Effect of amino acid enriched diets on hemolymph amino acid composition in honey 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 ß-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 ß-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 ß-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.

Bombus terrestris terrestris (Linnaeus, 1758) and hybrids with the endemic Bombus xanthopus spotted on Capraia Island (Tuscan Archipelago, Italy): some conservation management implications.

Expansion of wild and managed allochthonous species leads to potential negative consequences for the endemic wildlife, such as resource competition, pathogens spread, hybridization and native species replacements. On Capraia Island, the last sighting of Bombus terrestris terrestris dates back to 1917. All subsequent surveys carried out on the island only reported the presence of B. xanthopus and B. pascuorum melleofacies with B. t. terrestris apparently no longer existing in the area. In 2021 B. t. terrestris was again detected on the island raising two main hypotheses: (i) B. t. terrestris has always been present with a low population density, such as not to be detected in previous investigations, or (ii) its presence is the result of a more recent recolonization. The recolonization event may be promoted by either intentional or unintentional introduction or it may be the result of a natural migration from the mainland or surrounding islands. Hybridization between B. t. terrestris × B. xanthopus on Capraia Island has been also ascertained by the detection of hybrid queens, workers and males. These new finding provides insight on the distribution range of B. t. terrestris in the Tuscan Archipelago and raise concern on the conservation of the endemic B. xanthopus population.

Detection and pH-Thermal Characterization of Proteinases Exclusive of Honeybee Worker-Fate Larvae (Apis mellifera L.).

The occurrence of the honeybee caste polyphenism arises when a change in diet is transduced into cellular metabolic responses, resulting in a developmental shift mediated by gene expression. The aim of this investigation was to detect and describe the expression profile of water-soluble proteases during the ontogenesis of honeybee worker-fate larvae. The extraction of insect homogenates was followed by the electrophoretic separation of the protein extract in polyacrylamide gels under semi-denaturing condition, precast with gelatin, pollen, or royal jelly protein extracts. The worker-fate honeybee larva showed a proteolytic pattern that varied with aging, and a protease with the highest activity at 72 h after hatching was named PS4. PS4 has a molecular weight of 45 kDa, it remained active until cell sealing, and its enzymatic properties suggest a serine-proteinase nature. To define the process that originates a queen-fate larvae, royal jelly and pollen were analysed, but PS4 was not detected in either of them. The effect of food on the PS4 was investigated by mixing crude extracts of queen and worker-fate larvae with pollen and royal jelly, respectively. Only royal jelly inhibited PS4 in worker-fate larvae. Taken together, our data suggest that PS4 could be involved in caste differentiation.

Preliminary investigation on enzymatic activity in saliva of Hystrix cristata L., 1758.

Mammal's saliva contains a variety of electrolytes and proteins. They carry out an important role in the digestion process, in the antibacterial and antiviral activity, in lubrication and maintenance of oral general health status. It may also contain several enzymes according to dietary habits and general wellness. Sialochemistry is a valid alternative to the haematochemical analysis for the evaluation of animal health and nutritional status. At present, very little knowledge is available on health status and pathology of crested porcupine (Hystrix cristata) and no data are yet available on salivary enzymes. Between 2018 and 2020, a preliminary investigation of enzymatic activity on saliva samples was carried out from captured porcupines. In crested porcupine saliva, enzymatic activity of trypsin, chymotrypsin, N-Aminopeptidase, amylase, lignin peroxidise, cellulase and chitinase were recorded. Superoxide dismutase, catalase, glutathione S-transferase and alkaline phosphatase activity was also detected. The superoxide dismutase activity resulted higher (3.13 SD 3.58 U/mg proteins) than those of catalase (130.80 SD 110.65 mU/mg proteins) and glutathione S-transferase (20.21 SD 16.62 mM/mg proteins). Alkaline phosphatase activity resulted lower (5.91 SD 6.12 mU/mg proteins) than acidic phosphatase (19.00 SD 16.16 U/mg proteins) with the highest values of saliva alkaline phosphatases recorded in young individuals. These preliminary data bring new knowledge on crested porcupine saliva enzymes and may provide a useful tool for further investigation on the adaptive response of crested porcupine to different environmental condition and diet. Additional investigation concerning a possible alternative use of saliva enzymes as indicator of health and nutritional status of this rodent are desirable.

Effects of Non-Protein Amino Acids in Nectar on Bee Survival and Behavior.

