J.C. van Lenteren
1and M.G. Tommasini
21Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH Wageningen, The Netherlands; 2CRPV (Centro Ricerche Produzioni Vegetali), Via
Vicinale Monticino 1969, 47020 – Diegaro di Cesena (FC), Italy
Introduction
Since the beginning of this century, the mass production of natural enemies has been considered as a means of improving biological control programmes, especially those based on inundative and seasonal inoculative releases. For general informa- tion on mass production and quality con- trol of insects and other arthropods, we refer the reader to Morrison and King (1977), King and Morrison (1984), Singh (1984), Singh and Moore (1985), van Lenteren (1986a) and various chapters in this book. For mass production related to commercially produced natural enemies, we refer the reader to van Lenteren (1986b), van Lenteren and Woets (1988), Nicoli et al.
(1994) and Bolckmans (1999). We shall not discuss the question of how to obtain a good stock colony to start a mass produc- tion, because this issue is addressed in Chapters 1, 6 and 7. In this chapter, we shall briefly summarize developments in the mass rearing of natural enemies for commercial biological control during the 20th century.
Mass production of beneficials is a ‘skil- ful and highly defined processing of an entomophagous species through insectary procedures which results in economical pro- duction of millions of beneficial insects’
(Finney and Fisher, 1964). This is true for most mass-rearing programmes, but there are important exceptions where mass pro- duction seems to be a fairly simple process.
© CAB International 2003. Quality Control and Production of Biological Control Agents:
Theory and Testing Procedures (ed. J.C. van Lenteren) 181
Abstract
Mass production of natural enemies started in the 1940s and quickly developed thereafter. These devel- opments were mainly triggered by trying to economize rearing and making biological control more com- petitive when compared with other pest-control methods. Storage of natural enemies is only possible for very short periods, with the exception of species for which it is known how to start and terminate dia- pause. Initially, the collection, shipment and release of biological control agents were rather amateurish, but enormous progress has also been made in this area. Many natural-enemy species can now be pro- duced at competitive costs, resulting in increased use of biological pest control.
The first step in a mass-rearing pro- gramme is a trial to rear the natural enemy on a natural host (the pest organism) in an economical way. Most natural enemies are reared in this way. However, several natural enemies are not mass-reared on their natural host because it is either too expensive or undesirable due to the risk of infection with the pest organism or concurrent infection with other pests or diseases when natural enemies are released on their natural sub- strate. In these cases a search is made for an opportunity to rear the natural enemy on an alternative host (and often an alternative host plant).
A subsequent step in making mass rear- ing more economical is to change from a nat- ural host medium (host plant) to an artificial medium for rearing the host. Rearing insects on artificial diets was developed earlier this century and considerable progress has been made recently. Rearing on artificial diets is considerably cheaper as less expensively cli- matized space is needed, but artificial rear- ing may create serious quality problems, which will be discussed later in this chapter.
Singh (1984) summarizes the historical development, recent advances and future prospects for insect diets as follows:
1. Some 750 species, mainly phytophagous insects, can be reared successfully on (semi-) artificial diets.
2. Only about two dozen species have been successfully reared for several generations on completely artificial diets.
3. Large-scale mass rearing on artificial media has been developed for fewer than 20 species of insects.
4. Quality control is essential, as there can be dietary effects on all critical performance traits of the mass-reared insect and also on the natural enemy produced on a host that was mass-reared on an artificial medium.
5. Suitable bioassays are important for answering the question ‘what is the ultimate effect of the diet on the reared insect?’
A final step when trying to minimize rear- ing costs is the search for ways to rear the natural enemy on an artificial diet. This has been attained for several ecto- and endopara- sitoids (e.g. Trichogramma) and a few preda-
tors (e.g. Chrysoperla). The technology for rearing natural enemies on diets is, however, far less developed than that for rearing pest species (Chapter 9; Grenier et al., 1994).
The rapid development of commercial biological control based on mass-produced natural enemies can be illustrated well with data from Europe. About 150 species of nat- ural enemies have been imported and released into Europe during the 20th century to control about 55 mite and insect pest species. Until 1970 this mainly concerned inoculative (classical) biological control. After 1970 many developments took place in greenhouses and annual field crops, and commercial biological control programmes for c. 50 pest species were developed by importing more than 60 species of natural enemies. In addition, more than 40 endemic species of natural enemies were employed in commercial biological control. For all these species, fine-tuned mass-production systems had to be developed. Our experience with the development of new biological control pro- grammes has shown that dogmatism is use- less when selecting natural enemies. This contrasts with the approach of earlier biocon- trol workers (see, for example, DeBach, 1964).
