DOI: https://doi.org/10.21203/rs.3.rs-1908868/v1
Insects, leading the richest group in terms of species diversity in the world of living organisms, display the largest diversity in terms of feeding, habitat and adaptation to different living environments. In addition to studies related to the use of insects in the search for alternative foods in the world in general, the focus has been on toxicologic and pathogenic research about insects. The habitats of aquatic beetles are basically the water sources used for both agricultural and drinking water, so aquatic beetles gain further importance. Aquatic insects were collected from different localities in Erzurum and surroundings. After species identification of the collected samples, digestive tracts were dissected under aseptic conditions and microbiological analyses were performed. Isolates were identified by analyzing molecular and conventional data together. In addition to conventional analyses, 16S rDNA gene sequences were amplified with PCR reaction and DNA sequencing analyses were performed. After BLAST analysis of the obtained DNA sequences, GenBank accession numbers were obtained. In this study, the aim was to identify definite and potential human pathogens in bacteria isolated from the intestinal microbiota cultures, characterized with conventional and molecular methods, of insects among the aquatic beetles.
Insects are within the Arthropoda phylum in the Insecta (Hexapoda) class and comprise the richest group of living organisms on the earth’s surface in terms of biological diversity and animal biomass with an estimated 5.5 million different species. The Coleoptera order, called beetles or hard wing case insects, is the insect group represented by most species and 40% of insects are included within this order (Engel and Maron et al. 2013; Bektaş et al.2015; Bertola et al. 2021).
At the moment on a global scale nearly 2000 insect species are consumed and insects from the Coleoptera order are included most (31%) within this group. Additionally, it is the insect group with highest fat content (33.4%) (Amiri et al. 2017; Ramos-Elorduy et al. 2009; Bertola et al. 2021).
In parallel to the rapid increase in global population, currently edible insects appear to comprise a market in the world for increasing nutrient needs and protein supplementation. However, as they involve a range of chemical and biological hazards like microtoxins, pathogenic microorganisms, pesticide residues and heavy metals, eating all insects is not safe in terms of human health. The microbiological safety for humans of this microbiota, comprising 1-10% of insect biomass, has not gained full clarity and there are few studies about this topic (Douglas et al. 2015; Amiri et al. 2017).
Insects are a candidate as a source of protein in the future for human nutrition. However, insect microbiota is a potential hazard as a biological vector for pathogenic microorganisms and for this reason is a topic of intensified research. In this study, the bacterial intestinal microbiota of species from the Helophoridae and Hydrophilidae families in the Coleoptera order were identified. The reason for choosing the Helophoridae and Hydrophiliae families is the lack of adequate research about these families.
Collection and Species Identification of insects
Aquatic beetles were collected in samples from lakes, springs, streams, ponds and water cells in regions close to the research area taken regularly every month from May-September 2020. The collected insect species and locality information are given in Tab. 1. After catching live insects, the locality information was noted and they were brought to Atatürk University, Faculty of Agriculture Entomology Laboratory for species identification under aseptic conditions. The general map of the research region and detailed information about the areas of the research are presented in Figure 1.
Dissected of insect digestive structre
For each trial, one insect was used from among adult and healthy insects collected from aquatic environments. Insect samples were first immobilized by being placed in closed boxes containing cotton soaked in ethyl acetate and then joints like the elytra and wings were removed and the outer surface of the insects was treated with 70% ethanol for 5 minutes to remove possible contaminant microorganisms. Then, alcohol was washed off by shaking in sterile distilled water and the digestive tract of the insects was dissected under a binocular microscope in an environment with aseptic conditions in the laboratory (Figure 2). The digestive structures had cultures taken for microbiological identification on the same day.
Inoculation of Samples and Microbiological Characterization with conventional methods
The insect digestive tract was pulverized with a sterile glass rod in 0.85% sterile NaCl and homogenized and serial dilutions were prepared. From the prepared dilutions (10-6) smear plate cultivated was performed according to aseptic rules on tryptic soy agar (TSA) medium. Samples with inoculation performed were incubated at 30 °C in both aerobic environments. Isolates with different features in terms of color, texture and colony morphology were placed on new media to obtain pure cultures and stored at -70 °C in Luria-Bertani (LB) containing 16% glycerol.
Characterization with API 20 E multi-test system
With the aim of identifying the metabolic enzyme profiles of bacteria, analytical profile index (API) 20 E test strips suitable for enteric bacteria were used. The selected 25 Gram (-) isolates were analyzed according to the kit protocol (Anonymous a). Twenty different biochemical properties were tested for the analyzed bacteria.
