Tick-Borne pathogens and defensin genes expression: A closer look at Ixodes ricinus and Dermacentor reticulatus.

The immune system of ticks, along with that of other invertebrates, is comparatively simpler than that of vertebrates, relying solely on innate immune responses. Direct antimicrobial defence is provided by the synthesis of antimicrobial peptides (AMPs), including defensins. The aim of this study was to investigate the differences in defensin genes expression between questing and engorged Ixodes ricinus (def1 and def2) and Dermacentor reticulatus (defDr) ticks, in the presence of selected pathogens: Borrelia spp., Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis in the natural environment. After pathogen screening by PCR/qPCR, the expression of defensin genes in pathogen positive ticks and ticks without any of the tested pathogens, was analysed by reverse transcription qPCR. The results showed an increased expression of defensin genes in I. ricinus ticks after blood feeding and I. ricinus and D. reticulatus ticks during in cases of co-infection. In particular, the expression of defensins genes was higher in questing D. reticulatus than in questing and engorged I. ricinus ticks, when borreliae were detected. This study contributes to uncovering the expression patterns of defensin genes in the presence of several known tick pathogens, the occurrence of these pathogens and possible regulatory mechanisms of defensins in tick vector competence.

Ticks and Tick-Borne Pathogens Circulating in Peri-Domestic Areas in Mainland Portugal.

Over the years, tick-borne pathogens (TBPs) have garnered significant interest due to their medical, veterinary and economic importance. Additionally, TBPs have drawn attention to how these microorganisms interact with their own vectors, increasing the risk to human and animal infection of emerging and reemerging zoonoses. In this sense, ticks, which are obligate hematophagous ectoparasites, have a key role in maintaining and transmitting TBPs among humans and animals. The aim of this study was to assess the prevalence of neglected TBPs in mainland Portugal, namely Anaplasma spp., Babesia spp., Ehrlichia spp. and Neoehrlichia mikurensis. DNA fragments were detected in questing ticks collected from five different ecological areas under investigation. To the best of the authors' knowledge, this study reports new worldwide findings, including B. bigemina infecting Ixodes frontalis, Ixodes ricinus and Rhipicephalus sanguineus sensu lato. Additionally, it presents new findings in Portugal of N. mikurensis infecting I. ricinus and of presumably Wolbachia endosymbionts being detected in I. ricinus. Overall, there were 208 tick samples that were negative for all screened TBPs. The results herein obtained raise concerns about the circulation of neglected TBPs in mainland Portugal, especially in anthropophilic ticks, highlighting the importance of adopting a One Health perspective.

Tick-borne pathogens in questing adults Dermacentor reticulatus from the Eastern European population (north-eastern Poland).

Dermacentor reticulatus is tick species with an expanding geographical range in Europe, which creates the possibility of spreading microorganisms of significant veterinary and medical importance. The study aimed to investigate the prevalence and genetic diversity of Rickettsia spp., Babesia spp., Borrelia spp. and Anaplasma phagocytophilum in adult D. reticulatus ticks from the Eastern European population in the urban and the natural biotopes of north-eastern Poland. Microorganisms were detected by PCR and identified by DNA sequencing. The overall infection rate of at least one of the pathogens was 29.6%. The predominantly was Rickettsia spp. (27.1%) (with R. raoultii-9.1%) followed by Babesia spp. (2.4%) with B. canis (1.5%) as the most frequent. Based on 18S rRNA gene sequence, three B. canis genotypes were revealed. The prevalence of R. raoultii and B. canis was significantly higher in ticks from natural biotopes. The infection rates of B. afzelii and A. phagocytophilum were determined at 0.9% and 0.3%, respectively. Co-infections were detected in 3.8% of infected ticks. In diagnosing tick-borne diseases in humans, tick-borne lymphadenopathy should not be excluded. The prevalence of different genotypes of B. canis suggests differences in the clinical picture of canine babesiosis in the area.

