1 

Dynamics and Cultivation of Gut Microbiota in 

Hermetia illucens Larvae 

 Dissertation to obtain the doctoral degree in natural sciences (Dr. rer. nat) 

 Faculty of Agricultural Sciences, Nutritional Sciences, and Environmental 

Management (Fachbereich 09)  

Justus Liebig Universität Gießen,  

Ludwigstraße 23, 35390 Gießen, Germany 

Submitted by 
M. Sc. Yina Alejandra Cifuentes Triana 



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The present work was carried out at the Institute of Applied Microbiology, Faculty of 

Agricultural Sciences, Nutritional Sciences and Environmental Management, Justus-Liebig- 

University, Gießen during the period from 2017 to 2024 under the supervision of Prof. Dr. Dr.-

Ing. Peter Kämpfer and guidance of Dr. Stefanie Glaeser.  

 

 

 

 

 

I Supervisor: Prof. Dr. Dr.-Ing. Peter Kämpfer  

Institute of Applied Microbiology  

Justus-Liebig-University 

Heinrich-Buff-Ring 26-32, 35392 Gießen, Germany  

 

 

 

 

 

II Supervisor: Prof. Dr. Andreas Vilcinskas 

Institute for Insect Biotechnology  

Justus-Liebig-University 

Heinrich-Buff-Ring 26-32, 35392 Gießen, Germany 

  



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Erklärung 

 

Hiermit erkläre ich, dass die hier vorgelegte Arbeit kein Material enthält, das zur Erlangung 

eines anderen Grades oder einer anderen Qualifikation an einer Universität oder akademischen 

Einrichtung eingereicht oder angenommen worden ist. Nach meinem besten Wissen und 

Gewissen ist diese Arbeit authentisch und enthält kein Material, das zuvor geschrieben und 

veröffentlicht wurde, es sei denn, ein entsprechender Verweis ist im Text angegeben. Alle von 

mir durchgeführten und in der Dissertation beschriebenen Arbeiten entsprechen den 

Grundsätzen guter wissenschaftlicher Praxis, wie sie in der „Satzung der Justus-Liebig-

Universität Gießen zur Sicherung guter wissenschaftlicher Praxis“ niedergelegt sind. 

 

Declaration 

Herewith, I declare that the thesis presented here contains no material, which has been 

submitted or accepted for obtaining any other degree or qualification at any university or 

academic institution. To the best of my knowledge, this thesis is genuine and contains no 

material previously written and published, unless an appropriate reference is stated in the text. 

All the work carried out by me and described in the dissertation adhered to the principles of 

good scientific practice as defined in the “Statutes of the Justus Liebig University Giessen for 

the Safeguarding of Good Scientific Practice”. 

 

 

 

 

 

 

                                                                                              ____________________________ 

Gießen, Hessen                                                                         Yina Alejandra Cifuentes Triana 

  



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“Experience is the name everyone gives to their mistakes.” 

Lady Windermere's Fan (1892) act 3, Oscar Wilde 

 

 

  



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Table of Contents 

Hypotheses ........................................................................................................... 6 

Summary ............................................................................................................. 7 

Zusammenfassung ................................................................................................ 9 

CHAPTER I ....................................................................................................... 12 

Review chapter: Hermetia illucens gut microbiota .......................................................... 12 

CHAPTER II ...................................................................................................... 38 

The gut and feed residue microbiota changing during the rearing of Hermetia illucens larvae
 ................................................................................................................................ 38 

CHAPTER III .................................................................................................... 61 

Isolation of Hermetia illucens larvae core gut microbiota by two different cultivation 
strategies ................................................................................................................... 61 

Annex  Chapter I, Supplementary material .............................................................. 79 

Annex  Chapter II, Supplementary material ............................................................. 87 

Abbreviations ..................................................................................................... 95 

List of publications .............................................................................................. 97 

Acknowledgments ................................................................................................ 98 
 

 

  



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Hypotheses  

 

Hypothesis 1: Hermetia illucens larvae possess a specific and stable core gut microbiome that 

remains consistent across different developmental stages. 

Hypothesis 2: The bacterial community composition within the feed residue of H. illucens 

larvae undergoes significant changes during the rearing process. 

Hypothesis 3: Rearing H. illucens can contribute to a reduction of potentially pathogenic 

bacteria in the feed substrate. 

Hypothesis 4: Within the stable core gut microbiome of H. illucens larvae, there exists a higher 

degree of strain-level diversity than previously recognized. 

Hypothesis 5: Diverse cultivation strategies, especially those incorporating a dilution-to-

extinction approach, enhance the genetic diversity of cultured bacteria isolated from the gut of 

H. illucens larvae. 

 

 

 

  



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Summary 

 

Understanding the intricate interplay between Hermetia illucens larvae, and their gut 

microbiome is crucial for using their potential in applications such as waste management and 

animal feed production. This study investigated the composition and dynamics of bacterial 

communities within the larval gut and the feed residue during H. illucens larvae rearing. It was 

found that H. illucens larvae gut harbors a consistent and stable core microbiome, composed 

mainly of specific bacterial genera, including Dysgonomonas, Morganella, Enterococcus, 

Providencia, Klebsiella, and others. This potential core microbiome exhibits a high degree of 

strain-level diversity, suggesting a complex ecosystem within the larval gut that may contribute 

to the adaptability of H. illucens larvae to varying diets. 

 

In contrast to the stable gut microbiome, the bacterial community in the feed residue undergoes 

significant alterations during rearing. This is likely due to the feeding and digestion processes 

of the larvae, which modify the composition of the residue, leading to a decrease in the 

abundance of certain bacterial taxa and an increase in others. Remarkably, a significant 

decrease was observed in the abundance of potential pathogens in the feed residue as rearing 

progressed. This indicates that H. illucens larvae rearing can contribute to a reduction in 

harmful bacteria within processed organic waste. 

 

To further elucidate the interplay between the larval gut microbiome and the feed residue, this 

study also assessed the presence and prevalence of antibiotic and disinfectant resistance genes 

within both environments. For instance, the alternative penicillin binding protein coding gene 

mecA, mainly found in Staphylococcus species, was highly abundant in the feed residue at the 

initial stage but decreased significantly in the last stages. This pattern mirrored the relative 

abundance of Staphylococcus in the residue, which was also abundant at the beginning and 

declined over time. In contrast, the extended-spectrum beta-lactamase gene, blaSHV, was 

consistently present in the gut microbiome of the H. illucens larvae and pupae throughout the 

rearing process. While blaSHV genes were also detected in the feed residue in the first stages, 

they were not detected at the final stage.  

The quaternary ammonium compound (QAC) resistance genes coding for transmembrane 

protein (qacE/qacEΔ1) were not detected in any of the larval gut samples but were found in 

the feed residue. Their abundance increased significantly from the initial to the final stage of 



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rearing. This finding implies that while the larvae themselves may not harbor these disinfectant 

resistance genes, the feed residue, potentially enriched with these genes, could pose a risk if 

applied as fertilizer. Despite significant shifts in bacterial community composition during H. 

illucens larvae rearing, the relative abundance of the tetracycline resistance gene (tetM) 

remained stable in both the larval gut and the feed residue. However, the relative abundance of 

the sulfonamide resistance gene (sul2) increased significantly in the feed residue, while 

remaining stable in the larval gut. The potential for the transfer of these antibiotic and 

disinfectant resistance genes from the feed residue to the environment, and possibly other 

organisms, demands more in-depth analysis. 

