How relevant are house dust mite-fungal interactions in laboratory culture to the natural dust system?

Both house dust and house dust mitesDermatophagoides pteronyssinus contained a wider range of fungi than laboratory mite cultures. In total, nine species of fungi were isolated fromD. pteronyssinus in house dust, and these included three xerophilic species (Eurotium amstelodami, Aspergillus penicillioides andWallemia sebi) commonly found in laboratory cultures ofD. pteronyssinus. It is concluded that mites do interact with a similar range of fungi in natural dust and in laboratory culture, but that the diversity of fungal species in the laboratory is reduced and the density of individual fungal species in culture exceeds that of house dust. In a second experiment, dust samples were incubated at room temperature with 75% relative humidity. The diversity of fungi invariably declined from up to 13 genera to the few species recorded in laboratory culture. This suggests that the dominance of xerophilic fungi in laboratory mite rearings is mediated primarily by low relative humidity, and the exclusion of air-borne spores.     

Population dynamics of mites of the family pyroglyphidae and micromycetes in laboratory cultures

Original data on the study of the population dynamics in allergenic house dust mites of the family Pyroglyphidae (Dermatophagoides pteronyssinus (Trouessart 1897) and D. farinae Hughes 1961) during long (37 weeks) joint cultivation with the micromycetes Aspergillus penicillioides Speg. in simple periodical cultures without addition of food (SPC) with different initial population density of mites are given. The micromycete A. penicillioides Speg., dominating in laboratory cultures of pyroglyphids, was cultivated in parallel without mites. It was found that during joint cultivation of A. penicillioides and mites, population dynamics of D. pteronyssinus and D. farinae in SPC depended on the initial population density of mites, which affected the duration of mite developmental stages and the degree of the maximal population density. Cultures of D. farinae developed more rapidly than cultures of D. pteronyssinus, independently of the initial population density (50 or 200 specimens per gram of the substrate). A high degree of the initial population density in both mite species resulted in the shortening of the lag-phase, in more rapid reaching of the maximal population density, and in the higher degree of the maximal population density. Population density of the fungus A. penicillioides did not depend on the presence of both mite species. On the basis of our own data and literary analysis, we assume that A. penicillioides can affect the ability of mites to explore the trophic substrate, the rate of the population development, and the degree of their maximal population density. Mites, in their turn, did not significantly affect the development of A. penicillioides in our experiments.     

Do the microorganisms from laboratory culture spent growth medium affect house dust mite fitness and microbiome composition?

The interaction of house dust mites(HDM)and microorganisms is the key factor in the survival of these mites in human-made environments.Spent growth medium(SPGM)provides the rest of the dict,along with dead mite bodies and microorganisms.SPGM represents a source of microorganisms for the recolonization of mite food and the mite digestive tract.An experiment was performed to observe how adding SPGM to the HDM diet affects HDM population growth,the microbiome composition and the microbial respiration in microcosms.We analyzed American house dust mite(Dermatophagoides farinae)and European house dust mite(Dermatophagoides pleronyssinus)originating from control diets and diets treated with an extract of SPGM from 1-and 3-month-old mite cultures.The microbiome was described using 16S and 18S barcode sequencing.The composition of the bacterial and fiungal microbiomes differed between the HDM species,but the SPGM treatment influenced only the bacterial profile of D.farinae.In the D.farinae microbiome of specimens on SPGM-treated dicts compared to those of the control situation,the Lactobacillus profile decreased,while the Candinium,Staphylococ-cus,Acinetobacter,and Sphingomonas profiles increased.The addition of SPGM extract decreased the microbial respiration in the microcosms with and without mites in almost all cascs.Adding SPGM did not influence the population growth of D.farinae,but it had a variable effect on D.pteronyssimus.The results indicated that the HDM are marginally influenced by the microorganisms in their feces.     

