Imidacloprid Pesticide Causes Unexpectedly Severe Bioelement Deficiencies and Imbalance in Honey Bees Even at Sublethal Doses.

Pesticides impair honeybee health in many ways. Imidacloprid (IMD) is a pesticide used worldwide. No information exists on how IMD impact the bees' body bioelement balance, which is essential for bee health. We hypothesized that IMD disturbs this balance and fed the bees (in field conditions) with diets containing 0 ppb (control), 5 ppb (sublethal considered field-relevant), and 200 ppb (adverse) doses of IMD. IMD severely reduced the levels of K, Na, Ca, and Mg (electrolytic) and of Fe, Mo, Mn, Co, Cu, Ni, Se, and Zn, while those of Sn, V, and Cr (enzymatic) were increased. Levels of P, S, Ti, Al, Li, and Sr were also decreased, while only the B content (physiologically essential) was increased. The increase in Tl, Pb, and As levels (toxic) was alarming. Generally, IMD, even in sublethal doses, unexpectedly led to severe bioelement malnutrition in 69% of bioelements and to a stoichiometric mismatch in the remaining ones. This points to the IMD-dependent bioelement disturbance as another, yet unaccounted for, essential metabolic element which can interfere with apian health. Consequently, there is a need for developing methods of bioelement supplementation of the honey bee diet for better preventing bee colony decline and protecting apian health status when faced with pesticides.

Activities of Antioxidant and Proteolytic Systems and Biomarkers in the Fat Body and Hemolymph of Young Apis mellifera Females.

The proteolytic and antioxidant systems are important components of humoral immunity, and these biomarkers indicate the immune status. These compounds are synthesized in the bees' fat body and released into the hemolymph. Their functions maintain the organism's homeostasis and protect it against adverse environmental factors (including pathogens). We determined the activities of acidic, neutral, and alkaline proteases and their inhibitors, as well as superoxide dismutase (SOD), catalase (CAT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and the level of total antioxidant potential (TAC). These compounds were investigated in the fat body and hemolymph in the females with increased reproductive potential, i.e., queens and rebels, and in normal (non-reproductive sterile non-rebel) workers. The phenoloxidase (PO) activities were determined in the hemolymph. The normal workers had higher activities of proteases and their inhibitors, SOD and CAT, in the fat body and hemolymph, compared to the queens and rebels. The protease inhibitors were not usually active in the queens. As we predicted, the rebels revealed values between those of the queens and normal workers. The highest activities of proteases and antioxidants were identified in the fat body from the third tergite in comparison with the sternite and the fifth tergite. These results are important for oxidative stress ecology and give a better understanding of the functioning of the fat body and the division of labor in social insects.

Morphology of Nasonov and Tergal Glands in Apis mellifera Rebels.

Social insect societies are characterized by a high level of organization. This is made possible through a remarkably complex array of pheromonal signals produced by all members of the colony. The queen's pheromones signal the presence of a fertile female and induce daughter workers to remain sterile. However, the lack of the queen mandibular pheromone leads to the emergence of rebels, i.e., workers with increased reproductive potential. We suggested that the rebels would have developed tergal glands and reduced Nasonov glands, much like the queen but contrary to normal workers. Our guess turned out to be correct and may suggest that the rebels are more queen-like than previously thought. The tergal gland cells found in the rebels were numerous but they did not adhere as closely to one another as they did in queens. In the rebels, the number of Nasonov gland cells was very limited (from 38 to 53) and there were fat body trophocytes between the glandular cells. The diameters of the Nasonov gland cell nuclei were smaller in the rebels than in the normal workers. These results are important for understanding the formation of the different castes of Apis mellifera females, as well as the division of labor in social insect societies.

Reproductive Potential Impacts Body Maintenance Parameters and Global DNA Methylation in Honeybee Workers (Apis mellifera L.).

