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STORAGE PROTEINS IN WINTER HONEY BEES
G. W. Otis1
; D. E. Wheeler2
; N. Buck2
; H. R. Mattila1
1
University of Guelph, Guelph, Ontario, Canada N1G 2W1 2
University of Arizona, Tucson, Arizona, USA 85718
ABSTRACT
Honey bee colonies in temperate climates begin brood rearing in late winter before
floral resources are available. Protein required for brood rearing comes from pollen
stored in combs as well as proteins stored internally in the bees' bodies. We studied
the changes in amounts of several proteins that could serve as internal storage
compounds in fall-emerging bees that contributed to the wintering population.
Vitellogenin, a known storage protein, increased from none in newly emerged bees to
~60 (range: 10-200)ug/abdomen in winter bees (60-day-old bees sampled in late
November). Probable jelly proteins from bee heads were present in minor amounts.
A third previously unreported protein from adult honey bee abdomens was a six-unit
storage protein, probably arylphorin. Like vitellogenin, it was nonexistant in newly
emerged bees and reached an average of 75 ug/bee by late November.
Although the two hives from which we sampled bees were randomly selected, there
were highly significant colony-related differences in amounts of protein in the bees.
Bees from one colony consistently had higher levels of all proteins studied.
We have demonstrated the existence of a previously unknown storage protein in
wintering bees that is probably arlyphorin. Although wintering bees have significantly
elevated amounts of vitellogenin and arylphorin, the amounts of protein present in the
bodies of wintering bees are not sufficient to rear large amounts of brood. We
suggest that their primary function may be to allow colonies to continue brood rearing
when extremely cold periods prevent bees from leaving the cluster to feed on pollen.
Other possible functions are suggested.
Keywords: storage proteins, vitellogenin, winter bees, arylphorin
INTRODUCTION
A key adaptation that enabled tropical Apis to colonize temperate regions was the
ability to survive cold winters. Several traits must co-occur to make this possible:
regulation of the cessation of brood rearing in fall and its initiation in the late winter
prior to the availability of floral food resources; the ability of individual worker bees to
live for many months; storage of large amounts of honey for thermoregulation; and
storage of sufficient protein for late winter and early spring brood-rearing (Seeley
1985).
All protein in a colony of bees is ultimately derived from floral pollen. Some pollen is
fed directly to older larvae. Additional pollen is ingested by adult worker bees,
converted to jelly, and fed to larvae of all castes as well as to older adult workers,
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drones, and queens (Crailsheim 1992). Protein from pollen is required for the
development of glands (e.g., hypopharyngeal glands, wax glands) during the
behavioural ontogeny of worker bees. Finally, some protein is stored internally within
worker bees, primarily in the fat body, haemolymph, and hypopharyngeal glands
(Amdan and Omholt 2002).
In late summer and fall in temperate regions, bees store pollen along with honey.
They also consume pollen and develop hypertrophied hypopharyngeal glands and
large fat bodies containing globules of protein, traits related to the development of
long-lived winter bees (Ribbands, 1953). Vitellogenin, a known internal storage
protein, increases in amount in wintering bees along with several other proteins
(Amdan and Omholt 2002). When brood rearing commences in mid-winter, bees
must obtain protein for larvae from stored pollen and internal protein reserves.
A wide variety of insects synthesize special storage proteins during periods of
resource abundance. These are retained until the time and setting required for egg
production or other functions including cuticle formation, transport of organic
compounds, and humoral immune defense (Burmester 1999). For example, queen
ants of several species have large amounts of large hexameric (e.g., comprised of
six subunits) proteins that are digested to produce food for their first larvae during
claustral colony founding (Wheeler and Martinez 1994, 1995, Wheeler and Buck
1995). Adult worker honey bees have been documented as containing Hex70a in
relatively large amounts (Danty et al. 1998), but details about the amounts and its
function are lacking. In addition to hexamerins, there are additional proteins that may
serve as protein storage molecules, including vitellogenin which is a very high density
lipoproteins (VHDLs) (Wheeler and Buck 1995, Amdan and Omholt 2002). There has
been little research into internal sources of protein in honey bees with the recent
exception of Amdan and Omholt (2002). Our study investigated the amounts of
stored protein in developing winter bees, and discusses their likely usage within the
colony.
