Interference with the retinoic acid signalling pathway inhibits the initiation of teeth and caudal primary scales in the small-spotted catshark Scyliorhinus canicula.

The retinoic acid (RA) pathway was shown to be important for tooth development in mammals, and suspected to play a key role in tooth evolution in teleosts. The general modalities of development of tooth and "tooth-like" structures (collectively named odontodes) seem to be conserved among all jawed vertebrates, both with regard to histogenesis and genetic regulation. We investigated the putative function of RA signalling in tooth and scale initiation in a cartilaginous fish, the small-spotted catshark Scyliorhinus canicula. To address this issue, we identified the expression pattern of genes from the RA pathway during both tooth and scale development and performed functional experiments by exposing small-spotted catshark embryos to exogenous RA or an inhibitor of RA synthesis. Our results showed that inhibiting RA synthesis affects tooth but not caudal primary scale development while exposure to exogenous RA inhibited both. We also showed that the reduced number of teeth observed with RA exposure is probably due to a specific inhibition of tooth bud initiation while the observed effects of the RA synthesis inhibitor is related to a general delay in embryonic development that interacts with tooth development. This study provides data complementary to previous studies of bony vertebrates and support an involvement of the RA signalling pathway toolkit in odontode initiation in all jawed vertebrates. However, the modalities of RA signalling may vary depending on the target location along the body, and depending on the species lineage.

Hot and cold waves decrease sperm production and bias sex ratio in the parasitoid wasp Cotesia typhae (Hymenoptera, Braconidae).

Parasitoid wasps are haplodiploid, meaning that sperm stored by egg laying females are only used to produce daughters. Thus, the sex ratio of the offspring depends on the availability of sperm after mating. In these insects, males are sensitive to temperature at the pupal stage. This stress leads to subfertility due to a drastic reduction in the number of sperm produced and transferred to females. Experiments were conducted under controlled conditions on the parasitoid wasp Cotesia typhae (Hymenoptera, Braconidae), a natural enemy of the invading pest Sesamia nonagrioides (Lepidoptera, Noctuidae). At 25-27 °C, sperm production was measured for 7 days, and found to reach a plateau at the third day of adult life. It leads to a final amount around 25,000 sperm per male. A male can successfully inseminate at least 10 females, producing predominantly female offspring. Sperm production decreased significantly after 1 day of pupal exposure to heat at 34 or 36 °C and 7 days of cold at 0, 5 or 10 °C. This highlights that both cold and heat are stressful. After mating with one male treated at 10 or 34 °C, females store fewer sperm than the control, and produce fewer daughters. The sex ratio of the offspring is male biased when males experienced temperature stresses during development, like other parasitoid wasps. In the field, C. typhae populations would be affected by heat and cold, at least at the pupal stage. This lowers overwintering risk in case this biological agent was introduced in Europe. This risk is both economical, as companies seek to establish costly continuous production to sell beneficial insects, and ecological as the introduced population would not settle in the ecosystem. Lastly, the transport and storage of this insect of agronomic interest would need to consider temperature variations to ensure successful application.

Investigating bracovirus chromosomal integration and inheritance in lepidopteran host and nontarget species.

Bracoviruses (BVs) are domesticated viruses found in braconid parasitoid wasp genomes. They are composed of domesticated genes from a nudivrius, coding viral particles in which wasp DNA circles are packaged. BVs are viewed as possible vectors of horizontal transfer of genetic material (HT) from wasp to their hosts because they are injected, together with wasp eggs, by female wasps into their host larvae, and because they undergo massive chromosomal integration in multiple host tissues. Here, we show that chromosomal integrations of the Cotesia typhae BV (CtBV) persist up to the adult stage in individuals of its natural host, Sesamia nonagrioides, that survived parasitism. However, while reproducing host adults can bear an average of nearly two CtBV integrations per haploid genome, we were unable to retrieve any of these integrations in 500 of their offspring using Illumina sequencing. This suggests either that host gametes are less targeted by CtBVs than somatic cells or that gametes bearing BV integrations are nonfunctional. We further show that CtBV can massively integrate into the chromosomes of other lepidopteran species that are not normally targeted by the wasp in the wild, including one which is divergent by at least 100 million years from the natural host. Cell entry and chromosomal integration of BVs are thus unlikely to be major factors shaping wasp host range. Together, our results shed new light on the conditions under which BV-mediated wasp-to-host HT may occur and provide information that may be helpful to evaluate the potential risks of uncontrolled HT associated with the use of parasitoid wasps as biocontrol agents.

