A single-step extraction and immobilization of soybean lipolytic enzymes by using a purpose-designed copolymer of styrene and maleic acid as a membrane lysis agent.

Approaches aiming to recover proteins without denaturation represent attractive strategies. To accomplish this, a membrane lysis agent based on poly(styrene-alt-maleic acid) or PSMA was synthesized by photopolymerization using Irgacure® 2959 and carbon tetrabromide (CBr4) as a radical initiator and a reversible chain transfer agent, respectively. Structural elucidation of our in-house synthesized PSMA, so-called photo-PSMA, was performed by using NMR spectroscopy. The use of this photo-PSMA in soybean enzyme extraction was also demonstrated for the first time in this study. Without a severe cell rupture, energy input or any organic solvent, recovery of lipolytic enzymes directly into nanometric-sized particles was accomplished in one-step process. Due to the improved structural regularity along the photo-PSMA backbone, the most effective protective reservoir for enzyme immobilization was generated through the PSMA aggregation. Formation of such reservoir enabled soybean enzymes to be shielded from the surroundings and resolved in their full functioning state. This was convinced by the increased specific lipolytic activity to 1,950 mU/mg, significantly higher than those of sodium dodecyl sulfate (SDS) and the two commercially-available PSMA sources (1000P and 2000P). Our photo-PSMA had thus demonstrated its great potential for cell lyse application, especially for soybean hydrolase extraction.

Tropilaelaps mercedesae Infestation Is Correlated with Injury Numbers on the Brood and the Population Size of Honey Bee Apis mellifera.

Tropilaelaps mercedesae, one of the most devastating parasitic mites of honey bee Apis mellifera hosts, is a major threat to honey products by causing severe damage to honey bee colonies. Here, we recorded injury numbers caused by T. mercedesae to different body parts of the larval, pupal, and crippled adult stages of honey bee A. mellifera. We evaluated the relationship between infestation rate and injury numbers per bee for both larvae and pupae. We also noted the total bee numbers per beehive and examined the relationship between the infestation rate and population size. T. mercedesae infested all developmental stages of honey bees, with the highest injury numbers in the abdomens of bee pupae and the antennas of crippled adult bees. Although larvae received more injury numbers than pupae, both infestation rate and injury numbers decreased as the larval stage progressed to the pupal stage. The infestation rate increased as the population size per beehive decreased. This study provided new perspectives to the understanding of changes in the effects of T. mercedesae infestations on different developmental stages of honey bees. It also showed useful baseline information for screening honey bee stock that might have high defensive behaviors against mite infestation.

Distinct gut microbiota profiles of Asian honey bee (Apis cerana) foragers.

Bee gut microbial communities have been studied extensively and linked to honey bee biology in terms of stages of bee development and behavior. Associations of bee gut microbiota in health and disease have also been explored. A large number of studies have centered on the gut microbiome of Apis mellifera, with similar investigations lagging far behind in Asian honey bee foragers. In this study, we characterized and compared the gut bacterial profiles of foragers and nurse bees of A. cerana and A. mellifera. Analysis of 16S rRNA partial gene sequences revealed significant differences in gut bacterial communities between the two honey bee species. Despite sharing dominant taxa, Bacteroides was more abundant in A. cerana, while Proteobacteria was higher in A. mellifera. Specific gut members are distinctly associated with hosts performing different tasks (i.e. nurse bees versus foragers). An exclusive abundance of Apibacter detected in Asian honey bee seemed to be a microbial signature of A. cerana foragers. Overall, our study highlights that variations in gut microbiota could be linked to task-specific (nurse bees and foragers) bacterial species associated with honey bees. Future investigations on the symbiotic relationship between host and the resident microbiota would be beneficial for improving honey bee health.

Bacterial Communities in Three Parts of Intestinal Tracts of Carpenter Bees (Xylocopa tenuiscapa).

