Antibacterial Activity of Extracts from Seven Insect Species Against Mediterranean Marine Aquaculture Bacterial Pathogens

Debailleul, Gatien; Gatouillat, Hugo; Chatzimpalasis, Dimitrios; Chatteleyn, Matteo; Chronopoulos, Petros; Nikoloudaki, Chrisanthi; Vasilaki, Antigoni; Kogiannou, Dimitra; Henry, Morgane

doi:10.3390/fishes11070389

Open AccessArticle

Antibacterial Activity of Extracts from Seven Insect Species Against Mediterranean Marine Aquaculture Bacterial Pathogens

by

Gatien Debailleul

^1,2

,

Hugo Gatouillat

^1,2,

Dimitrios Chatzimpalasis

^2,3

,

Matteo Chatteleyn

^2,4

,

Petros Chronopoulos

²

,

Chrisanthi Nikoloudaki

²

,

Antigoni Vasilaki

²

,

Dimitra Kogiannou

²

and

Morgane Henry

^2,*

¹

L’Institut Agro Rennes Angers, 65 Rue de St-Brieuc, FR-35000 Rennes, France

²

Institute of Marine Biology, Biotechnology and Aquaculture (IMBBC), Hellenic Centre for Marine Research (HCMR), 46.7 km Athens-Sounio Ave., GR-19013 Anavyssos, Greece

³

Department of Bio-Engineering, University of Technology of Compiegne, CS 60319, Cedex, FR-60203 Compiègne, France

⁴

Department of Zoology, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece

^*

Author to whom correspondence should be addressed.

Fishes 2026, 11(7), 389; https://doi.org/10.3390/fishes11070389

Submission received: 14 May 2026 / Revised: 18 June 2026 / Accepted: 23 June 2026 / Published: 30 June 2026

(This article belongs to the Special Issue Microbial Pathogens, Disease Control and Veterinary Drug Use in Aquaculture)

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Abstract

Beyond their role as fishmeal replacements in aquafeeds, insects may enhance fish resistance to microbial diseases and improve feed shelf life through the presence of antibacterial compounds. This study evaluated the antibacterial activity of extracts from silkworm pupae (Bombyx mori, BM), larval meals of black soldier fly (Hermetia illucens, HI), mealworm (Tenebrio molitor, TM), and superworm (Zophobas morio, ZM), and adults of house cricket (Acheta domesticus, AD), two-spotted cricket (Gryllus bimaculatus, GB) and weaver ant (Oecophylla smaragdina, OS). The extracts were tested against ten Gram-negative marine bacterial pathogens associated with aquaculture disease outbreaks, as well as the Gram-positive bacterium, Bacillus thuringiensis. The antibacterial activity of aqueous and solvent-soluble extracts was assessed using agar diffusion assays, turbidimetric growth kinetics, and MTT metabolic assays. Several insect extracts exhibited moderate to strong antibacterial activity. In particular, the aqueous extracts of BM and GB, the methanolic extract of HI, and the isopropanolic extracts of HI and OS were effective against multiple bacterial strains, including strains with reduced susceptibility to the reference antibiotics bacitracin, oxolinic acid, and streptomycin. These findings highlight the potential of HI, GB and especially OS as functional ingredients or feed additives for aquaculture.

Keywords:

IC₅₀; MIC; growth inhibition zone; bacteria; aquaculture

Key Contribution: Water, methanol and isopropanol effectively extract antibacterial compounds. High concentrations of silkworm extracts are effective against many fish pathogens. GB (water), HI (methanol and isopropanol) and OS (isopropanol) yield moderate to strong extracts. GBa (water) and OSa (isopropanol) extracts active against Vibrio harveyi resistant to bacitracin. HI extracts as potent as streptomycin against B. thuringiensis and V. splendidus.

1. Introduction

Aquaculture is one of the fastest-growing food production sectors and plays a crucial role in global food security by supplying high-quality animal protein to an increasing human population [1,2]. Mediterranean aquaculture contributes substantially to regional economy and food security, producing over 600,000 tons of finfish annually, with gilthead seabream (Sparus aurata), European sea bass (Dicentrarchus labrax), and meagre (Argyrosomus regius) representing the principal cultured species [2]. However, the sustainable expansion of the sector is increasingly constrained by infectious diseases, which reduce productivity, compromise animal welfare, and cause substantial economic losses [3,4].

Bacterial pathogens are among the most significant infectious agents affecting Mediterranean aquaculture [5,6]. Species of the genera Vibrio, Photobacterium, Aeromonas, and Pseudomonas are responsible for recurrent outbreaks associated with hemorrhagic septicemia, ulcerative lesions, systemic infections, and high mortality rates in cultured fish [5,6,7,8]. These pathogens are particularly problematic under intensive farming conditions and elevated seawater temperatures, conditions commonly encountered in Mediterranean production systems [5]. Disease management has traditionally relied on antibiotics such as oxytetracycline, florfenicol, and quinolones [5,6]. However, their extensive use has contributed to the emergence and dissemination of antimicrobial resistance (AMR), reducing treatment efficacy and raising concerns regarding environmental contamination, food safety, and public health [9,10]. Multidrug resistant Vibrio and Aeromonas isolates have already been reported from Mediterranean aquaculture environments [9,10], while global efforts to reduce antimicrobial use have led to stricter regulations, including restrictions on prophylactic antibiotic administration in the European Union. Consequently, the identification of alternative antimicrobial agents capable of controlling fish pathogens has become a research priority.

Natural products represent an important source of novel antimicrobial compounds, and insects have recently attracted considerable attention in this regard. Living in microbially rich environments, insects have evolved sophisticated innate immune systems that produce a wide range of bioactive molecules to combat pathogenic microorganisms. These include antimicrobial peptides (AMPs), such as defensins, cecropins, attacins, and coleoptericins, as well as medium-chain fatty acids—particularly lauric acid—chitin, chitosan, phenolic compounds, and other metabolites with antimicrobial and immunomodulatory properties [11,12,13,14,15,16,17]. Numerous studies have demonstrated antimicrobial activity of insect-derived extracts or purified compounds against human, veterinary, and aquatic pathogens, highlighting insects as promising reservoirs of natural antimicrobial agents [11,12,13,14,15,16,17,18]. Beyond their direct antibacterial effects, several insect-derived compounds have also been reported to stimulate host immunity and improve resistance to infection [18], suggesting potential applications in both disease prevention and functional feed development.

In parallel, insects have gained considerable interest as sustainable feed ingredients for aquaculture because of their high nutritional value and a relatively low environmental footprint [19,20]. They efficiently convert low-value organic substrates into high-quality biomass while requiring less land and water than conventional protein sources [19,20]. The European Union has progressively recognized their importance by authorizing several insect species for use in aquafeeds, including the black soldier fly (Hermetia illucens), yellow mealworm (Tenebrio molitor), house cricket (Acheta domesticus), and silkworm (Bombyx mori), among others. However, although the nutritional properties of these species have been extensively investigated, comparatively fewer studies have systematically evaluated and compared their antibacterial potential against Mediterranean fish pathogens. Furthermore, emerging edible insect species, such as the two-spotted cricket (Gryllus bimaculatus), the superworm (Zophobas morio), and the weaver ant (Oecophylla smaragdina), are increasingly attracting scientific and commercial interest because of their nutritional and medicinal properties. In particular, G. bimaculatus and O. smaragdina have long histories of use in traditional foods and medicine in Asia and have been reported to contain bioactive compounds with antimicrobial and antioxidant activities, although their activity against fish pathogens remains poorly characterized [21,22,23,24].

Previous studies have largely focused on individual insect species, specific antimicrobial peptides, or antibacterial activity against human pathogens, making direct comparisons between different edible insects and extraction methods difficult. Moreover, information regarding the antibacterial potential of insect extracts against bacterial pathogens relevant to Mediterranean aquaculture remains limited. Comparative screening studies are therefore needed to identify promising insect species and extraction approaches for future characterization of active compounds and potential application in sustainable aquaculture.

The present study aimed to comparatively evaluate the antibacterial activity of crude extracts obtained from seven insect species, including four species authorized for use in European aquafeeds (B. mori, H. illucens, T. molitor, A. domesticus) and three additional edible insect species of emerging interest (Z. mori, G. bimaculatus, O. smaragdina). Aqueous, methanolic, and isopropanolic extracts were prepared and screened against a panel of Mediterranean fish bacterial pathogens, including Aeromonas veronii, Bacillus thuringiensis, Pseudomonas aeruginosa, Photobacterium damselae subsp. damselae, P. damselae subsp. piscicida, Vibrio alginolyticus, V. anguillarum, V. harveyi, V. splendidus). By providing a comparative assessment of the antibacterial activity of crude insect extracts, this study seeks to identify promising candidates for future isolation of bioactive compounds and to support the development of insect-derived functional ingredients for sustainable aquaculture.

