Abstract

Background: Stored food products are highly susceptible to fungal contamination, posing significant risks to food safety and public health. Insects that infest stored food act as carriers of mycotoxigenic fungi, further exacerbating contamination. Knowledge Gap: While fungal contamination in stored food is well-documented, the role of storage insects in fungal transmission and aflatoxin production remains underexplored. Aims: This study aimed to isolate and identify fungi associated with storage insects and assess their potential for aflatoxin production. Methods: A total of 750 fungal isolates were obtained from four insect species collected from stored food across Basra Governorate. Fungal identification was conducted based on morphological and chemical characteristics, and aflatoxin production was analyzed using ELISA. Results: The dominant fungal genera included Aspergillus, Cladosporium, Penicillium, Rhizopus, and Yeast spp., with Aspergillus niger being the most prevalent (44.66%). All tested Aspergillus species, Cladosporium sp., and Penicillium sp. exhibited aflatoxin production, with the highest concentration (0.075 ppb) observed in Penicillium sp. Novelty: This study highlights the significant role of storage insects in fungal transmission and aflatoxin contamination, emphasizing A. niger's adaptation to storage conditions. Implications: These findings underscore the need for improved storage management and mycotoxin control strategies to mitigate health risks associated with contaminated food.

Highlights:

1. Storage insects contribute to fungal contamination and aflatoxin production.

2. Aspergillus niger dominates; multiple fungi produce aflatoxins in stored food.

3. Strengthen storage management to reduce mycotoxin-related health risks.

Keywords: Aspergillus, Penicillium, Cladosporium Storage insects, Aflatoxin, ELISA.

Introduction

Stored grains are one of the most important sources of food for humans. Wheat covers 21% of human energy needs and 20% of protein needs. due to its high nutritional value [1]. Dates also have high nutritional and economic value. They are also susceptible to many insect pests after harvest and during transportation and storage. It has been recorded that they are infected with many insects belonging to the order Coleoptera, which includes the largest number of pests that attack foodstuffs. They cause direct and indirect damage that causes them to lose their nutritional value [2]. Storage insects thats cause significant contamination of foodstuffs, according to their waste and molting skins cause contamination, and so They affect the quality of the food, and leading to lose its natural properties. They emit unpleasant odors and therefore the food became unsuitable for human consumption, putting them at risk to human health [3].The activity of insects contaminating stored materials, represented by movement and feeding, provides suitable and enhanced conditions for the growth of fungi that may be harmful. and their dangerous lies in the production of mycotoxins [4]. Fungi are among the most widespread eukaryotic organisms on the surface of the Earth. They include about 144,000 species and are characterized by their vegetative growth. They grow in the form of, hyphae of threads that grow the food material, and digest the organic materials present in the food and absorb it without swallowing [5]. Many fungi can produce mycotoxins, as [6] mentioned. The three fungal genera Aspergillius, Penicillium and Fusarnium are responsible for the production of more than two-thirds of the number of known mycotoxins, which vary in their physical and chemical properties and in their ability to cause harmful health effects on humans and animals. The consumption of nuts, including pistachios, has increased recently due to their health benefits and being an excellent food source. However, they have been associated with chemical risks. Contamination with some fungi has led to contamination with mycotoxins, which are secondary metabolites of fungi. The most important of these toxins are aflatoxins, which are the most common in pistachios and the most toxic to humans, because they have toxic effects on the liver. Due to the effects caused by these toxins at low concentrations, there must be highly sensitive and rapid techniques to identify them. Immunological testing methods are useful because of their simplicity and rapid detection [7].Many researchers have been interested in studying aflatoxins. Due to their carcinogenic potential to humans and animals. Most cases of acute and chronic poisoning with these toxins are due to contamination of food with A. flavus fungi in the field or during harvest and storage, especially when stored under conditions of humidity and temperature suitable for the growth of these fungi [8]. Aflatoxin B1 is the most dangerous toxin among the types of aflatoxin, it is carcinogenic and causes liver damage to humans and animals. According to the recommendations of international organizations, the permissible limit for the presence of aflatoxin in food should not exceed 10 micrograms/kg, therefore, protecting grains, stored foods and from aflatoxin contamination has become important [9]. The effect of these toxins is not only on humans, but it was found that when fish are fed on food contaminated with aflatoxin B1, it causes liver enlargement, tumor-like swelling, necrosis, bleeding, heart enlargement and abdominal swelling [10]. So mycotoxins represent a threat to food safety all over the world. They are among the most widespread and dangerous toxins, as they can affect any part of the food chain [11]. The current study aimed to identify fungi associated with stored insects and to confirm their ability to produce mycotoxins and the presence of these toxins in stored foodstuffs and insects, which are the main carriers of these fungi.