 

Two different cultivation strategies, a dilution-to-extinction cultivation approach, and direct 

plating approach, were used to further characterize H. illucens larvae gut microbiome. Both 

methods successfully cultured a diverse array of bacterial species. The dilution-to-extinction 

approach yielded a total of 341isolates and the direct plating approach 138 isolates. A total of 

18 different phylotypes based on 16S rRNA gene sequence analysis were identified. Six 

phylotypes were found exclusively in the dilution-to-extinction approach: Pseudomonas, 

Alcaligenes, Providencia, Serratia, Brucella, and Micrococcus. Only the Mammaliicoccus 

phylotype was unique to the direct plating method. Genomic fingerprinting further revealed a 

high degree of genetic diversity at the strain level within several phylotypes, particularly for 

Enterobacteriaceae, Providencia, Enterococcus, and Morganella. These findings highlight the 

importance of utilizing diverse cultivation techniques to gain a more comprehensive 

understanding of the complex microbial diversity present within the H. illucens larvae gut.



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Zusammenfassung 

 

Das Verständnis der komplexen Wechselwirkungen zwischen Hermetia illucens-Larven und 

ihrem Darmmikrobiom ist entscheidend für die Ausschöpfung ihres Potenzials in 

Anwendungsbereichen wie Abfallwirtschaft und Tierfutterproduktion. Diese Studie 

untersuchte die Zusammensetzung und Dynamik der bakteriellen Gemeinschaften im 

Larvendarm und im Futterrückstand während der Aufzucht von H. illucens-Larven. Es wurde 

festgestellt, dass der Darm von H. illucens-Larven ein konsistentes und stabiles 

Kerndarmmikrobiom beherbergt, das hauptsächlich aus spezifischen Bakteriengattungen wie 

Dysgonomonas, Morganella, Enterococcus, Providencia, Klebsiella und anderen besteht. Das 

Kerndarmmikrobiom zeichnet sich durch eine hohe Diversität auf Stammebene aus, was auf 

ein komplexes Ökosystem im Larvendarm hinweist. Dieses könnte einen wesentlichen Beitrag 

zur Anpassungsfähigkeit der H. illucens-Larven an verschiedene Diäten leisten. 

 

Im Gegensatz zu dem stabilen Darmmikrobiom unterliegt die bakterielle Gemeinschaft im 

Futterrückstand während der Aufzucht erheblichen Veränderungen. Diese Veränderungen sind 

vermutlich auf die Fütterungs- und Verdauungsprozesse der Larven zurückzuführen, welche 

die Zusammensetzung des Rückstands beeinflussen und zu einer Abnahme der Häufigkeit 

bestimmter Bakterienarten sowie einer Zunahme anderer führen. Bemerkenswerterweise 

wurde ein signifikanter Rückgang der Häufigkeit potenzieller Krankheitserreger im 

Futterrückstand im Laufe der Aufzucht beobachtet. Dies deutet darauf hin, dass die Aufzucht 

von H. illucens-Larven möglicherweise zur Reduzierung schädlicher Bakterien im 

verarbeiteten organischen Abfall beitragen könnte. 

 

Um die Wechselwirkungen zwischen dem Larvendarmmikrobiom und dem Futterrückstand 

weiter zu untersuchen, wurden in dieser Studie auch das Vorhandensein und die Häufigkeit 

von Antibiotika- und Desinfektionsmittelresistenzgenen in beiden Umgebungen bewertet. Zum 

Beispiel war das alternative Penicillin-Bindungsprotein-codierende Gen mecA, das 

hauptsächlich in Staphylococcus Arten nachgewiesen wird, zu Beginn der Aufzucht im 

Futterrückstand stark vertreten, nahm jedoch im Laufe der Zeit signifikant ab. Dieses Muster 

spiegelte sich in der relativen Häufigkeit von Staphylococcus im Rückstand wider, die ebenfalls 

zu Beginn hoch war und im Laufe der Zeit abnahm. Im Gegensatz dazu war das Gen für die 



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Extended-Spectrum Beta-Laktamase, blaSHV, im Darmmikrobiom der H. illucens-Larven und 

Puppen während des gesamten Aufzuchtprozesses konstant vorhanden. Während blaSHV Gene 

auch in den initialen Stadien im Futterrückstand nachgewiesen wurden, erfolgte in der 

Endphase kein Nachweis mehr. 

 

Die Resistenzgene für quaternäre Ammoniumverbindungen (QAV), die für 

Transmembranproteine (qacE/qacEΔ1) kodieren, wurden in keinem der Larvendarmproben 

nachgewiesen. Allerdings konnte ein Vorliegen dieser Gene im Futterrückstand bestätigt 

werden. Ihre Häufigkeit nahm von der Anfangs- zur Endphase der Aufzucht signifikant zu. 

Diese Erkenntnis deutet darauf hin, dass, obwohl die Larven selbst diese 

Desinfektionsmittelresistenzgene womöglich nicht beherbergen, der Futterrückstand, der 

möglicherweise mit diesen Genen angereichert ist, ein Risiko darstellen könnte, wenn er als 

Dünger verwendet wird. 

 

Trotz signifikanter Veränderungen in der Zusammensetzung der bakteriellen Gemeinschaft 

während der Aufzucht von H. illucens-Larven blieb die relative Häufigkeit des Tetracyclin-

Resistenzgens (tetM) sowohl im Larvendarm als auch im Futterrückstand stabil. Dagegen 

jedoch nahm die relative Häufigkeit des Sulfonamid-Resistenzgens (sul2) im Futterrückstand 

signifikant zu, während sie im Larvendarm stabil blieb. Das Potenzial für den Transfer dieser 

Antibiotika- und Desinfektionsmittelresistenzgene vom Futterrückstand in die Umwelt und 

möglicherweise auf andere Organismen erfordert eine eingehendere Analyse. 

 

Zur weiteren Charakterisierung des Darmmikrobioms von H. illucens-Larven wurden zwei 

verschiedene Kultivierungsstrategien angewandt: eine Kultivierung durch Verdünnung bis 

zum Aussterben und eine direkte Plattierung. Die Anwendung beider Methoden führte zur 

erfolgreichen Kultivierung einer vielfältigen Reihe von Bakterienarten. Im Rahmen des 

Verdünnungs-zu-Aussterben-Ansatzes wurden insgesamt 341 Isolate generiert, während der 

Direktplattierungsansatz 138 Isolate ergab. Insgesamt wurden 18 verschiedene Phylotypen 

basierend auf der 16S rRNA Gen Sequenzanalyse identifiziert. Sechs Phylotypen wurden 

ausschließlich im Verdünnungs-zu-Aussterben-Ansatz gefunden: Pseudomonas, Alcaligenes, 

Providencia, Serratia, Brucella und Micrococcus. Lediglich der Phylotyp Mammaliicoccus 

war einzigartig für die Direktplattierungsmethode. Die Analyse genomischer Fingerabdrücke 

zeigte weiterhin einen hohen Grad an genetischer Vielfalt auf Stammebene innerhalb mehrerer 

Phylotypen, insbesondere bei Enterobacteriaceae, Providencia, Enterococcus und 



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Morganella. Diese Ergebnisse heben die Bedeutung der Verwendung vielfältiger 

Kultivierungstechniken hervor, um ein umfassenderes Verständnis der komplexen 

mikrobiellen Vielfalt im Darm der H. illucens-Larven zu gewinnen.



