Identification and evaluation of Trichogramma parasitoids for biological pest control

Egg parasitoids of the genus Trichogramma are used as biological control agents against lepidopterous pests. From the 180 species described world-wide, only 5 have large scale application. The development of better methods to select other Trichogramma species/strains is necessary for a more effective use of these wasps against target pests. The main aim of this thesis is to investigate criteria and methods for identification and selection of Trichogramma species/strains for biological control. In chapter 1, I give a short review on biology, research and use of Trichogramma , state the research aims and present the outline of the thesis.One of the first problems researchers are confronted with when working on Trichogramma is their identity. Along with the classical methods, simple and practical methodologies of species identification are needed. The use of molecular methods to characterise and identify five Portuguese Trichogramma species is described in chapter 2 . These methods are: 1) electrophoresis of PCR amplified DNA internal transcribed spacer 2 (ITS-2), followed by digestions by means of restriction enzymes and 2) esterase electrophoresis. Five Trichogramma species occurring in Portugal, T. bourarachae, T. cordubensis, T. evanescens, T. pintoi and T. turkestanica can be easily distinguished from each other by means of dichotomous keys that were constructed based on the two methods.The two methods may be used together with other identification methods for field specimens or be used on its own for detection of possible laboratory contaminations. The specificity of mating attempts was investigated as an additional tool to quickly distinguish between Trichogramma species ( chapter 3 ). Although mating attempts occurred more often between conspecifics, they were not always species-specific. Two compounds that might be involved in mate-specific recognition in Trichogramma were characterised. These two compounds are produced by virgin females and were not found in mixed groups of males and presumably mated females or in groups of males only. We concluded that the specificity of mating attempts cannot be used as a general taxonomic tool for Trichogramma .From the point of view of biological control only the females are useful, because they kill the host eggs. We know of several Trichogramma species in which virgin females produce female offspring (thelytoky). In most of these cases thelytoky is related to the presence of Wolbachia , an endosymbiotic bacterium. In some cases all female wasps in a population are infected with the bacteria (fixed population), while in other cases infected and uninfected females coexist (mixed population). The hypothesis that in fixed populations the effect of the bacteria on Trichogramma female fitness is less negative than in mixed populations was tested in chapter 4 . In these experiments two fixed Trichogramma lines of T. cordubensis and T. oleae and four mixed lines, one of T. deion and three of T. kaykai , were tested for several female fitness parameters.Each of these original thelytokous lines was compared with a laboratory-generated sexual (=arrhenotokous) line, which had been produced by killing the Wolbachia bacteria using antibiotics. Daughter production was higher for the thelytokous fixed lines than for their sexual counterparts, while the opposite was found for three of the four mixed lines studied. As expected, the negative impact of Wolbachia on total offspring numbers was much more severe for the mixed lines than for the fixed ones.In addition to the infection status of the wasp populations, fixed versus mixed, there are other factors that can influence the reproductive success of the wasps. One of them is the females' ability to search for eggs to parasitize. The dispersal of two thelytokous lines of T. cordubensis was assessed in a laboratory chamber and in a greenhouse ( chapter 5 ). This chamber was especially designed and tested as a tool for Trichogramma screening. The Trichogramma line which dispersed more in the chamber also dispersed more in the greenhouse, suggesting that the chamber might be a useful tool for a pre-evaluation of Trichogramma strains. Next, the biological control potential of Wolbachia -infected and of uninfected Trichogramma was investigated ( chapter 6 ).Fecundity and dispersal of T. cordubensis and T. deion were measured in the laboratory and in the greenhouse. Sexual lines had a higher fecundity than their thelytokous counterparts. The arrhenotokous lines dispersed more in the laboratory chamber but the opposite effect or no difference was found in the greenhouse. Calculations show that by releasing hundred adult wasps of both lines, thus including arrhenotokous males in the sexual line, more eggs are parasitised by the thelytokous wasps. Therefore, in spite of their lower individual female fecundity, thelytokous lines have a better potential for biological control than their sexual counterparts.Trichogramma females do not only parasitise lepidopteran eggs but also parasitise eggs of some beneficial insects. To determine which Trichogramma species is the least detrimental to the beneficial insect Chrysoperla carnea , the degree of successful parasitisation of Helicoverpa armigera (a pest insect) and Chrysoperla carnea , by the five Portuguese Trichogramma species was studied in the laboratory ( chapter 7 ). The fraction H. armigera/C. carnea eggs parasitised differed between Trichogramma species. The average number of parasitised eggs per female in 24 h by T. pintoi and T. bourarachae was 10 of H. armigera and about 0.5 of C. carnea . For the other three Trichogramma species ( T. cordubensis , T. evanescens and T. turkestanica ) these averages varied from 6 to 11 H. armigera eggs and from 3 to 4 C. carnea eggs.Total adult offspring production, contacts with hosts, secondary clutch size and sex ratio of each Trichogramma line were determined as well. The results obtained show that sympatric Trichogramma may parasitise target and non-target species in different proportions. If this difference corresponds to the field situation, simple laboratory tests could be performed to select not only effective biological control agents, but also species that are the least detrimental to non-target hosts. Trichogramma bourarachae and T. pintoi are the most promising candidates for the control of H. armigera in agroecosystems where Chrysopidae form an important part of the natural enemy assemblage. In chapter 8 , the most important research findings are discussed in a broader context.</p