Methods

Insect collection: Insects were collected from flour, rice, beans, sugar, sesame, lentils and other infested stored food items. The collection was from local markets and homes in the districts of Basra Governorate during the period from November 2023 to October 2024. Samples were brought to the laboratory. Insects were collected using a brush. They were placed in small bottles.After that the insects were identified and classifified according to their morphological characteristics[12].These bottles were placed in the refrigerator at a temperature 15°C for 30 minutes [13]. After superficially sterilizing them by immersing them in a plate containing 5% ethanol alcohol, every 5 insects were placed in a Petri dish containing one sterile and moist filter paper. The plates were incubated in the incubator at a 25°C for 7-14 days. Then, the insects were transferred to Petri dishes containing potato dextrose agar (PDA) medium with the addition of 250 mg/L [14].

Fungal isolation:

a. Fungi were isolated from the plates on which the insects were placed and transferred to different culture media. These were potato dextrose agar (PDA), Malt Extract agar (MEA), and Zapecks agar (CZA),with the antibiotic chloramphenicol added to prevent bacterial growth.

b. The plates were incubated at 25°C for 7 days, then examined and purified to obtain pure culture of fungal isolates .

c. The pure isolates were examined, diagnosed and classified depending on the following references (15,16) and based on the morphological and chemical characteristics of the colonies.

Calculating the percentage of fungal occurrence:The percentage of fungal isolates occurrence (Occurrence%) as calculated according to the following equation:

View rate = Number of appearances of one type/ Total number of samples X 100%

Aflatoxin Detection

I-Sample Preparation :Samples were prepared according to the manufacturer's instructions (Elabscience) using the Enzyme-Linked Immunosorbent Assay (ELISA) Kit.

A - Dry materials, including sesamum , Anacardium and occidentale, were prepared by the following steps:

1. The samples were ground very well by the grinder and then sieved to obtain a homogeneous material.

2. 2 g were taken from each sample and placed in a centrifuge tube. Then, 8 ml of n-hexane and 10 ml of 70% ethanol were added and shaken for 5 minutes.

3. The tubes were placed in the centrifuge at 4000 rpm for 10 minutes at room temperature.

4. 0.5 ml of the supertant solution was taken to another centrifuge tube, and 0.5 ml of distilled water was added to it.

5. 50 µl were taken from the above s0lution for analysis.

B- Insects and dry materials are represented by flour, rice, and wheat. They were prepared as in the previous steps, without adding n-hexane but only adding 5 ml of ethanol alcohol at a concentration of 70%.

C- Fungal extract: Random isolates were selected from each fungal species and planted on a liquid medium of Potato Dextrose Broth (PDB). Fungal extracts were prepared by taking 2 ml of each isolate and placing it in a centrifuge tube, and adding 5 ml of ethanol alcohol at a concentration of 70%. Shake for five minutes and, place in a centrifuge, and follow the same steps as method (A).

II –The detection of aflatoxin in the samples:The presence of aflatoxin in the samples was studied using the enzyme-linked immunosorbent assay (ELISA).