Neoehrlichia mikurensis-A New Emerging Tick-Borne Pathogen in North-Eastern Poland?

Neoehrlichia mikurensis is a new emerging tick-borne Gram-negative bacterium, belonging to the family Anaplasmataceae, the main vector of which in Europe is the tick Ixodes ricinus. N. mikurensis is responsible for neoehrlichiosis, occurring mostly in patients with underlying diseases. In the present study, a total of 348 I. ricinus and Dermacentor reticulatus ticks collected in north-eastern Poland were analyzed for the prevalence of N. mikurensis. A total of 140 questing ticks (124 of I. ricinus ticks and 16 D. reticulatus) collected with the flagging method and 208 ticks (105 and 103 I. ricinus and D. reticulatus, respectively) removed from dogs were selected for the study. cDNA (questing ticks) and total DNA (questing and feeding ticks) were analyzed by qPCR targeting the 16S rRNA gene of N. mikurensis. Positive samples were further analyzed by nested PCR and sequencing. The prevalence differed between ticks collected from vegetation (19.3%; 27/140) and ticks removed from dogs (6.7%; 14/208). The presence of the pathogen in questing and feeding D. reticulatus ticks was proven in Poland for the first time. In summary, our research showed that infections of ticks of both the most common tick species I. ricinus and D. reticulatus in north-eastern Poland are present and ticks collected from urban areas were more often infected than ticks from suburban and natural areas. The detection of N. mikurensis in I. ricinus and D. reticulatus ticks from north-eastern Poland indicates potential transmission risk for tick-bitten humans at this latitude.

Cystidicola farionis, a Swim Bladder Parasite of European Smelt: Characterization of the Nematode Trehalose Strategy.

The molecular identification of Cystidicola farionis (a swim bladder nematode of European smelt from the Vistula Lagoon in Poland) was performed. Their prevalence level was determined, and changes in the trehalose synthesis pathway in larvae and adult nematodes were demonstrated. The trehalose level was almost four times higher in adult nematodes than in larvae. In contrast, the activity of both enzymes (trehalose 6-phosphate synthase, TPS and trehalose 6-phosphate phosphatase, TPP) involved in the synthesis of trehalose was higher in larvae than in adults under optimal conditions. The optimum pH for TPS isolated from larvae and adults was pH 7.0. The optimum pH for TPP from larvae and adults was pH 7.0 and pH 8.0, respectively. The optimal temperature was 20 °C, and Mg2+ ions were an activator for trehalose-synthetizing enzymes from both sources. Enzymes isolated from adult nematodes were less susceptible to divalent ion chelator and inorganic phosphate than larval enzymes. The dynamic transformation of trehalose in the nematode developing inside the swim bladder of the smelt appears to be an important metabolic pathway in the nematode survival strategy. These studies are aimed at a better understanding of the issue of the metabolic adaptation of parasites, which, in the future, may indirectly contribute to the elimination of the parasite from aquacultures, which will impact public health.

Abundance of Ixodes ricinus Ticks (Acari: Ixodidae) and the Diversity of Borrelia Species in Northeastern Poland.

Monitoring the abundance of ticks and the prevalence of pathogens in ticks is an important activity in assessing the risk of tick-borne diseases and helps to develop preventive measures. This study aimed to estimate the density of Ixodes ricinus, the prevalence of Borrelia species, and their diversity in northeastern Poland. The overall mean I. ricinus density was 9.7 ticks/100 m2. There were no differences between years, subregions, or habitats of study. The Borrelia infection rate was higher in females (22.6%) and males (14.3%) than in nymphs 5.5% (MIR). The most infected ticks came from the eastern subregion (10.1%) where the incidence of borreliosis among the inhabitants was over 20% higher than in the other subregions. In the infected ticks, B. afzelii (38.3%) and B. garinii (34.5%) were predominant. B. bavariensis was confirmed in I. ricinus in Poland for the first time. The most polymorphic was B. garinii. B. miyamotoi (belonged to the European type) was identified as a mono-infection in 0.9% of ticks and in 1.5% as a co-infection with B. afzelii and with B. garinii. Besides the risk of borreliosis and co-infections with different Borrelia species, physicians should also be aware of B. miyamotoi infections among patients.

