Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Lactobacillus mucosae DPC 6426 as a bile-modifying and immunomodulatory microbe
Ryan, Paul M. ; Stolte, Ellen H. ; London, Lis E.E. ; Wells, Jerry M. ; Long, Sarah L. ; Joyce, Susan A. ; Gahan, Cormac G.M. ; Fitzgerald, Gerald F. ; Ross, R.P. ; Caplice, Noel M. ; Stanton, Catherine - \ 2019
BMC Microbiology 19 (2019)1. - ISSN 1471-2180
Bile acid - Bile salt hydrolase (BSH) - CVD - Exopolysaccharide - Hypercholesterolaemia

Background: Lactobacillus mucosae DPC 6426 has previously demonstrated potentially cardio-protective properties, in the form of dyslipidaemia and hypercholesterolemia correction in an apolipoprotein-E deficient mouse model. This study aims to characterise the manner in which this microbe may modulate host bile pool composition and immune response, in the context of cardiovascular disease. Lactobacillus mucosae DPC 6426 was assessed for bile salt hydrolase activity and specificity. The microbe was compared against several other enteric strains of the same species, as well as a confirmed bile salt hydrolase-active strain, Lactobacillus reuteri APC 2587. Results: Quantitative bile salt hydrolase assays revealed that enzymatic extracts from Lactobacillus reuteri APC 2587 and Lactobacillus mucosae DPC 6426 demonstrate the greatest activity in vitro. Bile acid profiling of porcine and murine bile following incubation with Lactobacillus mucosae DPC 6426 confirmed a preference for hydrolysis of glyco-conjugated bile acids. In addition, the purified exopolysaccharide and secretome of Lactobacillus mucosae DPC 6426 were investigated for immunomodulatory capabilities using RAW264.7 macrophages. Gene expression data revealed that both fractions stimulated increases in interleukin-6 and interleukin-10 gene transcription in the murine macrophages, while the entire secretome was necessary to increase CD206 transcription. Moreover, the exopolysaccharide elicited a dose-dependent increase in nitric oxide and interleukin-10 production from RAW264.7 macrophages, concurrent with increased tumour necrosis factor-α secretion at all doses. Conclusions: This study indicates that Lactobacillus mucosae DPC 6426 modulates both bile pool composition and immune system tone in a manner which may contribute significantly to the previously identified cardio-protective phenotype.

