Antonio Felicioli

In honey bees, queen–worker differentiation is regulated by diet and begins after the first 3 days post‐hatching. Several molecules and processes appear to be involved in caste polyphenism, including juvenile hormone, royalactin, and DNA methylation. A protease (PS4) was recently detected in worker larvae at 72 h post‐hatching and shown to be inhibited by royal jelly, suggesting a potential role in this process. This study investigated the expression and tissue localization of nucleoplasmin during caste polyphenism in Apis mellifera larvae. A protein spot corresponding to a previously described caste‐specific proteolytic activity was separated by 2DE and identified by mass spectrometry as a nucleoplasmin‐like protein. Based on this result, a polyclonal antibody was raised against a conserved nucleoplasmin peptide and subsequently used for immunoproteomic and immunohistochemical analyses in worker‐ and queen‐destined larvae. At this instar, Western blot analysis using a polyclonal anti–nucleoplasmin‐like protein antibody revealed bands between 43–88 kDa and 74–104 kDa in worker‐ and queen‐destined larvae, respectively, which may correspond to different polymeric conformations of the nucleoplasmin‐like protein. Differences in the molecular mass of nucleoplasmin‐like protein spots between worker and queen larvae suggest caste‐specific roles during differentiation. Immunohistochemistry showed a ubiquitous nuclear distribution of the protein in undifferentiated larvae. Worker‐fate larvae diverge from this pattern as early as Day 4, whereas queen‐fate larvae retain a distribution like that of undifferentiated larvae, possibly representing the baseline developmental condition. Further studies examining temporal changes in nucleoplasmin‐like protein expression between the third and fourth larval instars and during post‐larval stages are desirable.

Saliva is an important biological matrix that allows the investigation of various welfare parameters; in ruminants, it is abundant and can be easily collected without requiring professional veterinary intervention. The aim of the investigation was to provide additional information on both digestive and antioxidant enzymes of sheep farmed in good welfare conditions, assessed with the Animal welfare indicators (AWIN) protocol. Small surgical forceps holding a Salivette^® device (Sarstedt, Nümbrecht, Germany) were gently inserted into the sheep’s mouths without any force. The sheep chewed the swabs independently for a few seconds, allowing saliva collection. Seventeen enzymes from the saliva of 15 sheep were analyzed: antioxidant enzymes (catalase, glutathione S transferase), proteolytic enzymes (trypsin, chymotrypsin, N-aminopeptidases, carboxypeptidase A and B), carbohydrases (glucose oxidase, amylase, cellulase, lignin peroxidase, chitinase and α-glycosidase), and esterases (alkaline and acidic phosphatases, lipase and esterase). Esterase activity showed the highest value (12.95 ± 1.25 U/mg of proteins), whereas lignin peroxidase activity showed the lowest (2.23 ± 0.37 µU/mg of proteins). The activity of all enzymes was observed except for glutathione S transferase and α-glycosidase. Among the enzymes, lipase activity has already been identified as a biomarker of stress in sheep saliva. This investigation may represent a basis for further investigations into the diet and adaptive responses of sheep to different environmental conditions. Furthermore, samples collected using the Salivette^® device can be easily obtained without requiring specialized staff and without causing any stress to the animals. Further investigations into the origin of individual enzymes using a proteomic approach are desirable.

Brucellosis is a zoonosis that affects domestic and wild animals, causing reproductive disorders and significant economic losses in livestock. Brucella melitensis, B. abortus, and B. suis are the main agents of brucellosis in livestock and humans, thereby their control and eradication are crucial. Serological tests based on identification of antibodies against Brucella smooth lipopolysaccharides (sLPS) in the serum of infected animals are traditionally used. This approach shows two main limits: (i) tests can give false positives due to the similarity of Brucella sLPS with the LPS of other Gram-negative bacteria; (ii) antigen production represents a possible risk of zoonoses. In this work, a proteomic approach, starting from B. melitensis Brucellergene, was employed to identify possible Brucella antigenic proteins useful for a more specific and safe serological diagnosis. Four proteins binding to the infected swine serum were identified: (i) “probable sugar-binding periplasmic protein B. abortus str 2308A”; (ii) “peptide ABC transporter substrate-binding protein B. melitensis”; (iii) “GntR family transcriptional regulator B. melitensis”; (iv) “conserved hypothetical protein B. melitensis M28”. These proteins could be promising specific antigens for serological investigations in swine. In the near future, these antigenic proteins could be synthesized in vitro and used to produce a safer and more specific diagnostic kit.

Despite its global importance for the maintenance of healthy ecosystems and its contribution in providing economic benefits to human society, the clade Anthophila is in severe decline worldwide. In this context, counteracting the decline in Apoidea is of fundamental importance. Honey bee density and beekeeping are believed to negatively impact wild bee populations, mostly through competition for food sources. In this study, interspecific trophic competition was investigated using the still seldom exploited approach of a palynological analysis combined with a metabarcoding analysis of the pollen gathered by both managed honey bees and wild bees in three Italian national parks. The entire trophic network was identified as highly specialized (H2′ = 0.933). The results obtained suggest that, overall, wild bee species are sustained by different pollen sources than honey bees. This low sharing of resources could be due to the natural trend occurring in natural populations, where species tend to minimize the competitive overlap through niche differentiation or niche complementarity as a result of coevolution. National parks play a fundamental role in animal and plant species protection and conservation. Therefore, plans should focus on evaluating honey bee densities in the interests of achieving less intensive, more traditional, and sustainable beekeeping, as well as habitat restoration, to promote the survival and reproduction of wild bee populations.

