Evaluation of Morphometric Characteristics to Discriminate Inbred Lines of the Honeybee Apis mellifera

INTRODUCTION

Honeybees (A. mellifera) are generalist pollinators, thus important insects in agriculture, ecology and basic research(Jensen et al., 2005). Honeybee not only pollinator but also provide honey, royal jelly, propolis and pollen. In Korea, the economic value of honeybees as pollinators is reported to be more than over 29 billion dollars (Jung, 2008) and the demand for pollination also has increased drastically. However, honeybees face challenges, including pesticides, predators (such as hornets), pathogens and parasites (such as mites) have contributed to their decline (Bakonyi et al., 2002; Bailey and Ball, 1991; Ingram et al., 1996; Oldroyd, 2007).

Honeybee queens are polyandrous (Laidlaw and Page, 1985; Adams et al., 1977), meaning mating with two or more males and this behaviour contributes higher genetic diversity. Genetic diversity directly influences on colony productivity, disease and pest resistance to parasites and pathogens and resultantly enhances colony fitness via an enhanced task allocation system (Oldroyd and Fewell, 2008). Therefore, the loss of genetic diversity is of grave concern. It has been shown that colonies with reduced genetic diversity are less capable of controlling hive temperature and more prone to develop diseases when challenged by parasites (Tarpy, 2003). From the breeding perspective view, however, this behaviour makes selection of desired characters difficult, because several male traits are inherited simultaneously to the offspring in a colony. Moreover, honeybees are particularly sensitive to inbreeding (Seeley and Tarpy, 2007). Thus, there is a widely recognized need to encourage regional breeding efforts to preserve local adaptation and to maintain local strains in isolated conservation apiaries.

Morphometric characteristics of honeybees have also been well studied for the identification of geographic races or subspecies, based on multiple measurements of several individuals (Alpatov, 1929). Ruttner et al. (1978) chose 42 characteristics for analysis of honeybee workers from a wide range of geographic locations. Since then, more accurate bases for determining geographical races of bees using biometrics have been proposed by other researchers (Ruttner, 1988; Ruttner et al., 2000; Radloff et al., 2003; Diniz-Filho et al., 2000). The standard morphometric characteristics of honeybees include distances of body parts, venation angles and discrete classes of pigmentation. Recently, some researchers have been conducted discriminant analysis based on the morphometric characteristics. Discriminant analysis is a technique in which measurements of two or more characters are weig-hted and combined linearly to give maximal separation of two or more groups (Blackith and Reyment, 1971; Van de Geer, 1971). DuPraw (1964, 1965a, 1965b) first applied discriminant analysis to measure features of the wings and after that many researchers has been conducted to classifying samples of various subspecies led to other multivariate applications.

In a previous study, we have been conducting a breeding project for Apis mellifera at the National Academy of Agricultural Science (NAAC) in Korea (Kim et al., 2015). According to the data, the 6 inbred lines have been maintained under the controlled production of queens and artificial insemination. In this study, we evaluated morphometric characteristics based on different body parts and conducted discriminant analysis in 5 inbred lines, A, C, D, E and F.

MATERIALS AND METHODS

Investigation of morphometric characteristics

The standard morphometric characteristics were analyzed in 10 workers bees of each of 3 colonies of 5 inbreed lines, A, C, D, E and F. These characteristics are described in Ruttner (1988) and Alpatov (1929), and include the following: number of the hooks (Ha), basitarsus length (ML), proboscis length (PL), forewing length (FWL), Rs vein, distance (a), and Rs vein, distance (b). Fig. 1 shows standard morphometric characteristics of honeybees. Adult workers from 15 colonies were sampled from an apiary in NAAS and 10 workers were usually measured from each colony and constitute a collection in this study. Honeybees were preserved in 95% ethanol, and were then dissected and subsequently mounted in diaphane or permount on a slide and measured using Leica DM2500 (Leica microsystems, Germany) microscope. Wing venation and size characteristics were analyzed using a microscope and Leica Application Suite (LAS) V4.7 software.

Fig. 1.

Different body parts for measuring morphometric characteristics used in this study. A; the number of the hooks (Ha), B; basitarsus length (ML), C; proboscis length (PL), D; fore wing length (FWL) and E; Rs vein, distance (a), Rs vein, distance (b).