Virulence aspects of Listeria monocytogenes LO28 high pressure-resistant variants
Boeijen, K.H. van; Casey, P.G. ; Hill, C. ; Moezelaar, R. ; Zwietering, M.H. ; Gahan, C.G.M. ; Abee, T. - \ 2013
Microbial Pathogenesis 59-60 (2013). - ISSN 0882-4010 - p. 48 - 51.
high hydrostatic-pressure - ctsr - stress - kinetics - environment - strains - scott - acid - food
High pressure treatment is a novel food processing approach for reducing pathogens in foods and food ingredients. However, relatively little is known about the pathogenic potential of organisms that survive the treatment. Twelve previously isolated and characterized variants of Listeria monocytogenes LO28 obtained after a high pressure treatment were assessed for their virulence potential and antibiotic susceptibility. Ten variants showed attenuated virulence while two variants retained full virulence in a mouse model of infection. Seven of the attenuated variants demonstrated a reduction in virulence factor activity. Compared to the wild type, all variants exhibited similar or increased susceptibility to multiple antibiotics commonly used in listeriosis treatment. Keywords: High hydrostatic pressure; Mouse model; Diversity; Pathogenicity; Antibiotics; CtsR
Quantifying microorganisms during biooxidation of arsenite and bioleaching of zinc sulfide
Dinkla, I.J.T. ; Gonzalez Contreras, P.A. ; Gahan, C.S. ; Weijma, J. ; Buisman, C.J.N. ; Henssen, M.J.C. ; Sandström, A. - \ 2013
Minerals Engineering 48 (2013). - ISSN 0892-6875 - p. 25 - 30.
acidithiobacillus-ferrooxidans - iron oxidation - ferrous iron - ferric iron - leptospirillum - bacteria - cultures - thiobacillus - chalcopyrite - arsenate
The development of molecular tools for the detection and quantification of both species as well as functional traits, aids in a better understanding and control of microbial processes. Presently, these methods can also be used to assess the activity of these organisms or functions, even in complex ecosystems and difficult matrices such as ores and low pH samples. In this paper we present the versatility of one of these tools, Q-PCR, to allow accurate and fast insight in changes in two types of microbial processes representing two ways in which microbes can interact with metals, bioleaching and bioprecipitation. Using the Q-PCR technique it was possible to identify and quantify the thermoacidophilic archaeon Acidianus sp. to be the main microbial strain responsible for biooxidation of arsenite in a low pH reactor. The method was also used to study the dynamics between the iron oxidizing and sulfur oxidizing acidophiles during bioleaching of a zinc concentrate in a batch reactor system and showed that the iron oxidizer Leptospirillum ferriphilum that dominated the starting culture disappeared upon addition of the concentrate. Gradually, bacterial activity was regained starting with growth of sulfur oxidizers and at later stage iron oxidizers started to grow. Molecular analysis can be used to direct research to the relevant organisms involved and concentrate on improving their application (in the arsenite case Acidianus sp.) or in understanding appearances and disappearances of microorganisms (during leaching of zinc concentrate the disappearance of Leptospirillum after high inoculation levels) in order to allow optimization of leaching efficiencies at the lowest (oxygen) costs.
Human Microbiota in Health and Disease
Vos, W.M. de; Engstrand, L. ; Drago, L. ; Reid, G. ; Schauber, J. ; Hay, R. ; Mendling, W. ; Schaller, M. ; Spiller, R. ; Gahan, C.G.M. ; Rowland, I. - \ 2012
SelfCare Journal 3 (2012)6. - ISSN 2042-7018 - p. 1 - 68.
Each human body plays host to a microbial population which is both numerically vast (at around 1014 microbial cells) and phenomenally diverse (over 1,000 species). The majority of the microbial species in the gut have not been cultured but the application of culture-independent approaches for high throughput diversity and functionality analysis has allowed characterisation of the diverse microbial phylotypes present in health and disease
Biological control of Trialeurodes vaporariorum by Encarsia formosa on tomato in unheated greenhouses in the high altitude tropics
Vis, R.M.J. de; Lenteren, J.C. van - \ 2008
Bulletin of Insectology 61 (2008)1. - ISSN 1721-8861 - p. 43 - 57.
parasite-host relationship - commercial glasshouse - population-dynamics - aphelinidae - aleyrodidae - netherlands
Biological control of Trialeurodes vaporariorum (Westwood) by Encarsia formosa Gahan was tested during three consecutive production cycles (16-28 weeks) on a beef tomato (Solanum lycopersicum L.) crop in a glasshouse and a plastic greenhouse on the Bogota Plateau in Colombia. During the course of this study over the period 1997-1999, the mean temperature was around 16 °C in the plastic greenhouse and around 17 °C in the glasshouse. E. formosa was introduced at a rate of 3 adults per m2 per week in the 1997 production cycle, and at a rate of 3 and 5 pupae per m2 per week in 1998 and 1999, respectively. In 1997, the adult whitefly population increased exponentially to a peak of 76 adults per plant in the plastic greenhouse, while the whitefly population in the glasshouse reached a peak of only 12 adults per plant. The percentage parasitism fluctuated between 42 and 82% in the glasshouse and between 28 and 47% in the plastic greenhouse. In 1998, the T. vaporariorum population could not be brought under control in both greenhouses and reached a peak of 80 and 53 T. vaporariorum adults per plant in the plastic greenhouse and the glasshouse, respectively. Parasitism fluctuated between 55 and 97% in the glasshouse and between 32 and 84% in the plastic greenhouse. In 1999, biological control was successful in both greenhouses. Most of the time, populations of T. vaporariorum were lower than 1.2 adults per plant and parasitism by E. formosa was 80% or higher. We suggest that the higher temperature is the main reason for better parasitism in the glasshouse when compared to the plastic greenhouse. The successful results of 1999 show that biological control is possible under the short day and low temperature conditions of greenhouses situated in the high altitude tropics such as the Bogota Plateau. Recommendations are given for the application of E. formosa based on the results of these experiments.
A PrfA-regulated bile exclusion system (BilE) is a novel virulence factor in Listeria monocytogenes
Sleator, R.D. ; Wemekamp-Kamphuis, H.H. ; Gahan, C.G.M. ; Abee, T. ; Hill, C. - \ 2005
Molecular Microbiology 55 (2005)4. - ISSN 0950-382X - p. 1183 - 1195.
osmolyte transporters betl - stress-response - salt hydrolase - escherichia-coli - bacterial stress - gene - identification - growth - opuc - expression
The ability to colonize the gall bladder has recently been shown to be an important feature of virulent Listeria monocytogenes (J. Hardy, K. P. Francis, M. DeBoer, P. Chu, K. Gibbs, C. H. Contag. Science 303: 851853, 2004). We suggest that the cytotoxic effects of bile may be increased upon release from the gall bladder into the upper small intestine, and report the identification of a novel bile exclusion system which plays an essential role in intestinal colonization and virulence of L. monocytogenes. In silico analysis of the L. monocytogenes EGDe genome revealed a two-gene operon (formerly opuB) exhibiting significant sequence similarity to members of the betaine carnitine choline transporter (BCCT) family. The operon, herein designated bilE (bile Exclusion) is preceded by consensus A- and B-dependent promoter-binding sites and is transcriptionally upregulated at elevated osmolarities and reduced temperatures (stresses known to induce sigB). Furthermore, a significant reduction in the level of bilE transcription was observed in the absence of B. In addition, we demonstrate an important role for PrfA, the master regulator of virulence potential in L. monocytogenes, in coordinating bilE expression. Computational structural analysis suggests that, rather than functioning as a compatible solute uptake system as was previously believed, BilE is more likely to be an exclusion system, a conclusion substantiated by radiolabelled bile accumulation studies. In addition, functionally inactivating BilE resulted in a five-log reduction in the ability of the bacterium to tolerate lethal concentrations of bovine bile (oxgall) and also significantly increased sensitivity to physiological concentrations of human bile, a phenotype which translates to a significant reduction in virulence potential when administered to a murine model by the oral route. Thus, this novel bile exclusion locus bilE, coordinately regulated by B and PrfA, represents a new and important virulence factor in L. monocytogenes