This study investigated different bacterial communities in three intestinal parts (foregut, midgut and hindgut) of Xylocopatenuiscapa to understand the roles of gut bacteria. Our phylogenetic analysis revealed that X. tenuiscapa is closely related to Xylocopa latipes. The 16S rRNA gene in the genomic DNA samples from the gut was examined by illumina (Solexa) and a total of 998 operational taxonomic unit (OTUs) clusters were found. Taxonomic classification identified 16 bacterial phyla and unclassified bacteria. The dominant bacteria taxa in the three parts of X. tenuiscapa gut were Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. In the foregut, Lactobacillales and Enterobacteriaceae were predominantly found. The population in the midgut was similar to that in the foregut, with the addition of Gilliamella, which was also abundant. The most dominant bacteria identified in the hindgut were similar to those in the midgut and Lactobacillales, Enterobacteriaceae, Gilliamella, Bifidobacteriaceae and Flavobacteriaceae appeared in abundance. Moreover, our results suggest that a community structure of bacteria in different parts of X. tenuiscapa's gut may be an important indicator of carpenter bees' health. This functional study of bacterial communities revealed significant differences among the three intestinal parts and is the first report of the gut bacteria structure in solitary bees.

Feeding by Tropilaelaps mercedesae on pre- and post-capped brood increases damage to Apis mellifera colonies.

Tropilaelaps mercedesae parasitism can cause Apis mellifera colony mortality in Asia. Here, we report for the first time that tropilaelaps mites feed on both pre- and post-capped stages of honey bees. Feeding on pre-capped brood may extend their survival outside capped brood cells, especially in areas where brood production is year-round. In this study, we examined the types of injury inflicted by tropilaelaps mites on different stages of honey bees, the survival of adult honey bees, and level of honey bee viruses in 4th instar larvae and prepupae. The injuries inflicted on different developing honey bee stages were visualised by staining with trypan blue. Among pre-capped stages, 4th instar larvae sustained the highest number of wounds (4.6 ± 0.5/larva) while 2nd-3rd larval instars had at least two wounds. Consequently, wounds were evident on uninfested capped brood (5th-6th instar larvae = 3.91 ± 0.64 wounds; prepupae = 5.25 ± 0.73 wounds). Tropilaelaps mite infestations resulted in 3.4- and 6-fold increases in the number of wounds in 5th-6th instar larvae and prepupae as compared to uninfested capped brood, respectively. When wound-inflicted prepupae metamorphosed to white-eyed pupae, all wound scars disappeared with the exuviae. This healing of wounds contributed to the reduction of the number of wounds (≤10) observed on the different pupal stages. Transmission of mite-borne virus such as Deformed Wing Virus (DWV) was also enhanced by mites feeding on early larval stages. DWV and Black Queen Cell Virus (BQCV) were detected in all 4th instar larvae and prepupae analysed. However, viral levels were more pronounced in scarred 4th instar larvae and infested prepupae. The remarkably high numbers of wounds and viral load on scarred or infested developing honey bees may have caused significant weight loss and extensive injuries observed on the abdomen, wings, legs, proboscis and antennae of adult honey bees. Together, the survival of infested honey bees was significantly compromised. This study demonstrates the ability of tropilaelaps mites to inflict profound damage on A. mellifera hosts. Effective management approaches need to be developed to mitigate tropilaelaps mite problems.

Neonicotinoids and ectoparasitic mites synergistically impact honeybees.

The Western honeybee, Apis mellifera, is the most important managed pollinator globally and has recently experienced unsustainably high colony losses. Synergistic interactions among stressors are believed to be primarily responsible. However, despite clear evidence of strong effect on honeybee longevity of widely-employed neonicotinoid insecticides and of the ubiquitous ectoparasitic mite Varroa destructor, no data exist to show synergistic effects between these two stressors. Even though neonicotinoids had no significant impact by themselves, we here show for the first time a synergistic time-lag interaction between mites and neonicotinoids that resulted in significantly reduced survival of long-lived winter honeybees. Even though these mites are potent vectors of viruses, the virus-insecticide interaction had no significant impact. The data suggest a previously overlooked mechanism possibly explaining recent unsustainably high losses of managed A. mellifera honeybee colonies in many regions of the world. Future mitigation efforts should concentrate on developing sustainable agro-ecosystem management schemes that incorporate reduced use of neonicotinoids and sustainable solutions for V. destructor mites.

Phylogenetic Revision of Savoryellaceae and Evidence for Its Ranking as a Subclass.

