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The production of insects on an industrial scale has attracted the attention of the research and agricultural industry as novel protein sources. To detect the presence of Gryllodes sigillatus (GS) in feed and food, a real-time PCR method based on the mitochondrial cytochrome b (CYB) gene is proposed by this study. Forty DNA samples of animal and plant origin were used to confirm the specificity of the qPCR system. The detection method’s performance was evaluated on different processed GS matrices including native GS (UnGS) and different commercial products: crunchy roasted samples (RoGS), insect meal mixtures (ACGS) and energetic snacks containing GS (GSS). Data on sequencing were aligned with the reference gene to confirm the PCR products. The regression curve (y = −3.394 x + 42.521; R2 = 0.994, d.f. 14) between Ct values and Log DNA concentrations of Gryllodes sigillatus resulted in an efficiency of 96.4%. The severity of the technological processing treatments and the matrix structure affected the intensity of the PCR signal with the same amount of insect DNA as observed by different y-intercepts of the three-regression lines for RoGS, ACGS, and GSS. The real-time PCR method resulted in robust and sensitive outcomes able to detect low amounts of GS DNA (5 g/100 g) in a complex matrix, making it suitable for detecting the presence or absence of labeled Gryllodes sigillatus material both in feed and food.

For the development of a bio-sensor based on fluorescence excitation in transmission, a plastic substrate with excellent optical characteristics, such as high optical transmission in the visible range and low self-fluorescence, is required. Polyethylene (PE) films have been found to meet these optical characteristics. However, the adhesion of short sequences of oligonucleotides used as bio-probes, which are needed to develop a genosensor, has not proven ideal on this substrate, due to its olefinic composition. In order to overcome this challenge, a physical-chemical surface modification of PE films has been performed using a corona treatment, followed by the attachment of functional organic compounds. The surface modified substrates have been characterized for their wettability, surface energy, zeta potential and surface topography. The bio-probes’ adhesion on the treated surfaces has been tested by monitoring the fluorescence of dye-conjugated complementary sequences of the oligonucleotides deposited on the modified PE substrates. Finally, two modified PE substrates, comprising the best adhesion of the bio-probes, have been used to prepare a genosensor for the detection of the DNA of the insect Hermetia illucens. As expected, the better adhesion of bio-probes on the functionalized PE substrate has allowed a significant improvement of the bio-sensor’s limit of detection (LOD).

Despite a growing interest in microalgae and cyanobacteria as potential sources of nutrients in aquafeeds, little information is presently available on their nutritive value for carnivorous fish species. The aim of this study was to evaluate chemical composition and nutrient digestibility of a panel of microalgae and cyanobacteria dried biomasses (MACB), using rainbow trout (Oncorhynchus mykiss W.) as a fish model. Nine test diets were obtained by mixing 80 parts of a reference diet, added with 20 g/kg of acid insoluble ash as an indigestible marker, to 20 parts of each of the following dried whole-cell biomass: Arthrospira platensis, Nostoc sphaeroides, two strains of Chlorella sorokiniana, Nannochloropsis oceanica, Tisochrysis lutea, Phaeodactylum tricornutum, Porphyridium purpureum and Tetraselmis suecica. The digestibility measurements were conducted with rainbow trout (52.4 ± 1.5 g) kept in six tank units each including three 60-L vessels singularly stocked with 12 fish and fitted with a settling column for faecal recovery. Per each diet, faeces were collected over three independent 10-day periods. Apparent digestibility coefficients (ADCs) of dry matter, crude protein (CP), organic matter and gross energy (GE) of single MACB were calculated by difference relative to those of the reference diet. The MACBs had heterogeneous chemical composition (CP, from 20 to 69%; Lipid, 5-27%; GE, 12.5--22.6 MJ/kg dry matter basis) reflecting their overall biodiversity. Most of them can be considered as virtually good sources of minerals and trace elements and exhibit an essential amino acid profile comparable or even better than that of soybean meal commonly used in fish feeds with P. purpureum showing the best protein profile. The digestibility results obtained with rainbow trout allowed ranking the MACBs into two major groups. A first one, including C. sorokiniana, N. oceanica and T. suecica, resulted in markedly lower (P < 0.05) crude protein and energy ADC (64-73%; 51-59%, respectively) compared to a second group including P. purpureum, T. lutea and cyanobacteria (CP-ADC, 83-88%; GE-ADC, 74-90%) while P. tricornutum resulted in intermediate values. Overall, the present study confirms the consistently reported role of cell-wall structure/composition in affecting accessibility of nutrients to digestive enzyme. Based on the overall outcomes, only T. lutea and cyanobacteria actually meet the requirements for being used as protein sources in aquafeeds provided their mass production becomes more feasible and cost-effective, hence attractive for the feed-mill industry in the near future.