Importance of host feeding for parasitoids that attack honeydew-producing hosts
Burger, J.M.S. ; Komany, A. ; Lenteren, J.C. van; Vet, L.E.M. - \ 2005
Entomologia Experimentalis et Applicata 117 (2005)2. - ISSN 0013-8703 - p. 147 - 154.
lifetime reproductive success - encarsia-formosa - trialeurodes-vaporariorum - aphytis-melinus - insect parasitoids - bemisia-tabaci - egg-production - hymenoptera - wasp - strategies
Insect parasitoids lay their eggs in arthropods. Some parasitoid species not only use their arthropod host for oviposition but also for feeding. Host feeding provides nutrients to the adult female parasitoid. However, in many species, host feeding destroys an opportunity to oviposit. For parasitoids that attack Homoptera, honeydew is a nutrient-rich alternative that can be directly imbibed from the host anus without injuring the host. A recent study showed that feeding on host-derived honeydew can be an advantageous alternative in terms of egg quantity and longevity. Here we explore the conditions under which destructive host feeding can provide an advantage over feeding on honeydew. For 5 days, Encarsia formosa Gahan (Hymenoptera: Aphelinidae) parasitoids were allowed daily up to 3 h to oviposit until host feeding was attempted. Host feedings were either prevented or allowed and parasitoids had ad libitum access to honeydew between foraging bouts. Even in the presence of honeydew, parasitoids allowed to host feed laid more eggs per hour of foraging per host-feeding attempt than parasitoids that were prevented from host feeding. The higher egg-laying rate was not compromised by survival or by change in egg volume over time. In conclusion, host feeding can provide an advantage over feeding on honeydew. This applies most likely under conditions of high host density or low extrinsic mortality of adult parasitoids, when alternative food sources cannot supply enough nutrients to prevent egg limitation. We discuss how to integrate ecological and physiological studies on host-feeding behavior
Host feeding in insect parasitoids: why destructively feed upon a host that excretes an alternative?
Burger, W. ; Reijnen, T.M. ; Lenteren, J.C. van; Vet, L.E.M. - \ 2004
Entomologia Experimentalis et Applicata 112 (2004)3. - ISSN 0013-8703 - p. 207 - 215.
trialeurodes-vaporariorum homoptera - encarsia-formosa hymenoptera - alfalfa weevil coleoptera - aphytis-melinus - honeydew sugars - aphelinidae - aleyrodidae - food - strategies - evolutionary
Host feeding is the consumption of host tissue by the adult female parasitoid. We studied the function of destructive host feeding and its advantage over non-destructive feeding on host-derived honeydew in the whitefly parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae). We allowed parasitoids to oviposit until they attempted to host feed. We either prevented or allowed host feeding. Parasitoids had access to sucrose solution, with or without additional access to honeydew. Parasitoids that were allowed to host feed did not have a higher egg load 20 or 48 h after host feeding than parasitoids prevented from host feeding. Host feeding did not increase the number of eggs matured within these periods, nor did the time spent host feeding positively affect any of these response variables. On the other hand, the presence of honeydew did have a positive effect on egg load 20 and 48 h after host feeding compared with parasitoids deprived of honeydew. Parasitoids with access to honeydew matured more eggs within these periods than honeydew-deprived parasitoids. Host feeding increased life expectancy, but this effect was nullified when honeydew was supplied after the host-feeding attempt. In conclusion, feeding on honeydew could be an advantageous alternative to host feeding in terms of egg quantity and longevity. This applies especially to parasitoids exploiting Homoptera, because these parasitoids can obtain honeydew from the host itself. It is possible that destructive host feeding has evolved to enable females to sustain the production of high-quality anhydropic eggs, which may be important in the parasitoid's natural environment. We argue that future studies should take natural alternative food sources into more consideration.
Reproduction now or later: optimal host-handling strategies in the whitefly parasitoid Encarsia formosa
Burger, J.M.S. ; Hemerik, L. ; Lenteren, J.C. van; Vet, L.E.M. - \ 2004
Oikos 106 (2004)1. - ISSN 0030-1299 - p. 117 - 130.
trialeurodes-vaporariorum homoptera - gerbera-jamesonii leaves - foraging behavior - egg load - aphytis-melinus - hymenoptera-aphelinidae - gahan hym - evolutionary argument - lethal interference - feeding strategies
We developed a dynamic state variable model for studying optimal host-handling strategies in the whitefly parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae). We assumed that (a) the function of host feeding is to gain nutrients that can be matured into eggs, (b) oogenesis is continuous and egg load dependent, (c) parasitoid survival is exponentially distributed and (d) parasitoids encounter hosts randomly, are autogenous and have unlimited access to non-host food sources to obtain energy for maintenance and activity. The most important prediction of the model is that host feeding is maladaptive under field conditions of low host density (0.015 cm(-2)) and short parasitoid life expectancy (maximum reproductive period of 7 d). Nutrients from the immature stage that can be matured into eggs are sufficient to prevent egg limitation. Both host density and parasitoid life expectancy have a positive effect on the optimal host-feeding ratio. Parasitoids that make random decisions gain on average only 35% (0.015 hosts cm(-2)) to 60% (1.5 hosts cm(-2)) of the lifetime reproductive success of parasitoids that make optimal decisions, independent of their life expectancy. Parameters that have a large impact on lifetime reproductive success and therefore drive natural selection are parasitoid life expectancy and the survival probability of deposited eggs (independent of host density), the number of host encounters per day (when host density is low) and the egg maturation rate and number of host types (when host density is high). Explaining the evolution of host-feeding behaviour under field conditions requires field data showing that life expectancy in the field is not as short as we assumed, or may require incorporation of variation in host density. Incorporating variation in walking speed, parasitised host types or egg resorption is not expected to provide an explanation for the evolution of host-feeding behaviour under field conditions.
The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence
Karatzas, K.A.G. ; Wouters, J.A. ; Gahan, C.G.M. ; Hill, C. ; Abee, T. ; Bennik, M.H.J. - \ 2003
Molecular Microbiology 49 (2003)5. - ISSN 0950-382X - p. 1227 - 1238.
high hydrostatic-pressure - gram-positive bacteria - escherichia-coli - sensitive strains - gene-expression - clpc atpase - in-vivo - proteins - inactivation - temperature
A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183-3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive. Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.
Comparison of foraging behavior, interspecific host discrimination, and competition of Encarsia formosa and Amitus fuscipennis
Vis, R.M.J. de; Mendez, H. ; Lenteren, J.C. van - \ 2003
Journal of Insect Behavior 16 (2003). - ISSN 0892-7553 - p. 117 - 152.
trialeurodes-vaporariorum homoptera - hymenoptera-aphelinidae - bemisia-argentifolii - life-history - biological-control - tomato leaflets - sex-ratio - aleyrodidae - parasite - platygasteridae
