Selezione di razze di Bombyx mori ad alta efficienza nutrizionale e relative problematiche

The silkworm (Bombyx mori L.) is an insect belonging to the lepidopteran order, family Bombycidae: it is one of the two unique species belonging to the genus Bombyx, together with its ancestor – Bombyx mandarina Moore. In the past, silkworm exploitation in Europe was particularly related to the agricultural sector and the production of the silk thread but this type of activity has being disappearing from the old continent, in favour of some Asian countries, especially China, where cost of labour is low. In spite of the decline of these traditional activities the silkworm started to be used as a tool in a lot of technological applications and as a model in scientific research in various fields. Silk is regarded as a promising biomaterial because of its physical and chemical features both on the macro and microscale as well as in nano-technologies. Furthermore biology and biotechnology are interested in silkworm, or in its cell cultures, as a bioreactor to produce recombinant proteins which should otherwise be produced in bacteria or yeasts. The silkworm has a suitable size even for complex experiments with respect to other organisms used for the same aims, however it does not require large rearing rooms or high quantity of food, it can be easily reared under controlled conditions and its biological cycle is short (it lasts about 45 days), representing thus, also a model for basic research in life sciences. Moreover, to use the silkworm as a technological platform, important steps were achieved by sequencing the whole genome and by developing new optimized artificial diets for this insect (strictly monophagous), which freed rearing from seasonal constraints coming from mulberry leaf production in temperate climates. Thus, the above-mentioned new potentialities and a revived interest from the textile industry, about a silk supply chain outside Asia, establish the background of the research work described in this thesis. In fact, performed experiments relate with relaunching basic research activities about B. mori and aimed at meeting the needs of a silk industry, possibly developing in temperate countries. The starting material, that is the B. mori strains, belongs to the germplasm collection of Consiglio per la Ricerca e la sperimentazione in Agricoltura – Unità di ricerca di Apicoltura e Bachicoltura (CRA-API) of Padua and consists of about 200 strains, which differ in their phenotypic and genotypic characteristics. From the aforementioned collection, a selection process based on a mixed rearing strategy on both mulberry leaves and artificial diet has been started. The final goal was to select new strains characterized by high nutritional efficiency in order to optimize the cost-benefit ratio in all those application oriented to productivity. As already stated selection was based on a mixed approach: the first and more traditional step consisted in rearing silkworm larvae on mulberry leaf without any limitation in its quantity, by selecting only for the most important commercial parameters. On the other hand, the second step was a rearing cycle on artificial diet with a limited food amount fed to fifth instar larvae, followed by a selection process according to the most important productive traits. After three years and six generations of selection and genetic improvement, the obtained strains were compared among each other and with the unselected populations on the base of nutritional indexes which permitted an assessment of their conversion efficiency of proteins contained in the food matrix to body mass and silk. The final evaluation allowed to highlight the efficacy of the adopted strategy in selecting the desired features, although a difference related to diverse adaptability of the strains to the diet was recorded. In particular, for some of them, an additional preparatory step of selection on diet appears to be appropriate. During the period when experiments aimed at selection of highly efficient strains were performed, other relevant experimental data were produced. First of all, a new micro-organism which proved itself to be pathogenic for the silkworm reared on artificial diet was isolated and characterized; then, in-depth measurements about cold hardiness of eggs of a polyvoltine silkworm strain belonging to CRA-API’s collection were carried out. Described results to date do not represent the final goal but, an intermediate stage of a selection process that will continue at CRA-API and also, the starting point for the production of new hybrids with improved performances and suited to be reared on both mulberry leaf and artificialdiet

Genome sequence of Enterococcus mundtii EM01, isolated from Bombyx mori midgut and responsible for flacherie disease in silkworms reared on an artificial diet

The whole genome sequence of Enterococcus mundtii strain EM01 is reported here. The isolate proved to be the cause of flacherie in Bombyx mori. To date, the genomes of 11 other E. mundtii strains have been sequenced. EM01 is the only strain that displayed active pathological effects on its associated animal species

Silkworm and Silk: Traditional and Innovative Applications

The various subjects covered in the present Special Issue "Silkworm and Silk: Traditional and Innovative Applications" demonstrate how sericulture, a practice deeply rooted in human history, can act as a bridge to bring together an exceptionally wide range of scientific and technical expertise in both conventional topics and cutting-edge technologies [...]

