Variability and Number of Circulating Complementary Sex Determiner (Csd) Alleles in a Breeding Population of Italian Honeybees under Controlled Mating.

In Apis mellifera, csd is the primary gene involved in sex determination: haploid hemizygous eggs develop as drones, while females develop from eggs heterozygous for the csd gene. If diploid eggs are homozygous for the csd gene, diploid drones will develop, but will be eaten by worker bees before they are born. Therefore, high csd allelic diversity is a priority for colony survival and breeding. This study aims to investigate the variability of the hypervariable region (HVR) of the csd gene in bees sampled in an apiary under a selection scheme. To this end, an existing dataset of 100 whole-genome sequences was analyzed with a validated pipeline based on de novo assembly of sequences within the HVR region. In total, 102 allelic sequences were reconstructed and translated into amino acid sequences. Among these, 47 different alleles were identified, 44 of which had previously been observed, while 3 are novel alleles. The results show a high variability in the csd region in this breeding population of honeybees.

Genome-wide association study for antibody response to Mycobacterium avium ssp. paratubeculosis in goats.

Mycobacterium avium ssp. paratuberculosis (MAP), causes Johne's disease (JD), or paratuberculosis, a chronic enteritis of ruminants, which in goats is characterized by ileal lesions. The work described here is a case-control association study using the Illumina Caprine SNP50 BeadChip to unravel the genes involved in susceptibility of goats to JD. Goats in herds with a high occurrence of Johne's disease were classified as healthy or infected based on the level of serum antibodies against MAP, and 331 animals were selected for the association study. Goats belonged to the Jonica (157) and Siriana breeds (174). Whole-genome association analysis identified one region suggestive of significance associated with an antibody response to MAP on chromosome 7 (p-value = 1.23 × 10-5 ). These results provide evidence for genetic loci involved in the antibody response to MAP in goats.

Immunohistochemical Distribution of Serotonin Transporter (SERT) in the Optic Lobe of the Honeybee, Apis mellifera.

Visual information is processed in the optic lobes, which consist of three retinotopic neuropils. These are the lamina, the medulla and the lobula. Biogenic amines play a crucial role in the control of insect responsiveness, and serotonin is clearly related to aggressiveness in invertebrates. Previous studies suggest that serotonin modulates aggression-related behaviours, possibly via alterations in optic lobe activity. The aim of this investigation was to immunohistochemically localize the distribution of serotonin transporter (SERT) in the optic lobe of moderate, docile and aggressive worker honeybees. SERT-immunoreactive fibres showed a wide distribution in the lamina, medulla and lobula; interestingly, the highest percentage of SERT immunoreactivity was observed across all the visual neuropils of the docile group. Although future research is needed to determine the relationship between the distribution of serotonin fibres in the honeybee brain and aggressive behaviours, our immunohistochemical study provides an anatomical basis supporting the role of serotonin in aggressive behaviour in the honeybee.

Analysis of Complementary Sex-Determiner (csd) Allele Diversity in Different Honeybee Subspecies from Italy Based on NGS Data.

Sexual regulation in Apis mellifera is controlled by the complementary sex-determiner (csd) gene: females (queens and workers) are heterozygous at this locus and males (drones) are hemizygous. When homozygous diploid drones develop, they are eaten by worker bees. High csd allelic diversity in honeybee populations is a priority for colony survival. The focus of this study is to investigate csd variability in the genomic sequence of the hypervariable region (HVR) of the csd gene in honeybee subspecies sampled in Italy. During the summer of 2017 and 2018, worker bees belonging to 125 colonies were sampled. The honeybees belonged to seven different A. mellifera subspecies: A. m. ligustica, A. m. sicula, A. m cecropia, A. m. carnica, A. m. mellifera, Buckfast and hybrid Carnica. Illumina genomic resequencing of all samples was performed and used for the characterization of global variability among colonies. In this work, a pipeline using existing resequencing data to explore the csd gene allelic variants present in the subspecies collection, based on de novo assembly of sequences falling within the HVR region, is described. On the whole, 138 allelic sequences were successfully reconstructed. Among these, 88 different alleles were identified, 68 of which match with csd alleles present in the NCBI GenBank database.

Automated image analysis to assess hygienic behaviour of honeybees.

Focus of this study is to design an automated image processing pipeline for handling uncontrolled acquisition conditions of images acquired in the field. The pipeline has been tested on the automated identification and count of uncapped brood cells in honeybee (Apis Mellifera) comb images to reduce the workload of beekeepers during the study of the hygienic behavior of honeybee colonies. The images used to develop and test the model were acquired by beekeepers on different days and hours in summer 2020 and under uncontrolled conditions. This resulted in images differing for background noise, illumination, color, comb tilts, scaling, and comb sizes. All the available 127 images were manually cropped to approximately include the comb area. To obtain an unbiased evaluation, the cropped images were randomly split into a training image set (50 images), which was used to develop and tune the proposed model, and a test image set (77 images), which was solely used to test the model. To reduce the effects of varied illuminations or exposures, three image enhancement algorithms were tested and compared followed by the Hough Transform, which allowed identifying individual cells to be automatically counted. All the algorithm parameters were automatically chosen on the training set by grid search. When applied to the 77 test images the model obtained a correlation of 0.819 between the automated counts and the experts' counts. To provide an assessment of our model with publicly available images acquired by a different equipment and under different acquisition conditions, we randomly extracted 100 images from a comb image dataset made available by a recent literature work. Though it has been acquired under controlled exposure, the images in this new set have varied illuminations; anyhow, our pipeline obtains a correlation between automatic and manual counts equal to 0.997. In conclusion, our tests on the automatic count of uncapped honey bee comb cells acquired in the field and on images extracted from a publicly available dataset suggest that the hereby generated pipeline successfully handles varied noise artifacts, illumination, and exposure conditions, therefore allowing to generalize our method to different acquisition settings. Results further improve when the acquisition conditions are controlled.

