Rapid method for the detection of storage mites in cereals: feasibility of an ELISA based approach

This paper describes the development of rapid immunodiagnostic tests for the detection of storage mite infestations in cereals and cereal products. The study's first phase (proof of concept) involved the production of a species-specific enzyme-linked immunoassay (ELISA) for the flour mite, Acarus siro (L.), a major pest of stored commodities. The specificity of this new assay was assessed against key stored product contaminants (13 species of mites of which three were predatory, five species of insects and five species of fungi) in the presence and absence of grain. The assay was species-specific (no cross-reactivity to other storage contaminants) and was unaffected by the presence of cereal antigens in the extract. In the study's second phase, species- and genera-specific ELISAs were developed for a range of key storage mite pests: the cosmopolitan food mite (Lepidoglyphus destructor), the grocers' itch mite (Glycyphagus domesticus), the grainstack mite (Tyrophagus longior), mites of the Tyrophagus and Glycyphagus generas, and all storage mites. All tests were demonstrably specific to target species or genera, with no cross-reactions observed to other storage pest contaminants or cereals. The final, validation phase, involved a comparative assessment of the species-specific A. siro and the genus-specific Tyrophagus ELISAs with the flotation technique using laboratory and field samples. Both ELISAs were quantitative (0-30 mites per 10 g wheat) and produced good comparative data with the flotation technique (A. siro r(2)=0.91, Tyrophagus spp. r(2)=0.99).     

Molecular and biochemical properties of storage mites (except Blomia species)

In recent years, the allergological importance of different mite species not belonging to the family Pyroglyphidae has been demonstrated. These mites, commonly named storage mites, include Lepidoglyphus destructor, Glycyphagus domesticus, Tyrophagus putrescentiae, Acarus siro, Aleuroglyphus ovatus, Suidasia medanensis and Thyreophagus entomophagus. Several allergens from these species have been purified, sequenced and cloned. Many of these allergens have shown sequence homology and a biological function similar to those previously described in Blomia tropicalis and the Dermatophagoides spp. The main allergens described in storage mites include fatty acid binding proteins, tropomysin and paramyosin homologues, apoliphorine like proteins, alfa-tubulines and other, such as group 2, 5 and 7 allergens, which definitive biological function has not been described yet. Besides the purification and characterization of allergens, the allergenicity of other species such as Acarus farris, Austroglycyphagus malaysiensis, Blomia kulagini and B. tjibodas, Cheyletus eruditus, Chortoglyphus arcuatus, Gohieria fusca, Thyreophagus entomophagus and Tyrophagus longior has been investigated. Research has also been conducted to identify allergens in parasitic mites, such as Psoroptes ovis, Sarcoptes scabiei, Varroa jacobsoni, Diplaegidia columbae and Hemisarcoptes cooremani. The allergenicity of mites present in agricultural environments has been investigated. Crossreactivity studies have also been performed to elucidate to what extent all these mites share common, or species specific epitopes. Herein we present a comprehensive review of the allergenicity of mite species which have been implicated in human respiratory and/or dermatological diseases.     

Suppressive potential of bean (Phaseolus vulgaris) flour against five species of stored-product mites (Acari: Acarididae)

Previous research has demonstrated that legume proteins have insecticidal activity against stored-product pests, but activity against stored-product mites has not been tested. A study was therefore conducted to explore the potential of bean, Phaseolus vulgaris L., flour as novel botanical acaricide against five species of storage and dust mites: Acarus siro L., Aleuroglyphus ovatus (Troupeau), Caloglyphus redickorzevi (Zachvatkin), Lepidoglyphus destructor (Schrank), and Tyrophagus putrescentiae (Schrank). The effect of wheat, Triticum aestivum L., grain enriched with bean flour to eight concentrations (0, 0.01, 0.1, 0.5, 1, 2.5, 5, and 10%) on population growth initiating from the density of 50 mites per 100 g of wheat was recorded for 21 d under laboratory conditions (grain moisture 14.6% moisture content and 25 degree C in darkness). The enrichment of grain with bean flour suppressed the population growth of all tested species: 0.01% concentration reduced population growth of all tested species to >50% in comparison with the control population. The most sensitive species were A. siro and L. destructor, followed by T. putrescentiae and C. redickorzevi. The least sensitive species was A. ovatus. The terminal (i.e., after 21 d) density of mites positively correlated with bean flour concentration. The suppressive effect of bean flour was not linear but rather asymptotic. The results of this study are discussed in the context of the application of bean flour in integrated control of stored-product mites and the elimination of stored-product mite allergens.     