We have, for example, had excellent control results by releasing endemic natural enemies against exotic pests and vice versa: all combi- nations are worth trying (Table 12.1).
The most important species of natural ene- mies that are mass-reared in Europe are given in Table 11.2 of Chapter 11, and an overview of all available natural enemies is presented in Table 11.1 of the same chapter. Although on- farm production of natural enemies is possi- ble, most growers purchase them from commercial suppliers. Many of the mass-pro- duction companies are, understandably, reluc- tant to provide information on many aspects of mass production. Our experience is that most of the natural enemies produced for bio- control in protected cultivation are reared on their natural hosts (the pests) and host plants.
Rearing on purely artificial media (without organic additives) is very rare, primarily because this technology is insufficiently developed for mass production and because this way of production may lead to poor per- formance of natural enemies when exposed to 182 J.C. van Lenteren and M.G. Tommasini
their target hosts (for details, see van Lenteren, 1986a, and Chapters 1 and 2).
Rearing conditions should be as similar as possible to the conditions under which the natural enemies will have to function in the field or greenhouse. Two examples of mass- production schemes, one for the predator Oriusand the other for the parasitoid Encarsia, are presented in Figs 12.1 and 12.2.
Storage of Natural Enemies It is necessary to have storage methods and facilities available to meet the requirements for good planning for a mass-production
unit and because of the difficulty of accu- rately predicting demand from clients (both delivery dates and quantities). This is rela- tively simple for microbial biocontrol agents, such as fungi, viruses and bacteria, because they can often be stored in a resting stage for months or even years. Many preda- tors and parasitoids can only be stored for a short time. This usually involves placing the natural enemies as immatures at tempera- tures between 4 and 15°C. Normally, storage only lasts several weeks, but even then reduction in fitness is the rule (Posthuma- Doodeman et al., 1996). Storage of para- sitoids at a low temperature (8°C) for 2 and 16 days, respectively, gives similar percent- Mass Production of Natural Enemies 183
Table 12.1. Commercial biological control of endemic or exotic arthropods with endemic or exotic natural enemies in Europe (the numbers reflect the number of combinations in which a certain natural enemy is used for control of a certain pest;
situation in 1999).
Different combinations of natural-enemy use in Europe
Use of endemic natural enemies for the control of endemic pests:
example:Chrysoperla carneafor control of endemic aphid species 61 Use of endemic natural enemies for the control of exotic pests:
example:Diglyphus isaeafor control of exotic Lyriomyzaspecies 40 Use of exotic natural enemies for the control of endemic pests:
example:Harmonia axyridisfor control of endemic aphid species 44 Use of exotic natural enemies for the control of exotic pests:
example:Encarsia formosafor control of exotic whitefly species 47
Put bean pods with Orius eggs into cage with dispersal material andEphestia eggs as prey
Harvesting of newly emerged adults
Storage for a few days if necessary
Collection and change of bean pods, and addition of food (2–3 times per week)
New prey is added twice a week
Put adults into new cage, supply with prey and bean pods
Packaging and shipment
Old adults are eliminated 17 days
28 days
Fig. 12.1. Production scheme for the thrips predator Oriussp.
ages of emergence, but the ability to fly is much lower for the parasitoids that were stored for 16 days (Fig. 12.3). This test was done with the short-range flight cylinder as described in Chapter 19. Storage during the adult stage leads to even higher and faster reduction in fitness than with storage of immatures. The pupal stage seems to be most suitable for short-term storage.
Data on long-term storage of natural ene- mies or their hosts are limited. Host material (e.g. eggs of Sitotroga cerealellaand Grapholita lineatum) stored for long periods (in the case of Grapholitafor up to 5 years) in liquid nitro- gen could still be used for production of Trichogramma and Trissolcus simoni, respec- tively (Gennadiev and Khilistovskii, 1980).
Eggs of Ephestia kuehniella can be sterilized by ultraviolet (UV) radiation or freezing, and then be stored at low temperature for several months without losing their value as alterna- tive food for mass production of predators such asChrysoperlaand Orius.
The parasitoid Diglyphus isaea can be stored at a low temperature for at least 2 months, during which time mortality does not increase and fecundity remains the same (Burgio and Nicoli, 1994). Hagvar and Hofsvang (1991) reported that some species of Aphidiidae (e.g. Aphidius matricariae) can be stored at low temperatures for several weeks.
The possibility of storing beneficials in the diapausing stage has been studied, but most of this work has not yet led to practical application, because unacceptably high mor- tality occurred during the artificially induced diapause. There are, however, some positive exceptions. Diapausing adults of the preda- tor Chrysoperla carneacan be stored at a low temperature for about 30 weeks while main- taining an acceptable level of survival and reproduction activity (Tauber et al., 1993).