The widely accepted hypothesis in life history evolution about the trade-off between fecundity and longevity is not confirmed by long-living and highly fecund queens in eusocial insects. The fact that the queens and facultatively sterile workers usually arise from genetically identical eggs but differ in DNA methylation makes them a good model for studies on senescence, eusocial evolution, and epigenetics. Therefore, honeybees seem to be especially useful here because of long living rebel-workers (RW) with high reproductive potential recently described. Longevity, ovariole number, nosema tolerance, and global DNA methylation have been assayed in normal workers (NW) versus RW in hives and cages. RW always lived longer than NW and unexpectedly extended longevity of NW when they were together, similarly as the presence of a queen did. RW lived longer despite the fact that they had higher Nosema spore load; surprisingly they became infected more easily but tolerated the infection better. Global DNA methylation increased with age, being lower in RW than in NW. Therefore, RW are queen-like considering global DNA methylation and the link between fecundity, longevity, and body maintenance. Presented features of RW expands possibilities of the use of honeybees as a model for studies on senescence, nosemosis, eusocial evolution, and epigenetics.

Reproductive Potential Accelerates Preimaginal Development of Rebel Workers in Apis mellifera.

Rebel workers develop from eggs laid by the previous queen, before it went swarming and left the colony orphaned, until the emergence of a new queen. In contrast to normal workers developing in the queen's presence, rebels are set to reproduce and avoid rearing of successive bee generations. They have more ovarioles in their ovaries, as well as more developed mandibular glands and underdeveloped hypopharyngeal glands, just like the queen. We posited that rebels are not only similar to queens in some anatomical features, but also develop in a shorter time in comparison to normal workers. Therefore, the aim of this study was to compare preimaginal development duration in rebel and normal workers. The results show that rebels, i.e., workers with a higher reproductive potential, had a significantly shorter preimaginal development period (mean ± SD, 19.24 ± 0.07 days) than normal workers (22.29 ± 0.32 days). Our result confirmed that workers who develop in a queen-less colony undergo a shorter preimaginal development than those in a queen-right colony.

Humoral and Cellular Defense Mechanisms in Rebel Workers of Apis mellifera.

The physiological state of an insect depends on efficiently functioning immune mechanisms such as cellular and humoral defenses. However, compounds participating in these mechanisms also regulate reproductive caste formation and are responsible for reproductive division of labor as well as for labor division in sterile workers. Divergent reaction of the same genotype yielding reproductive queens and worker castes led to shaping of the physiological and behavioral plasticity of sterile or reproductive workers. Rebels that can lay eggs while maintaining tasks inside and outside the colony exhibit both queen and worker traits. So, we expected that the phagocytic index, JH3 titer, and Vg concentration would be higher in rebels than in normal workers and would increase with their age. We also assumed that the numbers of oenocytes and their sizes would be greater in rebels than in normal workers. The rebels and the normal workers were collected at the age of 1, 7, 14, and 21 days, respectively. Hemolymph and fat bodies were collected for biochemical and morphological analyses. The high levels of JH, Vg, and the phagocytic index, as well as increased numbers and sizes of oenocytes in the fat body cells demonstrate the physiological and phenotypic adaptation of rebels to the eusocial life of honeybees.

Segmentation of the subcuticular fat body in Apis mellifera females with different reproductive potentials.

Evolution has created different castes of females in eusocial haplodiploids. The difference between them lies in their functions and vulnerability but above all in their reproductive potentials. Honeybee queens are highly fertile. On the other hand, the workers are facultatively sterile. However, rebel workers, i.e. workers that develop in a queenless colony, reproduce more often than normal workers. As a result, the fat body of these bees, which apart from acting as the energy reserve, is also the site of numerous metabolic processes, had to specialize in different functions perfected over millions of years of eusocial evolution. Assuming that the variety of functions manifests itself in the pleomorphic structure of the fat body cells, we predicted that also different parts of the fat body, e.g. from different segments of the abdomen, contain different sets of cells. Such differences could be expected between queens, rebels and normal workers, i.e. females with dramatically different reproductive potentials. We confirmed all these expectations. Although all bees had the same types of cells, their proportion and segmental character corresponded with the caste reproductive potential and physiological characteristics shaped in the evolutionary process. The females with an increased reproductive potential were characterized by the presence of oenocytes in the third tergite and high concentrations of compounds responsible for energy reserves, like glucose, glycogen and triglycerides. Queens had very large trophocytes, especially in the third tergite. Only in workers did we observe intercellular spaces in all the segments of the fat body, as well as high protein concentrations-especially in the sternite. As expected, the rebels combined many features of the queens and normal workers, what with other findings can help understand the ways that led to the origin of different castes in females of eusocial Hymenoptera.