Nectar mediates complex interactions between plants and animals. Recent research has focused on nectar secondary compounds that may play a role in regulating some of these interactions. These compounds may affect the behavior of nectar feeders by interacting with their neurobiology. Non-protein amino acids (NPAAs) can constitute a large portion of the amino acid content of floral nectar, but their ecological function has, to date, not been investigated. In this study, we tested the effects of diets with low and high concentrations of γ-amino butyric acid (GABA) and ß-alanine on the survival and behavior of Bombus terrestris and Apis mellifera. The most apparent effect on longevity was observed for B. terrestris workers that fed on high concentration of GABA, with longevity increased. By contrast, neither of the two NPAAs (at either concentration) had an affect on A. mellifera longevity. At the low NPAA concentration, only B. terrestris workers showed a difference in consumption, consuming more ß-alanine solution than the other two solutions. By contrast, at the high NPAA concentration, only A. mellifera workers showed a difference in consumption, consuming more ß-alanine solution. The effects of the NPAAs on behavior differed between the two species, with B. terrestris appearing more sensitive to the NPAAs than A. mellifera. After consuming NPAAs, B. terrestris showed changes in three (walking, flying, stationary) of the four behaviors recorded, although the effects varied with concentration and compound. In contrast, honey bees only showed a change in feeding behavior, with consumption of both NPAAs (at low concentrations) resulting in a decrease. Thus, pollinator intake of NPAAs may have important behavioral/ecological implications.

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography.

American foulbrood disease (AFB) is the main devastating disease that affects honeybees' brood, caused by Paenibacillus larvae. The trend of the research on AFB has addressed the mechanisms by which P. larvae bacteria kill honeybee larvae. Since prepupae could react to the infection of AFB by increasing protease synthesis, the aim of this work was to compare protease activity in worker prepupae belonging to healthy colonies and to colonies affected by AFB. This investigation was performed by zymography. In gel, proteolytic activity was observed in prepupae extracts belonging only to the healthy colonies. In the prepupae extracts, 2D zimography followed by protein identification by MS allowed to detect Trypsin-1 and Chymotrypsin-1, which were not observed in diseased specimens. Further investigations are needed to clarify the involvement of these proteinases in the immune response of honeybee larvae and the mechanisms by which P. larvae inhibits protease production in its host.

Odorant-binding proteins and olfactory coding in the solitary bee Osmia cornuta.

Solitary bees are major pollinators but their chemical communication system has been poorly studied. We investigated olfactory coding in Osmia cornuta from two perspectives, chemical and biochemical. We identified (E)-geranyl acetone and 2-hexyl-1,3-dioxolane, specifically secreted by females and males, respectively. A transcriptome analysis of antennae revealed 48 ORs (olfactory receptors), six OBPs (odorant-binding proteins), five CSPs (chemosensory proteins), and a single SNMP (sensory neuron membrane protein). The numbers of ORs and OBPs are much lower than in the honeybee, in particular, C-minus OBPs are lacking in the antennae of O. cornuta. We have expressed all six OBPs of O. cornuta and studied their binding specificities. The best ligands are common terpene plant odorants and both volatiles produced by the bee and identified in this work.

Oxalic Acid Treatment: Short-Term Effects on Enzyme Activities, Vitellogenin Content, and Residual Oxalic Acid Content in House Bees, Apis mellifera L.

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.

Diversity, abundance, and sex-specific expression of chemosensory proteins in the reproductive organs of the locust Locusta migratoria manilensis.

Chemosensory proteins (CSPs) are small soluble proteins often associated with chemosensory organs in insects but include members involved in other functions, such as pheromone delivery and development. Although the CSPs of the sensory organs have been extensively studied, little is known on their functions in other parts of the body. A first screening of the available databases has identified 70 sequences encoding CSPs in the oriental locust Locusta migratoria manilensis. Applying proteomic analysis, we have identified 17 of them abundantly expressed in the female reproductive organs, but only one (CSP91) in male organs. Bacterially expressed CSP91 binds fatty acids with a specificity for oleic and linoleic acid, as well as medium-length alcohols and esters. The same acids have been detected as the main gas chromatographic peaks in the dichloromethane extracts of reproductive organs of both sexes. The abundance and the number of CSPs in female reproductive organs indicates important roles for these proteins. We cannot exclude that different functions can be associated with each of the 17 CSPs, including delivery of semiochemicals, solubilization of hormones, direct control of development, or other unknown tasks.

