The present invention relates to the field of biological crop protection. According to a first aspect the invention relates to a method for rearing a zoophytophagous insect. Further aspects of the invention relate to a method for crop protection, a method for reducing crop damage caused by a zoophytophagous insect, the use of a carbohydrate source as a nutritional source for a zoophytophagous insect, compositions comprising a carbohydrate source for specific uses in relation to the development of a zoophytophagous insect and the use of a carbohydrate source as a food source for a zoophytophagous insect.

Zoophytophagous insects are insects which have omnivorous feeding habits and can feed on both prey and plant tissue. The dependency on prey and plant tissue may differ between the species of zoophytophagous insect and for a certain species may differ depending on the environmental conditions, such as availability of prey. Species having a prominent predatory behaviour have a beneficial utility in agriculture and horticulture as biological crop protection tools, due to their preying on crop pests. Species having a prominent feeding on plant tissue are potential pests for crops produced in agriculture and/or horticulture.

Nesidiocoris tenuis Reuter (Hemiptera: Miridae) is an example of a zoophytophagous mirid with biological control potential that commonly appears in tomato crops in the Mediterranean (Arno et al. 2010b). This predator is mass-reared and it has been primarily released to control whiteflies (Hemiptera: Aleyrodidae) in greenhouses (Calvo et al. 2009, Urbaneja et al. 2005, Gabarra et al. 2008, Castane et al. 2008). Nesidiocoris tenuis was also detected preying upon the invasive pest, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), immediately after its detection in the Spanish Mediterranean coastal area (Desneux et al. 2010), on which it has been shown to be able to regulate its populations (Molla et al. 2011, Gonzalez-Cabrera et al. 2011). In addition to these two important tomato pests, N tenuis has also been observed to contribute to the control of thrips, leafminers, spidermites and other lepidopteran pests in greenhouses (Jacas et al. 2008). Nesidiocoris tenuis can make significant contributions to control of tomato greenhouse pests mentioned above. However, by feeding directly on vegetative and reproductive parts of the plant it may cause necrotic rings on stems and leaf petioles, flower abortion. In addition it can also puncture fruits, such as tomato fruits, possibly reducing yields (Sanchez 2008).

The intensity of plant feeding by N. tenuis on tomato crops under Mediterranean conditions has been addressed by several authors (Sanchez and Lacasa 2008, Sanchez et al. 2009, Calvo et al. 2009, Arno et al. 2010a), who basically conclude that damage inflicted by this mirid on tomato plants was directly related to the abundance of N. tenuis and inversely to the interaction between the number of N. tenuis and the number of prey.

In tomato greenhouses, inoculative releases of N. tenuis (1-2 individuals/m ² ) are usually conducted several weeks after transplanting. This strategy has been successfully used to control whitefly populations once a certain number of N. tenuis is present in the crop. However, establishment of this number requires five to eight weeks from the release in spring- summer crops.

It should be noted that this strategy does not work in all crop conditions. For example, in late crop cycles without heating, the mirid reproduction rate is not high enough to establish the desired population levels (Gabarra et al. 2008). To shorten the installation period and improve the distribution of N. tenuis in the crop, especially when weather conditions are less favorable to them, releases of predators have been made in seedling nurseries (predator in first) (Calvo et al. 2010). In Almeria (Spain), during the tomato growing season of 2010-2011, inoculation of N. tenuis in such nurseries was shown to be a good strategy for controlling T. absoluta and whiteflies, and was successfully implemented in more than 300 ha of commercial greenhouses (Koppert B.V. 2011).

This strategy entails transplanting tomato plants on which N. tenuis individuals have already laid eggs in the nursery into the production crop. In the nursery one to two N. tenuis individuals per plant are released with an alternative egg food source from Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). It is known that N. tenuis feeds on plants when there is a lack of prey. The possibility to substitute carnivory by phytophagy is a general characteristic of mirids (Naranjo and Gibson 1996). Thus the availability of alternative prey/food supports both a correct population establishment and minimization of crop damage. To date, the standard alternative food source used to enhance establishment of N. tenuis and other phytophagous insects such as mirids is E. kuehniella eggs, which can be rather expensive. The problem of costs of E. kuehniella eggs is also encountered in the rearing of phytophagous insects, such as mirids. In the rearing processes for these organisms E. kuehniella eggs are also used as a food source. For this reason, providing further alternative diets for mirids would be beneficial.