2. Materials and Methods

2.1. Insects

Silkworm pupae (B. mori, BMp) were purchased from Reptilia Nostra (Athina, Greece), larval meals from yellow mealworm (T. molitor, TMl), black soldier fly (H. illucens, HIl), and superworm (Z. morio, ZMl), were kindly provided by Dr. I. Karapanagiotidis (Aquaculture Laboratory, Department of Ichthyology and Aquatic Environment, Faculty of Agricultural Sciences, University of Thessaly, Volos, Greece). Adults of weaver ant (O. smaragdina, OSa), house cricket (A. domesticus, ADa) and two-spotted cricket (G. bimaculatus, GBa), were purchased from Next-Food (Ras Al Khaima, United Arab Emirates).

The insects were milled using a food blender and subsequently freeze-dried. Their proximate composition was analyzed in triplicate according to standardized analytical methods [25]. Moisture content was determined by drying pre-weighed samples in porcelain cups at 104 °C for 24 h (Method number 950.46). Ash content was determined by combustion at 500 °C for 12 h (Method number 920.153). Crude protein (CP) was quantified using to the Kjeldahl method (Method number 988.05) and calculated using a nitrogen-to-protein conversion factor (Kp) of 4.76 rather than the conventional coefficient of 6.25 commonly used for animal proteins, in order to avoid overstimulation due to nitrogen associated with chitin [26]. Total lipid content (ether extract, EE) was determined using a Soxhlet system (HT, 1043 Extraction unit Foss Tecator, Foss Analytical, Hilleroed, Denmark) according to the ISO method 6492:1999. Nitrogen-free extract (NFE) and gross energy (GE) were calculated as follows:

NFE = 100 − (Moisture + Ash + CP + EE)

GE = (CP × 23.6 + EE × 39.5 + NFE × 17.3)/100

2.2. Extraction Protocol

A preliminary experiment was conducted to select the most effective solvents for extracting antibacterial compounds from BMp, following a standard maceration protocol [27]. Briefly, 3 g of the milled freeze-dried BMp were extracted with 30 mL of different solvents (1:10 w/v) of increasing polarities (n-hexane, ethyl acetate, dichloromethane, chloroform, chloroform/methanol (1:1), isopropanol, methanol or distilled water) in sealed glass tubes (Parafilm). Extractions were performed for 48 h at room temperature under continuous magnetic stirring in a fume hood.

The resulting extracts were clarified by sequential centrifugation at 1000× g for 15 min followed by 2700× g for 10 min. Supernatants were collected, and solvents were removed according to their physicochemical properties: aqueous extracts were freeze-dried, methanol and isopropanol extracts were evaporated at 40 °C under a nitrogen stream (TurboVap LV system, Caliper Life Sciences, Hopkinton, MA, USA); and other organic extracts were concentrated using rotary evaporation at 35 °C.

Based on these results, three solvents (distilled water, methanol and isopropanol) were selected for further investigation of seven insect species, including silkworm pupae (BMp), three insect larval meals (HMl, TMl and ZMl) and three whole adult insects (ADa, GBa and OSa). Freeze-dried insect powders were macerated in each of the selected solvents (1:10 w/v) following the same procedure described above. The dry weight of each extract was determined, and extraction yield was calculated using the following equation:

Yield (%) = 100 × dried extract weight (g)/initial freeze-dried insect weight (g)

Concerning BMp extracts in the preliminary experiment, extracts were diluted either in distilled water (aqueous extracts) or in acetone (all other extracts), and working solution were adjusted with distilled water to 100–150 mg/mL depending on extract availability: methanol yielded high amount of extracts that were tested at 150 mg/mL, whereas extracts obtained with all other solvents were tested at 100 mg/mL. The final acetone concentration in the working solutions was ≤20% to avoid a turbidity that could interfere with optical density (OD) measurements. This acetone concentration did not exhibit antibacterial activity, in contrast to other tested solvents such as dimethyl sulfoxide (DMSO), dimethyl sulfide (DMS), dimethylformamide (DMF), or polysorbate 20 (Tween 20), which showed antibacterial activity even at low concentrations that were insufficient to fully solubilize the extracts.

Based on the preliminary solvent screening results using BMp, further experiments compared the seven insect species in standardized conditions. Stock solutions were prepared at 250 mg/mL, using either distilled water (for aqueous extracts) or acetone (for methanolic and isopropanolic extracts). Sonication in a water bath at 35 °C for 10 min was applied to facilitate dissolution of the extracts. Working solutions of all insects were adjusted to 50 mg/mL with distilled water. As before, the acetone concentration in all extracts adjusted to 50 mg/mL was 20%, except for the aqueous extracts, which were dissolved in water. Finally, all extracts were sterilized by filtration through 0.2 µm Whatman syringe filters (Whatman, Maidstone, UK) under a laminar flow hood to prevent microbial contamination. Samples were aliquoted, protected from light, and stored at −20 °C for a maximum of 2 months before analyses.

2.3. Bacterial Strains and Culture Conditions

Eleven strains of pathogenic bacteria commonly associated with disease outbreaks in Mediterranean fish farms were selected from the microbiological collection of the Microbiology Laboratory of the Hellenic Centre for Marine Research (Gournes, Greece). These strains were originally isolated from diseased fish or shellfish in Greek aquaculture systems and are maintained as glycerol stocks within the HCMR collection (Table 1).

Table 1. List of tested bacteria with their host origin and organ when known.

Code	Gram **	Bacterium Species	Origin
Av	−	Aeromonas veronii	European seabass, Dicentrarchus labrax
Bt	+	Bacillus thuringiensis	Gilthead seabream, Sparus aurata
Pa	−	Pseudomonas aeruginosa	unknown
Pdd	−	Photobacterium damselae damselae	Gilthead seabream, S. aurata
Pdp	−	Photobacterium damselae piscicida	European seabass, D. labrax
Val V1	−	Vibrio alginolyticus	Gilthead seabream, S. aurata
Val	−	Vibrio alginolyticus	Kidney European seabass, D. labrax
Va	−	Vibrio anguillarum	Spleen European seabass, D. labrax
Vh	−	Vibrio harveyi	Spleen European seabass, D. labrax
Vh2 *	−	Vibrio harveyi	Greater amberjack, Seriola dumerilii
Vs	−	Vibrio splendidus	Mussels

* Vibrio harveyi VH2 is extensively described in a recent publication [28]; ** Gram negative (−), Gram positive (+).

All experiments were conducted under aseptic conditions in a laminar-flow hood (BioAura 2000, BioAir, Siziano, Italy). Bacterial strains were cultured at 22 °C in tryptic soy broth supplemented with 2% NaCl (TSB-2%) for 24 h for most bacteria, or 48 h concerning P. aeruginosa and P. damselae piscicida, to reach the exponential growth phase. Cultures were then centrifuged at 2300× g for 10 min, washed in phosphate-buffered saline (PBS), and resuspended in PBS to 0.5 McFarland, corresponding to an optical density of 0.10 ± 0.02 at 625 nm and approximately 1.5 × 10⁸ CFU/mL [29,30].

2.4. Antibacterial Assays

Negative controls consisted of the solvent-only treatments, which were included in each agar plate and microplate. Positive controls consisted of antibiotics from different classes, including bacitracin (50 mg/mL), streptomycin (25 mg/mL), gentamycin (10 mg/mL), and oxolinic acid, flumequine, florfenicol and oxytetracycline (50 μg/mL). The reference antibiotic concentrations were selected based on preliminary optimization experiments. Each assay was repeated at least three times on different bacterial culture batches giving 3 to 6 biological replicates to ensure reproducibility.

2.4.1. Agar Well Diffusion Method

A pour-plate technique was performed following standard microbiological procedures [31]. Briefly, 1 mL of each logarithmic-phase bacterial suspension adjusted to 0.5 McFarland was mixed with 20 mL of freshly autoclaved tryptic soy agar supplemented with 2% NaCl (TSA-2%), cooled to 48 °C in a water bath, and poured into sterile Petri dishes. After complete solidification of the agar, wells of 2 mm diameter were aseptically punched using a sterile Pasteur pipette. Subsequently, 2 μL of each extract at the highest tested concentration or antibiotic at concentrations stated in Section 2.4 was dispensed into the wells. Plates were incubated at 22 °C for 24 h, or 48 h for P. aeruginosa and P. damselae piscicida). The diameters of the growth inhibition zones (GIZs) were then measured in millimeters.

