Use in Biological Control
S. Grenier 1 and P. De Clercq 2
1UMR INRA/INSA de Lyon, Biologie Fonctionnelle, Insectes et Interactions, Institut National des Sciences Appliquées, Bât. Pasteur, 20 av. A. Einstein, 69621 Villeurbanne Cedex, France; 2Laboratory of Agrozoology, Department of Crop Protection, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653,
B-9000 Ghent, Belgium
Introduction
The main concerns for artificial mass culture of entomophagous insects, after production of effective natural enemies at acceptable cost, are the maintenance of insect quality
and reliable methods for measuring quality.
Relevant and robust quality control pro- grammes are necessary for mass-produced natural enemies, particularly if they are reared on factitious or artificial diets (Sorati et al., 1996). Cost-effective production of
© CAB International 2003. Quality Control and Production of Biological Control Agents:
Theory and Testing Procedures (ed. J.C. van Lenteren) 115
Abstract
Quality assessment of entomophagous arthropods used in augmentative biological control is one of the main concerns after their mass production. The quality-testing procedures for natural enemies reared on artificial diets largely remain to be defined. As a first approach, comparisons of some relevant parameters between in vivo- and in vitro-reared entomophages are presented in this chapter. Results from experi- ments with different kinds of diets with or without insect components, developed for parasitoids and predators, are examined. Morphological traits as well as development and reproduction parameters used for comparisons between in vivo- and in vitro-grown arthropods are discussed. Morphological characters include body size and weight and the occurrence of abnormalities. Immature development is assessed by measuring duration and survival rates of the different stages. Sex ratio and symbiont association, fecun- dity–fertility and longevity are compared as reproduction parameters. It is important to consider bio- chemical parameters, such as protein, lipid and carbohydrate content, for quality control. These parameters may also indicate the deficiency or excess in a particular nutritional component. Behavioural and genetic parameters are considered as well. The establishment of relationships between certain para- meters, e.g. between body size and fecundity or longevity, may help in simplifying quality control proce- dures. The ultimate quality criterion for an artificially reared natural enemy is its capacity to reduce pest populations, which can be evaluated by measuring the predation efficiency or the parasitization rate under laboratory or field conditions.
high-quality natural enemies is a prerequi- site for increasing the use of entomophagous insects for pest control. For biological control strategies, the ultimate quality criterion for a mass-produced natural enemy is excellent field performance against the target pest insect (Thompson and Hagen, 1999).
However, very few field evaluations have been done for artificially reared beneficials.
Egg parasitoids, especially Trichogramma, are used worldwide for biological control on many crops (van Lenteren, 2000). In China, Trichogramma spp. and Anastatus spp. pro- duced at an industrial scale on artificial eggs have been released on thousands of hectares of different crops, with parasitism rates of usually more than 80% (Dai et al., 1991; Liu et al., 1995). In the USA, biocontrol companies have started to integrate artificial diets in their production process, and some preda- tors are at least partially being reared on var- ious artificial diets (P.D. Greany, Gainesville, USA, 2001, personal communication), but there are no reports on their field perfor- mance. Field assessments of artificially reared natural enemies are only useful, how- ever, when basic parameters during the pro- duction process in the laboratory are strictly controlled and changes in the rearing proce- dure are carefully monitored.
Quality control of mass-produced ento- mophagous insects can be based on pro- cedures developed earlier for non- entomophagous insects (Chapter 2; Leppla and Ashley, 1989; Leppla and Fischer, 1989;
Williams and Leppla, 1992). For example, quality control during storage, packaging and shipment of all kinds of mass-reared insects could be similar. There are already quality control processes recommended for in vivo production that could be used as a guideline for in vitro production (Bigler, 1994). Comparative analyses of the repro- ductive attributes of commercially in vivo- produced Trichogramma species were described by Kuhlmann and Mills (1999).
Both these latter authors and Liu and Smith (2000) chose fecundity, longevity, sex ratio and adult size as quality parameters.
