Heliothine caterpillars differ in abundance of a gut lumen aminoacylase (L-ACY-1)—Suggesting a relationship between host preference and fatty acid amino acid conjugate metabolism

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Abstract

Fatty acid amino acid conjugates (FACs) in the oral secretions of Lepidopteran larvae are responsible for eliciting plant defense responses. FACs are present despite fitness costs which suggests that they are important for larval survival. In previous work, an aminoacylase (L-ACY-1) was identified as the enzyme responsible for hydrolysis of FACs within the larvae gut. This gene is present in three related Heliothine species: Heliothis virescens, Helicoverpa zea, and Heliothis subflexa. Transcript levels in gut tissues are predictive of protein abundance and enzyme activity in the frass. H. zea has the least amount of L-ACY-1 present in gut tissue and frass, while H. virescens has intermediate protein levels and H. subflexa has the highest amount of L-ACY-1 in gut tissue as well as in frass samples. These species differ in their host range and protein intake targets, and recently, it has been shown that FACs, the substrates of L-ACY-1, are involved in nitrogen metabolism. The correlation between protein intake and degree of host range specialization suggests that this aminoacylase may allow specialized larvae to obtain nitrogen requirements despite limitations in diet heterogeneity.

Highlights

► Quantitative real time PCR was used to compare the abundance of mRNA of L-ACY-1 in three related Lepidopteran larvae. ► Transcript levels are higher in H. subflexa than H. zea and H. virescens. ► PAGE techniques suggest transcript levels reflect expression of protein. ► Banding patterns in native PAGE and Western blot suggest three isoforms differing in post translational modifications. ► Activity levels may be associated with host specificity and may play a role in obtaining target nutrient optimum intake.

Introduction

Recently a Lepidopteran aminoacylase, L-ACY-1, has been identified as a hydrolase of fatty acid-amino acid conjugates (FACs) (Kuhns et al., 2012). Presumably L-ACY-1 cleaves the FACs into free fatty acids and glutamine for reabsorption into the gut tissues (Kuhns et al., 2012, Mori et al., 2001). These FACs, present in the larva of many pest species of Lepidoptera (Alborn et al., 1997, Pohnert et al., 1999, Turlings et al., 2000, Yoshinaga et al., 2010), initiate plant defenses during herbivory (Alborn et al., 1997, Turlings et al., 2000). Therefore, FACs must have an essential, fundamental role in larval metabolism to offset the fitness costs of triggering plant defense. Understanding the metabolism of FACs will provide insight into larval metabolism which may facilitate development of eco friendly pest management practices. Investigation of the enzymes involved in FAC synthesis and hydrolysis is a prerequisite for understanding this complex metabolism.

Mori et al. (2001) showed that the amount of FACs present in the gut lumen is similar in two closely related species, Heliothis virescens and Helicoverpa zea but that the hydrolysis of FACs in the regurgitant of these species is significantly different. Furthermore, the hydrolysis was due to an enzyme within the gut of the larvae (Mori et al., 2001), which has recently been identified and the gene sequenced in several noctuid larvae (Kuhns et al., 2012). Mori’s observation raises the question, why does the hydrolysis rate differ when the species are closely related, have similar quality and quantity of FACs, and, as we later discovered, both possess the gene for the hydrolase?

Although these species are closely related, the breadths of their host ranges differ considerably. H. zea, is one of the most polyphagus Lepidopteran larvae known and is a major pest caterpillar of agricultural crops (Mitter et al., 1993). H. zea preferentially feeds on the fruits of many host plants including corn and tomato (Waldbauer and Friedman, 1991). H. virescens is also polyphagous and a major pest of agricultural crops, but its host range is narrower than H. zea (Mitter et al., 1993) preferring the leaves of Solanaceae plants. In contrast to H. zea and H. virescens, Heliothis subflexa is a specialist on Physalis, preferring to feed on the fruits, although it will also feed on other Solanaceous plants (Mitter et al., 1993). It is commonly believed that a narrower host range limits nutrient availability, although this hypothesis has not been explicitly tested.

The nutritional heterogeneity hypothesis states that specialists are less likely to encounter acceptable food items when compared to generalists; therefore they will consume smaller amounts and process the nutrients with higher efficiency (Behmer, 2009, Simpson et al., 2002). Efficiency in nutrient absorption is linked to the suite of digestive enzymes within the gut (Kolkovski et al., 1993, Kolkovski et al., 1997), thus it is expected that specialists should possess either specialized or more efficient digestive enzymes. Recently, FACs have been shown to be involved in nitrogen metabolism in Lepidopteran larvae (Yoshinaga et al., 2008). Therefore, a significantly different rate of hydrolysis of FACs may allow for more efficient absorption of nitrogen. Since FACs, and therefore L-ACY-1, have been implicated in nitrogen metabolism, we hypothesize that L-ACY-1 may be differentially required in related species with differing nutrition requirements. As a first step in understanding these relationships, we extended Mori’s (2001) study by comparing L-ACY-1 transcript levels, protein abundance, and L-ACY-1 activity, in H. virescens, H. subflexa, and H. zea.

Section snippets

Insect rearing

H. virescens and H. zea eggs were purchased from Benzon Company (Carlisle, PA). H. subflexa individuals were a gift from Dr. T.C. Baker, PSU. Eggs were maintained at room temperature until hatching. Neonates were transferred into two ounce cups containing about 1 ml of artificial corn earworm diet from Southland Products (Lake Village, AR). The larvae were reared with a 14:10 [L:D] photoperiod at 25:23 °C [L:D] temperatures.

Sample preparation

Fifth instar larvae were anesthetized on ice for 30 min. Each larva was

In-gel activity assay

The in-gel activity assay is an excellent tool to visualize enzyme activity. Protein mixtures are separated on native polyacrylamide gels and then stained for activity of the enzyme of interest. The native PAGE gels used in this experiment to separate frass proteins were stained for hydrolysis of free glutamine from N-linolenoyl-l-glutamine (Fig. 2A) based on a modified method from Gade and Brown (1981). Quantification of pixel density and subsequent statistical analysis using a t-test

Discussion

Here we have shown that an aminoacylase in the gut of Lepidopteran larvae, L-ACY-1, is present at different levels among three related Heliothine moths. The general trend between activity assays, protein abundance, and mRNA levels for L-ACY-1 suggests that these Lepidopteran larvae utilize L-ACY-1 to different extents.

Furthermore, analysis by PAGE and in-gel assays revealed potential quaternary structural characteristics of L-ACY-1. The presence of doublets and triplets in the western blots and

Acknowledgements

We thank Dr. Barb McGrath for assisting in this project, both in the use of equipment and technical expertise. Dr. T.C. Baker and Bryan Banks provided insects that were instrumental for completion of this work. EHK thanks her husband for his support through her graduate work. Pennsylvania State University College of Agriculture Competitive Grant provided partial funding for this research. This study was funded in part by a grant from USDA, CSREES, NRI.

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1

Present address: Citrus Research and Education Center, University of Florida, 700 Experiment Station Rd Lake Alfred, FL 33850, USA.

2

Present address: 122 Chemical Ecology Laboratory, University Park, PA 16802, USA.

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