Larval Extracts Research Articles

Effects of Apilarnil and Queen Bee Larvae on Larval Mortality and Longevity in Drosophila melanogaster

Various functional food sources are being researched to overcome nutritional deficiencies, imbalances and protein deficiencies. As a result of the researches, it has been reported that drone larvae (apilarnil) and queen larvae support the protection of health due to their high nutritional value. Apilarnil and queen larvae extracts added to standard D. melanogaster medium at different concentrations (0.5; 1.0; 2.5 and 5.0 mg/ml medium) were studied separately in male and female populations for treatment and control groups. Apilarnil showed the best effect at a concentration of 2.5 mg/ml, while the lifespan of flies exposed to queen larvae extracts increased in parallel with the increase in concentration in both populations and these results were statistically significant at the p

Evidence of Cuticle Chemicals of Heortia vitessoides (Lepidoptera: Crambidae) Larvae Influencing the Aggregation Behavior of Conspecific Larvae.

Heortia vitessoides (Lepidoptera: Crambidae) is a severe pest of Aquilaria plants, which produce high-priced agarwood. The larval stage of this pest is gregarious, usually forming large aggregates during young instars and becoming solitary during the fifth instar. We hypothesize that the cuticle chemicals of young-instar H. vitessoides larvae could promote larval aggregating, whereas the cuticle chemicals of late-instar larvae would no longer attract young-instar larvae. In this study, two-choice tests were conducted to evaluate the effect of cuticle extracts of second- and fifth-instar H. vitessoides larvae on the aggregation preference of second-instar larvae. Results show that significantly more larvae aggregated on the leaves treated with the hexane extract of second-instar H. vitessoides larvae than on untreated leaves. However, the hexane extract of fifth-instar larvae had no significant effect on the aggregation preference of the second-instar conspecific larvae. Interestingly, acetone extracts of both second- and fifth-instar H. vitessoides larvae repelled the second-instar conspecific larvae throughout the 8 h experiment. Our study shows that cuticle chemicals of H. vitessoides larvae may play a role in the group dynamics of this pest, which may contribute to screening novel attractants and repellents for H. vitessoides. Detailed chemical analyses of the extracts and identification of the compounds involved in larval attracting and repelling would be valuable in future studies.

Exploring the potential of Hermetia illucens larvae extracts: A promising approach for dermocosmetic formulations

Globally, the yearly disposal of 1.3 billion tonnes of food raises environmental and public health concerns. Black soldier fly (BSF) larvae present a sustainable solution, converting organic waste into nutrient-rich biomass. The extracted oil from BSF larvae, rich in fatty acids (FA), offers an eco-friendly alternative for the cosmetic industry. In this study, larvae sourced from a Portuguese company were fed olive pomace, a by-product of olive oil production.The lipidic sample extracted revealed a composition high in oleic acid, valuable for cosmetics. Investigating the biological activity of lipid extractions from larvae fed with olive pomace is a novel approach. Notably, the n-hexane ultrasound-assisted extraction method demonstrated potent antioxidant properties, and some extracts displayed antimicrobial activity. Five non-cytotoxic extracts; three with no relevant activity (IC50 from 236 to >400 μg/mL).These findings highlight BSF larvae as an environmentally friendly source of fatty acids, offering promising alternatives for diverse applications.

Anti-muscle atrophy effect of fermented Tenebrio molitor larvae extract by modulating the PI3K-Akt-mTOR/FoxO3α pathway in mice treated with dexamethasone