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CHAPTER I 
 

Review chapter: Hermetia illucens gut microbiota 

Yina Cifuentes 

  



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Introduction 

 

 

Insects, encompassing more than 50% of all described species, exhibit remarkable adaptability 

across diverse environments, including terrestrial, freshwater, and near-coastal marine habitats, 

as well as deserts and hot springs (Redak, 2023; Schowalter, 2022). They respond quickly to 

environmental changes, affecting other species and ecosystem parameters (Schowalter, 2022). 

Their diversity is reflected in various attributes such as feeding behavior, developmental 

patterns, ecological roles, habitat preferences, social structures, mouthparts, flight capabilities, 

coloration, size, lifespan, geographic distribution, and reproductive strategies (Capinera, 2008; 

Chapman & de Boer, 1995; Schowalter, 2022). This adaptability enables them to inhabit nearly 

every ecological niche (Schowalter, 2022). 

 

A critical aspect of this adaptability lies in the insect microbiota, the community of 

microorganisms residing primarily in the gut (Girard et al., 2022; Jones et al., 2013). 

Scavengers like H. illucens, which consume a varied diet of decaying organic matter, exemplify 

this remarkable adaptability, where the gut microbiota helps digest various organic sources and 

detoxifies toxic compounds, which efficiently convert into valuable products through a process 

known as bioconversion (De Smet et al., 2018; Eke et al., 2023; Smetana et al., 2019). This 

process offers solutions for rising concerns about organic waste management and the need for 

sustainable protein sources (Bruno et al., 2019; Eke et al., 2023; Spranghers et al., 2017). The 

ability to thrive on many types of organic waste, including food scraps, agricultural byproducts, 

and manure, due to the contribution of its gut microbiota, underscores the potential for 

optimizing mass rearing and bioconversion of H. illucens for even large-scale processes 

(Chavan et al., 2022; Eke et al., 2023).  

 

Microbial communities are strategically dispersed across various anatomical structures of 

insects, encompassing the exoskeleton, gut, blood cavity, salivary gland, and other organs. 

Remarkably, they constitute a substantial portion of the insect biomass, ranging from 1% to 

10% (Zhao et al., 2022). Their presence and distribution play crucial roles in numerous features 

of insect biology and physiology, ultimately facilitating essential functions and contributing to 

the insect overall survival and adaptive capabilities (Douglas, 2015; Zhao et al., 2022).  

 



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Despite the well-recognized presence of bacteria in other organs, the gut is the most studied 

protagonist since bacteria are predominantly present in the digestive tract, where they can act 

as modulators of the diverse lifestyles of their insect host (Gupta & Nair, 2020). The gut 

microbiota is well recognized for facilitating feeding even on recalcitrant food and 

compensating for poor diets by supplying essential amino acids. Consequently, insects become 

highly dependent on their gut microbiota for survival and development (Douglas, 2015; Engel 

& Moran, 2013; Gupta & Nair, 2020). 

 

Nutrient digestion and metabolism 

 

The gut microbiota is vital in aiding insects with nutrient digestion and metabolism, especially 

given the limitations of their intrinsic digestive systems. Many insects lack the specific 

enzymes needed to break down complex carbohydrates like cellulose and hemicellulose found 

in plants. They have formed symbiotic relationships with gut bacteria to counteract this, 

significantly contributing to their nutritional needs and overall physiology (Douglas, 2009; 

Engel & Moran, 2013). 

 

Insects with cellulose-rich diets also utilize bacterial cellulases to degrade these complex 

carbohydrates. H. illucens larvae illustrate this relationship by efficiently converting various 

organic substrates into protein-rich and fat-rich supplements, supporting the biosynthesis of 

polysaccharides, membrane transport, and energy metabolism attributed to a stable gut bacteria 

community, including Actinomycetes, Dysgomonas, Enterococcus, Providencia, and Proteus 

(Cifuentes et al., 2020, 2022; Klammsteiner et al., 2020; Shelomi et al., 2021; Tegtmeier, 

Hurka, Mihajlovic, et al., 2021; Xiao et al., 2018).  

Nitrogen is an essential nutrient for insect growth and development. H. illucens larvae gut 

bacteria contribute to nitrogen metabolism in several ways, such as nitrogen fixation and urea 

hydrolysis, which is strongly correlated to bacteria such as Klebsiella, Enterobacter, Proteus, 

and Providencia (Behar et al., 2005; Cifuentes et al., 2022; Gold et al., 2020; Klammsteiner et 

al., 2020). In addition to carbohydrate and nitrogen metabolism, these members have been 

linked to the breakdown of sulfur-containing amino acids and other organic molecules 

(Cifuentes et al., 2020; Jiang et al., 2019). 

Although several studies have focused on identifying bacterial taxa in H. illucens larvae gut, a 

comprehensive understanding requires going beyond taxonomy. The actual functions of these 



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microbes within the H. illucens larvae gut are often inferred from their known activities in other 

systems and require further experimental validation (Cifuentes et al., 2022; Eke et al., 2023). 

Immune system modulation and development 

 

Insects possess innate immunity without memory cells yet may exhibit a memory-like 

mechanism called immune priming, which microorganisms can regulate (Girard et al., 2022; 

Prakash & Khan, 2022). Insect immune priming showcases specificity similar to vertebrate 

adaptive immunity. For example, flour beetles exhibit priming specific to Bacillus 

thuringiensis strains, showing no survival advantage with different pathogen strains (Ferro et 

al., 2019; Khan et al., 2016; Prakash & Khan, 2022; Roth et al., 2010). This suggests that 

priming evolves against natural pathogens using strain-specific information. Understanding 

specific priming responses remains challenging due to limited insect immunity knowledge 

(Prakash & Khan, 2022). Traditionally, insect immunity was seen as broad, but recent findings 

show that antimicrobial peptides (AMPs) like Diptericins and Drosocin can offer complete 

protection against specific pathogens (Hanson et al., 2019; Prakash & Khan, 2022). 

The microbiota significantly impacts insect immunity by regulating inflammatory pathways 

(Prakash & Khan, 2022; Thaiss et al., 2016), reducing oxidative damage (Belkaid & Hand, 

2014; Prakash & Khan, 2022; Ray & Kidane, 2016), and potentially influencing immune 

strategies (Futo et al., 2017; Martínez et al., 2020; Prakash & Khan, 2022; Rodrigues et al., 

2010). The microbiota can also induce physicochemical changes for pathogen resistance. For 

instance, Kosakonia cowanii in tsetse flies acidifies the midgut, inhibiting trypanosome growth 

and increasing survival post-Serratia marcescens infection (Schmidt & Engel, 2021; Weiss et 

al., 2019). 

The gut microbiota and the immune system of H. illucens point to a complex interplay that 

contributes to the larvae ability to thrive in environments rich in diverse microorganisms, 

including potential pathogens (Cifuentes et al., 2020; C. Lalander et al., 2013; Vogel et al., 

2018). Inhabiting decaying organic matter, a niche with a wide array of microbes, H. illucens 

larvae have developed a robust immune system to survive this challenging environment, likely 

bolstered by their gut microbiota (Cifuentes et al., 2022; Eke et al., 2023; Tegtmeier, Hurka, 

Mihajlovic, et al., 2021; Vogel et al., 2018).  

