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PROCEEDINGS

ROYAL PHYSICAL SOCIETY OF EDINBURGH
SESSION 1915—1916.

VOL. XX.—Parr 1.

OBSERVATIONS AND EXPERIMENTS BEARING ON
“ISLE OF WIGHT” DISEASE IN HIVE BEES.

BY

JOHN ANDERSON, M. A, B. Se.,,
Lecturer in Bee- -keeping, North of Scotland College of Agpeultare,
AND - :
JOHN RENNIE, D.Sc, F.R.S.E.,.

., Leeturerin Parasitology, ‘University of Aberdeen:

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Isle of Wight Disease in Hive Bees. 23

IV—Observations and Experiments bearing on “Isle of Wight”
Disease in Hive Bees.! By John Anderson, M.A., B.Sc., Lecturer in
Bee-keeping, North of Scotland College of Agriculture, and John Rennie,
D.S8c., F.R.S.E., Lecturer in Parasitology, University of Aberdeen.

(With Plate.)

(Read 24th January 1916. MS, received 11th February 1916.)

ContTENTS, PAGE
I, Introduction ‘ 23
II. The Course of Isle of Wight nea as peer in a aistinet finuaiiog

(a) Isle of Lewis . , : . ‘ 3 ‘ : 24
Summary ‘ : : : ; 29
(6) Craibstone—Spontaneous Pecnyary i i é : 31
(c) Deeside ‘ 32

ITI. Investigations bearing on ite Hiden of oe apis to Tals of Wight
Disease : ; : : : ; ‘ 41
Nosema infection experiments i ‘ ; : 47
Conclusions : 51

IV. Observations and epevtinena bearing on fhe Infectivity of Isle a Wight
Disease as distinguished from Microsporidiosis _. 3 F 52
(a) Artificial Infection . F P ’ : ‘ : 52
(6) Natural Infection . ‘ 3 : : : : 59
Conclusion regarding Infectivity . ‘ F ‘ j ‘ 61
V. Explanation of Plate . F d ; ‘ ‘ : 61

L—INTRODUCTION ;

THESE observations were commenced in 1909 by one of us (J.A.), whose
journal contains a detailed record of the history of each stock of bees in the
Island of Lewis. In the autumn of 1913 JR. paid two visits to the Nicolson
Apiary at Stornoway, and subsequently arrangements were made, through
Professor J. Arthur Thomson, for continuing the research in association with
the Natural History Department of Aberdeen University.

The work here reported on was carried out with the aid of grants
from the Development Fund and the University of Aberdeen through
the Joint Committee on Research in Animal Nutrition of the University
of Aberdeen and North of Scotland College of Agriculture. In the
work we have been: assisted by Mr John Innes, BSc, M.B., who has
carried out most of the examinations of bees for the presence of Nosema, and

1 This constitutes a preliminary report, indicating the trend of the results so far
accomplished, Although the number of experiments is not large, stress is laid upon the
fact that they are the only ones on record in this country which have been made upon full

stocks of bees living under natural conditions, Further experiments are in progress, the
results of which will be recorded in a subsequent report.

24 Proceedings of the Royal Physical Society.

after his retiral on taking up military service, by Miss Beatrice Simpson, M.A.,
who continued this work. Mr Robert Ewen, M.A., Nicolson Institute,
Stornoway, has acted as local supervisor of the hives in Lewis since May
1915. We have also had the valued co-operation, in connection with the
Deeside epizootic, of Mr A. H. E. Wood, of Glassel House, and of Miss Nancy
Robinson, both of whom are bee experts of the British Bee-Keepers’
Association. The latter has provided a detailed record of the Deeside out-
break, which has proved of value as a basis for scientific study.

We are indebted also to various local observers in Lewis and elsewhere
who have taken charge of experimental hives, forwarded diseased bees, and
otherwise supplied helpful information regarding the disease.

@ 37945

IIl.—THE COURSE OF THE DISEASE, AS OBSERVED
IN THREE DISTINCT LOCALITIES.

(a) LEWIS OBSERVATIONS.

Prior to the 20th May 1909, all hive bees in Lewis had become extinct,
but on that date one stock was brought to Stornoway and placed in the
grounds of the Nicolson Institute. These were black bees obtained from
Wormit, Fife, and their previous history is unknown. In autumn of the
same year a second stock of black bees was imported from Duirinish, and
placed at Bayhead, Stornoway. Since 1909, other bees have been introduced
at various dates, as stocks or swarms, and some queens have also been imported.
At the present time there are bees in Stornoway, Lurebost, Sandwick, Bayble,
Tong, Tolsta, Barvas, Shawbost, Carloway, Breasclete, Achmore, Marybank,
Laxdale. The races include Blacks, Italians (Ligurians), Cyprians, Carniolans,
and certain hybrids. .

Lirst Appearance of Isle of Wight Disease-—On the 20th September 1910,
two lots of driven bees arrived in Stornoway from Sussex. They had come from
cottagers’ skeps, but had travelled to Lewis in swarm boxes on standard combs
containing some honey. These standard combs probably came from the
apiary of the bee-master who “drove” the bees, and it is possible that the
disease was derived from these combs. One box was sent unopened to Bayble,
5% miles from Stornoway, and the other lot was retained in Stornoway. The
bees in both cases were fed on syrup, settled down quietly, bred freely, and
gathered pollen up to 4th November 1910.

On 31st January 1911, bees were observed crawling in front of this stock,

Isle of Wight Disease in Hive Bees. 25

apiary number, A9. Next day a letter was received from Bayble stating that
the other stock of driven bees had begun to crawl on the same day. The
symptoms in both cases were those of Isle of Wight disease; bees emerged
on the alighting board and did not fly, some eventually fell off the board on
to the ground where they collected in little clusters, finally dying of cold and
hunger. Bees of this stock continued to die in this way at Stornoway till
2nd March. On this date the survivors were killed and the hive and ground
disinfected. Caustic soda was spread upon the soil, which was then turned
over, and all the combs and quilts were burned. The Bayble stock was also
killed off and the hive disinfected.

Second Attack. Two Stocks affected, B14, C13.—On the 6th October 1911,
two other stocks in Stornoway Apiary began to craw]. On this occasion,
samples of living bees were sent for diagnosis to Dr Graham-Smith, Cambridge,
who reported “young stages of Nosema apis in both stocks.” Spores were
apparently not observed, and it was not stated whether the examples seen
included intracellular forms. One of these stocks (B14) consisted of American
Golden Bees, and in their case crawling ceased after a few days; on 14th
October they were busily engaged once more carrying in pollen. They
wintered in excellent condition, built up rapidly in spring, and never showed
any further sign of Isle of Wight disease. They were not again tested for
Nosema. Unfortunately the queen died of old age, 15th June 1912, and the
strain became extinct. The noteworthy feature in the case of this stock is
that the symptoms, although associated with the presence of Nosema,
disappeared in a few days.

The other stock (C13) which consisted of black bees, became rapidly worse,
and, on 12th October 1911, it was destroyed. These two stocks stood side
by side.

D1 G9 B14 C13

Arrangement of Stocks, October 1911.

Third Attack. One Stock, No. D1.—Crawling bees were first observed in
front of this stock, the position of which is indicated above, on 20th January
1912. The bees were a black stock, numerous, and having abundance of
natural stores. It had wintered in excellent condition. Samples were again
sent to Cambridge, and Dr Graham-Smith reported the presence of all stages

of Nosema. :

26 Proceedings of the Royal Physical Society.

This stock was allowed to die out naturally, but the hive had been removed
to another site, and the original stance disinfected. A small remnant was
found stiff and motionless on one comb, on 29th February 1912. Thorough
disinfection of the site was effected by spraying crude carbolic acid, of such
strength that the worms came up and died, and the grass also was killed.
All inside fittings of the hive were burned, paraffin was also poured on the
hive site, and burned off.

Fourth Attack. Stock D4.—This stock, a strong one, crowded with bees
and brood, along with two others, came from Exeter on 21st May 1912,
where as yet the disease was unknown. It was placed on the stance
of D1, referred to above. Isle of Wight disease symptoms appeared on
28th May, exactly one week after the arrival of the stock upon the island,
and large numbers of crawling bees were observed in front of this hive on
every fine day. On 8th June, all the stores were exhausted and all the brood
was dead. Syrup was supplied, and the queen laid more eggs. On the
13th June there were still numerous bees, eggs, a few grubs, and food. At
this date samples were sent to Cambridge, and Dr Graham-Smith’s report
upon them is as follows :—

“The bees you sent all arrived dead. No spores were found by
microscopic examination, and it was impossible to say for certain whether
young stages of Wosema were present or not. I tried to infect some healthy
bees, but this experiment has turned out negative. 2/7/12.”

On the 17th bees were lying about in large numbers on the alighting
board and on the grass around, and dying off rapidly.

On the 11th July, the remaining bees, along with queen, were sent to
Dr Graham-Smith, who kept them for some time. His report regarding
them is :—

“The bees you sent have been doing moderately well, and nothing very
special has yet been found in those which have died.”

This must mean that Nosema was not found, yet the behaviour of the
bees, and the high mortality clearly indicated Isle of Wight disease.

In the case of this stock no disinfecting measures were adopted, nor in
any subsequent instance in the Nicolson Apiary, Stornoway. .

Fifth Attack. One Stock (K.M.).—This stock arrived in Stornoway from
the South of England on 31st May 1912. Its ten combs were crowded on
11th June and a super was added. The super was quite full of bees on
22nd June, and some sections were ready for sealing on 13th J uly. Latera

Isle of Wight Disease in Hive Bees. ‘ 27

second super was required, and some excellent sections were removed.
Crawling began on 10th September, and was very typical when observed on
the following day. On 12th October, all the bees were dead except the
queen and a handful of workers. This stock was situated about 200 yards
distant from the Nicolson Apiary.

