The need to manage insect pests has always been a key component in agricultural production systems. At the beginning of the twenty-first century, different strategies for pest management coexist and are sometime perceived as contradictory by the industry, farmers and consumers. On one hand, low-input and organic farmers base their pest management programmes on the integration of a series of cultural, biological and ecological tactics aimed at reducing pest survivorship and/or impact.
On the other hand, high-input conventional farmers rely on pesticides, mechanical control practices and transgenic crops to reduce pest impact. A transgenic plant is defined as one that has had foreign genetic material purposefully introduced and stably incorporated into the plant genome through means other than those that naturally occur in the environment, i.e. transgenic plants are those which carry additional stably integrated and expressed foreign gene(s), usually transform from unrelated organisms.
The whole process of introduction, integration, and expression of the foreign gene(s) in the host is called genetic transformation or transgenesis. Both approaches to pest management are reflected in the current scientific literature.
In recent years, several researchers have evaluated the potential of habitat manipulation and conservation of biological control to promote natural enemies of agricultural pests. The goal of these practices is to manipulate the environment to enhance the survivorship and/or performance of natural enemies.
Along-with the growing interest in ecological approaches, there has been an expansion of research into use of transgenic organisms as a component of high input conventional farming systems. The debate about ecological risks and benefits of using transgenic organisms to combat arthropod pests is very vast.
Further, the potential negative outcomes include evolution of resistant pests, impact on non-target insects, escape of transposable genes into wild relatives, cascading multi-trophic level impacts, and changes in farm management practices that could reduce the survivorship of natural enemies. But the potential benefits include high efficiency in pest population regulation, reduced environmental impact of pesticide applications, and increased crop yield.
The contributions of molecular technologies to pest management go beyond the development and release of transgenic organisms. Among the areas that could benefit are an increased ability to identify pests and natural enemies, evaluation of failure or success in the establishment of natural enemies, study of dispersal patterns, and assessment of genetic divergence among and within populations. Despite these advantages, few field ecologists have fully incorporated molecular tools into their research.
Molecular Techniques and Parasitoids:
Although parasitoids represent a valuable and diverse group of natural enemies of agricultural pests, their success in annual cropping systems has been limited. Among the factors constraining the effectiveness of parasitoids as biological control agents are their small size and their high species diversity that could hinder correct identification. Further, the harsh environment created by agricultural practices reduces parasitoid survivorship.
Parasitoids constitute an ubiquitous and diverse group of insects with enormous ecological and economic importance, particularly as biological control agents of agricultural pests. However, the small size of many parasitoids and their high species diversity has hampered their correct identification and selection for pest-management programmes.
The molecular methods can increase our ability to characterize 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.
Molecular methods can also help field entomologists better characterize 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 biological 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:
(1) Enzyme Electrophoresis:
The 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. Loxdale and 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. More recently, the use of allozymes has been supplemented with techniques generating director, 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. In addition, insects in the order Hymenoptera (most of the effective parasitoids used in agriculture) have exceptionally low allozyme variability.
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).
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 catalyze 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. 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.
The number of PCR-based techniques is expanding every year and they have been successfully 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.
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 disadvantage of using molecular methods is the cost involved.
Some of the PCR-based techniques commonly used are:
(1) Restriction fragment length polymorphism (RFLP) analysis
(2) Microsatellite analysis
(3) Single-strand conformation polymorphisms (SSCPs),
(4) Random amplified polymorphic DNA (RAPD) and
(5) Amplified fragment length polymorphism (AFLP) fingerprinting.
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 analyzed. 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.
However, RAPD markers have been criticized as a tool for molecular identification of species because their results can have poor reproducibility. 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.
Molecular Techniques Used in Biological Control:
(1) Cryptc Species Identification:
The unequivocal taxonomical identification of pests and natural enemies is important in biological control. Although evaluation of morphological features is the predominant tool to distinguish insect species, many parasitic groups are visually undistinguishable (cryptic species) despite their high genetic diversity. This genetic diversity, in turn, could be important to the success of parasitoids as biological control agents.
