Multi-trait evaluation and selection of superior Apis mellifera L. colonies based on brood survival, hygienic behaviour and colony productivity

Introduction

Honey bees are important pollinators and provide economically valuable hive products. In managed Apis mellifera populations, colony productivity is determined by interacting traits including worker population, queen performance, brood production, food reserves, foraging activity and behavioural resistance to pests and pathogens. Indian studies have demonstrated substantial variation among colonies in brood area, honey reserves, pollen stores, brood survival and other desirable traits, providing a basis for selection of superior colonies (Yadav et al., 2023; Sharma et al., 2020; Brar et al., 2018; Saini et al., 2018; Saini et al., 2018a) [30,19,3,17,18] . Brood survival is an important indicator of colony health because it integrates egg viability, larval care, food availability and the ability of worker bees to maintain developing brood. Colony strength and brood production are also closely related to honey and pollen reserves, particularly during periods of intensive colony development and honey flow (Kumar et al., 2024; Pande et al., 2011; Nadaf et al., 2020; Chhuneja et al., 1992) [8,15,13,6] . Selection based on several traits simultaneously can therefore identify colonies that are both productive and biologically robust. Hygienic behaviour is another important component of colony defence. Hygienic workers detect abnormal or dead brood, uncap affected cells and remove the brood, thereby limiting the persistence and spread of brood diseases and some parasites (Singh et al., 2007 [22,16,20] . Indian screening programmes have demonstrated substantial variability in hygienic behaviour among A. [27,28,14] . [12,11] . Recent work has further characterized A. mellifera pollen-foraging dynamics during litchi bloom in Muzaffarpur (Kaur et al., 2025) [7] . Thus, identifying robust colonies is relevant not only to hive productivity but also to pollination-service management.

Although several Indian investigations have evaluated individual colony traits, fewer studies combine brood survival, food reserves and hygienic behaviour into a single selection framework. Therefore, the present study evaluated ten A. mellifera colonies using these complementary traits and developed a derived multi-trait ranking to identify promising colonies for future breeding and validation.

Materials and Methods

Experimental colonies and study period

Brood survival and brood pattern

A marked area of 25 cm² was used for brood assessment. The numbers of eggs laid, eggs hatched and surviving larvae were recorded. Brood survival percentage was calculated from the experimental observations as the proportion of surviving brood relative to eggs laid. Colonies were classified as high, medium or low according to the supplied categories, and brood continuity was scored on the recorded 1-3 scale.

Honey and pollen stores

Honey stores were estimated from the number of squares filled with honey using the supplied conversion factor of 8.06 g per square. Pollen storage was recorded from pollenfilled cells and grid squares; pollen store area was calculated using the supplied factor of 6.45 cm² per grid square. These calculations were retained exactly as provided in the source dataset.

Hygienic behaviour

Two complementary indicators were used. First, the quantity of debris removed from each hive was recorded weekly on 24 March, 1 April, 8 April and 15 April 2024, and the mean was used to characterize nest-cleaning activity. Second, 25 pupae were experimentally killed in each colony and the number subsequently removed was recorded. Removal percentage was calculated as removed pupae divided by 25 × 100.

Multi-trait selection index

To provide a selection-oriented interpretation, five traits were standardized using min-max scaling across the ten colonies: brood survival, honey stores, pollen stores, pupalremoval percentage and debris removal. For debris, lower accumulation was treated as favourable and therefore reverse-scaled. The unweighted multi-trait index was calculated as the arithmetic mean of the five standardized scores. This derived index is an analytical ranking of the supplied data and is not an additional field observation.

Statistical presentation

The supplied dataset included means, standard errors, critical differences and coefficients of variation where reported. Results are presented descriptively by colony, followed by the derived multi-trait ranking. The manuscript does not claim an additional experimental replication or inferential test beyond the statistics contained in the supplied tables.

Results

Brood survival and brood continuity

The ten colonies differed in brood survival. 2 and Fig. 1B andC.

Hygienic behaviour based on debris removal

Mean debris removal/cleaning values ranged from 1.28 g in C 5 to 2.77 g in C 9 . C 3 (1.64 g) and C 10 (1.73 g) also showed low debris accumulation, while C 9 showed the highest value and was classified as poor cleaning. C 5 and C 3 were classified as very clean; the remaining colonies except C 9 were classified as clean presented in Table 3 and Fig. 1 D.