Sixth Attack. Stock E14.—This stock consisted of Swiss pedigreed bees
(blacks), and was imported from Exeter on 4th June 1913. When a super
was added on 9th June the hive was crowded with. bees and brood. When
examined nine days later the super was crowded with bees, the foundation
had been drawn out, and some honey stored in the cells. The stock had
been placed in a garden which had not previously been used for bee-keeping,
a short distance from the Nicolson Apiary.

On the 19th, bees were observed crawling in front of the hive, exactly a
fortnight after their arrival in Lewis. Numerous bees were examined on the
spot, their intestinal contents being searched microscopically. Nosema spores
were not found. Sick bees were sent to Cambridge, and regarding-these Dr
Annie Porter reported: “A very few spores in one bee, young stages in some
of the others, the rest negative.” Notwithstanding the apparent paucity of
Nosema-infected bees, the mortality amongst members of this stock continued,
and at this period was exceedingly heavy. On 7th July, the Swiss queen
was replaced by a Cyprian imported from Nicosia. The Swiss queen was
caged with some Cyprian bees, and died of injuries received from them.
She was sent to Dr Annie Porter, Cambridge, for examination, but no report
was obtained regarding her condition. By this time, not many blacks were
left alive; by September the stock was weak in numbers, but apparently
healthy, and very few blacks were present. On 16th September, one
of these was found to contain spores in abundance, but no spores were
found in the young Cyprians. A few of these were observed to “crawl”
on fine days, but the stock proved too weak to winter, and was allowed to
die out.

Seventh Attack. Two Stocks affected, Gp. and Gs—A stock of black
English bees had been brought to Stornoway from the mainland of Ross
(Duirinish) in the autumn of 1909. It prospered normally during 1910,
1911, and 1912, producing several swarms, and giving surplus honey. In
1913 it swarmed on 25th July. “Crawling” began in the swarm three days
after it was hived, and was well marked and typical when observed on 16th
August. On this date a few crawlers were observed in front of the parent
stock. On 6th September, when the apiary was again visited no crawlers

28 Proceedings of the Royal Physical Society. .

were found in front of the swarm, but a large number was seen in front of the
parent stock. Both hives were examined comb by comb on this occasion,
and found to have queens and brood in all stages. The swarm was small in
number, but lively, and apparently quite healthy. The parent stock was
more numerous, but a considerable number of bees showed dislocation of the
wings, an indication which, however, has little importance as a symptom of
the disease. On the 12th September, sample bees were taken from the
parent stock, These were picked off the combs, bees with dislocated wings
being specially selected. Owing to unfavourable weather conditions there
were no bees out of doors at this date. Microscopic examination showed no
recognisable traces of Nosema.

The hives in which the bees were housed had no previous history of Isle
of Wight disease. The bees were too few to keep warm in the hives during
winter, and, on this account alone, survival was not possible.

Eighth Attack. One Stock, F14,—Without any disinfecting measures,
Hive No. 14, which housed the previously mentioned infected stock (E14),
was used for a lot of Welsh driven bees procured in the late autumn of 1913.
The infected combs were also retained with contained honey. In early
spring, 1914, heaps of dead were found on the floor ; later, on fine days many
“crawlers” were observed. At the beginning of April, the bees covered only
a single comb, but they were carrying pollen freely, and there was a patch of
brood. Crawling had now ceased, the stock built up during the summer of
1914, wintered on natural stores, and was quite strong in the spring of 1915.
On 17th June 1915, J. R. examined six live bees, active after journey (posted
14th), all parts normal, no Wosema observed. Other samples of bees which
were sent to Aberdeen for examination arrived dead, and were not satisfactory
for Isle of Wight disease diagnosis. On the other hand, it can be stated
positively that Nosema spores were not present in the bees examined. Bees
were examined individually, and also pulped in groups. The colons con-
tained masses of pollen, but Nosema was not found. A stained smear was
also made of one bee, and this gave a negative result.

In August 1915, the stock was found weak and queenless. In October it
was quite extinct, and the local observer (Mr Gibson) reported that crawling
had been very marked.

In October the hive was restocked with a new lot of driven bees from Wales,
the old combs and stores being retained. Efforts were made to put this stock
in good condition for wintering, but the weather was rather cold while
feeding was going on.

This is now the third stock housed in the same hive in which two previous

Isle of Wight Disease in Hive Bees. 29

stocks had died of Isle of Wight disease. This new stock and hive is
named G14,

No further cases of Isle of Wight disease have arisen in the Nicolson
Apiary, although, as the records below show, Nosema could be obtained from
bees there at almost any time.

Outbreaks of Isle of Wight. Disease in Lewis,

Date of Nosema If
‘ Date of How :
Introduction Stock X, 0, or : Site, etc.
of Stock Outbreak ys. terminated disinfected
29/9/10 31/1/11 Blacks AQ _ Destroyed Yes
from Sussex and Bayble 2/3/11
6/10/11 American y.s. Recovered —
Goldens B14
6/10/11 Blacks C13 y.s. Destroyed Yes
12/10/11
20/1/12 Blacks D1 x Allowed to die Yes
out 29/2/12
21/5/12 28/5/12 Blacks D4 ? Remainder sent No
from Exeter to G-S.,
Cambridge
31/5/12 10/9/12 Blacks KM — Died out Yes
from 8. Eng. 12/10/12
4/6/18 18/6/13 Swiss Very few Died out No
from Exeter pedigreed |sporesin1;| before Winter
E14 numerous
in Sept.
Autumn 1909 28/7/18 Blacks Gp co) Died out No
from Duirinish Winter 1913-14
Autumn 1913 | Early Spring | Welsh F14 Co) Recovery in1914 No
from Wales 1914 Relapse in 1915
11/7/14 12/7/14 Black 0 Blacks died out =
from Exeter (at Bayble) in Autumn ;
remnant of
Yellows sur-
vived Winter

The number of stocks in the apiary varied from 4 to 14 during this period.
X—spores present. o—spores absent. y.s.—young stages,

Summary of Characteristics of Isle of Wight Disease as seen in
Lewis Outbreaks,

(1) In relation to area beyond the Island.
(a) Simultaneous outbreak of two cases 54 miles apart of stocks intro-
duced from same source (Sussex), 4 months previously. A9 and
Bayble stock.

30 Proceedings of the Royal Physical Society.

(b) D4. Disease appeared 1 week after stock arrived from Exeter.

E14. »” 2 weeks » ” ”
KM. Ke 34 months __,, a 8. England.
Fl4. 6 months after introduction from Wales.

»

See also under (2).

(2) In relation to other affected stocks in Lewis.

B14. ) More than 7 months after the termination of the previous

C13. \ outbreak.

D1. Three months after B14 and C13.

F14. Six months after introduction, upon which it had been placed
in hive in which a stock, E14, had just died out from
Isle of Wight disease.

(3) Duration, where not interfered with.

B14. <A few days, and recovered.

D1. 40 days, and died out.

KM. 32 days, and died out.

E14. June to beginning of winter, and died out.

Gp. July to winter, died out.

F14. Few weeks—recovery—recurrence in the following year,
lasting August to September 1915.

(4) Relation to Nosema apis.

1. Spores found in only two cases coincident with Isle of Wight
disease symptoms.

2. Young stages were found in two other affected stucks, one of which
completely recovered.

3. Healthy stocks known to have Nosema all the time in which no -
outbreak of Isle of Wight disease occurred.

( Placed on a new site when introduced.

Strong stock.

4. A particular case, | Mortality heavy.
E14, in detail. | Extreme paucity of Nosema in bees while this

mortality in progress.
Nosema fluctuations at different stages.

(5) Effect on Apiary as a whole. No more than two stocks ever displayed
symptoms at one time or in succession. During this period the apiary
contained various races and strains of bees. .

Isle of Wight Disease in Hive Bees. 31

(b) EXAMPLE OF NATURALLY OccURRING OUTBREAK oF ISLE OF WIGHT
DISEASE, SHOWING SPONTANEOUS RECOVERY.

CRAIBSTONE APIARY.

On 4th June 1915, three strong stocks of bees imported from Devonshire
were placed in this newly constituted apiary and housed in perfectly fresh
hives. During the fine weather of June they were very prosperous, each
‘stock swarming once and one of them twice. The swarms were hived
separately. Three races were represented, viz. pure Italian, pure Carniolan
(queen imported from Austria in 1914), and ordinary black bees.

Symptoms of Isle of Wight disease made their appearance on 30th June,
and continued, with fluctuations, till near the end of August. All the
stocks and swarms were affected more or less, but the heaviest mortality
occurred in the blacks (D4). Three of the seven stocks died out owing to
their failure to mate the virgin queens. In all three cases drone breeders
were developed. On 22nd September four stocks survived and no symptoms
of Isle of Wight disease were apparent, either in the behaviour of the
bees, which was perfectly normal, or in the condition of their internal
organs. At no stage of the disease were spores of Nosema found, although
repeated examinations of the contents of the alimentary canal were
made.

While the disease was at its height the number of affected bees was
very great. Their behaviour was of the usual character, and the walk leading
from the hives was littered with crawling bees to a distance of at least
25 yards, Heaps lay on the flower-beds, and a low box-hedge was full of
bees incapable of flight.

The mortality was, in consequence, very considerable, and the weather
conditions were exceedingly unfavourable to bee life. The recovery is
therefore all the more notable.

On 22nd September one of the stocks (D4) was decidedly weak. It was
headed by a young queen, had brood on two combs and plenty of store for
the number of bees. This is the stock which had the heaviest mortality
during the crawling period. Two other stocks (Al and C3) were in
excellent condition for wintering, headed by young queens, populous as
to bees, and with plenty of stores. The fourth stock (B2) is the only one
now that is not headed by a queen of this year. Possibly on this account,
and also on account of heavy losses during the crawling period, this stock
is also rather short of bees. The queens in D4 and B2 are black, C3 has
a young Carniolan queen. A1, originally pure Italian, is now filled with
dark bees, the offspring of a young queen.