Apart from N. tenuis other predatory species from the family of the Miridae are also used for biological crop protection. These related predators from the family of the Miridae have a similar developmental biology as N. tenuis. For example Macrolophus pygmaeus and Macrolophus caliginosus are also commercially available. The family of the Miridae has promise to provide additional commercially valuable predators, especially from the genera Nesidiocoris, Macrolophus, Engytatus, Dicyphus and Deraeocoris. Tests using the mirid N. tenuis as a model insect for zoophytophagous insects have surprisingly shown that when this model insect is provided with a carbohydrate source in the presence of alternative food sources known to be well accepted, such as E. kuehniella eggs or plant tissue, the consumption of these alternative food sources decreases. It has surprisingly been shown that provision of a carbohydrate source reduces the amount of E. kuehniella eggs required in mass rearing of this zoophytophagous mirid and/or for its establishment in a crop. Furthermore it has been found that provision of a carbohydrate source to the zoophytophagous mirid N. tenuis has a positive effect on parameters related to population development, such as fecundity. Consumption of sugar solutions provided from outside the natural habitat of zoophytophagous insects and the associated reduced consumption of other food sources known to be well accepted by these organisms has not been recorded prior to this invention. Instead the prior art aims at providing food sources and artificial diets particularly used as a sole food source not intended for combination with further food sources such as prey or plant tissue. For this W099/63814 and WOOO/49897 provide artificial diets, whereas WO2011/010308 provides processed medfly eggs.

The invention according to an aspect relates to a method for rearing a zoophytophagous insect comprising:

-providing a population of the zoophytophagous insect;

-providing to the population of the zoophytophagous insect a food source comprising protein, such as animal protein;

- providing to the population of the zoophytophagous insect a food source comprising a carbohydrate source.

Rearing should be understood to include any process aimed at and/or resulting in the increase of a population. Thus as a result from a rearing method the number of individuals of a population of the reared organism is increased. The population of the zoophytophagous insect provided preferably comprises sexually mature individuals of both sexes and/or comprises individuals of both sexes that can develop to sexual maturity. Individuals that can develop to sexual maturity include all pre-mature life stages of the zoophytophagous insect, including fertilized eggs and nymphs.

The method according to the invention is aimed at rearing a zoophytophagous insect. The skilled person will know that zoophytophagous insects are insects which have omnivorous feeding habits and can feed on both prey and plant tissue. Certain species of zoophytophagous insects are classified as beneficial, due to extensive preying on crop pests, other zoophytophagous insects are classified as pests due to extensive feeding on plant tissue.

Rearing of both beneficial and pest zoophytophagous insects is of interest to agriculture and horticulture. Beneficial zoophytophagous insects may be reared as biological crop protection tools. The zoophytophagous insects regarded as (potential) crop pests may be reared to conduct research into for example their behaviour, reproduction and control. Rearing of a zoophytophagous insect suitable for use as a crop protection agent is preferred for the present invention.

The zoophytophagous insect preferably is a zoophytophagous mirid, meaning that it is selected from the family of the Miridae. When selected from the family of the Miridae the zoophytophagous insect preferably is selected from the subtribe Dicyphina or from the tribe Deraeocorini, and from the subtribe Dicyphina for example is selected from the genus Nesidiocoris, such as Nesidiocoris tenuis, or from the genus Macrolophus, such as Macrolophus pygmaeus, Macrolophus caliginosus, Macrolophus praeclarus or Macrolophus melanotoma or from the genus Engytatus, such as Engytatus modestus, or from the genus Dicyphus, such as Dicyphus tamaninii, Dicyphus hesperus, Dicyphus hyalinipennis, and from the tribe Deraeocorini, for example is selected from the genus Deraeocoris, such as Deraeocoris brevis. The names and taxonomical ranking of the zoophytophagous insects as used in the context of this invention is as defined by Schuh and Slater (1995).

