Influence of honey bee (Apis mellifera) breeding on wing venation in Serbia and neighbouring countries.

In order to improve the productivity of honey bees (Apis mellifera), some of their traits are selected by breeding. On one hand, breeding is mainly based on the natural geographical variation of this species; on the other hand, mass production and distribution of artificially selected queens can significantly affect the natural geographic variation of honey bees. In this study, we have compared honey bee wings originating from breeding and non-breeding populations in Serbia. In the comparison, we have also used data from a large area of south-eastern Europe. The wings were measured using the 19 landmarks indicated on the wing images. The coordinates were analysed using the methodology of geometric morphometrics. We found that honey bees obtained from honey bee queen breeder differed in wing venation from surrounding populations, which are under natural selection. Therefore, we argue against including populations under artificial selection in the analysis of the natural geographical variation of honey bees. In our analysis of non-breeding samples, we found that in south-eastern Europe there is continuous variation in wing venation and no clear boundaries between A. m. carnica, A. m. cecropia, and A. m. macedonica.

In Search of Apis mellifera pomonella in Kazakhstan.

Apis mellifera pomonella is one of two subspecies that represent the eastern limits of honey bee (Apis mellifera) distribution, and it is important to understand its biogeography and evolution. Despite this, A. m. pomonella was not investigated after its discovery 20 years ago. In particular, it is not known if it was hybridized or not with exotic subspecies introduced by beekeepers. In this study, we analysed the variation in honey bee forewing shape in Kazakhstan. Bees were collected from apiaries, where the origin of the queens was not controlled by beekeepers; they formed a group called "local bees", and apiaries where queens declared as either A. m. carnica or A. m. mellifera were obtained from queen breeders. The two later groups were called "exotic bees". We showed that local bees are still distinct from exotic ones. However, some samples showed signs of introgression with exotic subspecies from lineage C. In terms of wing shape, the local bees were most similar to lineage O. We concluded that the local bees most likely represented A. m. pomonella. We provided wing images and identification data, which can help to identify A. m. pomonella and protect it in the future. It is interesting that the nearby honey bee population sourced from China, which is not separated by any clear barrier to gene flow, belonged to lineage M.

Honey bee (Apis mellifera) wing images: a tool for identification and conservation.

BACKGROUND: The honey bee (Apis mellifera) is an ecologically and economically important species that provides pollination services to natural and agricultural systems. The biodiversity of the honey bee in parts of its native range is endangered by migratory beekeeping and commercial breeding. In consequence, some honey bee populations that are well adapted to the local environment are threatened with extinction. A crucial step for the protection of honey bee biodiversity is reliable differentiation between native and nonnative bees. One of the methods that can be used for this is the geometric morphometrics of wings. This method is fast, is low cost, and does not require expensive equipment. Therefore, it can be easily used by both scientists and beekeepers. However, wing geometric morphometrics is challenging due to the lack of reference data that can be reliably used for comparisons between different geographic regions. FINDINGS: Here, we provide an unprecedented collection of 26,481 honey bee wing images representing 1,725 samples from 13 European countries. The wing images are accompanied by the coordinates of 19 landmarks and the geographic coordinates of the sampling locations. We present an R script that describes the workflow for analyzing the data and identifying an unknown sample. We compared the data with available reference samples for lineage and found general agreement with them. CONCLUSIONS: The extensive collection of wing images available on the Zenodo website can be used to identify the geographic origin of unknown samples and therefore assist in the monitoring and conservation of honey bee biodiversity in Europe.

Using the Software DeepWings© to Classify Honey Bees across Europe through Wing Geometric Morphometrics.

DeepWings© is a software that uses machine learning to automatically classify honey bee subspecies by wing geometric morphometrics. Here, we tested the five subspecies classifier (A. m. carnica, Apis mellifera caucasia, A. m. iberiensis, Apis mellifera ligustica, and A. m. mellifera) of DeepWings© on 14,816 wing images with variable quality and acquired by different beekeepers and researchers. These images represented 2601 colonies from the native ranges of the M-lineage A. m. iberiensis and A. m. mellifera, and the C-lineage A. m. carnica. In the A. m. iberiensis range, 92.6% of the colonies matched this subspecies, with a high median probability (0.919). In the Azores, where the Iberian subspecies was historically introduced, a lower proportion (85.7%) and probability (0.842) were observed. In the A. m mellifera range, only 41.1 % of the colonies matched this subspecies, which is compatible with a history of C-derived introgression. Yet, these colonies were classified with the highest probability (0.994) of the three subspecies. In the A. m. carnica range, 88.3% of the colonies matched this subspecies, with a probability of 0.984. The association between wing and molecular markers, assessed for 1214 colonies from the M-lineage range, was highly significant but not strong (r = 0.31, p < 0.0001). The agreement between the markers was influenced by C-derived introgression, with the best results obtained for colonies with high genetic integrity. This study indicates the good performance of DeepWings© on a realistic wing image dataset.