Temperature preference and respiration of acaridid mites

The thermal preferences in a grain mass and respiration at various temperatures in mites (Acari: Acarididae) of medical and economical importance [Acarus siro (L. 1758), Dermatophagoides farinae Hughes 1961, Lepidoglyphus destructor (Schrank 1871), and Tyrophagus putrescentiae (Schrank 1781)] were studied under laboratory conditions. Based on the distribution of mites in wheat, Triticum aestivum L., grain along a thermal gradient from 10 to 40 degrees C, L. destructor, D. farinae, and A. siro were classified as eurythermic and T. putrescentiae as stenothermic. The lowest preferred temperature was found for D. farinae (28 degrees C), followed by A. siro (28.5 degrees C), L. destructor (29.5 degrees C), and T. putrescentiae (31.5 degrees C). The relationship between the respiration rate and the temperature was similar for all four mite species. The highest respiration was found in the range from 31 to 33 degrees C. This is approximately 2 degrees C higher than the preferred temperature of these species. The lower temperature threshold of respiration ranged from 1 to 5 degrees C and the upper threshold ranged from 45 to 48 degrees C. Acclimatization of A. siro to temperature regimes of 5, 15, and 35 degrees C resulted in thermal preferences between 9 and 12 degrees C, 9 and 20 degrees C, and 28 and 35 degrees C, respectively. The respiration rate of acclimatized specimens increased with the temperature, reaching a maximum at 29.0 degrees C for mites acclimatized at 5 and 15 degrees C and a maximum at 33.7 degrees C for those acclimatized at 30 degrees C.     

Differential levels of mite infestation of wheat and barley in Czech grain stores

Abstract  While mites are able to utilize numerous food sources, the suitability of the food strongly influences population growth. The different suitabilities of various stored agricultural products will thus affect the level of infestation. In this study, we compared field mite infestation rates in two stored cereals: wheat and barley. We analyzed mite abundance, frequency and species composition in samples of grain obtained from 79 selected Czech grain stores. Stored barley seemed to be more vulnerable to mite attack than wheat, as we consistently found more infested samples, more species and higher mean and median mite abundance per sample in barley as compared to wheat. The mean mite abundance per sample were 55 and 506 individuals for wheat and barley, respectively. In barley, 10% of samples exceeded allergen risk threshold (i.e., 1 000 individuals per kg of grain). Altogether, 25 species were identified from approximately 35 000 individuals. The most frequently identified species were the same in wheat and barley, that is, Tydeus interruptus Sig Thor, Acarus siro L., Tarsonemus granarius Lindquist, Lepidoglyphus destructor (Schrank) and Tyrophagus putrescentiae (Schrank). Based on principal components analysis, we found a closer association of T. interruptus , T. putrescentiae , L. destructor and Cheyletus eruditus (Schrank) with barley samples, corresponding to the high frequency and abundance values of these mites. The probable reasons for the higher infestation, especially mite abundance in barley, are discussed in relation to the higher proportion of crushed parts, which may release favorable nutrient sources and amplify the abundance values.     

Detection and identification of species-specific bacteria associated with synanthropic mites

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2?×?10(2) to 1.4?×?10(3) per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of "Candidatus Cardinium hertigii" and as a separate novel cluster.     

OBSERVATIONS ON THE CONTROL OF MITE INFESTATIONS IN STORED WHEAT BY CHEYLETUS SPP. (ACARINA, CHEYLETIDAE)

An account is given of the control of the grain mite Acarus siro L. (= Tyroglyphus farinae L.) by the predatory mite Cheyletus sp. in imported wheat stored in bags or in bulk. After 12–18 months of storage Cheyletus sp. had developed in 75% of the parcels of wheat and in 67% of these it became dominant over other mites. This usually occurred in late summer for bagged wheat. Control of A. siro by Cheyletus sp. compared favourably with that achieved by fumigation with methyl bromide and on occasion was superior. Both development and dominance of Cheyletus sp. were adversely affected by earlier fumigation but there was some evidence that spraying wheat with γ-BHC favoured the development of this species and, in some circumstances, its dominance. Dominance by Cheyletus sp. in bulk grain often occurred in the winter months when the surface moisture content of the wheat was at its highest.
The development of Cheyletus eruditus Schr. is described in a bulk grain store where dense populations of A. siro and Glycyphagus destructor Schr. were eliminated by the predator. The numbers of C. eruditus in wheat tended to increase faster when the surface layers were disturbed than when the grain was undisturbed. Nevertheless, dominance over the other mites tended to occur first in undisturbed grain.     