32 Proceedings of the Royal Physical Society.

On 23rd September, a small lot (3 lbs.) of driven bees was united to
D4, and a new stock, E5, consisting of a small lot of Welsh driven bees
with their own queen, was placed between C3 and B2. On 10th October,
an Italian queen, supplied by Penna, was caged over D4, the original queen
having been removed, and this Italian queen was duly accepted. On the
same date it was observed that B2 (the weakest stock in the apiary) had
been entirely robbed out. D4 and E5 were fed with sugar syrup and duly
wintered down.

On 28th December, the four Craibstone stocks were examined. Except
at Al there were scarcely any dead bees to be seen, either on the ground,
on the alighting boards, or on the floors of the hives. Even at Al the number
of dead bees was not remarkable.

D4 was opened, and the bees were found active and in good condition
generally,

During the mild days in January 1916 the bees were moving freely and
even carrying pollen. The behaviour of C3, E5 and D4 was normal, but
Al showed very definite “crawling” symptoms and a high death-rate.

(c) IsLE oF WicHT DISEASE ON DEESIDE.

1.—HIstTory oF OUTBREAK.
By Nancy M. Roginsoy, First-Class Expert, B.B.K.A.

So far as can be ascertained, Isle of Wight disease was not recognised
anywhere on Deeside until 1913, when it appears to have broken out in
several distinct districts.

(1) On Lower Deeside, in Culter and the surrounding districts, the
disease was at its worst in 1914.

A,—A’s apiary was the first affected. He had bought bees from the
South of England in 1912. These stocks did well early in 1913, but they
had died out by 1914 after showing all the usual symptoms of Isle of
Wight disease.

In 1913 these stocks had been taken to the heather, east of the Hill of
Fare, in the Echt district, and several apiaries there, including two large
ones, were badly affected by the same disease in 1914 and 1915.

During 1913 and 1914 all bee-keepers within a radius of two miles or
more from A’s apiary found their bees affected or dying off rapidly, and
by the end of 1914 there were no bees left in that area.

Some apiaries were started again but with little or no success, owing
to the recurrence of the disease.

Isle of Wight Disease in Hive Bees. 33

In 1915 cases of the disease appeared still farther afield and embracing
a far wider area,

Drumoak,—In the parish of Drumoak, on the north side of the Dee, a
case of Isle of Wight disease occurred in 1913.

B—B had sent to England for driven bees in 1912, and in the spring
of 1913 the disease first began to show in his apiary.

During this year his six stocks gradually dwindled and died out. The
hives were cleaned up and disinfected.

As far as is known no further cases occurred in this parish in 1913,
unless it might be at H’s, which is mentioned later. But in 1914 all the
apiaries in B’s vicinity became affected.

C.—C lived about a quarter of a mile north of B. In June 1914, C
reported that his bees were not working, although the weather was good
and there was plenty of nectar to be had. The apiary, consisting of two
hives and a ruskie, was found to be in the final stages of the disease, and
in July there were very few bees left and less honey than in June. The
owner then destroyed his bees and disinfected his hives.

D.—D was another neighbour, living about three-eighths of a mile north-
east of B. He had only one stock, which was found to be very weak in
June 1914. The stock was left until August, when it was so much
reduced that the owner destroyed the few bees that were left and disinfected
his hive.

H#.—F’s apiary, situated three-quarters of a mile north-east of B, was
attacked in August 1914. The bees may have been suffering earlier, but
no record of this exists. E had sent samples of bees to the British Bee
Journal, which reported them to be suffering from Isle of Wight disease.
Incidentally, it may be mentioned that bees from the same source sent to
Aberdeen University investigators were reported as free from Nosema. Only
two stocks appeared to be infected, but in September 1914 the remainder
showed marked signs of the same trouble. In October three out of four
stocks were seriously affected, and between this time and April 1915 these
three stocks had died off, and the remaining one was so weak that there
was merely a handful of bees on the comb. These the owner treated
with candy and quinine without success, and the stock died out before the
end of the month.

F.—F lived about a mile north-east of B and owned four stocks. In August
1914 there were slight indications of Isle of Wight disease. In September
one stock became badly affected, and the three others all showed symptoms.
The worst of the affected stocks was removed to Aberdeen for experimental
purposes (60 DD. of this report). The British Bee Journal reported

VOL, XX. C

34 Proceedings of the Royal Physical Society.

on samples of bees sent: “It is Isle of Wight disease.” Dr Rennie
reported : “ Nosema can not be found in this stock.”

The remaining stocks were not destroyed; but all died out except one,
before April 1915. This stock was then very weak and was destroyed,
and the hives and appliances were disinfected.

G.—G kept two stocks of bees, about three-eighths of a mile east of B.
In June 1914 both were very strong; but at the end of June, drones were
being thrown out in great numbers, and later in the year the bees were suffer-
ing from Isle of Wight disease. Before April 1915 both stocks were dead.

H.—H’s house was quite close to G. In the spring of 1914 H had an
empty hive in his garden; it was filled with old combs and had been left
uncared for since the loss of a stock during the previous winter. (The
symptoms accompanying the loss of this stock were unmistakably those
of Isle of Wight disease. JR.) In June a swarm came and took
possession of this empty hive and flourished remarkably. 1914 proved a good
season for honey, and this stock came through the winter, 1914-1915, very
well. When visited in April 1915, the bees were found working vigorously
and very strong for that time of year.

A swarm came off at 9.30 A.M. on 22nd May (the earliest swarm known
on Deeside that year). H succeeded in skepping it, but unfortunately it rose
later and was lost.

In due time a second swarm came off, which was skepped successfully,
and stock and swarm both appeared to thrive.

Later, this swarm showed distinct signs of Isle of Wight disease during
two or three days, and then it seemed to recover. At the end of the honey
season, the two stocks were united. For further history of the united stock,
see p. 59 of this report.

J.—J, three-quarters of a mile south-east of B, kept two stocks of bees, No. 1
and No, 2. When visited in June 1914 both stocks were found very strong,
but No. 1 was especially good and was working well in the supers. In July
large numbers of drones were being thrown out of the hive, in ae of fine
weather and a good honey flow.

In August No, 1 showed marked signs of the disease, and J then destroyed
the bees. This stock had given him 126 sections.

No. 2 swarmed, and the old queen having been killed the swarm was
returned to the parent hive. A young queen mated successfully, and this
stock did very well in 1914, but died out during the winter 1914-1915.

It may be mentioned here that J had earlier in the season assisted some

of his neighbours in destroying their infected bees and cleaning and disinfect-
ing their hives.

Isle of Wight Disease in Ilive Bees. 35

In 1915 J resumed bee-keeping. He obtained a swarm and placed it in
one of his disinfected hives. This stock built up steadily at first, later it
began to show some symptoms of the trouble, and then partially recovered.

K.—K lived a mile south-west of B. He had only one stock, which became
seriously affected with Isle of Wight disease during the summer of 1914. It
was destroyed in September 1914.

In 1915 numerous outbreaks occurred farther afield from B's apiary.
There were several cases about 14 to 2 miles north of B. Many of the stocks
had died out during the winter, and not from starvation as there was plenty
of honey in the combs. Those stocks which survived soon began to show the
ordinary symptoms of Isle of Wight disease, and all died out. B’s stocks, it
may be noted, were cleaned out and disinfection effected in 1913.

On the south side of the Dee, opposite Drumoak, two cases were noted
in 1915.

L.—L lived rather over 2 miles south-east of B’s apiary, and owned six
hives and two ruskies. In June 1914, all were strong and apparently well,
but owing to the prevalence of Isle of Wight disease in the district, L was
warned to watch his bees carefully. No symptoms were recognised until
June 1916; all the stocks were then strong, but later they became seriously
affected and eventually died out before the end of the year. L dealt a good
deal in so-called “ remedies.”

M.—M’s apiary was 2 miles south of B. In May 1915, several of his
stocks were found to be affected. M was inclined to think the disease was
derived from some bees from Culter, which had been brought to the heather
near by, in August 1914, and which had died out while there.

(2) Banchory-Ternan District.

In this district the distance between the outbreaks has been greater than
in the others..

N.—A case occurred at Crathes in June 1914. N’s apiary lies about
three miles east of B. There were three stocks, Nos. 1, 2 and 3, The
following observations were made :—

13th June—-A warm, bright day. There were numerous dead bees
outside the hives, and a great many unable to fly and crawling on the ground
all over the garden. No clustering was seen, and no old bees observed
crawling, The wings and abdomens appeared to be normal, but the bees
seemed weak, Stocks all seemed strong.

15th June.—A fine day. Only a few bees were seen crawling, and very
few dead bees in front of the hives. Birds were seen to pick some up, and
possibly numbers may have disappeared in this way.

36 Proceedings of the Royal Physical Society.

16th June—A fine day. Bees appeared normal. No. 2 looks like
swarming. No crawling bees seen.

21st June.—A dull, rather cold day. Bees were clustering on the ground
near all three hives in small groups. No. 2 appeared to be the only normal
stock, and was strong. The bees from No. 1 and No. 3, both inside and
outside the hives, appeared weak and dormant and unable to sting. There
were very few dislocated wings. The abdomens and general appearance of
the bees, with a few exceptions, seemed normal. It is doubtful whether
any bees from No. 2 were clustering, although there were clusters under that
hive.

22nd June.—A fine day. The symptoms just as previous day. No. 1
and No. 3 stocks were destroyed and the hives disinfected.

24th June-——No 2 was working well and looked like swarming. The
ground was limed.

26th June.—No, 2 working well.

29th June.—Swarm came off, but got away and was lost.

9th July.—Bees working well, but some few drones observed crawling.

15th August.—No. 2 working well and seems strong. Supers nearly full.