2.4.2. Kinetic Microdilution Assay

Antibacterial activity was evaluated using a kinetic microdilution assay adapted from Clinical Laboratory Standard Institute (CLSI) guidelines [32,33], as reviewed by Balouiri and colleagues [29] in 384 wells flat-bottom transparent microplates (Greiner, Kremsmunster, Austria). Outer wells were filled with sterility controls (medium only or medium with solvent, i.e., water or 20% acetone), to both monitor the sterility of the assay and minimize evaporation. Growth controls (medium plus inoculum without extract) were also included on each plate. Serial two-fold dilutions of each insect extract were prepared in sterile distilled water and distributed 10 wells of the microplate. The 2-fold serial dilution of the positive controls (reference antibiotics) was done in 21 wells to cover the amplitude of antibacterial activities of these antibiotics against the various bacterial strains. The solvent control (medium plus inoculum plus 20% acetone) was also serially diluted in 10 wells of each microplate.

Each well contained 25 µL of extract solution, 50 µL of TSB-2%, and 25 µL of bacterial inoculum adjusted to 0.5 McFarland and diluted 1:150 in TSB-2% corresponding to 1·10⁶ CFU/mL (final volume: 100 µL per well). Bacterial growth kinetics were monitored at 22 °C every 15 min for 16 h for Aeromonas veronii (Av), Bacillus thuringiensis (Bt), Photobacterium damselae subsp. damselae (Pdd), Vibrio anguillarum (Va), V. splendidus (Vs), V. alginolyticus (Valg and Valg V1), 24 h for V. harveyi (Vh and VH2) or 48 h for Pseudomonas aeruginosa (Pa) and P. damselae subsp. piscicida (Pdp), by measuring the optical density at 600 nm (OD₆₀₀) using a microplate spectrophotometer (Genios Pro, Tecan, Mannedorf, Switzerland).

Antibacterial activity was expressed as the IC₅₀, defined as the lowest concentration of the serial dilution resulting in a ≥50% reduction in bacterial growth relative to the untreated solvent control. Subsequently, 10 µL of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/mL) were added in each well, incubated for 15 min, and absorbance was measured at 540 nm. Metabolically active bacteria reduce the yellow MTT to blue formazan crystals. The minimal metabolic inhibitory concentration (MIC) corresponded to the lowest concentration of the serial dilution of the extract or antibiotic that completely inhibited bacterial metabolic activity (i.e., absence of color change in the MTT assay). Antibacterial activity was interpreted using a semi-quantitative activity classification scale where MIC or IC₅₀ values ≤ 1 mg/mL indicate very strong activity, 1–5 mg/mL strong activity, 5–25 mg/mL moderate activity, and >25 mg/mL weak activity.

3. Results

3.1. Proximate Composition of the Seven Insects

The proximate composition of the seven insect species is presented in Table 2. Among the insects analyzed, superworm larval meal (ZMl) exhibited the highest lipid content (39.7% crude lipids), whereas adult house cricket (ADa) showed the highest protein content, reaching 44.7% crude protein (N × 4.76), equivalent to 58.7% when calculated using the conventional nitrogen-to-protein conversion factor of 6.25.

Table 2. Proximate composition (%) of the 7 insect species tested in the present study.

	BMp	HIl	TMl	ZMl	ADa	GBa	OSa
Moisture (%)	7.1 ± 0.1	8.0 ± 0.3	12.5 ± 0.2	8.7 ± 0.2	5.3 ± 0.1	5.4 ± 0.1	6.1 ± 0.2
Ash (%)	4.8 ± 0.1	10.0 ± 0.1	3.7 ± 0.2	2.6 ± 0.1	3.6 ± 0.1	3.5 ± 0.1	2.5 ± 0.1
CP (%) (Kp = 4.76)	40.4 ± 0.6	31.1 ± 0.7	39.4 ± 0.3	32.9 ± 0.4	44.7 ± 0.4	39.7 ± 0.2	37.5 ± 0.3
CP (%) (Kp = 6.25)	53.0 ± 0.7	40.9 ± 0.9	51.8 ± 0.4	43.2 ± 0.5	58.7 ± 0.6	52.1 ± 0.2	49.2 ± 0.3
Crude Lipids (%)	23.2 ± 0.6	29.6 ± 1.0	22.1 ± 0.5	39.7 ± 0.4	22.1 ± 0.1	31.6 ± 0.2	32.6 ± 0.1
NFE	24.6	21.3	22.2	16.1	24.3	19.8	21.5
GE (kJ/g)	22.9	22.7	21.9	26.3	23.5	25.3	25.4

Values represent mean ± standard deviation (n = 3). Abbreviations: CP: crude protein; NFE: nitrogen-free extract, GE: gross energy.

3.2. Preliminary Experiment on BMp

3.2.1. Extraction Yield of Silkworm Pupae

The extraction yields of BMp using eight different solvents are given in Table 3. The highest extraction yield of BMp was obtained with methanol (33.5%).

Table 3. Solvents used for the extraction of silkworm pupae; their polarity and extraction yields.

Solvent	Polarity	BMP
n-Hexane	0.009	24.0%
Ethyl-acetate	0.228	25.5%
Dichloromethane	0.234	24.2%
Chloroform	0.259	28.0%
Chloroform/Methanol	0.259/0.762	31.8%
Isopropanol	0.546	23.6%
Methanol	0.762	33.5%
DH₂O	1	7.3%

3.2.2. Antibacterial Activity of Silkworm Pupae Extracts

The results of the optimization experiment to identify the most effective extraction solvents for silkworm pupae, based on the growth inhibition zone (GIZ) assay, the minimum BMp extract concentration required to inhibit 50% of bacterial growth (IC₅₀), and the minimum metabolic inhibitory concentration (MIC), are presented in Table 4 and Table 5 and Table 6 respectively.

Table 4. Growth inhibition zone (in mm) of BMp extracts.

		Bacteria
		Av	Bt	Pa	Pdd	Pdp	Val V1	Val	Va	Vh2	Vs
Solvents	water	3.0 ± 0.0 *	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	methanol	4.5 ± 0.5 *	0.0	23.0 ± 5.9	0.0	20.0 ± 0.0	22.3 ± 1.5 *	22.5 ± 2.1 *	0.0	22.0 ± 2.8 *	0.0
	isopropanol	0.0	0.0	17.2 ± 5.5	0.0	20.0 ± 0.0	12.3 ± 2.6.0 *	14.0 ± 0.0 *	0.0	16.0 ± 1.0 *	0.0
	n-hexane	0.0	0.0	12.7 ± 2.9	4.0 ± 0.0	11.0 ± 1.4	10.7 ± 1.0.0 *	11.5 ± 0.7 *	0.0	10.7 ± 5.7 *	0.0
	chloroform	0.0	0.0	14.0 ± 4.9	0.0	10.0 ± 0.0	12.0 ± 2.5 *	12.0 ± 4.2 *	0.0	11.3 ± 2.3	0.0
	chloroform/methanol	0.0	0.0	13.3 ± 5.7	0.0	0.0	12.0 ± 2.5 *	16.0 ± 0.0 *	0.0	11.0 ± 1.4	0.0
	dichloromethane	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	ethyl acetate	0.0	0.0	14.7 ± 4.6	0.0	6.0 ± 1.4	7.7 ± 2.1 *	6.5 ± 0.7 *	0.0	8.0 ± 4.0 *	0.0

Growth inhibition zone (in mm) produced by 2 μL of at 100 mg/mL using different solvents (water, isopropanol, n-hexane, chloroform, chloroform/methanol, dichloromethane or ethyl acetate) or by the methanolic extract at 150 mg/mL against tested bacterial pathogens. * Asterisks indicate inhibition zones that were not fully translucent (i.e., blurred). Solvent-alone negative controls were included in each agar plate and exhibited no GIZ. Data are presented as mean ± standard deviation (S.D.), n = 3.

Table 5. Minimum BMp extract concentration inhibiting 50% of bacterial growth (IC₅₀ in mg/mL).