This study investigated the anti-sarcopenic effect of fermented Tenebrio molitor larvae (mealworms) extract (FME) in both dexamethasone (DEX)-treated C2C12 cells and mice. FME (100 µg/mL) increased the diameter of myotubes and inhibited the gene and protein expression of atrogin-1 compared to DEX- or non-fermented mealworms extract (ME)-treated C2C12 cells. Male C57BL/6N mice were divided into five groups: Normal Control (NC), DEX (10 mg/kg, intraperitoneal), and three groups of DEX+FME (100, 200, or 500 mg FME/kg/day, oral) for two weeks. FME at doses of 200 and 500 mg/kg effectively improved grip strength when compared to the DEX group. Histological analysis of the quadriceps muscle showed a larger muscle fiber size in the DEX+FME groups compared to DEX group. FME (200 and 500 mg/kg) significantly increased cross-sectional area of the muscle fiber compared to DEX group. FME (500 mg/kg) significantly decreased the ubiquitin, atrogin-1 and MuRF-1 protein levels, and increased levels of MHC and MyoG in DEX-treated mice. The puromycin labeling assay revealed that FME increased protein synthesis in DEX-induced muscle atrophy. The FME treatment demonstrated significant upregulation in phosphorylation levels, including mTOR, FoxO3α, Akt, and PI3K compared to DEX group. In conclusion, FME inhibited the increase in proteins associated with muscle atrophy, including, atrogin-1 and MuRF-1, by regulating the PI3K-Akt-FoxO3α pathway. FME improved the PI3K-Akt-mTOR signaling pathway, which was reduced by DEX. This study suggests that FME has the potential for use in sarcopenia therapy, possibly serving as a natural agent that counteracts the negative effects of DEX on muscle tissue.

Evaluating foliar insecticides and economic thresholds for Tychius picirostris (Coleoptera: Curculionidae) management in Oregon white clover seed production.

The clover seed weevil, Tychius picirostris Fabricius (Coleoptera: Curculionidae), is a major pest in Oregon white clover seed crops. Reliance on synthetic pyrethroid insecticides and limited availability of diverse modes of action (MoAs) has increased insecticide resistance selection in regional T. picirostris populations, emphasizing the need to evaluate novel chemistries and rotational strategies for effective insecticide resistance management (IRM). The efficacy of 8 foliar insecticide formulations for managing T. picirostris adult and larval life stages was determined in small and large-plot field trials across 2 crop years. In both years, bifenthrin (Brigade 2EC), the grower's standard, showed negligible adult and larval suppression. Insecticide formulations with isocycloseram and cyantraniliprole active ingredients reduced adult and larval populations when applied at BBCH 59-60 (prebloom) and BBCH 65-66 (full bloom) growth stages, respectively. While differences in T. picirostris abundance were observed among insecticide treatments, seed yield differences were not detected in large-plot trials. Larval abundance was correlated with reduced seed yield, and an economic threshold of ≥3 larvae per 30 inflorescences was determined as a conservative larval threshold to justify foliar applications of diamide insecticides. Additional commercial white clover seed fields were surveyed to compare larval scouting techniques, including a standard Berlese funnel and a grower's do-it-yourself funnel. Both larval extraction techniques were correlated and provided similar estimates of larval abundance. These findings demonstrate new MoAs, optimal insecticide application timing, and larval monitoring methods that can be incorporated into an effective T. picirostris IRM program in white clover seed crops.

Eco-friendly pH-responsive iron-doped insect larval frass extract as a hexaconazole nano-delivery system for controlling fungal disease and promoting crop growth

