Larval Protein Research Articles

Study on the mechanism of resistance to cypermethrin in Culex pipiens pallens using proteomics

To compare the differentially expressed proteins between cypermethrin-resistant and -sensitive Culex pipiens pallens, so as to unravel the mechanism underlying the resistance to cypermethrin in Cx. p. pallens. A quantitative proteomic analysis was performed among cypermethrin-sensitive and -resistant isolates of Cx. p. pallens using isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography with tandem mass spectrometry (LC-MS/MS). A total of 164 differentially expressed proteins were identified between cypermethrin-sensitive and -resistant isolates of Cx. p. pallens, including 54 up-regulated proteins and 110 down-regulated proteins. A large number of cuticular proteins, larval cuticular proteins, pupal cuticular proteins and cuticular structural constituent proteins, which are associated with cytoskeletal structure and components, were differentially expressed between cypermethrin-sensitive and -resistant isolates of Cx. p. pallens. Thirteen proteins, which were involved in energy production and conversion, translation, ribosomal structure and biogenesis, lipid transport and metabolism, post-translational modification, protein turnover, chaperones, cytoskeleton and intracellular transportation, were validated to be differentially expressed between cypermethrin-sensitive and -resistant isolates of Cx. p. pallens, which may serve as potential markers of cypermethrin resistance. Multiple insecticide resistance mechanisms contribute to the resistance to cypermethrin in Cx. p. pallens, including cuticular resistance and metabolic resistance, and the cuticular protein genes and cytochrome P450 enzymes may play an important role in the resistance of Cx. p. pallens to cypermethrin.

Release of Mediator Enzyme β-Hexosaminidase and Modulated Gene Expression Accompany Hemocyte Degranulation in Response to Parasitism in the Silkworm Bombyx mori.

In insects infections trigger hemocyte-mediated immune reactions including degranulation by exocytosis; however, involvement of mediator enzymes in degranulation process is unknown in insects. We report here that in silkworm Bombyx mori, infection by endoparasitoid Exorista bombycis and microsporidian Nosema bombycis activated granulation in granulocytes and promoted degranulation of accumulated structured granules. During degranulation the mediator lysosomal enzyme β-hexosaminidase showed increased activity and expression of β-hexosaminidase gene was enhanced. The events were confirmed in vitro after incubation of uninfected hemocytes with E. bombycis larval tissue protein. On infection, cytotoxicity marker enzyme lactate dehydrogenase (LDH) was released from the hemocytes illustrating cell toxicity. Strong positive correlation (R2 = 0.71) between LDH activity and β-hexosaminidase released after the infection showed parasitic-protein-induced hemocyte damage and accompanied release of the enzymes. Expression of β-hexosaminidase gene was enhanced in early stages after infection followed by down regulation. The expression showed positive correlation (R2 = 0.705) with hexosaminidase activity pattern. B. mori hexosaminidase showed 98% amino acid similarity with that of B. mandarina showing origin from same ancestral gene; however, 45-60% varied from other lepidopterans showing diversity. The observation signifies the less known association of hexosaminidase in degranulation of hemocytes induced by parasitic infection in B. mori and its divergence in different species.

Rhizopus oligosporus-Assisted Valorization of Coconut Endosperm Waste by Black Soldier Fly Larvae for Simultaneous Protein and Lipid to Biodiesel Production

Coconut endosperm waste (CEW) was treated by Rhizopus oligosporus via in situ and ex situ fermentations together with bioconversion into valuable black soldier fly larval biomass. The ex situ fermentation could overall enrich the nutritional compositions of CEW by hydrolyzing its complex organic polymers and exuding assimilable nutrients to enhance the black soldier fly larvae (BSFL) growth. Nevertheless, the larval gut bacteria were competing with Rhizopus oligosporus in in situ fermentation, derailing the hydrolysis processes and larval growth. Accordingly, the highest growth rates achieved were around 0.095 g/day, as opposed to only 0.065 g/day whilst using 0.5 wt% of Rhizopus oligosporus to perform ex situ and in situ fermentations, respectively. These were also underpinned by the greater amount of total CEW consumed when employing ex situ fermentation, with comparable metabolic costs to feeding on in situ-fermented CEW. The mature BSFL were subsequently harvested and the amounts of protein and lipid produced were assessed in terms of their feasibility for biodiesel production. While the statistical analyses showed that the larval protein yields derived from both fermentation modes were insignificant, the BSFL could attain higher lipid and protein productivities upon feeding with ex situ- rather than in situ-fermented CEW mediums. Better yet, the larval biodiesel quality measured in terms of the fatty acid methyl ester composition were not varied significantly by Rhizopus oligosporus through the fermentation process. Thereby, the presence of 1.0 wt% Rhizopus oligosporus was considered optimum to perform ex situ fermentation, giving rise to the acceptable growth of BSFL loaded with the highest lipid yield and productivity for producing biodiesel and protein simultaneously.