		Bacteria
		Av	Bt	Pa	Pdd	Pdp	Val V1	Val	Va	Vh2	Vs
Solvents	water	35.4 ± 16.6	31.8 ± 18.0	50 ± 0.0	87.5 ± 25.0	50 ± 0.0	34.4 ± 18.7	31.3 ± 21.7	44.6 ± 14.2	>100	>100
	methanol	100 ± 43.3	48.4 ± 18.9	125.0 ± 43.3	>150	18.6 ± 14.0	29.2 ± 19.1	32.5 ± 20.4	>150	>150	>150
	isopropanol	66.7 ± 28.9	>100	100 ± 0.0	83.3 ± 28.9	>100	100 ± 0.0	75.0 ± 28.9	>100	>100	>100
	n-hexane	>100	>100	>100	>100	>100	100 ± 0.0	>100	>100	>100	>100
	chloroform	>100	66.7 ± 28.9	100 ± 0.0	>100	>100	100 ± 0.0	>100	100 ± 0.0	>100	50.0 ± 0.0
	chloroform/methanol	>100	nd	50.0 ± 0.0	>100	>100	16.7 ± 7.2	>100	91.7 ± 14.4	20.8 ± 7.2	>100
	dichloromethane	>100	>100	100 ± 0.0	>100	>100	100 ± 0.0	83.3 ± 28.9	>100	100 ± 0.0	83.3 ± 28.9
	ethyl acetate	>100	>100	83.3 ± 28.9	>100	>100	100 ± 0.0	83.3 ± 28.9	>100	100 ± 0.0	66.7 ± 28.9

Extracts were serially 2-fold diluted in 10 wells from 100 mg/mL to 0.2 mg/mL for all extracts except for methanolic extracts which were diluted from 150 mg/mL to 0.3 mg/mL in a 384 wells-microplate where edges were filled with sterility controls to avoid evaporation. Data are presented as mean ± standard deviation (S.D.), n = 3.

Table 6. Minimum metabolic inhibitory concentration (MIC in mg/mL) of BMp extracts using various solvents against tested bacterial pathogens.

		Bacteria
		Av	Bt	Pa	Pdd	Pdp	Val V1	Val	Va	Vh2	Vs
Solvents	water	34.4 ± 18.7	44.8 ± 27.1	nd.	50.0 ± 0.0	nd.	50.0 ± 0.0	56.3 ± 12.5	59.7 ± 21.4	100.0 ± 0.0	83.3 ± 28.8
	methanol	23.4 ± 10.7	75.0 ± 0.0	nd.	46.8 ± 19.8	nd.	62.5 ± 21.7	62.5 ± 21.7	53.3 ± 5.8	75 ± 11.2	31.2 ± 10.8
	isopropanol	62.5 ± 15.6	100 ± 0.0	nd.	>100.0	nd.	100.0 ± 0.0	68.75 ± 23.9	75.0 ± 25.0	>100	41.6 ± 14.4
	n-hexane	>100	100 ± 0.0	nd.	>100	nd.	100.0 ± 0.0	>100	>100	>100	100.0 ± 0.0
	chloroform	100 ± 0.0	100 ± 0.0	nd.	>100	nd.	100.0 ± 0.0	100.0 ± 0.0	>100	>100	25.0 ± 0.0
	chloroform/methanol	100 ± 0.0	25.0 ± 0.0	nd.	100.0 ± 0.0	nd.	20.8 ± 7.2	25.0 ± 0.0	>100	20.8 ± 7.2	6.3 ± 0.0
	dichloromethane	100 ± 0.0	100 ± 0.0	nd.	>100	nd.	100.0 ± 0.0	100.0 ± 0.0	100 ± 0.0	>100	18.8 ± 8.8
	ethyl acetate	75.0 ± 35.4	100 ± 0.0	nd.	>100	nd.	100.0 ± 0.0	100.0 ± 0.0	100 ± 0.0	75.0 ± 35.4	50.0 ± 14.5

Extracts were serially 2-fold diluted in 10 wells from 100 mg/mL to 0.2 mg/mL for all extracts except for methanolic extracts which were diluted from 150 mg/mL to 0.3 mg/mL in a 384 wells-microplate where edges were filled with sterility controls to avoid evaporation. Data are presented as mean ± standard deviation (S.D.), n = 3. nd = not determined.

3.3. Comparison of Extracts from the 7 Insect Species

3.3.1. Extraction Yield of the Seven Insect Species

The extraction yields of the seven insect species using the three selected solvents (water, methanol, isopropanol) are presented in Table 7.

Table 7. Solvents used for insect extraction; their polarity and extraction yields for each insect tested.

		Extraction Yield
Solvent	Polarity	BMp	HIl	TMl	ZMl	ADa	GBa	OSa
DH₂O	1	7.3%	11.8%	42.9%	28.2%	9.3%	13.7%	18.7%
Methanol	0.762	33.5%	37.4%	41.9%	50.0%	24.9%	21.6%	19.2%
Isopropanol	0.546	23.6%	26.9%	24.9%	37.2%	38.1%	28.0%	27.3%

BMp = Bombyx mori pupae, HIl = H. illuscens larval meal, TMl = T. molitor larval meal, ZMl = Z. mori larval meal, ADa = A. domesticus adult, GBa = G. bimaculatus adult, OSa = O. smaragdana adult.

3.3.2. Antibacterial Activity of Extracts from the Seven Insects and Reference Antibiotics

Thereafter, insect extracts were adjusted to 50 mg/mL. The GIZ results are presented in Table 8. Photographs of two agar plates are given in the Supplementary Material S1. The IC₅₀ values of the extracts (mg/mL) and antibiotics (μg/mL) are shown in Table 9 and Table 10, respectively. The MIC values for the extracts (mg/mL) and reference antibiotics (μg/mL) are reported in Table 11 and Table 12, respectively.

Table 8. Growth inhibition zone (in mm) of insect extracts or by different antibiotics against tested bacterial pathogens.

		Bacteria
	Insect	Av	Bt	Pa	Pdd	Pdp	Val V1	Val	Va	Vh	Vh2	Vs
Water	BMp	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	HIl	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	TMl	0.0	0.0	0.0	8.0 ± 4.2	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	ZMl	0.0	0.0	0.0	7.0 ± 2.8	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	ADa	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	GBa	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	OSa	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Methanol	BMp	0.0	0.0	6.5 * ± 2.1	0.0	0.0	4.5 ± 0.7	0.0	0.0	0.0	0.0	0.0
	HIl	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	TMl	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	ZMl	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	ADa	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	GBa	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	OSa	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Isopropanol	BMp	0.0	0.0	5.5 * ± 4.9	0.0	0.0	6.0 ± 1.4	0.0	0.0	0.0	0.0	0.0
	HIl	0.0	0.0	7.5 * ± 4.9	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	TMl	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	ZMl	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	ADa	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	GBa	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	OSa	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Abiotics
Bacitracin		0.0	15.5 * ± 6.5	0.0	0.0	52.0 ± 22.0	3.0 ± 0.9	0.0	18.7 ± 10.1	15.2 ± 2.6	13.5 ± 2.7	10.0 ± 3.7
Streptomycin		8.8 ± 1.9	12.2 ± 5.2	11.3 ± 2.1	10.5 ± 3.5	22.5 ± 0.7	21.3 ± 5.5	10.0 ± 6.5	14.7 ± 7.0	11.7 ± 6.7	16.7 ± 2.5	17.0 ± 7.0
Gentamicin		20.5 ± 2.1	12.7 ± 4.5	16.3 ± 2.1	0	0.0	19.5 ± 0.7	16.5 ± 0.7	25.0 ± 12.7	23.5	22.5 ± 6.4	24.5 ± 0.7
Oxolinic Acid		0.0	0.0	0.0	2.0 ± 1.4	0.0	1.5 ± 0.7	0.0	0.0	0.0	0.0	0.0
Flumequine		0.0	0	0.0	6.5 ± 2.1	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Florfenicol		0.0	6.3 ± 2.1	0.0	7.0 ± 1.4	0.0	0.0	0.0	0.0	3.0 ± 2.8	5.5 ± 2.1	7.5 ± 4.9
Oxytetracycline		3.5 ± 0.7 *	2.0 ± 0.7	0.0	4.3 ± 0.6	14.0 ± 1.4	3.7 ± 2.1	3.5 ± 1.9	9.0 ± 0.0	6.3 ± 0.6	7.3 ± 1.5	8.0 ± 4.0

Growth inhibition zone (in mm) produced by 2 μL of insects extracts at 50 mg/mL using different solvents (water, methanol and isopropanol) or antibiotics (bacitracin at 50 mg/mL, streptomycin at 25 mg/mL, gentamycin at 10 mg/mL, and oxolinic acid, flumequine, florfenicol and oxytetracycline at 50 μg/mL) against tested bacterial pathogens. * Asterisks represent inhibition zones that were not totally translucid. Solvent-alone negative controls were included in each agar plate and exhibited no GIZ. Data are presented as mean ± standard deviation (S.D.), n = 3.