A Bombyx mori Infection Model for Screening Antibiotics against Staphylococcus epidermidis

The increasing number of microorganisms that are resistant to antibiotics is prompting the development of new antimicrobial compounds and strategies to fight bacterial infections. The use of insects to screen and test new drugs is increasingly considered a promising tool to accelerate the discovery phase and limit the use of mammalians. In this study, we used for the first time the silkworm, Bombyx mori, as an in vivo infection model to test the efficacy of three glycopeptide antibiotics (GPAs), against the nosocomial pathogen Staphylococcus epidermidis. To reproduce the human physiological temperature, the bacterial infection was performed at 37 °C and it was monitored over time by evaluating the survival rate of the larvae, as well the response of immunological markers (i.e., activity of hemocytes, activation of the prophenoloxidase system, and lysozyme activity). All the three GPAs tested (vancomycin, teicoplanin, and dalbavancin) were effective in curing infected larvae, significantly reducing their mortality and blocking the activation of the immune system. These results corroborate the use of this silkworm infection model for the in vivo studies of antimicrobial molecules active against staphylococci

Remote sensing imaging as a tool to support mulberry cultivation for silk production

In recent decades there has been an increasing use of remotely sensed data for precision agricultural purposes. Sericulture, the activity of rearing silkworm (Bombyx mori L.) larvae to produce silk in the form of cocoons, is an agricultural practice that has rarely used remote sensing techniques but that could benefit from them. The aim of this work was to investigate the possibility of using satellite imaging in order to monitor leaf harvesting in mulberry (Morus alba L.) plants cultivated for feeding silkworms; additionally, quantitative parameters on silk cocoon production were related to the analyses on vegetation indices. Adopting PlanetScope satellite images, four M. alba fields were monitored from the beginning of the silkworm rearing season until its end in 2020 and 2021. The results of our work showed that a decrease in the multispectral vegetation indices in the mulberry plots due to leaf harvesting was correlated with the different parameters of silk cocoons spun by silkworm larvae; in particular, a decrease in the Normalized Difference Vegetation Index (NDVI) and Soil Adjusted Vegetation Index (SAVI) had high correlations with quantitative silk cocoon production parameters (R2 values up to 0.56, p < 0.05). These results led us to the conclusion that precision agriculture can improve sericultural practice, offering interesting solutions for estimating the quantity of produced silk cocoons through the remote analysis of mulberry fields.This research was funded by the Veneto Region, Measure 16.1-2 Programme of Rural Development for the Veneto Region, 2014-2020-DGR 2175 del 23/12/2016, grant number: “Decree n. 55 of 4th December 2017 of financing of the project Serinnovation.”

Mechanical Processing of Hermetia illucens Larvae and Bombyx mori Pupae Produces Oils with Antimicrobial Activity

The aim of this work was to develop processing methods that safeguard the quality and antimicrobial properties of H. illucens and B. mori oils. We adopted a vegetable diet for both insects: leftover vegetables and fruit for H. illucens and mulberry leaves for B. mori. First, alternative techniques to obtain a good oil extraction yield from the dried biomass of H. illucens larvae were tested. Traditional pressing resulted to be the best system to maximize the oil yield and it was successfully applied to B. mori pupae. Oil quality resulted comparable to that obtained with other extraction methods described in the literature. In the case of B. mori pupae, different treatments and preservation periods were investigated to evaluate their influence on the oil composition and quality. Interestingly, agar diffusion assays demonstrated the sensitivity of Gram-positive Bacillus subtilis and Staphylococcus aureus to H. illucens and B. mori derived oils, whereas the growth of Gram-negative Pseudomonas aeruginosa and Escherichia coli was not affected. This study confirms that fat and other active compounds of the oil extracted by hot pressing could represent effective antimicrobials against bacteria, a relevant result if we consider that they are by-products of the protein extraction process in the feed industry

Phenotypic effects induced by knock-down of the period clock gene in Bombyx mori.