A Proteomic- and Bioinformatic-Based Identification of Specific Allergens from Edible Insects: Probes for Future Detection as Food Ingredients.

The increasing development of edible insect flours as alternative sources of proteins added to food and feed products for improving their nutritional value, necessitates an accurate evaluation of their possible adverse side-effects, especially for individuals suffering from food allergies. Using a proteomic- and bioinformatic-based approach, the diversity of proteins occurring in currently consumed edible insects such as silkworm (Bombyx mori), cricket (Acheta domesticus), African migratory locust (Locusta migratoria), yellow mealworm (Tenebrio molitor), red palm weevil (Rhynchophorus ferrugineus), and giant milworm beetle (Zophobas atratus), was investigated. Most of them consist of phylogenetically-related protein allergens widely distributed in the different groups of arthropods (mites, insects, crustaceans) and mollusks. However, a few proteins belonging to discrete protein families including the chemosensory protein, hexamerin, and the odorant-binding protein, emerged as proteins highly specific for edible insects. To a lesser extent, other proteins such as apolipophorin III, the larval cuticle protein, and the receptor for activated protein kinase, also exhibited a rather good specificity for edible insects. These proteins, that are apparently missing or much less represented in other groups of arthropods, mollusks and nematods, share well conserved amino acid sequences and very similar three-dimensional structures. Owing to their ability to trigger allergic responses in sensitized people, they should be used as probes for the specific detection of insect proteins as food ingredients in various food products and thus, to assess their food safety, especially for people allergic to edible insects.

Nutritional plasticity of the black soldier fly (Hermetia illucens) in response to artificial diets varying in protein and carbohydrate concentrations

Protein (P) and carbohydrate (C) concentrations present in food affect larval performance, larval body nutrient concentration, and fecundity of the black soldier fly (BSF). We substantially expanded the range of dietary P- and C-concentrations investigated thus far to assess the effects of nutritionally – unbalanced diets on BSF larval and adult life-history traits, and on larval body protein and lipid concentrations. Twenty five artificial diets varying in their P- and C-concentration and ratio were formulated. We tested five macronutrient (P+C) concentrations (5, 15, 25, 50 and 75%) and five P:C ratios (1:1, 1:2, 1:4, 2:1 and 4:1). BSF performance was affected by P+C-concentration rather than by P:C ratios. A P-concentration between 10 and 15% and a C-concentration between 10 and 60% supported high larval and adult performance. P-concentration is limiting for most of the performance variables, however, a P-concentration higher than 37% reduced larval survival. C-concentration affected egg production more strongly than P-concentration. Overall, at P+C values of 25 and 50%, and P:C ratios 1:2 and 1:4 resulted in the highest values of most of the larval and adult performance variables we measured. For the protein and carbohydrate sources tested, dietary macronutrient concentrations significantly affected larval and adult performance of BSF in different ways. These results show a remarkable degree of nutritional plasticity, and point to the relevance of differentiating the formulation of diets to achieve maximal larval yield, high body protein or high body lipid accumulation or high adult emergence and egg production.

Chironomus riparius Proteome Responses to Spinosad Exposure.

The potential of proteome responses as early-warning indicators of insecticide exposure was evaluated using the non-biting midge Chironomus riparius (Meigen) as the model organism. Chironomus riparius larvae were exposed to environmentally relevant concentrations of the neurotoxic pesticide spinosad to uncover molecular events that may provide insights on the long-term individual and population level consequences. The iTRAQ labeling method was performed to quantify protein abundance changes between exposed and non-exposed organisms. Data analysis revealed a general dose-dependent decrease in the abundance of globin proteins as a result of spinosad exposure. Additionally, the downregulation of actin and a larval cuticle protein was also observed after spinosad exposure, which may be related to previously determined C. riparius life-history traits impairment and biochemical responses. Present results suggest that protein profile changes can be used as early warning biomarkers of pesticide exposure and may provide a better mechanistic interpretation of the toxic response of organisms, aiding in the assessment of the ecological effects of environmental contamination. This work also contributes to the understanding of the sublethal effects of insecticides in invertebrates and their molecular targets.