Spatiotemporal distribution of insects and mites in horizontally stored wheat

Samples were taken from a flat storage facility located in central Greece, filled with approximately 45 tons of hard wheat, to assess the spatiotemporal distribution of stored-product insects and mites. The wheat was stored in a 1.5-m-deep bulk from June 2001 until March 2002. The samples were taken with a partitioned grain trier during the entire storage period, at 10-d intervals. The trier samples were examined separately for the upper, medial, and lower 0.5 m of the bulk. The spatial distribution of the insect and mite species found was examined by contour analysis based on the numbers of individuals in the trier samples. Nine insect and 20 mite taxa were found during the sampling period. The most abundant insect species were Tribolium castaneum (Herbst), Cryptolestes ferrugineus (Stephens), and Rhyzopertha dominica (F.); the most abundant mite species were Lepidoglyphus destructor (Schrank), Acarus siro L., and the predator Cheyletus malaccensis Oudemans. The highest population densities for the majority of the insect and mite species were recorded during autumn. The majority of the individuals of the most abundant insect and mite species were found in the upper 0.5 m of the bulk, with the exception of C. malaccensis, which was equally distributed in the upper and medial 0.5 m of the bulk. The spatiotemporal distribution during the entire experimental period was notably varied according to the insect and mite species.     

Mites and fungi in heavily infested stores in the Czech Republic

Toxigenic and allergen-producing fungi represent a serious hazard to human food and animal feed safety. Ninety-four fungal species were isolated from mite-infested samples of seeds taken from Czech seed stores. Fungi were isolated from the surface of four kinds of seeds (wheat, poppy, lettuce, and mustard) and from the gut and external surface of five species of mites (i.e., Acarus siro L., 1758, Caloglyphus rhizoglyphoides (Zachvatkin, 1973), Lepidoglyphus destructor (Schrank, 1781), Tyrophagus putrescentnae (Schrank, 1781) and Cheyletus malaccensis Oudemans 1903) separately. Multivariate analysis of fungi complex composition showed that the frequency of fungal was species significantly influenced by the kind of seed. Fungal frequencies differed between mites gut and exoskeleton surface and between the surfaces of mites and seeds. Three groups of fungal species were recognized: 1) mite surface-associated fungi: Penicillium brevicompactum, Alternaria alternata, and Aspergillus versicolor; 2) mite surface- and seed-associated fungi: Aspergillus niger, Penicillium crustosum, Penicillium aurantiogriseum, Penicillium chrysogenum, and Aspergillus flavus; and 3) seed-associated fungi: Cladosporium herbarum, Mucor dimorphosporus f. dimorphosporus, Botrytis cinerea, Penicillium griseofulvum, and Eurotium repens. Mite-carried species of microfungi are known to produce serious mycotoxins (e.g., aflatoxin B1, cyclopiazonic acid, sterigmatocystin, ochratoxin A, and nephrotoxic glycopeptides) as well as allergen producers (e.g., A. alternata and P. brevicompactum). Storage mites may play an important role in the spread of some medically hazardous micromycetes. In addition, these mite-fungi associations may heighten the risk of occurrence of mycotoxins in food and feed stuffs and cause mixed contamination by fungal and mite allergens.     

Molecular systematics of Acarus siro s. lat., a complex of stored food pests

The astigmatid mite Acarus siro (Linnaeus 1758) is an important agricultural pest and environmental allergen. However, it is likely that many mites described in the literature as A. siro, collected from both outdoor and stored product habitats, may belong to one of its sibling species, A. farris [Ent. Ber. Amst. 2 (26) (1905) 20] or A. immobilis [Bull. Br. Mus. Nat. Hist. 11 (1964a) 413; Acarologia. 6 (Suppl) (1964) 101]. The three species are difficult to separate morphologically, gene exchange between some of them is possible and, although each species displays environmental preferences, they occur together in some environments. This raises a question about their separate species status. In a pilot study, we investigated whether genetic data supported the separate species status of these forms. Both nuclear (the second internal transcribed spacer region [ITS-2] of the ribosomal cistron) and mitochondrial (cytochrome oxidase subunit I, mtcoxI hereafter) loci were employed for this purpose. Mtcox1 data does not conflict the differentiation into three separate species and while the ITS2 data were problematic for this group of mites it suggested that a congener, Acarus gracilis [Ann. Mag. Nat. Hist. 10 (1957) 753], is basal to the A. siro species complex.     