This stock died out during the winter 1914-1915, All stocks belonging
to three bee-keepers, who were near neighbours of N, were lost also.

0.—O’s apiary was 2 miles north of N. O had six stocks, and in July
1914 these were all found to be suffering from Isle of Wight disease. Only
one stock survived till 1915, and that eventually died out.

Several cases were reported at Raemoir in 1914 which were not verified
as the stocks had been destroyed before notice was received.

In 1915, there were two outbreaks, three-quarters of a mile distant from
each other, at this place.

P.—P had five stocks in good hives, clean and well kept. The stocks
were all strong. All appeared healthy in June 1915, but in July Isle of
Wight disease was very marked. Numerous bees were crawling. on the
ground and clustering; others were climbing up grass and leaves; many
had dislocated wings and distended abdomens. In those examined the colon
was very much enlarged and clogged. There were many dead lying about.

Three of the hives contained parent stocks, while the other two held
swarms. The parent stocks seemed to be more severely attacked. P
destroyed all his bees at the end of July and cleaned and disinfected his
hives and appliances.

@.—Q’s apiary was not so well kept, and his stocks were weak, con-
sequently the outbreak did not appear so marked, and the disease lingered
on for a long time. I have frequently noticed that when a really strong

Isle of Wight Disease in Hive Bees. 37

(ae. numerous) stock becomes affected, disease progresses much more rapidly
than in the case of weaker stocks,

&.—A serious outbreak occurred in July 1915, in R’s apiary, situated
about a mile and a half south-east of N’s apiary. R owned one hive and
five ruskies, and by the end of 1915 nearly all his bees were dead.

S.—This brings us within a mile of Banchory village where a case broke
out in July 1915 in S’s apiary. This was on the east side of the village and
within a mile of the last outbreak at R’s.

S owned five hives and a ruskie. In May three stocks were very weak.
Numerous dead bees lay near the hives, but this might have been the result
of robbing.

In July, Isle of Wight disease was marked in a swarm in this apiary. In
August all the other stocks but one began to show the same symptoms, and
S destroyed them. The one left was strong and gave two crates of sections,
which was good for a poor season.

At this date no cases farther west of Banchory and Raemoir have come
under notice until Dinnet and Tarland are reached—i5 miles away.

(3) Dinnet Area.

T.—In 1913, two bee-keepers at Dinnet, very near neighbours, found
their bees suffering from some trouble they were unable to recognise. They
consulted the North of Scotland College of Agriculture bee expert (Mr
Manson), who found the bees to be suffering from Isle of Wight disease. His
opinion was confirmed by the British Bee Journal expert, to whom
samples of bees were sent. The owners were recommended to destroy their
stocks in order to prevent the spread of infection. They not only did this,
but burnt up bees, hives, and all appliances. One bee-keeper, T, owned
fourteen stocks. Before the disease was recognised a swarm had come off
and settled in the roof of a cottage close at hand. This swarm seemed to be
strong and working well; and owing to the difficulty of removing the bees,
it was left alone. T had brought bees from England not very long before
the outbreak, and the disease was attributed locally to these imported
bees.

In 1914, T started keeping bees again. He had made himself new hives
and bought two stocks from a bee-keeper west of Ballater. In July 1914,
he had six hives and three ruskies; all were very strong and doing well in
the supers, and there was no appearance of disease.

In October 1914, the stocks seemed in splendid condition, and T was also
feeding two stocks of driven bees. These had been obtained locally.

38 Proceedings of the Royal Physical Society.

In the spring of 1915 he reported that several stocks had shown marked
signs of dysentery, but there were no crawlers. T disinfected his hives after
the attacks of dysentery.

In the spring of 1915 the swarm of bees already mentioned, which had
settled in the roof of the cottage in 1913, showed marked symptoms of
the disease. T, with the help of neighbours, promptly removed the bees,
disinfected the place, and blocked up the entrance.

In June 1915, T’s stocks were strong and working well.

In August 1915, one stock showed very slight signs ; there were very few
crawlers, and it is understood that T meant to destroy it.

U.—U’s apiary is situated about half a mile due east from T.

In 1914, he owned seven hives and five ruskies.

In October 1914, there were no signs of Isle of Wight disease; but
robbing was going on very vigorously. On this account some stocks were
probably weak.

During the following winter, 1914-1915, five stocks died out, and all showed
signs of dysentery in the early spring.

In June the remaining stocks were fairly strong, and all combs were badly
marked with dysentery. When visited the weather was dull and no crawlers
were seen, .

The following day was bright at intervals, and a few crawlers were then
observed. These were found to have the colon very much dilated and
clogged.

In August 1915 only one stock remained, viz. the one which had
appeared the weakest in June. It showed slight but distinct traces of
the trouble, and U decided to destroy the bees and clean up and disinfect
his hives. He did so in order to prevent the disease spreading among
his neighbours.

No other cases in the immediate neighbourhood have been reported,
although there are several apiaries near by.

V.—V’s apiary is situated about five miles north-east of T. It consisted
of ten hives and twenty-five ruskies.

In August 1915 all seemed very strong and well cared for, but examina-
tion showed the ground covered with bees unable to fly, with distended
abdomens, dislocated wings, and all the usual appearances of Isle of Wight
disease. I could not locate any special stocks as being the affected ones, for
the bees were distributed all over the grass, and not in front of any particular
hives. Samples of bees were sent to Aberdeen University investigators as
usual, who reported that the appearances were those associated with the
disease. The bees sent did not contain Nosema.

Isle of Wight Disease in Hive Bees. 39

A week later, I found only three hives and one ruskie left. The rest had
been destroyed on receipt of the above-mentioned report.

The surviving stocks were examined, but no symptoms of Isle of Wight
disease were apparent. The owner, as a precaution, however, destroyed
them.

&,—X, situated about one mile north of V, had originally come from
Strathdon, and his bees had never flourished, but had been continually dying
out. V had been down to work among them several times in 1915, and used
his own gloves, smoker, and veil.

Drones had been thrown out of one of X’s stocks abnormally early, and
V had come in order to advise. Later, a first swarm came off this stock, and
died out very shortly afterwards. A second swarm then came off, and these
bees also died off.

When the stock was visited in August 1915, there was merely a handful
of bees left. X then destroyed these, his last stock, and cleaned and dis-
infected his hives.

Y.—In 1914 X had sold an old hive to a neighbour Y, who lived about
two miles south of his apiary. The stock which was placed in this hive
died, and another stock beside it was reported dying out also.

No other cases within a radius of five miles are known.

2.—EXTRACTS FROM Diary oF VIsITS oF J. R. TO
DEESIDE BrE-KEEPERS.

23rd August 1915. Visited Anguston Apiary.—Found twenty-one hives
in small garden all affected with Isle of Wight disease. Ground littered
with dead bees so that it was not possible to walk in garden without treading
on them. Some had been treated with a syrup of sugar and jalap, and were
reported to be improved, but this was not permanent. Sample taken;
pulped. Negative result as regards Nosema spores.

Visited R’s Bees at Druwmoak.—Both stock and swarm active. Swarm had
required an eke. There were a few crawling bees about, but not many.
This is a case where intermittent crawling, moderate only, has occurred. Is
this an aspect of the resistance of this stock where the infection has laid hold
of a few, but has not overcome the majority of the stock ?

D. , Durris—When last seen, one stock had Isle of Wight disease ;
now, four or five have it. He has been spraying ground with Izal, and feeding
an advertised preparation to the diseased stock, but apparently without effect.
The disease has spread. Bees examined did not contain Nosema spores.
When visited again in January 1916, all the stocks were extinct.

40 Proceedings of the Royal Physical Society.

24th August. Visited Cullerlie, K’s Apiary.—Isle of Wight disease
prevalent in most stocks, No. 10 has got it. But the Italians, Nos. 25 and
18, are both going on well with no signs. Hybrids also are well, and these
have swarmed lately. This swarm has settled in the top of a hive at Wood-
side which has got Isle of Wight disease very badly. I saw both stocks
working (above and below) from the same hive. Took sample from No. 10.
Result—failure to find Nosema.

H—., Finnercy.—Has Isle of Wight disease in most of his stocks. Took
a sample from the worst, but did not find Nosema spores. Examined all K’s
sample, also H’s. All had congested colons with pollen in a pasty condition.
Most had chyle-stomachs, purplish ; some were quite dark.

Main Features of Deeside Epizootic.

This report deals with twenty-three apiaries extending from Culter,
near Aberdeen, to Dinnet, a stretch of about 30 miles along the Dee
valley, and covering 3 or 4 miles on either side.

I. The epizootic was observed first in 1913, in particular at Culter
and Dinnet, and in stocks introduced from the South of England.
In the Culter case one year at least elapsed between the time of
arrival and the commencement of the outbreak. The disease is
still prevalent in this district.

II. Within the area there has been a very considerable amount of
destruction of bees and appliances, of disinfecting generally, and
also of experimenting by the bee-keepers with advertised remedies.
All these efforts have failed to cure the disease or to affect
recognisably its continuance.

III. There are instances of stocks “passed over” within apiaries, though
in close proximity to sick stocks. In this area at least one example

is known of a stock prospering in a non-disinfected hive in which
a stock had died out of the disease.

IV. Bees, in a number of instances, were sent by bee-keepers to the
editors of the British Bee Journal, and were reported to be suffer-
ing from Isle of Wight disease. We have been unable to find
Nosema in the bees of Deeside generally.

Isle of Wight Disease in Hive Bees, 41

IIL—INVESTIGATIONS BEARING ON THE RELATION OF
NOSEMA APIS TO ISLE OF WIGHT DISEASE.

METHODS OF EXAMINATION oF BEES FOR THE PRESENCE or Nosema APIS.
As carried out by Jonn Innas, B.Sc., M.B,, Captain R.A,M.C,

Each bee was examined in a routine manner, and the following notes
were made :—

(1) Activity, as determined by movements in the postal cage—usually
a match-box,

(2) Power of flight on exposure in open space.

