 ± 1.29 0.68 ± 0.08 123.91 0.0001 0.961 Pupa 3.82 ± 0.84 0.40 ± 0.09 49.50 0.0009 0.908 Female 2.36 ± 0.62 0.69 ± 0.10 88.07 0.0002 0.946 Male 1.18 ± 0.47 0.78 ± 0.14 54.85 0.0007 0.917 y is %TBW lost and x is desiccation time (h). View Large Fig. 1. View largeDownload slide Percentage of total body water lost over time in (a) H. aequalis and (b) L. africanus. Fig. 1. View largeDownload slide Percentage of total body water lost over time in (a) H. aequalis and (b) L. africanus. Water Relation of Beetles in Relation to Habitats and Environmental Variables The eigenvalues for the first two axes of the ordinations were 0.8781 and 0.1219. Desert, woodland, and hardwood habitats are associated with low precipitation and low RH. Beetles inhabiting these environments exhibited low CP values (0.68–4.52 µg cm–2 h–1 mmHg–1) and low %TBW loss (1.25–17.27%) (Table 1, Fig. 2). Beetles inhabiting plantations where crops or vegetation were grown (Pf, Cb, and Dg) exhibited relatively higher CP values of 8.41–15.3 µg cm–2 h–1 mmHg–1 compared to beetles living in the desert environment. These beetles also lost an excessive amount of water as a consequence of dry conditions, illustrating their adaptiveness to habitats with high precipitation and high RH. Of the two wood-attacking beetles examined in our study, adult L. africanus showed similar environment adaptiveness with tenebrionid beetles that inhabit desert environments (Fig. 2). Fig. 2. View largeDownload slide Principal component analysis ordination diagram showing the water relation (CP and %TBW loss) of the beetles (circles) living in xeric to mesic habitats (triangles) in relation to environmental variables (arrow). First axis is horizontal, second axis is vertical. The beetle species are: Ca = Calosis amabilis, Cb = Charidotella bicolor, Ct = Ctenolepisma terebrans, Cm = Centrioptera municata, Cv = Cryptoglossa verrucosa, Dg = Deloyala guttata, Ea = Eleodes armata, Gm = Gyrosis moralesi, Ha = Heterobostrychus aequalis, Lya = Lyctus africanus, La = Lepidochora argentogrisea, Lp = Lepidochora porti, Ol = Onymaeris laeviceps, Or = Onymacris rugatipennis, Op = Onymaeris plana, Pf = Paederus fuscipes, Tsp = Trigonopus sp. Fig. 2. View largeDownload slide Principal component analysis ordination diagram showing the water relation (CP and %TBW loss) of the beetles (circles) living in xeric to mesic habitats (triangles) in relation to environmental variables (arrow). First axis is horizontal, second axis is vertical. The beetle species are: Ca = Calosis amabilis, Cb = Charidotella bicolor, Ct = Ctenolepisma terebrans, Cm = Centrioptera municata, Cv = Cryptoglossa verrucosa, Dg = Deloyala guttata, Ea = Eleodes armata, Gm = Gyrosis moralesi, Ha = Heterobostrychus aequalis, Lya = Lyctus africanus, La = Lepidochora argentogrisea, Lp = Lepidochora porti, Ol = Onymaeris laeviceps, Or = Onymacris rugatipennis, Op = Onymaeris plana, Pf = Paederus fuscipes, Tsp = Trigonopus sp. Discussion The powderpost beetles H. aequalis and L. africanus, which distributed predominantly in warm and moist regions, may be more widespread than is recognized, as they reportedly are able to establish themselves in temperate regions (Iwata 1982, Nobuchi 1986, Kawakami 1996, Mito and Uesugi 2004, Azmi et al. 2011). Creffield and Howick (1979) hypothesized that prolonged immature development during winter may increase the risk of desiccation in larvae and eventually results in development failure. However, the present study demonstrated that both H. aequalis and L. africanus larvae exhibited low CP. In particular, the CP value of H. aequalis larvae was twofold lower than that of adults. This feature allows the beetles to continue develop in desiccated environments throughout their relatively lengthy larval development process. These findings are another reminder of the many evolutionary solutions that allow insects to survive under adverse conditions. In general, our study showed that H. aequalis and L. africanus confined to hardwood shared similar water relation characteristics with beetles living in desert and woodland habitats (Fig. 2). When confined in wood with 12% moisture content (Ho 1995, Desch and Dinwoodie 1996), H. aequalis and L. africanus might obtain water solely from the wood they consume. However, the water gain may be insufficient to replace the amount of water lost if the beetles tend to lose excessive amounts of water. The beetles