Antioxidation Defenses of Apis mellifera Queens and Workers Respond to Imidacloprid in Different Age-Dependent Ways: Old Queens Are Resistant, Foragers Are Not.

We investigated how different antioxidant defenses (ADSs) were shaped by evolution in young/old Apis mellifera workers and queens to broaden the limited knowledge on whether ADSs are effective in contemporary pesticide environments and to complete bee oxidative-aging theory. We acquired 1-day-old, 20-day-old, and 2-year-old queens and 1-day-old and 20-day-old workers (foragers) fed 0, 5, or 200 ppb imidacloprid, a pesticide oxidative stressor. The activities of catalase, glutathione peroxidase, glutathione S-transferase, and superoxide dismutase and the level of total antioxidant potential were determined in hemolymph. The ADS was upregulated in workers with age but downregulated in queens. Imidacloprid suppressed the ADS in all workers, particularly in foragers with an upregulated ADS, but it did not affect the ADS in 1-day-old queens. In contrast to foragers, the downregulated ADS of 2-year-old queens was unexpectedly highly upregulated by imidacloprid, which has not been previously shown in such old queens. The principal component analysis confirmed that queen and worker ADSs responded to imidacloprid in opposite ways, and ADS of 2-year-queens was markedly different from those of others. Thus, evolutionary shaped ADSs of older queens and workers may be of the limited use for foragers dwelling in pesticide ecosystems, but not for old queens.

Seroprevalence of Toxoplasma gondii among Sylvatic Rodents in Poland.

Toxoplasma gondii is an intracellular Apicomplexan parasite with a broad range of intermediate hosts, including humans and rodents. Rodents are considered to be reservoirs of infection for their predators, including cats, felids, pigs, and wild boars. We conducted a multi-site, long-term study on T. gondii in northeastern Poland. The study aimed to monitor the seroprevalence of T. gondii in the four abundant vole species found in the region (Myodes glareolus, Microtus arvalis, Microtus agrestis, and Alexandromys oeconomus) and to assess the influence of both extrinsic (year of study and study site) and intrinsic (host sex and host age) factors on seroprevalence. A bespoke enzyme-linked immunosorbent assay was used to detect antibodies against T. gondii. We examined 577 rodent individuals and detected T. gondii antibodies in the sera of all four rodent species with an overall seroprevalence of 5.5% [4.2-7.3] (3.6% [2.6-4.9] for M. glareolus and 20% [12-30.9] for M. arvalis, M. agrestis, and A. oeconomus). Seroprevalence in bank voles varied significantly between host age and sex. Seroprevalence increased with host age and was higher in females than males. These results contribute to our understanding of the distribution and abundance of T. gondii in voles in Poland and confirm that T. gondii also circulates in M. glareolus and M. arvalis, M. agrestis and A. oeconomus. Therefore, they may potentially play a role as reservoirs of this parasite in the sylvatic environment.

Zoonotic Virus Seroprevalence among Bank Voles, Poland, 2002-2010.

Bank voles in Poland are reservoirs of zoonotic viruses. To determine seroprevalence of hantavirus, arenavirus, and cowpox virus and factors affecting seroprevalence, we screened for antibodies against these viruses over 9 years. Cowpox virus was most prevalent and affected by extrinsic and intrinsic factors. Long-term and multisite surveillance is crucial.

Seroprevalence of Trichinella spp. infection in bank voles (Myodes glareolus) - A long term study.

Rodents play an important role as reservoir hosts of zoonotic diseases. As a component of our long-term programme of monitoring parasitic infections in bank vole populations in three ecologically similar sites in NE Poland, we screened blood samples for signs of a serological response to the presence of Trichinella spp. The overall seroprevalence of Trichinella spp. was 1.52%, but prevalence was largely concentrated in one of our three study sites and confined to the oldest individuals in the study. Seroprevalence of Trichinella spp. did not differ between the sexes. Although a local prevalence of 1.52% may seem low, when this is extrapolated to the national population of bank voles in peak years, perhaps numbering hundreds of millions of animals, the number of infected bank voles on a country wide scale is likely to be huge. Our results suggest that bank voles may be reservoirs of Trichinella spp. However, on the basis of our results we consider their importance as epidemiologically significant hosts for Trichinella spp. to be moderate and their role in this context to require further investigation.

