Disentangling a Neotropical pest species complex: genetic diversity and population structure of the native rice stink bug Oebalus poecilus and the invasive O. ypsilongriseus.

BACKGROUND: A first step in any pest management initiative is recognizing the existing problem - identifying the pest species and its abundance and dispersal capacities. This is not simple and even more challenging when insidious (invasive) species are involved constituting a pest complex. Understanding a species' population diversity and structure can provide a better understanding of its adaptation and relative pest potential. Such is the need for the native rice stink bug Oebalus poecilus and the invasive O. ypsilongriseus in low and high flatlands of South America. RESULTS: The genetic structure differed between both rice stink bug species (FST  = 0.157, P = 0.001), where 84% of the overall genetic variability takes place within species and three genetic groups were recognized through Bayesian approach (K = 3). Oebalus poecilus exhibited slightly higher genetic diversity (HE  = 0.253) and structuring (FST  = 0.050, P = 0.001) than the invasive O. ypsilongriseus (HE  = 0.211; FST  = 0.038, P = 0.013). Nonetheless, only the former exhibited significant correlation between genetic and geographic distances (r = 0.48, P = 0.013). CONCLUSION: Despite the pointed peculiarities, the obtained results indicate overlap in both species' occurrence and similar genetic structure allowing for a compound problem to be dealt with as the complex requires managing without, as yet, a prevailing species or a niche specialization. © 2022 Society of Chemical Industry.

Signals of adaptation to agricultural stress in the genomes of two European bumblebees.

Human-induced environmental impacts on wildlife are widespread, causing major biodiversity losses. One major threat is agricultural intensification, typically characterised by large areas of monoculture, mechanical tillage, and the use of agrochemicals. Intensification leads to the fragmentation and loss of natural habitats, native vegetation, and nesting and breeding sites. Understanding the adaptability of insects to these changing environmental conditions is critical to predicting their survival. Bumblebees, key pollinators of wild and cultivated plants, are used as model species to assess insect adaptation to anthropogenic stressors. We investigated the effects of agricultural pressures on two common European bumblebees, Bombus pascuorum and B. lapidarius. Restriction-site Associated DNA Sequencing was used to identify loci under selective pressure across agricultural-natural gradients over 97 locations in Europe. 191 unique loci in B. pascuorum and 260 in B. lapidarius were identified as under selective pressure, and associated with agricultural stressors. Further investigation suggested several candidate proteins including several neurodevelopment, muscle, and detoxification proteins, but these have yet to be validated. These results provide insights into agriculture as a stressor for bumblebees, and signal for conservation action in light of ongoing anthropogenic changes.

High Genetic Diversity and Gene Flow Detected in Populations of Bombus morio from South Brazil.

Bumblebees are essential insects for the preservation of biodiversity in many ecosystems, as they can pollinate a wide variety of wild and cultivated plants. Knowledge of the genetic diversity of bumblebees can be used to understand and predict the health status of bee populations, enabling the development of strategies for crop management and conservation of this important group of pollinators. Here, we characterized the genetic diversity of B. morio populations from the Rio Grande do Sul state, Brazil, by amplification of the partial mitochondrial cytochrome oxidase I gene. The resulting data were then compared with genetic parameters of Bombus morio (Swederus 1787) obtained in populations from this species' full geographic range in South America. Our results revealed the presence of nine mitochondrial haplotypes in Rio Grande do Sul, three of which were novel haplotypes, and of significant genetic divergence among bumblebee populations from Brazil and South America. The mitochondrial haplotype BM01 was the most common and is probably the ancestral haplotype from which the others originated. There is also evidence that strong gene flow has taken place among Brazilian B. morio populations, explaining the sharing of haplotypes between distant populations. The populations of B. morio from Rio Grande do Sul present significant genetic diversity as the species is native to Southern/Southeastern Brazil and adapted to the ecological conditions in this wide range. Having well-connected populations with a large genetic potential will help this species to remain well adapted to the different environmental conditions within its native range.

Impact of intraspecific variation on measurements of thermal tolerance in bumble bees.

