Comparative Biochemistry and Physiology Part A 135 (2003) 625–634 Comparative water relations of adult and juvenile tortoise beetles: differences among sympatric species Helen M. Hull-Sanders*, Arthur G. Appel, Micky D. Eubanks Department of Entomology and Plant Pathology, Auburn University, 301 Funchess Hall, Auburn, AL 36849-5413, USA Received 5 February 2003; received in revised form 20 May 2003; accepted 20 May 2003 Abstract Relative abundance of two sympatric tortoise beetles varies between drought and ‘wet’ years. Differing abilities to conserve water may influence beetle survival in changing environments. Cuticular permeability (CP), percentage of total body water (%TBW), rate of water loss and percentage of body lipid content were determined for five juvenile stages and female and male adults of two sympatric species of chrysomelid beetles, the golden tortoise beetle, Charidotella bicolor (F. ) and the mottled tortoise beetle, Deloyala guttata (Olivier). There were significant differences in %TBW and lipid content among juvenile stages. Second instars had the greatest difference in CP (37.98 and 11.13 mg cmy2 hy1 mmHgy1 for golden and mottled tortoise beetles, respectively). Mottled tortoise beetles had lower CP and greater %TBW compared with golden tortoise beetles, suggesting that they can conserve a greater amount of water and may tolerate drier environmental conditions. This study suggests that juvenile response to environmental water stress may differentially affect the survival of early instars and thus affect the relative abundance of adult beetles in the field. This is supported by the low relative abundance of golden tortoise beetle larvae in a drought year and the higher abundance in two ‘wet’ years. ᮊ 2003 Elsevier Science Inc. All rights reserved. Keywords: Charidotella bicolor; Deloyala guttata; Cuticular permeability; Water relations; Desiccation; Body water composition 1. Introduction the cuticle itself, and active regulation by epider- mal cells influence the rate at which water is lost (Edney, 1977; Hadley, 1994). Many mechanisms Water relations are critical to insects and other influence water loss prevention, attesting to its arthropods because of their large surface area to importance to insect survival. body volume ratios. The cuticle is generally the ( Water loss or permeability of the cuticle can most important water efflux pathway Edney, ( ) also vary with environmental conditions temper- 1977 and is coated with a thin layer of epicuti- ) cular lipids that create a water-resistant barrier that ature and humidity , age and developmental stage. reduces cutaneous water loss (Neville, 1975; Typically, arthropods living in wet or humid habi- Gibbs, 1998). In addition, the chemical composi- tats often have greater cuticular permeabilities ( ) tion of these epicuticular lipids, the structure of CP than arthropods living in more xeric or dry habitats (Edney, 1977; Hadley, 1994). One reason for this conservation differential is that in humid *Corresponding author. Tel.: q1-334-844-2671; fax: q1- 334-844-5006. habitats, more water is available to diffuse pas- E-mail address: [email protected] (H.M. Hull-Sanders). sively into the body through the cuticle (Appel et 1095-6433/03/$ - see front matter ᮊ 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1095-6433(03)00142-9 626 H.M. Hull-Sanders et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 625–634 al., 1986) to replace what is lost due to diffusion increased cuticular water loss may prevent golden and respiration (Haverty and Nutting, 1976). tortoise beetles from developing or surviving dur- However, the environment in which the animals ing abnormally dry years and may partially account are found does not dictate CP values (sensu Cohen for differences in relative abundance. and Pinot, 1977). Many adult tortoise beetles in Specifically, this study determined CP, total the family Chrysomelidae, subfamily Cassidinae, body water composition (%TBW), rates of body have bright elytral colors that are lost following mass and percentage of total body water content desiccation related changes in the thickness of the loss in golden and mottled tortoise beetles. Each cuticle (Jolivet, 1994). Some adult tortoise beetles measure was compared among juvenile life stages are able to actively change cuticular colors and between male and female adults to determine (Mason, 1929; Neville, 1977) in response to host how water relations may change throughout the plant chemistry (Kirk, 1971), disturbance (Vascon- lifecycle of these beetles. In addition, whole body cellos-Neto, 1988) and sexual stimulation (Bar- lipid content (%lipid) was compared between spe- rows, 1979). These color changes are not related cies and among stages. Whole body lipid content to ambient relative humidity or photophase; there- affects cutaneous water loss and is inversely relat- fore, it is possible that the CP values in adult ed to CP (Loveridge, 1973; Tucker, 1977; Appel beetles may be related to regulation of their metal- et al., 1986). lic coloration and as well as adaptation to their environment. 