Box 31 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 nucleo-tide 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.

*After Altstein 2003; Hoy 2003.

are the ecdysteroids, the juvenile hormones, and the neurohormones (also called neuropeptides).

Ecdysteroid is a general term applied to any steroid with molt-promoting activity. All ecdysteroids are derived from sterols, such as cholesterol, which insects cannot synthesize de novo and must obtain from their diet. Ecdysteroids occur in all insects and form a large group of compounds, of which ecdysone and 20-hydroxyecdysone are the most common members. Ecdysone (also called a-ecdysone) is released from the prothoracic glands into the hemolymph and usually is converted to the more active hormone 20-hydroxyecdysone in several peripheral tissues. The 20-hydroxyecdysone (often referred to as ecdysterone or P-ecdysone in older literature) is the most widespread and physiologically important ecdysteroid in insects. The action of ecdysteroids in eliciting molting has been studied extensively and has the same function in different insects. Ecdysteroids also are produced by the ovary of the adult female insect and may be involved in ovarian maturation (e.g. yolk deposition) or be packaged in the eggs to be metabolized during the formation of embryonic cuticle.

Juvenile hormones form a family of related sesquiter-penoid compounds, so that the symbol JH may denote one or a mixture of hormones, including JH-I, JH-II, JH-III, and JH-0. The occurrence of mixed-JH-producing insects (such as the tobacco hornworm, Manduca sexta)

Fig. 3.8 The main endocrine centers in a generalized insect. (After Novak 1975.)

adds to the complexity of unraveling the functions of the homologous JHs. These hormones have two major roles - the control of metamorphosis and regulation of reproductive development. Larval characteristics are maintained and metamorphosis is inhibited by JH; adult development requires a molt in the absence of JH (see section 6.3 for details). Thus JH controls the degree and direction of differentiation at each molt. In the adult female insect, JH stimulates the deposition of yolk in the eggs and affects accessory gland activity and pheromone production (section 5.11).

Neurohormones constitute the third and largest class of insect hormones. They are generally peptides (small proteins) and hence have the alternative name neuropeptides. These protein messengers are the master regulators of many aspects of insect development, homeostasis, metabolism, and reproduction, including the secretion of the JHs and ecdy steroids. Nearly 150 neuropeptides have been recognized, and some (perhaps many) exist in multiple forms encoded by the same gene following gene duplication events. From this diversity, Table 3.1 summarizes a representative range of physiological processes reportedly affected by neurohormones in various insects. The diversity and vital co-ordinating roles of these small molecules continue to be revealed thanks to technological developments in peptide molecular chemistry (Box 3.1) allowing characterization and functional interpretation. Structural diversity among peptides of equivalent or related biological activity is a consequence of synthesis from large precursors that are cleaved and modified to form the active peptides. Neuropeptides either reach terminal effector sites directly along nerve axons or via the hemolymph, or indirectly exert control via their action on other endocrine glands (corpora allata and prothoracic glands). Both inhibitory and stimulatory signals are involved in neurohormone regulation. The effectiveness of regulatory neuropeptides depends on stereospecific high-affinity binding sites located in the plasma membrane of the target cells.

Hormones reach their target tissues by transport (even over short distances) by the body fluid or hemo-lymph. Hormones are often water-soluble but some may be transported bound to proteins in the hemo-lymph; for example, ecdysteroid-binding proteins and JH-binding proteins are known in a number of insects. These hemolymph-binding proteins may contribute to the regulation of hormone levels by facilitating uptake by target tissues, reducing non-specific binding, or protecting from degradation or excretion.

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