Also the predator Orius insidiosus maintains good longevity and reproduction rate after storage in diapause for up to 8 weeks 184 J.C. van Lenteren and M.G. Tommasini
Sow tobacco seeds in pots in greenhouse
ReleaseEncarsia formosa to parasitize whiteflies
Harvest part of the leaves with black pupae
1st generation of E.
formosa, remove black pupae from leaves, ship to grower
Unparasitized whiteflies emerge, fly to upper leaves of tobacco plants and oviposit
Harvest part of the leaves with black pupae
2nd generation of E.
formosa, remove from leaves and ship
Harvest part of the leaves with black pupae
3rd generation of E.
formosa, remove from leaves and ship
All developments take place on same tobacco plants in same greenhouse for several months
4–8 weeks
2–3 weeks
2–3 weeks
2–3 weeks 2–3 weeks
2–3 weeks
1–2 weeks
1–2 weeks
1–2 weeks
etc.
2–3 weeks
etc.
Release whitefly adults to infest tobacco plants
E. formosa emerge from black pupae, move up in plant and parasitize whitefly
E. formosa emerge from black pupae, move up in plant and parasitize whitefly Unparasitized whiteflies emerge, fly to upper leaves of tobacco plants and oviposit
Fig. 12.2. ‘Continuous’ production scheme for the whitefly parasitoid Encarsia formosa.
(Ruberson et al., 1998). The predator Aphidoletes aphidimyzacan survive periods of 3–8 months when stored at 10°C (Tiitanen, 1988). Long-term storage of the diapausing stage of the parasitoid Trichogramma, has been successful for periods of up to a year and is now commercially exploited (J.
Frandon, Biotop, Antibes, France, 1996, per- sonal communication).
Long-term storage capability is very desirable for production companies, because:
• continuous production of the same quan- tity of beneficial insects is often economi- cally more attractive than seasonal production of very large numbers;
• storage facilities enable them to build up reserve supplies of entomophages to com- pensate for periods of low production or periods of unexpected high demand;
• storage makes rearing possible at the best period of the year, e.g. at a period when host plants can be grown under optimal conditions.
Collection and Shipment of Natural Enemies
After production, the beneficials should be delivered to the growers as soon as possible.
If delivery is looked after by the producer
and occurs within 48 h of harvesting the organisms, no special shipment procedures are normally needed for parasitoids and non-cannibalistic predators other than pro- tection against excessive heat, cold or rough handling. When transport takes several days, climatized containers should be used and it may be necessary to add food (e.g.
honey in the case of parasitoids and pollen/prey for predators). A way to over- come problems with long times for transport of predators is for young stages to be pack- aged with food so that further development takes place during transport. Packaging of predators demands special attention when cannibalism is a common phenomenon.
Many of the commercially available preda- tors are generalists and exhibit cannibalism when kept at high densities, even if food is available in the containers for shipment. To reduce the risk of cannibalism, it is common to provide hiding-places for the natural enemy by using paper, buckwheat, vermi- culite or wheat bran in the container (see Table 12.2). In the early days of mass pro- duction, the biological control agents were often collected and shipped on the host plant on which they were reared. With the internationalization of biocontrol, shipment on or in inert media became a necessity.
Ingenious collection and shipping proce- dures have been developed.
Mass Production of Natural Enemies 185
100
80
60
40
20
0
% Parasitoids trapped
2 16
Days stored at 8°C
Fig. 12.3. Percentage Encarsia formosafemales capable of flying (= reaching trap in short-range flight test) when stored for 2 and 16 days at 8°C.