First, the reagents and samples must be at room temperature before use, and all reagents must be mixed well by stirring gently before withdrawal while avoiding the occurrence of foam and then follow the following steps:

1. ELISA Microliter: Standard toxins are placed in it at a rate of two replicates for each standard toxin.

2. Adding the sample: Add 2 µl of the sample to each hole of the plate, 50 µl of Horseradish Peroxidase (HRP) enzyme to each hole, and then 50 µl of Antibody working solution. The plate is covered with its cover to prevent contamination. Shake gently for 10 seconds until its contents are completely homogeneous. Then, incubate for 30 minutes at a of 25 °C.

3. Washing: Gently remove the cover of the plate and then gently turn the plate upside down on paper towels. Then, add 260 µl of washing solution (Solution 9) to each hole and wash it. The washing process is repeated four times with a time interval of 30 seconds/time, and the plate is also turned over on paper towels to remove the washing water.

4. Color change: Add 50 µl of Substrate Reagent A and B to the holes. Shake gently for 10 seconds to mix the solution well. Incubate in the dark for 15 minutes at a temperature of 25 °C.

5. Stop the reaction: Add 50 µl of Stop Solution to each hole and shake gently for 10 seconds until the solutions are well mixed.

6. Use the ELISA reader to measure the optical density of each hole at a wavelength of 450 nm.

Results and Discussion

Table (1) shows the types of fungi associated with insects that infect grains and stored food. This includes four species belonging to the genus Aspergillus that appeared during the months of the study. This genus can grow in low water contents and wide temperature ranges. This was agreed with the results by [17].

Figure 1. A . niger , B : A,flavus , C : A.terreus , D : A.fumigatus , E : Penicillium spp. F : Cladosporium spp.

50 fungal isolates were obtained from the stored insects under study. The results in Tabal(1) showed that A.niger was the most prevalent, accounting for 44.66% of the fungal isolates, compared to the rest of the fungal isolates during the study months. The results also showed that all insects were contaminated with many fungal isolates. These fungi are dangerous due to their ability to secrete a wide range of mycotoxins. The most prevalent fungi were Aspergillus spp. followed by Penicillium spp. Figer(1) shoes the most important fungal isolates during the current study. This is consistent with many studies that have focused on fungal contamination of stored crop grains. The studies also diagnosed the emergence of various fungi, including Aspergillus flavus, A.niger, Cladosporium spp., Penicillium spp., and Rhizopus spp. [18]. The results also showed that the dominance was for the species of the genus Aspergillus.

The explanation for its dominance is its ability to use a wide range of organic materials as well as adapt to a wide range of environmental conditions that enable it to exist in different environments. It can form large numbers of conidia that are highly tolerant to stressful environmental conditions. These conidia are characterized by their small size and lightness. Thus, they spread easily in the air [19]. The fungal isolates also showed their ability to secrete aflatoxin. This is consistent with what was proven by [20]. It was proven that Aspergillus can produce aflatoxin. This makes the grains contaminated with it unfit for consumption and causes them to lose their ability to germinate. The genus Penicillum also had a large presence in the study samples. This fungus is of great importance in terms of its presence and spread in stored grains and foodstuffs. This genus also can secrete mycotoxins. All isolates of Penicillum can produce aflatoxin, and this is consistent with [21,22]. The results also showed the presence of other fungi, including Cladosprium. Although these fungi were present in small proportions, they are considered one of the main fungi that cause seed rot and deterioration in their quality. Consequently, the production of mycotoxins is widespread and has a harmful effect on humans and animals [23]. The results of the enzyme-linked immunosorbent assay (ELISA) showed the ability of all fungal isolates isolated from stored grain insects to produce aflatoxin at different levels of productivity, ranging from high to low productivity. The ten isolates of A. fumigatus grown on PDB liquid medium showed the ability to produce aflatoxin at varying rates, as shown in Table (2). The Penicillium genus was the highest with a rate of 0.075ppb., while the lowest rate was in A.niger with a rate of 0.041ppb.as showen in Figure (2). The variation of isolates in the production of mycotoxins is attributed to the genetic ability and the preferential conditions suitable for each isolate to produce a specific toxin, mainly temperature and humidity during storage [24].