  

While the specific mechanisms are not yet fully elucidated, it has been shown that the presence 

of a gut microbiota can indeed influence the expression of immune-related genes in H. illucens 



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larvae. The absence of a microbiome has been shown to trigger significant changes in the 

transcriptional profile of H. illucens larvae during larval development, suggesting that the 

interaction with microbes induces intense regulation of host functional genes, including those 

involved in immune responses (Auger et al., 2023; Eke et al., 2023). Specific bacterial and 

fungal species residing in the gut have been shown to exhibit antimicrobial activity against 

pathogens. For instance, Trichosporon asahii, a yeast species frequently found in H. illucens 

larvae, has been shown to have antimicrobial activity against pathogenic yeasts (Tegtmeier, 

Hurka, Klüber, et al., 2021). Additionally, Bacillus strains isolated from H. illucens larvae 

exhibited potent antimicrobial activity against Staphylococcus aureus (Eke et al., 2023; Zhang 

et al., 2022). 

 

Furthermore, studies investigating microbial changes during the development of H. illucens 

larvae and the impact on the employed substrate have shown that certain pathogenic bacteria 

decrease while other bacterial abundances remain stable or increase (Cai, Ma, Hu, Tomberlin, 

Thomashow, et al., 2018; Cifuentes et al., 2020; Wynants et al., 2019). This phenomenon is 

mainly attributed to AMPs in H. illucens. Notably, H. illucens expresses approximately 50 

genes encoding putative AMPs, contributing to their ability to modulate microbial populations 

(Park & Yoe, 2017; Van Moll et al., 2022; Vogel et al., 2018). Such observations highlight the 

need for further research to fully comprehend immunological responses. Considering the 

dynamics of the different bacterial genotype profiles, as it has been stated, the diversity of 

genotypes in isolated bacteria associated with H. illucens (Cifuentes et al., 2022) and potential 

ignored mechanisms where colonization resistance could contribute to its defense.  

 

Overall host fitness  

 

There is a strong connection between the gut microbiota and the overall fitness of H. illucens 

larvae, with impacts extending beyond digestion to immune function and potentially even 

detoxification. The gut microbiota can be viewed as a key contributor to H. illucens larvae 

success in utilizing a wide range of organic substrates and surviving in challenging 

environments. 

A primary factor in H. illucens larvae fitness is their ability to convert organic waste into 

valuable biomass efficiently (Xiang et al., 2024). This efficient bioconversion relies heavily on 

the metabolic activities of their gut microbiota, since H. illucens larvae lack the necessary 

enzymes to break down complex molecules like cellulose and lignin, which are abundant in 



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many organic waste materials (Eke et al., 2023; Xiang et al., 2024). The gut microbiota fills 

this gap, providing diverse enzymes that contribute to the breakdown of complex 

polysaccharides, proteins, and lipids and influencing the downstream metabolic processes 

within the host (Eke et al., 2023; Y. Wang et al., 2024; Xiang et al., 2024). This symbiotic 

partnership enables H. illucens larvae to extract nutrients from various substrates, contributing 

to their remarkable dietary flexibility. 

 

Emerging research suggests that the gut microbiota of H illucens larvae may contribute to the 

detoxification of harmful compounds present in organic waste, such as antibiotics and heavy 

metals (Cai, Ma, Hu, Tomberlin, Yu, et al., 2018; Eke et al., 2023; C. Liu et al., 2020, 2021). 

This detoxification capability further enhances H. illucens larvae fitness by enabling them to 

tolerate substrates that might be toxic to other organisms. The specific mechanisms involved 

in detoxification by the gut microbiota are still under investigation, but the potential benefits 

for both H. illucens larvae and the environment are significant. 

 

Studies focused on identifying critical bacterial players in the development of H. illucens, have 

shed light on the importance of bacteria during different developmental stages (Cifuentes et 

al., 2020; Querejeta et al., 2022; Zheng et al., 2013). For instance, Comamonas is highly present 

in the egg stage of H. illucens, potentially aiding in lipid biosynthesis and supporting insect 

development (Querejeta et al., 2022). On the other hand, bacteria like Brevundimonas are 

abundant during the pupal stage and may be involved in oxidative protection due to their 

production of carotenoids (Cifuentes et al., 2020; Querejeta et al., 2022). 

 

The complex interplay between H. illucens and its gut microbiota has significant implications 

for its overall fitness, allowing it to thrive in challenging environments and efficiently convert 

waste into valuable biomass. This symbiotic relationship not only benefits the insect, but also 

has the potential for biotechnological applications, such as the development of probiotics for 

improving insect rearing and the discovery of novel enzymes for industrial uses. Further 

exploration of these interactions is crucial for unlocking the full potential of H. illucens in 

various fields, including waste management, animal feed production, and biotechnology. 

 

 

 

 



 18 

Gut Microbiota Composition in H. illucens Larvae: General Patterns and Variations 

 

The gut microbiota of H. illucens larvae exhibits notable patterns, including a potential core 

microbiota, as well as significant variations influenced by factors like diet, developmental 

stage, and rearing environment. 

 

Dominant Phyla and Genera 

 

The gut microbiota of H. illucens larvae is predominantly composed of four bacterial phyla, 

which are Pseudomonadota (formerly Proteobacteria), Bacillota (formerly Firmicutes), 

Actinomycetota (formerly Actinobacteria), and Bacteroidota (formerly Bacteroidetes) 

(Cifuentes et al., 2020; Eke et al., 2023). These phyla encompass several prevalent bacterial 

genera, including Morganella, Providencia, Dysgonomonas, Ignatzschineria, Enterobacter, 

Proteus, Enterococcus, Bacillus, Klebsiella, Citrobacter, Scrofimicrobium, and Actinomyces 

(Cifuentes et al., 2020; Eke et al., 2023; Tegtmeier, Hurka, Mihajlovic, et al., 2021; Zheng et 

al., 2013). This prevalent bacteria has been suggested to be part of a potential core microbiota 

in H. illucens larvae gut. 

 

Core Microbiota 

 

Stable bacterial communities, transmitted through vertical mechanisms, often exhibit a core 

microbiota fundamental to community stability. However, the precise characterization of this 

core microbiota remains the subject of ongoing discussion within the scientific community. 

This ongoing debate is attributed to the fact that most compositional studies have traditionally 

focused on genus-level discrimination, with scant consideration for strain-specific variations 

(Berg et al., 2020). 

 

In this context, the well-studied H. illucens larvae serve as an illustrative case study. Extensive 

research has explored the gut microbiota composition in these larvae, employing high-

throughput sequencing to analyze various growth stages and rearing conditions, with findings 

suggesting the existence of a potential core microbiota (Callegari et al., 2020; Cifuentes et al., 

2020; Gorrens et al., 2021; Jeon et al., 2011; Shelomi et al., 2021; Tegtmeier, Hurka, 

Mihajlovic, et al., 2021). Nevertheless, a more comprehensive analysis at the strain level, using 

fingerprinting techniques, reveals remarkable diversity within genera (Cifuentes et al., 2022).  



 19 

The genus Providencia, which is postulated to constitute a part of the core microbiota, 

demonstrated the presence of at least two distinct phylotypes, each characterized by unique 

genotypic profiles as elucidated by their BOX-Polymerase Chain Reaction (BOX-PCR) 

patterns (Cifuentes et al., 2022). Similarly, the genus Morganella exhibited a single phylotype 

accompanied by multiple genotypes (Cifuentes et al., 2022). These observations highlight the 

intricate complexity involved in definitively characterizing the core microbiota of H. illucens, 

as mentioned by Eke et al. in 2023. 

