|Significance and feeding of psocids (Liposcelididae, Psocoptera) with microorganisms
Kalinovic Irma,a* Rozman Vlatka,a Liska Anitaa
aFaculty of Agriculture in Osijek, Trg Sv. Trojstva 3, 31000 Osijek, CROATIA
After our investigations of psocids in the grain storages in Croatia, the most abundant were represenatives of genus Liposcelis, as following: Liposcelis decolor (Pearman), L. corrodens (Heymons), L. entomophila (Enderlein), L. pearmani Lienhard, L. bostrichophila Badonnel, L. paeta Pearman. L. mendax Pearman, L. brunnea Motschulsky, L. pubescens Broadhead, L. tricolor Badonnel. Feeding and food utilisation of L. corrodens and L. bostrichophila with pure culture of microoganisms (fungi – Mucor spp., Apergillus niger Tiegh., A. flavus Link ex Gray, Penicillium spp., Cladosporium spp., Fusarium spp. and bacteria – Esherichia coli, Bacillus subtilis, as well cellulolitic microorganisms of genera Cytophaga, Cellvibrio) were investigated. Also feeding with microorganisms causing plant diseases in the herbarium plant material (Uromyces pisi (Pers.) de Bary, Helminthosporium turcicum Pass., Ustilago maydis (DC) Corda, U. levis (Kell.et Sw.) Magn., U. nuda (Jens.) Kell. et Sw., Tilletia tritici (Bjerk.) Winter, Pseudoperonospora humuli (Miyabe et Takah.) G. Wilson, Peronospora schleideni Ung., Cercospora beticola Sacc.), was studied. Transporting of harmful microorganisms of stored wheat by bodies, hairs and in the faecal matter of Liposcelididae was investigated too.
Keywords: Liposcelididae; microorganisms; feeding; food utilisation; harmful microrganisms, transporting.
E-mail address: kirma@.pfos.hr (Irma Kalinovic); Fax: ++385 31 207 017
Psocids do not have economic importance for stored commercial wheat and corn in Croatia. Psocids are considered to be insect pests of seed products since they usually feed on the germ part of a whole seed and even the complete embryo (Rees, 2004). Psocids appear to cause a significant problem for packaged foodstuffs. According to our previous research, psocids were found in packaged pasta, rice, flour, wheat and corn grits, as well as in different kinds of tea (Kalinović et al., 1981). The highest number of psocid species was found in stored wheat and corn (Kalinović, 1979; 1995; Kalinović et al., 2006). Psocids were found in a small number in empty storehouses in Croatia (Kalinović and Rozman, 2000; Kalinović et al. 2006), whereas in Australia psocid species Lachesilla quercus Kolbe are considered to be harmful pests in coastal grain handling facilities (Rees, 2004). Moreover, Liposcelis spp. are known to cause allergic responses in sensitized people (Rees, 2004). There are still no extensive data published on feeding activities and food selection of Liposcelididae. Sinha and Srivastava (1970) identified L. entomophila in packaged rice and moldy rice stems in the fields, which indicates that they feed on fungi and bacteria that hydrolyze cellulose. Furthermore, Srivastava and Sinha (1975) found that L. entomophila showed a preference for flour, dead insects and bark when compared with wood fungi, straw and paper. Liposcelididae that live outdoors feed on mycelium of Ascomyceta fungi, organic matter and pollen of various plants (Günther, 1974). According to the research on L. bostrichophila food selection such as buckwheat, pot barley, brown rice flour, yellow millet and wheatgerm (Green and Turner, 2005), the results show that their most preferred choice were ground buckwheat and yellow millet impregnated with glucose and fructose.
2. Materials and methods
Booklice were collected from the samples of stored grains (wheat, barley, corn, oat, soybeans, sugar beet, sunflower, oil seed rape) and packaged foodstuffs (flour, rice, wheat and corn grits, different kinds of tea, dried fruit and vegetables) in the period between 1975 and 2005. The samples weighed 100g. They were sifted with automatic sieves that ranged in diameter from 0.2 - 2.5 mm (standard method of sample analysis). Booklice obtained from the sieved material were collected with camel hair brushes and were put in test tubes with 70% alcohol (Günther, 1974, Smithers, 198l). Booklice from empty storehouses were collected with camel hair brushes at the area of l m2. Also, booklice from herbarium material of plant species that cause plant diseases during vegetation period (Uromyces pisi, Helminthosporium turcicum, Ustilago maydis, U. nuda, Tilletia tritici, Pseudoperonospora humuli, Peronospora schleideni, Cercospora beticola) were collected with camel hair brushes and were put in 70% alcohol. Laboratory reared species L. corrodens and L. bostrichophila were placed on herbarium plant material to feed for 48 hours. Liposcelididae were macerated by Günther method (1974). Specimens obtained were identified using the keys of Günther (1974), Lienhard (1990), Lienhard and Smithers (2002) and Mockford (1993).