Climate change is an important driver of bee decline despite the fact that many species might respond to climate change differently. One method to predict how a species will respond to climate change is to identify its thermal tolerance limits. However, differences in thermal tolerance might also occur among distant populations of the same species based on their local environment or even among castes of social insects. Here, we investigated intraspecific differences in thermal tolerance among subspecies of the large earth bumble bee, Bombus terrestris (Apidae). We determined the critical thermal minima and maxima (CTmin and CTmax, respectively) of workers and queens from three lab-reared B. terrestris subspecies (B. t. terrestris, B. t. audax, and B. t. canariensis) which originated from different thermal environments. Our results showed that caste has an influence on critical thermal minima, with queens being most cold-tolerant, but the values of critical thermal maxima were not correlated to caste or size. The thermal tolerance of workers did not differ among the subspecies. Although heat tolerance was similar in queens, B. t. canariensis queens (originating from the warmest environments) were the least cold tolerant. Overall, we showed that B. terrestris may be generally robust against climate warming, but that particular subspecies and/or populations may be more vulnerable to extreme temperature variability. Future research should focus on responses of B. terrestris populations to short, extreme thermal events.

Bumblebee resilience to climate change, through plastic and adaptive responses.

Bumblebees are ubiquitous, cold-adapted eusocial bees found worldwide from subarctic to tropical regions of the world. They are key pollinators in most temperate and boreal ecosystems, and both wild and managed populations are significant contributors to agricultural pollination services. Despite their broad ecological niche at the genus level, bumblebee species are threatened by climate change, particularly by rising average temperatures, intensifying seasonality and the increasing frequency of extreme weather events. While some temperature extremes may be offset at the individual or colony level through temperature regulation, most bumblebees are expected to exhibit specific plastic responses, selection in various key traits, and/or range contractions under even the mildest climate change. In this review, we provide an in-depth and up-to-date review on the various ways by which bumblebees overcome the threats associated with current and future global change. We use examples relevant to the fields of bumblebee physiology, morphology, behaviour, phenology, and dispersal to illustrate and discuss the contours of this new theoretical framework. Furthermore, we speculate on the extent to which adaptive responses to climate change may be influenced by bumblebees' capacity to disperse and track suitable climate conditions. Closing the knowledge gap and improving our understanding of bumblebees' adaptability or avoidance behaviour to different climatic circumstances will be necessary to improve current species climate response models. These models are essential to make correct predictions of species vulnerability in the face of future climate change and human-induced environmental changes to unfold appropriate future conservation strategies.

Shift in size of bumblebee queens over the last century.

Species can respond differently when facing environmental changes, such as by shifting their geographical ranges or through plastic or adaptive modifications to new environmental conditions. Phenotypic modifications related to environmental factors have been mainly explored along latitudinal gradients, but they are relatively understudied through time despite their importance for key ecological interactions. Here we hypothesize that the average bumblebee queen body size has changed in Belgium during the last century. Based on historical and contemporary databases, we first tested if queen body sizes changed during the last century at the intraspecific level among four common bumblebee species and if it could be linked to global warming and/or habitat fragmentation as well as by the replacement by individuals from new populations. Then, we assessed body size changes at the community level, among 22 species, taking into account species population trends (i.e. increasing, stable or decreasing relative abundance). Our results show that the average queen body size of all four bumblebee species increased over the last century. This size increase was significantly correlated to global warming and habitat fragmentation, but not explained by changes in the population genetic structure (i.e. colonization). At the community level, species with stable or increasing relative abundance tend to be larger than declining species. Contrary to theoretical expectations from Bergmann's rule (i.e. increasing body size in colder climates), temperature does not seem to be the main driver of bumblebee body size during the last century as we observed the opposite body size trend. However, agricultural intensification and habitat fragmentation could be alternative mechanisms that shape body size clines. This study stresses the importance of considering alternative global change factors when assessing body size change.

Pattern of population structuring between Belgian and Estonian bumblebees.

Several population genetic studies investigated the extent of gene flow and population connectivity in bumblebees. In general, no restriction in gene flow is considered for mainland populations of common bumblebee species. Whether this assumption holds true for all species is not known. An assessment of bumblebee genetic structure in the context of their geographic distribution is needed to prioritize conservation and management needs. Here, we conducted a genetic study on seven bumblebee species occurring in Belgium and Estonia. Using 16 microsatellite markers, we investigated genetic diversity and population structuring in each species. This is the first study investigating population structuring of both declining and stable bumblebee species on both small and large geographic scales. Our results showed no or only low population structuring between the populations of the restricted and declining bumblebee species on both scales, while significant structuring was found for populations of the common species on the larger scale. The latter result, which may be due to human or environmental changes in the landscape, implies the need for the conservation of also widespread bumblebee species. Conservation strategies to improve gene flow and connectivity of populations could avoid the isolation and future losses of populations of these important species.