Table 9. Minimum extract or bacitracin concentration inhibiting 50% of the growth of tested bacterial pathogens (IC50 in mg/mL).

		Bacteria
	Insect	Av	Bt	Pa	Pdd	Pdp	Val V1	Val	Va	Vh	Vh2	Vs
Water	BMp	35.4 ± 16.6	31.8 ± 18.0	>50	>50	50 ± 0.0	34.4 ± 18.7	31.3 ± 21.7	44.6 ± 14.2	>50	>50	>50
	HIl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	TMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	ZMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	ADa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	GBa	3.4 ± 2.7	>50	9.4 ± 4.4	37.8 ± 26.3	>50	9.4 ± 4.4	25.0 ± 0.0	41.7 ± 14.4	7.3 ± 4.77	33.3 ± 14.4	20.8 ± 7.2
	OSa	>50	32.5 ± 24.0	>50	>50	>50	>50	>50	>50	>50	>50	>50
Methanol	BMp	>50	48.4 ± 18.9	>50	>50	18.6 ± 14.0	29.2 ± 19.1	32.5 ± 20.44	>50	>50	>50	>50
	HIl	0.5 ± 0.3	33.5 ± 28.6	>50	>50	1.6 ± 0.0	0.9 ± 0.7	0.7 ± 0.5	>50	>50	0.3 ± 0.1	1.3 ± 1.1
	TMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	ZMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	8.3 ± 3.6
	ADa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	GBa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	OSa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
Isopropanol	BMp	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	HIl	27.1 ± 21.9	1.7 ± 1.4	>50	>50	1.9 ± 0.8	1.0 ± 0.4	1.1 ± 1.0	>50	>50	1.8 ± 1.2	0.8 ± 0.7
	TMl	47.5 ± 33.5	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	ZMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	ADa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	GBa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	OSa	3.4 ± 2.7	37.5 ± 14.4	2.6 ± 0.9	8.3 ± 3.6	>50	6.8 ± 5.5	5.2 ± 1.8	29.7 ± 23.6	6.8 ± 5.5	16.7 ± 7.2	8.3 ± 3.6
Bacitracin		11.3 ± 2.8	40.0 ± 13.7	35.4 ± 25.3	10.9 ± 8.5	30.8 ± 25.5	11.7 ± 9.7	3.94 ± 3.86	43.8 ± 12.5	>50	5.9 ± 4.8	2.8 ± 2.2

Extracts and bacitracin were serially 2-fold diluted in 10 wells from 50 mg/mL to 0.1 mg/mL in a 384 wells-microplate where edges were filled with sterility controls to avoid evaporation. Solvent-alone negative controls were included. Data are presented as mean ± standard deviation (S.D.), n = 3.

Table 10. Minimum antibiotic concentration inhibiting 50% of the growth of tested bacterial pathogens (IC₅₀ in μg/mL).

	Bacteria
Antibiotics	Av	Bt	Pa	Pdd	Pdp	Val V1	Val	Va	Vh	Vh2	Vs
Streptomycin	91.5 ± 75.7	439.5 ± 245.8	162.8 ± 56.4	107.4 ± 64.8	81.4 ± 28.1	130.2 ± 56.4	162.8 ± 56.4	336.5 ± 279.9	146.5 ± 84.6	146.5 ± 69.1	3125.0 ± 0.0
Gentamicin	19.6 ± 13.7	58.6 ± 27.6	9.8 ± 6.9	29.3 ± 13.8	17.1 ± 4.8	17.6 ± 4.4	26.0 ± 10.1	97.7 ± 39.1	17.6 ± 4.4	86.1 ± 33.7	43.0 ± 21.4
Oxolinic Acid	9.5 ±8.2	14.1 ± 12.6	>50	15.6 ± 10.8	>50	29.2 ± 19.1	38.0 ± 19.5	30.6 ± 28.3	41.7 ± 14.4	>50	20.8 ± 7.2
Flumequine	5.4 ± 4.5	1.0 ± 0.4	10.0 ± 3.4	3.8 ± 4.3	11.2 ± 2.8	9.9 ± 4.2	8.2 ± 4.5	1.5 ± 1.0	11.7 ± 8.6	13.3 ± 9.0	10.4 ± 8.3
Florfenicol	0.7 ±0.5	0.3 ± 0.2	18.7 ± 7.2	0.5 ± 0.2	1.4 ± 0.4	0.6 ± 0.3	0.8 ± 0.4	0.4 ± 0.2	1.3 ± 0.4	1.6 ± 1.0	0.9 ± 0.5
Oxytetracycline	0.5 ± 0.3	0.2 ± 0.1	5.6 ± 1.4	0.3 ± 0.1	0.7 ± 0.2	0.5 ± 0.2	0.5 ± 0.2	0.2 ± 0.1	0.7 ± 0.2	0.6 ± 0.2	0.2 ± 0.1

Antibiotics (streptomycin at 25 mg/mL, gentamycin at 10 mg/mL, and oxolinic acid, flumequine, florfenicol and oxytetracycline at 50 μg/mL) were serially 2-fold diluted in 21 wells of a 384 wells-microplate where edges were filled with sterility controls to avoid evaporation. Solvent-alone negative controls were included. Data are presented as mean ± standard deviation (S.D.), n = 3.

Table 11. Minimum metabolic inhibitory concentration (MIC in mg/mL) of each insect extracts and bacitracin against tested bacterial pathogens.

		Bacteria
	Insect	Av	Bt	Pa	Pdd	Pdp	Val V1	Val	Va	Vh	Vh2	Vs
Water	BMp	34.4 ± 18.7	44.8 ± 27.1	>50	>50	>50	>50	>50	>50	>50	>50	>50
	HIl	50.0 ± 0.0	>50	>50	48.3 ± 18.1	>50	>50	>50	>50	>50	>50	>50
	TMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	ZMl	50.0 ± 0.0	>50	>50	>50	>50	>50	>50	44.0 ± 17.82	>50	>50	>50
	ADa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	GBa	3.4 ± 2.7	>50	8.3 ± 3.6	9.3 ± 4.4	12.5 ± 0.0	9.4 ± 4.4	25.0 ± 0.0	46.0 ± 19.2	14.6 ± 9.5	>50	20.8 ± 7.2
	OSa	>50	8.3 ± 3.6	>50	>50	>50	>50	>50	>50	>50	>50	>50
Methanol	BMp	23.4 ± 10.7	>50	>50	46.8 ± 19.8	>50	>50	>50	>50	>50	>50	31.2 ± 10.8
	HIl	8.3 ± 3.6	0.3 ± 0.1	>50	>50	>50	1.2 ± 0.7	1.5 ± 1.2	0.6 ± 0.5	>50	0.5 ± 0.4	2.2 ± 1.7
	TMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	ZMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	35.7 ± 24.5
	ADa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	GBa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	OSa	50.0 ± 0.0	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
Isopropanol	BMp	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	41.6 ± 14.4
	HIl	12.5 ± 10.8	0.5 ± 0.2	43.4 ± 23.3	40.8 ± 20.8	34.5 ± 19.7	7.0 ± 4.6	1.3 ± 0.6	0.9 ± 0.5	>50	1.0 ± 0.9	1.2 ± 0.6
	TMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	ZMl	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	ADa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	GBa	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
	OSa	4.2 ± 1.8	32.5 ± 16.8	2.6 ± 0.9	5.7 ± 4.5	3.1 ± 0.0	37.5 ± 17.7	13.5 ± 11.0	42.0 ± 16.0	6.8 ± 5.5	16.7 ± 7.2	10.4 ± 3.6
Antibiotics
Bacitracin		10.9 ± 3.1	33.3 ± 16.5	32.9 ± 26.8	31.0 ± 21.2	7.0 ± 3.9	15.0 ± 5.6	11.2 ± 2.8	37.5 ± 17.7	>50	40.9 ± 26.1	29.2 ± 19.1

Extracts were serially 2-fold diluted in 10 wells from 50 mg/mL to 0.1 mg/mL for all extracts in a 384 wells-microplate where edges were filled with sterility controls to avoid evaporation. Data are presented as mean ± standard deviation (S.D.), n = 3.