Beginning in 1994, two Ageniaspis (Hymenoptera : Encyrtidae) populations, one from Australia and one from Taiwan, were introduced into Florida as part of a classical biological control project against the Asian citrus leafminer Phyllocnistis citrella (Lepidoptera -Gracillariidae). Although taxonomic specialists identified the two populations as belonging to the same species, Ageniaspis citricola, there were clear biological and physiological differences between them.
By 1996, the Australian population of A. citricola had colonized most of Florida’s 344 250 ha of citrus groves and parasitism of citrus leafminer pupae was found to be as high as 99% in some sites. Alvarez and Hoy (2002) collected Ageniaspis individuals in 10 field sites at six Florida countries and took advantage of RAPDs to show no evidence that the Taiwan population had established in Florida by the end of 1999. After these results were obtained, only Australian A. citricola was sent to Caribbean and Latin American countries for biological control of the citrus leafminer, to ensure establishment.
(2) Differentiation between Exotic and Indigenous Parasitoids:
In biological control programmes where exotic strains of parasitoids are introduced into a new area, it is not always possible to distinguish between imported and native strains using morphological features. Molecular methods have been used to differentiate strains of indigenous and exotic biological control agents prior to their release.
For example, many strains of exotic parasitic Hymenoptera that are investigated in the US to control the Russian wheat aphid, Diuraphis noxia (Homoptera – Aphididae), are indistinguishable from the indigenous ones. Narang et al. (1994) took advantage of RAPD-PCR to identify and characterize four species of hymenopteran parasitoid introduced for the control of the Russian wheat aphid and develop an easy to use dichotomous key for parasitoid identification.
Zhu and Greenstone (1999), using the resulting PCR product in a gel of the amplified ribosomal ITS2 region, were able to distinguish three species and two strains of Aphelinus (Hymenoptera – Aphelinidae) endoparasitoids that had been released against the Russian wheat aphid. In South Africa, Prinsloo et al., (2002) used specific primers for both the ribosomal ITS2 and mitochondrial 16s DNA sequences of several morphologically similar parasitoid species and strains of Aphelinus, digested them with a restriction enzyme and showed in an agarose gel electrophoresis the differences between strains.
These procedures allowed a reliable separation of Aphelinus species and confirmation that the exotic aphid parasitoid A. hordei had successfully spread in wheat fields, suggesting it could be effective at controlling the Russian wheat aphid.
(3) Parasitoid Rearing:
The contamination problems are recurrent in laboratory colonies of parasitoids, especially when rearing small, morphologically similar species. In many cases, the biological control worker is not aware of the contamination but molecular methods can be used to monitor the taxonomic quality of parasitoid colonies or to assist in selecting desirable traits prior to release.
For example, Rosen and DeBach (1973) cited that cultures of Encarsia perniciosi (Hymenoptera – Aphelinidae) imported from Germany to control the San Jose scale, Quadraspidiotus perniciosus (Homoptera – Diaspididae) were contaminated with a related but ineffective species, E. fasciata. It is estimated that about 4 million E. fasciata were released, but never became established.
There is fairly extensive prior work on the use of molecular markers for identification and characterization of Trichogramma species and strains. For example, a number of workers have found specific markers for several morphologically similar Trichogramma strains and species with the use of RAPD, ITS2 sequences and RFLP analyses. These DNA markers could potentially be used in rearing laboratories for rapid species identification.
Agricultural Practices and Parasitoids:
The modern agricultural practices create a harsh environment for parasitoids. High-input conventional annual cropping systems usually depend on large amounts of off-farm chemical and mechanical inputs such as insecticide and herbicide applications, primary and secondary tillage, fertilization, cultivation and harvest. From an ecological point of view, these management practices represent disturbances that disrupt the characteristics of the ecosystem, the community or the population through a rapid change in resources, substrate availability or physical environment.