Genome Size Variation of Chagas Disease Vectors of the Rhodniini Tribe.

The genome size of five Rhodnius species (R. milesi, R. nasutus, R. neivai, R. prolixus, and R. robustus) and two Psammolestes species (P. coroedes and P. tertius) were estimated using flow cytometry and/or k-mer distributions in genome sequences. Phylogenetic generalized linear mixed models highlighted significant genome size variations among species and between sexes, with R. prolixus showing the largest genome. In this study we provide the first data on female genome size in Triatominae. For five species, female genome size did not differ from males, except for R. robustus, where females had smaller genomes. Genome size estimations based on the k-mer distribution method were less than those estimated from flow cytometry, but both methods exhibited the same pattern of sexual differences. Further genomic studies are needed to infer whether genome size variation could be an adaptive trait in Rhodnius.

Measuring the evolutionary potential of a winter-active parasitic wasp to climate change.

In temperate climates, as a consequence of warming winters, an increasing number of ectothermic species are remaining active throughout winter months instead of diapausing, rendering them increasingly vulnerable to unpredictable cold events. One species displaying a shift in overwintering strategy is the parasitoid wasp and biological control agent Aphidius avenae. The current study aimed to better understand the consequence of a changing overwintering strategy on the evolutionary potential of an insect population to adapt to the cold stress events, set to increase in frequency, even during milder winters. Using a parental half-sibling breeding design, narrow-sense heritability of the cold tolerance, morphology and longevity of A. avenae was estimated. The heritability of cold tolerance was estimated at 0.07 (CI95% = [0.00; 0.25]) for the Critical Thermal Minima (CTmin) and 0.11 (CI95% = [0.00; 0.34]) for chill coma temperature; estimates much lower than those obtained for morphological traits (tibia length 0.20 (CI95% = [0.03; 0.37]); head width 0.23 (CI95% = [0.09; 0.39]); wing surface area 0.28 (CI95% = [0.11; 0.47])), although comparable with the heritability estimate of 0.12 obtained for longevity (CI95% = [0.00; 0.25]). The heritability estimates obtained thus suggest that A. avenae possesses low adaptive potential against cold stress. If such estimates are indicative of the evolutionary potential of A. avenae cold tolerance, more emphasis may be placed on adaptive phenotypic plasticity at the individual level to persist in a changing climate, with potential implications for the biological control function they provide.

Quantitative trait loci involved in the reproductive success of a parasitoid wasp.

Dissecting the genetic basis of intraspecific variations in life history traits is essential to understand their evolution, notably for potential biocontrol agents. Such variations are observed in the endoparasitoid Cotesia typhae (Hymenoptera: Braconidae), specialized on the pest Sesamia nonagrioides (Lepidoptera: Noctuidae). Previously, we identified two strains of C. typhae that differed significantly for life history traits on an allopatric host population. To investigate the genetic basis underlying these phenotypic differences, we used a quantitative trait locus (QTL) approach based on restriction site-associated DNA markers. The characteristic of C. typhae reproduction allowed us generating sisters sharing almost the same genetic content, named clonal sibship. Crosses between individuals from the two strains were performed to generate F2 and F8 recombinant CSS. The genotypes of 181 clonal sibships were determined as well as the phenotypes of the corresponding 4,000 females. Informative markers were then used to build a high-quality genetic map. These 465 markers spanned a total length of 1,300 cM and were organized in 10 linkage groups which corresponded to the number of C. typhae chromosomes. Three QTLs were detected for parasitism success and two for offspring number, while none were identified for sex ratio. The QTLs explained, respectively, 27.7% and 24.5% of the phenotypic variation observed. The gene content of the genomic intervals was investigated based on the genome of C. congregata and revealed 67 interesting candidates, as potentially involved in the studied traits, including components of the venom and of the symbiotic virus (bracovirus) shown to be necessary for parasitism success in related wasps.

Under-Expression of Chemosensory Genes in Domiciliary Bugs of the Chagas Disease Vector Triatoma brasiliensis.