exhibited high %TBW content as well as an impressive ability to conserve water (low %TBW loss, except for the immature instar of L. africanus) due to their impermeable cuticle (CP value: 0–15.57 ± 4.90 µg cm–2 h–1 mmHg–1) at all life stages. The %TBW content and CP of these powderpost beetles within current result were consistent with those of the cowpea weevil, Callosobruchus maculatus (Fabricius) (Coleoptera: Chrysomelidae), and the bean weevil, Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae) (Prasantha et al. 2015). These weevils are host-specific and feed on legumes for development. Similar to the powderpost beetles, their high %TBW content (~50%) and low CP (4.5–7.4 µg cm–2 h–1 mmHg–1) enable them to maintain the body water level necessary to function properly and survive the dry conditions in the legumes. It is also possible that these confined microhabitats (cracks and excavated spaces in woods or regumes) create an ideal microclimate for the beetles or serve as insulator against low ambient temperatures. Thus, water loss through diffusion is decreased. In open macrohabitats with less or without vegetation like arid tropical areas, tenebrionid beetles possess extraordinary physiological features, particularly in terms of water conservation, as a way to survive in these extreme conditions where water and food are absent (Zachariassen 1996). This phenomenon explains the water conservation differential in beetles occupying macrohabitats with moisture- and vegetation-rich regions (e.g., Cb, Dg, and Pf in plantations) (Fig. 2), where more water is available to diffuse passively into the body through the cuticle (Appel et al. 1986) to replace what is lost due to diffusion and respiration (Haverty and Nutting 1976). Physiologically, Lyctus species are desiccation sensitive. Parkin (1943) and Bootle (1983) proved that the sympatric species L. brunneus only survived in wood with 10−50% moisture content, and larvae developed optimally in wood with 15% moisture content. Wood moisture contents below 10% disrupted embryo and larval development (Beeson and Bhatia 1937, Smith 1955). Although both test beetles in the present study share a similar habitat (i.e., residing in desiccated wood), the larvae and pupae of L. africanus lost approximately 50 and 15% of TBW, respectively. However, their CP value was comparably low. The high water loss can be explained by the small body size of Lyctus, which results in a high surface area to volume ratio, resulting in more water vaporizing from the larval cuticle relative to total water mass. A series of previous studies reported a significant association between body size and desiccation resistance (Hadley 1994). For instance, adults of C. maculatus from South India are larger than those of Brazilian strains and lose water slowly (Yoder et al. 2010). The fungus-growing termite Microtermes gilvus (Hagen), which has a high surface area-to-volume ratio (40.28–69.75) showed a higher rate of water loss and %TBW loss compared to the larger Macrotermes carbonarius (Hagen), which has a lower surface area to volume ratio (29.26–53.66) (Hu et al. 2012). The ability to reduce water loss rates in association with a reduced body size has often been implicated in the evolution of behavioral adaptation (Cohen and Pinto 1977, Hood and Tschinkel 1990, Hu et al. 2012), e.g., M. gilvus foraged only below ground or concealed within foraging mud tubes as a way to reduce water loss rates, while the larger termite, M. carbonarius that can tolerate desiccation stress was found foraging above-ground in the open air (Hu et al. 2012). Although the surface area to volume ratio of adult Lyctus in the present study was threefold higher than that of H. aequalis, the water loss rate remained comparable or even lower than that of the larger adult H. aequalis. Desiccation tolerance tests of 10 individuals per group for each L. africanus instar, suggest that the main function of evolved aggregative behavior in Lyctus is to reduce water loss rates in ways similar to those known for other arthropods. For instance, the interindividual distance of German cockroaches (Blattella germanica Linnaeus) decreased to form grouping when RH decreased (Dambach and Goehlen 1999). Adult giant Madagascar hissing cockroaches (Gromphadorhina portentosa (Schaum)) in a group of six retained more water content compared to isolated individuals (Yoder and Grojean 