Parasitization rate is another key parameter tested for quality evaluation (Losey and Calvin, 1995).
For quality control of insects no absolute criteria exist, but different criteria could be defined in relation to the objectives for which they are produced. The degree of difference between insects produced in vivoand in vitro that we can accept will depend on the goals of the production (Moore et al., 1985), in this case the pest-control efficiency to be obtained in the field. For in vitro rearing of ento- mophages we are only at the very first steps of developing quality control, except for a few species of parasitoids and some preda- tors (Table 9.1). The main question will be:
what kinds of parameters are reliable enough to be used in the assessment of qual- ity? In this chapter we shall not develop a quality control scheme. Instead, we shall conduct in vivo–in vitrocomparisons, which form the starting-point for establishment of quality control parameters for artificially reared entomophages.
Different Kinds of Artificial Diets for Parasitoids and Predators
It would be convenient if the different kinds of diets used in mass rearing could be typi- fied by simple terms. Long ago some terms were used based on the presence or absence of complex components, but they were not very clearly defined: holidic media (chemi- cal structure of all ingredients known), meridic media (holidic base to which at least one substance or preparation of unknown structure or uncertain purity is added) or oligidic media (crude organic materials). However, we are of the opinion that these distinctions are not very relevant, because only a complete description of the composition of a diet would be able to char- acterize it. Nevertheless, for practical con- siderations, a critical characteristic is the presence or the absence of insect compo- nents. Thus, considering that synthetic diets were supposed to replace the insect host or prey, it is worth distinguishing two main kinds of media: those including and those excluding insect components. Addition of insect materials implies the necessity to cul- ture not only the host but often also the host’s food plant, rendering entomophage 116 S. Grenier and P. De Clercq
production more expensive. But we have to emphasize that in some parts of the world, especially in China and some other Asian countries and in Latin America, insect com- ponents, such as haemolymph, could be by- products of the silk industry and thus cheap and easy to obtain.
Diets with insect additives
Insect additives can be used in different ways. Sometimes almost the whole host con- tents are used as scarcely diluted extracts.
The main elements used are whole-body tis- sue extracts or haemolymph from lepi- dopterous pupae in artificial diets for parasitoids. This is the case for larval para- sitoids, such as the chalcidid Brachymeria intermedia (Dindo et al., 1997), the ichneu- monid Diapetimorpha introita(Ferkovich et al., 1999, 2000) or the tachinid Exorista larvarum (Dindo et al., 1999), and for oophagous para- sitoids, such as Trichogramma spp. (for a review, see Grenier, 1994). Usually silkworm species (Antheraea pernyi, Philosamia cynthia)
and easily reared insects like Galleria mel- lonellaare used for these extracts.
Bee extracts or even whole pulverized bees or bee brood have been added in diets for coccinellid predators (Smirnoff, 1958;
Niijima et al., 1977, 1986).
Some diets for Trichogramma contain egg juice from a natural host (Consoli and Parra, 1996). For the egg parasitoid Edovum puttleri a homogenate of host eggs (Colorado potato beetle) was used (Hu et al., 1998).
In hymenopterous parasitoids, teratocytes play various roles (Dahlman, 1990), mainly in the exploitation of the host by the para- sitoid larva, through secretion of digestive enzymes attacking host tissues or proteins as food for the parasitoid larva (Falabella et al., 2000). In vitro, cell products or cell cultures were also used in lieu of haemolymph or host factors (Grenier et al., 1994).
Diets devoid of insect components Very few diets are chemically defined. The first defined diet concerning a true para- Quality of Artificially Reared Biocontrol Agents 117
Table 9.1. Proposed parameters for quality control of parasitoids and predators produced under artificial conditions.