Figure 2. T he average con centration of Aflatoxin in fungal isolates grown on a PDB medium.

Foodstuffs infected with insects, such as flour, nuts, wheat, rice, sesame and sugar, were taken and ground. Their ability to contain aflatoxins was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that they contained aflatoxins in varying proportions. The highest percentage was flour, at 0.075ppb. Then came nuts, wheat and rice, at 0.061, 0.01 and 0.006 ppb., respectively. While sesame and sugar were free of aflatoxins, Figure (3). The Food and Agriculture Organization (FOA) explained that various foods, such as grains, spices and nuts, can be contaminated with various mycotoxins. They are exposed to many microscopic organisms, such as fungi and bacteria, which cause significant damage to them [5]. [25] also found five main types of mycotoxins present in wheat. These types include aflatoxins, which are mostly produced during storage[26].found that the most common mycotoxin in spices, nuts, stored grains, and feed is aflatoxin.[27], also demonstrated the ability of fungi isolated from yellow corn to produce aflatoxin B1. and used the ELISA test to estimate the natural production of aflatoxin in yellow corn grains.

Figure 3.shows the concentration rate of Aflatoxin in stored food materials.

As for insects and the presence of toxins inside them, the results focus on examining the red rusty flour beetle Tribolium castaneum isolated from stored infected food items, represented by flour, nuts, wheat and rice. The food items were placed in bottles, refrigerated, and then ground. The possibility of containing aflatoxin was tested by enzyme-linked immunosorbent assay. The results proved that this insect isolated from the selected food items in the study contains aflatoxin in varying concentration. The highest proportion was in the insect isolated from nuts, at concentration of 0.08, while the proportions in the insect isolated from wheat, rice and flour were 0.071, 0.070 and 0.045ppb., respectively, Figure (4).

Figure 4.shows the concentration of Aflatoxin in the rusty red flour bug T. castaneum isolated from the studied food materials.

Conclusion

The study confirmed that there is a diversity of fungi associated with storage insects and the dominance of A.niger. This indicates its high adaptation to storage conditions. The results also confirmed the ability of most fungal isolates to produce aflatoxins in varying concentration. The study also indicated that insects play a major role in transmitting fungi and contaminating stored materials with them and the toxins they produce. Toxins were found in different concentrations inside the bodies of storage insects.