Laboratory rearing of Liposcelis bostrichophila (Figure 10) and L. corrodens for the study of feeding activity of booklice was performed by Wyniger method (1974).
The surface microflora from grain products as well as microorganisms from the bodies of booklice were isolated in the Petri dishes on the Czapek’s agar plate. The incubation was performed in the thermostat at 25ºC for the 7-day-period (standard method).
2.1. Feeding activity on pure fungi cultures
Feeding activity of L. bostrichophila and L. corrodens was studied with pure fungi cultures (Mucor spp., Aspergilus niger, A. flavus, Penicillium spp., Cladosporium spp. and Fusarium spp.) developed in the thermostat on the Czapek’s agar plate. Laboratory reared booklice were isolated in the sterile Petri dishes for 48-72 hours in order to cleanse their digestive system, as well as to clean their bodies, heads, legs and antennae from the agar i.e. from the fungi on their hair, which was examined under the stereomicroscope. Finally, starved and cleaned booklice (5 adult Liposcelididae) were placed in the Petri dishes with the prepared pure fungi cultures substrate for their feeding which was examined under the stereomicroscope. The Petri dishes with booklice were placed in the thermostat at 28ºC with the relative humidity of 80% which was accomplished with damp sterile filter paper inside the lid of each Petri dish. Liposcelididae were left to feed on pure fungi cultures (mycelium parts) for 72 hours. After 3 days of feeding, psocids were placed in an empty sterile Petri dish to examine removal of vegetative organs of fungi (spores or conidia) from their bodies under stereomicroscope. Next, Liposcelididae (3-5 adult specimens) were placed in sterile Petri dishes in order to perform mechanical maceration with sterile microbiological and Platinum needles and were poured over with nutrient Czapek’s plate. Developed fungi in the digestive system of Liposcelididae were studied after the incubation period in the thermostat at 25ºC during 7-20 days.
2.2. Liposcelididae feeding activity on bacteria
Having determined the presence of bacteria on the bodies and in the digestive system of psocids species in stored wheat and corn, the isolates of pure cultures of the dominant bacteria Escherichia coli and Bacillus subtilis were prepared. They were grown on 2% meat peptone agar slant in order to gain higher biomass. Bacterial biomass was removed from the agar slant with Platinum needle and placed into the sterile Petri dishes together with the observed Liposcelididae species (starved for 72 hours). The period of biomass feeding of psocids on pure bacteria cultures lasted 48 hours, after which they were chemically maceratated (surface sterilization), rinsed with 10% KOH, distilled water and 45%, 70%, 85% and 96% alcohol (Günther, 1974) and smeared onto a glass slide. The smear was dried and stained with base Fuchsin solution. Evaluation of bacterial cells consumption in the digestive system of psocids was carried out after 48 and 72 hours of their starvation. The same psocids were macerated mechanically in the sterile Petri dishes where they were poured over with 2% meat peptone agar cooled at 45ºC. Examination of the bacterial development was conducted after 72 hours of incubation at 24ºC.
2.3. Liposcelididae feeding activity on aerobic cellulose degraders
Studying of Liposcelididae feeding on aerobic cellulose degraders was performed with genera Cytophaga and Cellvibrio. Starved Liposcelididae were placed on the developed colonies of cellulolytic microorganisms to feed for 48 hours in the thermostat at 28ºC. The testing procedure was the same as with bacteria but the slide was stained with Methylene Blue solution. Likewise, cellulolytic microorganisms in the digestive system were determined with the same procedure as with bacteria after 72 hours, but for the development of bacteria, after maceration of psocids, liquid Waksman-Carey plate in the test tubes with filter paper was used. The incubation lasted 30 days at 30ºC.
2.4. Testing of Liposcelididae feces
Testing of Liposcelididae feces was carried out after their feeding on fungi, bacteria and cellulolytic microorganisms. Psocids were placed into empty sterile Petri dishes to examine their fecal excretion under the stereomicroscope. They were transferred into the sterile Petri dishes and poured over with the appropriate medium for the development of certain groups of microorganisms. The plates with the feces of psocids were placed in incubation for 7-30 days in the thermostat at an appropriate temperature, depending on the time needed for the complete development of the microorganisms. Adult specimens of Liposcelididae isolated from the wheat in silos were placed on the nutrient plate in the Petri dishes for the study of microorganisms from their bodies and hair.