Wing measurements are a possible tool for the identification of European forensically important Sarcophagidae.

The flesh flies are a group of insects well known for their forensic importance. Reliable identification of these flies relies on the use of either molecular markers or the morphology of the male genital apparatus. Identification of female flesh flies is more time consuming and less reliable than their male counterparts. This is particularly problematic for forensic entomology practitioners, because female flesh flies are more abundant than males in carrion arthropod assemblages. As such, it is critical that alternative methods for flesh fly identification are established that are equally effective for both sexes. One promising technique is the use of wing measurements, which have been shown to be reliable for the identification of some groups of necrophagous Diptera from several geographical regions. We applied this method to the European Sarcophagidae for the first time, using a dataset of 881 specimens representing 29 species and 5 genera. Species identifications were based on 15 landmarks located at wing vein junctions. We also combined our results with data from previous studies of Calliphoridae and Muscidae which utilised the same methodology, enabling the testing of family level identification using wing morphometrics. Species identifications using wing measurements had varied success. While some species were successfully identified without error, others, particularly from the genus Sarcophaga, were often misclassified. Notably, in several species wing measurements successfully identified both males and females. The results presented here suggest that wing measurements are a promising complementary method to other methods for the identification of necrophagous Sarcophagidae especially in material unsorted at the family level. It can also be used to double check identification performed by a taxonomist using traditional methods.

Geographical Variation of Honey Bee (Apis mellifera L. 1758) Populations in South-Eastern Morocco: A Geometric Morphometric Analysis.

In Morocco, there are two well-recognised honey bee (Apis mellifera L.) subspecies: A. m. intermissa in the north and A. m. sahariensis in the south-east. The latter subspecies is found in the arid and semiarid climates of the Sahara Desert. In this study, we used honey bees from four areas of south-eastern Morocco which are, to some degree, isolated by arid zones. We analysed the shape and size of the forewings, using the method of geometric morphometrics. The bees from the four areas of south-eastern Morocco differed significantly in terms of wing shape. Moreover, bees from traditional hives were smaller than those from modern hives. The bees from south-eastern Morocco were clearly different from the reference samples obtained from the Morphometric Bee Data Bank in Oberursel, Germany, representing most of the global variation in honey bees. Surprisingly, the bees were also different from A. m. sahariensis, which should occur in the study area, according to earlier studies. This difference could have been caused by introgression with non-native subspecies imported by beekeepers. The distinct honey bees from south-eastern Morocco deserve to be protected. We provide a method for identifying them, which can help protect them.

Historical Changes in Honey Bee Wing Venation in Romania.

The honey bee (Apis mellifera) is an ecologically and economically important species that provides pollination services to natural and agricultural systems. The biodiversity of the honey bee is being endangered by the mass import of non-native queens. In many locations, it is not clear how the local populations have been affected by hybridisation between native and non-native bees. There is especially little information about temporal changes in hybridisation. In Romania, A. m. carpatica naturally occurs, and earlier studies show that there are two subpopulations separated by the Carpathian Mountains. In this study, we investigated how the arrangement of veins in bees' wings (venation) has changed in Romanian honey bees in the last four decades. We found that in the contemporary population of Romanian bees, there are still clear differences between the intra- and extra-Carpathian subpopulations, which indicates that natural variation among honey bees is still being preserved. We also found significant differences between bees collected before and after 2000. The observed temporal changes in wing venation are most likely caused by hybridisation between native bees and non-native bees sporadically introduced by beekeepers. In order to facilitate conservation and the monitoring of native Romanian bees, we developed a method facilitating their identification.

Mitochondrial DNA Suggests the Introduction of Honeybees of African Ancestry to East-Central Europe.

In Europe, protecting the genetic diversity of Apis mellifera is usually perceived in the context of limiting the spread of the evolutionary C-lineage within the original range of the M-lineage. However, due to climate change and large-scale ongoing movement of breeding individuals, the expansion of bees from the African A-lineage could represent another threat. This issue has not yet been investigated in detail, although A-mitotypes occur in South-West and South Europe due to natural gene flow. Here, we determine the diversity of mtDNA in honey bees from East-Central Europe. We sequenced the COI-COII region in 427 bees sampled along two 900 km transects (17.5° N and 23° E). We found that 1.64% of bees (95% CI: 0.66-3.35 %) had A-mitotypes. It is unlikely that their presence in the area resulted from natural migration but instead human driven introductions of hybrids of African ancestry. This expansion deserves more attention, as it may contribute to the dissemination of undesirable traits, parasites and diseases.