The Risk of Exposure to Ticks and Tick-Borne Pathogens in a Spa Town in Northern Poland.

The aim of this study was to determine the potential risk of human exposure to tick-borne infection in a recreation areas in a spa town located in northern Poland. Questing Ixodes ricinus and Dermacentor reticulatus ticks were collected in the spring of 2018. Tick-borne microorganisms were detected by PCR. Species were identified based on RFLP and the sequencing of DNA. In total, 38.3% of the ticks (34.6% of I. ricinus and 48.6% of D. reticulatus) were infected. The prevalence was 14.9% for Borrelia spp., 10.6% for Babesia spp. and 17.7% for Rickettsia spp. No Anaplasma phagocytophilum was detected. Spirochaetes B. afzelii, B. garinii and B. burgdorferi s.s. were detected only in I. ricinus ticks (20.2%). The differences in the infection rates of Babesia spp. between I. ricinus (7.7%) and D. reticulatus (18.9%) were not significant. DNA of B. canis and B. venatorum were identified in both tick species. B. microti were detected in D. reticulatus ticks. The prevalence of Rickettsia spp. was significantly higher in D. reticulatus (37.8%) than that in I. ricinus (10.6%). R. raoultii was identified only in D. reticulatus and R. helvetica in I. ricinus. Co-infections of at least two pathogens were recognized in 13% of positive ticks.

Tick-Borne Pathogens in Ticks Collected from Wild Ungulates in North-Eastern Poland.

This study was carried out in north-eastern Poland during two hunting seasons between 2018 and 2020. Ticks (Ixodes ricinus and Dermacentor reticulatus) were removed from wild cervids and boars and examined for the presence of Borrelia spirochetes and Rickettsiales members: Rickettsia spp. and Anaplasma phagocytophilum. The present study contributes to the knowledge of even-toed ungulates, which are an important reservoir of the above-mentioned pathogens and a potential source of infections for humans through ticks as vectors. Almost 40% of the collected ticks (191 out of 484) were infected with the following pathogens: 3.3% with Borrelia spp., 19.2% with A. phagocytophilum and 26.9% with Rickettsia spp. Only the ticks collected from cervids carried Borrelia. Typing of the species DNA confirmed the presence of B. afzelii, B. garinii, B. lusitaniae and B. miyamotoi. An analysis of Rickettsia spp. sequences using the GenBank data revealed the presence of R. helvetica, R. raoultii and R. monacensis. Monoinfections (79.1%) dominated over co-infections (20.9%). Among co-infections, the most frequent was A. phagocytophilum/Rickettsia spp. (70%), however co-infections, including B. afzelii/A. phagocytophilum, B. afzelii/Rickettsia spp., B. miyamotoi/A. phagocytophilum and B. afzelii/B. garinii/B. lusitaniae, were also noted. Significant differences were observed in the affinity of some pathogens to their vectors. Thus, Borrelia spp. and A. phagocytophilum were more frequently detected in I. ricinus (5.3% and 23.1%) than in D. reticulatus (1.2% and 15.3%). Infection frequency with Rickettsia spp. was similar (approximately 25-29%) in both tick species. The prevalence of A. phagocytophilum and Rickettsia spp. in ticks removed from cervids was 19.8% and 27.1%, and in ticks from wild boars it was 13.3% and 24.4%, respectively.

Borrelia miyamotoi-An Emerging Human Tick-Borne Pathogen in Europe.

