CONCEPTUALIZACION DE COMPETENCIAS

Parasitoids constitute a ubiquitous and diverse group of insects with enormous ecological and economic importance, particularly as biological control agents of agricultural pests (Greathead 1986; Quicke 1997). However, the small size of many parasitoids and their high species diversity have hampered their correct identification and selection for pest-management programs. Molecular methods can increase our ability to characterise parasitoid biology, genetic diversity and ecological requirements. This in turn allows an unambiguous identification of parasitoid species and an improved selection of the best genotype to be released as a biological control agent (Ives and Hochberg 2000; Liu et al. 2000). Molecular methods can also help field entomol- ogists better characterise the parasitoid community already present in agricultural systems, understand host associations and assess the evolution of parasitoid behavioural traits that affect host selection. These factors could play important roles in the success of parasitoids as biologi- cal control agents. The following is a brief introduction to some of the molecular tools used to solve questions related to parasitoid ecology, biology and population genetics. For a detailed description of molecular methods we recommend Symondson and Hemingway (1997), Loxdale and Lushai (1998) and Parker et al. (1998).

Enzyme electrophoresis

Allozymes are variant proteins produced by allelic forms of the same locus that can be separated by electrophoresis. The analysis of variation in allozymes (a reflection of changes in their gene codes) was the earliest molecular method successfully used to determine genetic variation among insect species and for taxa identification (Berlocher 1979; Gonzalez et al. 1979; May 1992). Loxdale and den Hollander (1989) and Menken and Ulenberg (1987) provide a detailed summary of cases in which allozyme electrophoresis has been used in agricultural entomology. In the specific case of parasitoids, this technique allowed successful discrimination between

several species of Trichogramma (Hymenoptera: Trichogrammatidae), a genus of minute parasitoids of great importance in biological control (Symondson and Hemingway 1997). More recently, the use of allozymes has been supplemented with techniques generating direct or indirect estimates of nucleic acid variation.

Although enzyme electrophoresis is inexpensive and relatively easy to conduct, it investigates only some of the variation in the most conserved class of DNA (the slowly evolving coding DNA), underestimating the amount of genetic variation in the non-coding DNA. This non- coding DNA, also called ‘junk’, ‘parasitic’ or ‘selfish’ DNA, may constitute 30–90% of the insect genome (Hoy 1994). In addition, insects in the order Hymenoptera (most of the effective parasitoids used in agriculture) have exceptionally low allozyme variability (Graur 1985).

DNA molecular markers and nucleotide polymorphisms

The recent advent of molecular methods to directly investigate the DNA molecule has increased accuracy and resolution in genome analysis. DNA molecules are constructed of monomeric units called nucleotides consisting of a purine or pyrimidine base, a pentose and a phosphoric acid group. Molecular methods allow direct evaluation of genomes, determining the proportion of nucleotide sites differing between two or more DNA sequences (nucleotide polymorphisms). Genomes can also be compared indirectly by assessing the bands produced in an electrophoresis gel by DNA fragments obtained from known individuals (DNA molecular markers) (Alvarez 2000).

The number of molecular techniques available for entomological studies has greatly expanded since the advent of the polymerase chain reaction (PCR). Kary Mullins conceived the PCR method in 1983, receiving the Nobel Prize in chemistry for the invention. PCR is an in vitro method for amplifying a target region of DNA by means of enzymes that catalyse the formation of DNA from deoxyribonucleoside triphosphates, using single-stranded DNA as a template. The presence of conserved regions in DNA sequences such as the mitochondrial DNA and nuclear ribosomal DNA makes it possible to amplify fragments from organisms for which there is no specific sequence information available (Kocher et al. 1989). Although the part of the genome to be used will depend on the level of resolution needed by the researcher, several mitochondrial (COI, COII, 16S) and nuclear (ITS1, ITS2, 28S, D2, D3 and EF-1) regions have proved useful for discriminating parasitoid species (Alvarez 2000).

The number of PCR-based techniques is expanding every year and they have been success- fully used in ecological and entomological studies. Among the different areas that have benefited from PCR-based techniques are systematics, population genetics and insecticide resistance assessment (Loxdale and Lushai 1998; Parker et al. 1998). PCR-based techniques offer several advantages, including the possibility of working with extremely small insects, such as many of the effective parasitoids used in biological control. They are also unaffected by the life stage of insects and can potentially be used with stored, dry or old material. Currently, the main disad- vantage of using molecular methods is the cost involved.

Some of the PCR-based techniques commonly used are restriction fragment length polymorphism (RFLP) analysis, microsatellite analysis, single-strand conformation polymor- phisms (SSCPs), random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) fingerprinting (Hedrick 1992; Hughes and Queller 1993; Mueller et al. 1996; Symondson and Hemingway 1997). Table 6.1 presents a comparison of different molecu- lar methods in terms of their sensitivity, cost, efficiency, level of discrimination and application. The selection of any given molecular technique depends on the type of problem to be solved, the cost of the technique, its ease of use and the sample size to be analysed. The section of the genome to be investigated depends on the level of variation that the researcher needs to resolve. Among the different PCR-based techniques, RAPD-PCR has been very popular in entomological

studies perhaps because the technique does not require prior knowledge of DNA sequence, provides a rapid way of identifying genetic markers, is inexpensive and is easy to develop (Williams et al. 1990; Alvarez 2000). However, RAPD markers have been criticised as a tool for molecular identification of species because their results can have poor reproducibility (Ellsworth et al. 1993). Reineke et al. (1999) recommended amplifying the DNA in a second reaction in order to assess the reproducibility of the banding patterns produced by the RAPD primers used in a test.