Impact of hydraulic retention time on community assembly and function of photogranules for wastewater treatment.

Photogranules are dense, spherical agglomerates of cyanobacteria, microalgae and non-phototrophic microorganisms that have considerable advantages in terms of harvesting and nutrient removal rates for light driven wastewater treatment processes. This ecosystem is poorly understood in terms of the microbial community structure and the response of the community to changing abiotic conditions. To get a better understanding, we investigated the effect of hydraulic retention time (HRT) on photogranule formation and community assembly over a period of 148 days. Three laboratory bioreactors were inoculated with field samples from various locations in the Netherlands and operated in sequencing batch mode. The bioreactors were operated at four different HRTs (2.00, 1.00, 0.67, 0.33 days), while retaining the same solid retention time of 7 days. A microbial community with excellent settling characteristics (95-99% separation efficiency) was established within 2-5 weeks. The observed nutrient uptake rates ranged from 24 to 90 mgN L-1 day-1 and from 3.1 to 5.4 mgP L-1 day-1 depending on the applied HRT. The transition from single-cell suspension culture to floccular agglomeration to granular sludge was monitored by microscopy and 16S/18S sequencing. In particular, two important variables for driving aggregation and granulation, and for the structural integrity of photogranules were identified: 1. Extracellular polymeric substances (EPS) with high protein to polysaccharide ratio and 2. specific microorganisms. The key players were found to be the cyanobacteria Limnothrix and Cephalothrix, the colony forming photosynthetic eukaryotes within Chlamydomonadaceae, and the biofilm producing bacteria Zoogloea and Thauera. Knowing the makeup of the microbial community and the operational conditions influencing granulation and bioreactor function is crucial for successful operation of photogranular systems.

Habitat complexity reduces parasitoid foraging efficiency, but does not prevent orientation towards learned host plant odours.

It is well known that many parasitic wasps use herbivore-induced plant odours (HIPVs) to locate their inconspicuous host insects, and are often able to distinguish between slight differences in plant odour composition. However, few studies have examined parasitoid foraging behaviour under (semi-)field conditions. In nature, food plants of parasitoid hosts are often embedded in non-host-plant assemblages that confer both structural and chemical complexity. By releasing both naïve and experienced Cotesia glomerata females in outdoor tents, we studied how natural vegetation surrounding Pieris brassicae-infested Sinapis arvensis and Barbarea vulgaris plants influences their foraging efficiency as well as their ability to specifically orient towards the HIPVs of the host plant species on which they previously had a positive oviposition experience. Natural background vegetation reduced the host-encounter rate of naïve C. glomerata females by 47 %. While associative learning of host plant HIPVs 1 day prior to foraging caused a 28 % increase in the overall foraging efficiency of C. glomerata, it did not reduce the negative influence of natural background vegetation. At the same time, however, females foraging in natural vegetation attacked more host patches on host-plant species on which they previously had a positive oviposition experience. We conclude that, even though the presence of natural vegetation reduces the foraging efficiency of C. glomerata, it does not prevent experienced female wasps from specifically orienting towards the host-plant species from which they had learned the HIPVs.

Introgression study reveals two quantitative trait loci involved in interspecific variation in memory retention among Nasonia wasp species.

Genes involved in the process of memory formation have been studied intensively in model organisms; however, little is known about the mechanisms that are responsible for natural variation in memory dynamics. There is substantial variation in memory retention among closely related species in the parasitic wasp genus Nasonia. After a single olfactory conditioning trial, N. vitripennis consolidates long-term memory that lasts at least 6 days. Memory of the closely related species N. giraulti is present at 24 h but is lost within 2 days after a single trial. The genetic basis of this interspecific difference in memory retention was studied in a backcrossing experiment in which the phenotype of N. giraulti was selected for in the background of N. vitripennis for up to five generations. A genotyping microarray revealed five regions that were retained in wasps with decreased memory retention. Independent introgressions of individual candidate regions were created using linked molecular markers and tested for memory retention. One region on chromosome 1 (spanning ∼5.8 cM) and another on chromosome 5 (spanning ∼25.6 cM) resulted in decreased memory after 72 h, without affecting 24-h-memory retention. This phenotype was observed in both heterozygous and homozygous individuals. Transcription factor CCAAT/enhancer-binding protein and a dopamine receptor, both with a known function in memory formation, are within these genomic regions and are candidates for the regulation of memory retention. Concluding, this study demonstrates a powerful approach to study variation in memory retention and provides a basis for future research on its genetic basis.