186 J.C. van Lenteren and M.G. Tommasini
Table 12.2.Survey of collection, shipment and release methods for biological control agents. Stage at which Quality Natural enemycollectedSpecial handlingCountingPackingShippingMode of introductioncontrol Amblyseiusspp.All stagesEstimateIn wheat branPlastic containerSprinkling on plantsYes Aphidiusspp.PupaRemoval from leafWeightPlastic containerSmall numbers in Yes sheltered locations Aphidoletes aphidimyzaPupaWeightVermiculitePlastic containerSmall numbers in Yes sheltered locations Chrysoperla carneaEggRemoval from leafVolumetr.BuckwheatPlastic containerSprinkling on plantsYes Cryptolaemus montrouzieriAdultCountingPaper stripsPlastic containerSmall numbers on plants Dacnusa sibericaAdultCountingPlastic containerTapped from containerYes Diglyphus isaeaAdultCountingPlastic containerTapped from containerYes Encarsia formosaPupa on leavesRemoval from leafVolumetr.Glued on cardboardPaper boxCards hung in plantsYes Eretmocerus mundusPupa on leavesRemoval from leafVolumetr.Glued on cardboardPaper boxCards hung In plantsYes Harmonia axyridisLarvaeCountingPopcornPlastic containerTapped from container on plants Leptomastix dactylopiiAdultCountingPlastic containerTapped from containerYes Lysiphlebus testaceipesPupaRemoval from leafCountingPlastic containerSmall numbers in sheltered locations Macrolophus caluginosusNymphs and adultCountingBuckwheat + vermic.Plastic containerSmall numbers on plantsYes Neoseiulusspp.All stagesEstimateIn wheat branPlastic containerSprinkling on plantsYes Oriusspp.Nymphs and adultVolumetr.Buckwheat + vermic.Plastic containerSmall numbers on plantsYes Phytoseiulus persimilisAll moving stagesVolumetr.In wheat branPlastic containerSprinkling on plantsYes Trichogrammaspp.PupaVolumetr.Glued on cardPaper boxCards hung in plants or Yes by sprinkling on plants
Poor shipping conditions frequently led to natural enemies arriving either dead or in poor condition. Difficulties in shipping can be considerable in countries where crops with the same target pest are not concen- trated together and where distances are large. Most transport is still by truck, although an increasing quantity is sent by aircraft. Intercontinental transport problems are caused less by containerization than by the sometimes excessively long handling time at customs, which leads to high mortal- ity or decrease in fitness. The logistics of shipments remains one of the main problems for the commercialization of biological con- trol. Examples of the different techniques for collecting, counting, packaging and shipping of natural enemies are given in Table 12.2.
Release of Natural Enemies
Developmental stage at which organism is released
Entomophagous insects can be brought into greenhouses or the field in different stages of their development (see also Table 12.2):
• eggs (e.g. Chrysoperla);
• larvae or nymphs (e.g. Chrysoperla, Phytoseiulus, Amblyseius, Orius);
• pupae or mummies (e.g. Aphidius, Trichogramma, Encarsia);
• adults (e.g. Dacnusa, Diglyphus, Orius, Phytoseiulus);
• all stages together (e.g. Phytoseiulus, Amblyseius).
The stage in which the beneficials are intro- duced depends mainly on the ease of trans- port and manipulation in the field, but it is, of course, also important to release the nat- ural enemy at a stage when it is most active at killing the pest. Usually the stage that is least vulnerable to mechanical handling is chosen and therefore a non-mobile stage, often the egg or pupa, is most suited for transport and release. In situations where it is difficult, but essential, to distinguish the natural enemy from the pest, the only solu- tion is to introduce adults. Adult releases for parasitoids are advised only when younger
natural-enemy stages cannot be distin- guished or separated from the pest insect:
the handling and releasing of delicate adult parasitoids is very difficult and often a large reduction of fertility is observed compared with the fertility of parasitoids when released as immatures. When the natural enemy is released in one of the developmen- tal stages that do not prey upon or parasitize the host, the timing should be such that the active stage emerges at the right moment of pest population development. For some nat- ural enemies the stage of release depends on pest development: when pest density is low, release of first-instar C. carneasuffices; when the infestation with the pest organisms is already relatively high, it is better to release second-instar larvae, which have a much higher predation capacity.
Methods of introduction
Beneficials are introduced into the field in many ways (Table 12.2). Eggs and pupae are either distributed over the field on their nor- mal substrate (leaves of the host plant, e.g.
Chrysoperla and Encarsia) or glued on paper/cardboard cards (e.g. Encarsia, Trichogramma). These stages of the natural enemies can also be collected and put into containers, which are then brought into the field (e.g. Trichogramma).
The mobile stages of natural enemies, lar- vae or nymphs and adults, can be put into the field in containers from which they emerge (e.g. many adult parasitoids and predators) or the grower can distribute nat- ural enemies in these stages over the crop, for example by ‘sprinkling’ them on to the plant. In this case, the use of dispersal mater- ial (e.g. buckwheat, vermiculite) is often nec- essary in order to obtain a homogeneous distribution of small natural enemies. When natural substrates (e.g. buckwheat or wheat bran) are used as dispersal materials, they must be free of pesticides.
Instead of introducing the predator or parasitoid by itself, one can also introduce a whole ‘production unit’, e.g. ‘banker-plants’
containing the host insect and its natural enemy can be brought into a crop. When the Mass Production of Natural Enemies 187