Morphology, phylogeny, and molecular clock analyses were carried out on Savoryellaceae in order to understand the placements of taxa in this family. Ascotaiwania and Neoascotaiwania formed a well-supported separate clade in the phylogeny of concatenated partial SSU, LSU, TEF, and RPB2 gene data. These two genera share similar morphological features, especially in their asexual morphs, indicating that they are congeneric. Hence, we synonymize Neoascotaiwania under Ascotaiwania. Ascotaiwania hughesii (and its asexual morph, Helicoon farinosum) and Monotosporella setosa grouped in a clade sister to Pleurotheciales and are excluded from Ascotaiwania which becomes monophyletic. A novel genus Helicoascotaiwania is introduced to accommodate Ascotaiwania hughesii and its asexual morph, Helicoon farinosum. A novel species, Savoryella yunnanensis is introduced from a freshwater habitat in Yunnan Province, China. Comprehensive descriptions and illustrations are provided for selected taxa in this family. In addition, we provide evolutionary divergence estimates for Savoryellomycetidae taxa and major marine based taxa to support our phylogenetic and morphological investigations. The taxonomic placement of these marine-based taxa is briefly discussed. Our results indicate that the most basal group of marine-based taxa are represented within Lulworthiales, which diverged from ancestral Sordariomycetes around 149 Mya (91-209) and Savoryellomycetidae around 213 Mya (198-303).

Survival of Ectoparasitic Mites Tropilaelaps mercedesae in Association with Honeybee Hive Products.

The global trade of honeybee hive products imposes the risk of the introduction of exotic pests. However, data on the potential of specific products enabling pest survival are often lacking. This holds especially true for ectoparasitic mites Tropilaelaps spp., which are mandatory pests of honeybees in many countries. Here, we evaluated the longevity of Tropilaelaps mercedesae mites associated with empty honeycomb and dry pollen as two possible global import routes. Mites were able to survive up to three days in dry pollen and up to six days in empty honeycomb, thereby suggesting a sufficient time window for the potential introduction of T. mercedesae into mite-free countries via import of these hive products.

Tropilaelaps mite: an emerging threat to European honey bee.

The risk of transmission of honey bee parasites has increased substantially as a result of trade globalization and technical developments in transportation efficacy. Great concern over honey bee decline has accelerated research on newly emerging bee pests and parasites. These organisms are likely to emerge from Asia as it is the only region where all 10 honey bee species co-occur. Varroa destructor, an ectoparasitic mite, is a classic example of a pest that has shifted from A. cerana, a cavity nesting Asian honey bee to A. mellifera, the European honey bee. In this review, we will describe the potential risks to global apiculture of the global expansion of Tropilaelaps mercedesae, originally a parasite of the open-air nesting Asian giant honey bee, compared to the impact of V. destructor.

Successful reproduction of unmated Tropilaelaps mercedesae and its implication on mite population growth in Apis mellifera colonies.

Successful reproduction by unmated Tropilaelaps mercedesae is reported here for the first time. Of the eight mature daughters that did not have male mates within their natal cells, four produced both mature sons and daughters, and four produced mature daughters only. Overall, 78% of the new daughters that had no egg-laying experience, and 84% of the foundresses that had or had not laid previously reproduced. Both inoculum daughter and foundress mites were collected from tan-bodied pupae and inoculated immediately. Therefore, our results suggest that phoresy is not required for reproduction in tropilaelaps mites. The ability of virgin females to lay both males and females (deuterotoky), and to reproduce without spending a phoretic period on adult bees may play major roles in tropilaelaps mites' competitive advantage over varroa mites in Apis mellifera colonies.

Ecology, Life History, and Management of Tropilaelaps Mites.

Parasitic mites are the major threat to the Western honey bee, Apis mellifera L. For much of the world, Varroa destructor Anderson & Trueman single-handedly inflicts unsurmountable problems to A. mellifera beekeeping. However, A. mellifera in Asia is also faced with another genus of destructive parasitic mite, Tropilaelaps. The life history of these two parasitic mites is very similar, and both have the same food requirements (i.e., hemolymph of developing brood). Hence, parasitism by Tropilaelaps spp., especially Tropilaelaps mercedesae and Tropilaelaps clareae, also results in death of immature brood or wing deformities in infested adult bees. The possible introduction of Tropilaelaps mites outside their current range heightens existing dilemmas brought by Varroa mites. In this review, we provide historic, as well as current information on the taxonomic status, life history, distribution and host range, diagnosis, and control of Tropilaelaps mites. Because the biology of Tropilaelaps mites is not well known, we also suggest areas of research that demand immediate attention. Any biological information about Tropilaelaps mites will provide useful information for the development of control measures against them.