Comparison of the fecundity of three species of grain store mites on fungal diets

The egg production of isolated pairs ofAcarus siro, Glycyphagus destructor andTyrophagus longior fed on a control diet of wheatgerm and yeast was compared with that on mycelial pellets from shake cultures ofCladosporium cladosporiodes, Aspergillus repens, A. ruber andPenicillium cyclopium as well as spores ofP. cyclopium andA. repens. The mites always produced fewer eggs on the fungal diets than on the ideal diet.Tyrophagus longior usually did best of the three mite species on the fungal diets, andG. destructor worst.Aspergillus ruber proved the most suitable fungus for all mite species, whileC. cladosporiodes was the least suitable. Spores were a less suitable diet than mycelial pellets from shake cultures, which were predominantly mycelium.     

Tyrophagus putrescentiae (Sarcoptiformes: Acaridae) in the in vitro cultures of slime molds (Mycetozoa): accident,contamination, or interaction?

Experimental and Applied Acarology - Tyrophagus putrescentiae (Schrank), commonly known as the cereal mite, cheese mite, or ham mite, is a cosmopolitan species reported from various environments in...     

Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology.

The dust mite Lepidoglyphus destructor is a common species in Europe and a major cause of dust mite allergy in rural surroundings, but it also contributes to dust mite allergy in urban areas. One major allergen, Lep d 2, has been expressed as a recombinant protein and evaluated both in vivo and in vitro and shown to detect 60% or more of L. destructor-sensitized subjects. Additional recombinant allergens are needed to obtain a reliable diagnostic tool for L. destructor allergy. The aim of this study was to clone and express new allergens from L. destructor and determine their recognition frequency among sensitized individuals. A phage display cDNA expression library was constructed and screened with sera from L. destructor-sensitized individuals. The cDNAs encoding the allergens were cloned into the pET17b vector and subsequently expressed in Escherichia coli as C-terminal His6-tagged proteins. Immunoblotting of the recombinant proteins was performed using sera from 45 subjects allergic to L. destructor. Three new allergens from L. destructor, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13, were identified and recognized by 4/45 (9%), 28/45 (62%) and 6/45 (13%) sera from L. destructor-sensitized subjects, respectively.     

The effectiveness of organophosphate acaricides on stored product mites interacting in biological control

Three organophosphates (pirimiphos-methyl, chlorpyrifos-methyl, chlorpyrifos) were tested on a laboratory strain of Cheyletus eruditus (Schrank), a predatory mite used for biological control of stored food mites, and on tow species of acaroid mites, Acarus siro L. and Tyrophagus putrescentiae (Schrank). Biological control is often preceded by a chemical treatment with organophosphates and thus it is important to know how the acaricides affect the predators. It was found that chlorpyrifos-methyl was the most toxic organophosphate on C. eruditus. The effectiveness of pirimiphos-methyl and chlorpyrifos was approximately equal and was three times lower than the effectiveness of chlorpyrifos-methyl. The organophosphates were nearly equally effective on both acaroid mites but A. siro was slightly more susceptible than T. putrescentiae. On the basis of these results, the use of pirimiphos-methyl or chlorpyrifos rather than chlorpyrifos-methyl is recommended for protection of empty stores or stored grain against resiliant populations of stored food mites.     

Comparative analyses of proteolytic activities in seven species of synanthropic acaridid mites