Honey bee predisposition of resistance to ubiquitous mite infestations.

Host-parasite co-evolution history is lacking when parasites switch to novel hosts. This was the case for Western honey bees (Apis mellifera) when the ectoparasitic mite, Varroa destructor, switched hosts from Eastern honey bees (Apis cerana). This mite has since become the most severe biological threat to A. mellifera worldwide. However, some A. mellifera populations are known to survive infestations, largely by suppressing mite population growth. One known mechanism is suppressed mite reproduction (SMR), but the underlying genetics are poorly understood. Here, we take advantage of haploid drones, originating from one queen from the Netherlands that developed Varroa-resistance, whole exome sequencing and elastic-net regression to identify genetic variants associated with SMR in resistant honeybees. An eight variants model predicted 88% of the phenotypes correctly and identified six risk and two protective variants. Reproducing and non-reproducing mites could not be distinguished using DNA microsatellites, which is in agreement with the hypothesis that it is not the parasite but the host that adapted itself. Our results suggest that the brood pheromone-dependent mite oogenesis is disrupted in resistant hosts. The identified genetic markers have a considerable potential to contribute to a sustainable global apiculture.

Level of Genetic Diversity in European Bumblebees is Not Determined by Local Species Abundance.

Bumblebee species with declining population trends tend to show lower genetic diversity levels than stable species. The observed difference might be explained by abundance differences, with declining bumblebee species having lower genetic diversity levels simply due to their lower local species abundances. However, whether this holds true is not known. Here, we investigated whether bumblebee local abundances determines population genetic diversity levels. Therefore, local species abundances were measured for bumblebee species at four locations in Belgium and two locations in Estonia during bumblebee foraging years 2013-2017. These locations and countries were chosen to ensure the greatest possible variance in both local abundances and population trends for these species. Hence, genetic diversity levels were obtained for seven species by genotyping collected specimens with 16 microsatellites. Our results showed that the observed patterns in genetic diversity did not depend on local species abundance. So, although declining bumblebee species can become locally abundant, they will still show lower genetic diversity levels than stable species. This result implies that measuring bumblebees' local abundance cannot be used to directly determine the health status of a population. Furthermore, this result has also major impact on future conservation strategies as increasing the genetic diversity levels of declining species will be very difficult, and habitat quality should be high to maintain their populations, otherwise these species are doomed to disappear first.

Chemical reproductive traits of diploid Bombus terrestris males: Consequences on bumblebee conservation.

The current bumblebee decline leads to inbreeding in populations that fosters a loss of allelic diversity and diploid male production. As diploid males are viable and their offspring are sterile, bumblebee populations can quickly fall in a vortex of extinction. In this article, we investigate for the first time a potential premating mechanism through a major chemical reproductive trait (male cephalic labial gland secretions) that could prevent monandrous virgin queens from mating with diploid males. We focus our study on the cephalic labial gland secretions of diploid and haploid males of Bombus terrestris (L.). Contrary to initial expectations, our results do not show any significant differentiation of cephalic labial gland secretions between diploid and haploid specimens. Queens seem therefore to be unable to avoid mating with diploid males based on their compositions of cephalic labial gland secretions. This suggests that the vortex of extinction of diploid males could not be stopped through premating avoidance based on the cephalic labial gland secretions but other mechanisms could avoid mating between diploid males and queens.

A century of temporal stability of genetic diversity in wild bumblebees.

Since the 1950s, bumblebee (Bombus) species are showing a clear decline worldwide. Although many plausible drivers have been hypothesized, the cause(s) of this phenomenon remain debated. Here, genetic diversity in recent versus historical populations of bumblebee species was investigated by selecting four currently restricted and four currently widespread species. Specimens from five locations in Belgium were genotyped at 16 microsatellite loci, comparing historical specimens (1913-1915) with recent ones (2013-2015). Surprisingly, our results showed temporal stability of genetic diversity in the restricted species. Furthermore, both historical and recent populations of restricted species showed a significantly lower genetic diversity than found in populations of co-occurring widespread species. The difference in genetic diversity between species was thus already present before the alleged recent drivers of bumblebee decline could have acted (from the 1950's). These results suggest that the alleged drivers are not directly linked with the genetic variation of currently declining bumblebee populations. A future sampling in the entire distribution range of these species will infer if the observed link between low genetic diversity and population distribution on the Belgium scale correlates with species decline on a global scale.