The foraging behavior of Amitus fuscipennis MacGown & Nebeker and Encarsia formosa Gahan was studied on tomato leaflets with 20 Trialeurodes vaporariorum (Westwood) larvae in the first or third stage. Ten of the whitefly larvae were previously parasitized and contained a conspecific or a heterospecific parasitoid egg or larva. The host type (host stage and/or previous parasitization) did not influence the foraging behavior of either parasitoid species. The residence time on these tomato leaflets was about 0.9 h for A. fuscipennis and 1.9 h for E. formosa. Amitus fuscipennis hardly stood still and fed little, while E. formosa showed extensive standing still and feeding. As a result, the time walking while drumming was similar for both parasitoid species. The numbers of host encounters and ovipositions per leaflet were similar for both parasitoid species. However, the residence time of A. fuscipennis was half as long as that of E. formosa so the rate of encounters and ovipositions was higher for A. fuscipennis. Amitus fuscipennis is more efficient in finding and parasitizing hosts under these conditions. The walking activity and host acceptance of the synovigenic E. formosa diminished with the number of ovipositions, but not those of the proovigenic A. fuscipennis. Encarsia formosa is egg limited, while A. fuscipennis is time limited because of its short life span and high egg load. Both parasitoid species discriminated well between unparasitized larvae and self-parasitized larvae, but discriminated poorly those larvae parasitized by a conspecific and did not discriminate larvae parasitized by a heterospecific. Self-superparasitism, conspecific superparasitism, and multiparasitism were observed for both parasitoid species. Superparasitism always resulted in the emergence of one parasitoid and multiparasitism resulted in a higher emergence of one parasitoid of the species that had parasitized first. The data suggest that A. fuscipennis is a good candidate for use in biological control of high-density spots of T. vaporariorum when we consider its high encounter and oviposition rate.
Life history of Amitus fuscipennis (Hym., Platygastridae) as parasitoid of the greenhouse whitefly Trialeurodes vaporariorum (Hom., Aleyrodidae) on tomato as function of temperature
Vis, R.M.J. de; Fuentes, L.E. ; Lenteren, J.C. van - \ 2002
Journal of Applied Entomology 126 (2002)1. - ISSN 0931-2048 - p. 24 - 33.
Life history parameters of Amitus fuscipennis MacGown and Nebeker as parasitoid of Trialeurodes vaporariorum (Westwood) were determined at 15, 20, 25 and 30°C on tomato using three different methods. For each method, immature development, mortality, longevity, fecundity, oviposition frequency and post-oviposition period were determined and temperature-dependent relations were estimated. Oviposition frequency was also estimated as a function of parasitoid age. Immature development had a maximum of 61 days at 15°C that decreased to 22 days at 30°C. Mortality in the grey stage was less than 2% at temperatures lower than 30°C, where it was 60%. Longevity fluctuated between 3 and 18 days. Fecundity increased from 338 eggs/female at 15°C to a maximum of 430 eggs/female at 25°C and then decreased to 119 eggs/female at 30°C. Oviposition frequency varied between 3 and 46 eggs/female per day and had its maximum on the first day after emergence of the parasitoid. Net reproduction rate, generation time and intrinsic rate of increase were calculated. The intrinsic rate of increase increased from 0.090 at 15°C to a maximum of 0.233 at 25°C and then decreased to 0.159 at 30°C. The influence of the methods to determine the life history parameters on the results is discussed. The results are compared also with the life history of Encarsia formosa Gahan (Hym., Aphelinidae) a parasitoid of the same host. The advantages and disadvantages of the pro-ovigenic A. fuscipennis in comparison with the synovigenic E. formosa are discussed.
Multiple deletion of the osmolyte transporters BetL, Gbu and OpuC of Listeria monocytogenes affects growth at high osmolarity and virulence
Wemekamp-Kamphuis, H.H. ; Wouters, J.A. ; Sleator, R.D. ; Gahan, C.G.M. ; Hill, C. ; Abee, T. - \ 2002
Nederlands Tijdschrift voor Medische Microbiologie 10 (2002)suppl.. - ISSN 0929-0176 - p. S40 - S40.
Characterisation and properties of a Listeria monocytogenes isolate with increased tolerance to high hydrostatic pressure
Karatzas, K.A. ; Wouters, J.A. ; Vladramidis, V. ; Gahan, C.G.M. ; Hill, C. ; Abee, T. ; Bennik, M.H.J. - \ 2002
In: Frontiers in microbial fermentation and preservation : Joint meeting of The Society for Applied Microbiology and The Dutch Society for Microbiology, Wageningen, 2002 Society for Applied Microbiology - p. 41 - 42.
Multiple deletions of the osmolyte transporters BetL, Gbu, and OpuC of Listeria monocytogenes affect virulence and growth at high osmolarity
Wemekamp-Kamphuis, H.H. ; Wouters, J.A. ; Sleator, R.D. ; Gahan, C.G.M. ; Hill, C. ; Abee, T. - \ 2002
Applied and Environmental Microbiology 68 (2002). - ISSN 0099-2240 - p. 4710 - 4716.
The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the food environment and, after ingestion, within the animal host. Growth at high salt concentrations is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We characterized L. monocytogenes LO28 strains with single, double, and triple deletions in the osmolyte transport systems BetL, Gbu, and OpuC. When single deletion mutants were tested, Gbu was found to have the most drastic effect on the rate of growth in brain heart infusion (BHI) broth with 6dded NaCl. The highest reduction in growth rate was found for the triple mutant LO28BCG (ΔbetL ΔopuC Δgbu), although the mutant was still capable of growth under these adverse conditions. In addition, we analyzed the growth and survival of this triple mutant in an animal (murine) model. LO28BCG showed a significant reduction in its ability to cause systemic infection following peroral coinoculation with the wild-type parent. Altering OpuC alone resulted in similar effects (R. D. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001), leading to the assumption that OpuC may play an important role in listerial pathogenesis. Analysis of the accumulation of osmolytes revealed that betaine is accumulated up to 300 μmol/g (dry weight) when grown in BHI broth plus 6␗aCl whereas no carnitine accumulation could be detected. Radiolabeled-betaine uptake studies revealed an inability of BGSOE (ΔbetL Δgbu) and LO28BCG to transport betaine. Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 μmol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter.
Analysis of the role of OpuC, an osmolyte transport system, in salt tolerance and virulence potential of Listeria monocytogenes
Sleator, R.D. ; Wouters, J. ; Gahan, C.G.M. ; Abee, T. ; Hill, C. - \ 2001
Applied and Environmental Microbiology 67 (2001). - ISSN 0099-2240 - p. 2692 - 2698.
The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the external environment prior to ingestion and subsequently within the animal host. Growth at high salt concentrations and low temperatures is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We utilized a novel system for generating chromosomal mutations (based on a lactococcal pWVO1-derived Ori RepA- vector, pORI19) to identify a listerial OpuC homologue. Mutating the operon in two strains of L. monocytogenes revealed significant strain variation in the observed activity of OpuC. Radiolabeled osmolyte uptake studies, together with growth experiments in defined media, linked OpuC to carnitine and glycine betaine uptake in Listeria. We also investigated the role of OpuC in contributing to the growth and survival of Listeria in an animal (murine) model of infection. Altering OpuC resulted in a significant reduction in the ability of Listeria to colonize the upper small intestine and cause subsequent systemic infection following peroral inoculation.
Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28
Sleater, R.D. ; Gahan, C.G.M. ; Abee, T. ; Hill, C. - \ 1999
Applied and Environmental Microbiology 65 (1999). - ISSN 0099-2240 - p. 2078 - 2083.
Effect of the eggs and empty moulted cuticulae of larvae of Trialeurodes vaporariorum Westwood (Homoptera: Aleyrodidae) on the behaviour of Encarsia formosa Gahan (Hymenoptera: Aphelinidae).
Imre, S. ; Doodeman, C.J.A.M. ; Roermund, H.J.W. van; Lenteren, J.C. van - \ 1996
In: Abstract volume of 42th Plant Protection Days, Budapest - p. 27 - 28.
Understanding biological control of greenhouse whitefly with the parasitoid Encarsia formosa : from individual behaviour to population dynamics
Roermund, H.J.W. van - \ 1995
Agricultural University. Promotor(en): J.C. van Lenteren; R. Rabbinge. - S.l. : Van Roermund - ISBN 9789054854371 - 243