Climatic oscillations in Quaternary have shaped the co-evolutionary patterns between the Norway spruce and its host-associated herbivore.

During the Last Glacial Maximum in the Northern Hemisphere, expanding ice sheets forced a large number of plants, including trees, to retreat from their primary distribution areas. Many host-associated herbivores migrated along with their host plants. Long-lasting geographic isolation between glacial refugia could have been led to the allopatric speciation in separated populations. Here, we have studied whether the migration history of the Norway spruce Picea abies in Quaternary has affected its host-associated herbivorous beetle-Monochamus sartor. By using microsatellite markers accompanied by the geometric morphometrics analysis of wing venation, we have revealed the clear geographic structure of M. sartor in Eurasia, encompassing two main clusters: southern (Alpine-Carpathian) and eastern (including northeastern Europe and Asia), which reflects the northern and southern ecotypes of its host. The two beetles' lineages probably diverged during the Pleniglacial (57,000-15,000 BC) when their host tree species was undergoing significant range fragmentation and experienced secondary contact during post-glacial recolonization of spruce in the Holocene. A secondary contact of divergent lineages of M. sartor has resulted in the formation of the hybrid zone in northeastern Europe. Our findings suggest that the climatic oscillations during the Pleistocene have driven an insect-plant co-evolutionary process, and have contributed to the formation of the unique biodiversity of Europe.

Wing measurement can be used to identify European blow flies (Diptera: Calliphoridae) of forensic importance.

Correct species identification is a crucial step in research and the practice of forensic entomology. At the moment the most common and widely used tool for this purpose are taxonomic keys based on morphological characters. During recent decades molecular methods have been used more and more often for species identification. Automated species identification can also be based on wing measurements. This method proved to be reliable in the case of some groups of necrophagous Diptera from various geographical regions. Up to now, this method has not been applied to a wide range of species of European Calliphoridae. We have used 968 specimens representing 15 species and 6 genera. Identification was based on 15 landmarks located at wing vein junctions. Wing shape differed markedly between the studied species. The overall correct classification rate at the level of species was 97.1%. Seven species, including Calliphora vomitoria, Chrysomya albiceps, Phormia regina, Protophormia terraenovae and Lucilia sericata were identified without error. Lucilia caesar and Lucilia illustris, which are considered relatively difficult to distinguish, were correctly classified in 93.0% and 94.1% cases, respectively. The correct classification rate at the level of genera was 99.2%. The results presented here suggest that wing measurements are a promising alternative to other methods of identification of necrophagous Calliphoridae. This method can be particularly useful for identification of large samples of females collected during carrion succession studies. It can also be used to double check identification performed by a taxonomist using traditional methods.

Honey bees are larger and live longer after developing at low temperature.

Honey bees (Apis mellifera) are known to be temperature specialist and actively maintain brood temperature in a very narrow temperature range. Developing larvae are sensitive to changes of temperature in the nest. Temperatures lower than generally assumed as optimal have been shown to cause a number of negative developmental and behavioural changes in honey bees. We have reared both worker and drone larvae during the capped brood stage in cold (32 °C) and in warm temperatures (35 °C). Next, we measured their body mass at emergence and the longevity of individuals either caged in incubator (workers) or placed in maintaining colonies (drones). For drones, the reproductive caste, we also compared the mass and ratio of body parts (head, thorax, and abdomen) to body mass. As expected, both castes were heavier when reared in cold, but contrary to our expectations, both castes survived longer after emergence than bees reared in warm. Drones reared in cold were characterized by proportionally larger abdomens, in comparison to drones reared in warm.

Developmental Costs of Biological Invasion: The Exotic Wood Borer Tetropium fuscum (Coleoptera: Cerambycidae) is More Asymmetric and Smaller in Invaded Area.