On a temporal dimension, annual crop fields can be characterized as ephemeral and predictable habitats where early successional plants (the crops) are established as short-lived monocultures. These plants have been selected to have low chemical and mechanical defences and are readily attacked by herbivores.
Spatially, the current tendency of removing non-crop habitats has severely reduced and fragmented native habitats that could provide resources for parasitoids including over wintering sites, pollen and nectar, suitable environmental microsites and alternative hosts. In this way, industrialized agriculture generates a mosaic of small and isolated late-successional patches with high species diversity embedded in a matrix of extensive early successional agricultural fields with low species diversity.
The joint impact of disturbances, temporal predictability, resource allocation and habitat fragmentation has been identified as a limiting factor conditioning the abundance and effectiveness of parasitoids in annual agro-ecosystems. These unique biotic and abiotic characteristics, coupled with the relatively low cost and high effectiveness of pesticide applications, has discouraged the use of natural enemies, including parasitoids, as a tool to manage arthropod pests in annual cropping systems.
Improving Parasitoid Survivorship through Habitat Manipulation and Molecular Biology:
The habitat manipulation, defined as a series of environment manipulations to provide natural enemies with the necessary resources to improve their effectiveness at combating agricultural pests, emerges as a unifying theme to mitigate the negative impact of agricultural practices. The underlying concept of habitat manipulation is that provision of supplementary and complementary food, microclimate modification and existence of refuge habitats in close association with crop fields might help natural enemies to cope with the detrimental impact of agricultural practices.
For example, many species of adult parasitoids used wildflowers and aphid honeydew as food resources that are not provided in agricultural fields in which weeds are controlled. Practices such as no tillage and conservation tillage, cover cropping, crop residue conservation, intercropping and establishment of herbaceous strips in close spatial association with crop fields represent viable within-crop field approaches to provide the necessary resources to enhance parasitoid survival.
The molecular tools can be integrated with an in-depth knowledge of insect biological and ecological requirements to help entomologists improve habitat manipulation practices for parasitoid conservation. The integration of these tools and knowledge could be done at different spatial scales.
Within – Crop Field Activities and Molecular Based Techniques:
Pesticide applications can be the most important factors reducing parasitoid survivorship within crop fields. In the short term, pesticides can kill large numbers of parasitoids, which may lead to an outbreak of secondary pests. In the long term, repeated pesticide applications may select resistant biotypes and influence parasitoid population dynamics.
Ruberson et al. (1998) described different approaches to integrate pesticide applications with natural enemies. Among the recommended tactics are periodic scouting of crop fields, use of selective pesticides and sublethal doses, and spatial and temporal separation of pesticides and natural enemies.
The molecular technologies can help biological control practitioners to integrate pesticide use with natural enemies through the selection of pesticide-resistant strains of natural enemies. Although this approach appears to be more successful with predators than with parasitoids, recent progress in parasitoid selection programmes could reverse this situation.
For example, RAPD-PCR has proved to be a useful technique to distinguish between two populations of the walnut aphid parasitoid, Trioxys pallidus (Hymenoptera – Aphididae), differing in resistance to pesticides. Although the experiments were performed in the laboratory, the authors suggest that RAPD-PCR could be used to determine the fate of pesticide-resistant parasitoids released in the field.
Conserving Parasitoids at the Farm and Landscape Level:
While practices at the within – field level might enhance parasitoid survivorship, agricultural practices occurring at larger scales might negate such conservation efforts. Several studies have shown that field size, crop rotation and presence of non-crop habitats play a critical role in determining within-field parasitoid abundance. For example, Landis and Haas (1992) determined that parasitism of the European cornborer, Ostrinia nubilalis (Lepidoptera- Pyralididae) by its larval parasitoid, Eriborus terebrans (Hymenoptera- Ichneumonidae) was significantly higher at the borders of maize (also known as corn) fields than in field interiors.