insecten - plantenplagen - aleyrodidae - biologische bestrijding - nuttige insecten - chalcididae - eulophidae - trichogrammatidae - trialeurodes vaporariorum - insects - plant pests - aleyrodidae - biological control - beneficial insects - chalcididae - eulophidae - trichogrammatidae - trialeurodes vaporariorum
The greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Homoptera, Aleyrodidae), is a very common, highly polyphagous pest insect all over the world. Biological control of whiteflies with the parasitoid Encarsia formosa Gahan (Hymenoptera, Aphelinidae) was already applied in the 1920s in England, Australia, New Zealand and Canada. The use of the parasitoid was discontinued in the fourties and fifties when chemical pesticides were used extensively. In the seventies, when the first problems with pesticide resistance occurred in Western Europe, interest in using the parasitoid increased again. A reliable introduction scheme of the parasitoid was found by a 'trial and error' approach: natural enemies were released at different times and in different numbers, and their level of control was examined. In 20 of the 35 countries with a greenhouse industry, the parasitoid is used on about 5000 ha. Biological control with E. formosa is now used commercially in 90% of the tomato growing areas in the Netherlands. On several other important greenhouse crops such as cucumber and gerbera, biological control of whitefly is not so successful. This study aims at integrating existing knowledge on the major processes known to affect the whitefly-parasitoid interaction in a crop by means of an explanatory simulation model. The goal is to obtain quantitative understanding of the tritrophic system cropgreenhouse whitefly- E. formosa to explain failure or success of biological control. With the model we are able to (1) explain the ability of E. formosa to reduce whitefly populations in greenhouses on crops like tomato, (2) improve introduction schemes of parasitoids for crops where control is more difficult to obtain and (3) predict effects of changes in cropping practices (e.g. greenhouse climate, choice of cultivars) on the reliability of biological control. Direct observation experiments on foraging of E. Formosa. When the present research project started, the behaviour of E. formosa had been observed in various experiments. These experiments resulted in the following picture. E. formosa is a solitary larval parasitoid: females lay one egg per host during an oviposition. Like in other synovigenic parasitoids new eggs mature when the egg load of the parasitoid drops below the storage capacity, which is 8-10 mature eggs for E. formosa. About ten days after oviposition the immature parasitoid pupates in the host pupa, which (in case of greenhouse whitefly) then turns black and parasitism can easily be seen from the outward appearance of the whitefly. Female parasitoids produce daughters parthenogenetically. Males are rarely observed. The parasitoid searches for the sessile whitefly immatures by flying or hopping from leaf(let) to leaf(let) without distinguishing between infested and clean plants or leaves before landing. Once on the leaf she starts walking and drumming the leaf with her antennae. Hosts are encountered randomly and the walking pattern is not changed after an encounter with a host. After an encounter, four behaviours on that host can be distinguished: the parasitoid may reject the host after an inspection with the antennae (antennal rejection) or after insertion of the ovipositor (ovipositorial rejection), she may parasitize (oviposition) or she may use the host as a food source (host feeding). However, these earlier experiments did not lead to a complete picture of foraging behaviour. Quantitative data on some aspects were lacking, such as the parasitoids' searching or walking activity between host encounters, and the effect of temperature on the foraging processes. In many of the earlier experiments parasitoids were confined to an experimental arena, and therefore little was known about the time allocation of the parasitoid on leaves, such as the time until leaving, the time spent on upper and lower leaf side, and how these are affected by encounters with or ovipositions in hosts. In this thesis, these gaps in our knowledge are first identified and studied experimentally. Chapters 2, 3 and 4 describe experiments where individual parasitoids were observed continuously until they flew away, either on clean tomato leaflets, on
leaflets with honeydew, or on leaflets with unparasitized and parasitized whitefly larvae. In Chapter 2 the residence times of the parasitoids on leaflets are discussed. In Chapter 3 the leaving tendency of the parasitoid from the leaflet and effects of several
intra-patch experiences with hosts are quantified. In Chapter 4 other basic aspects of foraging are quantified, such as the parasitoids' walking speed and walking activity, the probability of each handling behaviour to occur after an encounter with a host and the host handling times. These data enable quantification of the foraging process of the parasitoid from landing on a leaf until departure. For an overview of the subsequent processes, see Figure 1 of Chapter 4. The work described in Chapters 2, 3 and 4 resulted in the following conclusions:
- The parasitoid E. formosa searches at random without a preference for the edge or the middle of a leaf, or for the upper or lower leaf side, whereas whitefly immatures (the hosts) are present only on the lower side of a tomato leaflet.
- The median residence time of the parasitoid on uninfested tomato leaflets (or giving up time, GUT) is 18.6 min at 20, 25 and 30°C and equal to that on infested leaflets on which no hosts are encountered.
- Parasitoids are arrested on the leaf by encounters with, and especially by ovipositions in, unparasitized hosts, by encounters with parasitized (unsuitable) hosts and by contact with honeydew. GUT since latest host encounter is again 18.6 min, also when the hosts were parasitized, but increases to 40 min after the first oviposition in an unparasitized host.
- Parasitoids are arrested on the lower leaf side by encounters with hosts, and especially by ovipositions in unparasitized hosts. The median time since the arrival on a particular leaf side or, if it occurred, since the latest host encounter on that leaf side until changing to the other side (TUC) is initially 11.6 min and drops to 5.7 min after both leaf sides have been visited. After the first
oviposition in an unparasitized host, TUC since latest host encounter on the lower leaf side (where hosts are present) becomes twice as long.
- Parasitoids usually leave from the upper leaf side, where no hosts are present. The patch-leaving behaviour of the parasitoid can be described by a stochastic threshold mechanism, which is characterized by a certain tendency (probability per time) to leave. The parasitoid leaves after the host encounter rate falls below a certain threshold (encounters per time, which is the reciprocal of GUT). This threshold is not fixed, however, but shows a great variation and is expressed as a probability.
- The parasitoids' walking speed increases linearly between 15 and 25-30°C.
- The parasitoids' walking activity is very low at temperatures below 18°C and increases to about 75% of the total time on the leaf at 20, 25 and 30°C. The walking activity is not affected by host encounters, but decreases with decreasing egg load after 4 ovipositions.
- The percentage of encounters resulting in an oviposition is about 75% for the most preferred stage (unparasitized L4 larva), but decreases with decreasing egg load.
- Host handling behaviour and handling time is not influenced by the host plant.
- The total handling time (including drumming etc.) for antennal rejection of an unparasitized hosts is about 20 s, for oviposition and for ovipositional rejection about 6 min, and for host feeding about 15 min. These handling times slightly differ when hosts are parasitized.
- Self-superparasitism is not observed. Conspecific-superparasitism occurs in 14% of the contacted hosts containing a parasitoid egg, but is not observed anymore when the parasitoid egg had hatched.
- No difference is observed in host handling behaviour between naive and experienced parasitoids.
- Many inactive parasitoids are observed when the barometric pressure had decreased over a time span of at least 12 h.