Biological invasions provide a unique opportunity to gain insight into basic biological processes occurring under new circumstances. During the process of establishment, exotic species are exposed to various stressors which may affect their development. Presence of the stressors is often detected by measurements of left-right body asymmetry, which consists of two main components: fluctuating asymmetry and directional asymmetry. Fluctuating asymmetry constitutes random differences between the two body sides, whereas directional asymmetry occurs when a particular trait is bigger on one of the sides. The relation between these two asymmetry components is still not fully understood. Our goal was to investigate the potential differences in asymmetry patterns between native and invasive populations of Tetropium fuscum (Fabr. 1787) (Coleoptera: Cerambycidae), a harmful forest pest native to Europe and introduced to North America. Wing asymmetry assessment was based on the geometric morphometrics of hind wings. We found that specimens from invaded area were markedly smaller and have more asymmetric wings than individuals from native population, suggesting some unfavorable conditions in the invaded area. Moreover, we found significant directional asymmetry in the native but not in the invasive population. On the other hand, differences between left and right hind wings were similar in the native and invasive populations, in terms of direction. This suggests that a high level of fluctuating asymmetry in the invasive population may blur the intrinsic directional asymmetry and hinder its detection. Our data show that fluctuating asymmetry has a potential as an indicator of developmental stress in invasive species.

DKey software for editing and browsing dichotomous keys.

Despite advances in computer technology and the increasing availability of multiple-access taxonomic keys, traditional dichotomous keys remain the most often used taxonomic identification tools. On the other hand, there seems to be a lack of an editor of dichotomous keys, which is both freely available and easy to use. The DKey software was developed in order to alleviate this problem. A taxonomic key in text format can be imported to the software in order to edit it. Various editing options are possible, including: moving couplets, removing couplets, combining keys and renumbering keys. The software can output the key either in the traditional text format, ready for publication in a scientific journal, or in hypertext linked format, which makes identification faster and easier, due to the fact that pointers can be clicked in order to move to the next couplet. The DKey software should be useful for both taxonomic experts creating keys and those who use them for identification. The DKey software is freely available and open source.

Elytra reduction may affect the evolution of beetle hind wings.

Beetles are one of the largest and most diverse groups of animals in the world. Conversion of forewings into hardened shields is perceived as a key adaptation that has greatly supported the evolutionary success of this taxa. Beetle elytra play an essential role: they minimize the influence of unfavorable external factors and protect insects against predators. Therefore, it is particularly interesting why some beetles have reduced their shields. This rare phenomenon is called brachelytry and its evolution and implications remain largely unexplored. In this paper, we focused on rare group of brachelytrous beetles with exposed hind wings. We have investigated whether the elytra loss in different beetle taxa is accompanied with the hind wing shape modification, and whether these changes are similar among unrelated beetle taxa. We found that hind wings shape differ markedly between related brachelytrous and macroelytrous beetles. Moreover, we revealed that modifications of hind wings have followed similar patterns and resulted in homoplasy in this trait among some unrelated groups of wing-exposed brachelytrous beetles. Our results suggest that elytra reduction may affect the evolution of beetle hind wings.

Forewing structure of the solitary bee Osmia bicornis developing on heavy metal pollution gradient.

Wild bees in natural conditions can develop under various environmental stressors. Heavy metal pollution of the environment is one of the most widely studied stressors in insects, yet its effect is poorly described in bees. We have measured how pollution of the environment along a zinc, cadmium and lead contamination gradient in Poland affects bee development, using red mason bees (Osmia bicornis) as a model and their forewing asymmetry measures to assess possible developmental instabilities. We have also described wing asymmetry measures in the red mason bee-an important managed pollinator species-for the first time. The development of bee larvae in a contaminated environment did not affect forewing asymmetry measures, but it did lead to a negative correlation of wing size with contamination in females. Bees also showed a clear change in their asymmetry measures between various seasons, suggesting other, unknown environmental factors affecting wing asymmetry more than pollution. Sexes were found to have different forewing shape and size, larger females having larger forewings than the smaller males. The direction of size asymmetry was in favour of the left side in both sexes and also shape differences between the left and right wings showed similar tendencies in males and females. The levels of forewing shape and size asymmetry were smaller in females, making them the more symmetrical sex.

Identification of Muscidae (Diptera) of medico-legal importance by means of wing measurements.

Cadavers attract numerous species and genera of Muscidae, both regular elements of carrion insect assemblages, and accidental visitors. Identification of adult Muscidae may be considered difficult, particularly by non-experts. Since species identification is a vital first step in the analysis of entomological material in any forensic entomology orientated experiment and real cases, various alternative methods of species identification have been proposed. We investigated possibility of semiautomated identification by means of wing measurements as an alternative for classic morphology and DNA-based approaches. We examined genus-level identification success for 790 specimens representing 13 genera of the most common European cadavers visiting Muscidae. We found 99.8% of examined specimens correctly identified to the genus-level. Without error, the following were identified: Azelia, Eudasyphora, Graphomya, Hydrotaea, Musca, Muscina, Mydaea, Neomyia, Polietes, Stomoxys and Thricops. Genus-level misidentifications were found only in Helina and Phaonia. Discrimination of examined material on the species level within Hydrotaea (318 specimens representing eight species) and Muscina (163 specimens representing four species) showed lower, yet still high average identification success, 97.2 and 98.8%, respectively. Our results revealed relatively high success in both genus and species identification of Muscidae of medico-legal importance. Semiautomated identification by means of wing measurements can be used by non-experts and does not require sophisticated equipment. This method will facilitate the identification of forensically relevant muscids in comparison to more difficult and more time-consuming identification approaches based on taxonomic keys or DNA-based methods. However, for unambiguous identification of some taxa, we recommend complementary use of identification keys.