Morphological parameters
Size or weight of last larval instar/pupa/adult
Percentage abnormalities (deformation of wings/abdomen) Development and reproduction parameters
Duration of egg/larval/pupal stage
Survival rate of egg/larval/pupal/adult stage
Sex ratio – presence/absence of symbionts influencing sex ratio Fecundity/fertility
Duration of preoviposition/oviposition/postoviposition period Longevity
Biochemical parameters
Protein, lipid, carbohydrate content Hormone titre
Behavioural parameters
Predation or parasitization efficiency Locomotion/flight activity
Host/prey localization capability Genetic parameters
Genetic variability Homozygosity rate
sitoid species was that for Itoplectis conquisi- tor(Yazgan, 1972). Diets whose entire chem- ical composition is known, even if the structure of some components is not fully defined (nucleic acids, proteins), can be con- sidered as chemically defined. A small num- ber of diets that fit such a definition were tested successfully for rearing ento- mophagous insects (Grenier et al., 1994). In such diets, many complex or ‘crude’ compo- nents can be added as host substitutes.
Irrespective of the species reared, whether parasitoids or predators, the most com- monly used components are hen’s egg yolk, chicken embryo extract, calf fetal serum, bovine serum, cow’s milk, yeast extract or hydrolysate, crude proteins or protein hydrolysates, meat or liver extracts and seed oils. For recent reviews of such diets, see Thompson (1999) and Thompson and Hagen (1999).
Success in Development of Some Species in Artificial Conditions
The main successes in artificial mass rearing have been obtained with hymenopterous egg and pupal parasitoids, tachinid larval parasitoids and some polyphagous preda- tors. Extensive general reviews of artificial diets for entomophagous arthropods have been published by Grenier et al. (1994), Thompson (1999) and Thompson and Hagen (1999).
Koinobiontic endoparasitic Hymenoptera (parasitoids that do not immediately kill their hosts and where the parasitoid larvae develop in the still living host) are the most difficult species to rear in vitro because the parasitoid has a close relationship with its living host, which probably supplies the par- asitoid with some specific growth factors nec- essary for normal development of the parasitoid larva (Greany et al., 1989).
Moreover, endoparasitoids, for which the diet is not only their food but also their envi- ronment for larval development, have special requirements compared with ectoparasitoids or predators. Thus, special attention has to be paid to factors such as osmotic pressure and pH (Grenier et al., 1994).
Comparison of Artificially and Naturally Reared Natural Enemies
Many parameters used as quality criteria are linked, such as adult body weight and longevity, fecundity, flight activity and searching ability (Kazmer and Luck, 1995).
Quality control procedures could be simpli- fied and could thus be made less costly if we were able to use one parameter that is easily measured (e.g. size) to predict the value of another trait that is more complex or time- consuming to determine (e.g. fecundity or field performance). In parasitoids, body size may be related to fecundity, longevity, rate of search and flight ability (Kazmer and Luck, 1995). Bigler (1994) pointed out that the female body size of a parasitoid could be used as an index of fitness or a quality para- meter, as in Trichogramma. But female size is not a reliable parameter for predicting field performance when the parasitoids are reared on factitious or artificial hosts. In Trichogramma, large-sized wasps developed from in vitro rearing showed characteristic abnormalities called ‘big belly’. Despite their large body size, such adults usually have a low viability. The size of a normally shaped Trichogrammaadult produced in vitrois also larger than that of a wasp that developed in the natural host (Nordlund et al., 1997). This is often found in oophagous parasitoids and is the result of a low number of parasitoid eggs developing in the large amount of food that is available to them (Grenier et al., 1995).
In general, the size of Trichogramma and other oophagous parasitoids varies accord- ing to the number of adults developing in the same host, which consume all the avail- able host material. Remains of the host pre- vent proper pupation of parasitoids, and parasitoid larvae that are excessively large cannot pupate. In a natural situation with a great many Trichogrammalarvae in one host, adult parasitoid size will be reduced accord- ingly. Under artificial rearing conditions, however, the quantity of food in the artificial host egg is usually very large compared with a natural host egg, and the number of para- sitoid eggs laid is often too low for the devel- opment of normal-sized Trichogramma (Grenier et al., 2001).
