Biomacromolecules 2008, 9, 3065–3069
3065
An Unlikely Silk: The Composite Material of Green Lacewing Cocoons Sarah Weisman,† Holly E. Trueman,† Stephen T. Mudie,‡ Jeffrey S. Church,§ Tara D. Sutherland,*,† and Victoria S. Haritos† CSIRO Entomology, Clunies Ross Street, Acton, ACT 2601, Australia, CSIRO Materials Science and Engineering, Normanby Road, Clayton, VIC 3168, Australia, and CSIRO Materials Science and Engineering, Henry Street, Belmont, VIC 3216, Australia Received May 26, 2008; Revised Manuscript Received July 31, 2008
Spiders routinely produce multiple types of silk; however, common wisdom has held that insect species produce one type of silk each. This work reports that the green lacewing (Mallada signata, Neuroptera) produces two distinct classes of silk. We identified and sequenced the gene that encodes the major protein component of the larval lacewing cocoon silk and demonstrated that it is unrelated to the adult lacewing egg-stalk silk. The cocoon silk protein is 49 kDa in size and is alanine rich (>40%), and it contains an R-helical secondary structure. The final instar lacewing larvae spin protein fibers of ∼2 µm diameter to construct a loosely woven cocoon. In a second stage of cocoon construction, the insects lay down an inner wall of lipids that uses the fibers as a scaffold. We propose that the silk protein fibers provide the mechanical strength of the composite lacewing cocoon whereas the lipid layer provides a barrier to water loss during pupation.
Introduction The egg-stalk silk of lacewings (Neuroptera: Chrysopidae) was one of the earliest silks to be characterized. The amino acid composition,1 secondary structure,2 and mechanical properties3 of the silk have been reported. X-ray fiber diffraction has demonstrated that egg-stalk silk has a well-defined cross-β-sheet structure.2,4 This is unusual because parallel β sheets are the typical conformation of described silks,5 and native cross-β structure is rare in any protein set. The primary sequence of the protein or proteins that compose the lacewing egg-stalk silk has not yet been determined. Lacewing egg-stalk silk is produced by the colleterial gland (part of the female insect reproductive system) of adult females during the egg-laying process. However, lacewings also use silk in another life stage: final instar larvae spin a silk cocoon in which they pupate. The cocoon silk proteins are produced by modified malphigian tubules (part of the insect excretory system) in larvae of both sexes.6 The marked differences in silk production sites and functions between adults and larvae beg the question of whether the cocoon silk and egg-stalk silk are the same material. Generally, it is considered that the production of multiple silks by the same individual occurs only in spiders. Spiders can produce up to seven different silks for different functions such as web construction, egg sacs, wrapping of their prey, or life lines.7 We were interested in discovering whether lacewings produce a single class of silk proteins that function as both cocoon and egg-stalk silk or whether two separate protein classes have been developed for the different roles. It was reported that lacewing cocoons are constructed from two layers: an outer fibrous silk and a solid inner wall.8 In agreement, LaMunyon9 found that the cocoons were composed of two different substances. Initially, an eosin-staining (proteinaceous) component was secreted by the malphigian tubules; * Corresponding author. E-mail:
[email protected]. † CSIRO Entomology. ‡ CSIRO Materials Science and Engineering, Clayton. § CSIRO Materials Science and Engineering, Belmont.
subsequently, a material that was not stained by easin was secreted by the hindgut epithelial cells.9 The protein component has not been characterized in any way, and the nature of the nonproteinaceous substance is unknown. We determined to identify and sequence the gene/s that encode the lacewing cocoon silk and to characterize the molecular structure and microstructure of the silk. This is the first example of a silk gene sequence from the insect order Neuroptera.
Methods Material Collection. Eggs from Mallada signata, a lacewing species that is endemic to Australia, were obtained from Bugsforbugs, QLD, Australia (www.bugsforbugs.com.au). The insects were raised in plastic containers that were filled with lucerne chaff and kept at 20-22 °C. The larvae were fed every 2 to 3 days on frozen moth eggs. Silk glands (modified malphigian tubules) were dissected from third instar larvae under phosphate-buffered saline, were placed directly into RNAlater (Gibco), and were stored at -20 °C. Other larvae were left to pupate, and the cocoon silk was collected. cDNA Library Construction. Total RNA (26 µg) was isolated from the malphigian tubules of approximately 50 larval lacewings by the use of the RNAqueous-4PCR kit (Ambion). Messenger RNA was isolated by the use of the Micro-FastTrack 2.0 mRNA isolation kit (Invitrogen). The cDNA library was constructed from the mRNA by the use of the CloneMiner cDNA kit (Invitrogen) with modifications of the standard protocol, as previously described.10 The cDNA library comprised approximately 6 × 106 colony forming units (cfu) with
