Diversity of Diptera in Estonian pig farms including their capability as vectors of the African swine fever

Magistritöö Vee ja maismaa ökosüsteemide rakendusbioloogia erialalMaailmas on kahetiivaliste mitmekesisust loomafarmides uuritud vähe. Eestis pole antud uuringut üheski farmitüübis teostatud. Loomakasvatusega seonduvad kahetiivalised on võimelised siirutama mitmesuguseid haigustekitajaid, tuues sellega kaasa olulist kahju majandusele. Seakasvatustele on sigade Aafrika katk üks raskemini kontrollitavaid ja majanduslikult laastavamaid haiguseid. Magistritöö eesmärkideks oli selgitada välja kahetiivaliste mitmekesisus seafarmides ning kas sigade Aafrika katkuga kokku puutunud kahetiivalistelt on võimalik viirust tuvastada. Lähtuvalt töö eesmärkidest on püstitatud järgnevad hüpoteesid: Hüpotees I: Kahetiivaliste arvukus ja liigiline mitmekesisus seafarmides sõltuvad nii looduskeskkonnast seafarmi ümbruses kui ka tootmissüsteemist. Hüpotees II: Sigade Aafrika katku nakatunud sigadega kokku puutunud kahetiivalistelt on võimalik leida viirust. Töö käigus uuriti 2016. aasta augustis ja septembris ning 2017. aastal maist–augustini liimiga kaetud kärbsepabereid kasutades, millised kahetiivaliste liigid esinevad Eesti seafarmides. Samuti uuriti nakatunud metssigade lähiümbrusest ja Saaremaal asuvast nakatunud seafarmist kogutud putukate DNA analüüsimisel otsest kokkupuudet sigade Aafrika katku viirusega. Töö tulemusena määrati uuritud seafarmidest kokku 23 liiki/ rühma kahetiivalisi. Kokku koguti kahe aasta proovikogumiskordade jooksul seafarmidest 186 701 lülijalgset, kellest 180 444 moodustasid kahetiivalised. Putukate koguhulka farmis ei mõjutanud seafarmi ümbritsev looduslik keskkond ega farmi tootmissüsteemi parameetrid. Käesoleva uuringu puuduseks on ilmselgelt liialt väike farmide valim ja farmide mittejärjepidev osalemine uuringus. Surnud metssigade pealt ja kohalt kogutud putukate hulgas osutusid kolm liiki SAK positiivseks, kellest omakorda õnnestus SAK viirusetüvi määrata seatäidelt. Saaremaal nakatunud seafarmis epidemioloogilise uuringu käigus kogutud putukatest osutusid SAK positiivsteks 4 isendit (27% kõikidest analüüsitud putukatest), kuid viirusetüve sekveneerimine ei õnnestunud mitte ühegi farmist püütud viiruspositiivse putuka puhul. DNA analüüsil saadud viiruspositiivsete putukate tulemused näitavad, et kahetiivalistel on potentsiaali sigade Aafrika katku levitada ning selle kinnitamiseks on oluline edasi uurida viiruse ülekandemehhanisme läbi kahetiivalise kui vektori.The diversity of two winged insects (Diptera) in animal farms has not yet been extensively studied. However, until now this kind of research has not been carried out in any farm type in Estonia. Several Diptera species associated with animal farms are known as vectors for variety of pathogens and therefore of economic importance for farmers. African swine fever virus (ASFV) is one of the most severe infections to control and economically devastating for pig farms. The aims of this master’s thesis were to find out the diversity of Diptera in pig farms and whether these insects could play a role in transmission of African swine fever (ASF). Based on the objectives of this work, the following hypotheses have been established. Hypothesis I: The abundance and species diversity of Diptera in the pig farms depends both on the natural surroundings of the farm as well as the production system. Hypothesis II: It is possible to detect ASFV in Diptera that has been exposed to infected pigs. The study, was carried out in August and September 2016 and from May to August 2017 using glue traps for flies, to examine which species of Diptera occur in Estonian pig farms. Insects were also sampled from infected wild boar carcasses and from an infected pig farm in Saaremaa, to test their vector potential for ASF. As a result, 23 species/groups of Diptera were identified in studied pig farms. In total, 186701 arthropods, from which 180444 were Diptera were collected from pig farms during two years of sampling. The total number of insects in the farm was not affected by the natural surroundings nor the parameters of the farm production system. These results may have been influenced by a too small sample size of farms and non-consecutive participation of farms in the study. Among the insects collected from the dead wild boar carcasses, there were three specimens infected with ASF, from these the exact strain of ASF was only possible to be determined from hog lice. The insects collected from the infected pig farm during the epidemiological inspection in Saaremaa, there were 4 ASF positive specimens (27% of all insects analysed), but virus strain sequencing failed for any virus–positive insect caught from the farm. The results of the virus–positive insects derived from DNA analysis indicate that Diptera have a potential to transmit ASF and there are needed further studies to investigate the ability of the Diptera as an ASFV vector

