Saija Piiroinen

Sequencing, De Novo assembly and annotation of the Colorado Potato Beetle, Leptinotarsa decemlineata, Transcriptome.

Background The Colorado potato beetle (Leptinotarsa decemlineata) is a major pest and a serious threat to potato cultivation throughout the northern hemisphere. Despite its high importance for invasion biology, phenology and pest management, little is known about L. decemlineata from a genomic perspective. We subjected European L. decemlineata adult and larval transcriptome samples to 454-FLX massively-parallel DNA sequencing to characterize a basal set of genes from this species. We created a combined assembly of the adult and larval datasets including the publicly available midgut larval Roche 454 reads and provided basic annotation. We were particularly interested in diapause-specific genes and genes involved in pesticide and Bacillus thuringiensis (Bt) resistance. Results Using 454-FLX pyrosequencing, we obtained a total of 898,048 reads which, together with the publicly available 804,056 midgut larval reads, were assembled into 121,912 contigs. We established a repository of genes of interest, with 101 out of the 108 diapause-specific genes described in Drosophila montana; and 621 contigs involved in insecticide resistance, including 221 CYP450, 45 GSTs, 13 catalases, 15 superoxide dismutases, 22 glutathione peroxidases, 194 esterases, 3 ADAM metalloproteases, 10 cadherins and 98 calmodulins. We found 460 putative miRNAs and we predicted a significant number of single nucleotide polymorphisms (29,205) and microsatellite loci (17,284). Conclusions This report of the assembly and annotation of the transcriptome of L. decemlineata offers new insights into diapause-associated and insecticide-resistance-associated genes in this species and provides a foundation for comparative studies with other species of insects. The data will also open new avenues for researchers using L. decemlineata as a model species, and for pest management research. Our results provide the basis for performing future gene expression and functional analysis in L. decemlineata and improve our understanding of the biology of this invasive species at the molecular level.

Chronic neonicotinoid pesticide exposure and parasite stress differentially affects learning in honeybees and bumblebees

Learning and memory are crucial functions which enable insect pollinators to efficiently locate and extract floral rewards. Exposure to pesticides or infection by parasites may cause subtle but ecologically important changes in cognitive functions of pollinators. The potential interactive effects of these stressors on learning and memory have not yet been explored. Furthermore, sensitivity to stressors may differ between species, but few studies have compared responses in different species. Here, we show that chronic exposure to field-realistic levels of the neonicotinoid clothianidin impaired olfactory learning acquisition in honeybees, leading to potential impacts on colony fitness, but not in bumblebees. Infection by the microsporidian parasite Nosema ceranae slightly impaired learning in honeybees, but no interactive effects were observed. Nosema did not infect bumblebees (3% infection success). Nevertheless, Nosema-treated bumblebees had a slightly lower rate of learning than controls, but faster learning in combination with neonicotinoid exposure. This highlights the potential for complex interactive effects of stressors on learning. Our results underline that one cannot readily extrapolate findings from one bee species to others. This has important implications for regulatory risk assessments which generally use honeybees as a model for all bees.

Pre-invasion history and demography shape the genetic variation in the insecticide resistance-related acetylcholinesterase 2 gene in the invasive Colorado potato beetle

BackgroundInvasive pest species offers a unique opportunity to study the effects of genetic architecture, demography and selection on patterns of genetic variability. Invasive Colorado potato beetle (Leptinotarsa decemlineata) populations have experienced a rapid range expansion and intense selection by insecticides. By comparing native and invasive beetle populations, we studied the origins of organophosphate (OP) resistance-associated mutations in the acetylcholinesterase 2 (AChE2) gene, and the role of selection and demography on its genetic variability.ResultsAnalysis of three Mexican, two US and five European populations yielded a total of 49 haplotypes. Contrary to the expectations all genetic variability was associated with a point mutation linked to insecticide resistance (S291G), this mutation was found in 100% of Mexican, 95% of US and 71% of European beetle sequences analysed. Only two susceptible haplotypes, genetically very differentiated, were found, one in US and one in Europe. The genetic variability at the AChE2 gene was compared with two other genes not directly affected by insecticide selection, diapause protein 1 and juvenile hormone esterase. All three genes showed reduction in genetic variability indicative of a population bottleneck associated with the invasion.ConclusionsStochastic effects during invasion explain most of the observed patterns of genetic variability at the three genes investigated. The high frequency of the S291G mutation in the AChE2 gene among native populations suggests this mutation is the ancestral state and thus, either a pre-adaptation of the beetle for OP resistance or the AChE2 is not the major gene conferring OP resistance. The long historical association with host plant alkaloids together with recombination may have contributed to the high genetic variation at this locus. The genetic diversity in the AChE2 locus of the European beetles, in turn, strongly reflects founder effects followed by rapid invasion. Our results suggest that despite the long history of insecticide use in this species, demographic events together with pre-invasion history have been strongly influential in shaping the genetic diversity of the AChE2 gene in the invasive beetle populations.