Microsatellite Analysis of Museum Specimens Reveals Historical Differences in Genetic Diversity between Declining and More Stable Bombus Species.

Worldwide most pollinators, e.g. bumblebees, are undergoing global declines. Loss of genetic diversity can play an essential role in these observed declines. In this paper, we investigated the level of genetic diversity of seven declining Bombus species and four more stable species with the use of microsatellite loci. Hereto we genotyped a unique collection of museum specimens. Specimens were collected between 1918 and 1926, in 6 provinces of the Netherlands which allowed us to make interspecific comparisons of genetic diversity. For the stable species B. pascuorum, we also selected populations from two additional time periods: 1949-1955 and 1975-1990. The genetic diversity and population structure in B. pascuorum remained constant over the three time periods. However, populations of declining bumblebee species showed a significantly lower genetic diversity than co-occurring stable species before their major declines. This historical difference indicates that the repeatedly observed reduced genetic diversity in recent populations of declining bumblebee species is not caused solely by the decline itself. The historically low genetic diversity in the declined species may be due to the fact that these species were already rare, making them more vulnerable to the major drivers of bumblebee decline.

Quantitative Trait Loci for Light Sensitivity, Body Weight, Body Size, and Morphological Eye Parameters in the Bumblebee, Bombus terrestris.

Bumblebees such as Bombus terrestris are essential pollinators in natural and managed ecosystems. In addition, this species is intensively used in agriculture for its pollination services, for instance in tomato and pepper greenhouses. Here we performed a quantitative trait loci (QTL) analysis on B. terrestris using 136 microsatellite DNA markers to identify genes linked with 20 traits including light sensitivity, body size and mass, and eye and hind leg measures. By composite interval mapping (IM), we found 83 and 34 suggestive QTLs for 19 of the 20 traits at the linkage group wide significance levels of p = 0.05 and 0.01, respectively. Furthermore, we also found five significant QTLs at the genome wide significant level of p = 0.05. Individual QTLs accounted for 7.5-53.3% of the phenotypic variation. For 15 traits, at least one QTL was confirmed with multiple QTL model mapping. Multivariate principal components analysis confirmed 11 univariate suggestive QTLs but revealed three suggestive QTLs not identified by the individual traits. We also identified several candidate genes linked with light sensitivity, in particular the Phosrestin-1-like gene is a primary candidate for its phototransduction function. In conclusion, we believe that the suggestive and significant QTLs, and markers identified here, can be of use in marker-assisted breeding to improve selection towards light sensitive bumblebees, and thus also the pollination service of bumblebees.

16S rRNA Amplicon Sequencing Demonstrates that Indoor-Reared Bumblebees (Bombus terrestris) Harbor a Core Subset of Bacteria Normally Associated with the Wild Host.

A MiSeq multiplexed 16S rRNA amplicon sequencing of the gut microbiota of wild and indoor-reared Bombus terrestris (bumblebees) confirmed the presence of a core set of bacteria, which consisted of Neisseriaceae (Snodgrassella), Orbaceae (Gilliamella), Lactobacillaceae (Lactobacillus), and Bifidobacteriaceae (Bifidobacterium). In wild B. terrestris we detected several non-core bacteria having a more variable prevalence. Although Enterobacteriaceae are unreported by non next-generation sequencing studies, it can become a dominant gut resident. Furthermore the presence of some non-core lactobacilli were associated with the relative abundance of bifidobacteria. This association was not observed in indoor-reared bumblebees lacking the non-core bacteria, but having a more standardized microbiota compared to their wild counterparts. The impact of the bottleneck microbiota of indoor-reared bumblebees when they are used in the field for pollination purpose is discussed.

Molecular variability and genetic structure of Chrysodeixis includens (Lepidoptera: Noctuidae), an important soybean defoliator in Brazil.

This study provides the first genetic characterization of the soybean looper, Chrysodeixis includens (Walker, 1857), an important defoliating pest species of soybean crops in Brazil. Population genetic variability and the genetic structure of C. includens populations were evaluated by using ISSR markers with samples from the major soybean producing regions in Brazil in the growing seasons 2011/2012. Seven different primers were applied for population characterization of the molecular variability and genetic structure of 8 soybean looper populations from 8 states of Brazil. The seven ISSR loci generated 247 bands in 246 individuals of C. includens sampled. The expected heterozygosity (HE) in the populations varied between 0.093 and 0.106, while the overall HE was 0.099, indicating low genetic diversity. The analysis of molecular variance indicated that 98% of the variability was expressed among individuals within populations (FST = 0.021, p = 0.001). The low level of polymorphism over all populations, the high levels of gene flow, and the low genetic structure are indicatives of the exchange of genetic information between the different sampled regions. Population structuring suggests the presence of two major groups which do not correlate with their geographic sampling location in Brazil. These results may indicate recent recolonization of C. includens in Brazil or migration patterns following source-sink dynamics. Furthermore, the presence of two groups within C. includens suggests that a study on development of resistance or any other genetic-based trait needs to be evaluated on both groups, and pest management in soybean fields should be aware that differences may come to the control strategies they use.