In the method according to the invention a food source comprising protein is provided to the zoophytophagous insect. The proteinaceous food source aids in supporting the protein requirement of the individuals of the zoophytophagous insect population. The protein source may be selected such that it comprises animal protein. Animal protein has a composition well adapted to the requirements of the zoophytophagous insect. According to a preferred embodiment the proteinaceous food source is a prey. A prey should be understood to mean a life stage of an animal organism that can be preyed by the zoophytophagous insect. Arthropod eggs, such as lepidopteran eggs, more particularly eggs from Ephestia kuehniella or Sitotroga cereallella, or eggs from other insect species such as from medfly (for example as disclosed in WO2011/010308) are particularly suitable prey that may be used as a protein containing food source.

Arthropod eggs preferably are provided in a frozen state or dried state. Alternative prey may be selected from Artemia cysts, preferably decapsulated Artemia cysts.

In the method according to the invention a food source comprising a carbohydrate source is also provided to the zoophytophagous insect. The food source comprising a carbohydrate source preferably is an artificial food source, meaning that at least one of its constituents and/or components is in a processed form. Preferably the carbohydrate in the carbohydrate source is an isolated carbohydrate, meaning that it is obtained by isolation and/or purification from a natural source. The skilled person will understand that the term carbohydrate embraces monosaccharides, disaccharides, oligosaccharides and polysaccharides. Within the present invention the use of all these forms of saccharides is envisaged. It is preferred to select the carbohydrate as a monosaccharide, a disaccharide, a trisaccharide or a tetrasaccahride. Suitable sugars may be selected from glucose, fructose, mannose, sucrose, lactulose, maltose, trehalose, cellobiose, melizitose, trehalulose, glucosucrose, stachyose or mixtures thereof.

The carbohydrate may be provided to the zoophytophagous insect in any form which renders it suitable as a food source for the insect. Suitable forms include solid forms and aqueous solutions. Solid forms are preferably provided as powder forms. Solutions, are preferably provided entrapped in a matrix and/or by a coating. The use of an aqueous carbohydrate solution is beneficial in view of the fact that it may also be used as a water source by the zoophytophagous insect.

Solutions entrapped in a matrix include gels. Gels may be obtained by using a gelling agent. Within the present invention the use of biological gelling agent is preferred. The selection of biological gelling agent is within the ambit of the knowledge of the skilled person. Examples of suitable biological gelling agents are agar, carrageenan, alginate, gelatine, starch.

Solutions entrapped by a coating may be obtained with known encapsulation techniques. Particularly useful are the methods disclosed in US 6,780,507. Products obtained with the methods disclosed herein are obtainable via HYDROCAPSULE ^® (Jasper, GA, USA).

If the carbohydrate source comprises a carbohydrate solution, the concentration of the carbohydrate in the carbohydrate solution may be in the range 0.2 -1.5 M, preferably 0.2-0.8 M, more preferably 0.3-0.6 M. The use of solutions, having a carbohydrate concentration, in particular a sugar concentration, within this range have proven to be well accepted by zoophytophagous insects while obtaining the surprising effects associated with the present invention.

If the carbohydrate source is provided in solid form it is preferred that the carbohydrate source comprises >15%, >20%, > 25%, > 30%, >35%, >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95% carbohydrate. In the rearing method it is further preferred to provide a water source to the

zoophytophagous insect. The source of water may for example be plant tissue such as tissue from tobacco plants as used in the rearing method disclosed by Castane and Zapata (2005).

Further requirements for rearing zoophytophagous insects are known to the skilled person and are for example disclosed by Cohen (2004) or Castane and Zapata (2005). Suitable standard conditions are 23-27°C, preferably 25 °C and 70 +/- 10% RH, and a 16:8 (L:D) photoperiod. In general tobacco plants are used to provide a water source and/or an oviposition substrate.