Resting metabolic rate can vary with age independently from body mass changes in the Colorado potato beetle, Leptinotarsa decemlineata.

Temperature and mass dependency of insect metabolic rates are well known, while less attention has been given to other factors, such as age. Among insect species that experience seasonal variation in environmental conditions, such as in temperate latitudes, age may also have indirect effects on the metabolic rate. We examined the effect of age on the resting metabolic rate of Leptinotarsa decemlineata during 11 days after adult emergence by using flow-through respirometry. Age had a significant mass-independent effect on metabolic rate of beetles. A twofold increase in metabolic rate occurred during the first 2 days of adult life after which metabolic rate decreased with age relatively slowly. Ten day-old adult beetles had a metabolic rate similar to newly emerged beetles. The beetles have to be able to complete their development and prepare for overwintering during the relatively short favourable summer periods. Therefore, the observed pattern in metabolic rate may reflect physiological changes in the pre-diapause beetles adapted to temperate latitudes.

Photoperiodic effects on diapause-associated gene expression trajectories in European Leptinotarsa decemlineata populations

Behavioural and physiological changes during diapause, an important strategy of insects for surviving harsh seasonal conditions, have been intensively studied. The genetic and molecular mechanisms underpinning diapause development are less well known. We took a candidate gene approach to study prediapause gene expression patterns in the Colorado potato beetle (Leptinotarsa decemlineata), an invasive insect that has rapidly spread northwards to high seasonality environments. Newly eclosed beetles originating from southern (Italy) and northern (Russia) Europe were reared under short‐ [12 h light (L):12 h dark (D)] and long‐day (18L:6D) photoperiods for 10 days. This time period includes the sensitive period for the photoperiodic induction and initiation of diapause. Gene expression trajectories of 12 diapause‐related genes (regulatory, metabolic and stress‐resistance) were analysed from 0‐, 5‐ and 10‐day‐old beetles. Gene expression differences increased with age, deviating significantly between populations and photoperiods in 10‐day‐old beetles. The gene expression profiles, particularly those related to energy metabolism and stress‐resistance, indicate that beetles originating from Russia also prepare for diapause under the long‐day photoperiod and show qualitative differences in the diapausing phenotype. Our study shows that population‐dependent differences seen in behavioural and physiological traits connected with diapause in L. decemlineata are also evident in the expression trajectories of diapause‐related genes.

Stress for invasion success? Temperature stress of preceding generations modifies the response to insecticide stress in an invasive pest insect

Adaptation to stressful environments is one important factor influencing species invasion success. Tolerance to one stress may be complicated by exposure to other stressors experienced by the preceding generations. We studied whether parental temperature stress affects tolerance to insecticide in the invasive Colorado potato beetle Leptinotarsa decemlineata. Field‐collected pyrethroid‐resistant beetles were reared under either stressful (17°C) or favourable (23°C) insecticide‐free environments for three generations. Then, larvae were exposed to pyrethroid insecticides in common garden conditions (23°C). Beetles were in general tolerant to stress. The parental temperature stress alone affected beetles positively (increased adult weight) but it impaired their tolerance to insecticide exposure. In contrast, offspring from the favourable temperature regime showed compensatory weight gain in response to insecticide exposure. Our study emphasizes the potential of cross‐generational effects modifying species stress tolerance. When resistant pest populations invade benign environments, a re‐application of insecticides may enhance their performance via hormetic effects. In turn, opposite effects may arise if parental generations have been exposed to temperature stress. Thus, the outcome of management practices of invasive pest species is difficult to predict unless we also incorporate knowledge of the evolutionary and recent (preceding generations) stress history of the given populations into pest management.

No effect of low-level chronic neonicotinoid exposure on bumblebee learning and fecundity

In recent years, many pollinators have declined in abundance and diversity worldwide, presenting a potential threat to agricultural productivity, biodiversity and the functioning of natural ecosystems. One of the most debated factors proposed to be contributing to pollinator declines is exposure to pesticides, particularly neonicotinoids, a widely used class of systemic insecticide. Also, newly emerging parasites and diseases, thought to be spread via contact with managed honeybees, may pose threats to other pollinators such as bumblebees. Compared to honeybees, bumblebees could be particularly vulnerable to the effects of stressors due to their smaller and more short-lived colonies. Here, we studied the effect of field-realistic, chronic clothianidin exposure and inoculation with the parasite Nosema ceranae on survival, fecundity, sugar water collection and learning using queenless Bombus terrestris audax microcolonies in the laboratory. Chronic exposure to 1 ppb clothianidin had no significant effects on the traits studied. Interestingly, pesticide exposure in combination with additional stress caused by harnessing bees for Proboscis Extension Response (PER) learning assays, led to an increase in mortality. In contrast to previous findings, the bees did not become infected by N. ceranae after experimental inoculation with the parasite spores, suggesting variability in host resistance or parasite virulence. However, this treatment induced a slight, short-term reduction in sugar water collection, potentially through stimulation of the immune system of the bees. Our results suggest that chronic exposure to 1 ppb clothianidin does not have adverse effects on bumblebee fecundity or learning ability.