Removal of experimentally killed pupae

The mean removal of experimentally killed pupae was 91.60%. C5 removed all 25 killed pupae (100%), while C 3 , C 6 and C 8 each removed 24 pupae (96%). C 1 , C 7 and C 10 removed 23 pupae (92%), whereas C 2 and C 4 removed 22 (88%). C9 removed 19 pupae, corresponding to 76%, the lowest value in Table 4and Fig. 1 E.

Multi-trait selection and identification of superior colonies

The derived multi-trait index integrated brood survival, honey, pollen, pupal removal and inverse debris accumulation. C 5 was ranked first because it simultaneously expressed the highest brood survival, the highest honey and pollen stores, the lowest debris accumulation and complete removal of killed pupae. C 3 and C 6 followed, reflecting consistently strong values across most traits. C 9 ranked last because it combined low brood survival, low honey and pollen stores, high debris accumulation and the lowest pupal-removal percentage.

Discussion

The present observations demonstrate clear colony-level variation in brood viability and resource accumulation. Such variation is expected in managed A. mellifera populations and is the basis for selecting breeding colonies rather than treating the apiary as a homogeneous population. Indian selection studies have similarly reported significant differences among colonies in bee strength, brood area, honey reserves, pollen reserves and brood survival, and have recommended ranking colonies across several traits rather than relying on a single measure colonies (Yadav et [ 30,19,3,17,18] . C 5 was consistently superior in the present dataset. Its brood survival of 97.06% was the highest, while its honey and pollen stores were also maximal (Singh et [8,15,13,6] . However, the present results should be interpreted as colony-level association rather than proof that one trait directly caused another.

The hygienic results provide an additional basis for selection. C 5 removed 100% of the experimentally killed pupae and had the lowest mean debris value. C 3 also showed high hygienic performance. Hygienic behaviour is widely recognized as a colony-level defence involving recognition, uncapping and removal of abnormal brood ( [27,28,14] . The strong performance of C 5 across productivity and hygiene is particularly valuable from a breeding perspective because selection based only on honey yield can overlook health-related traits. Conversely, selecting only for hygienic behaviour could favour colonies that are behaviourally resistant but not necessarily highly productive. A multi-trait approach reduces this risk and is consistent with Indian colony-selection programmes that use composite scoring systems (Yadav et al., 2023; Saini et al., 2018; Saini et al., 2018a) [30,17,18] . The litchi context is also important for the broader application of superior colonies in Bihar. Litchi is a major horticultural crop in the region, and studies from Muzaffarpur have documented A. mellifera as an important flower visitor and pollinator. Kumar and Kumar (2014) [9] reported that A. mellifera was the dominant recorded forager in their litchi observations and that proximity to colonies was associated with higher fruit set and yield (Mahindru et al., 2021) [12] . Srivastava et al. (2017) [27] documented a diverse pollinator complex and high A. mellifera activity on litchi flowers Mahindru et al., 2023) [11] . Recent pollenforaging research during litchi bloom has further demonstrated the importance of A. mellifera colonies for understanding pollen resources in Muzaffarpur orchards (Kaur et al., 2025) [7] . These findings support the practical importance of maintaining strong, healthy colonies for pollination services. Nevertheless, the present selection index is a derived analytical tool based on one supplied dataset. It should not be interpreted as a finalized breeding value. Before C 5 or other colonies are used extensively as breeder colonies, their performance should be repeated over multiple seasons and locations, with queen age/genetic background, colony strength, disease and parasite load, honey yield, brood pattern and hygienic behaviour assessed under standardized conditions.

Conclusion

Ten Apis mellifera colonies showed substantial variation in brood survival, honey and pollen reserves and hygienic behavior on litchi crop. C 5 was the best-performing colony in the supplied dataset, recording 97.06% brood survival, 403.00 g honey, 37.41 cm² pollen stores, the lowest debris accumulation (1.28 g) and 100% removal of experimentally killed pupae. C 3 and C 6 were additional promising colonies. C 9 consistently displayed inferior values and ranked last in the derived multi-trait index. The study supports multi-trait colony evaluation as a practical approach for identifying candidate colonies for further breeding and apiary multiplication. Repeated evaluation across seasons and

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