Effects of Enzymatic Hydrolysis on the Functional Properties, Antioxidant Activity and Protein Structure of Black Soldier Fly (Hermetia illucens) Protein.

Simple SummaryAccording to the FAO, the world’s population will reach 9 billion by 2050, and in order to provide enough food, meat production must increase by 100% and food production by 70%. Furthermore, more than 80% of fresh water resources are being used for agriculture, and 40% of the total food produced annually, is wasted. One sustainable agricultural practice involves converting by-products from the food and agriculture industry into valuable biomass, such as black soldier flies. Black soldier fly larvae can feed on by-products, and convert them to protein, carbohydrates, and oil. Black soldier flies could be used for feed and food development using different processing methods including enzymatic hydrolysis.The effects of chemical protein extraction, and enzymatic hydrolysis with Alcalase, papain and pepsin, on the functional properties, antioxidant activity, amino acid composition and protein structure of black soldier fly (H. illucens) larval protein were examined. Alcalase hydrolysates had the highest degree of hydrolysis (p < 0.05), with the highest hydrolysate and oil fraction yield (p < 0.05). Pepsin hydrolysates showed the lowest oil holding capacity (p < 0.05), whereas no significant differences were observed among other enzymes and protein concentrates (p > 0.05). The emulsifying stability and foam capacity were significantly lower in protein hydrolysates than protein concentrate (p < 0.05). The antioxidant activity of protein hydrolysates from protein concentrate and Alcalase was higher than that with papain and pepsin (p < 0.05), owing to the higher hydrophobic amino acid content. Raman spectroscopy indicated structural changes in protein α-helices and β-sheets after enzymatic hydrolysis.

Phylogenetic comparisons reveal mosaic histories of larval and adult shell matrix protein deployment in pteriomorph bivalves

Molluscan shells are organo-mineral composites, in which the dominant calcium carbonate is intimately associated with an organic matrix comprised mainly of proteins and polysaccharides. However, whether the various shell matrix proteins (SMPs) date to the origin of hard skeletons in the Cambrian, or whether they represent later deployment through adaptive evolution, is still debated. In order to address this issue and to better understand the origins and evolution of biomineralization, phylogenetic analyses have been performed on the three SMP families, Von Willebrand factor type A (VWA) and chitin-binding domain-containing protein (VWA-CB dcp), chitobiase, and carbonic anhydrase (CA), which exist in both larval and adult shell proteomes in the bivalves, Crassostrea gigas and Pinctada fucata. In VWA-CB dcp and chitobiase, paralogs for larval and adult SMPs evolved before the divergence of these species. CA-SMPs have been taken as evidence for ancient origins of SMPs by their presumed indispensable function in biomineralization and ubiquitous distribution in molluscs. However, our results indicate gene duplications that gave rise to separate deployments as larval and adult CA-SMPs occurred independently in each lineage after their divergence, which is considerably more recent than hitherto assumed, supporting the “recent heritage and fast evolution” scenario for SMP evolution.

RNAi-mediated silencing of Trichinella spiralis serpin-type serine protease inhibitors results in a reduction in larval infectivity