Borrelia miyamotoi is classified as a relapsing fever spirochete. Although B. miyamotoi is genetically and ecologically distinct from Borrelia burgdorferi sensu lato, both microorganisms are transmitted by the same Ixodes tick species. B. miyamotoi was detected in I. persulcatus ticks in 1994 in Japan. A phylogenetic analysis based on selected sequences of B. miyamotoi genome revealed genetic differences between isolates from Asia, North America, and Europe, which are clearly separated into three genotypes. Symptomatic human cases of Borrelia miyamotoi disease (BMD) were first reported in 2011 in Russia and then in North America, Europe, and Asia. The most common clinical manifestation of BMD is fever with flu-like symptoms. Several differences in rare symptoms (thrombocytopenia, monocytosis, cerebrospinal fluid pleocytosis, or symptoms related to the central nervous system) have been noted among cases caused by Asian, European, and American types of B. miyamotoi. BMD should be considered in the diagnosis of patients after tick bites, particularly with meningoencephalitis, without anti-Borrelia antibodies in the cerebrospinal fluid. This review describes the biology, ecology, and potential of B. miyamotoi as a tick-borne pathogen of public health concern, with particular emphasis on Europe.

Sucking of human blood by Placobdella costata (O. F. Müller, 1846) (Hirudinida: Glossiphoniidae): Case study with notes on body form.

Four events of Placobdella costata sucking human blood are described.Human blood was sucked by both adult and juvenile specimens of P. costata.The feeding strategies of juveniles under parental care are presented.New data on juvenile specimens' body form are presented.Information on the potential role of mammals in dispersion and habitat preferences of leeches P. costata is considered.

S-Methyl-(2-Methoxycarbonylamino-Benzimidazole-5) Thiosulfonate as a Potential Antiparasitic Agent-Its Action on the Development of Ascaris suum Eggs In Vitro.

Ascaris suum is a soil-transmitted parasite causing ascariasis in pigs, largely limiting livestock production globally. Searching for new drugs affecting all stages of nematode development is necessary and widely postulated. The in vitro activity of S-methyl-(2-methoxycarbonylamino-benzoimidasole-5) thiosulfonate on A. suum developing eggs was studied. Five concentrations of the drug were used-0.625, 1.25, 2.5, 5 and 10 mM during 24, 48 and 72 h of exposure. After drug treatment, the eggs were washed and cultured in 0.05 M HCl at 27 °C for 20 days. Both the concentration and duration of the drug exposure had an inhibitory impact on the percentage of L2 larvae developed. The best effect was obtained after 72 h of incubation in 5 mM drug solution, only 1.9 ± 3.3% of the larvae developed to the L2 stage. Moreover, no SNP was detected at codon 167, which is correlated with benzimidazole resistance, in the tested samples. For the first time, it has been demonstrated that S-M-(2-MKA-BZ-5)TS seems to be a potential ovicidal anti-helminthic agent. It may lead to the elimination of parasites and reduce environmental contamination from roundworm eggs. The ovicidal effects of the drug should be additionally confirmed by further infection studies using experimental animals.

Molecular Detection of Borrelia burgdorferi Sensu Lato and Anaplasma phagocytophilum in Ticks Collected from Dogs in Urban Areas of North-Eastern Poland.

From 2016 to 2018, ticks were collected from 272 dogs admitted to veterinary clinics in the city of Olsztyn (north-eastern Poland). Among 522 collected ticks, 423 were identified as Ixodes ricinus (413 females and 10 males) and 99 as Dermacentor reticulatus (62 females and 37 males). Non-engorged (86 individuals) and engorged (436 individuals) ticks were screened for the presence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum DNA. Borrelia and A. phagocytophilum species detection was determined based on the sequence of the fla B and 16S RNA genes, respectively. DNA of B. burgdorferi s.l. was identified in 31.6% (165/522, 95% CI: 27.6-35.8%) of ticks (I. ricinus 151/423, 35.7%, 95% CI: 31.1-40.4%; D. reticulates 14/99, 14.1%, 95% CI: 7.9-22.6%). A. phagocytophilum was identified in 0.96% (5/522, 95% CI: 0.3-2.2%) of specimens. All positive samples were engorged I. ricinus females (5/402, 1.2%, 95% CI: 0.4-2.9%). In 85.4% (141/165, 95% CI: 79.1-90.4%) of Borrelia infected ticks, the DNA of one genospecies was revealed. The DNA of at least two different genospecies was detected in 14.5% of specimens (24/165, 95% CI: 9.5-20.8). The coexistence of B. burgdorferii s.l. and A. phagocytophilum was not detected.

