Medycyna Weterynaryjna - Summary Med. Weter. 68 (10), 599-602, 2012

Med. Weter. 2012, 68 (10)

599

Praca oryginalna

Original paper

The Varroa destructor mite is regarded as the main

causative agent of Colony Collapse Disorder (30, 31).

The mite body size (width 1708.9 µm, length 1167.3

µm) (2) enables it to parasitize bee broods in a way

that is difficult for bees to discover. The mite causes

deformations in bees and a decrease in their body

weight (6, 24, 26), which is the result of loss of the

fat-protein body in the brood (7, 8). Reduced body

weight leads to a decline in flight efficiency and

vita-lity, as well as in a loss of navigation ability in adult

bees (1, 6, 8, 9, 11, 12, 23, 27). Moreover, Varroa

destructor is a biological vector for viruses, which

replicate in their organisms (4, 19). Bees that are

infe-sted by the virus-infected mites exhibit high mortality

rates. Consequently, entire colonies usually die within

6 months up to 2 years (22, 33). At present, there are

no fully satisfactory methods of varrosis control. This

is because of increasing resistance of Varroa

destruc-tor to varroacides (10). Therefore, the continuous

moni-toring of bee colonies’ infestation by counting mites

that have died from both a natural death and upon

application of varroacidal agents is important.

Deve-loping of a scheme of alternate application of curative

agents is also of the greatest importance (3). One of

the ways to control mites is to employ of a biological

method, which involves use of 4.9 mm small-cell

combs (21). Unlike in standard sized cells (5.4 mm),

a considerably larger number of Varroa females seem

not to undergo the full developmental cycle in small

sized cells, as bigger numbers of immature mites have

been found among the specimens that died a natural

death (18).

Considerable genetic variability, including different

haplothypes was found in Varroa destructor (2, 16,

28). Therefore it is worth exploring whether mites

parasitizing bee colonies kept at the standard-cell

combs differ in their genotype from mites found in

colonies kept in the small-cell combs. Evaluation of

the genetic variation of Varroa destructor populations

is based on the mtDNA sequence analysis of the

cyto-chrome oxidase I gene (CO I) fragment, which

facili-tates membrane transport in the respiratory chain in

adult mites. The CO I gene is most frequently employed

in determination of Varroa haplotypes (2, 17).

Genetic and morphometric variation

of the Varroa destructor developing in standard

and small comb cells*

)

GRZEGORZ BORSUK, KRZYSZTOF OLSZEWSKI, ANETA STRACHECKA,

JERZY PALEOLOG, KORNEL KASPEREK

Department of Biological Bases of Animal Production, Faculty of Biology and Animal Breeding, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland

*) _{This work was supported by:}

– Ministry of Science and Higher Education grant No. N N311 632138 in 2010-2012,

– National Science Centre grant No. N N311 542140 in 2011-2014.

Borsuk G., Olszewski K., Strachecka A., Paleolog J., Kasperek K.

Genetic and morphometric variation of the Varroa destructor developing in standard

and small comb cells

Summary

The aim of the work was to check whether Varroa destructor mites parasitizing brood developing in

standard size cells (5.4 mm) (STC) and in small size cells (4.9 mm) (SMC) differed in the sequence of the

cytochrome oxidase I gene fragment (CO I) and body size.

Two mite groups were formed (100 specimens in each); the mites parasitized brood developing in STC

and SMC combs. Six mite specimens from the STC and 6 from SMC were subject to genetic analyses.

Morphometric measurements involved 94 mites both from STC and SMC groups.

The small cell size in the honeycomb (4.9 mm) did not affect the sequence of the CO I gene fragment in

Varroa destructor, but led to significant reduction in their body size, possibly as a response to the limited space

in the cell.

Med. Weter. 2012, 68 (10)

600

Reduction in the comb cell size leads to a decreased

body size of bees kept in the cells; however, these

chan-ges are not directly proportional. The investigations

carried out by McMullan and Brown (15)

demonstra-ted that a 7-8% reduction in the comb cell size led to

a decrease in the bee body size by a mere 1%.