Seroprevalence of TBEV in bank voles from Poland-a long-term approach.

Rodents are known to play a significant role as reservoir hosts for TBEV. During three sequential expeditions at 4-year intervals to three ecologically similar study sites in NE Poland, we trapped bank voles (Myodes glareolus) and then tested their blood for the presence of specific antiviral antibodies to TBEV. The strongest effects on seroprevalence were the extrinsic factors, site of capture of voles and year of sampling. Seroprevalence increased markedly with increasing host age, and our analysis revealed significant interactions among these three factors. Seroprevalence did not differ between the sexes. Therefore, based on the seroprevalence results, the dynamics of TBEV infection differ significantly in time, between local sub-populations of bank voles and with increasing host age. To fully understand the circulation of the virus among these reservoir hosts and in the environment, long-term monitoring is required and should employ a multi-site approach, such as the one adopted in the current study.

Changes in the bioelement content of summer and winter western honeybees (Apis mellifera) induced by Nosema ceranae infection.

Proper bioelement content is crucial for the health and wellness of all organisms, including honeybees. However, the situation is more complicated in these important pollinators due to the fact that they change their physiology during winter in order to survive the relatively harsh climatic conditions. Additionally, honeybees are susceptible to many diseases such as nosemosis, which during winter can depopulate an entire colony. Here we show that summer bees have a markedly higher content of important bioelements such as: Al, Cu, P, V, (physiologically essential); Ca, K, Mg, (electrolytic); Cr, Se, Zn, (enzymatic); As, Hg, (toxic). In contrast, a markedly higher content of: Fe (physiologically essential); Mn, Ni, (enzymatic); Cd (exclusively toxic) were present in winter bees. Importantly, N. ceranae infection resulted in an increased honeybee bioelement content of: S, Sr (physiologically essential) and Pb (exclusively toxic), whereas the Nosema-free worker-bees had higher amounts of B and Si (physiologically essential). We propose that the shortages of Fe, Mn, Ni, and Na observed in Nosema-infected bees, could be the reason for the higher mortality of Nosema-infected bees throughout overwintering. In addition, a shortage of bioelements such as B and Si may be a reason for accelerated aging in foragers that is observed following N. ceranae infection. Therefore, in winter, bioelement content was more strongly affected by N. ceranae infection than during summer. We found a strong correlation between the bioelement content of bees and seasons (summer or winter) and also with Nosema infection. We conclude that the balance of bioelements in the honeybee is altered by both seasonal affects and by Nosema infection.

Insights into the biochemical defence and methylation of the solitary bee Osmia rufa L: A foundation for examining eusociality development.

We examined age-related biochemical and histological changes in the fat bodies and hemolymph of Osmia rufa males and females. We analysed solitary bees during diapause, in October and in April; as well as the flying insects following diapause, in May and June. The trophocyte sizes, as well as the numbers of lipid droplets were the greatest at the beginning of diapause. Subsequently, they decreased along with age. Triglyceride and glucose concentrations systematically decreased in fat body cells but increased in the hemolymph from October to June. Concentrations/activities of (enzymatic and non-enzymatic) antioxidant and proteolytic systems, as well as phenoloxidase, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were constant during the diapause, usually lower in the males than the females. Prior to the diapause/overwintering, the concentrations/activities of all the compounds were higher in the fat bodies than in the hemolymph. Later in the spring and in the summer, they increased in the hemolymph and on the body surfaces, while decreasing in the fat bodies. The global DNA methylation levels increased with age. Higher levels were always observed in the males than in the females. The study will promote better understanding of bee evolution and will be useful for the protection and management of solitary bees, with benefits to the environment and agriculture.

A New Method for Quick and Easy Hemolymph Collection from Apidae Adults.