A further aspect of the invention relates to a method for crop protection. As the skilled person is aware of, a zoophytophagous insect may be suitable for use as a crop protection agent in view of its ability to predate on crop pests. However, as is known, under certain circumstances, such as low prey densities, beneficial zoophytophagous predators may also cause damage to crops by feeding on the plant tissue. The inventors of the present invention have found that providing a carbohydrate source on a crop reduces the crop damage caused by a zoophytophagous insect and/or supports the establishement of a population of the zoophytophagous insect in the crop.

The method for crop protection comprises:

-providing on the crop a population of a zoophytophagous insect;

-providing on the crop an amount of a food source, for the zoophytophagous insect, said food source comprising a carbohydrate source.

In the method of invention a zoophytophagous insect is provided which is suitable for use as a crop protection agent. The possible selections of zoophytophagous insect and further details concerning the zoophytophagous insect have already been discussed in connection to the preferred and alternative embodiments of the method of rearing.

The zoophytophagous insect may be provided on the crop by known methods. Suitable methods may be the methods known for releasing N. tenuis in a crop. In this respect reference may be made to the NESIBUG ^® product available from Koppert B. V. (Berkel an Rodenrijs, The Netherlands) and the guidelines for its use. In the method for crop protection a food source for the zoophytophagous insect is provided on the crop. Said food source comprises a carbohydrate source. Technical details about the food source comprising the carbohydrate source have already been discussed in connection to the method of rearing of the invention.

The method for crop protection may be aimed at protection against a pest selected from whiteflies (Hemiptera: Aleyrodidae), Tuta absoluta (Meyrick) (Lepidoptera:

Gelechiidae, thrips, leafminers, spidermites, lepidopteran pests.

According to a preferred embodiment of the method for crop protection a number of further bioactive compounds is provided to the zoophytophagous insect. A bioactive compound may be any compound which has an effect on the physiology of the zoophytophagous insect. Within this invention a number of is to be understood to mean one or more. The bioactive compound may have a positive or negative effect on the physiology of the zoophytophagous insect. Positively acting bioactive compounds promote or improve normal physiological conditions and/or functions of the

zoophytophagous insect and may for example be selected from from natural amino acids, natural proteins, vitamins. Negatively acting bioactive compounds may for example reduce or inhibit normal physiological conditions and/or functions of the zoophytophagous insect. It is preferred that the negatively acting bioactive compounds reduces the population development of the population of the zoophytophagous insect. Bioactive compounds reducing the population development of the zoophytophagous insect may for example be selected from agents that reduce fecundity, or reduce longevity or an insecticide, preferably a biological insecticide. The further provision of a bioactive compounds reducing the population development of the zoophytophagous insect, may be helpful at times where it is beneficial to decrease the number of zoophytophagous insects in the crop. For example at the end of the growing season or at times of explosive population development of the zoophytophagous insect. Under such circumstances the provision of an insecticide to the zoophytophagous insect may help to control its population development. Suitable insecticides may be selected from thiacloprid, pyrethrenins, piperonylbutoxide or imidacloprid. A further aspect of the invention relates to a method for reducing plant damage caused by a zoophytophagous insect. As already discussed above, both zoophytophagous insects regarded as beneficial and zoophytophagous insects regarded as pests can cause plant damage. According to this aspect of the invention plant damage by a

zoophytophagous insect may be reduced by providing on the plant an amount of a carbohydrate source.

For this aspect of the invention similar preferred and alternative embodiments, as discussed in connection to the method for rearing may be defined. The benefits of these preferred and alternative embodiments within this aspect of the invention will be evident from the discussion in connection to the method for rearing and/or the method for crop protection.

Yet another aspect of the invention relates to the use of a carbohydrate source as a food source for a zoophytophagous insect. The use of a carbohydrate source as a food source for a zoophytophagous insect has not been disclosed in the prior art, particularly not when used in the presence of plant tissue or prey, which are food source known to be well accepted by zoophytophagous insects. The inventors of the present invention have found that such use of a carbohydrate source as a food source for a zoophytophagous insect results in surprising effects. According to an embodiment of this aspect of the invention the surprising effect is faster establishment of a population of the

zoophytophagous insect in a crop. According to an alternative embodiment of this aspect of the invention the surprising effect is reduction of the immature development time and/or increasing the mean body weight of the zoophytophagous insect.

