Physiology of crapemyrtle bark scale, Acanthococcus lagerstroemiae (Kuwana), associated with seasonally altered cold tolerance
Graphical abstract
Introduction
Temperature is one of the most important abiotic factors influencing adaptation and diversification of animals, especially for poikilotherms like insects (Angilletta et al., 2003, Bale, 2002). To adapt to seasonal changes in temperature, diverse and complex physiological mechanisms have evolved to help insects prevent potential cold injury (Denlinger and Lee, 2010). Identification and quantification of biochemical composition of insects can help us better understand the mechanisms associated with cold tolerance such as seasonal changes in insects body water content, contents of cryoprotectants, such as polyols and sugars, and changes in lipids composition in order to maintain fluidity (Clark and Worland, 2008, Rozsypal et al., 2014, Slachta et al., 2002).
Water plays an important role in insect cold tolerance, especially in maintaining osmotic stasis. For example, loss of water increases the concentrations of cryoprotectants that maintain the insect in an unfrozen (or supercooled) state (MacMillan et al., 2015). Polyols or polyhydric alcohols, and sugars, usually function as cryoprotectants in insects (Salt, 1961), which mitigate ice crystal formation, help stabilize membrane and protein structures, enhance supercooling capacity, and maintain osmotic balance and cell volume (Bale, 2002, Teets and Denlinger, 2013). The type and amounts of cryoprotectants vary among insect species. Although glycerol is the most common type of cryoprotectant in insects (Bale, 2002, Storey, 1997), other cryoprotectants have been reported from different taxa, including myo-inositol (Koštál et al., 2007, Vesala et al., 2012, Watanabe and Tanaka, 1999), mannitol (Hendrix and Salvucci, 1998, Saeidi et al., 2012, Wang et al., 2006), and trehalose (Izumi et al., 2005, Khani et al., 2007, Lee et al., 2002).
Lipid composition changes such as in triacylglycerols (TAGs) and phospholipids (PLs) can help insects overwinter, as well as altering their fatty acid compositions (Clark and Worland, 2008, Rozsypal et al., 2014). TAGs function as energy reserves for many insects and are usually accumulated before winter and directly affect the insects’ overwintering capacity (Buckner et al., 2004, Ohtsu et al., 1995, Ohtsu et al., 1992). Phospholipids are basic structures of cell membranes (Lodish and Zipursky, 2000), and some insects have evolved to alter the lipid fatty acid composition for maintaining accessibility to stored lipids (Rozsypal et al., 2014), and preserve membrane fluidity when facing changing temperatures (Sinensky, 1974). Most changes in stored and membrane lipids, reported by previous studies, showed increased unsaturated fatty acids during winter (Rozsypal et al., 2014, van Dooremalen et al., 2011a, Vukašinović et al., 2015). Reduction of carbon length of fatty acids is a key strategy to reduce melting temperature and maintain lipid liquidity (Michaud and Denlinger, 2006). However, none of these cryoprotectants has been explored in scale insects (Hemiptera: Coccoidea).
The crapemyrtle bark scale, Acanthococcus lagerstroemiae (Kuwana) (Hemiptera: Eriococcidae), a newly introduced pest of crapemyrtles, Lagerstroemia spp. (Myrtales: Lythraceae) (Wang et al., 2016b), can be a good model system to understand the cold physiology of scale insects. This scale pest attacks one of the most popular summer-flowering ornamental shrubs and trees in the southeastern US (Chappell et al., 2012, NASS, 2014). Acanthococcus lagerstroemiae has sexual dimorphism: the male is winged and motile while the female is wingless and sessile on the bark. Nymphs are the main overwintering stages observed in the field, of which sex is difficult to differentiate. Crapemyrtles heavily infested with A. lagerstroemiae exhibit stress resulting in plant growth reduction (Luo et al., 2000, Ma, 2011) and fewer blossoms (Merchant, 2014). Similar to other scale pests (Gullan and Kosztarab, 1997), A. lagerstroemiae secretes honeydew which causes accumulation of unsightly sooty mold on trunks and branches, reducing the aesthetic value of the plant (Gu et al., 2014, Wang et al., 2016b).
Our previous experiments revealed that cold tolerance of A. lagerstroemiae nymphs changes over seasons (Wang et al., 2016a). The supercooling points of A. lagerstroemiae, defined as the temperature when the insect body spontaneously freezes (Sinclair et al., 2015), decreased from −21.2 °C in July 2015 to −26.6 °C in January 2016. Reduction of supercooling points suggests potential biochemical changes to enhance cold tolerance (Clark and Worland, 2008). In addition, mortality of nymphs collected in different seasons responded in a similar pattern when exposed to various low temperatures. When comparing mortality at 0 °C between nymphs collected in summer and winter, the exposure period required to cause 90% mortality increased from 9 h for the population collected in summer to 50 h for those collected in winter, with similar trends observed at other low temperatures. Because the temperatures leading to nymphal mortality were higher than the supercooling points in A. lagerstroemiae, chill-susceptible is likely a cold tolerance strategy of A. lagerstroemiae (Sinclair et al., 2015). To overcome low winter temperatures, physiological changes in A. lagerstroemiae nymphs could play important roles.
The goal of this study was to describe the biochemical and physiological changes in A. lagerstroemiae associated with seasonally altered cold tolerance. Specifically, we studied seasonal changes in water content, fatty acid composition in both TAGs and PLs, and four potential cryoprotectants (glycerol, d-mannitol, myo-inositol and d-trehalose) in A. lagerstroemiae nymphs collected in different seasons. Results obtained from this study provide a better understanding of the overwinter strategies of A. lagerstroemiae and the cold physiology of scale insects in general.
Section snippets
Insect source
Branches with length ranging from 4 to 8 cm were collected from crapemyrtle trees infested with A. lagerstroemiae nymphs in Shreveport, LA (32.55°N, 93.78°W) every other month from November 2015 to October 2016. Upon collection, branches were placed into a plastic bag, kept in an incubator (Series 101, Percival Scientific®, Perry, IA) at 25 °C, and examined under a microscope within 24 h. Nymphs were gently flipped over without injuring any appendages using a needle and gathered onto a 10 cm
Water content
The water content of A. lagerstroemiae nymphs was lower in samples collected in winter and early spring compared with those collected in summer and early fall (40.8% vs. 63.3%) (F(5, 30) = 14.0, P < 0.0001; Fig. 1).
Fatty acid composition
A total of twenty fatty acids with 6–30 carbon atoms were found present in A. lagerstroemiae nymphs (Fig. 2, Table 1, Table 2, Supplementary Material Fig. S2–S4). Most types of fatty acids found between PLs and TAGs were similar except that shorter chain fatty acids (C6:0 and C8:0)
Discussion
Insect adaptations to low temperatures are critical for their winter survival and population growth (Chown and Nicolson, 2004). Cold tolerance of insects is often studied to better understand their overwintering strategies, predict their potential distribution, and help design appropriate management solutions (Lapointe et al., 2007, Sunday et al., 2012). Seasonally altered cold tolerance is one common overwintering strategy for insects in temperate regions and is associated with complex
Acknowledgements
We thank Connie David for instructing the use of GC-MS, Madeline Rewards for assisting TLC experiments in the laboratory, and Madeline Gill for providing language help. We thank Dr. Spencer Behmer and two anonymous reviewers for providing important suggestions and comments. This work was supported in part by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2014-70006-22632; by the LSU Department of Entomology; and by the LSU Department of
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Present address: Department of Entomology, Michigan State University, East Lansing, MI 48824, USA.