Trichinella spiralis serpin-type serine protease inhibitors (TsSPIs) are expressed in adult worms (AW), newborn larvae (NBL) and muscle larvae (ML) of T. spiralis, with the ML stage demonstrating the highest expression level. This study aims to determine TsSPI functions in larval viability and invasion of intestinal epithelial cells in vitro, as well as their development, survival, and fecundity in vivo via RNAi. TsSPI-specific siRNAs and dsRNA were transfected into ML by incubation. The silencing effect of TsSPI transcription and expression was determined using qPCR and western blot, respectively. After incubation in 60 ng/μL dsRNA–TsSPI for 3 days, larval TsSPI mRNA and protein expression levels were reduced by 68.7% and 68.4% (P < 0.05), respectively. dsRNA-mediated silencing of TsSPI significantly impacted larval invasion into intestinal epithelial cells in vitro but did not affect the survival rate of larvae. After challenge with dsRNA–TsSPI-treated ML, mice exhibited a 56.0% reduction in intestinal AW burden and 56.9% reduction in ML burden (P < 0.05), but NBL production of female AW remained the same (P > 0.05). Our results revealed that RNAi-mediated silencing of TsSPI expression in T. spiralis significantly reduced larval infectivity and survival in the host but had no effect on the survival rate and fecundity. Furthermore, TsSPIs have no effect on the growth and reproduction of parasites but may be directly involved in regulating the interaction of T. spiralis and the host. Therefore, TsSPIs are crucial in the process of T. spiralis larval invasion and parasite survival in the host.

Rearing substrate impacts growth and macronutrient composition of Hermetia illucens (L.) (Diptera: Stratiomyidae) larvae produced at an industrial scale

Organic waste is a rapidly increasing problem due to the growth of the agricultural production needed to meet global food demands. Development of sustainable waste management solutions is essential. Black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae) (BSF), larvae are voracious consumers of a wide range of organic materials ranging from fruits and vegetables to animal remains, and manure. Thanks to this ability and considering the larval high protein and lipid content, BSF larvae are a useful additive in animal feeds and biodiesel production. Unfortunately, the feasibility of using the black soldier fly as a tool for waste valorization and feed production has primarily been investigated at the benchtop scale. Thus, mobilization of current practices to an industrial scale is challenging because scaling up from small laboratory studies to large industrial studies is not necessarily linear. The goal of this study was to demonstrate the ability of the BSF to recycle organic waste at an industrial scale. To accomplish this goal, three organic waste streams were used (e.g., apples, bananas, and spent grain from a brewery) to test six diet treatments (1) apple, (2) banana, (3) spent grain, (4) apple and banana, (5) apple and spent grain, and (6) banana and spent grain. Working at scale of 10,000 BSF larvae life history traits, waste valorization, protein and lipid profiles were measured for each diet treatment. Differences were recorded across all variables, except substrate conversion, for larvae fed on fruit and spent grain (alone or with fruit). Growth rate significantly differed across treatments; larvae reared on spent grain grew twice as fast as those fed apples alone, but those reared on the apple and spent grain mixture produced twice as much insect biomass. However, it should be noted that larvae resulting from the apple diet contained 50% more fat than larvae fed the fruit and spent grain mixtures. Commonly-available organic wastes were successfully used at an industrial scale to produce BSF larvae that have the potential to substitute other sources of protein and lipids in different industrial applications. Industrialization efforts are encouraged to assess these impacts when integrating diverse ingredients into larval diets as a means to more precisely predict output, such as larval development time and final larval biomass.

Protein Fe-S Centers as a Molecular Target of Toxicity of a Complex Transition Metal Oxide Nanomaterial with Downstream Impacts on Metabolism and Growth.

Oxidative stress is frequently identified as a mechanism of toxicity of nanomaterials. However, rarely have the specific underlying molecular targets responsible for these impacts been identified. We previously demonstrated significant negative impacts of transition metal oxide (TMO) lithium-ion battery cathode nanomaterial, lithium cobalt oxide (LCO), on the growth, development, hemoglobin, and heme synthesis gene expression in the larvae of a model sediment invertebrate Chironomus riparius. Here, we propose that alteration of the Fe-S protein function by LCO is a molecular initiating event leading to these changes. A 10 mg/L LCO exposure causes significant oxidation of the aconitase 4Fe-4S center after 7 d as determined from the electron paramagnetic resonance spectroscopy measurements of intact larvae and a significant reduction in the aconitase activity of larval protein after 48 h (p < 0.05). Next-generation RNA sequencing identified significant changes in the expression of genes involved in 4Fe-4S center binding, Fe-S center synthesis, iron ion binding, and metabolism for 10 mg/L LCO at 48 h (FDR-adjusted, p < 0.1). We propose an adverse outcome pathway, where the oxidation of metabolic and regulatory Fe-S centers of proteins by LCO disrupts metabolic homeostasis, which negatively impacts the growth and development, a mechanism that may apply for these conserved proteins across species and for other TMO nanomaterials.