Immature development may be measured as the number of days required for development from fresh hatchling (day of hatch from egg) to the last moult (last nymphal stage to adult). The mean body weight may be determined of freshly moulted adults. In view of differences in development of males and females it is preferred to determine the mean body weight per sex.

Also for this aspect of the invention similar preferred and alternative embodiments, as discussed in connection to the method for rearing and the method for crop protection may be defined. The benefits of these preferred and alternative embodiments within this aspect of the invention will be evident from the discussion in connection to these methods.

A further aspect of the invention relates to a composition comprising a carbohydrate source for use in reducing the immature development time and/or increasing the mean body weight of a zoophytophagous insect. The composition is used particularly as a food source for a zoophytophagous insect in the presence of a first food source, said first food source being selected from plant tissue or prey, which are food sources known to be well accepted by zoophytophagous insects.

Also for this aspect of the invention similar preferred and alternative embodiments, as discussed in connection to the method for rearing and the method for crop protection may be defined. The benefits of these preferred and alternative embodiments within this aspect of the invention will be evident from the discussion in connection to these methods.

The invention will now be further illustrated with the following experiments.

Experiments

General Methods

Plants

Pesticide-free tomato plants (30 cm high) variety "Optima" (from Seminis Vegetable Seeds, Inc., Almeria, Spain) were used in all the assays. Tomato plants were planted on 8 x 8 x 8 cm pots.

Nesidiocoris tenuis

Nesidiocoris tenuis individuals came from commercial sources (NESIBUG ^® ; Koppert Biological Systems, S.L.,Aguilas, Murcia, Spain). Each commercial bottle contained approximately 500 3-days old specimens. To obtain newly emerged nymphs, four tomato plants and 500 N. tenuis from a NESIBUG ^® bottle were placed in 40 x 25 x 25 cm methacrylate boxes. Eggs of Ephestia kuehniella Zeller (Lep.:Pyralidae) were added ad libitum to feed N. tenuis. The boxes were placed in a climatic chamber (25 ± 1°C, 60 ± 5 % HR, 16:8 h L:D photoperiod) during five days. Then, N. tenuis adults were removed and the aerial parts of the plants were checked for oviposition scars and cut into small sections each containing one egg of N. tenuis. Plant parts were placed individually in Petri dishes (6 cm in diameter) and examined daily until nymphs emerged. These first nymphal instars were used in the following assays. Experiment 1

Effect of sugar addition on nymphal development, fertility and prey consumption of the model organism N. tenuis.

Sugars added with ependorfs

To determine the effect of sugar (sucrose) addition in N. tenuis nymphal development, newly emerged nymphs were individualized in Petri dishes (6 cm in diameter) with a tomato leaf disc and fed with six different treatments: three sugar treatments (1 M sucrose solution, 0.5 M sucrose and water) with and without E. kuehniella eggs (Table 1). Each treatment was replicated 20 times. The sugar sources were added in 1.5 ml eppendorfs sealed with cotton and they were replaced every three days. Nesidiocoris tenuis used the cotton to feed on the sugars. Ephestia kuehniella eggs were added ad libitum and renewed daily. The number of eggs added each day depended on the nymphal instar of N. tenuis (O. Molla, IVIA Moncada, Spain; personal communication) (Ni -> 5 eggs, N ₂ -> 10 eggs, N ₃ -> 15 eggs, N ₄ -> 20 eggs and N ₅ -> 25 eggs). Eggs were offered in the adhesive part of a Post-it (1 cm ² ) (Post-it® 3M. Madrid, Spain), where the eggs stuck. A young tomato leaf disc per dish was added and renewed daily. The Petri dishes were placed in a climatic chamber (25 ± 1°C, 60 ± 5 % HR, 16:8 L:D photoperiod). The petri dishes were checked daily to determine the N. tenuis instar, egg consumption and mortality. These data were used to calculate the nymphal developmental time, the survivorship of each nymphal instar and the number of eggs consumed (the last only for those treatments where eggs had been added). Sugars added with Hydrocapsules

Once we had analyzed the previous results, we decided to determine whether N. tenuis was able to obtain the sugars from Hydrocapsules produced by Hydrocapsule Inc. (Jasper, Georgia (USA)) and the effect of their addition on the immature development of N. tenuis when it also feeds on E. kuehniella eggs. These microcapsules have a polymeric shell for the preservation, storage, and controlled delivery of sugars. The Hydrocapsules used in this assay were 1-2 mm in diameter. The core of the Hydrocapsules was 0.5 M sucrose solution (this concentration was selected from the previous results, in prep.).