Relatively high incidence of order Psocoptera observed during this multiyear research was shown in Figure 1. Obviuosly, the most frequently encountered suborder was Troctomorpha, infraorder Nanopsocetae – Liposcelididae, with the prevalence of 87%, suborder Trogiomorpha, infraorder Atropetae –Trogiidae, with the prevalence of 8%, followed by the same suborder, infraorder Psocoathropetae – Psyllipsocidae, with 4%, and suborder Psocomorpha, infraorder Homilopsocidea – Lachesillidae with the 1% prevalence. Figure 2 shows incidence of Psocoptera species in the analyzed samples of grain products and foodstuffs. There were 17 Psocoptera species identified with the most dominant genus Liposcelis and its following 10 species: Liposcelis decolor (Pearman) 20%, L. corrodens (Heymons) 18%, L. entomophila (Enderlein) 16%, L. pearmani Lienhard 13%, L. bostrichophila Badonnel 8%, L. paeta Pearman 4%, L. mendax Pearman 3%, L. brunnea Motschulsky 2%, L. pubescens Broadhead 2%, and L. tricolor Badonnel 1%. The genus Lepinotus was identified with its 2 species Lepinotus reticulatus Enderlein and L. inquillinus Heyden with the prevalence of 6% and 1% respectively. The genus Psyllipsocus was identified with 2 forms of 1 species Psyllipsocus ramburi Sellys-Longchamps f. destructor, P. ramburi f. troglodytes with the prevalence of 2% and 1% respectively. The genus Dorypteryx was encountered with species Dorypteryx domestica (Smithers) and genus Trogium with its 1 species Trogium pulsatorium (Linneaus) both with the prevalence rate of 1%. Finally, the genus Lachesilla was observed with the prevalence of 1% of its species Lachesilla pedicularia (Linneaus).
The Psocoptera prevalence rate was 77.2% in the stored grain products with the most dominant genus Liposcelis with the highest infestation of stored wheat and corn (Kalinović, 1979, 1995). Likewise, the multiyear research revealed the Psocoptera incidence of 22.8% in packaged foodstuffs also with the dominant genus Liposcelis. The highest infestation was detected in packaged pasta, rice and flour and less in wheat and corn grits and tea (Kalinović et al., 1981). Inspection of empty storehouses, mostly in silos basements identified the following psocids species Lachesilla pedicularia, L. quercus, Lepinotus raticulatus and Liposcelis bostrichophila. The relative humidity and air temperatures varied from 65–68 % and 22-25ºC respectively. Psocoptera were mostly found on moldy basement walls feeding on fungi and bacteria (Kalinović and Rozman, 2000).
Liposcelididae – Liposcelis corrodens and Liposcelis bostrichophila were identified on the herbarium material of plant species that cause the following plant diseases Uromyces pisi, Helminthosporium turcicum, Ustilago maydis, U. levis, U. nuda, Tilletia tritici, Psudoperonospora humuli, Peronospora shleideni and Cercospora beticola. Chemical maceration of the digestive system of laboratory reared Liposcelididae that were feeding on the herbarium plant material revealed mycelium and spores of plant diseases proving that those plant diseases were perfect source for their feeding (Figure 3 and 4).
For the purpose of studying psocids feeding activity, first the surface microflora of the stored wheat and corn grains from the samples taken from silos was tested. Next, the isolation of the surface microflora determined that the most prevalent fungi species were that of the genera Fusarium, Alternaria, Penicillium, Cladosporium, Mucor and Rhizopus, and bacteria Escherichia coli and Bacillus subtilis, as well as aerobic cellulose degraders – cellulolytic microorganisms of the genera Cytophaga and Cellvibrio. Pure fungi cultures were prepared for the laboratory reared Psocoptera species L. corrodens and L. bostrichophila that were starved in the Petri dishes. After a rather short time, psocids found their way to fungi colonies and started feeding on them. Their behavior and feeding activity were examined under stereomicroscope. It showed that the adult specimen moved rather quickly to the very dense but widespread fungi colony of Mucor app. It bit sporangiophore with its mouthparts all the way to sporangium which, according to its maturity phase, broke open. It was greedily consuming released sporangiospores with its mouthpart, settled down to chew and subsequently swallowed its food. It remained at the bottom of the colony probably due to the less light exposure and higher influence of humidity, where it continued to feed. Chemical maceration showed the digestive system of Psocoptera (Figure 5). Observation of feeding activity of Psocoptera on Aspergilus niger and A. flavus fungi that were quite widespread, revealed that the adult specimen climbed on their conidiophores and bit them in two in their middle part where they are softer, continued biting it towards conidiophore head with conidia which it swallowed after 3-4 bites (content of the digestive system was shown in Figure 7). In fungi species with more dense mycelium (Penicillium spp., Cladosporium spp., Fusarium spp.), adult specimens fed on conidia from the upper surface of the colony and then due to its density easily climb on its top and continued to feed on conidia and sterigmata (Figure 6, 8). Feeding of Liposcelididae on pure fungi cultures of E. coli and B. subtilis did not reveal any significant difference in bacterial intake into their digestive system. Chemical maceration and microscopic examination of the permanent slide determined the existence of big vegetative bacilli and coccoidal forms of Bacilus spp. spores (Figure 9). Evaluation of bacteria consumption in the digestive system of Liposcelididae, maceration of adult specimens and pouring over with meat peptone agar after the incubation determined only the development of pure cultures of Bacillus subtilis, whereas other bacteria did not develop. This proves that the adult digestive system completely absorbs asporogenous and vegetative bacteria forms, whereas conservation forms (spores) of sporogenous bacteria are excreted.