Identification and evaluation of Trichogramma parasitoids for biological pest control

Egg parasitoids of the genus Trichogramma are used as biological control agents against lepidopterous pests. From the 180 species described world-wide, only 5 have large scale application. The development of better methods to select other Trichogramma species/strains is necessary for a more effective use of these wasps against target pests. The main aim of this thesis is to investigate criteria and methods for identification and selection of Trichogramma species/strains for biological control. In chapter 1, I give a short review on biology, research and use of Trichogramma , state the research aims and present the outline of the thesis.One of the first problems researchers are confronted with when working on Trichogramma is their identity. Along with the classical methods, simple and practical methodologies of species identification are needed. The use of molecular methods to characterise and identify five Portuguese Trichogramma species is described in chapter 2 . These methods are: 1) electrophoresis of PCR amplified DNA internal transcribed spacer 2 (ITS-2), followed by digestions by means of restriction enzymes and 2) esterase electrophoresis. Five Trichogramma species occurring in Portugal, T. bourarachae, T. cordubensis, T. evanescens, T. pintoi and T. turkestanica can be easily distinguished from each other by means of dichotomous keys that were constructed based on the two methods.The two methods may be used together with other identification methods for field specimens or be used on its own for detection of possible laboratory contaminations. The specificity of mating attempts was investigated as an additional tool to quickly distinguish between Trichogramma species ( chapter 3 ). Although mating attempts occurred more often between conspecifics, they were not always species-specific. Two compounds that might be involved in mate-specific recognition in Trichogramma were characterised. These two compounds are produced by virgin females and were not found in mixed groups of males and presumably mated females or in groups of males only. We concluded that the specificity of mating attempts cannot be used as a general taxonomic tool for Trichogramma .From the point of view of biological control only the females are useful, because they kill the host eggs. We know of several Trichogramma species in which virgin females produce female offspring (thelytoky). In most of these cases thelytoky is related to the presence of Wolbachia , an endosymbiotic bacterium. In some cases all female wasps in a population are infected with the bacteria (fixed population), while in other cases infected and uninfected females coexist (mixed population). The hypothesis that in fixed populations the effect of the bacteria on Trichogramma female fitness is less negative than in mixed populations was tested in chapter 4 . In these experiments two fixed Trichogramma lines of T. cordubensis and T. oleae and four mixed lines, one of T. deion and three of T. kaykai , were tested for several female fitness parameters.Each of these original thelytokous lines was compared with a laboratory-generated sexual (=arrhenotokous) line, which had been produced by killing the Wolbachia bacteria using antibiotics. Daughter production was higher for the thelytokous fixed lines than for their sexual counterparts, while the opposite was found for three of the four mixed lines studied. As expected, the negative impact of Wolbachia on total offspring numbers was much more severe for the mixed lines than for the fixed ones.In addition to the infection status of the wasp populations, fixed versus mixed, there are other factors that can influence the reproductive success of the wasps. One of them is the females' ability to search for eggs