 

Other members of the potential core microbiota in H. illucens larvae include Dysgonomonas, 

Ignatzschineria, Enterobacter, Proteus, Enterococcus, Bacillus, Klebsiella, Citrobacter, 

Scrofimicrobium, and Actinomyces. Notably, while these genera are frequently found in 

H. illucens larval guts, no single genus has been shown to have 100% prevalence across studies 

(Bruno et al., 2019; Cifuentes et al., 2020, 2022; Eke et al., 2023; Klammsteiner et al., 2020). 

Furthermore, there is no evidence suggesting these microbes are vertically transmitted from 

parents to offspring (Eke et al., 2023). 

 

Although the specific functions of many potential core microbiota members remain largely 

unknown, they are hypothesized to play a crucial role in the capability to digest complex 

organic materials. H. illucens larvae inherently lack the enzymatic machinery necessary for the 

efficient degradation of complex compounds such as cellulose and lignin (Eke et al., 2023). 

The gut microbiota compensates for this enzymatic deficiency. For example, Dysgonomonas, 

known for its polysaccharide-degrading abilities, likely facilitates the breakdown of 

lignocellulose within the digestive tract (Cifuentes et al., 2020; Eke et al., 2023; Querejeta et 

al., 2022; Xiang et al., 2024). Enterococcus may contribute to the production of immune-

related antimicrobial peptides, engage in the degradation of plant polymers, and participate in 

nitrogen, hydrogen, and sulfur metabolism (Eke et al., 2023; Klammsteiner et al., 2020). 

Morganella might be involved in urea hydrolysis and phenol production (Eke et al., 2023). 

Providencia is thought to assist in protein and lipid conversion, antibiotic degradation, and may 

contribute to hemicellulose digestion via xylanase production (Eke et al., 2023; Xiang et al., 

2024). Additionally, Lactobacillus may exert protective effects by detoxifying pesticides and 

xenobiotics, and promoting the expression of antimicrobial peptides which inhibit pathogenic 

bacterial colonization (Eke et al., 2023; Y. Wang et al., 2024). 

 



 20 

A comprehensive characterization of the potential core microbiota in H. illucens larvae requires 

further rigorous research. The variability in experimental conditions, such as rearing 

temperatures, humidity levels, and substrate compositions, across different studies, 

complicates the comparative analysis and the extraction of definitive conclusions (Eke et al., 

2023). Additionally, the reliance on short-read sequencing techniques limits taxonomic 

resolution and the accurate inference of biological functions (Cifuentes et al., 2022; Eke et al., 

2023). The functional roles of core microbiota members require extensive experimental 

validation. Genomic analyses, in vitro phenotyping, and in vivo experiments are essential for 

elucidating the functional diversity and ecological roles of the gut microbiota in H. illucens 

larvae. 

 

 Diet and Environmental Conditions 

 

The diet plays an essential role in modulating the gut microbiota. Empirical evidence has shown 

that dietary variations significantly impact the metabolic activities and structural composition 

of gut bacterial communities (Colman et al., 2012; Yun et al., 2014). While one part of the gut 

microbiome of H. illucens larvae exhibits stability during development under a consistent diet 

(Cifuentes et al., 2020), notable shifts in microbial composition occur with dietary changes. 

This phenomenon is evidenced by studies demonstrating significant microbiome alterations in 

H. illucens larvae reared on diverse substrates, including food waste, cooked rice, and calf 

forage (Jeon et al., 2011; Tegtmeier, Hurka, Klüber, et al., 2021), as well as vegetable or fish 

meal (Bruno et al., 2019; Tegtmeier, Hurka, Klüber, et al., 2021). 

 

Recent studies, such as the one conducted by Bruno et al. (2019), have investigated the gut 

microbiota composition of H. illucens across various substrates with different nutrient contents. 

The results highlight the significant influence of diet, particularly in the midgut, on the 

composition of the gut microbiota. Diets rich in carbohydrates were associated with an 

increased abundance of bacteria like Sphingobacterium and Dysgomonas, indicating their 

potential role in polysaccharide degradation. On the other hand, diets based on fish led to an 

abundance of Providencia in the gut microbiota, suggesting a response to the specific dietary 

composition in H. illucens larvae.   

 

The functional consequences of these dietary-driven microbial shifts extend beyond simple 

nutrient digestion C. Liu et al., 2021 suggest that the H. illucens larvae gut microbiota could 



 21 

play a critical role in detoxifying harmful compounds in various organic wastes. For instance, 

larvae feeding on oxytetracycline-enriched diets exhibit increased antibiotic-resistant bacteria, 

suggesting a potential for bioremediation of pharmaceutical waste (Eke et al., 2023; C. Liu et 

al., 2021). Similarly, heavy metals like cadmium and copper found in animal manure have been 

shown to alter the gut microbiota composition, although without apparent negative effects on 

larval development (Eke et al., 2023; Wu et al., 2020). 

 

Among the various factors influencing the gut microbiota of H. illucens larvae, rearing 

temperature stands out by altering the relative abundance of bacterial taxa (Raimondi et al., 

2020). Increasing the rearing temperature has been shown to decrease the relative abundance 

of Providencia while increasing the abundance of other genera like Bacillus, Proteus, 

Bordetella, and Alcaligenes (Eke et al., 2023; Raimondi et al., 2020). These temperature-

induced shifts could have implications for bioconversion efficiency, as well as the potential for 

pathogen multiplication (Eke et al., 2023). 

 

Developmental Stage 

 

Insect development includes several stages, each characterized by significant physiological and 

morphological changes, including the structure and its microenvironmental conditions, which 

are closely linked to the composition and function of the gut microbiota (Girard et al., 2022). 

These transformations significantly impact extracellular symbionts, which reside on the surface 

of tissues that change during development, compared to their intracellular counterparts (Girard 

et al., 2022; Hammer & Moran, 2019). 

 

Insect development is a progressive transformation that culminates in the adult form. During 

this process, most organs undergo modifications in response to endocrine regulations. The 

digestive tract is a significant carrier of microbial diversity and density among these organs. 

The gut microbiota, in particular, experiences substantial changes due to the elimination of the 

gut epithelium and shifts in physicochemical conditions. These changes profoundly impact the 

microbial communities residing in the gut (Girard et al., 2022) 

 

Insects that undergo complete metamorphosis experience an even more drastic change. During 

pupation, the gut is replaced, leading to a shift in bacterial composition from the larval stage to 

the adult stage (Girard et al., 2022; Manthey et al., 2023).  



 22 

H. illucens, as a holometabolous insect, undergoes complete metamorphosis, including a pupal 

stage. Significant anatomical changes occur during this transformation, particularly in the 

digestive system (Bonelli et al., 2020; Nguyen et al., 2013; Querejeta et al., 2022). The bacterial 

community of H. illucens changes throughout its life cycle in a stage-specific manner, 

influenced by factors such as gut remodeling, dietary shifts, and host-microbe interactions 

(Cifuentes et al., 2020; Eke et al., 2023; Querejeta et al., 2022; Zheng et al., 2013). Despite 

these stage-specific variations, consistent evidence suggests the presence of a potential core 

microbiota that persists throughout the life cycle of H. illucens. This core set of abundant taxa 

remains relatively stable and is independent of the developmental stage (Cifuentes et al., 2020; 

Eke et al., 2023). 