Simulation models of foraging behaviour of E. formosa

The information described above is used as input in the simulation models of E . formosa's foraging behaviour, which is described in Chapters 5, 6 and 7 . Here, foraging behaviour is analyzed using Monte Carlo simulation at three spatial scales: in a small experimental arena, on a tomato leaflet and on a tomato plant. For an overview of the models, see the flow diagrams (Figure 1) of these chapters. Foraging behaviour is first studied at these small spatial scales, to better understand the quantitative effects of parasitoids on whitefly populations as observed in a crop.

The above simulation models are mechanistic, that is, they explain how parasitoids, in terms of searching efficiency, host handling and available eggs, realize the observed level of parasitism. Mechanistic explanations can help to understand failure or success of biological control in practice. The models do not explain why the parasitoids choose to behave in this way, in terms of the selection pressure acting on them. Thus, they do not provide a functional explanation of the observed behaviour. This is subject of study in optimal foraging models.

The simulated number of hosts encountered, parasitized and killed by host feeding, and the residence times on leaflets are validated with experimental data and the simulation results agree well with these observations. According to the model, E. formosa can parasitize 16 hosts per day on average at 25°C on a tomato leaflet if they start searching with a full batch of mature eggs and if host density is not limiting. Thus about 7 new eggs mature during the day (16 h) at that temperature. From the second day onwards, the parasitoid can parasitize I I hosts per day, due to egg limitation: if the parasitoid laid all eggs the preceding day, only 4 eggs mature during the night (of 8 h) at 25°C, so the parasitoids do not have a full batch of mature eggs the next morning. The model shows that at a density of I L3 larva per tomato leaflet, 15.7% of the parasitoids discover the larva before they leave. Also at higher host densities, not all hosts are encountered and patches (leaflets) are not depleted after one visit. Variation in number of encounters and ovipositions between parasitoids is considerable, mainly caused by the random encounter of hosts, the variation in handling behaviour of an encountered host and by the variation in GUT and TUC.

In greenhouses, whiteflies show a clustered distribution over plants and leaves and average numbers are usually very low. The models show that at such conditions the number of parasitizations on tomato leaflets or plants is strongly affected by the leaf area, the parasitoids' walking speed and walking activity, the probability of oviposition after encountering a host, the initial egg load (egg load at the beginning of the experiment) and the ratio of search times on both leaf sides. At extremely high host densities, the egg storage capacity and the initial egg load of the parasitoid are most important, and on plants with a clustered host distribution also the parasitoids' GUT.