Bio-analysis of insects is increasingly dependent on highly sensitive methods that require high quality biological material, such as hemolymph. However, it is difficult to collect fresh and uncontaminated hemolymph from adult bees since they are very active and have the potential to sting, and because hemolymph is rapidly melanized. Here we aimed to develop and test a quick and easy method for sterile and contamination-free hemolymph sampling from adult Apidae. Our novel antennae method for hemolymph sampling (AMHS), entailed the detachment of an antenna, followed by application of delicate pressure to the bee's abdomen. This resulted in the appearance of a drop of hemolymph at the base of the detached antenna, which was then aspirated using an automatic pipetter. Larger insect size corresponded to easier and faster hemolymph sampling, and to a greater sample volume. We obtained 80-100 µL of sterile non-melanized hemolymph in 1 minute from one Bombus terrestris worker, in 6 minutes from 10 Apis mellifera workers, and in 15 minutes from 18 Apis cerana workers (+/-0.5 minutes). Compared to the most popular method of hemolymph collection, in which hemolymph is sampled by puncturing the dorsal sinus of the thorax with a capillary (TCHS), significantly fewer bees were required to collect 80-100 µL hemolymph using our novel AMHS method. Moreover, the time required for hemolymph collection was significantly shorter using the AMHS compared to the TCHS, which protects the acquired hemolymph against melanization, thus providing the highest quality material for biological analysis.

Nosema ceranae Infection Promotes Proliferation of Yeasts in Honey Bee Intestines.

BACKGROUND: Nosema ceranae infection not only damages honey bee (Apis melifera) intestines, but we believe it may also affect intestinal yeast development and its seasonal pattern. In order to check our hypothesis, infection intensity versus intestinal yeast colony forming units (CFU) both in field and cage experiments were studied. METHODS/FINDINGS: Field tests were carried out from March to October in 2014 and 2015. N. ceranae infection intensity decreased more than 100 times from 7.6 x 108 in March to 5.8 x 106 in October 2014. A similar tendency was observed in 2015. Therefore, in the European eastern limit of its range, N. ceranae infection intensity showed seasonality (spring peak and subsequent decline in the summer and fall), however, with an additional mid-summer peak that had not been recorded in other studies. Due to seasonal changes in the N. ceranae infection intensity observed in honey bee colonies, we recommend performing studies on new therapeutics during two consecutive years, including colony overwintering. A natural decrease in N. ceranae spore numbers observed from March to October might be misinterpreted as an effect of Nosema spp. treatment with new compounds. A similar seasonal pattern was observed for intestinal yeast population size in field experiments. Furthermore, cage experiments confirmed the size of intestinal yeast population to increase markedly together with the increase in the N. ceranae infection intensity. Yeast CFUs amounted to respectively 2,025 (CV = 13.04) and 11,150 (CV = 14.06) in uninfected and N. ceranae-infected workers at the end of cage experiments. Therefore, honey bee infection with N. ceranae supported additional opportunistic yeast infections, which may have resulted in faster colony depopulations.

Evaluation of Anthelmintic Activity and Composition of Pumpkin (Cucurbita pepo L.) Seed Extracts-In Vitro and in Vivo Studies.

UNLABELLED: A significant number of studies report growing resistance in nematodes thriving in both humans and livestock. This study was conducted to evaluate the in vitro and in vivo anthelmintic efficiency of Curcubita pepo (C. pepo) L. hot water extract (HWE), cold water extract (CWE) or ethanol extract (ETE) on two model nematodes: Caenorhabditis elegans (C. elegans) and Heligmosoides bakeri (H. bakeri). METHODS: Raman, IR and LC-MS spectroscopy analyses were performed on the studied plant material to deliver qualitative and quantitative data on the composition of the obtained extracts: ETE, HWE and CWE. The in vitro activity evaluation showed an impact of C. pepo extracts on C. elegans and different developmental stages of H. bakeri. The following in vivo experiments on mice infected with H. bakeri confirmed inhibitory properties of the most active pumpkin extract selected by the in vitro study. All of the extracts were found to contain cucurbitine, aminoacids, fatty acids, and-for the first time-berberine and palmatine were identified. All C. pepo seed extracts exhibited a nematidicidal potential in vitro, affecting the survival of L1 and L2 H. bakeri larvae. The ETE was the strongest and demonstrated a positive effect on H. bakeri eggs hatching and marked inhibitory properties against worm motility, compared to a PBS control. No significant effects of pumpkin seed extracts on C. elegans integrity or motility were found. The EtOH extract in the in vivo studies showed anthelmintic properties against both H. bakeri fecal egg counts and adult worm burdens. The highest egg counts reduction was observed for the 8 g/kg dose (IC50 against H. bakeri = 2.43; 95% Cl = 2.01-2.94). A decrease in faecal egg counts (FEC) was accompanied by a significant reduction in worm burden of the treated mice compared to the control group. CONCLUSIONS: Pumpkin seed extracts may be used to control of Gastrointestinal (G.I.) nematode infections. This relatively inexpensive alternative to the currently available chemotherapeutic should be considered as a novel drug candidate in the nearest future.