 

While most research focuses on bacterial diversity, a shift in the fungal composition of the 

H. illucens larvae gut microbiota across different life stages has been observed. For instance, 

Trichosporon asahii, from the family Trichosporonaceae, becomes significantly enriched in 

the larval gut in later stages (Tegtmeier, Hurka, Klüber, et al., 2021). 

 

Gut Regionalization and Host Genetics 

 

The H. illucens larval midgut is anatomically and functionally compartmentalized, with 

different regions exhibiting distinct pH levels and hosting different microbial communities  

(Bonelli et al., 2020; Bruno et al., 2019). The anterior midgut harbors a greater microbial 

diversity than the middle and posterior regions (Bruno et al., 2019). This regionalization 

emphasizes the need to investigate the microbiota composition at a finer scale, considering the 

specific conditions and functions of each gut region. Additionally, the genetic nature of the 

host can influence the composition of the gut microbiota, potentially through variations in 

immune responses or other physiological factors. Studies have shown that H. illucens strains 

originating from different geographical locations, and therefore possessing genetic variations, 

harbor distinct bacterial communities (Eke et al., 2023; Khamis et al., 2020). 

 

Beyond Bacteria: Mycobiota and Virobiota 

 

While the majority of research on the H. illucens gut microbiota centers around bacteria, few 

studies provide some information about mycobiota (fungi) and virobiota (viruses) (Klüber et 

al., 2022; Pienaar et al., 2022; Tegtmeier, Hurka, Klüber, et al., 2021). H. illucens hosts a 



 23 

variety of fungi, mainly from the phylum Ascomycota, including genera like Pichia, Candida, 

Diutina, Kluyveromyces, Trichosporon, and Fusarium. While a core mycobiota has not been 

established, Pichia and Candida are frequently associated with the species (Eke et al., 2023; 

Klüber et al., 2022; Tegtmeier, Hurka, Klüber, et al., 2021). The mycobiota is hypothesized to 

contribute to detoxification processes, enzyme production, and provision of essential nutrients 

(Eke et al., 2023; Klüber et al., 2022). However, concerns exist regarding the potential presence 

of pathogenic fungi, like Fusarium solani, and the risk of mycotoxin production under specific 

conditions (Klüber et al., 2022; Schrögel & Wätjen, 2019). 

 

The virobiota of H. illucens remains largely unexplored. In silico analysis suggests the presence 

of Totiviridae viruses, but the nature of these interactions is unknown (Pienaar et al., 2022). 

While H. illucens larvae have shown the ability to reduce viral loads in contaminated substrates 

(Eke et al., 2023; C. H. Lalander et al., 2015), more research is needed to understand the 

composition and structure of the virobiota, particularly in the context of potential risks 

associated with mass rearing. 

 

Impact of Rearing on the Feed Residue Microbiome 

 

Knowing the microbial composition of the residual substrate after H. illucens larvae 

bioconversion is crucial for several reasons, ranging from optimizing bioconversion efficiency 

to addressing safety concerns. Analyzing the microbial composition of the residual substrate 

can help researchers understand how effectively H. illucens larvae and their gut microbiota 

have broken down the organic matter (Y. Wang et al., 2024; Xiang et al., 2024). On the other 

hand, the residual substrate may harbor potentially pathogenic microorganisms, including 

bacteria, fungi, and viruses (Y. Wang et al., 2024). Knowing the microbial composition allows 

researchers to assess the safety risks associated with the residual substrate, particularly if it is 

intended for use as animal feed or fertilizer. 

 

Various studies have reached different conclusions regarding the impact of H. illucens larvae 

on the microbial community of feed residues. Some investigations report no significant 

alteration in the bacterial community composition from the initial to the final phase of feeding 

(Bruno et al., 2019; Cifuentes et al., 2020). Conversely, other studies indicate a progressive 

modification of the microbial community, particularly noting a reduction in potential human 

pathogens within the residual substrate (Cai, Ma, Hu, Tomberlin, Thomashow, et al., 2018; 



 24 

Cifuentes et al., 2020). These discrepancies highlight the complex and context-dependent 

nature of microbial interactions within the H. illucens rearing environment. 

 

Antibiotic Resistance and Safety Considerations 

 

The use of H. illucens larvae as a sustainable protein source for animal feed and potentially 

even human consumption is gaining traction. However, the concern about antibiotic resistance 

and other safety aspects related to the bacteria inhabiting the larval gut is growing. A variety 

of antibiotic resistant genes (ARGs) in both H. illucens larvae and their frass (excrement and 

substrate residue) has been detected. These genes confer resistance to major antibiotic classes, 

including tetracyclines, erythromycin, vancomycin, β-lactams, and aminoglycosides 

(Cifuentes et al., 2020; C. Liu et al., 2021; Milanović, Cardinali, et al., 2021; Milanović, 

Roncolini, et al., 2021). 

 

Several studies have suggested that the composition of the larvae feed can significantly 

influence the prevalence of ARGs. For example, substrates enriched with the microalgae 

Isochrysis galbana were linked to a higher incidence of ARGs in larvae and their frass 

(Milanović, Roncolini, et al., 2021). This highlights the potential role of the feed in shaping the 

resistome of the larvae. Moreover, specific bacterial genera within the gut microbiome, such 

as Morganella, Paenibacillus, Lysinibacillus, and Enterococcus, have been identified as 

potential hosts for ARGs, particularly those conferring resistance to tetracyclines and 

erythromycin (Milanović, Cardinali, et al., 2021; Milanović, Roncolini, et al., 2021) 

 

Beyond antibiotic resistance, the presence of potentially pathogenic bacteria in the gut 

microbiome of H. illucens larvae, including those belonging to the Enterobacteriaceae family, 

if not adequately controlled, could pose a risk to animal or human health (Cifuentes et al., 

2020). A deeper understanding of strain-level variations within the gut microbiota is crucial to 

assess the functional implications of bacterial changes during larval development (Cifuentes et 

al., 2020, 2022). This includes identifying specific strains carrying ARGs and their potential 

for horizontal gene transfer. 

 

The use of H. illucens larvae in applications like animal feed and waste management holds 

immense promise, but the potential risks posed by pathogenic bacteria and ARGs necessitate 

the development and implementation of effective decontamination strategies (Cifuentes et al., 



 25 

2020; Xiang et al., 2024). By optimizing rearing practices, such as using controlled 

environments and appropriate insect densities, the incidence of contamination can be reduced 

(Eke et al., 2023; R Caparros et al., 2024). Careful substrate selection, including pretreatments 

like composting, can minimize the introduction of harmful microbes (Eke et al., 2023; 

Klammsteiner et al., 2020; R Caparros et al., 2024; Y. Wang et al., 2024). Furthermore, 

manipulating feed composition to include balanced diets and natural antimicrobial compounds 

can foster a healthy gut microbiota that contributes to pathogen control and ARG degradation. 

Implementing post-harvest processing methods, such as heat treatment, fermentation, and 

extraction techniques, is crucial to ensure the safety and quality of H. illucens products 

(Callegari et al., 2020; Eke et al., 2023).  

 

Methodological Approaches for Studying Insect Gut Microbiota  

 

The first step in studying the gut microbiota of insects involves careful sampling and 

experimental design. This could include field studies, semi-field studies, or laboratory studies. 

The choice of study type depends on the research question and the insect species being studied 

(Dada et al., 2021; De Cock et al., 2019). Metadata collection is another essential aspect of 

studying insect gut microbiota. This involves collecting information about the habitat of the 

insect, diet, life stage, and other factors that could influence the gut microbiota (Dada et al., 

2021). 