At all spatial scales tested, the number of encounters, ovipositions and host feedings increase with host density with a decelerating rate until a maximum level is reached. This shape of the curves resemble a Holling Type II functional response, which is caused by the parasitoids' decreasing walking activity and probability of oviposition after encountering a host when egg load decreases. This is predominant at all levels, and even a change in GUT from 18.6 to 40 min after the first oviposition on the leaf does not result in an accelerating increase of the curve. The shape of the curves, describing the effect of host density on parasitism as a result of the basic processes, helps to understand the dynamics of the host-parasitoid interaction at the population level. In case of a Type II functional response, percentage parasitism declines with increasing host density and parasitism is inversily density dependent. A high host density thus reduces the per capita parasitization pressure caused by one parasitoid. According to theory, inversely density dependence tends to have a destabilizing effect on the dynamics of host and parasitoid. However, the functional response or the parasitization pressure caused by one parasitoid is only one factor in determining the dynamics at the population level. Another factor is the number of parasitoids on the leaf. For E. formosa, the effect on the population level depends on the balance between the parasitization pressure caused by one parasitoid and the arrestment and subsequent aggregation of parasitoids on leaves with high host density (see Chapter 10).

Life-history parameters of greenhouse whitefly and E. formosa

In Chapters 8 and 9, life-history parameters of the greenhouse whitefly and E. formosa are reviewed. Data from literature were selected on development rate of each immature stage, percentage mortality of each immature stage, sex ratio, longevity, preoviposition period, period of increase of daily oviposition, fecundity and oviposition frequency. Most of these experiments have focused on the effect of temperature with little attention to other environmental factors such as humidity or light. With these data, the relationship between the life-history parameters and temperature are assessed by non-linear regression. Five mathematical equations were fitted, the best being selected on the basis of the coefficient of determination (r 2) and on visual comparison of the curves, which was necessary to check whether a curve was biologically realistic, particularly the tails. Coefficients to describe the mean of each life-history parameter as a function of temperature are summarized in these chapters. Coefficients of variation (cv: sd/mean) among individuals are also given. These coefficients are used as input in the submodels of population development of whitefly and parasitoid (see Chapter 10).

For greenhouse whitefly, the life-history parameters depend very much on the type of host plant. For E. formosa , data for several host plants were combined. The high r2 values indicate that host plant effects can be disregarded for the parasitoids' life-history parameters, except for oviposition at low host densities, which is caused by differences in the parasitoids' walking speed and walking activity on leaves with a different morphology. The host stage originally parasitized strongly affects the immature development rate and immature mortality of the parasitoid.

The development rate is calculated as the reciprocal of the stage duration. For all immature stages of whitefly and parasitoid the relationship between development rate and temperature is described by the Logan curve: just above the lower threshold temperature, the development rate increases exponentially to an optimum, whereafter it declines sharply until the upper lethal temperature has been reached. The relationships of longevity, fecundity, and oviposition frequency with temperature are described by the Weibull curve: they increase exponentially from the lower lethal temperature to an optimum, whereafter they decrease exponentially. Only for E. formosa, the longevity decreases exponentially with temperature and an optimum was not found at greenhouse conditions. No relationship with temperature is found for the immature mortality, the sex ratio and the cv values of the life-history parameters of whitefly and parasitoid.

Simulation model of whitefly-parasitoid interaction in a crop

The final model simulates the population dynamics of the pest insect-parasitoid interaction in a tomato crop and is described in Chapter 10. The model is based on the parasitoids' searching and parasitization behaviour and on developmental biology of the two insect species. This model comprises several submodels, such as the submodel for whitefly population development, for parasitoid population development, for the parasitoids' foraging behaviour on tomato leaflets (model of Chapter 6), for spatial distribution of whitefly and parasitoid in the canopy, for dispersion of adult whiteflies and parasitoids from leaf to leaf, for leaf production and a submodel for checking simulation errors. Life-history parameters of Chapters 8 and 9 are used as input in the submodels for population development of whitefly and parasitoid on tomato. For an overview of the model, see the relational diagrams (Figures 1, 2 and 3) of Chapter 10. The model is unique in that it is an individual-based model which simulates local searching and parasitization behaviour of a large number of individual parasitoids in a whitefly-infested crop. The model includes stochasticity and spatial structure which is based on location coordinates of plants and leaves.

The model is validated with population counts from experiments on tomato with and without introduction of E.formosa in small greenhouse compartments and in a large commercial greenhouse. The simulated population increase of greenhouse whitefly in the absence of parasitoids agree well with the observations. This result can for an important part be explained by the accurate estimates of the life-history parameters, which are based on many experiments at a wide temperature range (see Chapters 8 and 9).

With these life-history parameters as input in the model, the intrinsic rate of increase (r m ) of both insect species is simulated. The (r m ) of E. formosa is much higher than that of the greenhouse whitefly above 14°C. The r m of a parasitoid however, plays a limited role in biological control, because it is only valid when all parasitoids can lay their daily egg load. This can only happen at extremely high host densities when the parasitoids do not have to spent much time searching for hosts. In greenhouses whitefly densities are usually much lower and the realized whitefly density depends on the parasitoids' searching efficiency. Therefore, to evaluate and understand success or failure of biological control, (r m ) values are inappropiate and it is essential to built models which include searching and parasitization behaviour of the natural enemy at very low host densities.

Also in the presence of parasitoids, the simulation results agree well with greenhouse observations on tomato. Apparently, the hypothesized random host encounter of E. formosa in a tomato crop is reliable. In the model, the parasitoid does not distinguish between uninfested and infested leaflets before landing, the parasitoid searches randomly for hosts once on the leaflet, and shows a strong arrestment effect: it stays longer on the leaflet once a host is encountered. Simulations show that the adult parasitoid-whitefly ratio is very high and can even reach 250:1. As a result, whiteflies are suppressed rather than regulated by the parasitoids at extremely low host densities (<0.3 unparasitized pupae per plant), but never become extinct. These whitefly densities are much lower than the economic damage threshold for greenhouse whitefly. Percentage black pupae fluctuates between 40 and 70%. According to the model, the parasitoid adults reach high densities of 7.4 per plant, but due to the low whitefly density not more than 1% of the parasitoids is searching on infested leaflets.