BACKGROUND: In Latin America, the bloodsucking bugs Triatominae are vectors of Trypanosoma cruzi, the parasite that causes Chagas disease. Chemical elimination programs have been launched to control Chagas disease vectors. However, the disease persists because native vectors from sylvatic habitats are able to (re)colonize houses-a process called domiciliation. Triatoma brasiliensis is one example. Because the chemosensory system allows insects to interact with their environment and plays a key role in insect adaption, we conducted a descriptive and comparative study of the chemosensory transcriptome of T. brasiliensis samples from different ecotopes. METHODOLOGY/PRINCIPAL FINDING: In a reference transcriptome built using de novo assembly, we found transcripts encoding 27 odorant-binding proteins (OBPs), 17 chemosensory proteins (CSPs), 3 odorant receptors (ORs), 5 transient receptor potential channel (TRPs), 1 sensory neuron membrane protein (SNMPs), 25 takeout proteins, 72 cytochrome P450s, 5 gluthatione S-transferases, and 49 cuticular proteins. Using protein phylogenies, we showed that most of the OBPs and CSPs for T. brasiliensis had well supported orthologs in the kissing bug Rhodnius prolixus. We also showed a higher number of these genes within the bloodsucking bugs and more generally within all Hemipterans compared to the other species in the super-order Paraneoptera. Using both DESeq2 and EdgeR software, we performed differential expression analyses between samples of T. brasiliensis, taking into account their environment (sylvatic, peridomiciliary and domiciliary) and sex. We also searched clusters of co-expressed contigs using HTSCluster. Among differentially expressed (DE) contigs, most were under-expressed in the chemosensory organs of the domiciliary bugs compared to the other samples and in females compared to males. We clearly identified DE genes that play a role in the chemosensory system. CONCLUSION/SIGNIFICANCE: Chemosensory genes could be good candidates for genes that contribute to adaptation or plastic rearrangement to an anthropogenic system. The domiciliary environment probably includes less diversity of xenobiotics and probably has more stable abiotic parameters than do sylvatic and peridomiciliary environments. This could explain why both detoxification and cuticle protein genes are less expressed in domiciliary bugs. Understanding the molecular basis for how vectors adapt to human dwellings may reveal new tools to control disease vectors; for example, by disrupting chemical communication.

MtDNA COI-COII marker and drone congregation area: an efficient method to establish and monitor honeybee (Apis mellifera L.) conservation centres.

Honeybee subspecies have been affected by human activities in Europe over the past few decades. One such example is the importation of nonlocal subspecies of bees which has had an adverse impact on the geographical repartition and subsequently on the genetic diversity of the black honeybee Apis mellifera mellifera. To restore the original diversity of this local honeybee subspecies, different conservation centres were set up in Europe. In this study, we established a black honeybee conservation centre Conservatoire de l'Abeille Noire d'Ile de France (CANIF) in the region of Ile-de-France, France. CANIF's honeybee colonies were intensively studied over a 3-year period. This study included a drone congregation area (DCA) located in the conservation centre. MtDNA COI-COII marker was used to evaluate the genetic diversity of CANIF's honeybee populations and the drones found and collected from the DCA. The same marker (mtDNA) was used to estimate the interactions and the haplotype frequency between CANIF's honeybee populations and 10 surrounding honeybee apiaries located outside of the CANIF. Our results indicate that the colonies of the conservation centre and the drones of the DCA show similar stable profiles compared to the surrounding populations with lower level of introgression. The mtDNA marker used on both DCA and colonies of the conservation centre seems to be an efficient approach to monitor and maintain the genetic diversity of the protected honeybee populations.

Defensive behaviour of Apis mellifera against Vespa velutina in France: testing whether European honeybees can develop an effective collective defence against a new predator.

We investigated the prey-predator interactions between the European honeybee, Apis mellifera, and the invasive yellow-legged hornet, Vespa velutina, which first invaded France in 2004 and thereafter spread to neighbouring European countries (Spain, Portugal and Italy). Our goal was to determine how successfully honeybees are able to defend their colonies against their new predator in Europe. Experiments were conducted in the southwest of France-the point of entry of the hornet in Europe-under natural and semi-controlled field conditions. We investigated a total of eight apiaries and 95 colonies subjected to either low or high levels of predation. We analyzed hornet predatory behaviour and collective response of colonies under attack. The results showed that A. mellifera in France exhibit an inefficient and unorganized defence against V. velutina, unlike in other regions of Europe and other areas around the globe where honeybees have co-evolved with their natural Vespa predators.

High-resolution linkage map for two honeybee chromosomes: the hotspot quest.

