Box 3.1. Molecular genetic techniques and their application to neuropeptide research

Molecular biology is essentially a set of techniques for the isolation, analysis, and manipulation of DNA and its RNA and protein products. Molecular genetics is concerned primarily with the nucleic acids, whereas research on the proteins and their constituent amino acids involves chemistry. Thus, genetics and chemistry are integral to molecular biology. Molecular biological tools provide:

• a means of cutting DNA at specific sites using restriction enzymes and of rejoining naked ends of cut fragments with ligase enzymes;
  
• techniques, such as the polymerase chain reaction (PCR), that produce numerous identical copies by repeated cycles of amplification of a segment of DNA;
  
• methods for rapid sequencing of nucleotides of DNA or RNA, and amino acids of proteins;
  
• the ability to synthesize short sequences of DNA or proteins;
  
• DNA—DNA affinity hybridization to compare the match of the synthesized DNA with the original sequence;
  
• the ability to search a genome for a specific nucleotide sequence using oligonucleotide probes, which are defined nucleic acid segments that are complementary to the sequence being sought;
• site-directed mutation of specific DNA segments in vitro;
• genetic engineering — the isolation and transfer of intact genes into other organisms, with subsequent stable transmission and gene expression;
• cytochemical techniques to identify how, when, and where genes are actually transcribed;
  
• immunochemical and histochemical techniques to identify how, when, and where a specific gene product functions.

Insect peptide hormones have been difficult to study because of the minute quantities produced by individual insects and their structural complexity and occasional instability. Currently, neuropeptides are the subject of an explosion of studies because of the realization that these proteins play crucial roles in most aspects of insect physiology (see Table 3.1), and the availability of appropriate technologies in chemistry (e.g. gas-phase sequencing of amino acids in proteins) and genetics. Knowledge of neuropeptide amino acid sequences provides a means of using the powerful capabilities of molecular genetics. Nucleotide sequences deduced from primary protein structures allow construction of oligonucleotide probes for searching out peptide genes in other parts of the genome or, more importantly, in other organisms, especially pests. Methods such as PCR and its variants facilitate the production of probes from partial amino acid sequences and trace amounts of DNA. Genetic amplification methods, such as PCR, allow the production of large quantities of DNA and thus allow easier sequencing of genes. Of course, these uses of molecular genetic methods depend on the initial chemical characterization of the neuropeptides. Furthermore, appropriate bioassays are essential for assessing the authenticity of any product of molecular biology. The possible application of neuropeptide research to control of insect pests is discussed in section 16.4.3.

Table 3.1. Examples of some important insect physiological processes mediated by neuropeptides.

(After Keeley & Hayes 1987; Holman et al. 1990; Gäde et al. 1997; Altstein 2003.)

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Tracheal hypertrophy in mealworms at low oxygen concentrations

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