Providing rigor in bee colony strength auditing methods.

The primary method used to audit honey bee (Apis mellifera Linnaeus, 1758 [Hymenoptera: Apidae]) colony strength for almond pollination services, Nasr et al.'s (1990) frame-top cluster count method, is a subjective visual audit that relies on an auditor's spot assessment and may lack rigor and repeatability. We created novel, open-source software for the analysis of frame-top cluster count photographic assessments to improve methodological rigor and repeatability. We evaluated 2 existing visual audit methods, created 3 novel audit method variations, and determined between-method conversion factors using linear modeling. The software has potential applications in apiological research, apiarist and orchardist colony auditing, as well as training future generations of apiarists in auditing techniques. The software enhances the rigor and repeatability of Nasr et al.'s (1990) frame-top cluster count population assessment. In this article, we introduce the novel open-source software and between-method regression equations and review the tested visual assessment methods and their application.

16S Amplicon Metabarcoding of the Nest Materials of Native Australian Stingless Bees.

We present 16S amplicon data derived from the nest materials of three species of Australian stingless bees (Meliponini). This data set reveals the diversity of bacteria associated with these materials. It will serve as a valuable baseline for further study of the nest microbiome and comparison with the stingless bee microbiota.

Temperature Sensing and Honey Bee Colony Strength.

Strength auditing of European honey bee (Apis mellifera Linnaeus, 1758 [Hymenoptera: Apidae]) colonies is critical for apiarists to manage colony health and meet pollination contracts conditions. Colony strength assessments used during pollination servicing in Australia typically use a frame-top cluster-count (Number of Frames) inspection. Sensing technology has potential to improve auditing processes, and commercial temperature sensors are widely available. We evaluate the use and placement of temperature sensing technology in colony strength assessment and identify key parameters linking temperature to colony strength. Custom-built temperature sensors measured hive temperature across the top of hive brood boxes. A linear mixed-effect model including harmonic sine and cosine curves representing diurnal temperature fluctuations in hives was used to compare Number of Frames with temperature sensor data. There was a significant effect of presence of bees on hive temperature and range: hives without bees recorded a 5.5°C lower mean temperature and greater temperature ranges than hives containing live bees. Hives without bees reach peak temperature earlier than hives with bees, regardless of colony strength. Sensor placement across the width of the hive was identified as an important factor when linking sensor data with colony strength. Data from sensors nearest to the hive geometric center were found to be more closely linked to colony strength. Furthermore, a one unit increase in Number of Frames was significantly associated with a mean temperature increase of 0.36°C. This demonstrates that statistical models that account for diurnal temperature patterns could be used to predict colony strength from temperature sensor data.

Thermal Impacts of Apicultural Practice and Products on the Honey Bee Colony.

Hive design and apicultural processes have not been fundamentally changed since the design and commercialization of the Langstroth moveable frame hive in 1854. Colonies of Apis mellifera Linnaeus (Hymentoptera: Apidae) (the honey bee) maintain a brood nest temperature within the narrow range of 34.5-35.5°C, critical for brood development. Apis mellifera invest considerable energy to maintain hive homeostasis through behavioral modification of the hive environment. Human honey-harvesting processes and removal of the honey-filled comb (a source of thermal mass) have a detrimental impact on hive temperature that requires an increased investment of energy to rectify. This additional energy demand on the bees is a form of stress to the colony and diverts workers away from other essential tasks to that of environmental management. We investigated the thermal energy loss resulting from the removal and extraction of honey, the rate of thermal loss of an Australian standard Langstroth 10 frame hive, and the effect of honey and wax as a thermal mass in unoccupied bee hive. The results demonstrate that considerable energy expenditure would be required to rectify the hive thermal environment after honey harvesting or honeycomb frame addition. Honey provides thermal mass in the beehive, acting as a thermal buffer to external temperature change, which may mediate part of the thermal losses from the simplistic design of the Langstroth hive. Identification of these impacts in current apicultural practice and hive design allows for the improvement in the design of beehives and associated practices. These improvements may reduce stress to the bee colony, increasing colony efficiency for pollination and nectar foraging.

MetaGaAP: A Novel Pipeline to Estimate Community Composition and Abundance from Non-Model Sequence Data.

Next generation sequencing and bioinformatic approaches are increasingly used to quantify microorganisms within populations by analysis of 'meta-barcode' data. This approach relies on comparison of amplicon sequences of 'barcode' regions from a population with public-domain databases of reference sequences. However, for many organisms relevant 'barcode' regions may not have been identified and large databases of reference sequences may not be available. A workflow and software pipeline, 'MetaGaAP,' was developed to identify and quantify genotypes through four steps: shotgun sequencing and identification of polymorphisms in a metapopulation to identify custom 'barcode' regions of less than 30 polymorphisms within the span of a single 'read', amplification and sequencing of the 'barcode', generation of a custom database of polymorphisms, and quantitation of the relative abundance of genotypes. The pipeline and workflow were validated in a 'wild type' Alphabaculovirus isolate, Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV-AC53) and a tissue-culture derived strain (HaSNPV-AC53-T2). The approach was validated by comparison of polymorphisms in amplicons and shotgun data, and by comparison of predicted dominant and co-dominant genotypes with Sanger sequences. The computational power required to generate and search the database effectively limits the number of polymorphisms that can be included in a barcode to 30 or less. The approach can be used in quantitative analysis of the ecology and pathology of non-model organisms.

Comparative Analysis of HaSNPV-AC53 and Derived Strains.

Complete genome sequences of two Australian isolates of H. armigera single nucleopolyhedrovirus (HaSNPV) and nine strains isolated by plaque selection in tissue culture identified multiple polymorphisms in tissue culture-derived strains compared to the consensus sequence of the parent isolate. Nine open reading frames (ORFs) in all tissue culture-derived strains contained changes in nucleotide sequences that resulted in changes in predicted amino acid sequence compared to the parent isolate. Of these, changes in predicted amino acid sequence of six ORFs were identical in all nine derived strains. Comparison of sequences and maximum likelihood estimation (MLE) of specific ORFs and whole genome sequences were used to compare the isolates and derived strains to published sequence data from other HaSNPV isolates. The Australian isolates and derived strains had greater sequence similarity to New World SNPV isolates from H. zea than to Old World isolates from H. armigera, but with characteristics associated with both. Three distinct geographic clusters within HaSNPV genome sequences were identified: Australia/Americas, Europe/Africa/India, and China. Comparison of sequences and fragmentation of ORFs suggest that geographic movement and passage in vitro result in distinct patterns of baculovirus strain selection and evolution.

Complete Genome Sequences of Four Isolates of Plutella xylostella Granulovirus.

Granuloviruses are widespread pathogens of Plutella xylostella L. (diamondback moth) and potential biopesticides for control of this global insect pest. We report the complete genomes of four Plutella xylostella granulovirus isolates from China, Malaysia, and Taiwan exhibiting pairs of noncoding, homologous repeat regions with significant sequence variation but equivalent length.

Complete Genome Sequences of Seven Helicoverpa armigera SNPV-AC53-Derived Strains.

Wild-type baculovirus isolates typically consist of multiple strains. We report the full genome sequences of seven alphabaculovirus strains derived by passage through tissue culture from Helicoverpa armigera SNPV-AC53 (KJ909666).

Complete Genome Sequences of Helicoverpa armigera Single Nucleopolyhedrovirus Strains AC53 and H25EA1 from Australia.

We report here the genome sequences of two alphabaculoviruses of Helicoverpa spp. from Australia: AC53, used in the biopesticides ViVUS and ViVUS Max, and H25EA1, used in in vitro production studies.