Seasonal abundance of xerophilic fungi and house-dust mites (Acarida: Pyroglyphidae) in mattress dust

Summary Arthropods and xerophilic fungi in dust from 5 mattresses and air-borne fungi were identified and counted every 4 weeks from January 1976 to October 1977.The arthropod fauna consisted mainly of the pyroglyphid mites Dermatophagoides pteronyssinus (58%) and Euroglyphus maynei (30%). The fungal flora consisted of Aspergillus restrictus (48%), A. glaucus (16%), Wallemia sebi (3%) and Penicillium spp. (25%). Air-borne fungi belonged to the same taxa but in different frequencies, 30, 7, 21 and 27%, respectively. Mattresses differed in quantities of mites and Penicillium.In July 1977, the highest population density of pyroglyphid mites was encountered: 69 specimens/g of dust. In the same month the numbers of A. restrictus rose significantly, reaching a maximum of 3.8×10⁴ diaspores/g of dust. Most air-borne fungi were isolated in the winter period of 1976/1977. No positive correlation was found between the numbers of air-borne and mattress-dust fungi. The summer of 1976 was exceptionally dry resulting in both a premature decline of the mite populations and a low level of A. restrictus diaspores.The seasonal peaks of A. restrictus and pyroglyphid mites correspond and suggest a synergistic cooperation which may result in an increased house-dust allergen production in the environment of asthmatic patients.Supported by grant no. 230 of the Dutch Asthma Foundation     

Interference in foraging behaviour of European and American house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) by catmint, Nepeta cataria (Lamiaceae)

The European and American house dust mites, Dermatophagoides pteronyssinus and D. farinae, have a huge impact upon human health worldwide due to being the most important indoor trigger of atopic diseases such as asthma, rhinitis and atopic dermatitis. Preceding studies have shown that the behavioural response of house dust mites towards volatile chemicals from food sources can be assessed using a Y-tube olfactometer assay. In the current study, we used this assay to investigate, for the first time, the ability of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), known to repel other ectoparasites affecting human and animal health, to interfere with the attraction of D. pteronyssinus and D. farinae towards a standard food source (fish flakes). Two distinct chemotypes (A and B), enriched in the iridoid compounds (4aS,7S,7aR)-nepetalactone and (4aS,7S,7aS)-nepetalactone, and the sesquiterpene (E)-(1R,9S)-caryophyllene, were used. Initial assays with a hexane extract of fish flakes (FF extract) confirmed attraction of mites to this positive control (P < 0.001 and P < 0.05 for D. pteronyssinus and D. farinae respectively), but when presented in combination with either N. cataria chemotype, tested across a range of doses (10, 1, 0.1 and 0.01 μg), decreasing attraction of mites to their food source was observed as the dose augmented. Our study shows that N. cataria, enriched in iridoid nepetalactones and (E)-(1R,9S)-caryophyllene, exhibits potent repellent activity for house dust mites, and has the potential for deployment in control programmes based on interference with normal house dust mite behaviour.     

Seasonal prevalence of allergenic mites in house dust of Kolkata Metropolis,India

During the past few decades, house dust mites have attracted worldwide interest among medical entomologists and acarologists because of their importance in causing nasobronchial allergic disorders in human beings. House dust mites are present throughout the year; however, their relative densities differ in different seasons and habitats. Because the prevalence of house dust mite allergen is important epidemiologically and clinically, detailed knowledge on the seasonal abundance of important allergenic mites is of great importance for better understanding of the pathogenesis of the disease. In view of this, a systematic survey was carried out on the prevalence of total mites and four common allergenic mites in the city of Kolkata for two consecutive years. Both bed and bedroom floor dust were collected separately from homes inhabited by asthmatic patients situated in different corners of the city on monthly basis from January 2004 to December 2005. The population levels of total mites and four common allergenic mites, namely Dermatophagoides pteronyssinus, D. farinae, Austroglycyphagus geniculatus, and Blomia tropicalis separately, were highest during the pre-monsoon period (March–May), irrespective of habitat, whereas densities were low in all cases during winter (December–February). The study indicates that season had the most significant effect on the relative abundance of house dust mites except Dermatophagoides farinae, irrespective of habitat.     