Developing environmentally friendly pesticides and improving pesticide utilization efficiency are urgent needs for modern agriculture. Herein, a pH-responsive biodegradable dual pesticide-fertilizer was fabricated using extracts from naturally occurring Protaetia brevitarsis larval frass (PBLF) material. Iron ions (Fe3+) were integrated as metal nodes into PBLF, and hexaconazole (Hex), a triazole fungicide, was encapsulated in Fe-PBLF with a loading rate of 22.93 %. Hex@Fe-PBLF nanoparticles (NPs) presented a sphere-like construction with a hydrated size of 715.3 ± 5.0 nm and exhibited acid-responsive controlled release behavior, gradually self-degrading in acidic environments within 120 h. The NPs showed good foliar adhesion and were effectively absorbed and translocated by pathogenic fungal mycelium and the rice seedling. Compared with Hex Technical control (TC), the control effects of Hex@Fe-PBLF against Rhizoctonia solani (R. solani) and Botrytis cinerea (B. cinerea) increased by 9.9 % and 12.73 %, respectively, after 7 days. This naturally sourced nanocarrier not only promoted the growth of rice seedlings as a fertilizer but also enhanced their stress resistance. The low mobility of Hex@Fe-PBLF NPs in soil effectively reduced environmental risks. The excellent biosafety of the Fe-PBLF nanocarrier was further evaluated. Overall, this environmental-friendly multifunctional nano pesticide-fertilizer has promising application potential in agriculture.

Protaetia brevitarsis larvae extract ameliorated obesity in high fat diet-fed mice by modulating gut microbiota and lipid metabolism

<em>Protaetia brevitarsis</em> larvae extract ameliorated obesity in high fat diet-fed mice by modulating gut microbiota and lipid metabolism

Antibacterial properties of oil extracts of black soldier fly larvae reared on bread waste

Context Farming black soldier fly larvae (BSFL) has become an emerging agricultural sector for upcycling food waste into high-quality protein and oil biomass. Depending on the chemical composition of the food waste, the oil extracted from BSFL can reach high levels of lauric acid, a natural antimicrobial compound. Aims We aimed to evaluate whether feeding a bakery waste-based substrate can enhance the lauric acid synthesis of BSFL. In addition, we aimed to test the antibacterial activity of these BSFL oil extracts against E. coli in vitro. Methods In a 12-day-long feeding trial, we reared BSFL either on a ground corn-based control substrate (n = 6) or a substrate based on bread waste sourced from a local bakery in Hong Kong (n = 6). We studied the growth performance, crude fat and lauric acid content, and agar well-diffusion assay-based antibacterial activity of oil samples extracted from the larvae. Key results The BSFL reared on the bread waste had a higher average weight (P &lt; 0.001) and average daily gain (P &lt; 0.001), similar crude fat content (P = 0.17), and higher lauric acid content (P &lt; 0.001) than did the control larvae. Both oil samples moderately inhibited the growth of E. coli in vitro; the inhibition zones were 1.92 ± 1.0 mm and 1.25 ± 0.75 mm (means ± s.d.) big in the bread waste-based and the control oil samples respectively (P = 0.28). Conclusions Our results indicated that locally sourced bread waste is a suitable substrate for farming BSFL, providing a sustainable alternative for waste management. Implications The inclusion of locally produced BSFL oil as a feed ingredient for the local livestock farming sector might be an avenue not only to achieve circular economy, but also to reduce antimicrobial drug use. However, the effects on animal health and productive performance warrant further in vivo experiments.

Trichinella spiralis Larval Extract as a Biological Anti-Tumor Therapy in a Murine Model of Ehrlich SolidCarcinoma.

Trichinella spiralis (T. spiralis) is an immunomodulating parasite that can adversely affect tumor growth and extend host lifespan. The aim of this study was to elucidate the mechanisms by which T. spiralis larval antigens achieve this effect using Ehrlich solid carcinoma (ESC) murine model. Assessment was done by histopathological and immunohistochemical analysis of caspase-3, TNF-α, Ki-67 and CD31. Additionally, Bcl2 and Bcl2-associated protein X (Bax) relative gene expression was assessed by molecular analysis for studying the effect of T. spiralis crude larval extract (CLE) antigen on tumor necrosis, apoptosis, cell proliferation and angiogenesis. We found that both T. spiralis infection and CLE caused a decrease in the areas of necrosis in ESC. Moreover, they led to increased apoptosis through activation of caspase-3, up-regulation of pro-apoptotic gene, Bax and down-regulation of anti-apoptotic gene, Bcl2. Also, T. spiralis infection and CLE diminished ESC proliferation, as evidenced by decreasing Ki-67. T. spiralis infection and CLE were able to suppress the development of ESC by inhibiting tumor proliferation, inducing apoptosis and decreasing tumor necrosis, with subsequent decrease in tumor metastasis. T. spiralis CLE antigen may be considered as a promising complementary immunotherapeutic agent in the treatment of cancer.