118 S. Grenier and P. De Clercq
All parameters related to reproduction are important, and sometimes reproduction capacity can be estimated by a simple mea- surement, such as the body size of the para- sitoid, as in Encarsia formosa (van Lenteren, 1999). In predators as well, body size is often believed to be a good predictive index of fecundity, but the relationship between the two parameters is not always clear. For instance, females of the predatory pentatomid Podisus maculiventris reared on an artificial diet were significantly smaller than those fed larvae of Tenebrio molitor, but their fecundities were similar (De Clercq et al., 1998a). Rojas et al.(2000) obtained females of Perillus biocula- tuson artificial diet with similar size to that of those offered Leptinotarsa decemlineata larvae, but their fecundity was only 10% of that of prey-fed controls. Establishing a relationship between the size and predation capacity of a laboratory-produced predator has been shown to be even more problematic, even when it is produced on live prey (e.g. De Clercq et al., 1998b). Cohen (2000a) reported that Geocoris punctipesreared for over 6 years on artificial diet were significantly smaller than feral specimens but had similar preda- tion capacities. Chocorosqui and De Clercq (1999) found that, despite their smaller size, artificially reared nymphs of P. maculiventris even showed significantly greater predation rates than prey-fed controls.
Several morphological traits and develop- mental and reproductive parameters that have been used to assess the quality of artifi- cially reared parasitoids and predators will be reviewed below. For clarity we shall treat the most widely used parameters separately, but, where applicable, links between differ- ent traits will be discussed.
Morphological parameters Size or weight of different developmental
stages
Size or weight of eggs deposited by in vitro- reared natural enemies has only rarely been monitored. Fresh weights of eggs laid by females of the heteropteran predators G.
punctipes and P. maculiventris cultured on bovine meat diets were similar to those of
eggs from females maintained on insect prey (Cohen, 1985; De Clercq and Degheele, 1992).
Only occasionally have workers monitored fresh weights of larvae during their develop- ment on artificial diets. Nevertheless, such studies may reveal where nutritional deficien- cies are most crucial. Larval weights of preda- tors fed on artificial diets have often been found to be inferior to those of their prey-fed peers (Hagen and Tassan, 1965; Hussein and Hagen, 1991; Chocorosqui and De Clercq, 1999; Wittmeyer and Coudron, 2001).
Puparial weight was surprisingly higher in the tachinid fly E. larvarumreared in vitroon artificial diets than in vivo in G. mellonella (Dindo et al., 1999). Availability of food ad libi- tum in artificial rearing might explain this result. Vanderzant (1969) also reported greater pupal weights for artificially reared lacewings, Chrysoperla carnea, compared with controls fed Sitotroga cerealella eggs. Hassan and Hagen (1978) succeeded in rearing larvae of C. carnea on an artificial diet and obtained pupae with similar weights to those in controls fed moth eggs. Likewise, no difference was observed between pupal weight of Chrysoperla rufilabris fed a meat-based artificial diet and those fed Ephestia kuehniella eggs (Cohen and Smith, 1998). Pupal weights of the colydiid beetle Dastarcus helophoroidesreared on artificial diet were not different from those of predators reared on live prey (Ogura et al., 1999), but pupal weights of in vitro-reared Trogossita japonica were only half of those of beetles reared in vivo(Ogura and Hosada, 1995).
In both parasitoids and predators, adult body weight and size have often been used to evaluate the effectiveness of a diet (see above). Adult weights of D. introita devel- oped in vitrowere lower than those of indi- viduals fed host pupae (Spodoptera frugiperda), and they were not significantly improved by adding some conditioned tis- sue-culture media (Ferkovich et al., 1999), but they were improved when the diet was sup- plemented with certain host lipid extracts (Ferkovich et al., 2000). Usually larger hosts produce larger wasps showing a greater egg load and a higher longevity, as described in the encyrtid Metaphycus sp. (Bernal et al., 1999). As in many insects, puparial weight in tachinid flies is closely correlated with adult Quality of Artificially Reared Biocontrol Agents 119
weight (Grenier and Bonnot, 1983). As a con- sequence, differences in pupal weights between in vitro- and in vivo-reared E. lar- varum are reflected in the adult stage (see also below).