2. Materials and methods In this study, we examined the water relations of two sympatric tortoise beetles, the golden tor- 2.1. Insects toise beetle, Charidotella bicolor (F. ) and the mottled tortoise beetle, Deloyala guttata (Olivier), Tortoise beetle adults and larvae were collected (Chrysomelidae: Cassidinae), across life stages. from Lee and Macon counties in Alabama, USA Both golden and mottled tortoise beetles can be during the summers of 2000 and 2001 to establish significant pests of sweet potatoes and tortoise continually breeding colonies. Colonies were beetles may be used as biological control agents maintained at 25.0"2 8C, 80"10% RH, and a for pest plant species in the family Convolvulaceae photoperiod of 16:8 (L:D) h, fed 3–10 leaves of (Rosenthal and Carter, 1977; Baloch, 1977).As morning glories, Ipomoea spp., 3 d per wk, and adults, golden and mottled tortoise beetles inhabit provided water on a cotton ball ad libitum. Beetles very similar habitats and feed on the same species were separated by species and kept in 0.95-l plastic of plants (Rausher et al., 1993; Capinera, 2001). containers with lids that had been modified by Larvae feed in a similar manner as adults (Capi- cutting out the central portion and hot gluing Tight nera, 2001), but the larvae of these two species Weave No-See-Um mosquito netting over the are not found on the same host plant at the same opening. Ten to 15 adults were reared together. time (pers. obs.). During an unusually dry year All animals used in this study were reared in the (2000) in Lee and Macon Counties, Alabama, colony from eggs. First instar larvae were collected USA average rainfall for the months May through from the colony within 2 h after handing and August was 4.06 cm of rain per month (AWIS, before their first feeding or production of their 2002). Mottled tortoise beetle larvae abundance first fecal mass. Larvae of tortoise beetles retain ranged from 15 to 20 individuals per square meter their feces on small anal projections called uro- and were collected from the underside of leaves at gomphi. These fecal masses are used as defense field sites in Lee and Macon Counties, Alabama, shields against predators. Second instars, third USA. However, during the next 2 years rainfall instars, pre-pupae and pupae were selected from averages for the same time period were 8.41 cm the colony and fecal masses were removed with per month in 2001 and 7.60 cm per month in fine forceps from the urogomphi thereby ensuring 2002. Golden tortoise beetle larvae abundance all water loss recorded was from body tissue. All ranged from 18 to 30 individuals per m2 at the specimens of each life stage were within 2 d of same field sites and mottled tortoise beetle larvae age of each other. Adult females and males were were absent or patchily distributed (3–7 per m2) identified and removed from the colony during (Hull-Sanders, unpublished data). If mottled and copulation. Sexes can only be accurately identified golden tortoise beetle larvae have different CPs, during copulation. Females and males were placed H.M. Hull-Sanders et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 625–634 627 in separate containers for at least 24 h prior to pods found in mesic environments usually have experiments. permeabilities between 31 and 60 mg cmy2 hy1 mmHgy1 and those found in hygric 2.2. Water relations experiments (water saturated) habitats have permeabilities )60 mg cmy2 hy1 mmHgy1 (Edney, 1977). One measure of water loss through the cuticle, Percentage of TBW was calculated as the dif- or CP, can be quantified under a constant set of ference between initial mass and the mass after conditions as the mass of water lost per unit body drying, dividing by the initial mass and then surface area (cm2) per unit time (e.g. h) per unit multiplying by 100. Percentage of lipid content saturation deficit (Torr or mmHg)(Edney, 1977), was calculated as the difference between dry mass but see Noble-Nesbitt (1991) for additional units. and the mass after lipid extraction divided by the A total of 20 adults (ten of each gender) and 50 initial mass and then multiplied by 100. Mean juveniles (ten of each stage of each species) were hourly mass loss and %TBW were used to calcu- selected randomly. Water content, CP late rates of mass and %TBW loss. (mg cmy2 hy1 mmHgy1), rate of percentage of total body water loss (%TBW), and percentage of 2.3. Data analysis body lipid content of freshly killed tortoise beetles were determined gravimetrically with a digital Experiments were conducted in a randomized balance (0.01 mg sensitivity)(Appel et al., 1983; complete block design to evaluate differences in Mack and Appel, 1986). Beetles were killed by a initial masses, %TBW, percentage of lipid content, 15 min exposure to HCN gas.