Age structure and dynamics of house dust mite populations

Age structure—the relative numbers of eggs, immatures and adults—in populations of the house dust mitesDermatophagoides pteronyssinus andEuroglyphus maynei was investigated in four sequential monthly samples taken from mattresses in each of eight homes in Glasgow, Scotland. Additionally, age structure ofD. pteronyssinus was determined in samples taken bimonthly for 6 months from nine quadrats of a double mattress. It was found that although age structure varied considerably with time, forD. pteronyssinus in different homes the most common structure was one in which immatures were dominant, then eggs and then adults (31% of samples). Immatures or eggs were dominant in 75% of samples. ForE. maynei the age structure was quite different: the most common structure was one in which adults were dominant, then immatures and then eggs (69% of samples). In different quadrats of a double mattress, mean age structure ofD. pteronyssinus underwent a shift towards higher proportions of immatures and then eggs during the sampling period, which reflected the increase in population density detected during this period.Life and fecundity tables were constructed forD. pteronyssinus andE. maynei using previously-available in vitro data on fecundity and survivorship rates and hypothetical values based on means derived from a number of studies. From the tables the stable age distributions were calculated and compared with the age structures of the natural populations. It was found that mean age structure of natural populations ofD. pteronyssinus was fairly close to the predicted stable age distribution, but those ofE. maynei indicated the populations were in decline during the sampling period, a fact confirmed by abundance data. The concept that the rate of increase of house dust mite populations can be estimated by determining age structure of mites isolated from dust samples was explored using the hypothetical population parameters ofD. pteronyssinus. It was predicted that quite large differences in fecundity and mortality would not drastically alter the proportions of eggs, immatures and adults in stable populations.Eggs as components of the house dust mite population are considered seriously for the first time. Those ofD. pteronyssinus andE. maynei were identified and differentiated by allometry. It is stressed that forD. pteronyssinus, during the sampling period, half or more of the mites in a dust sample may be represented as eggs, and to ignore them is to deliberately make a less accurate estimate of population density than could be otherwise achieved.     

Identification of Glycycometus malaysiensis (for the first time in Brazil), Blomia tropicalis and Dermatophagoides pteronyssinus through multiplex PCR

Blomia tropicalis and Dermatophagoides pteronyssinus play an important role in triggering allergy. Glycycometus malaysiensis causes IgE reaction in sensitive people, but is rarely reported in domestic dust, because it is morphologically similar to B. tropicalis making the identification of these species difficult. The identification of mites is mostly based on morphology, a time-consuming and ambiguous approach. Herein, we describe a multiplex polymerase chain reaction (mPCR) assay based on ribosomal DNA capable to identify mixed cultures of B. tropicalis, D. pteronyssinus and G. malaysiensis, and/or to identify these species from environmental dust. For this, the internal transcribed spacer 2 (ITS2) regions, flanked by partial sequences of the 5.8S and 28S genes, were PCR-amplified, cloned and sequenced. The sequences obtained were aligned with co-specific sequences available in the GenBank database for primer design and phylogenetic studies. Three pairs of primers were chosen to compose the mPCR assay, which was used to verify the frequency of different mites in house dust samples (n?=?20) from homes of Salvador, Brazil. Blomia tropicalis was the most frequent, found in 95% of the samples, followed by G. malaysiensis (70%) and D. pteronyssinus (60%). Besides reporting for the first time the occurrence of G. malaysiensis in Brazil, our results confirm the good resolution of the ITS2 region for mite identification. Furthermore, the mPCR assay proved to be a fast and reliable tool for identifying these mites in mixed cultures and could be applied in future epidemiological studies, and for quality control of mite extract production for general use.

     

Effect of certain socio-ecological factors on the population density of house dust mites in mattress-dust of asthmatic patients of Calcutta, India

Allergy to house dust mites, particularly tothe genus Dermatophagoides is a fairlycommon problem in Calcutta and its adjoiningareas since last two decades. Both the commonspecies of the genus Dermatophagoidesi.e. D. farinae and D.pteronyssinus are found to be abundant in thedust samples collected from beds of patientssuffering from nasobronchial allergic disordes.The presence of these mites in quite a goodnumber in the patients' beds are clinicallycorrelated with the aetiopathology of variousallergic manifestations like bronchial asthma.Dermatophagoides mites may occupydifferent niches in the homes of asthmatics andare more common in beds than elsewhere in thehouse, however, the distribution and abundanceof these mites are influenced by somesocio-ecological factors. The aim and objectiveof the present study is to assess the impact ofsome common socio-ecological managementpractices like the age of house, age ofmattress, type of mattress, frequency ofcleaning of the mattress and even the economicstatus of the patients on the growth,multiplication and finally accumulation ofthese mites in the patients' surroundings.Proper identification of offending allergensand subsequent reduction of load of these miteallergens from the patient's environment may behelpful for the prophylactic management ofthese diseases in Calcutta metropolis.     