Questing Ixodes ricinus ticks (Acari, Ixodidae) as a vector of Borrelia burgdorferi sensu lato and Borrelia miyamotoi in an urban area of north-eastern Poland.

Green areas located within large cities, as natural ecotypes, are a convenient habitat for ticks and their use as recreational areas is associated with the potential risk of acquiring tick-borne diseases. This study estimated the I. ricinus tick density, prevalence of infection with Borrelia species and the diversity of these bacteria in a green urban area (Olsztyn) of north-eastern Poland, an endemic region of tick-borne diseases. The ticks were collected during spring and autumn of 2015, at sites differing in the degree of human pressure and habitat. Borrelia species detection, typing and a molecular phylogenetic analysis were carried out based on the sequenced flaB gene. The overall mean abundance of I. ricinus was 2.0 ± 1.55 ticks per 100 m2. The density of I. ricinus did not vary significantly between sites. According to semi-qualitative tick abundance categories, the collection sites were classified as 'very low' and 'low' tick abundance category. The overall infection rate of I. ricinus with Borrelia spirochaetes was 27.4%. The infection rate of adult ticks (42.0%) was three times higher than with nymphs (14.3%). Based on the restriction patterns and sequencing, B. afzelii (93.1%; 27/29), B. valaisiana 3.5% (1/29) and B. miyamotoi (3.5%; 1/29), related to the relapsing fever (RF) spirochaetes, were detected. No co-infections were found. Borrelia miyamotoi, detected for the first time in ticks in the north-eastern urban areas of Poland, was identical to isolates described as European-type. The Borrelia spirochaete infection rate of I. ricinus ticks in an urban area indicated a high risk of LB. Physicians should also be aware of B. miyamotoi infections among patients with a history of tick-bites in north-eastern Poland.

Infective larvae of Anisakis simplex (Nematoda) accumulate trehalose and glycogen in response to starvation and temperature stress.

Anisakis simplex L3 larvae infect fish and other seafood species such as squid or octopi; therefore, humans consuming raw or undercooked fish may become accidental hosts for this parasite. These larvae are induced to enter hypometabolism by cold temperatures. It is assumed that sugars (in particular trehalose and glycogen) are instrumental for survival under environmental stress conditions. To elucidate the mechanisms of environmental stress response in A. simplex, we observed the effects of starvation and temperature on trehalose and glycogen content, the activity of enzymes metabolizing those sugars, and the relative expression of genes of trehalose and glycogen metabolic pathways. The L3 of A. simplex synthesize trehalose both in low (0°C) and high temperatures (45°C). The highest content of glycogen was observed at 45°C at 36 h of incubation. On the second day of incubation, tissue content of trehalose depended on the activity of the enzymes: TPS was more active at 45°C, and TPP was more active at 0°C. The changes in TPP activity were consistent with the transcript level changes of the TPP gene, and the trehalose level, while glycogen synthesis correlates with the expression of glycogen synthase gene at 45°C; this suggests that the synthesis of trehalose is more essential. These results show that trehalose plays a key role in providing energy during the thermotolerance and starvation processes through the molecular and biochemical regulation of trehalose and glycogen metabolism.

Composition of fatty acids in the Varroa destructor mites and their hosts, Apis mellifera drone-prepupae.