Molecular analysis and DNA isolation
For identification of bacterial isolates, the region synthesizing 16S rRNA was selected and proliferated in an in vitro environment. The master mix and PCR program are given in Tab. 2. Analyses of the rDNA sequences proliferated with PCR were obtained and after general assessment, they were compared with virtual library data for BLAST analysis (Anonymous b.) Isolates were assessed using molecular and conventional data together and names and GenBank registration numbers were obtained. The image of amplicons and single band are given in Figure 3.
In this study, inoculations were performed for the intestinal content of 17 insects collected and identified from the relevant regions. As a result of isolations from all samples, a total of 140 bacterial isolates were purified. As a result of simple characterization procedures for these, 66 isolates assessed as different were chosen and identified. The results of the study identified 32 different isolates including pathogens and potential pathogens from 18 different families. Of these, 29 microorganisms were different species, and 3 were microorganisms from different species that could not be identified at species level. Test results are shown API 20 E analysis results in Tab. 3, the molecular diagnosis results for the isolates are given in Tab. 4, while the distribution of isolated bacteria according to density and family are given in Figure 4.
Increasing protein needs, in parallel with the increase in the world’s population, has led people toward the innovative protein source of insects. Insects are one of the most promising alternative sources that can meet global protein requirements (Jantzen da Silva Lucas et al. 2020), because insects were proven in studies to be better protein and fat sources compared to other nutrient sources.
Consumption of insects, defined as entomophagy, is chosen because most insects are rich in high-quality proteins, good lipids, vitamins, minerals (like calcium, iron and zinc), fiber and chitin. Compared with traditional farm animals, farmed insects proliferate more rapidly, are more effective in converting feed into protein, require less space for reproduction and produce less greenhouse gas and ammonium emissions. However, in addition to all these positive features, entomophagy involves a range of chemical and biological hazards. Some microbial agents in the insect microbiota may be potential disease vectors in humans through consumption of insects. For this reason, it is important to research the microbiota of insects, especially those offered for human consumption (Garofalo et al. 2017; Van der Spiegel et al.2013; Makkar et al. 2014).
The intestinal microbiota of insects may contain water-derived pathogens and have the potential to be distributary agents for these pathogens. Migrating insects, especially, may act as a mechanism for the derivation and spread of diseases carried in water (Wooldridge and Wooldridge et al. 1972; Evariste et al. 2019).
Bacteria are common microorganisms in insect microbiota. The insect-bacteria interaction may be symbiotic and pathogenic. Most symbiont bacteria in the insect intestine comprise environmental samples. Literature information obtained from studies about insect intestinal assemblages reported that the Pseudomonas and Bacillus taxa are dominant, dependent to a large extent on diet (Broderick et al. 2004, Robinson et al. 2010)In this study, environmental isolates like Acinetobacter, Aeromonas, Pseudomonas, and Bacillus were densely observed. Considering the microenvironmental conditions of the insect intestine, it is a predicted outcome that environmental isolates with water and soil origin are frequently observed.
Bacteria in the Enterobacteriaceae family are known as pathogens/opportunistic pathogens and are a frequent parameter for assessment of enteric contamination in food (Barco et al. 2014; Stoops et al. 2016) The presence of these bacteria in insects indicates that the intestines cannot be cleaned or pollution of the aquatic habitat. E. coli, included in the enteric bacteria group and an element in human intestinal flora, was isolated from 2 different insect species (Laccobius syriacus Guillebeau, 1896, Hydrophilus piceus Linnaeus 1758) in this study. This bacteria, in a compatible relationship with the host organism, may cause disease in situations with emplacement in organs outside the intestines or in the intestines of another host.
Enterococcus species bacteria include species adapting at high rates to different habitats like humans, animals, insects, plants, soil, water and fermented foods. Bacteria from this family are important as they have shown antibiotic resistance in nosocomial infections since the 1970s to the present day. The inclusion of insects as a reservoir for antibiotic resistant enterococci is worrying. In our study, the E. rivorum species from this family was identified (Lebreton et al. 2014).
Two Vibrio cholerae were identified with both API 20E and molecular methods isolated from the Laccobius syriacus Guillebeau, 1896 and Helophorus brevipalpis Bedel, 1881 insect species. Vibrio are found in abundant amounts in sea water and river mouth environments. They may cause sporadic gastroenteritis and severe disease by proliferating on phytoplankton and zooplankton surfaces (Traore et al. 2014; Cabral et al. 2010). Consumption of insects from water containing sewage waste, especially, is a risk factor in terms of V. cholerae.