The influence of household pets on the composition and quantity of allergenic mite fauna within Irish homes: a preliminary investigation

Allergenic mites are responsible for inducing hypersensitive reactions in genetically predisposed people worldwide. Mites in dust from 30 Irish homes with pets (dogs, n = 23; cats, n = 7) were compared with those in 30 homes without pets. House dust mites constituted 78% of all mites recorded, with Dermatophagoides pteronyssinus (Acariformes: Pyroglyphidae) representing 57–72% of mites in furniture and mattresses in both home types compared with only 22% of mites in pet beds. Although storage mites accounted for just 13% of all mites recorded, they represented 46% of mites recorded in pet beds. Median levels of the dust mite allergen Der p 1 (µg/g) in dust samples from mattresses in homes without pets were significantly greater than in mattresses from homes with pets, reflecting the greater densities of D. pteronyssinus found in the former home category. Mite species richness was greater in homes with pets (17 species) than in homes without pets (13 species). This suggests that although the presence of pets can result in a wider variety of epidemiologically important mite species within households, increased competition among mite species may result in a more balanced mite fauna in the home, inhibiting the dominance of any one species and hence lowering allergen‐associated risks.     

Ecological relationships between xerophilic fungi and house-dust mites (Acarida: Pyroglyphidae)

Summary At. 75 and 80% relative humidity (RH), on a wheat germ flake medium, Aspergillus penicilloides grew abundantly and suppressed the population growth of Dermatophagoides pteronyssiunus. At 71% RH, A. penicilloides grew moderately and was only antagonistic to D. pteronyssinus when the fungus was previously incubated on the medium.On a human dander medium and on mattress dust, A. penicilloides grew moderately at 71% and 75% RH and stimulated the development of D. pteronyssinus populations. Also a moderate growth of Eurotium repens on human dander positively influenced D. pteronyssinus. Wallemia sebi and Penicillium brevicompactum grew slightly or did not grow at all at 75% RH. No effect was observed on D. pteronyssinus.It appears that xerophilic fungi may stimulate, and occasionally may reduce, the growth of house-dust mite populations in the natural environment.     

Identification of house-dust mites

The term house-dust mite usually refers to those species of the mite family Pyroglyphidae, that are known to commonly occur, although sometimes regionally, in the dust of human dwellings. These species belong to five genera:Dermatophagoides, Euroglyphus, Hirstia, andMalayoglyphus. Related species ofDermatophagoides have the most world-wide occurrence, the commonest being:D. farinae, D. microceras, andD. pteronyssinus. A correct taxonomic identification of house-dust mites is very important, not only from a biological stand point, but also regarding the consequences of their respective allergenical properties. Several immuno-chemical studies revealed differences between the products of two hard to distinguish sibling species. A preliminary practical taxonomic key for the most common and important house-dust mites is presented.     

Skin-associated Bacillus, staphylococcal and micrococcal species from the house dust mite, Dermatophagoides pteronyssinus and bacteriolytic enzymes

Dust mites produce bacteriolytic enzymes, one of which belongs to the NlpC/P60 superfamily comprising bacterial and fungal proteins. Whether this enzyme is derived from the mite or from mite-associated microbes is unclear. To this end, the bacteriology of mites per se, and carpet and mattress dust from a group of asthmatic children and their parents was investigated. Dust from parents’ and children’s mattresses yielded significantly more colony forming units compared with dust from their corresponding carpets. Zymography demonstrated some dusts contained bacteriolytic enzymes, and in nine of the twelve dust samples from three of five houses examined, a prominent bacteriolytic band was obtained that corresponded to the mite band, although in one home, other lytic bands were detected. Fifty bacterial isolates were obtained from surface-sterilised, commercially obtained Dermatophagoides pteronyssinus. 16S rRNA, tuf and rpoB gene sequencing of nine Gram-positive isolates identified them as Bacillus cereus, B. licheniformis, Staphylococcus aureus, S. epidermidis, S. capitis and Micrococcus luteus, known human skin commensals. 16S rRNA sequence homologies of four of the nine isolates identified as B. licheniformis formed a distinct phylogenetic cluster. All species secreted lytic enzymes during culture although the lytic profiles obtained differed between the rods and the cocci, and none of the bands detected corresponded to those observed in dust or mites. In conclusion, mites harbour a variety of bacterial species often associated with human skin and house dusts contain bacteriolytic enzymes that may be mite-derived. The identification of a novel cluster of B. licheniformis isolates suggests an ecological adaptation to laboratory-reared D. pteronyssinus. It remains to be determined whether the previously described mite-associated 14 K lytic enzyme is derived from a microbial source.     