Identification of Immune Response to Sacbrood Virus Infection in Apis cerana Under Natural Condition.

Chinese sacbrood virus (CSBV) is a serious threat to eastern honeybees (Apis cerana), especially larvae. However, the pathological mechanism of this deadly disease remains unclear. Here, we employed mRNA and small RNA (sRNA) transcriptome approach to investigate the microRNAs (miRNAs) and small interfering RNAs (siRNAs) expression changes of A. cerana larvae infected with CSBV under natural condition. We found that serine proteases involved in immune response were down-regulated, while the expression of siRNAs targeted to serine proteases were up-regulated. In addition, CSBV infection also affected the expression of larvae cuticle proteins such as larval cuticle proteins A1A and A3A, resulting in increased susceptibility to CSBV infection. Together, our results provide insights into sRNAs that they are likely to be involved in regulating honeybee immune response.

Changes in composition and levels of hemolymph proteins during metamorphosis of Manduca sexta

The tobacco hornworm, Manduca sexta, is a lepidopteran model species widely used to study insect biochemical processes. Some of its larval hemolymph proteins are well studied, and a detailed proteomic analysis of larval plasma proteins became available in 2016, revealing features such as correlation with transcriptome data, formation of immune complexes, and constitution of an immune signaling system in hemolymph. It is unclear how the composition of these proteins may change in other developmental stages. In this paper, we report the proteomes of cell-free hemolymph from prepupae, pupae on day 4 and day 13, and young adults. Of the 1824 proteins identified, 907 have a signal peptide and 410 are related to immunity. Drastic changes in abundance of the storage proteins, lipophorins and vitellogenin, for instance, reflect physiological differences among prepupae, pupae, and adults. Considerably more proteins lacking signal peptide are present in the late pupae, suggesting that plasma contains relatively low concentrations of intracellular components released from remodeling tissues during metamorphosis. The defense proteins detected include 43 serine proteases and 11 serine protease homologs. Some of these proteins are members of the extracellular immune signaling network found in feeding larvae, and others may play additional roles and hence confer new features in the later life stages. In summary, the proteins and their levels revealed in this study, together with their transcriptome data, are expected to stimulate focused explorations of humoral immunity and other physiological systems in wandering larvae, pupae, and adults of M. sexta and shed light upon functional and comparative genomic research in other holometabolous insects.

Biochemical Factors Associated with Resistance to Spotted Pod Borer, Maruca vitrata (Fabricius) in Green Gram

Background: Legume pod borer or spotted pod borer, Maruca vitrata is one of the serious insect pest to the green gram, which causes damage mainly at the reproductive phase of the crop growth. The larvae of spotted pod borer are known to cause damage by webbing the leaves, bud, flower and pods together and feed from inside on them. Due to its webbing nature, it is very difficult to enter inside by natural enemies and chemicals cannot directly reach inside webbing. Host plant resistance to insect pests is an economically and ecologically preferred alternative as compared to other pest management tactics, particularly the synthetic pesticides. The present investigation was undertaken to examine the role of certain bio-chemical constituents of the immature pods of green gram in the expression of damage by the spotted pod borer. Methods: The experiments on screening were conducted during the kharif 2014 and 2015 at the Agricultural Research Farm, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, (U.P.) to define the occurrence of M. vitrata on 20 promising varieties/genotypes of green gram. Pods were collected at the immature stage from different varieties/genotypes and biochemical constituents such as total soluble sugar, total chlorophyll, protein were estimated. Result: The maximum protein content was found in genotype IPM 306-6 (36.17 mg/g) and minimum in genotype PM-5 (20.53 mg/g). Highest total sugar content was reported from genotype ML 1256 (16.71 mg/g) and lowest total sugar content was recorded in PM-5 (10.94 mg/g).The phenol content in genotype PM-5 (9.00 mg/g) was significantly higher than others whereas, the minimum phenol content was reported in IPM 306-6 (5.14 mg/g). The higher amount of total chlorophyll content found in genotype IPM 306-6 (3.22 mg/g) and lowest in PM-5 (1.85 mg/g). The significant and positive correlation was observed between M. vitrata larval population, pod damage and protein (r=0.954** and r=0.952**, respectively) and total sugar content (r=0.986** and r=0.986**, respectively) and total chlorophyll content (r=0.994** and r=0.993**, respectively) in immature pods, while, the significant and negative correlation was found between phenol (r= -0.916** and r= -0.919**, respectively).