Neonicotinoid insecticides can serve as inadvertent insect contraceptives.

There is clear evidence for sublethal effects of neonicotinoid insecticides on non-target ecosystem service-providing insects. However, their possible impact on male insect reproduction is currently unknown, despite the key role of sex. Here, we show that two neonicotinoids (4.5 ppb thiamethoxam and 1.5 ppb clothianidin) significantly reduce the reproductive capacity of male honeybees (drones), Apis mellifera Drones were obtained from colonies exposed to the neonicotinoid insecticides or controls, and subsequently maintained in laboratory cages until they reached sexual maturity. While no significant effects were observed for male teneral (newly emerged adult) body mass and sperm quantity, the data clearly showed reduced drone lifespan, as well as reduced sperm viability (percentage living versus dead) and living sperm quantity by 39%. Our results demonstrate for the first time that neonicotinoid insecticides can negatively affect male insect reproductive capacity, and provide a possible mechanistic explanation for managed honeybee queen failure and wild insect pollinator decline. The widespread prophylactic use of neonicotinoids may have previously overlooked inadvertent contraceptive effects on non-target insects, thereby limiting conservation efforts.

The ectoparasitic mite Tropilaelaps mercedesae reduces western honey bee, Apismellifera, longevity and emergence weight, and promotes Deformed wing virus infections.

Historically an ectoparasite of the native Giant honey bee Apis dorsata, the mite Tropilaelaps mercedesae has switched hosts to the introduced western honey bee Apis mellifera throughout much of Asia. Few data regarding lethal and sub-lethal effects of T. mercedesae on A. mellifera exist, despite its similarity to the devastating mite Varroa destructor. Here we artificially infested worker brood of A. mellifera with T. mercedesae to investigate lethal (longevity) and sub-lethal (emergence weight, Deformed wing virus (DWV) levels and clinical symptoms of DWV) effects of the mite on its new host. The data show that T. mercedesae infestation significantly reduced host longevity and emergence weight, and promoted both DWV levels and associated clinical symptoms. Our results suggest that T. mercedesae is a potentially important parasite to the economically important A. mellifera honey bee.

Differential viral levels and immune gene expression in three stocks of Apis mellifera induced by different numbers of Varroa destructor.

The viral levels and immune responses of Italian honey bees (IHB), Russian honey bees (RHB) and an outcross of Varroa Sensitive Hygienic bees (POL) deliberately infested with one or two foundress Varroa were compared. We found that the Deformed wing virus (DWV) level in IHB inoculated with one or two foundress Varroa increased to about 10(3) or 10(5) fold the levels of their uninfested brood. In contrast, POL (10(2) or 10(4) fold) and RHB (10(2) or l0(4) fold) supported a lower increase in DWV levels. The feeding of different stages of Varroa nymphs did not increase DWV levels of their pupal hosts. Analyses of their corresponding Varroa mites showed the same trends: two foundress Varroa yielded higher DWV levels than one foundress, and the addition of nymphs did not increase viral levels. Using the same pupae examined for the presence of viruses, 16 out of 24 genes evaluated showed significant differential mRNA expression levels among the three honey bee stocks. However, only four genes (Defensin, Dscam, PPOact and spaetzle), which were expressed at similar levels in uninfested pupae, were altered by the number of feeding foundress Varroa and levels of DWV regardless of stocks. This research provides the first evidence that immune response profiles of different honey bee stocks are induced by Varroa parasitism.

An evaluation of the associations of parameters related to the fall of Varroa destructor (Acari: Varroidae) from commercial honey bee (Hymenoptera: Apidae) colonies as tools for selective breeding for mite resistance.