Effect of Ageing in the Mating Behaviour Sequence of Osmia cornuta Latr. (Hymenoptera: Megachilidae).

Osmia cornuta Latr. is largely managed worldwide for the pollination of orchard crops, playing a key role in the maintenance of healthy ecosystems and ensuring economic and social benefits for human society. The management techniques of this pollinator include the possibility of delaying emergence from cocoons after diapause, allowing for the pollination of later-blooming fruit crops. In this study, the mating behaviour of bees emerging at the natural time (Right Emergence Insects) and of late-emerged bees (Aged Emergence Insects) was described in order to test if a delay in emergence could affect the mating sequence of O. cornuta. Markov analysis of the mating behaviour revealed the occurrence of antenna motion episodes that were repeated in a stereotyped manner at regular intervals during the mating sequence of both Right Emergence Insects and in Aged Emergence Insects. Pouncing, rhythmic and continuous emission of sound, motion of antennae, stretching of the abdomen, short and long copulations, scratching, inactivity, and self-grooming were identified as the stereotyped behavioural units of a behavioural sequence. The occurrence of short copulations, the frequency of which increased with the age of bees, could lead to a failure in the reproduction of the mason bee.

The colors of Tuscan bee pollen: phytochemical profile and antioxidant activity.

Bee pollen's nutritional and beneficial health properties depend on the botanical origin and storage conditions. Palynological analysis determines the botanical composition of the multiflora and colour fractions. This study aimed to characterize the phytochemical profile and antioxidant activity of Tuscan bee pollen stored at freezing temperature for 2 years to verify the preservation of nutraceutical properties of the multiflora and colour fractions. Polyphenols, flavonoids content, antioxidant activity and volatile compounds profiles were measured. Non-terpene derivatives (acids and aldehydes) represented the main class of volatile compounds in most analysed samples. Among the colour fractions, coral showed significant differences in the antioxidant compounds. In the multiflora were also determined the soluble sugar content (128.33 mg/g of fresh weight) and mineral content, with the prevalence of K, organic N and Ca. The results suggest that the freezing storage of bee pollen for a long period can be still used as food.

Host-Parasitoid Relationship between Apis mellifera (Linnaeus, 1758) and Senotainia tricuspis (Meigen, 1838) (Diptera, Sarcophagidae): Fly Aggression Behavior and Infestation Rates of Senotainiosis.

Senotainia tricuspis (Meigen, 1838) is a Sarcophagid dipteran endoparasitoid of Apis mellifera L., and myiasis, caused by this fly, is reported in several European, North African and Middle Eastern countries. Nevertheless, very little knowledge concerning the aggression and parasitisation behavior of S. tricuspis toward A. mellifera is available in the scientific literature, and the temporal pattern of aggression remains unclear. The aim of this investigation was to describe the aggressive behavior of S. tricuspis and to provide data on pupation and adult emergence in order to identify further tools for the control of senotainiosis in beekeeping. Data were collected in an apiary in Pisa province (Tuscany, Italy), where observations of aggressive behavior were conducted indirectly by using a VHS camera and also directly by an observer. Four behavioral categories of the attack were described. A total of 55 aggressions, 21 beecatchers, 104 chases and 6 parasitization events were recorded with the camera. Slow-motion recording analyses of the parasitization episodes resulted in contact of at least 1/6 s between the parasitoid and the host. Through four days of direct observations, a total of 1633 aggression events were recorded. The daily temporal pattern of the number of aggressions showed two main peaks: one during the morning hours (i.e., from 10:00 to 11:00) and one in the afternoon (i.e., from 15:00 to 17:00). The morphometric data on the first-instars of S. tricuspis allowed us to hypothesize a penetration in the bee through its prothoracic spiracle as a modality of entrance in the host body. Third-instars successfully pupate when sinking in topsoil or clay soil, and adults emerge when left to a 4 °C overwintering period of six months. Furthermore, the high mortality rate of those larvae that did not sink and did not pupate successfully suggests that reaching a certain depth in the soil is a determining factor for larvae survival and that mulch and/or minimum soil tillage could prevent severe senotainiosis in apiaries.

Porcupettes Management at Wildlife Rescue Centers and Liberation into the Wild: Implications for Post-Liberation Success.