Effects of protein extract from the housefly larvae (Musca domestica vicina) on the hyperlipidemic mice induced by Triton WR-1339

Hypercholesterolemia is a chief risk reason for atherosclerosis. Triton-WR 1339 (Tyloxapol) was used by several investigators to initiate hypercholesterolemia in animals. Housefly maggots contain high larval protein extract (PE) that is used as an anti-atherosclerosis. The goal of the current study was to evaluate the possible protective effects of the PE of houseflies against Triton-WR 1339-induced hyperlipidemia, oxidative damage, and histopathological changes of the liver and dorsal aorta of male mice. In this study, four groups of experimental mice, including ten mice each, were classified into: Group I, mice were administered with 0.5 ml saline solution, and considered as a positive control; Group II, mice were administered day after day by gavage with 0.5 ml of 200 mg/kg bw PE; Group III, hyperlipidemic mice were taken no treatment till the end of experiment and Group IV, the hyperlipidemic mice were administered day after day by gavage with 200 mg/kg bw PE. On day 40, the mice were anesthetized, blood samples were collected, the livers and the aorta were dissected for various biochemical tests and histopathological examinations. The biochemical parameters, triglycerides (TGs), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and lipid peroxidation (LPO) were increased, and reduction in high-density lipoprotein-cholesterol (HDL-C), the total glutathione (GSH) and the glutathione peroxidase (GPx) levels were observed following Triton WR-1339 treatment in mice. After administration of the PE (200 mg/kg bw), the results revealed significant reduction in serum TC and TGs while HDL-C, oxidative stress levels in liver tissue, LPO, GSH and GPx were significantly increased. Histopathological examinations of the liver tissue and the aorta of mice showed alterations afterwards Triton WR-1339 administration. Treatment with 200 mg/kg bw PE showed beneficial effects with lower capsular thickening, enlargment of hepatocytes and much lower inflammatory cell infiltration.

Quality characteristics and protein digestibility of Protaetia brevitarsis larvae.

Herein, the in vitro protein digestibility of lyophilized Protaetia brevitarsis larvae flour with and without defatting using 70% ethanol was compared with beef loin. Proximate analysis showed that the defatted larvae contained the highest protein content (p < 0.05). The viable counts of total aerobic bacteria, Escherichia coli, and coliform bacteria decreased significantly after defatting the larval samples with 70% ethanol (p < 0.05). Measurement of α-amino group content and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed higher amounts of low molecular weight proteins in the larvae compared to beef loin (p < 0.05). After in vitro digestion, the degree of protein hydrolysis of the digesta was higher for both larvae samples compared to beef loin (p < 0.05). No change was observed in the in vitro larval protein digestibility after defatting. These results highlight the excellent protein digestibility of P. brevitarsis larvae with high protein content. Defatting insect flour with 70% ethanol could enhance microbial safety while maintaining excellent protein digestibility.

Protein Stores Regulate When Reproductive Displays Begin in the Male Caribbean Fruit Fly.

Many animals exhibit reproductive behavior that requires expenditure of valuable nutrients. In males of many species, competitive energetically demanding displays and the development of sexual ornaments require prior accumulation of nutrient stores. Males must coordinate nutrient stores with ornament development and reproductive displays or they risk depleting their resources mid-development or mid-display, reducing their chance of mating. Males may use nutrient stores to regulate their reproductive behavior. Amino acid reserves may be important for reproduction, but the roles of amino acid stores in initiating maturation and reproductive behavior are less studied than fat stores. Insects store amino acids as hexamerin storage proteins. Many fly species use a specific hexamerin, larval serum protein 2 (LSP-2), as both a juvenile storage medium and to store protein consumed after adult eclosion. Protein stored as LSP-2 has previously been suggested to regulate reproduction in females, but no role has been proposed for LSP-2 in regulating male maturation. We use males of the Caribbean fruit fly, Anastrepha suspensa, a species with nutrient-intensive male sexual displays to test whether LSP-2 stores regulate male reproductive displays. We fed adult A. suspensa males a diet with or without protein, then assayed these males for lsp-2 transcript abundance via qRT-PCR, LSP-2 protein abundance via Western blot, and reproductive display behavior via observation. We found that adult males with ad libitum dietary protein had greater lsp-2 transcript and protein abundance, earlier sexual display behavior, and were more likely to exhibit sexual display behavior than protein-deprived adult males. We show that lsp-2 knockdown via RNAi decreases the proportion of males exhibiting reproductive displays, particularly early in the onset of reproductive behavior. Our results suggest circulating LSP-2 protein stores regulate reproductive behavior in A. suspensa males, consistent with protein stores modulating reproduction in males with expensive reproductive strategies. Our results are consistent with hexamerin storage proteins performing dual roles of protein storage and protein signaling. Our work also has substantial practical applications because tephritid flies are a pest group and the timing and expression of male reproductive displays in this group are important for control efforts using the sterile insect technique.