(3) Dislocation of wings; if present or absent.

(4) Gait; whether co-ordinated or paretic.

(5) Response to stimuli, such as handling, ete.

The chyle-stomach and colon of each bee were examined systematically,
and the removal of these parts from the body was carried out in the following
manner. The head of the bee was firmly crushed between fairly stout
dissecting forceps, and while held thus, the last segment of the abdomen
was grasped with straight fine-pointed forceps, and gentle traction applied.
In this way the colon was carefully pulled out and the chyle-stomach also
came away with it. These organs were placed on a slide, separated at
their anatomical junction, and teased. Cover-glasses were then applied, and
the preparation examined microscopically in the wet, unstained condition.
If the spores of NVosema apis were present they were readily identified by
their shape, size, and refractility. Young, intracellular stages and planonts
were also observed in like manner.

Stained preparations from smears of the colon, and of the chyle-
stomach, were made from every sample of bees examined, and the stain
which was almost exclusively used was the iron-hematoxylin of Heideinhain.
Although requiring longer time for staining, this was found to give the
most reliable and most definite results. The fixative used was a hot alcoholic
solution of corrosive sublimate.

Various other stains were tried, such as Giemsa, Romanowsky, Erhlich’s
triacid, etc., but the varying results led to their abandonment in favour of
the more stable hematoxylin preparation.

No stain, however, was found which would show up the spores clearly.
Various experiments in this connection were tried, but with no success.
Heat had very little effect in helping the stain to penetrate the resistant
spore capsule. For its identification in stained preparations we could

42 Proceedings of the Royal Physical Society.

rely only upon the surrounding structures being stained and the spore,
left untouched, appearing as a clear structure.

The number of bees examined from each stock was, on an average,
about eight. Where more than eight bees were available from a stock, then
these were also examined.

The amount of infection was reported as slight, heavy, or very heavy,
according to the number of spores found.

Where young stages were found, these were recorded as such, but it
was rare to find a heavy young stage infection without the presence of a
fairly heavy infection with spores.

The routine here described was that followed by Dr Innes in the
laboratory, in all cases of bees which were sent for Nosenva diagnosis
from the Lewis stocks, from apiaries on Deeside, and several other
districts, Examinations of bees for Nosema by the other workers was
mostly, though not exclusively, confined to the searching for spores. These
were obtained by examining bees in the fresh state and by the pulping
method described later. Miss Simpson and Dr Rennie also prepared a
number of sections of the alimentary canals of Nosema-infected bees
(apparently normal in behaviour), of Isle of Wight diseased bees, and of
healthy bees, some of which are utilised in illustration of the present work,

Diagnosis oF NoseMA INFECTION IN DEAD BEES.

Graham-Smith, Fantham and Porter, in their report upon Nosema as the
cause of Isle of Wight disease, express the opinion that dead bees are
unsuitable for Nosema diagnosis (p. 41). This opinion is reflected in the
Board of Agriculture Leaflet, where it is stated that “it is not possible as
a rule to express any opinion from an examination of dead bees.” Leaving
out of the question altogether the problem of Isle of Wight disease, and
considering only that of Nosema infection, this statement has not been
borne out by our experience. Undoubtedly, the most reliable means of
diagnosis of Nosema infection is the recognition of the spores, and our
experience has been that these are recoverable from bees at least as long as
seven months after their death. We have in our possession at the present time
(January 1916), a number of dead bees which were artificially infected
with Nosema early in June 1915, in which we are still able to obtain spores,
not only from numbers pulped together, but readily from individual bees.

Graham-Smith, Fantham and Porter are of opinion that spores are not
often formed in the warmer months of the year. They state: “ Undoubtedly

Isle of Wight Disease in Hive Bees. 43

the most certain evidence of the presence of Nosema apis is afforded by
finding the characteristic spores. Unfortunately these are not always
present. . . . Our observations show that during the warmer months the
parasites often do not reach the spore stage, but seem to kill the affected
bees before this stage is reached” (p. 48 of 1913 report),

Our evidence is unfortunately rather contradictory, as we have been
able to find abundance of spores in the months of May, June, J uly, August
and September as readily as in other months. Our illustration of a section
of a chyle-stomach in which every visible cell is heavily infected with
spores (Pl. I. fig. 2) is from a summer bee. And we are a little doubtful
of any heavy mortality resulting entirely from infection in the period of
the bees’ maximum vigour, at an early stage of the parasites’ growth. This is
especially so since we have found, throughout the summer, living bees
without any obvious signs of disease, which proved to be very heavily
infected with spores. Further it is claimed that the chyle-stomach of the
bee is, even under normal conditions, constantly shedding and renewing
its secreting epithelium.

PuLPing EXPERIMENTS.

The routine of examining individual bees for Nosema diagnosis, although
valuable and most reliable as regards the single bee in question, has several
serious disadvantages. The first of these is due to the amount of time
required to examine thoroughly even one bee, and the second, which
depends upon this fact, is that one is led to base conclusions regarding
a whole stock upon the results obtained from the examination of relatively
insignificant numbers. A small sample of bees taken at random from a
stock does not correspond, say, to a sample of a homogeneous fluid or solid
substance.

This difficulty has been largely overcome in our later work by the
introduction of the method of pulping collections of bees designed for
examination for the presence of Nosema spores. Our method is simply to
cut off the abdomens of the bees, letting them drop into an ordinary mortar.
They are then gently pulped in sufficient water to render the contents fluid.
Sometimes we have removed the alimentary canals from behind the honey
sac and teased these collectively in a little water in a large watch-glass.
Samples of the resulting fluid in either case are taken up with a pipette, and
if Nosema spores are present in the pulped preparation, they are usually found
in the first drop examined. Our first experiment of this nature was as
follows.

+4 Proceedings of the Royal Physical Society.

Four samples of dead bees were taken. Two of these were from collections
of bees which had been infected by feeding with Nosema spores nearly three
months previously, and the remaining two consisted of bees from Isle of
Wight diseased stocks.

(a) Pulp of twenty-six Mosema-infected bees yielded, on examination,
numerous spores in the first and every succeeding drop of fluid taken
from the mass.

(o) Pulp of a second (separate) lot of MNosema-infected bees—about a
dozen bees. Result as in case of (a).

(c) Pulp of bees, “crawlers,” from two stocks—different apiaries on
Deeside—same stocks as were used for infection in second Shawbost
experiment (p. 5£)—about twenty bees. Result ; No Nosema spores
observed after repeated examinations.

(d) Pulp made of about two thousand bees from a stock which had died
out from Isle of Wight disease at. Banchory. The fluid, after
draining off from the solid residue, was tested extensively (twenty
Separate examinations), and Nosema spores were not found.

Other similar tests are given below:

1. Thirty “crawlers” from DES1 stock (p. 57) were picked up on 10th
September. They were allowed to die in a small cage, and on 14th were
pulped in a little water in the usual way. In this stock there had been found
previously an unidentified organism. The first field examined in the first
small drop contained numerous spores of this organism. Nosema spores were
not seen.

2. Sample from Anguston, consisting of nineteen “ crawlers,’ had their
abdomens pulped and examined for Nosema spores. Negative result.

3, On 11th September 1915, visited Anguston and found nineteen of the
stocks had succumbed to Isle of Wight disease since last visit, Took a
sample of dead bees from the site of one of the dead stocks.

On 20th September these bees, sixty in number, were pulped, and
examined. Nineteen samples were dealt with and no Nosema found.

4. On 25th September 1915, seven bees which had died in transit from
Glenhouse, Stornoway, were pulped. Negative result as regards Nosema
spores. These were “crawlers” from the front of the hive, but we have no
other record of Isle of Wight disease in connection with this stock. Seven
live ones which survived the journey were similarly treated. Result: Negative
as regards Nosema.

5. Six bees which remained of the lot of Carniolans (No. 7) from
Stornoway were pulped on 25th September. No Nosema was found.

Isle of Wight Disease in Hive Bees. 45

6. Pulped 27 chyle-stomachs of Welsh driven bees, which were hived
to-day in Desswood apiary (29th September 1915), and examined twelve
drops for Nosema, Result: Negative.

Numerous further tests of this nature have been made, and in every case
where Nosema was known from previous experience to be present in the
collection from which the bees were taken, Nosema spores were found.

As early as 1913 we had become doubtful of the conclusion that Nosema
apis stood in direct causal relation to Isle of Wight disease. From February
of that year we regularly examined bees for Mosema spores, especially any
that crawled or seemed sickly. We found such spores very frequently,
particularly in old bees and not necessarily in association with unusual
mortality in a stock. We have found bees with a heavy spore infection able
to fly perfectly and even to carry loads of pollen and nectar. We have had
under observation for three years, 1913-1915, a stock of Ligurians, G9 (Nicolson
Apiary), which has stood in proximity to several of the forementioned stocks
throughout their periods of crawling. This stock has never displayed any
noticeable crawling symptoms. Yet all the time Nosema has been present in
it, sometimes in enormous quantities (see Pl]. I. fig. 4—a microphotograph,
taken in the present year, of the colon contents of an apparently healthy bee
from this stock).

It is significant that, while G9 contained Nosema over a prolonged period,
it failed in any marked way to infect the apiary generally. We have not
found Nosema distributed in the bees of all the stocks, but only in a few
stocks at any one time, and where it occurred, Isle of Wight disease was not
present,

History of certain Stocks in Lewis—with special reference to the alleged
connection between Nosema apis and Isle of Wight Disease.