In many cases, adults of predatory insects from various orders reared on artificial food are smaller than those obtained on insect prey (e.g. Racioppi et al., 1981; Hussein and Hagen, 1991; De Clercq and Degheele, 1992;
Hattingh and Samways, 1993; Ogura and Hosada, 1995; Zanuncio et al., 1996; De Clercq et al., 1998a; Cohen, 2000a). However, Rojas et al. (2000) and Arijs and De Clercq (2001) obtained female adults of the preda- tory bugs P. bioculatus and Orius laevigatus, respectively, with equal weights to those of predators maintained on insect prey.
Abnormalities
Abnormalities observed in artificial rearing are usually deformations of wings (expanded or not) and of the abdomen. The percentage of deformed females, i.e. mainly unexpanded wings, is a criterion for measuring abnormal- ities in Trichogramma (Cerutti and Bigler, 1995). Bloated larvae with unexpanded wings are produced in vitrodue to too few eggs per artificial host egg (Grenier et al., 1995). Wing and abdominal deformations occurred in Trichogrammareared on artificial diet, but no differences in the morphology of their genital apparatus were detected (Consoli and Parra, 1996). The same wing and body anomalies were recorded after Trichogramma was pro- duced for ten generations in vitro(Nordlund et al., 1997). Suboptimal diets have also been reported to cause deformation of wings and abnormal pigmentation in some predatory coccinellids (Atallah and Newsom, 1966;
Racioppi et al., 1981).
Development and reproduction parameters
Duration of different developmental stages Many workers have assessed developmental rates of immature stages to evaluate the nutritional value of a diet. Usually the imma- ture development of parasitoids (from egg or
first-instar larva to adult emergence) is longer under artificial conditions than in nat- ural hosts. This was observed in the tachinid fly E. larvarum(Dindo et al., 1999) and in the ichneumonid D. introita (Greany and Carpenter, 1998; Ferkovich et al., 1999). The development from egg to adult on artificial diets was delayed for Trichogramma pretiosum compared with natural hosts, but no differ- ences were observed for Trichogramma galloi (Consoli and Parra, 1996). The development of the egg stage was not very often tested, but for Trichogramma australicum it took longer in artificial diet vs. natural-host eggs (Dahlan and Gordh, 1998). In T. australicum also the larval stages develop more slowly in artificial diet vs. natural-host eggs (Dahlan and Gordh, 1998). Total development time is not different in Bracon mellitorand Catolaccus grandis reared in vivo vs. in vitro (Guerra et al., 1993; Guerra and Martinez, 1994).
Usually, when the whole preimaginal devel- opment is longer, only the larval develop- ment is delayed, the embryonic and pupal development being less dependent on the diet composition.
Likewise, developmental times in preda- tors are usually longer on artificial diets.
Some workers have even reported total developmental periods two to three times longer than those observed in controls on natural food (Hagen and Tassan, 1965;
Vanderzant, 1969; Racioppi et al., 1981;
Hussein and Hagen, 1991). Whereas in a number of studies differences in develop- mental times were consistent across all larval instars, differences in some species became more important in later instars (Racioppi et al., 1981; Zanuncio et al., 1996; Wittmeyer and Coudron, 2001). Because Wittmeyer and Coudron (2001) found differences in develop- mental durations between in vitro- and in vivo-reared P. maculiventristo be most marked in the fifth (final) nymphal stage, they sug- gested selecting this stage to evaluate the suitability of a diet. Similar developmental periods for artificially and naturally reared predators have occasionally been reported, e.g. by Grenier et al. (1989) for Macrolophus caliginosus, by De Clercq et al.(1998a) for indi- vidually reared P. maculiventrisand by Arijs and De Clercq (2001) for O. laevigatus.
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