The fatty acid (FA) profile of lipids extracted from the Varroa destructor parasitic mite and its host, drone-prepupae of Apis mellifera, was determined by gas chromatography (GC). The percentages of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) were generally similar in parasites and their hosts. Fatty acids were arranged in the following descending order based on their content: MUFAs (ca. 52-55%), SFAs (ca. 41%) and PUFAs (ca. 3%). The predominant fatty acids were oleic acid (46% in mites, 44% in prepupae) and palmitic acid (23% and 30%, respectively). Varroa parasites differed from their hosts in the quantity of individual FAs and in their FA profiles. Three PUFAs noted in the host were not observed in parasitic mites, whereas the presence of C21:0, C24:0 and C22:1 FAs was reported in mites, but not in drones.

The in vitro effect of ivermectin on the activity of trehalose synthesis pathway enzymes and their mRNA expression in the muscle of adult female Ascaris suum (Nematoda).

The in vitro effect of ivermectin lethal dose on the activity of trehalose-6-phosphate synthase (TPS) and phosphatase (TPP) and the expression of their mRNA (tps1, tps2, and tpp genes) in the muscle of adult female Ascaris suum was investigated. The presence of ivermectin in the medium caused a decrease in TPS and TPP activities during the experiment compared with the start and control groups. The exception was the group of worms grown for 8 hours in a IVM solution, in which there was a little higher TPS activity than in the control. Real-time qPCR analysis showed reduced expression of tps1 and tps2, and unchanged tpp expression after 20 hours of incubation relative to the expression at time zero. Relative to the appropriate control groups, the expression of tps2 gene was slight increased but the other two genes were reduced after 8-hours of IVM-treatment. Then the expression of all three genes was lower at the end of cultivation. The level of gene expression was positively correlated with the activity of specific enzymes. In the case of tpp gene there was only a weak correlation. Prolonged exposure to ivermectin was effective in lowering TPS and TPP activity and their mRNA expression. However, the drug did not block the pathway.

Supplementing with vitamin C the diet of honeybees (Apis mellifera carnica) parasitized with Varroa destructor: effects on antioxidative status.

We studied a total of eight developmental stages of capped brood and newly emerged workers of Apis mellifera carnica colonies naturally parasitized with Varroa destructor. During winter and early spring four colonies were fed syrup containing 1.8 mg vitamin C kg(-1) (ascorbic acid group; group AA) while four colonies were fed syrup without the vitamin C (control group C). Selected elements of the antioxidative system were analysed including total antioxidant status (TAS), glutathione content and antioxidative enzyme activities (superoxide dismutase, catalase, peroxidase and glutathione S-transferase). Body weight, protein content and indices of infestation were also determined. The prevalence (8.11%) and intensity (1·15 parasite per bee) of the infestation were lower in group AA compared with group C (11.3% and 1.21, respectively). Changes in the indicators of antioxidative stress were evidence for the strengthening of the antioxidative system in the brood by administration of vitamin C. In freshly emerged worker bees of group AA, despite the infestation, protein content, TAS, and the activity of all antioxidative enzymes had significantly higher values in relation to group C.

Esterases of Varroa destructor (Acari: Varroidae), parasitic mite of the honeybee.