A new detection method for a newly revealed mechanism of pyrethroid resistance development in Varroa destructor.

The Varroa destructor mite has recently displayed an ever increasing resistance to new drugs, contributing to CCD proliferation. This work was aimed at determining new viable methods for identifying the pyrethroid resistance of V. destructor and DNA methylation in resistant and sensitive mites. DNA was extracted from Varroa mites. Nucleotide changes in the DNA of pyrethroid-resistant, pyrethroid-sensitive, and control mites were identified with polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in the case of five mitochondrial gene fragments. More bands were observed in the drug-resistant mites than in the other two groups. Sequencing confirmed these observations. Decreased global DNA methylation levels were observed in the pyrethroid-resistant mites. There exists a previously undescribed mechanism of pyrethroid resistance development in Varroa mites. The PCR-SSCP methods can be considered and further developed as useful tools for detecting V. destructor resistance.

Coenzyme Q10 treatments influence the lifespan and key biochemical resistance systems in the honeybee, Apis mellifera.

Natural bioactive preparations that will boost apian resistance, aid body detoxification, or fight crucial bee diseases are in demand. Therefore, we examined the influence of coenzyme Q10 (CoQ10, 2,3-dimethoxy, 5-methyl, 6-decaprenyl benzoquinone) treatment on honeybee lifespan, Nosema resistance, the activity/concentration of antioxidants, proteases and protease inhibitors, and biomarkers. CoQ10 slows age-related metabolic processes. Workers that consumed CoQ10 lived longer than untreated controls and were less infested with Nosema spp. Relative to controls, the CoQ10-treated workers had higher protein concentrations that increased with age but then they decreased in older bees. CoQ10 treatments increased the activities of antioxidant enzymes (superoxide dismutase, GPx, catalase, glutathione S-transferase), protease inhibitors, biomarkers (aspartate aminotransferase, alkaline phosphatase, alanine aminotransferase), the total antioxidant potential level, and concentrations of uric acid and creatinine. The activities of acidic, neutral, and alkaline proteases, and concentrations of albumin and urea were lower in the bees that were administered CoQ10. CoQ10 could be taken into consideration as a natural diet supplement in early spring before pollen sources become available in the temperate Central European climate. A response to CoQ10 administration that is similar to mammals supports our view that Apis mellifera is a model organism for biochemical gerontology.

Proteolysis on the body surface of pyrethroid-sensitive and resistant Varroa destructor.

The aim of this work was to determine the activity of proteases and protease inhibitors sampled from the body surface of tau-fluvalinate-sensitive and resistant V. destructor. Proteins were isolated from the tau-fluvalinate-sensitive and resistant mites, while mites untreated with tau-fluvalinate constituted the control. Subsequently, the following methodology was applied: protein concentration assay by the Lowry method - as modified by Schacterle and Pollack; assay of proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; identification of proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee and Lin method; identification of acidic, neutral and basic protease activities by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method and for protease inhibitor detection with the Felicioli method. The highest value of protein concentration was found in the tau-fluvalinate-sensitive V. destructor, while the highest activity levels of acidic, neutral and alkaline proteases were observed in the tau-fluvalinate-resistant mites. Aspartic, serine, thiolic and metallic proteases were found in the drug-resistant and drug-sensitive Varroa mites. The control samples were found to contain aspartic and serine proteases. In an acidic and alkaline environment, the results revealed a complete loss of inhibitor activities in the in vitro analyses and electrophoresis. Serine protease inhibitor activities (at pH 7.0) were high, especially in the group of tau-fluvalinate-resistant mites.