Investigating Genetic and Phenotypic Variability of Queen Bees: Morphological and Reproductive Traits.

The quality of the honeybee queen has an important effect on a colony's development, productivity, and survival. Queen failure or loss is considered a leading cause for colonies' mortality worldwide. The queen's quality, resulting from her genetic background, developmental conditions, mating success, and environment, can be assessed by some morphological measures. The study aims to investigate variability for traits that could assess the quality of the queen. Related animals were enrolled in this study. Variance components were estimated fitting a mixed animal model to collected data. Heritabilities of body and tagmata weights ranged from 0.46 to 0.54, whereas lower estimates were found for the tagmata width and wing length. Heritabilities estimated for the spermatheca diameter and volume, number of ovarioles, and number of sperms were 0.17, 0.88, 0.70, and 0.57, respectively. Many phenotypic correlations related to size were high and positive, while weak correlations were found between morphology and reproductive traits. Introducing a queen's traits in a selection program could improve colonies' survivability. Further research should focus on better defining the correlations between the individual qualities of a queen and her colony's performance.

Using Omics Approaches in the Discovery of Biomarkers for Early Diagnosis of Johne's Disease in Sheep and Goats.

Johne's disease (JD) is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is an important and emerging problem in livestock; therefore, its control and prevention is a priority to reduce economic losses and health risks. Most JD research has been carried out on cattle, but interest in the pathogenesis and diagnosis of this disease in sheep and goats is greatest in developing countries. Sheep and goats are also a relevant part of livestock production in Europe and Australia, and these species provide an excellent resource to study and better understand the mechanism of survival of MAP and gain insights into possible approaches to control this disease. This review gives an overview of the literature on paratuberculosis in sheep and goats, highlighting the immunological aspects and the potential for "omics" approaches to identify effective biomarkers for the early detection of infection. As JD has a long incubation period before the disease becomes evident, early diagnosis is important to control the spread of the disease.

Whole-Genome Sequence Analysis of Italian Honeybees (Apis mellifera).

At the end of the last glaciation, Apis mellifera was established in northern Europe. In Italy, Apis melliferaligustica adapted to the mild climate and to the rich floristic biodiversity. Today, with the spread of Varroa destructor and with the increasing use of pesticides in agriculture, the Ligustica subspecies is increasingly dependent on human action for its survival. In addition, the effects of globalization of bee keeping favored the spread in Italy of other honeybee stocks of A. mellifera, in particular the Buckfast bee. The purpose of this study was to characterize the Italian honeybee's population by sequencing the whole genome of 124 honeybees. Whole genome sequencing was performed by Illumina technology, obtaining a total coverage of 3720.89X, with a mean sample coverage of 29.77X. A total of 4,380,004 SNP variants, mapping on Amel_HAv3.1 chromosomes, were detected. Results of the analysis of the patterns of genetic variation allowed us to identify and subgroup bees according to their type. The investigation revealed the genetic originality of the Sicula, and in A.m. ligustica limited genetic introgression from the other breeds. Morphometric analysis of 5800 worker bees was in agreement with genomic data.

Combinatorial Discriminant Analysis Applied to RNAseq Data Reveals a Set of 10 Transcripts as Signatures of Exposure of Cattle to Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis or Johne's disease in cattle is a chronic granulomatous gastroenteritis caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). Paratuberculosis is not treatable; therefore, the early identification and isolation of infected animals is a key point to reduce its incidence. In this paper, we analyse RNAseq experimental data of 5 ELISA-negative cattle exposed to MAP in a positive herd, compared to 5 negative-unexposed controls. The purpose was to find a small set of differentially expressed genes able to discriminate between exposed animals in a preclinical phase from non-exposed controls. Our results identified 10 transcripts that differentiate between ELISA-negative, clinically healthy, and exposed animals belonging to paratuberculosis-positive herds and negative-unexposed animals. Of the 10 transcripts, five (TRPV4, RIC8B, IL5RA, ERF, CDC40) showed significant differential expression between the three groups while the remaining 5 (RDM1, EPHX1, STAU1, TLE1, ASB8) did not show a significant difference in at least one of the pairwise comparisons. When tested in a larger cohort, these findings may contribute to the development of a new diagnostic test for paratuberculosis based on a gene expression signature. Such a diagnostic tool could allow early interventions to reduce the risk of the infection spreading.