Once the samples have been collected, they need to be processed to know the microbial 

composition, which could involve different approaches. In microbial community analysis, it 

has become increasingly evident that exploring diversity at the genus level is often insufficient, 

especially when searching for deeper classifications, such as at the strain level (S. Liu et al., 

2022). The establishment of a core microbiota, for instance, exemplifies the necessity for 

analyses beyond the genus level (Shade & Handelsman, 2012).  

 

Microbial communities exhibit inherent genetic diversity, and understanding this diversity at 

the level of community properties and functions is essential (Ackermann, 2015; S. Liu et al., 

2022). This underexplored diversity holds the potential for unearthing novel biosynthetic 

pathways and previously unknown biochemical characteristics with applications in various 

industries (Overmann et al., 2017).  

 



 26 

Cultivation-based approaches provide invaluable insights into the phenotypic and genotypic 

attributes of bacterial isolates, enabling taxonomic identification and classification at varying 

levels of resolution (Hahn et al., 2019). This becomes particularly crucial when metagenomic 

sequencing fails to distinguish closely related bacterial taxa (Lema et al., 2023). Characterizing 

bacteria or microbial communities at the genotypic level is of fundamental importance in 

diverse sectors, including medical, industrial, and environmental, offering insights into the 

ecology and taxonomy of microbiota (Emerson et al., 2008; Nocker et al., 2007).  

 

Selecting the appropriate cultivation technique depends on the desired bacteria to be isolated. 

Traditional methods often rely on plate-based techniques, but it has been reported that the 

employed media may not accurately represent the natural environment. Consequently, some 

bacteria fail to thrive. Additionally, the presence of symbiosis between cells or viable but non-

colony-forming cells can pose challenges. Notably, gut microbiota are anaerobic organisms, 

and they may require complex media in an anaerobic environment (Xu et al., 2024).  

 

To address these challenges, culturomics has emerged as an approach where diverse culture 

conditions are employed for studying gut microbiota. However, one of the major drawbacks is 

its time-consuming nature (S. Wang et al., 2020; Xu et al., 2024). High-throughput droplet 

microfluidic systems, where bacteria are encapsulated within droplets, allow the study of a 

large number of colonies in a single experiment. These systems also facilitate the growth of 

slow growers and rare taxa. However, maintaining such systems can be challenging due to 

specialized equipment requirements (Watterson et al., 2020; Xu et al., 2024).   

 

In situ cultivation is a technique that aims to replicate the natural growth conditions of bacteria. 

This method offers advantages over traditional lab techniques because it closely resembles the 

environments where these microorganisms thrive. However, challenges remain in designing 

equipment that can effectively mimic these environments and in purifying bacterial colonies 

from these complex setups (Xu et al., 2024). Another approach, known as dilution-to-extinction 

cultivation, has been successfully employed to isolate and study a wide variety of bacteria. This 

method, pioneered by Button et al. (1993), was initially used to culture slow-growing marine 

bacteria that were difficult to isolate using conventional methods (Button et al., 1993; Cifuentes 

et al., 2022). Dilution-to-extinction involves repeatedly diluting a sample of bacteria in a low-

nutrient medium until no further growth is observed (Button et al., 1993; Cifuentes et al., 2022). 

This process ensures that only a small number of cells are present in each culture well, 



 27 

increasing the likelihood of obtaining pure cultures of individual bacterial species (Button et 

al., 1993; Cifuentes et al., 2022). 

 

This technique has proven particularly useful in isolating bacteria that were previously 

considered "unculturable." Button et al. (1993) achieved success in isolating Candidatus 

Pelagibacter ubique (SAR11) using this approach (Button et al., 1993). Candidatus 

Pelagibacter ubique is a highly abundant marine bacterium that plays a significant role in ocean 

ecosystems but was notoriously difficult to culture using standard laboratory techniques 

(Button et al., 1993). The success in culturing Candidatus Pelagibacter ubique highlighted the 

potential of dilution-to-extinction cultivation in uncovering the hidden diversity of microbial 

life. Building on these successes, researchers have adapted dilution-to-extinction cultivation to 

study the human gut microbiota, a complex community of microorganisms that plays a crucial 

role in human health (Lagier et al., 2018).  

 

The utility of the dilution-to-extinction technique was evaluated in the study of the gut 

microbiota of H. illucens larvae (Cifuentes et al., 2022). This method facilitated the isolation 

of a wide variety of bacteria from the larvae gut, including genera such as Alcaligenes, 

Providencia, Serratia, Brucella, Micrococcus, and Enterococcus (Cifuentes et al., 2022). 

Significantly, even specific strains of Pseudomonas, a bacterium typically considered easy to 

cultivate, suggesting that dilution-to-extinction can uncover a higher level of diversity within 

bacterial genera (Cifuentes et al., 2022). This finding underscores the importance of this 

technique in revealing the full spectrum of microbial diversity. 

 

Implications and Future Directions 

 

The use of H. illucens larvae to convert organic waste into animal feed is a promising approach 

to address sustainability challenges. However, potential risks associated with the transfer of 

ARGs and pathogenic bacteria need to be addressed. 

 

One implication of the findings is the need for a comprehensive risk assessment to ensure the 

safety of H. illucens larvae and their residue as a food source. This assessment should consider 

various factors, such as the types and abundance of ARGs present, their potential for transfer, 

and the risks associated with pathogenic bacteria. While H. illucens larvae effectively reduce 

the levels of certain pathogenic bacteria, the presence of ARGs raises concerns. The widespread 



 28 

use of disinfectants in animal husbandry may increase the risk associated with these genes. 

Therefore, decontamination technologies should be considered to eliminate or reduce hazards 

in the final product. 

Future research should focus on elucidating the dynamics of ARG transfer within the gut 

microbiome and between the larvae and their environment. This includes understanding the 

mechanisms of horizontal gene transfer, the factors influencing transfer rates, and the 

persistence of ARGs in the surrounding environment. Studying the impact of different rearing 

conditions and substrate types on ARG profiles would provide valuable insights into mitigating 

these risks. 

 

Genomic studies of H. illucens gut microbes are crucial for comprehending their diversity, 

shared genetic traits, and adaptations to the larval gut environment. This includes analyzing the 

metabolic functions of various strains, their roles in nutrient digestion, waste degradation, and 

their impact on the larval resistome. Building extensive strain collections is essential for 

thorough genomic analysis and understanding species-specific traits. 

 

Recognizing a potential core microbiome that can be manipulated to enhance performance is 

vital. Promoting beneficial bacteria could improve nutrient digestion and pathogen 

suppression, thus boosting rearing practices and larval growth. The strain-level diversity within 

this potential core microbiome underscores the importance of considering strain-specific traits, 

as different strains might possess unique functional capacities that influence digestive 

processes and interactions with their environment. 

 

However, the difficulty in cultivating certain core taxa, like Lachnospiraceae and Actinomyces, 

underscores the need for developing and refining cultivation techniques. Expanding the range 

of culturable bacteria would enable researchers to perform more detailed functional studies, 

uncovering novel metabolic pathways and interactions within the gut ecosystem, potentially 

leading to the discovery of new enzymes or bioactive compounds.  

 

Finally, exploring the intricate interactions between different bacterial taxa within the 

H. illucens gut microbiome and their influence on the overall ecosystem dynamics is essential. 

Network analysis and co-culture experiments could reveal the synergistic or antagonistic 

relationships between microbes, providing a deeper understanding of the complexity and 

functionality of the gut microbiome.  