We visited Bayble on 7th May 1914, and took samples of a stock of bees
which had no history of crawling. Spores of Mosema were found in several
of the bees, the infection being very heavy in a number of cases. On 4th
September, when again visited and examined, this stock was in a highly
prosperous condition ; it filled the brood box of ten combs and crowded also
a super of ten shallow frames filled with honey. On 14th August 1913, this
stock was still prosperous and had never shown any crawling symptoms.
Sample bees from this stock were taken in September, and these were found
to be free from spores. Twenty bees were examined: these were lively,
vigorous, and flew readily after two days in transit. The chyle-stomachs of

46 Proceedings of the Royal Physical Society.

five examined individually were of a healthy appearance; the colons were
not markedly congested, and contained watery material consisting almost
entirely of indigestible pollen residues. The abdominal parts of the
remaining bees were pulped, and drops of the fluid examined as offering the
most reliable means of detecting the presence of Nosema spores. This stock
remained healthy alongside the next-mentioned.

Meanwhile there had been in the same apiary, and in close proximity to
this stock, an independent outbreak of Isle of Wight disease. A swarm of
black bees arrived from the South of England on Saturday, 11th July 1914,
and was duly hived. The bees began to crawl next day, and the symptoms
were very marked when we visited the apiary on 13th July. Then, nearly
half of the stock was to be seen crawling or dead upon the ground.!

Bees taken on this occasion were examined in Stornoway by J. A. and
J. R. with negative results, and stained preparations were afterwards made
without young stages being found. This swarm was headed by an Italian
queen, which had been supplied just before the stock was despatched to
Lewis. A little patch of brood was hatched, and the yellow bees did not
“crawl.” The blacks were all dead in autumn and the yellows were not
strong enough to winter.

Stock G9: Nicolson Apiary.—This stock has no history of Isle of Wight
disease and no abnormal death-rate. A few “crawlers” have occasionally
been observed, but nothing to indicate disease, yet this stock is known to
have harboured Nosema for at least three years. The original queen was a
pure Ligurian imported from Bologna (apiary of Enrico Penna), in the
autumn of 1911. A black stock on each side of this hive (the stocks already
referred to as C13 and D1) died of Isle of Wight disease during that autumn
and winter, but the Ligurians showed no signs of trouble. These extinct
stocks were replaced in 1912 by two other black stocks (D4 and H12) which
died out—D4 certainly, and H12 probably, from Isle of Wight disease
before the following winter. But still the Ligurians showed no signs of this
disease.

1st September 1912 was very windy, and the hive of G9 was blown right
over, the combs being scattered. The bees were gathered up and the queen
found uninjured, but this accident handicapped the bees for getting into
condition for wintering. The spring of 1913 found them very weak and
hardly any of the young bees were able to fly.

Examination for Nosema spores gave negative results at this stage. The
queen eventually ceased laying, and she was transferred to a queenless black
stock. In due course brood appeared, and in this case most of the young bees

* Bees from this stock were used in Tolsta experiment (see p. 52).

Isle of Wight Disease in Hive Bees. 47

could fly. A number of those which “ crawled” were sent to Dr Annie Porter,
together with a description of the symptoms, and she reported as follows :—
“Every bee contained spores of - Nosema.” This was in July 1913. The
crawling referred to here was not typical of Isle of Wight disease, and
lasted for quite a short time. Also the number involved was very limited,
and consisted entirely of young Italian bees, At intervals during June, weak
and aged bees, both blacks and Italians, had been picked up in front of this
hive and examined on the spot, by one of us (J.A.). These were full of spores
of Nosema, but none were found in the young Italians examined at the same
time.

A neighbouring queenless black stock was now united with this Ligurian
stock, and soon there was a prosperous colony with plenty of brood, worker
and drone. On 12th September 1913, bees taken at random off the frames
were examined and no spores of Nosema were seen. During the winter of
1913-14 frequent samples were taken, and a large proportion of the bees
examined were found heavily infected with spores. Yet those bees were quite
active and healthy and could fly perfectly.

On 19th December 1913, the old queen died and stock G9 continued
queenless and broodless for over three months. On 28th March 1914, we
added a small lot of Italians which had been found starving in another hive.
The queen was a Penna Italian imported in 1913. Bees of this stock were
found to be heavily infected with Nosema on 2nd June.

When we examined the stock on 20th June 1914, it was quite prosperous
with a good show of brood. The weather was wet and cold, and diligent
search in front of the hive resulted in the finding of eight benumbed bees.
Seven of these were full of spores of Mosema, Hight bees were also picked
off the combs at random, and three of these contained spores of Nosema.
Again, on 13th July, one bee out of six examined contained an abundance of
spores. This stock is still alive at the present time.

Nosema INFECTION EXPERIMENTS.
(a) Infection of a full Stock of Bees with Nosema apis.

On 11th July 1914, a small lot of Cyprian bees which were known
previously to harbour Nosema were placed in confinement. On 28th July
most had died, and the remainder appeared weak. Four were examined, two
live and two dead. All contained Nosema spores in quantity. The whole
lot of bees, ninety-one in number, were mashed with honey, and fed to a full
stock of bees at Achmore, Lewis. These were ordinary black bees imported

48 Proceedings of the Royal Physical Society.

as a swarm from the South of England in June 1914. They had been examined
on 30th June, and again on 13th July, and found negative as regards Nosema
spores and young stages. There were no other hive bees within a radius of
5} miles.

On 19th of August, the stock was visited and examined. It covered nine
combs, and clustered on the dummy. Not a “crawler” nor dead bee was to
be seen in front of the hive, although the weather was very favourable for
this. Ten bees, taken at random, were examined for Nosema, and spores
were found in four, two of these showing a very heavy infection.

On 7th September, a second sample consisting of twelve bees was
examined, again taken at random from within the hive, and five were found
infected, three of them heavily. The day was fine, and there were no indica-
tions of Isle of Wight disease.

On 10th October, the bees were again visited and samples taken, from
inside the hive as formerly. It was a fine October day, and the behaviour of
the bees was normal.

On 13th October nine of the sample bees were examined. They were
active, flying readily, and their intestines were normal in appearance. No
spores of Mosema were found. On the 14th, eight more bees were examined,
again with negative results. On the 22nd, three bees of another sample were
taken. These arrived alive and active. On examination they were found to
have the intestine normal, and to contain no spores of Nosema.

From the foregoing it appears certain that this stock of bees was definitely
infected with Nosema, but this infection failed to produce Isle of Wight
disease.

The following are illustrations of numerous artificial infections with
Nosema carried out on small lots of bees confined in cages.

(b) Laboratory Laperiments.

I. On 14th June 1915.—Nosema spores derived from bees of Stornoway,
stock No. 7 (Carniolans), were used to infect some bees taken from an
observation hive at Marischal College, Aberdeen.

On the 17th, Nosema spores were found in plenty in five bees taken at
random from the infected lot. These spores were in alimentary canal; no
intra-cellular forms were seen. Our view is that those seen were the spores
ingested.

19th June 1915.—Numerous spores were found in the colon of one dying

bee, and a few in the chyle-stomach. Young stages were observed in cells of
chyle-stomach (J. A.).

Isle of Wight Disease in Hive Bees. 49

21st June—A dead bee from infected lot was examined; spores were
found in colon, but not in chyle-stomach. Many cells of the chyle-stomach
showed intra-cellular stages (J. A. and J. R.).

Ist July 1916—On 30th June, two bees were found dead. When
examined on this date a few spores were found in both, and a great many
young stages were seen, especially in one bee. A stained smear showed
many meronts within the cells, there being frequently four parasites in one
cell (J. R. and J. A.).

This infection experiment, through pressure of other duties, was allowed
to come to an end about this time. But the dead bees were preserved, and
in September they were found to contain Nosema spores in great abundance.
At this latter date, however, several attempts to produce infection by means
of these spores failed.

II. 20th September 1915.—On this date thirty bees from Craibstone
No. Al (a stock originally Italian, but now consisting entirely of black bees)
were confined in a small cage, and fed on spore-infected material mixed with
honey. The spore material was obtained from the 14th June experiment.
Five bees were killed and their chyle-stomachs pulped and examined, before
infection, for Nosema spores. Result—negative (J. A. and J. R.).

On 21st, the bees were again fed with the spore-laden material. Up
to 25th the behaviour of the bees was normal; ‘they fed freely, and
were quite active. There had been no mortality except in the case of
three, which died on the 21st. In one of these ingested spores were found.

On 26th, one bee died and one was killed. No spores were found in
either of these.

On 28th September, two died and two were in a weakly state. No spores
were found in any of these.

On 29th, one died and no spores were found. The bees were fed on
Scholtz candy.

The intestine in the last examined bee was whitish, and not unlike the
appearance of a Nosema infection, but, as stated, spores were not observed.

On 7th October, fourteen bees which had died were pulped. These were

black bees: no Nosema spores were found.
It is clear from the foregoing that the spores used in this experiment

failed to infect.

III. On 30th September, a small lot of Welsh bees were caged and fed on
Nosema-infected material of 15th June. Spores were present in the material
fed. A similar number of bees were caged as a control, and fed on clean
candy. Feeding with the infected material, in the case of the first lot, was

continued on the 1st and 2nd October, after which clean candy was given.

VOL, XX. D

50 Proceedings of the Royal Physical Society.

On 7th October, it was found that sixteen bees of the infected lot had died,
These were pulped (abdomens) and examined for Nosema spores. Free spores
in moderate numbers were found ; two or three in every field examined of
the first drop taken. One live bee examined gave a negative result.. In the
control, two bees had died. These were pulped (abdomens only), but no spores
were found.

16th October 1915.—Five bees dead. These were pulped on 18th, and
Nosema spores were found. Other three were examined, and spores found in
all. Other six were pulped, and spores were again found. All the control
bees had died at this date.

From these results it seems probable that no infection had taken place.

IV. 4th January 1916.—On this date all the Desswood stocks were in
flight. The opportunity was taken to procure a number for experiment, and
samples were captured on the alighting board of the “Kelly” hive. These
were fed with candy, which on the same day was well smeared with pulped
matter from bees of the June experiments. This matter was found on
preliminary tests to contain Vosema spores in plenty.