Varroa destructor (Anderson and Trueman) trapped on bottom boards were assessed as indirect measurements of colony mite population differences and potential indicators of mite resistance in commercial colonies of Russian and Italian honey bees (Apis mellifera L.) by using 35 candidate measurements. Measurements included numbers of damaged and nondamaged younger mites, nymphs, damaged and nondamaged older mites, fresh mites, and all mites, each as a proportion of total mites in the colonies and as a proportion of all trapped mites or all trapped fresh mites. Several measurements differed strongly between the stocks, suggesting that the detailed characteristics of trapped mites may reflect the operation of resistance mechanisms in the Russian honey bees. Regression analyses were used to determine the relationships of these candidate measurements with the number of mites in the colonies. The largest positive regressions differed for the two stocks (Italian honey bees: trapped mites and trapped younger mites; Russian honey bees: trapped younger mites and trapped fresh mites). Also, the regressions for Italian honey bees were substantially stronger. The largest negative regressions with colony mites for both stocks were for the proportion of older mites out of all trapped mites. Although these regressions were statistically significant and consistent with those previously reported, they were weaker than those previously reported. The numbers of mites in the colonies were low, especially in the Russian honey bee colonies, which may have negatively influenced the precision of the regressions.

Observations on the removal of brood inoculated with Tropilaelaps mercedesae (Acari: Laelapidae) and the mite's reproductive success in Apis mellifera colonies.

This study assessed the response of Apis mellifera to brood deliberately infested with Tropilaelaps mercedesae. The reproductive success of T. mercedesae in mite-inoculated and naturally infested brood was also compared. The presence of T. mercedesae inside brood cells significantly affected brood removal. Thai A. mellifera removed 52.6 ± 8.2 % of the brood inoculated with T. mercedesae as compared to 17.2 ± 1.8 and 5.7 ± 1.1 % removal rates for the groups of brood with their cell cappings opened and closed without mite inoculation and the control brood (undisturbed, no mite inoculation), respectively. Brood removal peaked during the second and third days post inoculation when test brood was at the prepupal stage. Overall, non-reproduction (NR) of foundress T. mercedesae was high. However, when NR was measured based on the criteria used for Varroa, the naturally infested pupae (NIP) supported the highest NR (92.8 %). Newly sealed larvae inoculated with Tropilaelaps collected from newly sealed larvae (NSL) had 78.2 % NR and those inoculated with Tropilaelaps collected from tan-bodied pupae (TBP) had 76.8 % NR. Since Tropilaelaps is known to have a short development period and nearly all progeny reach adulthood by the time of host emergence, we also used two Tropilaelaps-specific criteria to determine NR. Foundresses that did not produce progeny and those that produced only one progeny were considered NR. Using these two criteria, NR decreased tremendously but showed similar trends with means of 65, 40 and 33 % for NIP, NSL and TBP, respectively. High NR in the NIP group may indicate increased hygienic behavior in Thai A. mellifera colonies. The removal of infested prepupae or tan-bodied pupae will likely decrease the reproductive potential of Tropilaelaps. Our study suggests that brood removal may be one of the resistance mechanisms towards T. mercedesae by naturally adapted Thai A. mellifera.

Susceptibility of four different honey bee species to Nosema ceranae.

In this study, we investigated the infectivity of Nosema ceranae and the immune response of the European honey bee, Apis mellifera and the Asian honey bee species, Apis cerana, Apis dorsata and Apis florea when inoculated with two isolates of N. ceranae isolated from different climates (Canada and Thailand), using cage experiments. The results indicated that the local isolate of N. ceranae (Thailand) had high infectivity in A. mellifera, A. cerana and A. dorsata but only a few spores were observed in A. florea. However, we found that only two honey bee species, A. mellifera and A. dorsata became infected when inoculated with N. ceranae isolated from Canada. Finally, our results showed that transcript levels of antimicrobial peptides (AMPs) in Asian honey bees were significantly higher than that of A. mellifera in both the control and N. ceranae inoculated bee groups. Comparing the expression of AMPs between the control and inoculated bees in each species, it was evident that N. ceranae inoculations did not affect the expression level of abaecin in all four honey bees species investigated in this experiment. Nevertheless, we found a significant up-regulation of apidaecin in A. cerana and A. florea when inoculated with N. ceranae (Canadian isolate). Also, the mRNA levels of hymenoptaecin were significantly increased in A. cerana after inoculation by N. ceranae isolated from Canada as compared with the Thai isolate.