Table 12. Metabolic minimum inhibitory concentration (MIC in μg/mL) of antibiotics against tested bacterial pathogens.

	Bacteria
Antibiotics	Av	Bt	Pa	Pdd	Pdp	Val V1	Val	Va	Vh	Vh2	Vs
Streptomycin	48.8 ± 42.3	455.7 ± 298.3	130.2 ± 56.4	115.4 ± 51.8	390.3 ± 0.0	195.3 ± 0.0	227.9 ± 149.2	415.0 ± 280.5	211.6 ± 171.5	97.7 ± 69.1	4687.5 ± 2209.7
Gentamicin	18.3 ± 15.1	55.8 ± 20.9	15.5 ± 12.5	29.1 ± 13.8	19.5 ± 0.0	52.7 ± 34.9	32.6 ± 10.1	82.0 ± 48.6	29.3 ± 10.7	39.1 ± 27.6	25.6 ± 16.6
Oxolinic Acid	17.7 ± 12.6	28.8 ± 20.5	44.7 ± 19.6	39.7 ± 22.7	>50	37.5 ± 21.6	45.4 ± 19.1	39.0 ± 19.2	>50	>50	>50
Flumequine	8.6 ± 6.8	7.0 ± 6.0	32.0 ± 26.3	1.8 ± 1.2	12.5 ± 0.0	20.0 ± 6.0	16.7 ± 6.5	5.0 ± 4.6	17.5 ± 6.8	11.3 ± 9.0	6.9 ± 3.4
Florfenicol	1.1 ± 0.7	0.7 ± 0.7	12.8 ± 9.0	0.4 ± 0.2	1.4 ± 0.3	1.6 ± 0.0	1.4 ± 0.4	0.8 ± 0.6	1.7 ± 0.8	2.3 ± 0.9	1.4 ± 1.0
Oxytetracycline	0.7 ± 0.6	0.3 ± 0.3	4.4 ± 2.6	0.3 ± 0.1	0.8 ± 0.0	1.1 ± 0.4	0.6 ± 0.4	0.5 ± 0.3	0.9 ± 0.3	0.9 ± 0.3	0.3 ± 0.2

Antibiotics (streptomycin at 25 mg/mL, gentamycin at 10 mg/mL, and oxolinic acid, flumequine, florfenicol and oxytetracycline at 50 μg/mL) were serially 2-fold diluted in 21 wells of a 384 wells-microplate where edges were filled with sterility controls to avoid evaporation. Solvent-alone negative controls were included. Data are presented as mean ± standard deviation (S.D.), n = 3.

4. Discussion

4.1. Proximate Composition

This study evaluated the proximate composition of seven insect species for their suitability as alternative ingredients in aquafeeds. Lipid and ash contents were consistent with previously reported ranges, although lipid levels varied considerably, likely due to differences in insect diets, except for the more specialized silkworm. Protein contents were generally at the lower end of published ranges, because a revised nitrogen-to-protein conversion factor (Kp = 4.76) [26] was used accounting non-protein nitrogen sources, such as chitin, chitosan, and other nitrogenous compounds commonly present in insects [34,35]. This adjustment reduces the overestimation of protein and the corresponding underestimation of nitrogen-free extract (NFE) common with the traditional Kp of 6.25 used for animal protein. After correction, protein values largely aligned with published ranges of 35.1–55.1% for BMp [35,36,37,38], 24.1–45.7% for HIl [34,39,40,41,42,43], 27.6–56.4% for TMl [44,45,46], 27.7–40.6% for ZMl [47,48,49,50] and 31.8.5–54.6% for ADa [21,36,46,51,52]. Values for OSa (39.2–42.1%) [22,23] and GBa (42.0–52.2%) [21,24,53] showed slightly lower protein and higher lipid contents, possibly reflecting limited available data. Variability in nutrient composition across studies can largely be attributed to differences in insect diet and processing methods [35,45,51,54].

Among the species analyzed, ADa combined high protein content and low moisture, indicating good potential for feed stability and fish growth performance. However, elevated NFE levels in species ADa and BMp may reduce their nutritional value due to the generally poor digestibility of carbohydrates in fish. The larvae of ZM exhibited the highest lipid content and are known to be rich in mono-unsaturated and poor in poly-unsaturated fatty acids, suggesting potential advantages of using defatted forms [47]. The larvae of HI were notably rich in mineral (ash content) (Table 2), likely related to elevated potassium, calcium, and phosphorus levels reported in the literature [55]. Overall, the findings confirm the favorable nutritional profile of the commonly studied HIl and highlight the potential less-explored, protein-rich species such as ADa and GBa for aquaculture feeds.

4.2. Efficacy of the Extraction Process and Selection of Optimal Solvents for Antibacterial Compound Extraction

Eight solvents of increasing polarity were tested on BMp, with extraction yields ranging from 7.3% (water) to 33.5% (methanol) (Table 3). Lipid-targeting solvents (ethyl acetate and n-hexane) produced yields (25.5 and 24% respectively, Table 3) comparable to BMp lipid content (23.17%; Table 2), whereas methanol achieved the highest recovery due to its ability to extract both hydrophilic and lipophilic compounds [56]. Growth inhibition zone assays revealed that most BMp extracts—except those obtained with dichloromethane—exhibited inhibitory activity against the majority of tested bacteria, with the exception of V. anguillarum, V. splendidus and B. thuringiensis (Table 4). Regarding the minimal extract concentration required to inhibit 50% of bacterial growth (IC₅₀) and the minimum inhibitory concentration (MIC), BMp extracts obtained with methanol, a chloroform/methanol mixture, and to a lesser extent water, exhibited mostly weak antibacterial activity against six fish pathogens, whereas isopropanol extracts were active against strains, but with relatively lower potency (Table 6). In contrast, chloroform and dichloromethane extracts showed relatively higher metabolic inhibition against V. splendidus but limited effect on other bacteria. These results indicate that both polar and nonpolar compounds contribute to the observed antimicrobial activity. Despite the higher efficacy of the chloroform/methanol mixture, subsequent analyses focused on methanol, isopropanol, and water due to chloroform’s toxicity and environmental concerns [57]. Methanol and isopropanol were selected for their efficiency in extracting antibacterial compounds and their relatively lower environmental persistence, despite known toxicity at high exposure levels [56,57,58]. Water, although less effective, was included as a green solvent for recovering hydrophilic bioactive compounds. Across the seven insect species, extraction yields varied widely, ranging from 7.3–42.9% for water, 19.2–50.0% for methanol, and 23.6–38.1% for isopropanol (Table 7). The 48 h extraction period substantially improved yields compared with previous 24 h extractions of HI, which achieved ≤2% yields [14].

4.3. Antibacterial Activity of Common Antibiotics Against Marine Aquaculture Pathogens

In the context of global rise of antimicrobial resistance and public health risks associated with the wide use of antibiotics [16,59], insect-derived feed ingredients enriched in antibacterial bioactive compounds may represent a more sustainable disease management strategy. The present study represents a preliminary screening of antibacterial potential of seven insect species. Although the antibacterial assays used in the present study do not distinguish between antibiotic-sensitive and resistant bacterial strains, they enable a comparative evaluation of the efficacy of insect extracts and conventional antibiotics. Most antibiotics tested showed very strong antibacterial activity (IC₅₀ and MIC < 1 mg/mL) against all fish pathogens. An exception was the case of streptomycin which exhibited a lower but still strong activity against V. splendidus (1 mg/mL < IC₅₀ and MIC < 5 mg/mL) (Table 10 and Table 12). This aligns with widespread reports of streptomycin resistance in aquaculture pathogens, including Aeromonas, Bacillus, Pseudomonas, and Vibrio species [10,59,60,61,62,63,64]. Similarly, oxolinic acid showed weak activity (IC₅₀ and/or MIC > 25 mg/mL) against several pathogens, including P. aeruginosa, P. damselae subsp. piscicida, and Vibrio spp., in agreement with previously documented resistance in aquaculture environments [10,59,65,66]. In contrast, florfenicol and oxytetracycline demonstrated the highest efficacy (IC₅₀ and MIC < 3 μg/mL) against most tested bacteria, except P. aeruginosa (Table 10 and Table 12), although resistance to these two antibiotics has been widely reported in the Mediterranean region [10]. Notably, some insect-derived compounds investigated in the present study showed antibacterial activity comparable to that of bacitracin and, in certain cases, streptomycin, as discussed below.