Meiotic recombination is a fundamental process ensuring proper disjunction of homologous chromosomes and allele shuffling in successive generations. In many species, this cellular mechanism occurs heterogeneously along chromosomes and mostly concentrates in tiny fragments called recombination hotspots. Specific DNA motifs have been shown to initiate recombination in these hotspots in mammals, fission yeast and drosophila. The aim of this study was to check whether recombination also occurs in a heterogeneous fashion in the highly recombinogenic honeybee genome and whether this heterogeneity can be connected with specific DNA motifs. We completed a previous picture drawn from a routine genetic map built with an average resolution of 93 kb. We focused on the two smallest honeybee chromosomes to increase the resolution and even zoomed at very high resolution (3.6 kb) on a fragment of 300 kb. Recombination rates measured in these fragments were placed in relation with occurrence of 30 previously described motifs through a Poisson regression model. A selection procedure suitable for correlated variables was applied to keep significant motifs. These fine and ultra-fine mappings show that recombination rate is significantly heterogeneous although poorly contrasted between high and low recombination rate, contrarily to most model species. We show that recombination rate is probably associated with the DNA methylation state. Moreover, three motifs (CGCA, GCCGC and CCAAT) are good candidates of signals promoting recombination. Their influence is however moderate, doubling at most the recombination rate. This discovery extends the way to recombination dissection in insects.

Native Prey and Invasive Predator Patterns of Foraging Activity: The Case of the Yellow-Legged Hornet Predation at European Honeybee Hives.

Contrary to native predators, which have co-evolved with their prey, alien predators often benefit from native prey naïveté. Vespa velutina, a honeybee predator originating from Eastern China, was introduced into France just before 2004. The present study, based on video recordings of two beehives at an early stage of the invasion process, intends to analyse the alien hornet hunting behaviour on the native prey, Apis mellifera, and to understand the interaction between the activity of the predator and the prey during the day and the season. Chasing hornets spent most of their time hovering facing the hive, to catch flying honeybees returning to the hive. The predation pressure increased during the season confirming previous study based on predator trapping. The number of honeybee captures showed a maximum peak for an intermediate number of V. velutina, unrelated to honeybee activity, suggesting the occurrence of competition between hornets. The number of honeybees caught increased during midday hours while the number of hornets did not vary, suggesting an increase in their efficacy. These results suggest that the impact of V. velutina on honeybees is limited by its own biology and behaviour and did not match the pattern of activity of its prey. Also, it could have been advantageous during the invasion, limiting resource depletion and thus favouring colonisation. This lack of synchronization may also be beneficial for honeybee colonies by giving them an opportunity to increase their activity when the hornets are less effective.

Fine scale analysis of crossover and non-crossover and detection of recombination sequence motifs in the honeybee (Apis mellifera).

BACKGROUND: Meiotic exchanges are non-uniformly distributed across the genome of most studied organisms. This uneven distribution suggests that recombination is initiated by specific signals and/or regulations. Some of these signals were recently identified in humans and mice. However, it is unclear whether or not sequence signals are also involved in chromosomal recombination of insects. METHODOLOGY: We analyzed recombination frequencies in the honeybee, in which genome sequencing provided a large amount of SNPs spread over the entire set of chromosomes. As the genome sequences were obtained from a pool of haploid males, which were the progeny of a single queen, an oocyte method (study of recombination on haploid males that develop from unfertilized eggs and hence are the direct reflect of female gametes haplotypes) was developed to detect recombined pairs of SNP sites. Sequences were further compared between recombinant and non-recombinant fragments to detect recombination-specific motifs. CONCLUSIONS: Recombination events between adjacent SNP sites were detected at an average distance of 92 bp and revealed the existence of high rates of recombination events. This study also shows the presence of conversion without crossover (i. e. non-crossover) events, the number of which largely outnumbers that of crossover events. Furthermore the comparison of sequences that have undergone recombination with sequences that have not, led to the discovery of sequence motifs (CGCA, GCCGC, CCGCA), which may correspond to recombination signals.

A third-generation microsatellite-based linkage map of the honey bee, Apis mellifera, and its comparison with the sequence-based physical map.

BACKGROUND: The honey bee is a key model for social behavior and this feature led to the selection of the species for genome sequencing. A genetic map is a necessary companion to the sequence. In addition, because there was originally no physical map for the honey bee genome project, a meiotic map was the only resource for organizing the sequence assembly on the chromosomes. RESULTS: We present the genetic (meiotic) map here and describe the main features that emerged from comparison with the sequence-based physical map. The genetic map of the honey bee is saturated and the chromosomes are oriented from the centromeric to the telomeric regions. The map is based on 2,008 markers and is about 40 Morgans (M) long, resulting in a marker density of one every 2.05 centiMorgans (cM). For the 186 megabases (Mb) of the genome mapped and assembled, this corresponds to a very high average recombination rate of 22.04 cM/Mb. Honey bee meiosis shows a relatively homogeneous recombination rate along and across chromosomes, as well as within and between individuals. Interference is higher than inferred from the Kosambi function of distance. In addition, numerous recombination hotspots are dispersed over the genome. CONCLUSION: The very large genetic length of the honey bee genome, its small physical size and an almost complete genome sequence with a relatively low number of genes suggest a very promising future for association mapping in the honey bee, particularly as the existence of haploid males allows easy bulk segregant analysis.