Northward range expansion requires synchronization of both overwintering behaviour and physiology with photoperiod in the invasive Colorado potato beetle (Leptinotarsa decemlineata).

Photoperiodic phenological adaptations are prevalent in many organisms living in seasonal environments. As both photoperiod and growth season length change with latitude, species undergoing latitudinal range expansion often need to synchronize their life cycle with a changing photoperiod and growth season length. Since adaptive synchronization often involves a large number of time-consuming genetic changes, behavioural plasticity might be a faster way to adjust to novel conditions. We compared behavioural and physiological traits in overwintering (diapause) preparation in three latitudinally different European Colorado potato beetle (Leptinotarsa decemlineata) populations reared under two photoperiods. Our aim was to study whether behavioural plasticity could play a role in rapid range expansion into seasonal environments. Our results show that while burrowing into the soil occurred in the southernmost studied population also under a non-diapause-inducing long photoperiod, the storage lipid content of these beetles was very low compared to the northern populations. However, similar behavioural plasticity was not found in the northern populations. Furthermore, the strongest suppression of energy metabolism was seen in pre-diapause beetles from the northernmost population. These results could indicate accelerated diapause preparation and possibly energetic adjustments due to temporal constraints imposed by a shorter, northern, growth season. Our results indicate that behavioural plasticity in burrowing may have facilitated initial range expansion of L. decemlineata in Europe. However, long-term persistence at high latitudes has required synchronization of burrowing behaviour with physiological traits. The results underline that eco-physiological life-history traits of insects, such as diapause, should be included in studies on range expansion.

Sublethal effects of deltamethrin exposure of parental generations on physiological traits and overwintering in Leptinotarsa decemlineata

Although the evolution of insecticide resistance has received a lot of attention, less is known about sublethal effects of insecticide stress experienced by the preceding generations on the performance of pest populations. We investigated whether three generations of parental exposure to a deltamethrin insecticide influences physiological traits and overwintering success of offspring in Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Beetles descending from insecticide‐exposed parents had lower adult body mass but higher relative lipid content and resting metabolic rate than those descending from non‐insecticide‐exposed parents. Also, a higher proportion of beetles descending from insecticide‐exposed parents than from control parents overwintered on the soil surface, which was associated with very low overwintering survival. When burrowed into the soil for overwintering, both groups had similar probability to survive the overwintering period. Parental insecticide exposure can reduce overall overwintering survival in the next generation by disturbing overwintering behaviour. Although beetles descending from insecticide‐exposed parents were small, the overwintering conditions seem to select for high lipid content, which could override the negative effects of small weight, and possibly improve fitness in the following growing season. Thus, insecticide application may have unintended consequences, which should be considered in pest management.

Responses in metabolic rate to changes in temperature in diapausing Colorado potato beetle Leptinotarsa decemlineata from three European populations

Many insects survive adverse periods in seasonal environments by entering diapause, a deep resting stage, during which energy consumption is typically low and gas exchange is in the form of a discontinuous gas exchange cycle (DGC). Because insects in high‐latitude environments are severely time constrained during summer, an effective diapause termination with careful regulation of metabolic rate is important. The present study examines whether diapausing Colorado potato beetles Leptinotarsa decemlineata Say originating from three latitudinally different regions in Europe differ in their quantitative or qualitative gas exchange patterns in response to an increasing temperature. Overall production of gaseous CO2, as well as qualitative patterns relating to the DGC, are measured at a late stage of diapause at four different temperatures in increasing order from 13, 18, 23 to 28 °C. Overall CO2 production is found to be lower in the two northern populations (61°49′N and 55°75′N) compared with the southernmost population (45°48′N) but increases as a function of temperature in all populations in a similar way. However, in the northern populations, raising the temperature increases the amount of CO2 discharged during single DGC peaks, whereas the DGC frequency remains relatively unchanged. By contrast, in the southernmost population, the amount of CO2 discharged during individual DGC peaks remains relatively unchanged, whereas the DGC frequency increases as a function of temperature. The observed differences may relate to water retention benefits or, alternatively, energetic benefits relating to heightened gas exchange efficiency in hypoxic or hypercapnic environments. Overall, the results suggest that, although populations of L. decemlineata may have similar thermal sensitivities, they have different qualitative strategies to regulate metabolic re‐activation at diapause termination.