The bees were kept in a small wooden cage with glass cover. By the 6th
several had died. Nine were removed at this date, but of these, two were
dead shortly after caging, having probably been hurt.

6th January 1916.—Two were allowed out of the cage, and these flew
quite strongly, and one of them defecated.

Sth January 1916.—Other six bees were found dead. One released
proved unable to fly, although it was quite active. When lifted it used its
sting. This bee had chyle-stomach, pale in colour, and colon dilated with a
pasty mass of pollen, Search in the dead bee and in the surviving one failed
to find any trace of Nosema spores. Another live bee very active was taken,
killed and examined. This bee had a pale chyle-stomach and inflated watery
colon. Numerous free spores were found in the latter, but a very thorough
search failed to discover any trace of infection in the chyle-stomach.

12th Januarg 1916.—On the 12th all the bees had died. These
(thirteen) were pulped, and examination showed free Nosema spores in
moderate numbers.

V. 24th June—Infected two boxes of “ Kelly” bees with Nosema
material from No. 7 Stornoway hive.

1st July—Examined two bees which had died. Planonts seen in both
bees and intra-cellular forms in stained film. No free spores. seen.

8th July—On this date the chyle-stomach of a bee of this experiment,
taken alive, was found on examination to contain enormous numbers of
spores and planonts. Spore clusters common.

Isle of Wight Disease in Hive Bees. 51

VI. 20th June.—Fresh spores of Nosema were, on this date, mixed with

honey and fed to a number of bees in a small cage. On the 26th several of
the bees were killed, and an examination was made of the alimentary canal.
Planonts were seen in very large numbers. A further examination on the
30th revealed the presence of both planonts and meronts. Spores were not
seen.
In connection with the foregoing experiments control lots of bees were
in most cases kept. Further we have had a very considerable experience with
caged bees under the same conditions as those of the experiments. We have
not been able to detect significant differences in mortality amongst such caged
bees. No conclusions are therefore drawn with reference to the cause of death.
Our experience is that captivity in small cages without a queen is eventually
fatal to the healthiest of bees.

With regard to the behaviour of the bees while under experiment, it
was not possible to decide whether Isle of Wight was present. The
condition of the intestine which we have learned to associate with Isle of
Wight disease, and which we find has been described by Imms (Jour. B. of A.,
xiv. pp. 133-4), has never been observed in those caged bees.

The experiments numbered II.-IV. were made in September and January
with spores obtained from bees which died in June. These dead bees were
kept in a laboratory cupboard under ordinary conditions, and the results
would suggest that in some instances at least the vitality of the spore is
brief.

CONCLUSIONS REGARDING RELATION BETWEEN NOSEMA APIS AND
IsLE OF WIGHT DISEASE.

Our main conclusion regarding Nosema apis is that so far we have
been unable to recognise any causal relation between the presence of this
parasite and the disease. We have found it to be present over prolonged
periods in healthy stocks, while we were unable to find it in other stocks
in the apiary, nor did Isle of Wight disease spread under these conditions
although various races of bees were present. Deliberate infection of a stock
with Nosema did not produce the disease. It is well established also that
the disease occurs where the parasite cannot be found. We have numerous
instances of this on Deeside.

From known facts regarding Nosema in other countries, eg. in Germany
and in Victoria, Australia, it appears that Mosema apis is just as common in
healthy bees as in diseased. They are practically all “ parasite carriers.”

52 Proceedings of the Royal Physical Society.

We recognise that Mosema may be a contributing weakening factor
favouring in certain cases the development of this disease. But we have
not found that it is an essential factor.

IV.—OBSERVATIONS AND EXPERIMENTS BEARING ON THE
INFECTIVITY OF ISLE OF WIGHT DISEASE. As

DISTINGUISHED FROM MICROSPORIDIOSIS.

(a) Arremprs TO Propuce IsLE or WicuT DISEASE BY ARTIFICIAL
INFECTION.

No. 1. Tolsta Experiment. July 1914.

The stock utilised in this experiment was a small swarm of unknown
race, originally imported from the South of England. It was transferred
for the purpose of this experiment from the Nicolson apiary to Tolsta
which is six miles from the nearest hive of bees. A control examination
gave a negative result as regards Nosema.

On 18th July 1914, three large lots of “crawling” bees, numbering
hundreds, were taken from the stock at Bayble suffering from Isle of Wight
disease (see p. 46). These were beaten into a compost with honey and
spread over the tops of the combs of the Tolsta bees. When the hive was
visited on the 27th July everything had been cleaned up by the bees. There
was no trace of the honey nor of the mashed-up bodies. The bees were
multiplying and required extra combs.

On 19th August, they covered fully eleven combs and had gathered
considerable stores of honey. Not a “crawler” was visible, nor even a
single dead bee to be seen in front of the hive. Samples at this date
were examined by Innes who reported :—“ Bees alive, active. No Nosema
spores found.”

On 12th September, the bees were found to fully occupy twelve combs,
and not a dead bee was to be seen. There were no indications of any
kind suggestive of Isle of Wight disease.

On 17th October, one fully sealed outside comb of honey was
removed leaving eleven combs loaded with honey. The bees were in
excellent order.

On 7th November, the Tolsta bees were again visited. It was a fine
mild day and the bees were flying freely. ‘Several drones were observed in
flight, but these were being attacked by the workers. The presence of
drones so late in the season probably indicated that the queen was

Isle of Wight Disease in Hive Bees. 53

failing. There was no crawling or other symptoms of Isle of Wight
disease.

On 2nd January 1915, the stock was visited and found normal.

On 13th March, the stock was found lively and vigorous.

On 28th April, the bees were found numerous and lively; there was a
patch of drone brood in worker cells suggesting the presence of a drone-
laying queen. Probably the aged queen had died during the winter leaving
eggs and young brood, from which the bees were able to raise a young
queen. This queen could not be mated, however, at this season. This
drone breeder was actually found on 3rd May and removed. After some
difficulty a fertile queen was again established in the hive and the stock
continued its prosperous career, giving, in July 1915, a natural swarm,
which was lost.

On 8th August, the swarmed stock was found queenless and broodless
and much reduced in numbers, but lively and vigorous, and with much
stored honey. The stock was re-queened on 10th August with a young
American Golden queen bred in Stornoway.

This stock, in spite of a heavy contamination with fresh Isle of Wight
material, remains free from the disease to this date, January 1916.

No. 2. Shawbost. First Experiment. October 1914.

The bees which were the subject of this experiment were Carniolans,
and the stock had swarmed in July 1914. It was headed, therefore, by a
young queen, and occupied seven combs.

On lst October, two ordinary match boxes of “crawlers” from an
infected stock (60 DD, see p. 56) were sent to Lewis from Aberdeen. The
crawlers were taken from a lot which came out in great numbers on a
sunny afternoon when the “Isle of Wight” signs were most typical.
They were picked up after having been out over night, and despatched by
post alive, with food. These crawlers were mashed up on arrival, mixed
with honey, and fed to the stock at Shawbost, on 3rd October.

On the 10th October, the bees were reported to be in fine order with
brood on two combs. All infected material had been cleaned up by the
bees, and not a dead bee was to be seen.

On 24th October, the local observer reported: “ No sign of any disease.”

On 20th February 1915, the stock on examination was found to be
in good condition, and no dead were visible.

On 27th April, the stock was reported to be in fine condition, the
bees were lively and “ vindictive,” and there was a good deal of brood.

At the beginning of June 1915, the stock was reported to be rather

54 Proceedings of the Royal Physical Society.

weak in numbers but it built up rapidly, and on 12th August the report
was as follows: “The seven original combs are now quite inadequate,
and the bees have passed behind a double dummy and have built a
considerable mass of new comb which is filled with honey. This is a
highly prosperous stock.”

On 4th September, the stock was still in excellent condition. At this
date eleven months had elapsed since the Isle of Wight disease material
was served up to the bees, and throughout the whole of that period the
bees had been perfectly healthy, and no infection had resulted from the
experiment.

No, 3. Shawbost. Second Experiment. September 1915.

On 24th August 1915, the colons and chyle-stomachs of twenty-five crawl-
ing bees were taken from front of hives No. 10 of K and the worst stock
of H, Finnercy. Both stocks were very bad with Isle of Wight disease. The
day was warm and sunny, and there were many hundreds of “crawlers”
about in both cases. The intestines of all the bees (twenty-five in number)
were drawn out, and in every case the colon was congested with pollen in
a pasty condition. Most of the chyle-stomachs were purplish. These
were laid out in a row and allowed to dry—and on the following day
heaped together.

The above-mentioned bee-material was mixed in a little water and
twelve samples examined. It consisted chiefly of pollen grains, a good
many of which were digested. A few epithelial cells of the chyle-stomach
in various stages of disintegration, some still showing the zymogen granules
present, were seen, also some which seemed intact. None of these showed
any sign of Nosema infection, and no free spores were observed.

Bacilli, long and short, and also cocci were seen in the preparations.
This material was mixed with candy and sent to Lewis the same day, viz.
30th August. The local observer (Ewen) reported that on 4th September
he fed this to Shawbost bees, and describes the bees at this date thus :—
“TI placed the compost on the top of the frames. The day was fine and
the bees were very active. The stock in the beginning of June was very
weak in numbers, but has done very well during the summer.” This stock
is still under observation, January 1916, and-apparently healthy.

No. 4. Back Bees, Stornoway, June 1915,

In this experiment old combs and hives were used. The bees originally
occupied only one of two hives, the other being left open in the hope that

Isle of Wight Disease in Hive Bees. 55

any swarm might hive itself. The nearest bees to Back are at Tong, about
2 miles away, and the Tong bees have always been quite healthy.