Newly emerged nymphs were individualized in Petri dishes (6 cm in diameter) with a tomato leaf disc and fed with two treatments: E. kuehniella with and without Hydrocapsules. Each treatment was replicated 50 times. The eggs and the tomato leaf disc were added and renewed as above. The hydrocapsules were added ad libitum (0.6 g per dish and day) and renewed daily. The Petri dishes were undisturbed placed in a climatic chamber (25 ± 1°C, 60 ± 5 % HR, 16:8 h L:D photoperiod).

The petri dishes were checked daily to determine N. tenuis instar, egg consumption and mortality. These data were used to calculate the nymphal developmental time, the survivorship of each nymphal instar and the number of eggs consumed. Once they reached the adult stage, they were transferred in couples to a plastic cup (370 cm ³ ) with an apical shoot of tomato (14 cm approximately) with E. kuehniella eggs (300-400 eggs) and Hydrocapsules (0.6 g) ad libitum. To maintain the shoot turgor, the plastic cup was introduced in a smaller plastic glass (230 cm ³ ) with water. Tomato shoots reached the water through a hole in the base of the first glass (Sanchez et al. 2009). The glass with tomato and N. tenuis was sealed with muselin and a rubber band. Each couple was transferred to a new glass with food and a new tomato shoot every five days during the 20 days that last the assay. Once the adults had been removed, the plastic glasses with the tomato shoot were kept undisturbed under the same climatic conditions than the immatures during ten days. Then, the number of nymphs per glass (replicate) was counted. These data were used to determine the fertility of both treatments.

Parameters from the studies on the effect of sugar on N. tenuis biology and N. tenuis phytophagy were compared using one-way ANOVA, and the means were compared using the Tukey test (P<0.05). For the biology study with Hydrocapsules, treatments were compared using Student's t-test (.Ρ<0.05). Results

As shown in figure 1 the number of E. kuehniella eggs preyed by N. tenuis to complete their nymphal development was significantly lower in the treatment where the 0.5 M sucrose Hydrocapsules were added (T ₈₈ = 14.00, P <0.0001) .

The offspring of adults emerged from the nymphs fed on E. kuehniella eggs 0.5 M sucrose Hydrocapsules and kept during their oviposition period with the same diet was significantly higher than adults without access to 0.5 M sucrose Hydrocapsules (t ₃₆ = 6.601, P <0.0001) (Figure 2).

Experiment 2

Effect of sugar addition on N. tenuis phytophagy

To determine whether the addition of sugar sources on tomato plants may influence the intensity of phytophagy by zoophytophagous organisms N. tenuis, was used as a model organism. The number of necrotic rings per plant was counted in plants with and without sugars added. Nesidiocoris tenuis produces necrotic rings when it feeds on tomato plants (Calvo et al. 2009, Arno et al. 2010a, Sanchez and Lacasa 2008). This parameter has been used to evaluate the damages produced by N. tenuis under field conditions (Calvo and Urbaneja 2004). This assay was conducted in a greenhouse at Instituto Valenciano de Investigaciones Agrarias (IVIA) (Montcada, Valencia). The environmental conditions were 25 ± 2 °C, 65 ± 10% RH and natural photoperiod.

Two amounts of sugar sources (one or three eppendorfs per plant) and a control (without sugar addition) were compared. Sugar sources were placed in the apical part of the tomato plant. Eppendorfs were filled with 0.5 M sucrose solution and tapped with cotton as in the previous assay.