Distribution and abundance of house-dust mites (Acarina: Pyroglycphidae) in Rome,Italy

For the first time in Rome, house-dust mite infestation was studied in 90 randomly selected houses. In each house, mite infestation was assessed in three sites: mattress, bedroom and living room. In total, 87.8% of the sampled houses were positive for dust mites. In the houses infested, 11.4% showed densities of >100 mites/g of dust, 15.2% registered densities between 50 and 99, and in the remaining houses (73.4%), the densities were between 1 and 49 mites/g dust. The percentages of infested houses were positively correlated with the relative humidity (RH) values (r=0.89,P=0.02). At the lowest range of RH (between 46 and 50), the infestation was 50% and at the highest range of RH (between 73 and 78) it was 100%. The mattress was significantly the most infested (71.1%) of the tested sites. Only wool and spring mattresses were infested, and they did not show any significant differences in mite concentrations.Dermatophagoides farinae was the most abundant species (53.1%), followed byGlycyphagus domesticus (34.5%),D. pteronyssinus (5.2%), andEuroglyphus maynei (0.2%);D. farinae was also the most frequent species (56.9%). The remaining specimens (7.0%) were predator species commonly found in houses. The prevalence ofD. farinae in Rome is discussed.     

Structural biology of mite allergens

Mite allergens contribute to a significant proportion of human allergic symptoms, including asthma and rhinitis. The development of therapies to treat and prevent these symptoms depends largely on our understanding of the properties of these allergens. Much effort has been devoted to determining the structure and organization of mite allergens, particularly of the house dust mites, toward understanding their activities and how they elicit immunological responses in humans. Here, we review the structural biology of the major allergens from two species of house dust mites, Dermatophagoides farinae and D. pteronyssinus, as well as allergens from a storage mite, Blomia tropicalis. The knowledge gained from the structural biology of these allergens will enable progress in producing novel, more effective treatments for mite allergies based on specific immunotherapy approaches.     

Clinical significance of dust mite allergens

Dust mites are an important cause of allergic diseases worldwide. The traces of Dermatophagoides farinae and Dermatophagoides pteronyssinus can be found all over the world, while Blomia tropicalis are more common allergenic mites in tropical areas. A variety of allergenic components in dust mites have been found, apart from the different positive rates of IgE reactions in dust mite allergic patients, their biological characteristics, effects on the innate immune system, and especially their distribution characteristics in patients are different. Studying the relationship between dust mite allergens and clinical significance will help for diagnosis of patients and formulation of corresponding Allergen Specific Immunotherapy.

     

Effectiveness of vacuum cleaning and wet cleaning in reducing house-dust mites,fungi and mite allergen in a cotton carpet: A case study

In order to evaluate the effect of continuous, thorough vacuuming on house dust organisms and mite allergen, a cotton carpet was vacuumed every other day, six times in total. Seven weeks later, the carpet was cleaned by means of spray extraction. Samples were taken before and after this wet cleaning.In total 1150 g of dust was collected, containing approximately 174 000 arthropods (dead and alive) and 9000×10⁶ fungal spores. In the course of the experiment, the amount of dust collected at each vacuuming decreased. The number of extracted house dust mites did not decrease significantly, but that of the predator miteCheyletus did. The number of extracted fungal spores showed a significant decrease (from 142 to 16×10⁶ spores/m² per minute vacuuming), as did the extracted mite allergen per m². After 7 weeks the number of mite eggs and complete house dust mites had increased enormously. After cleaning by spray extraction another increase in the number of complete mites and mite eggs was found, while the amount of mite allergen was diminished.The population growth of the house dust mite between the 6th and the 7th vacuuming is probably due to the decrease of their most important predator,Cheyletus. After the wet cleaning a number of extra eggs hatched, probably due to the high humidity in the carpet. The procedures used in this study to combat house dust mites may have an adverse effect in the long run.     