SummaryThe lepidopteranBombyx moriis an insect of considerable scientific and economic importance. Recently, theB. moricircadian clock geneperiodhas been molecularly characterized. We have transformed aB. moristrain with a construct encoding aperioddouble-strand RNA in order to knock-downperiodgene expression. We observe that this post-transcriptional silencing produces a small but detectable disruption in the egg-hatching rhythm, as well as a reduction in egg-to-adult developmental time, without altering silk production parameters. Thus we show that both circadian and non-circadian phenotypes can be altered by changingperexpression, and, at a practical level, these results suggest thatperknock-down may provide a suitable strategy for improving the efficiency of rearing, without affecting silk productivity

Selezione di razze di Bombyx mori ad alta efficienza nutrizionale e relative problematiche

The silkworm (Bombyx mori L.) is an insect belonging to the lepidopteran order, family Bombycidae: it is one of the two unique species belonging to the genus Bombyx, together with its ancestor – Bombyx mandarina Moore. In the past, silkworm exploitation in Europe was particularly related to the agricultural sector and the production of the silk thread but this type of activity has being disappearing from the old continent, in favour of some Asian countries, especially China, where cost of labour is low. In spite of the decline of these traditional activities the silkworm started to be used as a tool in a lot of technological applications and as a model in scientific research in various fields. Silk is regarded as a promising biomaterial because of its physical and chemical features both on the macro and microscale as well as in nano-technologies. Furthermore biology and biotechnology are interested in silkworm, or in its cell cultures, as a bioreactor to produce recombinant proteins which should otherwise be produced in bacteria or yeasts. The silkworm has a suitable size even for complex experiments with respect to other organisms used for the same aims, however it does not require large rearing rooms or high quantity of food, it can be easily reared under controlled conditions and its biological cycle is short (it lasts about 45 days), representing thus, also a model for basic research in life sciences. Moreover, to use the silkworm as a technological platform, important steps were achieved by sequencing the whole genome and by developing new optimized artificial diets for this insect (strictly monophagous), which freed rearing from seasonal constraints coming from mulberry leaf production in temperate climates. Thus, the above-mentioned new potentialities and a revived interest from the textile industry, about a silk supply chain outside Asia, establish the background of the research work described in this thesis. In fact, performed experiments relate with relaunching basic research activities about B. mori and aimed at meeting the needs of a silk industry, possibly developing in temperate countries. The starting material, that is the B. mori strains, belongs to the germplasm collection of Consiglio per la Ricerca e la sperimentazione in Agricoltura – Unità di ricerca di Apicoltura e Bachicoltura (CRA-API) of Padua and consists of about 200 strains, which differ in their phenotypic and genotypic characteristics. From the aforementioned collection, a selection process based on a mixed rearing strategy on both mulberry leaves and artificial diet has been started. The final goal was to select new strains characterized by high nutritional efficiency in order to optimize the cost-benefit ratio in all those application oriented to productivity. As already stated selection was based on a mixed approach: the first and more traditional step consisted in rearing silkworm larvae on mulberry leaf without any limitation in its quantity, by selecting only for the most important commercial parameters. On the other hand, the second step was a rearing cycle on artificial diet with a limited food amount fed to fifth instar larvae, followed by a selection process according to the most important productive traits. After three years and six generations of selection and genetic improvement, the obtained strains were compared among each other and with the unselected populations on the base of nutritional indexes which permitted an assessment of their conversion efficiency of proteins contained in the food matrix to body mass and silk. The final evaluation allowed to highlight the efficacy of the adopted strategy in selecting the desired features, although a difference related to diverse adaptability of the strains to the diet was recorded. In particular, for some of them, an additional preparatory step of selection on diet appears to be appropriate. During the period when experiments aimed at selection of highly efficient strains were performed, other relevant experimental data were produced. First of all, a new micro-organism which proved itself to be pathogenic for the silkworm reared on artificial diet was isolated and characterized; then, in-depth measurements about cold hardiness of eggs of a polyvoltine silkworm strain belonging to CRA-API’s collection were carried out. Described results to date do not represent the final goal but, an intermediate stage of a selection process that will continue at CRA-API and also, the starting point for the production of new hybrids with improved