Validation of vacuum extraction protocol for larval sampling of spotted-wing drosophila in blueberries

For spotted-wing drosophila, Drosophila suzukii Matsumura, the standard larval sampling protocol from the fruits involves immersing the fruits in hot water, salt, or sugar solution followed by enumerating the larvae that exit from the fruit. In this study, using the fruit samples collected from commercial blueberry farms, we validated the efficacy of a novel D. suzukii larval sampling technique, the vacuum extraction protocol. The larval extraction efficacy of the vacuum extraction method was compared with the salt extraction method using paired fruit samples collected from three different fields for four consecutive weeks. Overall, larval counts detected through the vacuum extraction were significantly higher than those detected through salt extraction in two out of three blueberry fields. Additionally, among the five blueberry fruit sample volumes tested (118, 177, 237, 355, and 473 mL), we identified 355 mL (12 fl. oz.) as the optimum fruit sample volume for vacuum extraction that maximized sampling precision for unit sampling cost. Since D. suzukii larvae exited from fruits after vacuum extraction remain alive and active, a delay in separating the larvae from the sample with coffee filter extraction may result in larval movement back into the fruit flesh. However, compared with immediate processing, delaying coffee filter extraction up to 60 minutes after vacuum incubating the fruit samples at -98 kPa for 60 minutes did not significantly reduce the larval count. Overall, the results of validation trials indicate that vacuum extraction is a promising sampling method for growers to detect D. suzukii larval infestation in blueberries.

Edible insects as a novel source of lecithin: Extraction and lipid characterization of black soldier fly larvae and yellow mealworm

Edible insects with high fat and phosphorus content are a potential novel source of lecithin, however, studies on their minor lipids are limited. In this study, lecithin was extracted from black soldier fly larvae and yellow mealworm. Herein, the effects of lecithin extraction method, matrix and ultrasound pretreatment were explored based on the fatty acid composition and phospholipid profile with soy lecithin as a reference. The use of a wet matrix and ultrasound pretreatment increased the extraction efficiency of total PLs from both insects. Insect lecithin contained a considerable amount of sphingomyelin compared to soy lecithin. In insect lecithin, a total of 47 glycerophospholipid and sphingomyelin molecular species, as well as four molecular species of fatty acyl esters of hydroxy fatty acid, were detected. This study is the first comprehensive investigation of insects as a new source of lecithin with applications in food, cosmetics and in the pharmaceutical industry.

Antiproliferative activity &lt;i&gt;in vitro&lt;/i&gt; of &lt;i&gt;Trichinella spiralis&lt;/i&gt; larval extracts depending on the infection stage following experimental infection of rats

Relevance.The aim is evaluation in vitro antiproliferative activity of T. spiralis larvae extracts obtained from experimentally infected rats, depending on the duration of infection.Methods. The experiment was carried out on outbred male rats infected with T. spiralis larvae at the rate of 10 larvae per 1 g of body weight. The antiproliferative activity of larval extracts obtained 40 days, 3 and 12 months after infection was determined on tumor cells of the A549 line (human lung carcinoma) using an MTT test during cultivation for 24, 48 and 72 hours in concentrations from 32 to 1000 micrograms/ml. Antiproliferative activity was expressed as % inhibition and IC50.Results. There was direct dependence of Trichinella extract antiproliferative effects on time of cultivation with A549 cells and protein concentration in extracts. At all stages of invasion, the antiproliferative activity of the extract increased from 24 to 72 hours. at a protein concentration of 250 to 1000 mcg/ml. The extract antiproliferative activity expressed in tumor cell % inhibition and IC50 increased from day 40 to month 12. The most intense dynamics was observed between day 40 and month 3. The possible reason of enhancement in extract antiproliferative effect at months 3 and 12 is alteration in quantitative and qualitative composition of proteins comprised the extracts.