Conse-quently, the developing bee fills the cell more tightly,

which may exert a negative effect on the reproduction

of mites. This was corroborated by various studies (14,

21). Therefore we decided to explore whether mites

from standard-cell combs differ in terms of morphology

from mites present in small-cell combs.

The aim of this work was to assess whether Varroa

destructor mites parasitizing broods in standard-cell

combs (5.4 mm) and those in small-cells (4.9 mm)

differ genetically and morphologically.

Material and methods

Buckfast bee colonies were kept in the standard-cell

combs (5.4 mm) (STC) and in the small-cell combs (4.9

mm) (SMC). The SMC colonies were kept for four years.

In the fourth year one comb of the capped brood in the

pupa stage was taken from each of the 5 STC and each of

the 5 SMC colonies. 20 mature (dark brown) V. destructor

females were collected from each comb. The mites were

placed in Eppendorf tubes and frozen. Two mite groups

(100 specimens in each) were obtained from the STC and

SMC combs in this way.

Genetic analyses. The genetic analyses were performed

on six mite specimens from the STC group and six from

the SMC group. DNA was isolated from single V.

destruc-tor specimens using the standard protocol for the tissue

DNA isolation; Qiagen DNeasy Blood & Tissue Kit. A 280

bp fragment of the mitochondrial cytochrome oxidase I

(COI) gene (29) was amplified with the PCR method using

the Qiagen Taq PCR Core Kit and the following primers:

V51:

5’-GTAATTTGTATCAAA-GAGGG-3’

V1400:

5’-CAATATCAATAGA-AGAATTAGC-3’

The reaction mixture for one

sam-ple contained 6 µl DNA; 3.5 µl 10 ×

PCR buffer, 7 µl Q buffer, 5.33 µl

MgCl

₂

, 0.58 µl of each dNTP, 0.33 µl

primer V51 and V1400 and 1 U

poly-merase. The final volume of the

sample was 30 µl. The amplification

of the PCR products was carried out

in a MJ Research PTC-225 Tetrad

thermocycler in accordance with the

following thermal-temporal profile:

preliminary denaturation at 94°C for

3 min; subsequently, a program of 36

repetitive cycles was employed –

de-naturation at 94°C for 1 min,

attach-ment of the primers at 45°C for 1 min.

and annealing of the primers at 72°C

for 1 min. The final annealing of the

primers was conducted at 72°C for 10

min.

The PCR matrices were purified with the ExoSap –

Exo-nuclease I and Shrimp Alkaline Phosphatase kits. The PCR

products were directly sequenced using a BigDye

Termi-nator Cycle Sequencing Mix v3.1 in an ABI3730xl

auto-mated DNA sequencer (Life Technologies).

Morphometric analyses. Morphometric measurements

involved 94 mites from STC and 94 mites from SMC. The

mites were photographed using a digital camera connected

to the Olympus SZX 12 microscope. The photographs were

analyzed using the MultiScanBase v 14.02 software for

image analysis. The photographs had to be scaled as they

may have been taken at various magnifications.

Statistics. The genetic analysis was performed with the

MEGA 4.0.2 program. The morphometric results were

statistically analyzed with the SAS software (SAS Institute

2002-2003 SAS/STAT User’s Guide Version 9.13, Cary,

NC, Statistical Analysis System Institute) using the

one-way ANOVA (a group effect was the experimental factor)

and the HSD (honestly significant difference) test (25).

Results and discussion

In the Varroa destructor mites collected from the

SMC combs, no change was detected in the base

sequence in the fragment of the cytochrome oxidase I

gene (CO I) compared with the reference sequence –

Tab. 1 (2). A mutation at the 993 bp locus was found

in one of the Varroa destructor specimens collected

from the STC comb (T/A underwent a transversion).

This random mutation caused a post-translational

change in the amino acid frequency in favor of Leu

(Tab. 2), which may have resulted in the protein

con-formation changes in the respiratory chain. Similarly,

the point mutation (Leu to Phe) in the sodium channel

can produce para-homologous proteins that are

res-ponsible for knockdown resistance to pyrethroids in

the insect pest species (5, 13, 16, 20, 32).