Pan-european assessment, monitoring, and mitigation of stressors on the health of bees

As part of the activities foreseen by the POSHBEE project, we performed experiments to verify the toxicokinetics of three pesticides (the insecticide sulfoxaflor, the fungicide azoxystrobin and the herbicide glyphosate) in the bodies of social bees (Apis mellifera and Bombus terrestris) and solitary bees (Osmia bicornis). For each species all castes and sexes were studied. Based on the results from dose-response assays, sublethal doses were used to treat individuals, which were exposed to the chosen pesticides orally and by contact. Bees were then sampled at multiple time points post- exposure, to capture the breakdown of the active ingredients in the bodies of the organisms. Results of the chemical analyses on bee samples were used for the evaluation of the dynamics of the oral and contact acute exposure in the three species. Overall, the active ingredients sulfoxaflor, azoxystrobin and glyphosate degrade in all species, sex or caste. Nevertheless, there were some exceptions: in honey bee workers, glyphosate administered topically and azoxystrobin administered via the oral route seemed to remain stable in the bees’ bodies even 10 days after exposure (11% and 13% of degradation rate, respectively). We also observed a low degradation of sulfoxaflor following topical exposure in bumble bee queens (26% of degradation rate) and in Osmia bee females (22% of degradation rate). In bumble bees exposed topically to glyphosate degradation was lower than 50% for all sexes and castes. These results deserve further attention by researchers to understand the destiny of these molecules in the bee body and their effects therein.Prepared under contract from the European Commission; Grant agreement No. 773921; EU Horizon 2020 Research and Innovation action.Prepared under contract from the European Commission; Grant agreement No. 773921; EU Horizon 2020 Research and Innovation actio

Intra-specific variation in sensitivity of <i>Bombus terrestris</i> and <i>Osmia bicornis</i> to three pesticides

There is growing evidence that pesticides may be among the causes of worldwide bee declines, which has resulted in repeated calls for their increased scrutiny in regulatory assessments. One recurring concern is that the current frameworks may be biased towards assessing risks to the honey bee. This paradigm requires extrapolating toxicity information across bee species. Most research effort has therefore focused on quantifying differences in sensitivity across species. However, our understanding of how responses to pesticides may vary within a species is still very poor. Here we take the first steps towards filling this knowledge gap by comparing acute, lethal hazards in sexes and castes of the eusocial bee Bombus terrestris and in sexes of the solitary bee Osmia bicornis after oral and contact exposure to the pesticides sulfoxaflor, Amistar (azoxystrobin) and glyphosate. We show that sensitivity towards pesticides varies significantly both within and across species. Bee weight was a meaningful predictor of pesticide susceptibility. However, weight could not fully explain the observed differences, which suggests the existence of unexplored mechanisms regulating pesticide sensitivity across bee sexes and castes. Our data show that intra-specific responses are an overlooked yet important aspect of the risk assessment of pesticides in bees

Pan-european assessment, monitoring, and mitigation of stressors on the health of bees

Within the PoshBee Project, we have worked out precise protocols for testing the toxicological endpoints and metabolisation rate of agrochemicals on and in bees. Three agrochemical classes (insecticides, fungicides and herbicides) were tested on three bee groups (honey bees, bumble bees and solitary mason bees). The specific protocols, different for each combination of the pesticide class and bee group, were designed based on the existing toxicity testing methods and modified according to the specificities of the different bee groups, their castes and the different exotoxicological features of the compounds. Sulfoxaflor, Azoxystrobin, and Glyphosate were used respectively as examples of the three pesticide classes. Apis mellifera, Bombus terrestris and Osmia bicornis were chosen as the examples of the three bee groups. The protocols for honey bees and bumble bees were designed based on the official existing guidelines edited by OECD. Nevertheless, given that the OECD guidelines provide recommendations to fit with most agrochemicals, many of the parameters are defined with low precision in order to be flexible and adapt to different categories of molecules. While OECD has no guidelines for testing solitary bees, we used information from the unpublished ring test protocols for Osmia bicornis by the ICPPR non- Apis working group.Prepared under contract from the European Commission; Grant agreement No. 773921; EU Horizon 2020 Research and Innovation action.Prepared under contract from the European Commission; Grant agreement No. 773921; EU Horizon 2020 Research and Innovation action

Pan-european assessment, monitoring, and mitigation of stressors on the health of bees