The genome of Apis mellifera: dialog between linkage mapping and sequence assembly.

Two independent genome projects for the honey bee, a microsatellite linkage map and a genome sequence assembly, interactively produced an almost complete organization of the euchromatic genome. Assembly 4.0 now includes 626 scaffolds that were ordered and oriented into chromosomes according to the framework provided by the third-generation linkage map (AmelMap3). Each construct was used to control the quality of the other. The co-linearity of markers in the sequence and the map is almost perfect and argues in favor of the high quality of both.

Exceptionally high levels of recombination across the honey bee genome.

The first draft of the honey bee genome sequence and improved genetic maps are utilized to analyze a genome displaying 10 times higher levels of recombination (19 cM/Mb) than previously analyzed genomes of higher eukaryotes. The exceptionally high recombination rate is distributed genome-wide, but varies by two orders of magnitude. Analysis of chromosome, sequence, and gene parameters with respect to recombination showed that local recombination rate is associated with distance to the telomere, GC content, and the number of simple repeats as described for low-recombining genomes. Recombination rate does not decrease with chromosome size. On average 5.7 recombination events per chromosome pair per meiosis are found in the honey bee genome. This contrasts with a wide range of taxa that have a uniform recombination frequency of about 1.6 per chromosome pair. The excess of recombination activity does not support a mechanistic role of recombination in stabilizing pairs of homologous chromosome during chromosome pairing. Recombination rate is associated with gene size, suggesting that introns are larger in regions of low recombination and may improve the efficacy of selection in these regions. Very few transposons and no retrotransposons are present in the high-recombining genome. We propose evolutionary explanations for the exceptionally high genome-wide recombination rate.

Whole-genome scan in thelytokous-laying workers of the Cape honeybee (Apis mellifera capensis): central fusion, reduced recombination rates and centromere mapping using half-tetrad analysis.

While workers of almost all subspecies of honeybee are able to lay only haploid male eggs, Apis mellifera capensis workers are able to produce diploid female eggs by thelytokous parthenogenesis. Cytological analyses have shown that during parthenogenesis, egg diploidy is restored by fusion of the two central meiotic products. This peculiarity of the Cape bee preserves two products of a single meiosis in the daughters and can be used to map centromere positions using half-tetrad analysis. In this study, we use the thelytokous progenies of A. m. capensis workers and a sample of individuals from a naturally occurring A. m. capensis thelytokous clone to map centromere position for most of the linkage groups of the honeybee. We also show that the recombination rate is reduced by >10-fold during the meiosis of A. m. capensis workers. This reduction is restricted to thelytokous parthenogenesis of capensis workers and is not observed in the meiosis of queen within the same subspecies or in arrhenotokous workers of another subspecies. The reduced rate of recombination seems to be associated with negative crossover interference. These results are discussed in relation to evolution of thelytokous parthenogenesis and maintenance of heterozygosity and female sex after thelytoky.

A microsatellite-based linkage map of the honeybee, Apis mellifera L.

A linkage map for the honeybee (Apis mellifera) was constructed mainly from the progeny of two hybrid queens (A. m. ligustica x A. m. mellifera). A total of 541 loci were mapped; 474 were microsatellite loci; a few were additional bands produced during PCRs, one of the two rDNA loci (using ITS), the MDH locus, and three sex-linked markers (Q and FB loci and one RAPD band). Twenty-four linkage groups were estimated of which 5 were minute (between 7.1 and 22.8 cM) and 19 were major groups (>76.5 cM). The number of major linkage groups exceeded by three the number of chromosomes of the complement (n = 16). The sum of the lengths of all linkage groups amounts to 4061 cM to which must be added at least 320 cM to link groups in excess, making a total of at least 4381 cM. The length of the largest linkage group I was 630 cM. The average density of markers was 7.5 cM and the average resolution was about one marker every 300 kb. For most of the large groups, the centromeric region was determined genetically, as described in (accompanying article in this issue), using half-tetrad analysis of thelytokous parthenogens in which diploid restoration occurs through central fusion. Several cases of segregation distortion that appreared to result from deleterious recessives were discovered. A low positive interference was also detected.