A STUDY OF IMMUNOTHERAPEUTIC EFFICACY OF TRICHINELLA SPIRALIS EXCRETORY-SECRETORY PROTEINS IN MURINE TRICHINELLOSIS

Trichinellosis is a serious zoonotic parasitic and globally endemic disease. Benzimidazole derivativesare apparently unable to kill encapsulated larvae and its effectiveness depends on solubility, dosage oftherapy, host biotransformation, selectivity patterns as well as onset of treatment after infection. Excretory-secretory (ES) proteins released by Trichinella induce a strong immune response when tested as avaccine. The current study investigated both therapeutic and protective effects of adult worm excretorysecretory protein (AW/ES), or larval excretory secretory protein (LES) on T. spiralis infected mice,conducted by parasitological, histopathological, immunohistochemical, serological and molecular investigations.Larval excretory secretory protein achieved more significant therapeutic and protectiveeffects than adult worm excretory secretory protein in experimentally infected mice, causing reductionin larval counts and decrease in pathological changes of both muscular and intestinal tissues. It increasedserum IgG OD values, decreased Foxp3 expressions and increased the mean cycle thres- holdvalues of muscular tissues but it had lesser effects on adult worm counts than albendazole.

The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda.

It is well known that insect larval midgut cadherin protein serves as a receptor of Bacillus thuringiensis (Bt) crystal Cry1Ac or Cry1Ab toxins, since structural mutations and downregulation of cad gene expression are linked with resistance to Cry1Ac toxin in several lepidopteran insects. However, the role of Spodoptera frugiperda cadherin protein (SfCad) in the mode of action of Bt toxins remains elusive. Here, we investigated whether SfCad is involved in susceptibility to Cry1Ab or Cry1Fa toxins. In vivo, knockout of the SfCad gene by CRISPR/Cas 9 did not increase tolerance to either of these toxins in S. frugiperda larvae. In vitro cytotoxicity assays demonstrated that cultured insect TnHi5 cells expressing GFP-tagged SfCad did not increase susceptibility to activated Cry1Ab or Cry1Fa toxins. In contrast, expression of another well recognized Cry1A receptor in this cell line, the ABCC2 transporter, increased the toxicity of both Cry1Ab and Cry1Fa toxins, suggesting that SfABCC2 functions as a receptor of these toxins. Finally, we showed that the toxin-binding region of SfCad did not bind to activated Cry1Ab, Cry1Ac, nor Cry1Fa. All these results support that SfCad is not involved in the mode of action of Cry1Ab or Cry1Fa toxins in S. frugiperda.

Silkworm pupae as source of high-value edible proteins and of bioactive peptides.

To characterize the high‐value protein content and to discover new bioactive peptides, present in edible organisms, as silkworm pupae, semiquantitative analytical approach has been applied. The combination of appropriate protein extraction methods, semiquantitative high‐resolution mass spectrometry analyses of peptides, in silico bioactivity and gene ontology analyses, allowed protein profiling of silkworm pupae (778 gene products) and the characterization of bioactive peptides. The semiquantitative analysis, based on the measurement of the emPAI, revealed the presence of high‐abundance class of proteins, such as larval storage protein (LSP) class. This class of proteins, beside its nutrient reservoir activity, is of great pharmaceutical interest for their efficacy in cardiovascular diseases. Potential allergens were also characterized and quantified, such as arginine kinase, thiol peroxiredoxin, and Bom m 9. This powerful bioanalytical approach proved the potential industrial applications of Bombyx mori pupae, as source of high‐value proteins in a green and “circular” economy perspective.