They further determined that the greatest levels of parasitism were observed at wooded field edges. In accordance, it has been observed that access to plant nectar, aphid honey dew or sugar improved E. terebrans survival in crop and non-crop habitats. These studies suggested that the absence of food resources in large maize fields, combined with high temperatures before canopy closure, was responsible for the low abundance of parasitoids observed in field interiors.
It has been determined that short rotation of periodical cutting of hedges and clover/grass grazing areas spread among crop fields forming a series of annual, biannual and semi-perennial habitats correlated with an increase in parasitism of the pest aphid Sitobion avenae (Homoptera – Aphididae). These and other studies highlight the importance of semi-natural habitats established in close association with crop fields for natural enemy conservation.
Ideally, agricultural landscapes should contain a series of non-crop habitats interspersed with crop fields to provide shelter and food for parasitoids. Mid- and late-successional habitats provide parasitoids with adult food resources, alternative hosts, overwintering sites and shelter for adverse conditions. It is interesting to note that the parasitoids use semi-chemicals in host location, some of which emanate from plants non crop vegetation interspersed with crop fields can also affect parasitoid behaviour.
Understanding changes in parasitoid behaviour to plant assemblage composition is a key element in the development of habitat practices aimed at increasing vegetational diversity of agricultural systems. Yet due to their small size, vagility and the inherent difficulty of mark and recapture experiments, very little is known about parasitoid dispersal behaviour.
The molecular-based techniques may aid entomologists to assess the impact of agricultural landscape structure on parasitoid population and community structure. Vaughn and Antolin (1998) used RAPD-PCR markers visualized by SSCP analysis to study the population structure of Diaretiella rapae (Hymenoptera – Braconidae), a parasitoid of several aphid species. The authors found that larger genetic variation occurred between fields separated by short distances than between areas separated by longer distances. They also argued that the reduced genetic exchange among subpopulations indicates that released D. rapae would not disperse between fields.
Althoff and Thompson (2001) used the mtDNA cytochrome oxidase 1 (CO1) gene sequence and nuclear rDNA RFLPs to compare patterns of host search behaviour among six populations of Agathis n. sp. (Braconidae – Agathidinae) located in a relatively large geographic area in south eastern Washington, USA. The results showed no isolation by distance, suggesting long distance dispersal among populations.
The authors argued that phenotypic differences in host parasitoid interactions such as time allocated to searching, ovipositor length and place of searching appear to be driven by local plant characteristics. Although this study was not developed within the context of annual cropping systems, it provides a framework that could be used to foster the understanding of how local, mid- and large- scale agricultural landscape affect parasitoid abundance, searching behaviour and distribution.
Concluding Remarks:
Despite the large potential of biological control in sustainable agriculture, the commercial development and success of this approach has been extremely limited. This is particularly true in annual cropping systems where disturbances, resource paucity and habitat simplification reduce natural enemy survivorship. More successful biological control may be possible if agricultural entomologists take advantage of newly developed concepts and techniques deriving from a wide variety of areas ranging from molecular based technologies to landscape ecology.
The molecular technologies are among the fastest growing sectors in modern agriculture with the potential of transforming farming systems. To date, much of the debate on this issue has been on the benefits and risks of releasing transgenic organisms. These newly developed technologies can also increase the adoption and success of biological control agents through greater taxonomic accuracy, higher precision in insect identification and a better understanding of the genetic and population structure of natural enemy populations.
There is a rich collection of evidence that ecological diversity plays a key role in the resilience of farm systems. More specifically, increasing diversity appears to be a cornerstone of habitat manipulation to enhance parasitoid survivorship.
Further, encouraging diversity however, might not be enough and sometimes it may even have negative consequences. Instead, it is necessary to identify the key elements of diversity needed for the particular set of parasitoid species under consideration. Undoubtedly, correct insect identification is crucial.
Parasitoids and alternative hosts must be accurately identified before any existing information on their environmental needs could be used in the selection of within field, farm and landscape level practices that will enhance their survivorship and effectiveness. Molecular techniques provide a useful tool to advance the accuracy and ease of parasitoid and host identification.