RESULT

Measurement of morphometric characteristics

Table 1 and Fig. 2 show measured morphometric characteristics in this study. The five inbreed lines exhibited statistically significant differences in fore wing length (FWL), varying from 8.2 to 9.1mm (one-way ANOVA and post hoc Tukey’s HSD test (p<0.05)). The highest fore wing length was observed in line D (9.1mm, N=35) and the lowest was observed in line C (8.2mm, N=26). The average basitarsus length (ML) in the five inbreed lines examined ranged from 1.7 to 2.0mm (p<0.05). The highest basitarsus length was found same for line A (2.0mm, N=37), D (2.0mm, N=35) and F (2.0mm, N=35), and the lowest in line C (1.7mm, N=26). The number of hooks (Ha) ranged from 20.6 to 22.5 (p<0.05). The highest number of hooks observed in line C (22.5mm, N=26) and the lowest was line A (20.6mm, N=37). The mean proboscis length (PL) showed no significant differences (p>0.05) among the 5 inbred lines, and it ranged from 5.3 to 5.7mm. The highest length of proboscis was observed in line A (5.7, N=37) and D (5.7mm, N=35) and the lowest was line C (5.3mm, N=26). The mean rs vein, distance (a) showed no significant differences (p>0.05) among the 5 inbred lines, ranging from 1.98 (F, N=35) to 2.87mm (D, N=35). However, the mean of rs vein, distance (b) showed significant differences (p<0.05). The ratio of rs vein distances a and b ranged from 1.98 (F, N=35) to 2.87mm (D, N=35).

Table 1.

Morphometric characteristics from 5 inbreed lines in Apis mellifera

Fig. 2.

Measured morphometric characteristics from different body parts. (A); Number of the hooks (Ha), (B); Measurement of the basitarsus length (ML), (C); Measurement of the proboscis length (PL), (D); Measurement of the fore wing length (FWL), (E); Rs vein (distance a in Fig. 3) and (F); Rs vein (distance b in Fig. 3). The boxplot diagram shows the median (the line in the box), means (+), interquartile range and outliers (○, *).

Discriminant function analysis

A discriminant function analysis of the five inbreed lines carried out on the data analyzed morphometric characteristics showed that the characteristics evaluated statistically significant differences among individuals of 5 inbred lines. The result of the discriminant function analysis of individuals indicated that the 5 inbred lines accurately classified 77.4% in their original groups. Among the 164 honeybees, 127 honey bees were assigned to their actual groups, whereas, 37 honey bees were scattered (Table 2). Specifically, line C was assigned to actual groups by 100% and line F was classified F line by 90%, but line A, D and E were assigned to actual groups by 45.9%, 88.9% and 71.4%, respectively. According to mahalanobis distance, the centroids of lines A, D and E showed partial overlapping, which found to be morphologically the closest. However, the mahalanobis distance between the centroids of lines C and F was separated, which mean the two lines morphologically difference (Fig. 3). The result of that five inbreed lines based on the six morphological characteristics was found separated for 3 groups (P<0.001). The relationship among 5 inbred lines is shown with the help of a dendrogram (Fig. 4). According to data from cluster analysis, the lines A, D, and E clustered closely together in the dendrogram.

Table 2.

Classification results of discriminant analysis for five inbreed lines

Fig. 3.

Distribution of five inbreed lines Apis mellifera using a canonical discriminant function.

Fig. 4.

Dendrogram demonstrates the relationship among the five inbreed lines based on the measured morphometric characteristics using cluster analysis.

DISCUSSION

As part of a breeding project, five Apis mellifera inbreed lines (named lines A, C, D, E and F) were bred and cultivated for several years (Kim et al., 2015). In this study, we discriminated maintained 5 inbreed lines based on standard morphometric characteristics. According to the results, the means of the measurements of each body part showed considerable differences among the 5 inbred lines. Of these, significant differences were found in fore wing length, hook number, rs vein, distance (b), and basitarsus length. Discriminant function analysis resulted in the clear separation of 5 inbred lines when 6 morphometric variables was utilized (Fig. 3). Among them line C was line F was classified to actual groups by 100% and 90%, respectively, which determined more accurately classified than other groups. Discriminant function analysis of 5 inbred lines was also supported by the Dendrogram based on Mahalanobis distances (Fig. 4). The C and F lines separated from other lines. The result shows that the two inbreed lines controlled mating and presence of a more pure population. In general, the standard morphometric characteristics that are used to discriminate honeybee species and subspecies. However, the discriminance among same subspecies are difficult by analyzing only a few morphological characteristics. But, in this study, the validated characteristics showed differences among the 5 lines, especially, line C and F showed morphologically distinguishable.

Acknowledgments

This study was carried out with the support of "Next-Generation BioGreen 21 Program (PJ01117001)", Rural Development Administration, Republic of Korea.

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