Varroa destructor is an ectoparasite that causes serious damage to the population of the honeybee. Increasing resistance of the parasite to acaricides is related, among others, to metabolic adaptations of its esterases to facilitate decomposition of the chemicals used. Esterases are a large heterogeneous group of enzymes that metabolize a number of endogenous and exogenous substrates with ester binding. The aim of the present study was to determine the activity of esterases in the body extracts (BE) and excretion/secretion products (E/SP) of the mite. The enzymes contained in the E/SP should originate mainly from the salivary glands and the alimentary system and they may play a particularly important role in the first line of defence of the mite against acaricides. Activity of cholinesterases (ChEs) [acetylcholinesterase (AChE) and butyrylcholinesterase], carboxylesterases (CEs) and phosphatases [alkaline phosphatase (AP) and acid phosphatase (AcP)] was investigated. The activity of all the enzymes except AChE was higher in the E/SP than in the BE. ChEs from the BE and from the E/SP reacted differently on eserine, a ChE inhibitor. Eserine inhibited both enzymes from the BE, increased decomposition of acetylcholine, but did not influence hydrolysis of butyrylcholine by the E/SP. Activity of the CEs from the BE in relation to the esters of carboxylic acids can be presented in the following series: C10 > C12 > C14 > C8 > C2 > C4 = C16, while activity of the CEs from the E/SP was: C4 > C8 > C2 > C14 > C10 > C12 > C16. The inhibitor of CEs, triphenyl phosphate, reduced the activity of esterases C2­C8 and C14­C16; however, it acted in the opposite way to CEs C10 and C12. The activity of both phosphatases was higher in the E/SP than in the BE (AcP about twofold and AP about 2.6-fold); the activities of AP and AcP in the same material were similar. Given the role of esterases in resistance to pesticides, further studies are necessary to obtain complete biochemical characteristics of the enzymes currently present in V. destructor.

The mutual influence of proteins from Varroa destructor extracts and from honeybee haemolymph on their proteolytic activity--in vitro study.

The influence of extracts from Varroa destructor, a parasitic mite of the honeybee Apis mellifera, on the proteinase activity of worker bee haemolymph was analysed in vitro, along with the influence of bee haemolymph on the proteolytic activity of V. destructor extract. The study was conducted in three different environments: pH 7.5 (high activity of bee enzymes and very low activity of parasite enzymes), pH 5 (moderate activity of enzymes from both sources) and pH 3.5 (limited activity of bee proteinases and high activity of mite proteinases). Based on electrophoretic studies, the inhibition of the activity of bee haemolymph proteinases by V. destructor extracts was observed at each pH. The study at pH 7.5 with commercial inhibitors of the 4 main classes of proteinases (pepstatin A, ethylenediaminetetraacetic acid (EDTA), E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), soybean trypsin inhibitor and Kunitz inhibitor) suggested that parasite extracts mainly inhibited serine proteinases and, to a lower degree, cysteine and aspartyl proteinases. At pH 3.5 and pH 5, a decrease of approximately 40% in parasite proteinase activity was also observed in the presence of bee haemolymph. The result points to the presence of aspartyl proteinase inhibitors in bee haemolymph, which may be an important defence element for bees during food intake by a mite. It was demonstrated that trypsin and trypsin inhibitors are active in the excretion/secretion products of V. destructor, the proteinases of which may assist the parasite in food suckling by preventing haemolymph coagulation, among other things.

The activity of carbohydrate-degrading enzymes in the development of brood and newly emerged workers and drones of the Carniolan honeybee, Apis mellifera carnica.

The activity of glycogen Phosphorylase and carbohydrate hydrolyzing enzymes α-amylase, glucoamylase, trehalase, and sucrase was studied in the development of the Carniolan honey bee, Apis mellifera carnica Pollman (Hymenoptera: Apidae), from newly hatched larva to freshly emerged imago of worker and drone. Phosphorolytic degradation of glycogen was significantly stronger than hydrolytic degradation in all developmental stages. Developmental profiles of hydrolase activity were similar in both sexes of brood; high activity was found in unsealed larvae, the lowest in prepupae followed by an increase in enzymatic activity. Especially intensive increases in activity occurred in the last stage of pupae and newly emerged imago. Besides α-amylase, the activities of other enzymes were higher in drone than in worker broods. Among drones, activity of glucoamylase was particularly high, ranging from around three times higher in the youngest larvae to 13 times higher in the oldest pupae. This confirms earlier suggestions about higher rates of metabolism in drone broods than in worker broods.