Microplate assays with 96 wells were optimized to screen proteolytic activities in mite homogenates. Whole-mite extracts of Acarus siro, Aleuroglyphus ovatus, Tyrophagus putrescentiae, Tyroborus lini, Carpoglyphus lactis, Lepidoglyphus destructor, and Dermatophagoides farinae exhibited non-specific proteolytic activity in buffers from pH 2 to 12, and three peaks of highest activity at pH 3, 5-6, and 10 were distinguished. The reducing agent Tris(2-carboxyethyl)phosphine hydrochloride decreased general proteolytic activity on azocasein at pH 5 and 6. The results obtained on two non-specific substrates, azocasein and azoalbumin, showed highly different ranks of the species at pH 5 and 6. Proteolytic activities toward N(α)-Benzoyl-D,L-arginine 4-nitroanilide hydrochloride, N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide, N-Succinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide, Benzyloxycarbonyl-L-arginine-L-arginyl 4-nitroanilide, and N-Methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-methionine 4-nitroanilide (MAAPMpNA) were highest at alkaline pH, but the activity toward MAAPMpNA was also high at pH 5 and 6. In contrast, N-Succinyl-L-alanyl-L-alanyl-L-phenylalanine 4-nitroanilide (AAPpNA) and L-arginyl 4-nitroanilide (ArgpNA) had the highest activity recorded at pH 6. The high activities observed on AAPpNA, ArgpNA, and MAAPMpNA at digestive pH suggest that enzymes present in these extracts could have the majority of proteolysis in the mite gut. Evidence of the presence of proteolytic activities on all tested substrates and in all the tested mite homogenates suggests that the proteolytic activities may contribute to allergenicity. Poor or undetected hydrolytic activities of mite extracts toward substrates for keratin and collagen at digestive pH underline the importance of ecological interactions between mites and microorganisms in the utilization of such substrates.     

The importance of starch and sucrose digestion in nutritive biology of synanthropic acaridid mites: α‐Amylases and α‐glucosidases are suitable targets for inhibitor‐based strategies of mite control

The adaptation of nine species of mites that infest stored products for starch utilization was tested by (1) enzymatic analysis using feces and whole mite extracts, (2) biotests, and (3) inhibition experiments. Acarus siro, Aleuroglyphus ovatus, and Tyroborus lini were associated with the starch‐type substrates and maltose, with higher enzymatic activities observed in whole mite extracts. Lepidoglyphus destructor was associated with the same substrates but had higher activities in feces. Dermatophagoides farinae, Chortoglyphus arcuatus, and Caloglyphus redickorzevi were associated with sucrose. Tyrophagus putrescentiae and Carpoglyphus lactis had low or intermediate enzymatic activity on the tested substrates. Biotests on starch additive diets showed accelerated growth of species associated with the starch‐type substrates. The inhibitor acarbose suppressed starch hydrolysis and growth of the mites. We suggest that the species with higher starch hydrolytic activity in feces were more tolerant to acarbose, and α‐amylase and α‐glucosidase of synanthropic mites are suitable targets for inhibitor‐based strategies of mite control. © 2009 Wiley Periodicals, Inc.     

The efficacy of Dryacide, an inert dust, against two species of Astigmatid mites, Glycyphagus destructor and Acarus siro, at nine temperature and moisture content combinations on stored grain

Dryacide, an inert silicaceous dust, was tested for efficacy on wheat after 14 and 28 days exposure against the mites Acarus siro and Glycyphagus destructor at doses of 1, 3 and 5 g kg–1, moisture contents (MCs) of 14.5, 15.5 and 16.5% and temperatures of 10, 17.5 and 25°C. After 28 days at 10°C, all doses were effective against A. siro with the exception of the lowest dose at the highest MC, but against G. destructor complete control only occurred at 3 g kg–1 and 14.5% MC and at 5 g kg–1 and 14.5 and 15.5% MC. After 28 days at 17.5°C, the dust was fully effective against A. siro at 3 and 5 g kg–1 but only at 14.5% MC. Glycyphagus destructor was only completely controlled after 28 days at 5 g kg–1 and 14.5% MC. After 14 days at 25°C, A. siro was completely controlled at 3 and 5 g kg1 and 14.5% MC as was G. destructor after 28 days. Neither species appeared to ingest the dust but considerable quantities adhered to their cuticles. The high mortalities observed under the range of experimental conditions, particularly the lowest temperature, suggest that a dose of 3 g kg–1 may be effective as a replacement for organo-phosphorous (OP) pesticide surface treatments in an integrated storage strategy based on grain cooling. © Rapid Science Ltd. 1998     

Comparison of detection methods for Acarus siro (Acari: Acaridida: Acarididae) contamination in grain