High-throughput olfactory conditioning and memory retention test show variation in Nasonia parasitic wasps.

Most of our knowledge on learning and memory formation results from extensive studies on a small number of animal species. Although features and cellular pathways of learning and memory are highly similar in this diverse group of species, there are also subtle differences. Closely related species of parasitic wasps display substantial variation in memory dynamics and can be instrumental to understanding both the adaptive benefit of and mechanisms underlying this variation. Parasitic wasps of the genus Nasonia offer excellent opportunities for multidisciplinary research on this topic. Genetic and genomic resources available for Nasonia are unrivaled among parasitic wasps, providing tools for genetic dissection of mechanisms that cause differences in learning. This study presents a robust, high-throughput method for olfactory conditioning of Nasonia using a host encounter as reward. A T-maze olfactometer facilitates high-throughput memory retention testing and employs standardized odors of equal detectability, as quantified by electroantennogram recordings. Using this setup, differences in memory retention between Nasonia species were shown. In both Nasonia vitripennis and Nasonia longicornis, memory was observed up to at least 5 days after a single conditioning trial, whereas Nasonia giraulti lost its memory after 2 days. This difference in learning may be an adaptation to species-specific differences in ecological factors, for example, host preference. The high-throughput methods for conditioning and memory retention testing are essential tools to study both ultimate and proximate factors that cause variation in learning and memory formation in Nasonia and other parasitic wasp species.

CREB expression in the brains of two closely related parasitic wasp species that differ in long-term memory formation.

The cAMP/PKA signalling pathway and transcription factor cAMP response element-binding protein (CREB) play key roles in long-term memory (LTM) formation. We used two closely related parasitic wasp species, Cotesia glomerata and Cotesia rubecula, which were previously shown to be different in LTM formation, and sequenced at least nine different CREB transcripts in both wasp species. The splicing patterns, functional domains and amino acid sequences were similar to those found in the CREB genes of other organisms. The predicted amino acid sequences of the CREB isoforms were identical in both wasp species. Using real-time quantitative PCR we found that two low abundant CREB transcripts are differentially expressed in the two wasps, whereas the expression levels of high abundant transcripts are similar.

Diploid males sire triploid daughters and sons in the parasitoid wasp Cotesia vestalis.

In the Hymenoptera, males develop as haploids from unfertilized eggs and females develop as diploids from fertilized eggs. In species with complementary sex determination (CSD), however, diploid males develop from zygotes that are homozygous at a highly polymorphic sex locus or loci. We investigated mating behavior and reproduction of diploid males of the parasitoid wasp Cotesia vestalis (C. plutellae), for which we recently demonstrated CSD. We show that the behavior of diploid males of C. vestalis is similar to that of haploid males, when measured as the proportion of males that display wing fanning, and the proportion of males that mount a female. Approximately 29% of diploid males sired daughters, showing their ability to produce viable sperm that can fertilize eggs. Females mated to diploid males produced all-male offspring more frequently (71%) than females mated to haploid males (27%). Daughter-producing females that had mated to diploid males produced more male-biased sex ratios than females mated to haploid males. All daughters of diploid males were triploid and sterile. Three triploid sons were also found among the offspring of diploid males. It has been suggested that this scenario, that is, diploid males mating with females and constraining them to the production of haploid sons, has a large negative impact on population growth rate and secondary sex ratio. Selection for adaptations to reduce diploid male production in natural populations is therefore likely to be strong. We discuss different scenarios that may reduce the sex determination load in C. vestalis.