to parasitize. The dispersal of two thelytokous lines of T. cordubensis was assessed in a laboratory chamber and in a greenhouse ( chapter 5 ). This chamber was especially designed and tested as a tool for Trichogramma screening. The Trichogramma line which dispersed more in the chamber also dispersed more in the greenhouse, suggesting that the chamber might be a useful tool for a pre-evaluation of Trichogramma strains. Next, the biological control potential of Wolbachia -infected and of uninfected Trichogramma was investigated ( chapter 6 ).Fecundity and dispersal of T. cordubensis and T. deion were measured in the laboratory and in the greenhouse. Sexual lines had a higher fecundity than their thelytokous counterparts. The arrhenotokous lines dispersed more in the laboratory chamber but the opposite effect or no difference was found in the greenhouse. Calculations show that by releasing hundred adult wasps of both lines, thus including arrhenotokous males in the sexual line, more eggs are parasitised by the thelytokous wasps. Therefore, in spite of their lower individual female fecundity, thelytokous lines have a better potential for biological control than their sexual counterparts.Trichogramma females do not only parasitise lepidopteran eggs but also parasitise eggs of some beneficial insects. To determine which Trichogramma species is the least detrimental to the beneficial insect Chrysoperla carnea , the degree of successful parasitisation of Helicoverpa armigera (a pest insect) and Chrysoperla carnea , by the five Portuguese Trichogramma species was studied in the laboratory ( chapter 7 ). The fraction H. armigera/C. carnea eggs parasitised differed between Trichogramma species. The average number of parasitised eggs per female in 24 h by T. pintoi and T. bourarachae was 10 of H. armigera and about 0.5 of C. carnea . For the other three Trichogramma species ( T. cordubensis , T. evanescens and T. turkestanica ) these averages varied from 6 to 11 H. armigera eggs and from 3 to 4 C. carnea eggs.Total adult offspring production, contacts with hosts, secondary clutch size and sex ratio of each Trichogramma line were determined as well. The results obtained show that sympatric Trichogramma may parasitise target and non-target species in different proportions. If this difference corresponds to the field situation, simple laboratory tests could be performed to select not only effective biological control agents, but also species that are the least detrimental to non-target hosts. Trichogramma bourarachae and T. pintoi are the most promising candidates for the control of H. armigera in agroecosystems where Chrysopidae form an important part of the natural enemy assemblage. In chapter 8 , the most important research findings are discussed in a broader context

Laboratory bioassay and greenhouse evaluation of Trichogramma cordubensis strains from Portugal

A simple, inexpensive chamber was developed and tested as an evaluative tool to monitor Trichogramma cordubensis dispersal in the laboratory. The chamber consisted of a continuous, winding channel which was cut into an aluminum block. Wasps were released at one end of the channel and allowed to walk in the channel for 21 h and to parasitize Mamestra brassicae eggs placed 3.4 m from the point of wasp introduction. Comparisons between two T. cordubensis populations demonstrated that one population (TCM) dispersed more in the chamber and located host eggs more successfully than the other population (TCD). Subsequent greenhouse releases confirmed that the TCM population dispersed more readily and had significantly higher parasitism rates on sentinel Ephestia kuehniella eggs on tomato plants. The potential utilization of this chamber as a tool to evaluate quality of Trichogramma populations, mainly dispersal activity, is discussed