MATERIALS AND METHODS
For this study two colonies (#77 and #91) were selected at random in late summer at
the University of Guelph, Ontario. Frames of emerging bees were removed,
incubated overnight, and large cohorts of bees <1 day old were paint marked and
returned to their colonies of origin on 4 September and 30 September, 2000.
Previous studies indicated that these would become “summer” and “winter” bees,
respectively. Samples of ten bees from each cohort were collected at various ages
(e.g., 0, 2, 5, 7, 11, 14 days of age) and frozen at –80 C. On November 30, when all
bees in the colonies were >60 days of age, an additional sample of “winter bees” was
obtained. The bees were shipped on dry ice to the University of Arizona where the
methods previously reported by Wheeler and Martinez (1994) were used to analyze
proteins present in the bees. In summary, the method involved removal of the gut
and sting, after which the head, thorax and abdomen were homogenized individually
in tris-buffered saline in the presence of several protease inhibitors. After
centrifugation, the supernatent was analyzed with SDS-PAGE (polyacrylamide gel
electrophoresis). After electrophoresis, a numerous bands were evident on the gels.
By their size and location in the bee, several of these were identified and quantified:
lipophorin, vitellogenin, a putative storage protein (probably arylphorin), an abundant
thorax protein (probably muscle-related), and a putative jelly protein.
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RESULTS
LIPOPHORIN
Lipophorin is a lipid-carrying protein that is generally a good indicator of the physical
condition of an insect/colony. There was a significant colony effect (p=0.02),
indicating that bees from Colony 77 had significantly more lipophorin than bees from
Colony 91. There was no effect of age (p=0.59) or age*colony effect (P=0.86).
Although we selected colonies at random within the home apiary, this analysis and all
others (below) indicate that Colony 77 was nutritionally better off.
VITELLOGENIN
Vitellogenin (VG) is a VHDL that acts as a storage protein that appears to be
involved in various metabolic functions including the production of hypopharyngeal
gland secretions (Amdan and Omholt, 2002). We quantified a significant increase in
the amount of total VG as bees aged in the fall (p<0.0001), from very small amounts
upon emergence to an overall mean of ~60 цg/bee. There was also a significant
colony effect (p<0.0001), with the winter bees in late November in Colony 77 having
much more VG (~75 цg) than bees in Colony 91 (~30 цg). The age*colony
interaction was also highly significant (p<0.0001). There were very large differences
in the amounts of VG per bee. For example, in bees sampled at the end of
November amounts ranged from 0-220 цg/bee.
PUTATIVE STORAGE PROTEIN
This protein was found only in abdomens and is likely stored in the fat body. The
molecular size was indicative of other hexameric storage proteins in insects, and is
probably an arylphorin protein as that is the only class of storage protein that has
been identified in adult Apis (Danty et al., 1998). It will be referred to as arylphorin
subsequently.
The patterns in the amounts of arylphorin are almost identical to those discussed for
VG. There were highly significant effects of age (p<0.0001), colony (p<0.0001), and
age*colony interactions (p=0.015). Amounts of arylphorin were generally low at
emergence (~20 цg/bee), and increased gradually with age to wintering levels of ~75
цg/bee (range from 0-195 цg/bee).
THORAX PROTEIN (MUSCLE)
We are unsure what this protein is, but its abundance in the thorax suggests it is
related to thoracic musculature. This putative muscle protein increased quickly with
age to a maximum at around age 35 days, after which it appeared to decline slightly
with further increases in age.
PUTATIVE JELLY PROTEIN
This protein was detected in bee heads, suggesting it may have been a jelly protein.
Amounts were relatively small, with the amounts in bees from Colony 77 (41.0
цg/bee) and Colony 91 (25.7 цg/bee) being significantly different (p=0.003). There
was no significant effect of bee age on the amounts of this protein, although it
appeared that the amount of this protein in bees in colony 91 gradually increased
until they matched the levels found in bees from colony 77.