4.4. Antibacterial Activity of the Extracts of Each Insect Species Tested

Insect extracts—particularly GB aqueous extracts, HI methanolic extract and isopropanolic extracts of HI and OS exhibited notable antibacterial activity against a broad range of bacteria, including strains with relatively reduced sensitivity to reference antibiotics such as B. thuringiensis, P. damsela piscida, V. harveyi and V. splendidus, which showed relatively low susceptibility to streptomycin and/or oxolinic acid. Compared to the polypeptide antibiotic bacitracin produced by Bacillus licheniformis, aqueous extracts of OS and GB, as well as methanolic extracts of HI and isopropanolic extractsof HI and OS, showed lower IC₅₀ and MIC values against most tested bacteria. Methanolic and isopropanolic extracts of BM also produced larger growth inhibition zones (GIZs) against Pa and ValV1. In addition, aqueous extracts of TM and ZM generated a larger GIZ than bacitracin against Pdd (Table 8).

Insect-derived bioactive compounds with known antibacterial activity include antimicrobial peptides (AMPs), which often outperform conventional antibiotics against multidrug-resistant strains [67,68], medium-chain saturated fatty acids, including lauric (C12:0), capric (C10:0), and myristic (C14:0) acids [13], and chitosan, a derivative of chitin [69,70]. Details for each insect extracted with the three selected solvents are further discussed below.

The antibacterial activity of silkworm pupae (BMp) extracts obtained using solvents of varying polarity, with the exception of dichloromethane, was evidenced by growth inhibition zones against multiple fish pathogens (A. veronii, P. aeruginosa, P. damselae subspecies, and V. spp.), as well as measurable IC₅₀ and MIC values, albeit generally at concentrations above 6.3 mg/mL. This bioactivity is likely attributable to AMPs, such as moricin, BM-ponericin-L1, cecropins, defensins, enbocins, gloverins, and lebocins, which are well-documented in B. mori. [71,72,73], polysaccharide silkrose [11,74]. Previous studies have also reported moderate antibacterial activity of BM extracts rich in fatty acids against resistant bacteria [75], although results were variable depending on extraction methods [76]. Overall, these findings support the potential use of silkworm pupae as a functional ingredient or additive in aquafeeds, particularly in Mediterranean regions where sericulture by-products are readily available.

The antibacterial activity of HIl extracts obtained using the three selected solvents was evaluated against a range of fish pathogenic bacteria. In the growth inhibition zone assay, only the isopropanolic extract exhibited weak activity against P. aeruginosa (7.5 mm), although the diffuse inhibition zone suggested a primarily bacteriostatic rather than bactericidal effect. This observation was supported by MTT assays, which showed a weak inhibitory activity against P. aeruginosa. The aqueous HIl extract displayed little or no antibacterial activity, contrasting with previous reports of broader efficacy [42]. In contrast, the methanolic and isopropanolic HIl extracts exhibited strong antibacterial activity, against several fish pathogens, including A. veronii, B. thuringiensis, P. damselae subsp. piscida, and multiple Vibrio species (V. alginolyticus and V. harveyi). Their activity generally exceeded that of bacitracin and, in the case of V. splendidus, those of both bacitracin and streptomycin. These findings agree with earlier reports of antibacterial activity of HI methanolic extracts against both Gram-positive and Gram-negative bacteria, in contrast to extracts using other solvents, including n-hexane, chloroform, ethanol and water [14,30,77,78,79]. The broad antimicrobial activity of HIl is likely due to its diverse array of bioactive compounds, including antimicrobial peptides (defensins, cecropins, attacins, diptericins), polysaccharides (dipterose), fatty acids (palmitic acid, lauric acid), sterols, and chitin/chitosan [13,42,75,78,80,81,82,83,84,85,86,87], which help protect larvae from microorganisms developing in the moist organic substrates. Moreover, antimicrobial compound production can be enhanced through immune stimulation [18,87,88,89,90], and dietary HIl has consistently improved disease resistance in aquaculture species [28,91,92,93,94]. Overall, these results highlight HIl as a highly promising insect species for aquaculture applications owing to its broad-spectrum antimicrobial properties.

The larval extracts of yellow mealworm (TMl) and superworm (ZMl), generally exhibited weak to no antibacterial activity. Unlike HIl, these species develop on relatively dry substrates, such as flour, wheat bran or cereal grains, which may reduce the need for antimicrobial defenses. Nevertheless, the aqueous extracts produced notable GIZ against P. damselae subsp. damselae, a pathogen resistant to the other insect extracts and to bacitracin and gentamycin, although this activity was not confirmed by microdilution assays. TMl isopropanolic extract showed weak activity against A. veronii, while ZMl methanolic extract moderately inhibited V. splendidus, a strain with low sensitivity to bacitracin and streptomycin. Previous studies have reported broader antibacterial activity of ZMl extracts, particularly using acidified methanol [95]. The antibacterial properties of TMl are attributed to bioactive compounds, including antimicrobial peptides (e.g., tenecin, coleoptericin, attacin, and defensins; reviewed by Errico [96]), chitosan and lipid-derived molecules. TMl-derived chitosan has demonstrated greater antibacterial activity than commercial preparations [97,98], while its weaker lipid-associated activity likely arises from compounds other than lauric acid, which is present at much lower levels than in HIl [13,80]. As with HIl, immune stimulation can enhance the production of bioactive compounds in both TMl and ZMl [95,99]. Moreover, the stability of TMl bioactive compounds under a wide range of processing conditions (temperature, pH, and salinity) [99] and evidence that dietary TMl enhances the resistance of red seabream (Pagrus major) to Edwardsiella tarda infection [100] further support its potential for aquaculture applications.

Concerning ADa, none of the aqueous, methanolic or isopropanolic extracts exhibited antibacterial activity under the tested conditions, as determined by the three assays (GIZ, IC₅₀, MIC). Although ADa is authorized for use in animal feed within the European Union, available literature on the antimicrobial properties of ADa remains scarce. Aqueous extracts have previously demonstrated antibacterial activity against several Gram-negative bacteria, although the concentrations tested were not specified [101]. Additionally, chitin derived from ADa has shown antibacterial effects against both Gram-positive and Gram-negative bacteria [102].

Another cricket species, GBa, is commonly used in animal nutrition (pets, reptiles, birds, and livestock) and in traditional medicine due to its high protein content and richness in functional bioactive compounds [103]. However, it has not yet been authorized by the European Commission for use in aquafeeds. Previous studies have demonstrated that dichloromethane extracts enriched in sterols exhibit antibacterial activity against Staphylococcus aureus [75], while chitosan derived from this species shares key physicochemical and potentially antimicrobial properties with shrimp-derived chitosan [104]. In the present study, the aqueous extract of GBa showed antibacterial activity against 9 of the 11 bacterial strains tested, with IC₅₀ and MIC values as low as 3.4 mg/mL against Av (Table 9 and Table 11). In contrast, the methanolic and isopropanolic extracts exhibited no detectable antibacterial activity. The pronounced activity of the aqueous extract is likely associated with hydrophilic bioactive compounds, such as chitin/chitosan derived molecules or antimicrobial peptides [104]. Collectively, these findings highlight GBa as a promising functional ingredient for aquafeeds, combining nutritional value with a diverse range of bioactive properties.

Weaver ants (OSa) are known for their nest-building behavior and production of antiseptic formic acid, and have long been used in traditional Southeast Asian medicine to treat wounds and infections [105]. Previous studies have reported variable antimicrobial activity, with methanolic extracts active against S. aureus and Escherichia coli and aqueous extracts attributed to benzoic acid derivatives [22,28,106]. In the present study, OSa extracts did not produce growth inhibition zones (GIZs) at 50 mg/mL against the tested bacteria. However, the aqueous extract exhibited weak inhibitory activity against B. thuringiensis, the methanolic extract showed only marginal effect, and the isopropanolic extract exhibited moderate to strong antibacterial activity against 8 out of the 11 bacterial strains, with IC₅₀ and MIC values usually lower than those of bacitracin. Given the growing interest in commercially available ant-derived compounds [107], OSa represents a very promising natural source of novel antibacterial agents in the fight against bacterial pathogens.