Diabrotica v. virgifera Seems Not Affected by Entomotoxic Protease Inhibitors from Higher Fungi.

Certain soil insects, such as the root-damaging larvae of the maize pest Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), are increasingly difficult to control because of recent bans of some insecticides. An alternative and safer approach may be the development of biopesticides based on entomotoxic defense proteins of higher fungi. Many of these potentially interesting proteins are protease inhibitors, and some have been shown to adversely affect insects. We examined the effects of the cysteine protease inhibitors macrocypin 1, 3, and 4 from Macrolepiota procera, clitocypin from Clitocybe nebularis, and cocaprin 1 and the serine protease inhibitor cospin 1 from Coprinopsis cinerea on D. v. virgifera. We confirmed the inhibition by mycocypins of the cysteine catalytic-type proteolytic activities in gut extracts of larvae and adults. The inhibition of pGlu-Phe-Leu-hydrolyzing activity was stronger than that of Z-Phe-Arg-hydrolyzing activity. Mycocypins and cospin resisted long-term proteolytic digestion, whereas cocaprin 1 was digested. Bioassays with overlaid artificial diet revealed no effects of proteins on neonatal mortality or stunting, and no effects on adult mortality. Immersion of eggs in protein solutions had little effect on egg hatching or mortality of hatching neonates. Microscopic analysis of the peritrophic matrix and apical surface of the midguts revealed the similarity between larvae of D. v. virgifera and the chrysomelid Leptinotarsa decemlineata, which are sensitive to these inhibitors. The resistance of D. v. virgifera to fungal protease inhibitors is likely due to effective adaptation of digestive enzyme expression to dietary protease inhibitors. We continue to study unique protein complexes of higher fungi for the development of new approaches to pest control.

Hermetia illucens as a Source of Antimicrobial Peptides – A Review of in vitro and in vivo Studies

Abstract Antimicrobial peptides (AMPs) are the focus of this manuscript, as bioconservative molecules that constitute a major defense mechanism in many organisms. Various antimicrobial peptides have been isolated and identified, but AMPs derived from Hermetia illucens (HI) will be the focus of this review. The review focuses mainly on cecropins, defensins, and attacins. Hermetia illucens is a remarkable organism adapted to life in a diverse, often highly polluted environment, and its resilience is largely attributed to amps. Amps are active against many bacterial and fungal species and also act to induce the osmotic lysis of protozoa. They attack pathogenic microorganisms without damaging host cells in the process. Given the increasing antibiotic resistance of many bacterial strains in animal production, amps appear to be a tempting alternative as substances that limit and inhibit the growth and development of pathogens, as well as an option in veterinary medicine as potential substitutes for antibiotics, the administration of which must be limited in the european union. The amp content of HI larvae extracts, which determines their activity, depends on the larval diet and the solvent used. This review presents in vitro studies on the effects of amps from Hermetia illucens on microorganisms and in vivo studies on the potential of Hi larvae meal as a feed supplement.

Protaetia brevitarsis larvae extract protects against lipopolysaccharides-induced ferroptosis and inflammation by inhibiting acid sphingomyelinase.