As reported by World Health Organization (WHO), most than half of the word population is potentially at risk to be infected with Dengue virus. This virus is a single strained RNA virus of the flaviviridae family that presents four different serotypes. Secondary infections with different serotypes can induce very dangerous complications, like Dengue haemorrhagic fever and shock syndrome. The tropical and sub-tropical areas are the most affected by this infection. Therefore, there is a strong need of a cheap, portable, fast and sensitive diagnostic test also for economic reasons. In this paper, the development of a Point-of-Care multiparametric system using an organic light emitting diode (OLED)-based biochip for the simultaneous detection of the four different Dengue serotypes, is presented. The system exploits the fluorescence detection of a low density matrix of protein antigens and secondary fluorophore tagged antibodies, excited by an OLED source. A low cost CMOS camera, allowing low light detection, to perform multiparametric quantitative analysis has been used in combination with a specifically developed image processing software. The software pipeline adopted is described as well. Fourty Dengue patient sera, previously characterized by standard diagnostic techniques, have been tested, using 3 μl of sera and an incubation time of 30 minutes. The results of these analysis demonstrate the capability of this diagnostic system to perform early Dengue diagnosis and to recognize the Dengue serotype with very high precision, in particular in convalescent patients.

Recently, the insects meal has been released by the European Union as an ingredient in aquafeeds and only seven insect species have been allowed to be used for these preparations. To avoid misleading results, a suitable method for the detection of these insect species in food/feed, would be highly desirable. In this paper, we present an innovative, sensitive, specific and low-cost Point-of-Care apparatus, based on DNA probes for the detection of one of the most popular insects used in aquafeed preparation, Hermetia illucens (HI), commonly known as black soldier fly. The DNA probes adopted for this genosensor have been designed to enable a very specific insect recognition which were derived from DNA primers. The DNA primers were first tested with qPCR. Forty different samples (animal species and common aquafeed ingredients) have been tested to check the possible cross-reactivity, confirming the adequate primers specificity. The primers have been used in a practical application, like the detection of HI in complex matrices of practical aquafeeds. The DNA probes derived by these primers have been then applied to implement an Organic Light Emitting Diode (OLED) based genosensor. The developed genosensor has been tested with HI DNA directly extracted from the insect, without any DNA amplification; a good test sensitivity has been obtained, being 0.75 ng/μl its detection (LOD) limit. Then, the genosensor has been tested with the DNA extracted from the same aquafeeds used in the qPCR experiments. The results obtained by these two methods have then been compared giving very good consistency and both allowed to detect HI meal up to a minimum threshold of 5 g/100gr feed.

Dittrichia viscosa is a Mediterranean ruderal species that over the last decades has expanded into new habitats, including coastal salt marshes, ecosystems that are per se fragile and threatened by human activities. To assess the potential risk that this native-invasive species represents for the genuine salt marsh vegetation, we compared its distribution with that of Inula crithmoides, a taxonomically related halophyte, in three salt marshes located in “La Albufera” Natural Park, near the city of Valencia (East Spain). The presence of D. viscosa was restricted to areas of low and moderate salinity, while I. crithmoides was also present in the most saline zones of the salt marshes. Analyses of the responses of the two species to salt and water stress treatments in controlled experiments revealed that both activate the same physiological stress tolerance mechanisms, based essentially on the transport of toxic ions to the leaves—where they are presumably compartmentalized in vacuoles—and the accumulation of specific osmolytes for osmotic adjustment. The two species differ in the efficiency of those mechanisms: salt-induced increases in Na+ and Cl− contents were higher in I. crithmoides than in D. viscosa, and the osmolytes (especially glycine betaine, but also arabinose, fructose and glucose) accumulated at higher levels in the former species. This explains the (slightly) higher stress tolerance of I. crithmoides, as compared to D. viscosa, established from growth inhibition measurements and their distribution in nature. The possible activation of K+ transport to the leaves under high salinity conditions may also contribute to salt tolerance in I. crithmoides. Oxidative stress level—estimated from malondialdehyde accumulation—was higher in the less tolerant D. viscosa, which consequently activated antioxidant responses as a defense mechanism against stress; these responses were weaker or absent in the more tolerant I. crithmoides. Based on these results, we concluded that although D. viscosa cannot directly compete with true halophytes in highly saline environments, it is nevertheless quite stress tolerant and therefore represents a threat for the vegetation located on the salt marshes borders, where several endemic and threatened species are found in the area of study., Work in the UPV laboratories was partly funded by a grant to OV from the Spanish Ministry of Science and Innovation (Project CGL2008-00438/BOS), with contribution from the European Regional Development Fund.

[EN] We have isolated full-length cDNA clones of the NHX1 gene – encoding the vacuolar Na+ /H+ antiporter – from three Plantago species (P. crassifolia, P. coronopus and P. major) differing in salt tolerance. A phylogenetic tree of their deduced protein sequences and those of NHX1 antiporters previously isolated from other species suggested the presence of some structural features characteristic of the proteins of salt tolerant taxa. The expression patterns of the NHX1 genes in the presence of salt correlated with the relative degree of salt tolerance of the three species: no induction was detected in the least tolerant P. major – and in the glycophyte A. thaliana – and the highest and quickest induction was observed in the most tolerant P. crassifolia. These data support the hypothesis that the NHX1 antiporters play a functional role in the mechanisms of salt tolerance in plants.