performances and suited to be reared on both mulberry leaf and artificialdiet.Il baco da seta (Bombyx mori L.) è un insetto appartenente all’ordine dei lepidotteri, famiglia Bombycidae; è una delle due uniche specie rappresentanti il genere Bombyx, assieme a Bombyx mandarina M., suo progenitore ancestrale. In passato, in Europa, il suo utilizzo era legato soprattutto al mondo agricolo ed alla produzione della fibra serica ma questo tipo di attività è andata scomparendo dal vecchio continente, a favore di alcuni Paesi asiatici, fra cui soprattutto la Cina, con basso costo della manodopera. A dispetto del declino di questo tipo di attività tradizionali, il baco da seta ha però cominciato ad essere utilizzato per applicazioni tecnologiche di alto profilo legate al mondo della ricerca scientifica e della bio-industria. Si sono indagati, da un lato, utilizzi che impiegano la seta come biomateriale e che mirano allo sfruttamento delle sue proprietà fisiche e chimiche sia su scala macroscopica sia su scala microscopica come nel campo delle nanotecnologie. Dall’altro lato, le scienze biologiche e le biotecnologie sono in grado di utilizzare l’insetto stesso, oppure colture cellulari da esso derivate come bio-fabbriche per la produzione di proteine altrimenti sintetizzate in batteri o lieviti. Poiché il baco da seta, rispetto ad altri organismi utilizzati per gli stessi scopi, è di dimensioni adeguate a sperimentazioni anche complesse, pur non richiedendo ampi spazi d’allevamento o elevate quantità di cibo, come ad esempio gli animali superiori, è facilmente allevabile in laboratorio e possiede un ciclo biologico breve (circa 45 giorni), rappresenta un ottimo modello per la ricerca di base nei campi delle scienze della vita. In tal senso, per rendere il B. mori un’appetibile piattaforma tecnologica, importanti passi avanti sono stati fatti con il sequenziamento del genoma e con la formulazione di diete artificiali in grado di svincolare l’allevamento dell’insetto, strettamente monofago, dalla produzione della foglia di gelso, reperibile solo stagionalmente nelle regioni a clima temperato. Sullo sfondo di queste potenzialità tecnologiche e anche in relazione ad un rinato interesse da parte dell’industria del settore serico per una filiera produttiva extra-asiatica, va inquadrato il lavoro di ricerca presentato nella seguente tesi. La sperimentazione descritta è, infatti, inquadrabile in una più generale attività di rilancio della ricerca di base riguardante il baco da seta ed in sintonia con le necessità di un potenziale settore produttivo localizzato in paesi climaticamente simili all’Italia. Il materiale di partenza, ossia le razze di B. mori, appartengono infatti alla banca di germoplasma del Consiglio per la Ricerca e la sperimentazione in Agricoltura – Unità di ricerca di Apicoltura e Bachicoltura (CRA-API) di Padova; la collezione di accessioni è composta da circa 200 razze distinguibili sulla base di diverse caratteristiche fenotipiche e genotipiche. Partendo dalla suddetta collezione, è stato compiuto un lavoro di selezione basato su una strategia di allevamento misto dieta-foglia finalizzato alla selezione di razze, caratterizzate da un’elevata efficienza alimentare sia su foglia di gelso sia su dieta sostitutiva, in modo da ottimizzare il rapporto costi/benefici in tutti quegli ambiti interessati ad aspetti di produttività. Come sopra ricordato, la strategia era basata su un approccio misto: un primo step, più tradizionale e orientato all’incremento dei parametri produttivi della seta in senso classico, era incentrato sull’allevamento con foglia, fornita senza limitazioni di quantità. Un secondo step prevedeva, invece, un ciclo d’allevamento su una dieta sostitutiva della foglia di gelso in cui la selezione per la produttività avveniva a fronte di un’alimentazione contingentata. Dopo tre anni in cui si sono svolte sei generazioni di selezione, le popolazioni originali sono state confrontate sia tra loro sia con le selezioni ottenute; per tale comparazione, sono stati utilizzati indici alimentari in grado di qualificare le larve secondo la loro efficienza nel convertire le proteine contenute nella matrice alimentare in massa corporea e seta. La valutazione finale ci ha permesso di evidenziare come la strategia adottata, si sia rivelata efficace nel permettere la selezione dei caratteri desiderati sebbene, alcune differenze legate alla diversa adattabilità delle varie razze alla dieta artificiale, suggeriscano l’utilità dell’introduzione di una selezione preliminare per questo carattere. Nell’ambito della sperimentazione finalizzata alla selezione delle razze ad alta efficienza sono stati condotti alcuni esperimenti complementari che hanno portato alla produzione di rilevanti dati scientifici. In primo luogo è stato isolato e caratterizzato un microorganismo, dimostratosi patogeno per il baco da seta allevato su dieta artificiale ed, in secondo luogo, sono state effettuate approfondite misurazioni sulla resistenza alle basse temperature da parte di uova di una razza polivoltina appartenente alla collezione del CRA-API. I risultati esposti nel presente lavoro rappresentano, allo stato attuale, non un punto d’arrivo ma la tappa intermedia di un processo di selezione che verrà proseguito presso CRA-API oltre che la base di partenza per la produzione di nuovi ibridi dalle prestazioni migliorate ed allevabili, indifferentemente, su foglia e dieta sostitutiva con buone performances