Honeybees (Apis mellifera L.) have to face many challenges, including Varroa destructor infestation, associated with viral transmission. Oxalic acid is one of the most common treatments against Varroa. Little is known about the physiological effects of oxalic acid, especially those on honeybees’ immune systems. In this study, the short-term effects (0–96 h) of oxalic acid treatment on the immune system components (i.e., glucose oxidase, phenoloxidase, glutathione S-transferase, catalase activities, and vitellogenin contents) of house bees were preliminarily investigated. Oxalic acid contents of bee bodies and haemolymphs were also measured. The results confirm that oxalic acid is constitutively present in bee haemolymphs and its concentration is not affected by treatment. At 6 h after the treatment, a maximum peak of oxalic acid content was detected on bees’ bodies, which gradually decreased after that until physiological levels were reached at 48 h. In the immune system, the oxalic acid treatment determined a peak in glucose oxidase activity at 48 h, indicating a potential defence response and an increase in vitellogenin content at 24 h. No significant changes were recorded in phenoloxidase, glutathione S-transferase, and catalase activities. These results suggest a time-dependent response to oxalic acid, with potential immune system activation in treated bees.

Amino acids (AAs) are an abundant class of nectar solutes, and they are involved in the nectar attractiveness to flower visitors. Among the various AAs, proline is the most abundant proteogenic AA, and γ‐amino butyric acid (GABA) and β‐alanine are the two most abundant non‐proteogenic AAs. These three AAs are known to affect insect physiology, being involved in flight metabolism and neurotransmission. The aim of this study was to investigate the effects of artificial diets enriched with either β‐alanine, GABA, or proline on consumption, survival, and hemolymph composition in honey bees belonging to two different ages and with different metabolism (i.e., newly emerged and foragers). Differences in feed intake among diets were not observed, while a diet enriched with β‐alanine improved the survival rate of newly emerged honey bees compared to the control group. Variations in the hemolymph AA concentrations occurred only in newly emerged honey bees, according to the diet and the time of hemolymph sampling. A greater susceptibility of young honey bees to enriched diets than older honey bees was observed. The variations in the concentrations of hemolymph AAs reflect either the accumulation of dietary AAs or the existence of metabolic pathways that may lead to the conversion of dietary AAs into different ones. This investigation could be an initial contribution to studying the complex dynamics that regulate hemolymph AA composition and its effect on honey bee physiology.

Searching for artificial diets positively affecting the survival, immune and antioxidant systems of honey bees is one of main challenges occurring in beekeeping. Among nutrients, lipids play a significant role in insect nutrition as structural components in cell membranes, energy sources and reserves, and are involved in many physiological processes. In this context, the aim of this work was to investigate the effect of 0.5% and 1% coconut oil-enriched diet administration on newly emerged and forager bees survival rate, feed intake, immune system, antioxidant system and both fat and vitellogenin content. In newly emerged bees, supplementation with 1% coconut oil determined a decrease in feed consumption, an increase in survival rate from the 3rd to 14th day of feeding, a short-term decrease in phenoloxidase activity, an increase in body fat and no differences in vitellogenin content. Conversely, supplementation with 0.5% coconut oil determined an increase in survival rate from the 3rd to 15th day of feeding and an increase in fat content in the long term (i.e., 20 days). Regarding the forager bee diet, enrichment with 0.5% and 1% coconut oil only determined an increase in fat content. Therefore, supplementation with coconut oil in honey bee diets at low percentages (0.5 and 1%) determines fat gain. Further investigations to evaluate the use of such supplement foods to prevent the fat loss of weak families during winter are desirable.

Flower nectar, a sugar‐rich solution containing amino acids as major secondary solutes, is the primary energy source for bees. Proline is one of the most abundant protein nectar amino acids, showing several effects on bee physiology. Mason bees are outstanding pollinators, often preferred to Apis mellifera for pollination of fruit trees. Among them, Osmia cornuta (Latreille) is one of the most successfully managed species on a commercial scale. In this work, the effects of 10 mM proline‐enriched diet administration on O. cornuta feed consumption, survival, behaviour and haemolymph amino acid composition were investigated. Feed intake was higher for the proline diet, while survival rate was not affected. Behaviour was affected only in the senescence of the mason bees with a reduction in locomotor activity induced by proline intake. Proline diet also affected the haemolymph amino acid composition, decreasing concentration of tyrosine, methionine, leucine and phenylalanine after 10 days of feeding. These results were discussed in relation to the potential involvement of proline in oxidative stress in insects and proline's ability to be converted to other amino acids. Further investigations are necessary to better understand the molecular mechanisms of proline effects on locomotion and oxidative stress.