Inflammation and ferroptosis are implicated in various diseases and lipopolysaccharides (LPS) have been linked with these disorders. Recently, many edible insects, such as Gryllus bimaculatus, Protaetia brevitarsis larvae (PB) and Tenebrio molitor larvae, have been recommended as alternative foods because they contain lots of nutritional sources. In this study, we explored the potential of PB extract in preventing LPS-induced inflammation and ferroptosis in Hep3B cells. PB powder was extracted using 70% ethanol and applied to Hep3B cells. Co-treatment with LPS was conducted to induce ferroptosis and inflammation. The anti-inflammatory and anti-ferroptosis mechanisms of the PB extract were confirmed using Western blot, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction analysis. PB extract effectively prevented LPS-induced cell death and restored LPS-induced inflammatory cytokine production, NF-κB signaling, endoplasmic reticulum (ER) stress and ferroptosis. Interestingly, PB extract reduced LPS-induced ceramide increase and acid sphingomyelinase (ASMase) expression. The use of the ASMase inhibitor, desipramine, also demonstrated a reduction in these pathways, highlighting the pivotal role of ASMase in inflammation and ferroptosis. Treatment with each inhibitor revealed that ferroptosis causes ER stress and that NF-κB and MAP kinase pathways are involved in inflammation. PB emerges as a potential functional food with inhibitory effects on LPS-induced inflammation and ferroptosis, making it a promising candidate for nutritional interventions.

Rhynchophorus ferrugineus larvae: A novel source for combating broad-spectrum bacterial and fungal infections.

Antimicrobial resistance is a growing concern due to the growth of antibiotic-resistant microorganisms, which makes it difficult to treat infection. Due to its broad-spectrum antimicrobial properties against a diverse array of bacteria, both Gram-positive and Gram-negative bacteria, and fungi, Rhynchophorus ferrugineus larval antimicrobial peptides (AMPs) have demonstrated potential as antimicrobial agents for the treatment of microbial infections and prevention of antibiotic resistance. This study emphasizes the unexplored mechanisms of action of R. ferrugineus larvae against microorganisms. Among the most widely discussed mechanisms is the effect of AMPs in larvae in response to a threat or infection. Modulation of immune-related genes in the intestine and phagocytic capacity of its hemocytes may also affect the antimicrobial activity of R. ferrugineus larvae, with an increase in phenoloxidase activity possibly correlated with microbial clearance and survival rates of larvae. The safety and toxicity of R. ferrugineus larvae extracts, as well as their long-term efficacy, are also addressed in this paper. The implications of future research are explored in this paper, and it is certain that R. ferrugineus larvae have the potential to be developed as a broad-spectrum antimicrobial agent with proper investigation.

Identification, characterization and hypolipidemic effect of novel peptides in protein hydrolysate from Protaetia brevitarsis larvae

The proteins were mainly derived from Protaetia brevitarsis larval extracts obtained using two empty intestine methods (traditional static method: TSM or salt immersion stress method: SISM) and extraction solvents (water: W or 50 % water–ethanol: W:E), and the proteins were used as objects to investigate the effect of emptying intestine methods on hypolipidemic peptides. The results revealed that the F-2 fractions of protein hydrolysate had stronger in vitro hypolipidemic activity, with the peptides obtained by SISM possessing a stronger cholesterol micelle solubility inhibition rate, especially in SISM-W:E-P. Moreover, a total of 106 peptides were tentatively identified, among which SISM identified more peptides with an amino acid number < 8. Meanwhile, five novel peptides (YPPFH, YPGFGK, KYPF, SPLPGPR and VPPP) exhibited good hypolipidemic activity in vitro and in vivo, among which YPPFH, VPPP and KYPF had strong inhibitory activities on pancreatic lipase (PL) and cholesteryl esterase (CE), and KYPF, SPLPGPR and VPPP could significantly reduce the TG content in Caenorhabditis elegans. Thus, P. brevitarsis can be developed as a naturally derived hypolipidemic component for the development and application in functional foods.