Each replicate consisted of a plastic cage (55 x 30 x 30 cm) with two lateral holes and one in the upper part (10 X 10 cm) sealed with muslin to permit ventilation. One of the lateral holes had a muslin sleeve to manipulate the interior part of the cage. In each cage, we placed a tomato plant with the eppendorfs (0, 1 or 3 depending on the treatment) and a couple of N. tenuis (3-days old). Every two days, the plants were watered and the eppendorfs renewed. Each treatment was replicated 16 times (a total of 48 cages). Seven days later, eight plants of each treatment were taken to the lab. Once there, the number of necrotic rings and N. tenuis nymphs per plant were counted under binocular. This procedure was repeated seven days later with the remaining plants. The number of nymphs and adults of N. tenuis and the number of necrotic rings were analyzed using a Generalized Linear Mixed Model with repeated measurements using IBM® SPSS® statistics, version 19.0.0 (SPSS Inc IBM Company 2010) assuming a Poisson probability distribution. Treatment was considered as a fixed factor and date as a random one. When significant differences were found, pairwise comparisons of the fixed factor levels were performed with the least significant difference (LSD) post hoc test (PO.05).

Results

Seven days after the N. tenuis release, the number of necrotic rings in the treatment where sucrose was not offered was significantly higher (~ 4 rings per plant) than in treatments where 1 and 3 ependorfs filled with 0.5 M sucrose were placed (~ 1 and ~ 2 rings, respectively) (t ₂₃ = 9.620, P = 0.0011) (Figure 7a). These differences were maintained in the second sampling at day 14 (t ₂₃ = 6.790, P = 0.0053) (Figure 7.b).

Significant differences between treatments were also found in the offspring of N. tenuis at day 14 (Figure 8) (t ₂₃ = 5.764, P = 0.0101); with a higher number of N. tenuis nymphs in the treatment with 3 ependorfs (~ 5 nymphs) than in the treatments with 1 (~ 2) and 0 ependorfs (~ 1).

Experiment 3

Effect of sugar availability in the establishment of N. tenuis

It was tested whether sugar addition influences consumption of the present diet used to establish N. tenuis in the field (E. kuehniella eggs). In this test four treatments were assayed:

1 Addition of 0.1 grams of E. kuehniella eggs per plant (recommended to establish N. tenuis in the field (Calvo et al. 2010).

2 Addition of 0.05 gr of frozen E. kuehniella eggs and 0.6 g of Hydrocapsules (sucrose 0.5M) per plant. This treatment will determine whether sugar addition may reduce the amount of E. kuehniella eggs needed to establish N. tenuis. 3. Addition of 0.6 g of Hydrocapsules (0.5 M sucrose solution) per plant.

4. Control: without addition of E. kuehniella eggs and Hydrocapsules.

For this assay, 60 ^χ 60 ^χ 60 cm white BugDorm-2 cages (MegaView Science Education Services Co., Taiwan) held on a greenhouse bench were used. Five tomato plants and five couples of N. tenuis (3-days old) were introduced per cage (one couple per plant). Each treatment was replicate four times (a total of 16 cages).

After the release and after 14 days, one plant of each replicate was taken to the lab. Once there, the number of N. tenuis nymphs per plant were counted under binocular. The number of nymphs and adults of N. tenuis were analyzed using a Generalized Linear Mixed Model with repeated measurements using IBM® SPSS® statistics, version 19.0.0 (SPSS Inc IBM Company 2010) assuming a Poisson probability distribution. Treatment was considered as a fixed factor and date as a random one. When significant differences were found, pairwise comparisons of the fixed factor levels were performed with the least significant difference (LSD) post hoc test (P<0.05).

Results

The results of the counts of N. tenuis nymphs per plant (mean ± SE) are presented in figure 4.

Experiment 4

Effect of sugar addition on prey consumption of the model organism M. pygmaeus.