Inter-individual differences in pesticide sensitivity may trigger variability in the risk posed by pesticides. Therefore, to better inform pesticide risk assessment for bees, we studied the variability of responses to several pesticides based on endogenous (developmental stage, genetic background, caste) and exogenous factors (pesticide co-exposure). We mainly investigated the toxicity of the insecticide sulfoxaflor, the fungicide azoxystrobin and the herbicide glyphosate. We first used LD50 tests to determine the acute oral and contact toxicity of these pesticides across the different bee species, developmental stages (larva vs adult in honey bees), castes (honey bee and bumble bee workers, queens and drones), and genetic backgrounds (honey bee subspecies). We then considered the risks posed by chronic and sublethal exposures to pesticides by implementing behavioural and reproductive endpoints in the screening of pesticide toxicity. Data showed that azoxystrobin and glyphosate under the test conditions were mildly toxic to bees. However, a large variability in bee sensitivity to sulfoxaflor was found, especially across species and individuals of different castes or sex. This variability is therefore important to consider for increasing the safety margin of the risk posed by insecticides in bees. Several effects induced by sublethal concentrations or doses of pesticides are also described, such as the occurrence of a Non-Monotonic Dose-Response (NMDR) and delayed effects in honey bees, impairment of reproductive performances in bumble bees, and a decreased longevity of Osmia adult females (although no effects were found on larval development). Finally, an interaction between pesticides was found when exposure was by contact, but not under oral exposure. In conclusion, the range of effects described here provides very useful insights for better understanding the toxicity of pesticides and therefore the risks they might pose to bees.Prepared under contract from the European Commission; Grant agreement No. 773921; EU Horizon 2020 Research and Innovation action.Prepared under contract from the European Commission; Grant agreement No. 773921; EU Horizon 2020 Research and Innovation action

Pan-european assessment, monitoring, and mitigation of stressors on the health of bees

Within the PoshBee Project we have tested three bee species – honey bees Apis mellifera, bumble bees Bombus terrestris and solitary bees Osmia bicornis – for their sensitivity to pesticides and analysed the clearance of pesticides from bees. For each species, all castes and sexes were studied. We synthesised the mortality data (LD50 or results of limit tests) with the toxicokinetic patterns and analysed this against the background of inter- and intraspecific variation in life-histories of the tested bees. The clearance of sulfoxaflor is relatively similar across all bee species tested and in females after contact treatment it tends to be retained. The toxicity increases over time independently of the clearance from the body. The clearance of azoxystrobin was rapid in Osmia and bumble bees, as well as in honey bee queens, but in honey bee workers there was very little clearance. Similar to sulfoxaflor the toxicity increased over time, although the residues were detected at very low levels. Glyphosate tended to be retained in bumble bees after contact treatment but cleared rapidly after oral treatment. For Osmia bees only in males after contact treatment was the glyphosate almost lost. The toxicity of a pesticide is dependent on the exact dosage, but also the exposure route and time, as well as the speed of detoxification and clearance from a body. The assessment for the hazard that a less toxic pesticide might pose, can be largely dependent on the exposure route. The effects of pesticide toxicity can increase even after the molecules have been cleared out of the body.Prepared under contract from the European Commission; Grant agreement No. 773921; EU Horizon 2020 Research and Innovation action.Prepared under contract from the European Commission; Grant agreement No. 773921; EU Horizon 2020 Research and Innovation action