Protective effects of an aqueous extract of Protaetia brevitarsis seulensis larvae in an ovalbumin-induced murine model of asthma

ABSTRACT Asthma is a disease-related allergic response characterized by the accumulation of inflammatory cells in airway tissue. Protaetia brevitarsis seulensis larvae are known for their nutritional value and have been used in food; however, it is unknown whether they exert protective effects in a murine ovalbumin (OVA)-induced asthmatic model. We investigated the protective effects of an aqueous extract of P. brevitarsis seulensis larvae (PBE) against OVA-induced asthma by performing histopathological analysis and measuring inflammatory mediators in bronchoalveolar lavage fluid using enzyme-linked immunosorbent assay and flow cytometry. Asthmatic symptoms were attenuated by PBE, as indicated by decreased inflammation in lung tissue and decreased levels of inflammatory cells, inflammatory cytokines, and Th2 cell activation. Additionally, four out of six major metabolites isolated from P. brevitarsis seulensis attenuated Th2 cell activation, indicating that PBE attenuates asthmatic inflammation by regulating Th2 cell activation. Therefore, our findings support PBE as a pharmacological candidate for asthma treatment.

Study of metabolism and potential toxicity of nine synthetic opioid analogs using the zebrafish larvae model.

The use of novel psychoactive substances (NPSs) has dramatically increased worldwide, and among them, synthetic opioids are one of the fastest growing groups, where cinnamylpiperazines and 2-benzylbenzimidazoles represent two of the most relevant subclasses. However, the data on their toxicity and metabolism are still limited. The aim of the present study was to evaluate the toxicity and metabolic pathways of some compounds belonging to these families, namely, AP-237, 2-methyl AP-237, isotonitazene, flunitazene, etodesnitazene, metonitazene, metodesnitazene, N-pyrrolidino etonitazene, and butonitazene. The study was performed using a zebrafish early life stages model. In fact, zebrafish (Danio rerio) embryos and larvae have recently been recognized as a suitable animal model in alternative to mammals, because they require less time and resources and do not need complex procedures for ethics approval. The cellular toxicity after a single administration was assessed at the fourth day post-fertilization with acridine orange staining. Possible morphological defects were evaluated with a light microscope after 24 h of exposure to 1μmol/L concentration of each drug. Subsequently, the larvae were euthanized and underwent analysis of drug metabolites using UPLC coupled to an Orbitrap high-resolution mass spectrometer. High rates of morphological defects, as well as of cellular death, were detected, but no significant difference in mortality between treatment and control groups was observed. In addition, several metabolites, mainly produced through monohydroxylation, N-dealkylation, and O-dealkylation, were identified in the larvae extracts.

Assesment of Hypoderma infestation in a wild population of Cervus elaphus from mountains Atlantic ecosystems in southwestern Europe (Spain)

Hypodermosis in Cervus elaphus was studied in the Riaño Regional Hunting Reserve, Province of León, north-western Spain. One hundred and ten red deer were examined for the presence of warble fly larvae. They were analyzed by PCR analysis of the COI region of mt-DNA and identified as Hypoderma actaeon. The prevalence of larvae was 42.7% with a mean intensity of 12.5 ± 18 (range 1–80) warbles/deer infested. The distribution of larvae in the infested animals showed an aggregated/overdispersed pattern (aggregation index = 25.84), where the larvae are not randomly or uniformly distributed, but strongly aggregated among their hosts. Larvae were found in all three states. First and second-instars were observed mainly in the autumn until the end of winter (November-March) and third-instars in late winter until mid-spring (March–May). The adult animals and the males had a higher prevalence than the young and the females, finding statistically significant differences only according to the sex of the animals. Seasonal variations were observed in the prevalence with the highest number of infested animals in winter and autumn, but not in terms of the mean intensity of parasites. Additionally, we assessed the presence of anti-Hypoderma antibodies in serum by means of indirect ELISA tests, using a crude larval extract (CLE) and a purified fraction the hypodermin C (HyC) obtained from first instars of Spanish isolates of Hypoderma lineatum (cattle). These findings confirm that H. actaeon is widely distributed in northern Spain, and provide new information about its chronobiology in mountainous Atlantic ecosystems from southwestern Europe.