The giving up times (GUT) of E. formosa vary to a large extend. The degree of whitefly control is very sensitive to those GUT's lower than 800 s of the parasitoids. The whiteflies are suppressed at much lower densities when the parasitoids stay at least five minutes on each leaflet (infested or uninfested) and after each host encounter. This minimum time increases the arrestment effect and the resulting percentage of parasitoids on infested leaflets, thereby reducing the chance that clustered hosts escape from parasitism. When variation in GUT is excluded in the model, the whitefly population becomes less stable and nearly goes extinct. Variation in GUT on leaflets induces host refuges from parasitoid attack. Also from more theoretical studies, host refuges are known to stabilize populations.

Whitefly adults migrate to young leaves in the top of the plant. A slower leaf production results in a longer stay and more ovipositions of whitefly adults on a particular leaflet. Thus, the same number of hosts are distributed over fewer leaflets, resulting in a more aggregated host distribution. Whiteflies are then suppressed by E. formos a to much lower numbers, according to the model. Parasitism of one E. formosa female on a tomato leaflet is inversely density-dependent, which is caused by a decreasing walking activity and probability of oviposition after encountering a host (Chapters 5, 6 and 7). Host aggregation thus 'dilutes' the per capita parasitization pressure caused by one parasitoid on the leaflet. The effect on the population level however, depends on the balance between this 'dilution' effect and the strength of the arrestment and aggregation of E. formosa . Therefore, the stronger whitefly reduction when whiteflies are more aggregated is caused by a stronger parasitoid arrestment and subsequent increase in the relative number of parasitoids searching on infested leaflets.

This shows that differences in whitefly distribution among crops are one factor in causing differences in success of biological control. Other factors are the size, number and surface (hairiness) of leaves in the canopy. Leaf size and total leaf area have a strong effect on whitefly control according to the model, caused by their direct effect on host density. Furthermore, leaf size and leaf surface strongly affect the efficiency of E. formosa by changing the parasitoids' arrestment effect (GUT) and the walking speed and activity, respectively.

Another important factor is the whitefly development duration on the crop. The model shows that plant resistance breeding aimed at an increase in egg-to-adult duration of the whiteflies is very efficient in causing a severe reduction of whitefly numbers, when biological control is applied. Observed development times of whitefly differ very little between tomato genotypes, and a much larger difference is found for whitefly longevity, oviposition rate and immature mortality. These parameters have a smaller effect on whitefly population development.

The important factors or crop properties affecting the success of biological control cannot be compared independently. For instance crops with large leaves usually have lower number of leaves which are produced at a lower rate than crops with small leaves or leaflets. It is particularly the combined effect of these important factors that can be tested with this model for different crops or plant varieties.

In biological control programs, parasitoids are usually tested in small-scale experiments at high host densities before introduction in the field. As a result, maximum daily oviposition of parasitoids is measured, whereas this study shows that egg storage capacity and egg maturation rate of E. formosa is not important for the level of whitefly control. In commercial greenhouses, whitefly densities have to be very low for biological control to be judged successful, therefore effective host searching is the most essential process. When selecting parasitoids for biological control, attention should be focused on the parasitoids' arrestment effect (minimum GUT), walking speed, walking activity, the probability of oviposition after encountering a host, the ratio of search times on both leaf sides and on longevity, when comparing different synovigenic and solitary parasitoid species with random search. These characteristic attributes of parasitoids are easily measured in laboratory studies. Again, they cannot be compared independently, however, because the attributes of natural enemies are often found in particular combinations. The combined effect of these important attributes of a parasitoid can be tested with this model.

Epilogue
The present study aimed at integrating existing knowledge on the major processes known to affect the whitefly-parasitoid interaction in a crop. Because of the multitude of relationships between the three trophic levels (crop-pest-parasitoid), it was decided to follow a combined experimental-simulation approach. The goal was to obtain quantitative understanding of the tritrophic system to explain failure or success of biological control. With the model we now unravelled the ability of E. formosa to reduce whitefly populations on greenhouse tomato. The study resulted in increased understanding of the relative importance of basic processes that affect the population interaction of whitefly and natural enemy. The life-history of parasitoids, often summarized in a (r m ) value, are less important than the parasitoids' searching capacity. This shows that in addition to the traditional selection criteria, a criterion based on searching efficiency is essential. The study has further generated knowledge on foraging behaviour of E. formosa . The tremendous effect of variation in patch times of individual parasitoids on the whitefly population in the greenhouse shows that individual-based models which include stochasticity and local searching behaviour are a necessity when developing models of host-parasitoid interaction at extremely low host densities and aggregated host distributions.

One of the main questions was to identify the main causal factors for differences among crops in success of biological control of whitefly. The parasitoid is more successful on tomato than on cucumber or gerbera. The present study showed that attention should be focused on differences in the parasitoids' arrestment effect (GUT), the parasitoids' walking speed and activity, the whitefly development duration and the number, size and production of leaves in the canopy. These parameters have also been quantified for cucumber and gerbera and some are very different from those of tomato (see Chapters 2, 3 and 8). The next step in the research is to use the simulation model presented in Chapter 10 for the other two crops and evaluate the main causal factors for success or failure of biological control.

When adapting the parameters in the model for gerbera and cucumber we are able to (1) explain the lower ability of the parasitoid to reduce whitefly populations on these crops, (2) improve introduction schemes of parasitoids for these crops, and (3) predict effects of changes in cropping practices (e.g. greenhouse climate, choice of cultivars) on the reliability of biological control. Furthermore, with the model we can identify the characteristics which compose an efficient natural enemy. These characteristics can later be used as evaluation criteria in natural enemy selection programs. In fact ideotypes of natural enemies may be designed, tailored to crop, whitefly and environmental conditions. In that way, a new field of ecological engineering may be explored. The present study already pointed at important selection criteria when comparing different synovigenic, solitary parasitoids showing random search. The model can be adapted for other parasitoids with different foraging strategies or for other natural enemies of whitefly.

Residence times of the whitefly parasitoid Encarsia formosa Gahan (Hym. Aphelinidae) on tomato leaflets
Roermund, H.J.W. van; Lenteren, J.C. van - \ 1995
Journal of Applied Entomology 119 (1995). - ISSN 0931-2048 - p. 465 - 471.
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