Complementary sex determination in the parasitoid wasp Cotesia vestalis (C. plutellae).

In the Hymenoptera, single locus complementary sex determination (sl-CSD) describes a system where males develop either from unfertilized haploid eggs or from fertilized diploid eggs that are homozygous at a single polymorphic sex locus. Diploid males are often inviable or sterile, and are produced more frequently under inbreeding. Within families where sl-CSD has been demonstrated, we predict that sl-CSD should be more likely in species with solitary development than in species where siblings develop gregariously (and likely inbreed). We examine this prediction in the parasitoid wasp genus Cotesia, which contains both solitary and gregarious species. Previous studies have shown that sl-CSD is absent in two gregarious species of Cotesia, but present in one gregarious species. Here, we demonstrate CSD in the solitary Cotesia vestalis, using microsatellite markers. Diploid sons are produced by inbred, but not outbred, females. However, frequencies of diploid males were lower than expected under sl-CSD, suggesting that CSD in C. vestalis involves more than one locus.

Linking spatial processes to life-history evolution of insect parasitoids.

Understanding the evolutionary transition from solitary to group living in animals is a profound challenge to evolutionary ecologists. A special case is found in insect parasitoids, where a tolerant gregarious larval lifestyle evolved from an intolerant solitary ancestor. The conditions for this transition are generally considered to be very stringent. Recent studies have aimed to identify conditions that facilitate the spread of a gregarious mutant. However, until now, ecological factors have not been included. Host distributions and life-history trade-offs affect the distribution of parasitoids in space and thus should determine the evolution of gregariousness. We add to current theory by using deterministic models to analyze the role of these ecological factors in the evolution of gregariousness. Our results show that gregariousness is facilitated through inversely density-dependent patch exploitation. In contrast, host density dependence in parasitoid distribution and patch exploitation impedes gregariousness. Numerical solutions show that an aggressive gregarious form can more easily invade a solitary population than can a tolerant form. Solitary forms can more easily invade a gregarious, tolerant population than vice versa. We discuss our results in light of exploitation of multitrophic chemical cues by searching parasitoids and aggregative and defensive behavior in herbivorous hosts.

Behavioural responses of diamondback moth Plutella xylostella (Lepidoptera: Plutellidae) to extracts derived from Melia azedarach and Azadirachta indica.

The impact of three different doses of botanical insecticide derived from the syringa tree, Melia azedarach and the neem tree, Azadirachta indica was tested on the behaviour of the diamondback moth, Plutella xylostella (Linnaeus). Both botanical insecticides had a significant impact on larval behaviour. At higher doses the extracts showed feeding deterrent activity, with larvae preferring the untreated sides of cabbage leaves and consuming less of the treated half of cabbage leaves. The botanical insecticides had less of an effect on the oviposition behaviour of P. xylostella moths. In laboratory and glasshouse trials, significantly fewer eggs were oviposited on the plants that had been treated with syringa extracts. Therefore, the syringa extracts appear to have a repellent effect. In contrast, when exposed to the neem extracts the moths did not discriminate between control plants and treated plants. Behavioural observation indicated that, despite the lower number of eggs oviposited on cabbage treated with syringa extracts, the moths chose cabbage treated with the highest dose of syringa more often than they chose control cabbage plants. Similar observations were found in cabbage plants treated with neem, moths chose the medium dose more often than they chose the control. Oviposition and feeding deterrent properties are important factors in pest control, and results from this study indicate that botanical insecticides have the potential to be incorporated into control programmes for P. xylostella in South Africa.

Variation in plant volatiles and attraction of the parasitoid Diadegma semiclausum (Hellén).

