Morphological and molecular characterization of eastern honey bee (Apis cerana Fabricius) in Kerala

Abstract

An investigation on “Morphological and molecular characterization of eastern honey bee (Apis cerana Fabricius) in Kerala” was carried out at the Department of Entomology, College of Agriculture, Vellayani during 2017-2022 with the objective to identify the morphological and genetic diversity of eastern honey bee (Apis cerana Fabricus) in southern, central and northern zones of Kerala. Survey was conducted in three zones of Kerala viz., southern, central and northern zones. Twenty worker bees each were collected from 75 colonies (feral /natural colonies as well as managed/ domesticated hives) of twenty five localities representing each zone. The nesting habitat, nesting site, nest height and nest characteristics of honey bee colonies from forest areas in Kerala were examined. Feral colonies of cavity nesting honey bees were found to inhabit the primary forests, disturbed and undisturbed lands as well as agricultural lands in Kerala. Tree hollows, rock crevices, mud wall, termite mound, letter box and mud pot were the nesting sites of cavity nesting bees in Kerala and majority of the bees were found to occur at a nest height of 0 cm to 200 cm. The number of combs in the colony varied from five to twelve. An exploration of foraging sources of honey bees in forest areas were done for the first time in Kerala via metabarcoding of rbcL (ribulose- bisphosphate carboxylase) gene. Plants belonging to 256 genera were identified as foraging sources of honey bees in forest areas of Kerala with Gossypium as the abundant genus in southern zone, Elaeis in central zone and Drypetes in northern zone. Seventeen genera of plants were common in all the three zones of Kerala i.e. Pterocarpus, Cannabis, Prunus, Ceiba, Ulex, Galatella, Myosotidium, Vachellia, Valeriana, Cassia, Androsace, Senna, Luisia, Cocos, Peliosanthes, Esmeralda, and Parkia among which Pterocarpus, Cannabis, Prunus, and Ceiba were the prevalent genera as the foraging source of honey bees in forest areas of Kerala. As part of the morphological studies, five worker bees from each colony were dissected and subjected to standard morphometric analysis using 29 parameters. No significant differences were observed in most of the morphological parameters among the zones. Based on elevation, the populations were classified as ‘Plain’ (<300 m above MSL) and ‘Hill’ (>300m above MSL) populations and ‘t test’ analysis among the populations showed highly significant differences across most of the size related morphometric characters viz., ratio of head length and width (HLW), distance between ocelli (DBO), ocello-ocular distance (OOD), proboscis length (PL), antennal length (AL), ratio of length and width of fore wing (FWLW), cubital index (CI), wing angles (A 32, A 38), length of third tergite (3TEL) and length of fourth tergite (4 TEL) and ratio of length and width of wax plate (WPLW). Moreover, the ‘Hill’ populations of honey bees exhibited higher values for most of the size related as well as wing related characters. Factor analysis yielded six factors with Eigen value > 1. The major factor (F1) was derived from honey bee size related characters (HLW, PL, AL, CI, 3TEL, 4TEL and WPLW) with high factor loadings in between 0.66 to 0.92 accounting for 35 per cent of the variation among the samples. Hence, these characters were found to be the main indices that could be used to discriminate among the populations. The hierarchical cluster analysis (HCA) of 75 populations with 29 morphological characters revealed the presence of two main clusters, ‘Plain’ group cluster and ‘Hill’ group cluster, the same being confirmed in linear discriminant analysis (LDA). The correlation analysis of morphological traits with elevation revealed that proboscis length (PL), antennal length (AL), cubital index (CI), ratio of head length and width (HLW), ratio of length and width of wax plate (WPLW), length of third tergite (3 TEL) and length of fourth tergite (4 TEL) exhibited positive linear correlation with topographic elevation. The ‘Hill’ and ‘Plain’ populations of honey bees exhibited variations in the colour and pigmentation pattern of different morphological traits viz., second tergite, third tergite, third sternite and abdomen. The everted endophallus of honey bee drones showed variations in the vestibulum, cervix and bulb indicating reproductive isolation between ‘Plain’ and ‘Hill’ populations of cavity nesting bees in Kerala. Surface pattern of bulb and vestibulum were detected through scanning electron microscopy (SEM) imaging wherein they exhibited variations in the surface structures. Molecular characterization of 75 populations of cavity nesting bees in Kerala was also carried out by amplifying the mitochondrial cytochrome oxidase I (COX I) gene. Phylogenetic analysis of the 75 bee populations from Kerala using maximum likelihood method identified the ‘Plain’ group as A. indica Fabricus, 1798 and the ‘Hill’ group as A. cerana Fabricus, 1793. Haplotype analysis of the populations revealed the existence of fifteen COX I haplotypes in Kerala, out of which fourteen were new haplotypes. Pair wise genetic distance of the samples revealed high nucleotide divergence (5.1% - 7.0%) between A. indica and A. cerana. The network analysis of haplotypes also supports the divergence between A. indica and A. cerana in Kerala. These fifteen nucleotide sequences of the haplotypes were submitted to NCBI Genbank and BOLD. Comparative metagenomic analysis of gut microbiome of A. indica and A. cerana revealed variations in the abundance of gut microbiome between the two species. The abundant genus recorded in A. indica was Lactobacillus while in A. cerana Gilliamella was the prominent genus. Morphological and molecular analysis of 75 populations from Southern, Central and Northern zones revealed the existence of two discrete populations of cavity nesting honey bees in Kerala i.e. ‘Plain’ population and ‘Hill’ population. Significant variations in the morphometric parameters of the bee populations were observed at elevations rather than the zones. Studies on the external genitalia revealed reproductive isolation between ‘Plain’ and ‘Hill’ populations of cavity nesting bees in Kerala. Haplotype analysis revealed fifteen COX I haplotypes of cavity nesting bees in Kerala, wherein fourteen haplotypes were new from Kerala. Thus, the present study revealed the existence of two species of cavity nesting bees in Kerala i.e. the Plain group, A. indica and Hill group, A. cerana.