Genetic variability of the neogregarine Apicystis bombi, an etiological agent of an emergent bumblebee disease.

The worldwide spread of diseases is considered a major threat to biodiversity and a possible driver of the decline of pollinator populations, particularly when novel species or strains of parasites emerge. Previous studies have suggested that populations of introduced European honeybee (Apis mellifera) and bumblebee species (Bombus terrestris and Bombus ruderatus) in Argentina share the neogregarine parasite Apicystis bombi with the native bumblebee (Bombus dahlbomii). In this study we investigated whether A. bombi is acting as an emergent parasite in the non-native populations. Specifically, we asked whether A. bombi, recently identified in Argentina, was introduced by European, non-native bees. Using ITS1 and ITS2 to assess the parasite's intraspecific genetic variation in bees from Argentina and Europe, we found a largely unstructured parasite population, with only 15% of the genetic variation being explained by geographic location. The most abundant haplotype in Argentina (found in all 9 specimens of non-native species) was identical to the most abundant haplotype in Europe (found in 6 out of 8 specimens). Similarly, there was no evidence of structuring by host species, with this factor explaining only 17% of the genetic variation. Interestingly, parasites in native Bombus ephippiatus from Mexico were genetically distant from the Argentine and European samples, suggesting that sufficient variability does exist in the ITS region to identify continent-level genetic structure in the parasite. Thus, the data suggest that A. bombi from Argentina and Europe share a common, relatively recent origin. Although our data did not provide information on the direction of transfer, the absence of genetic structure across space and host species suggests that A. bombi may be acting as an emergent infectious disease across bee taxa and continents.

Recruitment to forage of bumblebees in artificial low light is less impaired in light sensitive colonies, and not only determined by external morphological parameters.

Bumblebees of Bombus terrestris are essential pollinators in natural and managed ecosystems. Their foraging ability relies on the individual morphology, task allocation within the colony, and external factors, such as light intensity. The foraging activities of commercial bumblebees can sometimes be impaired, especially in the artificial and weak light intensities of greenhouses at high altitudes. Here we investigated whether the eagerness (or willingness) to forage of bumblebee colonies in different light conditions is correlated with the light sensitivity of bumblebees colonies and/or different external morphological parameters. The initial foraging capacity of bumblebee colonies correlated with their light sensitivity. However, light sensitive bumblebee colonies did not necessarily had a higher foraging activity at lower light intensities. Differences in initial foraging capacity and light sensitivity among colonies could not be explained by the external morphological parameters. In conclusion, our data illustrated that the recruitment to forage in artificial low light is less impaired in light sensitive colonies, and that not only the external morphology parameters determine the light sensitivity of bumblebees and their eagerness to forage in weak light conditions. The data obtained here create a better understanding of which criteria are able to select towards light sensitive bumblebees and their link with the foraging capacity of these bumblebees.

Evolution of the content of THC and other major cannabinoids in drug-type cannabis cuttings and seedlings during growth of plants.

In Europe, authorities frequently ask forensic laboratories to analyze seized cannabis plants to prove that cultivation was illegal (drug type and not fiber type). This is generally done with mature and flowering plants. However, authorities are often confronted with very young specimens. The aim of our study was to evaluate when the chemotype of cannabis plantlets can be surely determined through analysis of eight major cannabinoids content during growth. Drug-type seedlings and cuttings were cultivated, sampled each week, and analyzed by high-performance liquid chromatography with diode array detection. The chemotype of clones was recognizable at any developmental stage because of high total Δ(9)-tetrahydrocannabinol (THC) concentrations even at the start of the cultivation. Conversely, right after germination seedlings contained a low total THC content, but it increased quickly with plant age up, allowing chemotype determination after 3 weeks. In conclusion, it is not necessary to wait for plants' flowering to identify drug-type cannabis generally cultivated in Europe.