Acarus siro L. 1758 (Acari: Acaridida: Acarididae) is an important pest of stored grain because it contaminates the grain by allergens and transfers pathogenous microorganisms. Rapid detection of contamination enables to intercept an early grain infestation by the pest. In this study, we compared the usability and efficiency of various detection approaches. Under laboratory conditions, grain samples of various sizes were infested by different levels of the following contaminants: eggs, adults, and feces of A. siro. The samples were analyzed by enzyme-linked immunosorbent assay (ELISA) by using anti-A. siro polyclonal antibody (immunochemical method), extracted in Berlese-Tullgren funnels, sieved, and processed by filth-flotation (conventional methods). The adults or juveniles of A. siro could be detected by all the three tested conventional methods and ELISA with detection limits in the range from 221 to 1,157 mites/kg grain. Eggs were detected by filth-flotation only; the detection limit was 1,950 eggs/kg grain. The feces of A. siro were detectable by ELISA test, only. ELISA enabled the detection of the feces with the minimal threshold level of 1.04 microg feces/g grain; it means the assay allowed to trace less than one metabolically active mite per gram of grain. The study thus demonstrated that reliable A. siro detection in grain can only be achieved by combining different detection methods. European Union and U.S. administratives dictate zero or near-zero tolerance level for mite infestation in stored products. This demand is difficult to fulfill, because every detection method is limited by its detection limit; thus, it is hard to reliably detect infestation levels lower than obtained detection limits. This methodical limitation is discussed in context with the determined detection limits of the tested methods.     

Spatial associations of insects and mites in stored wheat

The spatial association pattern of insect and mite populations in a steel bin containing stored wheat, Triticum durum Desf., in central Greece, was studied using the Spatial Analysis by Distance IndicEs (SADIE). The monitoring was carried out for 7 mo by using grain trier samples and probe traps. The most abundant insect species were Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) and Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). For mites, the most abundant species were the phytophagous Lepidoglyphus destructor (Schrank) (Acari: Glycyphagidae) and the predator Blattisocius tarsalis (Berlese) (Mesostigmata: Ascidae). Both for P. interpunctella and C. ferrugineus, trap catches were associated with numbers of individuals in the trier samples, but the overall association index calculated among trap and sample counts was significant only in the 33% of trap-sample pairs of values. Generally, P. interpunctella had the main patch areas in the central part of the bin, with few exceptions, during the entire monitoring period. Similar trends also were noted in the case of C. ferrugineus, which was clearly aggregated in the center of the grain mass. Spatial association maps indicated a stable positive association in the central part of the bin, but in most of the other sampling zones the association was negative. However, distribution of L. destructor, based on trier samples, indicated increased presence in peripheral zones of the grain sampling area. Moreover, B. tarsalis presented the most dispersed distribution among all four species. For each species, the association between two consecutive samplings was significant in the majority of cases, indicating a stable spatial pattern. Finally, B. tarsalis was spatially associated to a higher degree with the insects found rather than with L. desctructor. Moreover, there was no association of insect and mite presence with grain temperature and moisture content. The results of the current study suggest that the coexistence of insects and mites in bulked grain follows a complex pattern, with significant interactions, especially in the case of mite predators, which are spatially associated with insect species.     

Proteolytic activities in body and faecal extracts of the storage mite, Acarus farris

Trypsin, chymotrypsin, cathepsins B and D, aminopeptidase and carboxypeptidases A and B were detected in body extracts of the storage mite Acarus farris (Oudemans) (Astigmata: Acaridae). Faeces-enriched medium exhibited higher (10-50-fold) specific protease activity rates than those measured with mite body extracts for trypsin, chymotrypsin and carboxypeptidases A and B, suggesting that they are involved in mite digestion. However, the activity of cathepsin B was only three-fold higher in faecal than in body extracts, indicating that its presence in the lumen of the digestive tract is low compared to that of serine proteases. The activity of aminopeptidases was higher in mite bodies, indicating that they might be membrane bound. Cathepsin D activity was only detected in body extracts, indicating that this enzyme is not a digestive protease in this species. Zymograms resolved three major bands of gelatinolytic activity, but at least one protease form was only present in body extracts. Protease inhibitors of different specificity were tested in vivo to establish their potential as control agents. The development of A. farris was significantly retarded when the immature stages were fed on artificial diet containing inhibitors of serine and cysteine proteases and aminopeptidases, whereas no such effect was found with inhibitors of aspartyl proteases and carboxypeptidases. Interestingly, the most significant effects on A. farris occurred when a combination of inhibitors targeting different enzyme classes was supplied mixed in the diet, suggesting a synergistic toxicity. Several plant lectins were also tested, but only wheat germ agglutinin and concanavalin-A affected development.