The Pseudomonadaceae family is commonly found in soil or water and has a role as pathogen for insects and opportunistic pathogen for humans. Additionally, it has clinical importance due to frequently developing resistance against antibiotics. It was identified in edible fresh locust and mealworm larvae samples marketed in Belgium (Stellato et al. 2015; Stoops et al. 2016). In the study, 4 Pseudomonas isolates were identified, with 1 identified at species level and 3 identified as P. putida. P. putida has both pathogenic and biotechnological importance. It is an opportunistic pathogenic microorganism in patients with suppressed immunity and was seen to colonize these individuals (Molina et al. 2016; Fernandez et al. 2015).
Aeromonas, a natural element in aquatic environments, comprised the most commonly isolated bacteria group in the study (18%). Within this group, A. hydrophila, A. caviae and A. veronii biotype sobria are defined as human pathogens. They may cause gastrointestinal and extraintestinal infectious diseases in humans. In this study, the 4 isolated A. veronii biotype sobria were frequently reported in clinical isolates and are reported to be pathogenic bacteria for traveler’s diarrhea (Al-Fatlawy and Al-Hadrawy et al. 2014). Consumption of raw shellfish was identified to be the main cause of transmission of this bacteria to humans. All species of Aeromonas, containing both mobile and immobile species, identified in this study were mobile species mostly found in freshwater, while immobile species were not encountered. A. hydrophila is a potential agent in gastroenteritis, septicemia, meningitis and wound infections, and was accepted as an opportunistic pathogen in recent years. A. hydrophila was reported to be the main cause of an epidemic occurring in people consuming oysters in Louisiana. Additionally, it was identified as a pathogenic bacterial species in fish and crab farms in China (De Silva et al.2021; Cabral JP et al. 2010) The A. rivipollensis and A. allosaccharophila species isolated in the study do not have pathogenic characteristics.
Acinetobacter was the bacteria identified most frequently after Aeromonas in this study (12%) due to being an isolate with environmental source. The clinically significant species are A. baumannii, A. nosocomialis, A. pittii and A. calcoaceticus and in this study, 3 A. pittii and 1 A. calcoaceticus were identified among pathogenic species. Attracting attention in recent years, especially as the most frequent cause of antibiotic resistance and nosocomial infections, Acinetobacter species are the most frequent causes of ventilator-associated pneumonia, blood circulation, urinary tract and intraabdominal infections in intensive care units (Esen et al. 2020).
Another isolated bacterium was Exiguobacterium sp. and species from this genus are rarely associated with human infections. Additionally, bacteremia and skin infection cases were documented. Additionally, as there is a tendency toward mistaken identification of the microorganism with routine commercial methods, deficient detection or reporting rates are high (Chen et al. 2017).
Tsukamurella inchonensis identified in Berosus spinosus (Steven, 1808) insect species was an environmental saprophyte firstly isolated from soil, arthropods, water and mud. It is an opportunistic pathogen in humans especially associated with medical devices and catheters (Safaei et al. 2018).
The most commonly used probiotic bacteria of Lactobacillus spp. is found in large scale habitats like stomach-intestinal systems and environmental media. In the study, Lactococcus lactis bacteria isolated from Laccobius syriacus Guillebeau, 1896 insect species is a microorganism accepted as having unknown virulence factors and being very safe. However, Rostagno et al. reported that these bacteria may be a source of a variety of infections (liver and brain abscess, cholangitis, peritonitis, osteomyelitis and deep neck infection) linked to consumption of raw milk and milk products by humans in the last twenty years (Rostagno et al. 2013).
Ignatzschineria larvae identified in Hydrobius fuscipes (Linnaeus 1758) insect species was first identified by isolation from larvae of Wohlfahrtia magnifica (spotted flesh fly) by Toth et al., in 2001. (Toth et al. 2007) Both pathogenic and antilarval effects of this species, which is closely related to larvae and causes bacteremia by reproducing in worm-infested wounds in humans, require further research.
Apibacter raozihei obtained from Helophorus aquaticus (Linnaeus, 1758) insect species is some facultative anaerobic bacteria. In this study, it reproduced on the 4th day in the aerobic environment and in a shorter duration (2 days) in the anaerobic environment. These bacteria were first isolated from honeybees in recent times and was defined as a microaerobic member of the bee intestine (Kwong et al. 2018). There is no literature information about pathogenicity.
Sphingobacterium sp. isolated from Helophorus aquaticus and Laccobius sulcatulus insect species has biotechnological importance in addition to being a bacterium that may be a rare infectious vector (Cai et al. 2019).