References

1. S.Congwei, D. Zhongdong, Z.Lei , R.Yan , Z.Ning and C.Feng," The Wheat 660K SNP array demonstrates great potential for marker-assisted selection in polyploid wheat" Plant Biotechnology Journal 18, 1354-1360.2020.
2. H.N. Al-Dosari," Insect pests on date palm fruits after harvest and during storage and methods of controlling them", Basra Journal of Palm Research, 22(2).2023.
3. M.Afifi," Insects and humans," Arab Press Agency, Egypt.2020.
4. G.Holighaus,and M.Rohlfs,"Fungal allelochemicals in insect pest management. Applied Microbiology and Biotechnology, 100(13), 5681–5689.2016.
5. U.Acaroz," Determination of the total aflatoxin in red pepper marketed in Afyonkarahisar", Turkey. Fresenius Environmental Bulletin. 28: 3276-3280.2019.
6. A.W.Al-Tahli,"Storage fungi are a health and economic problem". College of Agriculture. Journal of Agriculture, (51): 53-55.2016.
7. A.K.Pandey , M.K.Samota , A.Kumar , A.S.Silva, and N.K.Dubey, "Fungal mycotoxins in food commodities" : Present status and future concerns . 3 Biotech, 12(2),50.2022.
8. S.Kitigwa, "Occurrence of aflatoxins and associated risk factors in dairy value chain in selected districts of three agro-ecological zones in Tanzania" M.S.C. Thesis, Nelson Mandel African Institute of Science and Technology.3023.
9. FAO/WHO Expert Committee on Food Additives. Meeting, and World Health Organization, "Safety evaluation of certain mycotoxins in food" (Vol. 56). Food & Agriculture Org.2001.
10. E.W,Mwihia, P.G.Mbuthia, G.S. Eriksen, J.K.Gathumbi, J.G.Maina, S.Mutoloki, and J.L "Occurrence and levels of aflatoxins in fish feefs and their potential effects on fish in Nyeri, Kenya.Toxins,10(12):543.2018.
11. P.Li, R,Su, R.Yin, D.Lai, M.Wang, Y.Liu and L.Zhou, "Detoxification of Mycotoxins through Biotransformation". Toxins, 12(2), 12p.2020.
12. R.M.Mandali, "Stored grain pests and their identification National Institute of Plant" , (May).2020.
13. P.Fields, S.Bhadriraju, and R.Hulasare,"Extreme tem-Peratures,In: Stored Product Protection" ,eds. By D.W.Philip and G.Cuperus,pp. 179-190. K-State Research and Extension, Manhattan, USA.2012.
14. S.M.Nash, and W.C.Snyder, "Quantitative estimations by plant counts of propagules of the bean root Fusarium in field soils". Phytopathology 52:567-572.1962.
15. B.W.Nyongesa, S.Okoth, and V.Ayugi, "Identification key for Aspergillus species isolated from maize and soil of Nandi County, Kenya". Advances in Microbiology, 5(04), 205-229.2015.
16. G,S,Dehoogde and J.Guarro, "Atlas of clinical fungi Centeralbureauroor shimmel cultureUniversital Rovirai virgill",.Espan.720pp.1995.
17. S.H.Muhammad, "Study of contamination of some grains and their products with fungi producing mycotoxins", PhD thesis, College of Agriculture, University of Basra.142p.2003.
18. E.Marijani, H.Charo-Karisa, G.J.B.Gnonlonfin, E.Kigadye, and S.Okoth, "Effects of aflatoxin B1 on reproductive performance of farmed Nile tilapia". International Journal of Veterinary Science and Medicine, 7(1), 35-42.2019.
19. C.Lass-Flörl, A.M.Dietl, D.P.Kontoyiannis, and M.Brock, "Aspergillus terreus species complex". Clinical Microbiology Reviews, 34(4), e00311-20.2021.
20. G.A.Santos, I.A.D.S.Rodrigues, V. Starkl, K.Naehrer, U.Hofstetter, and P.Encarnação, "Mycotoxins in aquaculture: Occurrence in feeds ingredients and effects on animal performance".Aquaculture Europe , 35,6-10.2010.
21. L.Chen, W.Guo , Y.Zheng, J.Zhou, T,Liu, W,Chen, and J.Zhang, "Occurrence and characterization of fungi and mycotoxine in contaminated medicinal herbs" ,toxine, 12(1):30.2020.
22. S.Mutoloki, and J.L.Lyche, "Occurrence and levels of aflatoxins in fish feeds and their potential effects on fish in Nyeri, Kenya". Toxins, 10(12),543.2018.
23. G.N.Agrios," Plant pathology(5thed)." Elsevier. Academic press.2005.
24. M.Twaruzek, R.Kosicki, J. Kwiatkowska-Giżyńska, J.Grajewski, and I.Ałtyn, "Ochratoxin A and citrinin in green coffee and dietary supplements with green coffee extract. Toxicon": official journal of the International Society on Toxinology, 188, 172–177.2020.
25. D.Khodaei, F.Javanmardi and A,M.Khaneghah, "The global overview of the occurrence of mycotoxins in cereals: A three-year survey". Current Opinion in Food Science, 39, 36-42.2021.
26. D.Pickova, V.Ostry, J.Toman, and F.Malir, "Aflatoxins: History, significant milestones, recent data on their toxicity and ways to mitigation. Toxins", 13(6), 399.2021.
27. S.D,Al-Fahdawi,"Evaluation of the efficiency of some plant extracts in controlling the fungus Aspergillus flavus and reducing the production of aflatoxin B1 in yellow corn grains", M.S.C. thesis, College of Agriculture, University of Anbar.(105)p.2022.