1997). Broly et al. (2014) studied the terrestrial isopod Porcellio scaber Latreille and reported that the correlation between the water loss rate of an individual and group size was best fitted by a power function. Any addition of an individual to a small group (1–40 individuals) significantly reduces the individual water losses compared to a large group of more than 50 individuals (Broly et al. 2014). The aggregation pheromone of L. africanus may be responsible for attracting both females and males to promote courtship behavior and act as a signal to aggregate all members to a suitable resource to initiate boring activity, as shown in lesser and larger grain borer (Fadamiro and Wyatt 1996, Edde 2012). The evolved aggregative behavior in adult L. africanus could help maintain relatively low %TBW loss and water loss rates in adult stages to compensate for their small body size, which may contribute to a better fitness performance and ecological success, as has been shown in caterpillars (Klok and Chown 1999). In general, immature arthropods have been reported to lose water more rapidly than adults (Yoder et al. 1997, Bong et al. 2013). As a result of these observations, it is currently believed that significant high cuticular water loss in Lyctus beetles is compensated for by their relatively short immature developmental time (e.g., ~2 wk from egg eclosion to adult; Kartika and Yoshimura 2013), which minimizes prolonged exposure to dry conditions. In contrast, larval H. aequalis undergo an approximately 24-wk development period inside seasoned wood before turning into adults (Creffield and Howick 1979, Ho 1995, Bong et al. 2018). Under desiccated conditions, the larvae are exposed for months to variable temperatures, and, in general, CP and water loss of insects increase with increasing temperature (Ramlov and Lee 2000, Renault et al. 2005, Lyons et al. 2014). Thus, water loss in larval H. aequalis could be intense during summer months. However, there was no apparent increase water loss rate in the larval instar stage, as low water loss rate and %TBW loss were documented. The low surface area to volume ratio (large body size) of this species undoubtedly contributes to the low water loss in larval H. aequalis. In addition, the CP value of larval H. aequalis was two- to fivefold lower than that of the other development stages within the species as well as between test species in the study. The low CP value for immature H. aequalis is equivalent to that of the very drought-resistant larvae of the gall fly, Eurosta solidaginis (Cocquillet), which also have prolonged development time over extended periods during severely low humidity conditions (Ramlov and Lee 2000), and to that of tenebrionid beetle larvae (Mead-Briggs 1956). In summary, the results show the impressive abilities of H. aequalis and L. africanus to survive for months in desiccated wood due to their ability to enhance water conservation by resisting water loss and having an impermeable cuticle. The results demonstrate strategies that insects and other arthropods employ as a trade-off between body size, ontogenetic development, and insect sociality (aggregative and non-aggregative behavior) to maintain their water balance in xeric environments. The current study showed that these beetles, although predominantly distributed in tropical regions, are likely to become widespread in temperate regions due to their great ability to conserve water inside the wood. Acknowledgments We thank Akio Adachi (Research Institute for Sustainable Humanosphere, Kyoto University) for his helpful assistance in experiment preparations. L.-J.B. was a mission researcher at the Research Institute for Sustainable Humanosphere, K.-B.N. was an international research fellow at the Japan Society for the Promotion of Science. This study was partially supported by a grant from the Ministry of Science and Technology, Taiwan (MOST 105-2313-B-005-004-MY2). References Cited Ahearn , G. A . 1970 . The control of water loss in desert tenebrionid beetles . J. Exp. Biol . 53 : 573 – 595 . Google Scholar PubMed Appel , A. G. , and M. J. Tanley . 1999 . Water composition and loss by body color and form mutants of the German cockroach (Dictyoptera: Blattellidae) . 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Environmental EntomologyOxford University Press

Published: May 10, 2018

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