Differences in allelochemistry of plants may influence their ability to attract parasitoids. We studied responses of Diadegma semiclausum (Hellén), a parasitoid of the diamondback moth (Plutella xylostella L.), to inter- and intraspecific variation in odor blends of crucifers and a non-crucifer species. Uninfested Brussels sprout (Brassica oleracea L. gemmifera), white mustard (Sinapis alba L.), a feral Brassica oleracea, and malting barley (Hordeum vulgare L.) were compared for their attractivity to D. semiclausum in a Y-tube bioassay. Odors from all plants were more attractive to the parasitoid than clean air. However, tested against each other, parasitoids preferred the volatile blend from the three cruciferous species over that of malting barley. Wasps also discriminated between uninfested crucifers: mustard was as attractive as feral B. oleracea, and both were more attractive than Brussels sprout. Attractivity of uninfested plants was compared with that of plants infested by larvae of the host P. xylostella. Host-infested mustard and Brussels sprout were more attractive than uninfested conspecifics. Interestingly, the volatile blends of uninfested white mustard and infested Brussels sprout were equally attractive. We also compared the volatile composition of different plant sources by collecting headspace samples and analysing them with GC-MS. Similarities of volatile profiles were determined by hierarchic clustering and non-metric scaling based on the Horn-index. Due to the absence of several compounds in its blend, the volatile profile of barley showed dissimilarities from blends of crucifers. The odor profile of white mustard was distinctly different from the two Brassicaceae. Feral Brassica oleracea odor profile was different from infested Brussels sprout, but showed overlap with uninfested Brussels sprout. Odor blends from infested and uninfested Brussels sprout were similar, and mainly quantitative differences were found. D. semiclausum appears to discriminate based on subtle differences in volatile composition of odor blends from infested and uninfested plants.

Coexistence and niche segregation by field populations of the parasitoids Cotesia glomerata and C. rubecula in the Netherlands: predicting field performance from laboratory data.

Field experiments with foraging parasitoids are essential to validate the conclusions from laboratory studies and to interpret differences in searching and host selection behaviour of parasitoid species. Furthermore, field experiments can indicate whether the parameters measured in the laboratory are relevant to elucidation of the ecological processes under study, such as adaptation or species interactions. In previous extensive laboratory studies we studied plant- and host-searching behaviour, host acceptance, host suitability; host plant preference, and learning of two congeneric parasitoids of Pieris caterpillars: the generalist Cotesia glomerata, which has been reported to attack several Pieridae species, and C. rubecula, a specialist of the small cabbage white Pieris rapae. In the present field study our aim was to verify the importance of these previous laboratory findings for explaining the performance of these two species in the field. We investigated experimentally whether parasitism on three Pieris species varied with parasitoid species and with food plant of the caterpillars. We exposed different types of host plants, infested with different Pieris species, to parasitism by natural populations of Cotesia species, by setting the experimental plants out in Brussels sprouts cabbage fields. Furthermore we made direct observations of parasitoid foraging in the field. In general, the field results confirmed our predictions on the range of host plant and host species used in the field. The two Cotesia species appear to coexist through niche segregation, since C. glomerata was mainly recovered from P. brassicae and C. rubecula from P. rapae. Although C. glomerata is a generalist at the species level, it can be a specialist at the population level under certain ecological circumstances. Our study shows the importance of variation in host plant attraction and host species acceptance in restricting host plant and host diet in the field. Furthermore the results suggest that, at least in the Netherlands, specialisation of C. glomerata on P. brassicae may occur as a result of C. rubecula outcompeting C. glomerata in P. rapae larvae.

Development of the parasitoid, Cotesia rubecula (Hymenoptera: Braconidae) in Pieris rapae and Pieris brassicae (Lepidoptera: Pieridae): evidence for host regulation.