The crested porcupine is a frequent host species in wildlife rescue centers and no guidelines for its management, liberation and post-liberation monitoring are yet available. Here, captive-grown porcupines' behavior and survival in the wild after liberation were investigated and described for the first time. Management strategies adopted at the centers could affect porcupine adaptation to the natural environment. The detention of porcupettes in single cages and fed only human-supplied food may not ensure ethological welfare, nor a suitable behavioral development compatible with wildlife. The liberation of captive-grown porcupines should also be carefully planned to promote and increase the possibility of post-liberation success.

Gray Wolf (Canis lupus italicus) and Red Fox (Vulpes vulpes) Parasite Survey in Anthropized and Natural Areas of Central Italy.

Gastrointestinal nematodes and protozoa and other parasite occurrences were evaluated in free-ranging wolf (Canis lupus italicus) and red fox (Vulpes vulpes) populations from natural and anthropized areas of Central Italy. Analyzed fecal samples were collected from 60 foxes and 40 wolves in the anthropized areas, and 41 foxes and 39 wolves in the natural areas. In foxes, hookworm infections (p < 0.0001) were more frequently recorded in the anthropized environment, while coccidia (p < 0.05) and Cryptosporidium spp. (p < 0.0001) were more frequent in the natural area. In wolves, a higher frequency of hookworms (p < 0.0001) was observed in natural areas, while coccidia were more common in the anthropized area (p < 0.05). Moreover, in the natural environment, trichuroid nematodes (p < 0.0001) were significantly more frequent in wolves than in foxes, while Cryptosporidium (p < 0.001) and Giardia duodenalis (p < 0.001) were more common in foxes. In the anthropic area, the occurrence of hookworms was found to be significantly higher in foxes (p < 0.0001), while trichuroid nematodes were more common in wolves (p < 0.0001). The obtained data are indicative of a different diffusion of specific parasite taxa in wolves and foxes living in the natural and/or anthropized environments examined herein.

Effects of Virgin Coconut Oil-Enriched Diet on Immune and Antioxidant Enzymatic Activity, Fat and Vitellogenin Contents in Newly Emerged and Forager Bees (Apis mellifera L.) Reared in Cages.

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.

Variation of Circulating Brain-Derived Neurotrophic Factor (BDNF) in Depression: Relationships with Inflammatory Indices, Metabolic Status and Patients' Clinical Features.

This study seeks to offer a contribution to the method of subtyping major depressed patients by exploring the possible relationships between circulating brain-derived neurotrophic factor (BDNF), different peripheral inflammatory/metabolic markers in the blood and clinical characteristics. Thirty-nine patients, thoroughly diagnosed according to the DSM-5 criteria, underwent a comprehensive set of evaluations encompassing structured interviews, rating scales and a panel of blood tests. Correlation and comparison analyses were carried out by means of non-parametric statistical tests. Concurrently, a principal component analysis was performed to explain biochemical variance. The findings of our research unveiled that leukocyte counts, their ratios and other inflammatory parameters are positively correlated with depression scores. Moreover, we found variations within the BDNF pools of depressed patients. Specifically, higher levels of platelet-poor plasma BDNF (PPP-BDNF) were correlated with augmented inflammatory markers in patients showing specific episode characteristics, whereas reduced platelet BDNF (PLT-BDNF) provided a better indication of the changes that were linked to a diagnosis of long-term depression. Our findings suggest that PPP-BDNF and PLT-BDNF might differentiate depression conditions. They also imply usefulness in appraising peripheral biomarker profiles in patients for a deeper characterization of major depressive episodes. At the same time, it is plausible that they might constitute novel avenues for developing more tailored therapeutic strategies for patients with MDs.

Spatio-temporal inhabitation of settlements by Hystrix cristata L., 1758.