Estonian horse fly (Tabanidae) Species Diversity and their characterization

Eestis on parmlastega tegeletud vähe. Esimene terviklik nimestik parmlastest on koostatud H. Remmi poolt 1953. aastal ning neid andmeid ei ole 63. aasta jooksul uuendatud. Bakalaureusetöö eesmärkideks olid Eestis leidavate parmude liigilise koosseisu väljaselgitamine, nende uurimine ja kirjeldamine, illustreerimine fotodega ning parmude perekondade dihhotoomse ja interaktiivse määraja koostamine. Töö autor viis Eestis siiani kasutusel olevad parmlaste vananenud nimed kooskõlla valiidsete ning kogu maailmas kasutatavate nimedega. Määramiseks ja kirjeldamiseks koguti parmlaste valmikuid 2015. aastal juunist–septembrini ning samuti külastati parmlaste liigilise koosseisu välja selgitamiseks Eesti Maaülikooli ja Tartu Ülikooli zooloogilisi kogusid, kus leidus arvestatavalt siiani määramata materjali, mis määrati liigini. Töö tulemustes selgus, et laialdaselt kasutatava ja autoriteetse andmebaasi Fauna Europaea Eestis esinevate parmlaste nimestikus eksisteeris ainult 19 liiki, kellest kaks puuduvad ülejäänud allikate põhjal Eestist. Eesti Maaülikooli ja Tartu Ülikooli putukakogu lähemal uurimisel selgus aga, et lisaks H. Remmi poolt märgitutele, esineb Eestis veel üheksa liiki parmlasi kuid kogudest puudusid kolm parmlase liiki, kes esinevad Eestis Remmi (1953), Chvála et al. (1972) ja Olsufjevi (1977) andmetel. Kõikide andmete summeerimisel selgus, et Eestis on hetkeseisuga 36 liiki parmlasi. Analoogselt on hästi uuritud Eesti suurliblikate fauna pidevas muutumises: viimase paarikümne aastaga on Eestisse jõudnud üle 30 lõunapoolsema liigi ja kümne liigi puhul on täheldatud nende taandumist. Olukord, kus liikide tõendusmaterjali on vähe, nõuab kindlasti edasisi süvendatud uuringuid ja analüüsi. Selleks, et parmlaste täpsem liigiline koosseis kindlaks teha oleks vaja teostada mitmel järjestikusel aastal püüke Eesti eri paikadest, mis hõlmaksid endas nii saarte kui mandri osa. Samuti leiab autor, et töö edasiarendusena on vajalik koostada ka kõiki liike hõlmav interaktiivne määraja. See aitaks kiiremini ja lihtsamini Eestist leitud parme määrata ning teadvustaks nende olulisust laiemale üldsusele.Horse flies have not been extensively studied in Estonia. The first comprehensive list of flies was compiled by H. Remm in 1953 and this data had not been updated in 63 years. The aims of this Bachelor’s thesis were to identify the horse fly species of Estonia, to examine and characterize the material, illustrate them with photographs and create a dichotomous and an interactive identification key for the genera of horse flies. Outdated horse fly names that were still being used in Estonia were coordinated with valid and globally used ones. The research author collected horse fly adults between June and September of 2015 to determine the species and describe them. The author also visited the zoological collections of the Estonian University of Life Sciences and the University of Tartu to identify the horse fly species therein. There was still a considerable amount of undetermined material at the universities, which was ascertained to species. During this work, it became evident that in Fauna Europaea, in the list of Estonian horse flies, there were only 19 species, two of which were not present in Estonia according to other sources. A closer examination of the zoological collections at the Estonian University of Life Sciences and at the University of Tartu revealed that, in addition to those indicated by H. Remm there are nine more species of horse flies in Estonia. However, there were still three species that were missing from the collections, which should be present in Estonia according to the data of Remm (1953), Chvála et al. (1972) and Olsufjev (1977). The summation of all the data showed that at the moment there are 36 species of horse flies in Estonia. Similarly, the large butterfly fauna, which is well–studied in Estonia, is constantly changing: in the last twenty years, more than 30 southern species have reached Estonia and ten species have started to retreat. The current situation where there is little evidence of species (being present), certainly requires further in–depth research and analysis. In order to identify all the local horse fly species, catches from different parts of Estonia, both on islands and mainland, should be conducted in several consecutive years. The author of the thesis finds that, to streamline the work, it is necessary to develop an interactive identification key which includes all species of horse flies in Estonia. This would help to identify horse fly species faster and more easily and would make the general public more aware of their importance

Wild boar (Sus scrofa) carcasses as an attraction for scavengers and a potential source for soil contamination with the African swine fever virus

The wild boar (Sus scrofa) is a social animal species native to Eurasia. During the last decade, the wild boar population in Estonia has been severely affected by the African swine fever virus (ASFV), which has also affected domestic pig farming. The potential transmission routes of ASFV remain unclear and are currently under intensive investigation. This pilot study aimed to clarify the frequency and characteristics of contacts between living wild boars and the carcasses of their conspecifics, which could play a role in the transmission of ASFV. Wild animals' contact and scavenging behavior on wild boar carcasses were studied using trail cameras in an experimental setting on Hiiumaa, Western Estonia. Four legally hunted carcasses were used in the present study. This study aimed to determine whether intraspecies scavenging occurs in wild boars. The persistence of ASFV DNA in soil contaminated with infected wild boar carcasses was investigated separately. Among the 17 identified wildlife species that visited wild boar carcasses, the common raven (Corvus corax) was the most frequent one (37.26%), followed by raccoon dogs (Nyctereutes procyonoides; 4.25%), carcass conspecific/wild boars (3.16%), and red foxes (Vulpes vulpes; 2.14%). Regarding the direct contact with the carcass, the same species ranking was detected: common raven (74.95%), raccoon dogs (9.94%), wild boars (4.21%), and red foxes (4.21%). No clear signs of cannibalism were noted among the wild boars, although brief physical contact with the carcasses was evident. The persistence of ASFV DNA in soil contaminated by infected wild boar carcasses was investigated separately. This study revealed that ASFV DNA from infected carcasses could be detected in forest soil for prolonged periods, even after removing the carcasses. Hence, the carcasses of infected wild boars may play an important role in spreading the African swine fever virus in wild boar populations; thus, prompt removal and disinfection of the soil could be considered necessary to limit the spread of the infection