Accurate laboratory techniques have critical importance in identification of infectious syndromes linked to microorganisms with uncertain pathogenicity that are rarely isolated. 16S rRNA gene sequencing allows the opportunity to accurately identify potential pathogenic bacteria. Lysinibacillus, generally accepted as an environmental pollutant when isolated in clinical microbiology laboratories, was documented to have pathogenic potential in humans. Lysinibacillus sphaericus, identified in the study, was reported to cause 12 (2%) out of 469 bacteremia attacks in children with cancer during a 10-year duration in a pediatric cancer hospital in Italy (Wenzler et al. 2015).
Another bacterium obtained in the study was Kurthia gibsonii which is accepted as a zoonosis transmitted by sexual routes. It is commonly found in soil polluted by sewage, animal-sourced products and animal feces. This bacterium with the ability to survive in difficult living conditions was isolated from human feces in acute diarrhea, and is accepted as pathogenic in most cases due to causing gastroenteric diseases. However, the pathogenicity of this bacteria has not yet been confirmed with clinical evidence and the virulence factors are unknown (Pawar et al.2012; Cucini et al. 2020; Kövesdi et al. 2016).
In the study, 3 bacteria from Microbacterium species were identified. Microbacteria are found in soil, waste water, hospital pools and humidifiers and it is mainly known as an environmental isolate. In recent times, the frequency of reports as a potential pathogenic agent in humans, especially patients with weak immune systems, has increased (Woo et al. 2010).
Another group found densely in insect intestines and gaining importance due to spores and being pathogenic is Bacillus. In this study, 9 Bacillus species were isolated, with B. cereus isolated in 2 different insect species. The important human pathogen of B. cereus is a common cause of food-sourced gastroenteritis. Firstly, accepted as a harmless pollutant, this bacterium is known to be the etiologic agent for a variety of intestinal and extraintestinal diseases since the 1960s. (Tuipulotu et al.2020).
In spite of our study being an in vitro study characterizing cultured bacteria, bacterial density is notable. However, it is possible to identify more pathogenic microorganisms with metagenomic analyses, identifying the whole intestinal microbiome (Bektaş et al. 2021). For example, some fussy microorganisms defined with molecular methods in this study could not be isolated with traditional culture-based methods due to having specific growth requirements. For this reason, the use of methods independent of culture in research about pathogenic microorganisms in insect microbiota is very important.
The findings obtained in this study show that insect microbiota in the families studied contain many bacterial assemblages including microorganisms present in soil and water. While some of the bacteria in these microorganism assemblages, including permanent and temporary microbiota elements, protect the insect against a variety of pathogens, some may play the role of opportunistic pathogen in humans, while others have phytopathogenic properties. In the study, two bacteria (Pectobacterium cp., Bacillus mojavensis) without clear pathogenicity but accepted as phytopathogens were isolated.
Interest in insects as an alternative candidate for sustainable nutrient sources in the future will increase further in the future. Findings obtained in our study isolated both pathogenic and potentially pathogenic microorganisms and bacteria creating endospores like Bacillus sp. from insects in the Helophoridae and Hydrophilidae families. This makes it necessary to assess raw consumption of insects from these families in terms of being a risk factor for human health.
Additionally, the broad ecologic and taxonomic diversity of insects make it difficult to generalize about intestinal microbiota and there is a need for more research of this topic. The food safety of insect consumption has not been fully determined and it is thought the findings obtained in the study will provide current data about insect microbiota for the literature and contribute to future research.
Supplementary Information: This file does not contain contain supplementary material.
Author Contributions This study was carried out in collaboration between all authors has designed by all authors including the experimental process. MB: Collection of insects from the aquatic environment, species identification dissection of gut structures, FO & OB: conducted the experimental analyses. FO & MB & OB: Analysed the data wrote the manuscript. All authors read and approved the final manuscript.
Funding This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) with financial support (Project No: 119Z236) and Atatürk University Scientific Research Project FHD-2020-8649. The authors would like to thank the Scientific and Technological Research Council of Turkey for their financial support and Atatürk University Scientific Research Project Coordination Unit.
Data Availability The data sets supporting the results of this article are included within the article.
Declarations: The authors declare that they have no confict of interest.
Conflict of interest The authors have declared that no competing interests exist.
Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors. The authors state that the experiments were carried out under controlled conditions in the laboratory. All the aspects of the study were conducted in compliance with relevant Institutional biosafety and biosecurity protocols. Moreover, all authors read and approved the final manuscript.
Tables 1 to 4 are available in the Supplementary Files section