Settlements are usually shared at different times by semi-fossorial mammals. Porcupine reproductive pair shows high den-site fidelity, but no data are available on the spatio-temporal inhabitation of settlements. In this investigation, the spatio-temporal inhabitation of settlements by crested porcupine families was investigated using camera-trapping as well as the ethological factors affecting the settlements selection. The crested porcupine resulted to be the main inhabitant of settlements surveyed in the present study. Each settlement was inhabited exclusively by one porcupine family. Five out of six porcupine families, each alternatively and complementarily inhabited the same two settlements. In all the five monitored families, settlements selection doesn't follow a seasonal pattern. Settlement inhabitation of porcupines resulted positively affected by cohabitation with badger, while presence of porcupettes did not affect settlements selection. Long periods of settlement inhabitation were positively affected both by the presence of porcupettes and cohabitation with badger. The pattern of settlements inhabitation in relation to their availability and porcupine population density as well as factors promoting porcupine-badger cohabitation should be further investigated. New ethological knowledge obtained in this investigation could be involved in the evaluation of the ecological epidemiology of infectious diseases between porcupine and badger within a one health approach and may be a useful tool for a sustainable management of semi-fossorial mammals.

Effects of Two Commercial Protein Diets on the Health of Two Imago Ages of Apis mellifera L. Reared in Laboratory.

Protein-supplemented artificial diets are widely used by beekeepers during winter and whenever food availability is low, yet no data are available concerning their effects on bees' health. In this work, the effects of two commercial diets enriched with 1.7% and 7.7% protein concentration on feed intake, survival rate, glucose oxidase, phenoloxidase and glutathione S-transferase in newly emerged and forager bees were tested. Administration of a 7.7% protein-enriched diet significantly reduced the lifespan of both newly emerged and forager bees, while only in foragers a significantly higher feed intake was recorded. In newly emerged bees, administration of a high-protein-enriched diet stimulated glucose oxidase production at the 10th day of feeding, determined a reduction of phenoloxidase and did not affect glutathione S-transferase activity. In forager bees, a high level of protein inclusion did not determine any significant variation in either glucose oxidase, phenoloxidase or glutathione S-transferase activity. Therefore, the results obtained in this investigation suggest that administration of commercial protein diets negatively affect honey bee health, determining an increase in mortality. Further investigations on the effect of concentration and quality of proteins are desirable to provide beekeepers with scientific evidence on protein feeding.

Emerging Risk of Cross-Species Transmission of Honey Bee Viruses in the Presence of Invasive Vespid Species.

The increase in invasive alien species is a concern for the environment. The establishment of some of these species may be changing the balance between pathogenicity and host factors, which could alter the defense strategies of native host species. Vespid species are among the most successful invasive animals, such as the genera Vespa, Vespula and Polistes. Bee viruses have been extensively studied as an important cause of honey bee population losses. However, knowledge about the transmission of honey bee viruses in Vespids is a relevant and under-researched aspect. The role of some mites such as Varroa in the transmission of honey bee viruses is clearer than in the case of Vespidae. This type of transmission by vectors has not yet been clarified in Vespidae, with interspecific relationships being the main hypotheses accepted for the transmission of bee viruses. A majority of studies describe the presence of viruses or their replicability, but aspects such as the symptomatology in Vespids or the ability to infect other hosts from Vespids are scarcely discussed. Highlighting the case of Vespa velutina as an invader, which is causing huge losses in European beekeeping, is of special interest. The pressure caused by V. velutina leads to weakened hives that become susceptible to pathogens. Gathering this information is necessary to promote further research on the spread of bee viruses in ecosystems invaded by invasive species of Vespids, as well as to prevent the decline of bee populations due to bee viruses.

Impact of Different Storage Temperature on the Enzymatic Activity of Apis mellifera Royal Jelly.

Royal Jelly is a nutrient secretion of nurse bees and a high interest functional food in human nutrition. Very little information is available on its chemical composition integrity and enzymatic activity during shelf life and assessment of new freshness markers are desirable for its conservation. In this study, the activity of glucose oxidase, five proteases and two antioxidant enzymes in refrigerated and frozen Royal Jelly for different storage times was preliminary investigated. Refrigeration determined a significantly reduction in glucose oxidase and carboxypeptidase A-like activity in Royal Jelly after one year of storage while no differences were recorded in the activity of these enzymes in frozen samples. After one year of storage glucose oxidase and carboxypeptidase A-like activity resulted higher in frozen samples frozen than in refrigerate ones. Results obtained suggest that the activities of these enzymes may be good markers of Royal Jelly freshness within 1 year at refrigeration condition. Freezing could be a valid alternative storage method to ensure a higher preservation of glucose oxidase and carboxypeptidase A-like activities for at least 1 year. Further investigation to determine the timing of glucose oxidase inactivation/degradation under refrigerated conditions and the enzymatic activity trend under prolonged frozen conditions are desirable.