According to existing literature, Liposcelididae live in the nature in the places rich in cellulose i.e. under the tree bark, stumps, old paper and in rice straw (Sinha and Srivastava, 1970), which proves their ability to digest cellulose during their nutrition process. In natural conditions, cellulolytic microorganisms of the genera Cytophaga and Cellvibrio play the dominant role in cellulose transformation. Liposcelididae were very active on the grown pure cellulolytic microorganisms colonies, remained on mucilaginous colonies (Cytophaga) and had a longer lifespan as a result of proper nutrition (cellulose and cellulolytic microorganisms) and humidity level. Celluloid parts (pieces of filter paper) as well as cellulolytic microorganism cells were found in the digestive system of the dissected adult specimens. We believe that the majority of cellulose is digested during nutrition under the influence of cellulase ferment - aerobic cellulolytic microorganisms (Cytophaga and Cellvibrio) and transformed into glucoses, which attracts Liposcelididae to feed intensively.
Examination of Liposcelididae feces regarding the consumption of fungi and bacteria in the digestive system showed that sporangiospore or conidia in feces of psocids that were fed on fungi redeveloped on the nutrient plate. On the other hand, feces of psocids that were fed on bacterial flora showed that their digestive system completely absorbed asporogenous and vegetative bacterial forms, and even cellulolytic microorganisms, whereas conservation forms (spores) of sporogenous bacteria were excreted. Regeneration on the nutrient plate was observed only with Bacillus subtilis from the spores that redevelop.
The research proved that Liposcelididae feed on fungi (Mucor spp., Aspergilus niger, A. flavus, Penicillium spp., Cladosporium spp. and Fusarium spp.) and bacteria (Escherichia coli and Bacillus subtilis), as well as on cellulolytic microorganisms of genera Cytophaga and Cellvibrio, without preference for any particular specimen. Examination of undigested food content in the digestive system, as well as excreted feces of Liposcelididae revealed that all fungi species redevelop by germination of sporangiospores or conidia from feces on the artificial medium. Only sporogenous specimen of B. subtilis has the ability to regenerate, whereas the development of asporogenous bacteria of genus Chromopseudomonas did not occur. In other words, this proves that only sporogenous conservation forms regenerate since they are more resistant to fermentation activity of the Liposcelididae digestive system. Cellulolytic microorganisms are completely consumed under the influence of cellulase ferment so there is no regeneration. To sum up, Liposcelididae are considered to be insect pests not only since their presence, excrements and toxins infest foodstuff but also because they transfer microflora with their bodies. Timely preventive measures are necessary to avoid their rapid increase in number and the possible heavy infestation and damage of packaged products. Incidence of Liposcelididae in certain products indicates increased humidity and the presence of microflora.
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Fig 1 Relatively number of Psocoptera by family affiliation
Fig 2 Relatively number of Psocoptera species in analyzed samples
Fig. 4 Spores and parts of conidiophore of Uromyces pisi in digestive system of Liposcelis bostrichophila
(± 600x, photo I. Kalinović)
Fig. 5 Digestive system content of L. corrodens fed by clean culture of Alternaria spp.
(±600x, photo I. Kalinović)
Fig. 6 Digestive system content of L. corrodens fed by clean culture of Cladosporium spp.
(±600x photo I. Kalinović)
Fig. 7 Digestive system content of L. bostrichophila fed by clean culture of Aspergillus niger
(±600x photo I. Kalinović)
Fig. 8 Digestive system content of L. corrodens fed by clean culture of Penicillium spp.
(±600x photo I. Kalinović )
Fig. 9 Digestive system content of L. bostrichophila fed by clean culture of bacteria biomass Bacillus spp.
(±600x photo I. Kalinović)
Fig. 10 Liposcelis bostrichophila (photo E.C.N. Leong, S.H.Ho)