The effect of non-pathogenic phylloplane fungi on life-history traits of urticae (Acari: Tetranychidae)

The direct effect of three common non-pathogenic phylloplane fungi on the life-history traits of Tetranychus urticae Koch was investigated on intact leaves of the garden bean Phaseolus vulgaris L. (var. Tendergreen Improved), under conditions of low and high water-deficit stress. The survival rate of T. urticae was always reduced by an increase in water-deficit stress, whereas the effect of the fungi depended in part on the watering regime. On two of the three fungi tested, Alternaria alternata (Fr.) Keissler and Epicoccum nigrum Link, the mites showed a significant increase in net reproduction compared to those reared under control (i.e. no fungi added) conditions, independent of water-deficit stress, resulting in a higher intrinsic rate of increase. The third fungus tested, Cladosporium cladosporioides (Fres.) de Vries, had a similar response with respect to net reproduction when reared under low water-deficit stress but not when reared under a high water-deficit stress. There was, however, no evidence of an interactive effect with water-deficit stress and presence or absence of fungus affecting the net reproduction of the mites, for any of the three fungal species tested. An interactive effect between fungal species and water-deficit stress was observed for the intrinsic rate of increase of the mites. It is suspected that the fungi are acting as a source of an otherwise limiting resource and the differences observed between the fungal treatments is due to differences in consumption, based on the different sizes of their conidia.     

Population growth and allergen accumulation of <Emphasis Type="Italic">Dermatophagoides pteronyssinus</Emphasis> cultured at 20 and 25°C

The house dust mites, Dermatophagoides pteronyssinus and D. farinae are cultured commercially and in research laboratories and material is harvested from these cultures to make extracts that are used for diagnosis, immunotherapy and research. Temperature and other climatic conditions can influence population growth rates, dynamics of allergen production, and the associated endotoxin, enzyme and protein levels of the mite material harvested from these cultures. Here we determined how temperature affected these parameters. Dermatophagoides pteronyssinus was cultured at 20 and 25°C at 75% relative humidity, and at 2-week intervals the concentrations of mites, Der p 1 and Der p 2 allergens, endotoxin, and selected enzymes were determined. Mite density increased exponentially but growth rate and final population density were greater at 25°C compared to 20°C. The combined allergen (Der p 1 + Der p 2) concentrations accumulated in the cultures at about the same rate at both temperatures. However, individual Der p 1 and Der p 2 accumulation rates varied independently at the two temperatures. Der p 1 accumulated faster at 20°C whereas Der p 2 accumulated faster at 25°C. The amount of Der p 1 in whole cultures was greater than the amount of Der p 2. The concentration of allergen for washed mites harvested from the cultures was much less than for the whole cultures. Our study demonstrated that temperature is an important factor in population growth and the dynamics of allergen production in cultured mites.     

Molecular identification of house dust mites and storage mites

Mites are known causes of allergic diseases. Currently, identification of mites based on morphology is difficult if only one mite is isolated from a (dust) sample, or when only one gender is found, or when the specimen is not intact especially with the loss of the legs. The purpose of this study was to use polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) of the ITS2 gene, to complement the morphological data for the identification of mites to the species level. For this, six species were cultured: Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Tyrophagus putrescentiae, Aleuroglyphus ovatus and Glycycometus malaysiensis. Genomic DNA of the mites was extracted, quantified, amplified and digested individually with restriction enzymes. Hinf I and Ple I differentiated the restriction patterns of D. pteronyssinus and D. farinae. Bfa I and Alu I enzymes differentiated B. tropicalis and G. malaysiensis. Ple I enzyme was useful for the differentiation between T. putrescentiae and A. ovatus. Bfa I was useful for the differentiation of G. malaysiensis from the rest of the species. In conclusion, different species of mites can be differentiated using PCR–RFLP of ITS2 region. With the established PCR–RFLP method in this study, identification of these mites to the species level is possible even if complete and intact adult specimens of both sexes are not available. As no study to date has reported PCR–RFLP method for the identification of domestic mites, the established method should be validated for the identification of other species of mites that were not included in this study.