Hind wing morphology facilitates discrimination between two sibling species: Leiopus nebulosus and L. linnei (Coleoptera: Cerambycidae).

The study focused on two sibling beetle species: Leiopus nebulosus (Linnaeus, 1758) and L. linnei Wallin, Nylander & Kvamme, 2009. These species are very similar morphologically and their identification is difficult and possible only by experienced taxonomists. A supporting method for identification of L. nebulosus and L. linnei based on hind wings measurements was developed. The study was based on 115 specimens of L. linnei and 45 specimens of L. nebulosus. The correctness of identification of L. nebulosus amounted to 95.56%, and of L. linnei - 97.39%. The obtained model facilitates reliable identification of L. nebulosus and L. linnei also by less experienced entomologists. Geographical distributions of both species were summarized based on faunistic data from 39 scientific papers. The results show that both species have a Western-Palearctic distribution. Their distribution ranges are markedly overlapping. However, L. linnei is the species reported from larger number of localities, and observed more often.

Within- and Between-Species Variation of Wing Venation in Genus Monochamus (Coleoptera: Cerambycidae).

We have described the morphological variation of five Western Palaearctic species of Monochamus Dejean, 1821. The variation was assessed using wing measurements. Special emphasis was placed on the differences between Monochamus sartor (F., 1787) and Monochamus urussovii (Fischer-Waldheim, 1805). There was an interesting pattern of variation between the two species. Individuals of M. sartor from the Carpathians differed markedly from individuals of M. urussovii from Siberia, but individuals from north-eastern Poland were intermediate between those two populations. The intermediate individuals were more similar to the Siberian M. urussovii than to the Carpathian M. sartor despite the relatively large geographic distance between north-eastern Poland and Siberia. The occurrence of the intermediate individuals in north-eastern Poland may be the effect of hybridization between M. urussovii and M. sartor, which might have occurred after secondary contact between the two species in the Holocene.

Semiautomated Identification of European Corn Borer (Lepidoptera: Crambidae).

The European corn borer Ostrinia nubilalis (Hübner, 1796) is a serious and widely studied pest of corn. The most common method of its control is by means of insecticides. However, biological control is becoming more and more popular. The hymenopteran parasitoid Trichogramma sp. is the most promising and effective one among the biological agents and is now widely used in North America and Europe. Its application should occur at the time when the European corn borer is at the beginning of the eggs laying period. However, the discrimination between the European corn borer and some other species occurring in agricultural landscapes at the same time can be difficult, especially for farmers which are neither familiar with the morphological nor molecular methods of identification. The scope of this study is to test the ability of the automatic computer equipment to determine the European corn borer and to separate it from the most common Lepidoptera pests found in corn plantations. The experiment showed that the 97.0% of the 247 specimens belonging to four common pestlepidopterans were correctly classified by the use of a personal computer, desktop scanner, and the special software. The obtained results showed that this technique based on wing measurements can be an effective tool for monitoring of the European corn borer. In the future, this method can be used by farmers to identify this pest and apply control measures at optimal time.

Bourgeois behavior and freeloading in the colonial orb web spider Parawixia bistriata (Araneae, Araneidae).

Spiders of the tropical American colonial orb weaver Parawixia bistriata form a communal bivouac in daytime. At sunset, they leave the bivouac and construct individual, defended webs within a large, communally built scaffolding of permanent, thick silk lines between trees and bushes. Once spiders started building a web, they repelled other spiders walking on nearby scaffolding with a "bounce" behavior. In nearly all cases (93%), this resulted in the intruder leaving without a fight, akin to the "bourgeois strategy," in which residents win and intruders retreat without escalated contests. However, a few spiders (6.5%) did not build a web due to lack of available space. Webless spiders were less likely to leave when bounced (only 42% left) and instead attempted to "freeload," awaiting the capture of prey items in nearby webs. Our simple model shows that webless spiders should change their strategy from bourgeois to freeloading satellite as potential web sites become increasingly occupied.