 29 

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CHAPTER II 
The gut and feed residue microbiota changing during the rearing of Hermetia illucens 

larvae 

Yina Cifuentes . Stefanie P. Glaeser . Jacques Mvie . Jens-Ole Bartz . 

Ariane Müller . Herwig O. Gutzeit . Andreas Vilcinskas . Peter Kämpfer 

 

 

 

 

 

 

Contributions: 

 

SG, JM, YC designed the study, AM and HOG provided samples, YC, JM, J-OB, and AM 

performed research, YC, JM, and J-OB analysed data, SG, YC wrote the paper which was 

proofed by all co-authors, AV and PK received the funding. 

  



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CHAPTER III 
Isolation of Hermetia illucens larvae core gut microbiota by two different cultivation 

strategies 

 

Cifuentes, Y., Glaeser, S.-P., Vilcinskas, A., Kämpfer, P. 

 

 

 

 

 

 

Contributions: 

 

YC designed the study; YC, performed the experiments and analysed the data; SG, YC wrote 

the paper which was proofed by all co-authors; AV and PK received the funding. 

  



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Annex  

Chapter I, Supplementary material 

 



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Annex  

Chapter II, Supplementary material 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



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Supplementary Figures  

 

 
Supplementary Fig S1. A. BSFL before dissection. B. Dissected guts and resuspended guts in sterile 50 ml 

polypropylene tubes. 



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Supplementary Fig S2. Neighbour joining trees showing the phylotype assignment of the bacterial isolates 

cultured from BSFL gut samples based on partial 16S rRNA gene sequences. Trees were calculated for the 

Enterobacteriaceae clade (A), Firmicutes and Actinobacteria (B), and Alpha- and other Gammaproteobacteria 

(C). Analysis was performed in MEGA7 using the Jukes-Cantor distance correction as evolutionary model and 

100 replications for bootstrap analysis. Bootstrap values (>70 %) are given at the branch nodes. All isolated culture 

by the direct plating are in blue bold and those from the dilution-to-extinction in green bold. Accession numbers 

of isolates and type strains are given in brackets.  



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Supplementary Fig S3. Bacterial growth on TS agar after direct plating of serially diluted cell suspensions 

derived from the BSFL gut samples. An inhibition of swarming bacteria (identified as Proteus spp.) was 

obtained as clear inhibition zones around some colonies identifies as Bacillus spp.  



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Supplementary Fig S4. Neighbour joining trees showing the phylogenetic placement of the bacterial isolates 

from Callegari et al. (2020), Tegtmeier et al. (2021), and this study from BSFL gut microbiota. Trees were 

calculated based on partial 16S rRNA gene sequences in MEGA7 using the Jukes-Cantor distance correction as 

evolutionary model and 100 replications for bootstrap analysis. Bootstrap values (> 70 %) are given at the branch 

nodes. All isolates from this study are in blue bold for direct plating and green bold for dilution-to-extinction. 

Accession numbers are given in brackets. 

 

  



 95 

Abbreviations 

 

AMP: Antimicrobial Peptide 

ANOVA: Analysis of Variance 

ANOSIM: Analysis of Similarities 

ARG: Antibiotic Resistance Gene 

BHI: Brain Heart Infusion 

BLAST: Basic Local Alignment Search Tool 

BSFL: Black Soldier Fly Larvae 

BOX-PCR: BOX-Polymerase Chain Reaction 

Ct value: Cycle threshold 

DNA: Deoxyribonucleic acid 

ESBL: Extended Spectrum Beta-Lactamase  

g: gram 

µg: microgram 

LB: Lysogeny Broth 

MC: Monte Carlo 

MH: Mueller-Hinton 

mL: milliliter 

NMDS: Non-Metric Multidimensional Scaling 

OTU: Operational Taxonomic Unit 

PCR: Polymerase Chain Reaction 

PERMANOVA: Permutational Multivariate Analysis of Variance 

PERMDISP: Permutational analysis of multivariate dispersions 

QAC: Quaternary Ammonium Compound 

qPCR: Quantitative Polymerase Chain Reaction 

R2A: Reasoner's 2A Agar 

½ R2A: Half-concentrated Reasoner's 2A Broth 

16S rRNA: 16S Ribosomal RNA 

S1, S2, S3: These abbreviations refer to the three larval development stages examined in the 

study 

SIMPER: Similarity Percentages 

SRA: Sequence Read Archive 



 96 

TS: Tryptic Soy 

TSPP: Tetra-Sodium Pyrophosphate 

UPGMA: Unweighted Pair Group Method with Arithmetic Mean 

  



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List of publications 

 

Publication I 

 

Cifuentes, Yina, Stefanie P. Glaeser, Jacques Mvie, Jens-Ole Bartz, Ariane Müller, Herwig 

O. Gutzeit, Andreas Vilcinskas, and Peter Kämpfer. "The gut and feed residue microbiota 

changing during the rearing of Hermetia illucens larvae." Antonie Van Leeuwenhoek 113 

(2020): 1323-1344. 

 

Publication II 

 

Cifuentes, Yina, Andreas Vilcinskas, Peter Kämpfer, and Stefanie P. Glaeser. "Isolation of 

Hermetia illucens larvae core gut microbiota by two different cultivation strategies." Antonie 

Van Leeuwenhoek 115, no. 6 (2022): 821-837. 

 

Publication III 

 

Krause, Hans-Martin, Joe G. Ono-Raphel, Edward Karanja, Felix Matheri, Martina Lori, 

Yina Cifuentes, Stefanie P. Glaeser et al. "Organic and conventional farming systems shape 

soil bacterial community composition in tropical arable farming." Applied Soil Ecology 191 

(2023): 105054. 

 

 

 

  



 98 

Acknowledgments 

 

First and foremost, I extend my sincere gratitude to my supervisors for their invaluable 

guidance, insightful comments, and constructive suggestions throughout all stages of my Ph.D. 

journey. I am profoundly grateful to Prof. Dr. Peter Kämpfer for granting me the exceptional 

opportunity to join his esteemed research group and for his unwavering support throughout 

these years. Likewise, I wish to express my heartfelt appreciation to Prof. Dr. Andreas 

Vilcinkas for allowing me to participate in this fascinating project. My special thanks go to Dr. 

Stefanie Glaeser for her meticulous guidance and dedicated oversight of my daily work, as well 

as for the extensive knowledge transfer and enriching discussions that have significantly 

contributed to this work. 

 

Furthermore, I would like to extend my special thanks to all my friends, colleagues, and the 

staff of the Institute of Applied Microbiology: Angel, David, Santiago, Sanjana, Alessandra, 

Alex, Rita, Bellinda, Martina, and Katja. I am equally grateful to all the master and bachelor 

students whose fresh energy and enthusiasm have invigorated each project. 

 

Of course, my most profound appreciation goes to my family—my mother Yanira, sister Diana, 

niece Verónica, and father Jairo—whose support has been unwavering and invaluable despite 

the physical distance. To my husband Michael, who continuously inspired me to persevere, 

and to my daughter Celeste, who has been the final source of energy and motivation to bring 

this chapter of my life to a close, I owe my heartfelt thanks. 

 

I would also like to extend my gratitude to my furry companions in Germany and Colombia. 

Their presence has provided immense comfort and unwavering companionship, enriching my 

life throughout this journey.