Tab. 1. Comparison of the sequences at the 993 bp mutation locus

f e R AAT TCA TGG TTC TAT AGT TAA ATT AGA f o r e b m u N l a u d i v i d n i a o rr a V r o t c u rt s e d C T S . 1 ... ... ... ... ... ... ... ... ... C T S . 2 ... ... ... ... ..A ... ... ... ... C T S . 3 ... ... ... ... ... ... ... ... ... C T S . 4 ... ... ... ... ... ... ... ... ... C T S . 5 ... ... ... ... ... ... ... ... ... C T S . 6 ... ... ... ... ... ... ... ... ... C M S . 1 ... ... ... ... ... ... ... ... ... C M S . 2 ... ... ... ... ... ... ... ... ... C M S . 3 ... ... ... ... ... ... ... ... ... C M S . 4 ... ... ... ... ... ... ... ... ... C M S . 5 ... ... ... ... ... ... ... ... ... C M S . 6 ... ... ... ... ... ... ... ... ...

Explanations: Ref – Varroa destructor reference fragment of cytochrome oxidase

sub-unit I (CO-I) gene, partial cds; mitochondrial gene for mitochondrial product, National

Centre for Biotechnology Information (NCBI) database; STC – a standard-cell comb;

SMC – a small-cell comb; . – identity of bases

Med. Weter. 2012, 68 (10)

601

The mites collected from the SMC combs were

significantly smaller (width and length) than the

spe-cimens from the STC combs (Tab. 3). Reduction in

the cell size allows the developing bee to fill the small

cell more tightly (14, 15), which may limit the space

for development of V. destructor mites. The reduction

in the cell size decreased the mite size (Tab. 3). It is

difficult to claim, though, whether the reduced size of

mite bodies was a direct effect of the reduction in the

comb cell size or whether it was the result of

adapta-tion of mites to the reduced space in the comb cell. It

seems possible that the aggravated living conditions

led to coevolutional adaptation which last from four

year of mites to new environmental conditions, which

favored mites with a small size. The Varroa

destruc-tor specimens parasitizing the brood in the STC combs

exhibited a similar size to the mites described by

Anderson&Trueman (2) and Zhang (34).

Conclusions

The small size of the cell in the honeycomb (4.9

mm) did not lead to changes in the sequence of the

CO I gene fragment in Varroa destructor mites, but

resulted in a significant reduction in their body size,

which must have been a response to the reduction in

the free space in the cell.

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Tab. 2. Post-translational changes in the amino acid frequency

Explanations: as in Tab. 1

u l G Phe Gly His lIe Lys Leu Met Asn Pro Arg Ser Thr Trp f e R 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 f o r e b m u N l a u d i v i d n i a o rr a V r o t c u rt s e d C T S . 1 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C T S . 2 3.13 3.13 12.50 3.13 1.56 9.38 3.13 34.38 3.13 1.56 7.81 3.13 1.56 7.81 C T S . 3 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C T S . 4 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C T S . 5 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C T S . 6 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C M S . 1 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C M S . 2 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C M S . 3 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C M S . 4 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C M S . 5 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94 C M S . 6 3.17 3.17 12.70 3.17 1.59 9.52 1.59 34.92 3.17 1.59 7.94 3.17 1.59 7.94

Explanations: SE – standard error; Min – minimum value of the

features; Max – maximum value of the features; a, b – the

diffe-rences are statistically significant for comparisons in the columns

at P £ 0.05 (valid significant difference)

Tab. 3. Body widths and lengths (in µm) of Varroa destructor

females

f o y d o B r o t c u rt s e d a o rr a V Mean SE Min Max h t d i W SMC 1665.3a 0.013 1387.2 1844.1 C T S 1716.1b _{0.003 1387.2 2015.0} h t g n e L SMC 1121.4a 0.008 960.2 1211.3 C T S 1147.4b _{0.003 960.2 1855.7}

Med. Weter. 2012, 68 (10)

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