4.5. Feasibility of Insect-Derived Antibacterial Compounds in Aquafeeds

Extracts from three of the seven insect species evaluated in the present study (GBa, HIl and OSa) demonstrated moderate to strong antibacterial activity against fish pathogens. The extraction of antibacterial compounds from GBa using the most environmentally friendly solvent, i.e., water, was particularly interesting, but its relatively low extraction yield (13.7%) may limit practical application. In contrast, the methanolic and isopropanolic extracts of HIl, as well as the isopropanolic extract of OSa, combined higher extraction yields (>26.9%) with strong antibacterial activity across a broad range of pathogens, in some cases equivalent to antibiotics such as streptomycin and bacitracin.

The use of antibiotics in fish feeds for the treatment of bacterial infections typically requires concentrations ranging from 20 to 250 mg/kg body weight (BW) [59], corresponding to approximately 1–12.5 g/kg of feed when fish are fed at 2% of BW per day. Considering that bioactive compounds extracted from insects in the present study were, at best, approximately 500-fold less potent than oxytetracycline (as obtained for the methanolic extract of HIl and the isopropanolic extract of OSa), which is commonly used in aquaculture at 1–2 g/kg feed, the use of crude insects for therapeutic purposes is neither feasible nor practical. On the contrary, given the extraction yields calculated for each extract, the use of whole insects (adults or larvae) in functional feeds at 0.1 to 2% inclusion, or as a sustainable alternative to fishmeal at inclusion levels of up to 30%, would correspond to an estimated incorporation of approximately 0.0137–4.1% of the aqueous GBa extract, 0.037–11.2% of the methanolic HIl extract, and 0.027–8.2% of the isopropanol HIl or OSa extracts, equivalent to 137 mg to 112 g of insect-derived extract per kg of feed. Notably, all four extract types exhibited relatively higher activity than bacitracin against most of the tested bacterial pathogens and the recommended inclusion level of bacitracin as feed additive ranges from 50 to 275 mg/kg animal feed. However, the European Union has banned the use of bacitracin in 1998 (Council Regulation No 2820/98). Alternative antibacterial additives, such as medium-chain fatty acids, are usually administered at inclusion levels of 1–2 g/kg of aquafeed [107]. At realistic inclusion levels, namely 0.1–5.3 g HIl/kg feed, 0.4–14.6 g GBa/kg feed or 0.2–7.3 g OSa/kg feed, insect-derived ingredients could deliver biologically relevant concentrations of antibacterial compounds (approximately 50–2000 mg/kg feed), while aligning with current regulatory restrictions in aquaculture. This approach therefore represents a feasible nutritional strategy for antimicrobial intervention [108], either through direct inhibition of bacterial pathogens or indirectly via immunostimulation and increase of the fish resistance to bacterial diseases. From an economic and environmental perspective, insect meals can become competitive with fishmeal when considering not only ingredient costs, but also benefits such as improved growth performance, reduced disease-related losses, lower antibiotic usage, potential price premiums for sustainably produced seafood, and a reduced environmental footprint through decreased pressure on wild fish stocks, lower greenhouse gas emissions, and valorizing organic waste streams. However, the generation of precise mechanistic and applied knowledge remains essential to fully establish insects as viable and resilient innovative ingredients for aquafeeds.

5. Conclusions

The present study highlights the antibacterial activity of GBa and HIl and the untapped potential of the less-studied species OSa, as well as the potential for these three insect species to be sustainable ingredients for the development of next-generation aquafeeds reducing reliance on conventional antibiotics. Future research should include the purification and identification of active compounds, investigation of mechanisms of antibacterial action, assessment of toxicity and safety profiles, and validation of efficacy of dietary HIl, GBa and OSa in Mediterranean fish, focusing on growth performance, gut microbiota modulation, immune responses, and disease resistance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fishes11070389/s1, Suppementary Material S1: Photographs of two agar plates.

Author Contributions

Conceptualization: M.H.; methodology: M.H., A.V., D.K.; investigation: M.H., G.D., H.G., D.C., M.C., P.C., C.N.; data curation: M.H., G.D., H.G., C.N.; formal analysis: M.H., G.D., H.G., D.C., M.C.; writing—original draft: M.H., G.D., H.G.; writing review and editing: M.H., G.D., D.XC, D.K.; resources: M.H.; project administration: M.H.; supervision: M.H.; validation: M.H.; visualization: M.H., M.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to thank Panthelis Katharios and Constantina Kokkari of the HCMR for providing the bacteria used in the present study from their extensive microbial collection. The authors are also indebted to I. Karapanagiotidis of the Agronomical University of Volos for providing the insect meals. During the preparation of this manuscript, the authors used ChatGPT (version 5.5) for the purposes of improving language and readability. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:

ADa	House cricket (Acheta domesticus) adult
AMPs	Antimicrobial peptides
BMp	silkworm (Bombyx mori) pupae
CP	Crude protein
EE	Ether extract
GBa	two-spotted cricket (Gryllus bimaculatus) adult
GE	Gross energy
GIZ	Growth Inhibition Zone
HIl	Black soldier fly (Hermetia illuscens) larvae
IC₅₀	minimal concentration inhibiting 50% of bacterial growth
MIC	minimal metabolic inhibitory concentration
MTT	(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)
NFE	Nitrogen-free extract
OSa	weaver ant (Oecophylla smaragdina) adult
TSA	Tryptic soy agar
TSB	Tryptic soy broth
TMl	Yellow mealworm (Tenebrio molitor) larvae
ZMl	Superworm (Zophobas morio) larvae

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Debailleul, G.; Gatouillat, H.; Chatzimpalasis, D.; Chatteleyn, M.; Chronopoulos, P.; Nikoloudaki, C.; Vasilaki, A.; Kogiannou, D.; Henry, M. Antibacterial Activity of Extracts from Seven Insect Species Against Mediterranean Marine Aquaculture Bacterial Pathogens. Fishes 2026, 11, 389. https://doi.org/10.3390/fishes11070389

AMA Style

Debailleul G, Gatouillat H, Chatzimpalasis D, Chatteleyn M, Chronopoulos P, Nikoloudaki C, Vasilaki A, Kogiannou D, Henry M. Antibacterial Activity of Extracts from Seven Insect Species Against Mediterranean Marine Aquaculture Bacterial Pathogens. Fishes. 2026; 11(7):389. https://doi.org/10.3390/fishes11070389

Chicago/Turabian Style

Debailleul, Gatien, Hugo Gatouillat, Dimitrios Chatzimpalasis, Matteo Chatteleyn, Petros Chronopoulos, Chrisanthi Nikoloudaki, Antigoni Vasilaki, Dimitra Kogiannou, and Morgane Henry. 2026. "Antibacterial Activity of Extracts from Seven Insect Species Against Mediterranean Marine Aquaculture Bacterial Pathogens" Fishes 11, no. 7: 389. https://doi.org/10.3390/fishes11070389

APA Style

Debailleul, G., Gatouillat, H., Chatzimpalasis, D., Chatteleyn, M., Chronopoulos, P., Nikoloudaki, C., Vasilaki, A., Kogiannou, D., & Henry, M. (2026). Antibacterial Activity of Extracts from Seven Insect Species Against Mediterranean Marine Aquaculture Bacterial Pathogens. Fishes, 11(7), 389. https://doi.org/10.3390/fishes11070389

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Antibacterial Activity of Extracts from Seven Insect Species Against Mediterranean Marine Aquaculture Bacterial Pathogens

Abstract

1. Introduction

2. Materials and Methods

2.1. Insects

2.2. Extraction Protocol

2.3. Bacterial Strains and Culture Conditions

2.4. Antibacterial Assays

2.4.1. Agar Well Diffusion Method

2.4.2. Kinetic Microdilution Assay

3. Results

3.1. Proximate Composition of the Seven Insects

3.2. Preliminary Experiment on BMp

3.2.1. Extraction Yield of Silkworm Pupae

3.2.2. Antibacterial Activity of Silkworm Pupae Extracts

3.3. Comparison of Extracts from the 7 Insect Species

3.3.1. Extraction Yield of the Seven Insect Species

3.3.2. Antibacterial Activity of Extracts from the Seven Insects and Reference Antibiotics

4. Discussion

4.1. Proximate Composition

4.2. Efficacy of the Extraction Process and Selection of Optimal Solvents for Antibacterial Compound Extraction

4.3. Antibacterial Activity of Common Antibiotics Against Marine Aquaculture Pathogens

4.4. Antibacterial Activity of the Extracts of Each Insect Species Tested

4.5. Feasibility of Insect-Derived Antibacterial Compounds in Aquafeeds

5. Conclusions

Supplementary Materials

Author Contributions

Funding

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Acknowledgments

Conflicts of Interest

Abbreviations

References

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