Young nymphs of Macrolophus pygmaeus were offered either pure water or sugar- solutions, in addition to frozen eggs of the Lepidopteran Ephestia kuehniella. Survival and development of nymphs and weight of the offered eggs were determined twice a week (a control had no nymphs). With this method the relation between the

consumption rate of lepidopteran eggs and the diet offered, could be determined. Setup

Macrolophus pygmaeus has been mass-reared for several generations (>100) at the Koppert plant. The system supplies frozen Ephestia eggs as a food source and plant material as moisture source and oviposition substrate (replaced twice a week). Fresh nymphs (0-3 days old) were collected at the beginning of the trial from this mass- rearing. The trial was performed in a petri-dish (0 7,5 cm,h = 3 cm) with a ventilated gauze lid. 35 ±1 nymphs were placed in each unit on day 0, together with the treatment diet. Ephestia was offered on sticky labels (12x18 mm), to all rearing units. Each label prepared in this way contained an amount of ±40 mg. Twice a week the label was weighed and refreshed, and from day 10 onwards 2 labels were offered. Water or sugarwater was supllied as small packages covered with a polymer film. Normal tapwater (pH 7-8) was used and the sugars were saccharose (table sugar (produced from beet sugar) obtained from a local grocery store ) and trehalose (from Merck (108216)). Sugar-solutions of 0.5 M were made by dissolving the required amount of the sugar in tap water.

There were 3 replicates per treatment and the treatments were as follows: A. no nymphs + water (negative control to weigh dessication of eggs), B. water (control), C.

saccharose-solution and D. trehalose-solution. Twice a week the whole diet was replaced and evaluated (weighing of labels) and the nymphs were counted. Also, the number of adults was counted. Climatic conditions were 24±1°C , 65±5% RH and the light regime was 16L:8D. After 17 days the trial was stopped.

Calculation of consumption was as follows: the nett weight loss of Ephestia (= weight loss per unit - average weight loss of treatment A) was divided by the average number of live nymphs (= start + end number: 2) at each half week. The cumulative sum of consumption per unit was averaged per treatment and expressed as a percentage to obtain the shown graph. Data were statistically analysed using a one-way ANOVA (a = 0,05).

Results

The results are shown in figure 5. The results presented show that cumulative Ephestia consumption per treatment was highest for the water treatment (100%), followed by the trehalose treatment (73% ) and lowest for the saccharose treatment (70%).

Survival and development time were not significantly different. Experiment 5

Effect of sugar addition on fecundity of the model organism M. pygmaeus.

Fecundity of Macrolophus pygmaeus was assessed according to a direct method described by Callebaut et al (2004). The diet consisted either of pure frozen Ephestia kuehniella eggs or the same eggs supplemented with powdered saccharose sugar, both in combination with plant material as a moisture source and oviposition substrate. After 1 week of maturation the adults were dissected and the resulting egg counts provide a prediction of life-time fecundity.

Setup

Fresh nymphs (0-3 days old) were collected from this mass-rearing and reared until adulthood in the laboratory according the control in experiment 4. This experiment was started with fresh adults (0-3 days) and the experiment was performed in a petri-dish (0 7,5 cm,h = 3 cm) with a ventilated gauze lid. 10-15 adults of both sexes were placed in each unit on day 0, together with the treatment diet and plant material as a moisture source and oviposition substrate. Ephestia was offered on 2 sticky labels (prepared as in trial 1), to all rearing units and replaced twice a week. Powdered sugar was obtained from retail (Van Gilse) and contained 99.2 % sucrose and E341. The treatments were as follows: Ephestia eggs + powdered saccharose sugar (50:50 b/w), and Ephestia eggs.

Per treatment 26 or 27 females were dissected. Climatic conditions were 24±1°C , 65±5% RH and the light regime was 16L:8D. After 6 days of maturation under the treatment condition dissection was performed on all survived females. The number of oocytes per category (based on size and thus developmental stage: whole, half, quarter) were counted per female. An weighed average per female was calculated and the data were analysed using one-way ANOVA (a = 0,05). Results

The results are presented in figure 6. As shown in this figure the average oocyte counts per female for Ephestia eggs + powdered saccharose sugar was 16.9 and 12.6 for Ephestia eggs. The statistical significance of the results is indicated by the p-value op 0,015. The 34% higher egg-load of females on the diet supplemented with powdered sugar suggests a higher egg-laying capacity.

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