Several recent models examining the developmental strategies of parasitoids attacking hosts which continue feeding and growing after parasitism (=koinobiont parasitoids) assume that host quality is a non-linear function of host size at oviposition. We tested this assumption by comparing the growth and development of males of the solitary koinobiont endoparasitoid, Cotesia rubecula, in first (L1) to third (L3) larval instars of its preferred host, Pieris rapae and in a less preferred host, Pieris brassicae. Beginning 3 days after parasitism, hosts were dissected daily, and both host and parasitoid dry mass was determined. Using data on parasitoid dry mass, we measured the mean relative growth rate of C. rubecula, and compared the trajectories of larval growth of the parasitoid during the larval and pupal stages using non-linear equations. Parasitoids generally survived better, completed development faster, and grew larger in earlier than in later instars of both host species, and adult wasps emerging from P. rapae were significantly larger than wasps emerging from all corresponding instars of P. brassicae. During their early larval stages, parasitoids grew most slowly in L1 P. rapae, whereas in all other host classes of both host species growth to pupation proceeded fairly uniformly. The growth of both host species was markedly reduced after parasitism compared with controls, with the development of P. brassicae arrested at an earlier stage, and at a smaller body mass, than P. rapae. Our results suggest that C. rubecula regulates certain biochemical processes more effectively in P. rapae than in P. brassicae, in accordance with its own nutritional and physiological requirements. Furthermore, we propose that, for parasitoids such as C. rubecula, which do not consume all host tissues prior to pupation, that parasitoid size and host quality may vary independently of host size at oviposition and at larval parasitoid egression.

Larval parasitoid uses aggregation pheromone of adult hosts in foraging behaviour: a solution to the reliability-detectability problem.

Parasitoids that forage for herbivorous hosts by using infochemicals may have a problem concerning the reliability and detectability of these stimuli: host stimuli are highly reliable but not very detectable at a distance, while stimuli from the host's food are very detectable but generally not very reliable in indicating host presence. One solution to this problem is to learn to link highly detectable stimuli to reliable but not very detectable stimuli. Ample knowledge is available on how associative learning aids foraging parasitoids in the location of suitable microhabitats. However, in this paper we report on another solution to the reliability-detectability problem and present evidence for an essential, but as yet overlooked, aspect of Drosophila parasitoid ecology. For the first time it is shown that a parasitoid of Drosophila larvae spies on the communication system of adult Drosophila flies to locate potential host sites: naive parasitoids strongly respond to a volatile aggregation pheromone that is deposited in the oviposition site by recently mated female flies. Thus, the parasitoids resort to using highly detectable information from a host stage different from the one under attack (i.e. infochemical detour). The function and ecological implications of these findings are discussed.

Clutch size in a larval-pupal endoparasitoid : 1. Variation across and within host species.

Clutch size decisions by Aphaereta minuta (Nees) (Hymenoptera: Braconidae), a polyphagous, gregarious, larval-pupal endoparasitoid, were studied under laboratory conditions. This parasitoid attacks larvae of Diptera inhabiting ephemeral microhabitats such as decaying plant and animal material. Females oviposit in young larval stages, but the eventual size of the host pupa determines host food availability for competing offspring. The size of the pupa can differ greatly between host species. We questioned how A. minuta females deal with this delay between the moment of oviposition and eventual host food availability, and whether they make clutch size decisions that benefit their fitness. It was shown that females indeed vary their clutch size considerably and in an adaptive way: (1) females lay larger clutches in larvae of host species that produce larger pupae, even when the larvae are the same size at the moment of oviposition, and (2) females lay larger clutches in larger larvae than in smaller larvae of the same host species. The latter seems functional as larvae parasitized at an older stage indeed developed into larger pupae compared to larvae parasitized at a younger stage. Furthermore, mortality of parasitized young host larvae was greater than that of both unparasitized larvae and parasitized older larvae. Under field conditions the risk of mortality of young host larvae is expected to be even higher due to the limited period of microhabitat (host food) availability, strong scramble type competition between the host larvae, and the longer period of being exposed to predation.