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DISCUSSION
It has been shown recently that adult worker honey bees contain a hexameric
storage protein, Hex70a, but details relating to its rate of accumulation were lacking
(Danty et al. 1998). In the absence of more detailed information about hexameric
storage proteins, research attention has focussed on vitellogenin (VG) which is the
most abundant haemolymph protein and a good indicator of the protein status of the
bee (Cremonez et al. 1998; Amdan and Omholt 2002). In our study of wintering
bees, we found a putative storage protein (probably an arylphorin) that increased
gradually over the fall, had the same pattern of accumulation as VG, and exceeded
the total amount of VG by approximately 25%. If VG is a true storage protein, as
concluded my Amdan and Omholt (2002), its amount in bees in early winter is
exceeded by quantities of arylphorin. The other proteins quantified in this study
(lipophorin, putative muscle protein, putative jelly protein) did not increase with bee
age, suggesting that they are not involved in protein storage to the same extent.
Both VG and arylphorin exhibited very strong colony effects, indicating that the
nutritive state of colonies within the same apiary can differ tremendously. In addition
to the colony effects on amounts of VG and arylphorin, additional colony effects were
detected in the amounts of lipophorin and putative jelly protein. Despite these strong
differences between bees from the two colonies, there were no outward signs during
bee collection to suggest the colonies differed in their protein nutrition. Poor protein
nutrition cannot be observed directly except by assessing amounts of stored pollen
and even that is problematic as it depends on many additional factors. Protein
nutrition undoubtedly affects colony health quite considerably, but goes largely
undiagnosed by beekeepers and bee scientists alike. The very large variability in
amounts of VG and arylphorin within the bees of a colony also raises questions as to
the past and current behavioural roles of bees by early winter that differ so greatly in
protein status.
Even summing the amounts of VG, arylphorin, and putative jelly protein in “well- endowed” winter bees at the end of November, the amount of stored protein is still
relatively small (e.g., ~400 цg). It seems that proteins stored within the bodies of
wintering bees are insufficient to maintain brood rearing for long. To put this into
perspective, Alfonsus (1933; cited by Ribbands 1953) calculated that the amount of
protein required to rear a single bee was ~29 mg. From our study, the amount stored
within an individual bee’s tissues that can be devoted to feeding larvae is nearly an
order of magnitude smaller. Our initial idea was that internally stored protein would
enable colonies to continue brood rearing in spring when pollen stores were depleted
and ambient conditions prohibited the collection of pollen from flowers, but the
amount of protein stored within a bee’s tissues is clearly insufficient to achieve that
purpose.
Why then do worker bees synthesize large storage proteins when stored pollen is
available within their nest? There are several possible explanations. First, evidence
is accumulating that storage proteins are a regular feature of insect biology, in many
cases as a way to store proteins when amino acids are in abundance temporally
and/or spatially. The continued synthesis of storage proteins in an insect that does
not require them may reflect the evolutionary history of social honey bees from
solitary ancestors for which protein storage may have been more important. A
second possibility may relate to the fact that over time the quality of pollen stored in
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honey bee nests decreases. Converting the amino acids in stored pollen into high- quality, easily accessible storage proteins may enable the worker bee to always have
a ready source of high quality protein for the production of jelly fed to colony
members (Crailsheim, 1992). A third possibility may relate to the early brood rearing
by workers while it is still winter. During cold snaps, contraction of the bee cluster
may prevent individual bees from accessing the pollen required for feeding larvae.
Collectively, workers with good internal supplies of stored proteins could continue to
feed some larvae during such periods of time, thereby buffering the effects of the
cold temperatures. For colonies infested with parasitic mites, good quantities of
storage proteins may enable bees to tolerate the negative effects of haemolymph
loss to mites. Finally, good protein nutrition may enable good functioning of the bee’s
immune system, enabling it to resist microbial infections both in the absence and
presence of parasitic mites. These explanations are mutually compatible and
demand attention from the research community.
ACKNOWLEDGMENTS
Paul Kelly maintains the bee hives at the University of Guelph. At the time of this
research, G. Otis was receiving substantial infrastructure support from the Ontario
Ministry of Agriculture and Food.
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