At June 1915, the Back bees were so prosperous that they were supered.
On 28th June, the super was removed and the tops of the frames smeared
with a paste made by mashing diseased bees with honey. These bees were
crawlers picked off the ground in front of an infected Deeside stock. The
bees were sent alive from Aberdeen and pulped in Stornoway.

On 8th August the stock was visited, and it was found that the bees and
the two hives had been shifted from the original site to a less suitable
one in a very unskilful manner, by one who was not a bee-keeper. The
bees had previously swarmed, and the swarm had occupied the vacant hive
as had been expected. The combs and quilts had been disarranged, so that
the swarm got a very poor chance. The jar used in infection was still on
the parent stock and some comb had been built in it, but there was no trace
of the Isle of Wight material.

On 14th August, both lots of bees had to be hurriedly shifted to still
another site at a distance of about 400 yards, and during the process
a considerable number of bees was lost. There was no brood in either hive ;
presumably both were headed by virgin queens. This absence of fertile
queens, the two disastrous removals, and the swarming constituted a combina-
tion of factors which could not fail to weaken and discourage the bees.

On 19th June, the bees had been examined microscopically ; no spores of
Nosema were found.

On 11th September, bees from Back arrived in Aberdeen. They were
active, and some of them survived until 16th. On this date live bees were
examined individually for Nosema with negative results. The dead bees were
pulped, and samples examined were also found free from Nosema spores.

On 20th October, bees were received which had been dispatched on the
12th, with the following report:—“ Symptoms same as No. E14 in Nicolson
Apiary. The stock is almost exterminated. The bees sent represent
‘crawlers’ and remnants in the hive.”

Of these fifteen dead bees were pulped, and drops examined for Nosema
with negative results.

On 28th October, Back was visited and both stocks were found almost
extinct. A small knot of bees remained in each hive on the combs. The
queen of one was warmed into activity, and lived for a few hours. There
was plenty of food. The local observer (Morrison) reported that both lots
had displayed the typical “crawling” symptoms.

It is worth noting that while this experiment has culminated after four
months in the bees apparently contracting Isle of Wight disease, which did

56 Proceedings of the Royal Physical Society.

not occur in other two similar experiments, it is not clear that this was due
to the artificial infection. There were specially adverse circumstances in
the present case. In the other two instances of attempted infection, the bees
were maintained in good general external conditions. It should be noted
further that Nosema was not found at any time during the experiment.

The foregoing experiments in artificial infection were made in Lewis upon
isolated stocks. The following were carried out in Aberdeen.

No. 5. 60 DD. May 1916.

This stock, which stood in an apiary in a district suffering badly
from Isle of Wight disease, was found, on 24th September 1914, to have
large numbers of bees on the alighting board, and groups of “crawlers”
on the grass around. The weather conditions were fine and sunny. The
same thing was observed on the following day, and the hive and bees were
removed to an observation area in Aberdeen. For a few days after this the
stock seemed to be doing well enough and a good deal of pollen was being
taken in. On fine sunny days, however, “crawling” became very evident,
and on some occasions it was very extensive. As recorded elsewhere a
sample of the “crawling” bees was utilised in an infection experiment
(p. 53).

On 7th October, which was a fairly good day with a little sunshine,
“crawling” was again in evidence, but pollen was still being gathered.
About this time it was reported that three other stocks from the same apiary
were suffering from the disease. All these eventually perished (p. 34).

Up to 17th October, bees “crawled” every fine day, but the last of them
perished in the early winter, leaving abundance of stores in the hive.

On 19th May 1915, a fresh stock arrived from the South of England,
Italian hybrids, fully covering ten combs crowded with brood. The queen
had apparently been recently lost, but there were numerous queen cells on
the combs. The new stock was put in the hive in which the above-mentioned
stock, 60 DD, had been housed. There was no disinfection of any kind, but
the dead bees and the old combs were removed. The honey was drained
from some of those old combs and fed to the new stock.

On 3rd June, a swarm came off and was duly hived in a perfectly new
hive on frames with full sheets of foundation. This swarm was also fed on
honey drained from the old combs left by stock 60 DD.

On the afternoon of the following day a second swarm came off and was
hived in a straw skep. This second swarm received none of the honey from
the old combs, but was fed on a little honey bought in the open market.

a

Isle of Wight Disease in Hive Bees. 57

Eggs were found in the parent stock (DEP) on 14th June, and the queen
of the first swarm (DES 1) began to lay on the 15th.

The queen in the second swarm was long in being mated, and was balled
by the bees on 28th June. A black virgin queen was introduced on the 30th
June, and she began to lay in due course. The other two queens were as
yellow as pure Italians, and the young bees were also light coloured.

The population of the parent stock was much depleted by the double
swarming, and further, on 23rd June, foul brood was found to be present.
This was a variety which attacks the larva before sealing, and attempts were
made to treat the infected cells with dilute formalin. This treatment was
successful so far in that the disease appeared to be checked, but unfortunately
the queen was lost either during the manipulation, or poisoned by the formalin
vapour. Queen cells were made from the brood in the hive and a black queen
was produced, which ultimately began to lay. The stock was now very weak
in numbers but was active in raising brood, in defending the hive against
robbers, and in carrying pollen.

Up to 4th September it could not be said that there were any symptoms
in the apiary of Isle of Wight disease. From the nature of the experiment
here being recorded, a specially close watch was kept for indications of its
appearing. Only at one period were suspicions aroused. The month of July
was very wet and must have been very trying to the bees. On the 25th, a
few “crawling” bees were seen in front of the parent stock and also the first
swarm, as well as in front of another stock of black bees not related to the
bees of the present experiment. And again, on the 31st, it is recorded that there
were a few “crawlers,” most in front of the first swarm (DES 1). August was
also a wet month, but nothing of a suspicious nature was noticed. In spite of
the very adverse circumstances above narrated, all three stocks remained
perfectly healthy, and up to this date (20th September) the parent stock,
which is housed in the presumably infected hive, was fed with honey from this
hive, and was later given some of the original combs, is healthy and, as far as
the eye can judge, growing in prosperity.

On 30th September, a lot of driven bees from which the queen had been
removed was imported from North Wales and added to the little stock (DEP).
The bees were fed on sugar syrup boiled with vinegar and were packed down
for winter.

On 14th October, the first fine day since the addition of the Welsh bees,
large numbers of bees were seen out upon the alighting board. This was
probably related to robbing, which was being actively attempted at several
hives in the apiary.

On the 28th December, a mild day, with a few bees offering to fly, the hive

58 Proceedings of the Royal Physical Society.

was opened, and the quilts were found in a sodden condition owing to a defec-
tive roof. The bees, however, were in good condition with plenty of stores.

It has already been mentioned that “crawlers” were observed on two
occasions in July in front of the hive occupied by the first swarm. From
that date until the beginning of September there were no definite symptoms.
August, like July, was mostly wet, and the Italians with their usual improvi-
dence had converted practically the whole of their stores into brood. From
4th September onward this stock, however, now showed undoubted signs of
the disease. The first half of this month was uniformly fine, with a great
deal of sunshine, and on every occasion the alighting board and the grass in
front was covered with numbers of “crawling” bees. Some of these were
fairly active and could perform short flights but not sustained ones. Those
which left the board were, as far as we could see, unable to return to the hive
and crawled about, sometimes as far as twelve yards at least from the hive,
ultimately dying on the ground. Those near the hive tended to form small
clusters as the day advanced. The alighting board was always cleared in the
course of the afternoon. All this is of course typical of Isle of Wight disease.
Further, the internal appearance was that characteristic of this disease, although
all our microscopical examinations failed to reveal the presence of Nosema.
The appearance within the hive during these days was practically normal,
pollen was being carried in, and there were no indications that the disease
was making rapid progress. From 4th September sugar syrup was supplied
to all the stocks, and this was freely appropriated by the bees and stored.

The question naturally arises here, Had this outbreak any connection with
the proximity of the infected hive or the fact that this swarm came from it ?
In view of the prosperity of the parent stock which had continuously occupied
the hive for four months, and the length of time, viz. three months, since the
swarm constituting this stock left the hive, it does not seem likely. But we
must admit the possibility of infection having been transferred and remaining
latent until conditions favourable for its development within the bees had
arisen.

No further “crawling” symptoms have been observed since the beginning
of October. On 20th October, a large lot of Welsh bees (driven, queenless)
was added to this stock. It is probable that these killed the queen, for on
1st November several dead yellow queens were found on the floor of the hive,
and there were queen cells on one of the combs. No free virgin queen was
observed, and another queen was caged over the stock, This queen was found
on the alighting board a few days later. This stock, therefore, has a virgin
queen (grand-daughter of the original English queen).

Isle of Wight Disease in Hive Bees. 59

The second swarm, standing alongside the first, has never displayed any
symptoms of the disease.

(6) Naturat Occupation or IsLE or WIGHT AFFECTED SITE.
The Hive and its Contents as Possible Sowrces of Infection.

The following case is related to the foregoing experiment in respect that
it is a naturally occurring instance in which the same factors, viz. hive
and combs of an extinct Isle of Wight diseased stock, are involved.

Bees which occupied a frame hive began to exhibit “crawling” and
other symptoms of Isle of Wight disease in the autumn of 1913, and
the last survivors perished in the spring of 1914. The hive was not
disinfected nor the honey and dead bees removed, but the door was closed.
In June 1914, a vagrant swarm alighted beside the hive and was allowed to
enter. No particular attention was paid to them, for it was not expected
they would survive. But it was observed that the new-comers were very
diligent in removing the dead bees and getting the hive in order. The
bees wintered in good order, and in May 1915 they swarmed. This top
swarm was unfortunately lost, though followed for some distance, but a
second swarm on 7th June was duly hived in a skep and placed alongside
the parent stock. Both stocks were visited and examined for eggs on 12th,
15th, and 21st June, but none was observed. Up till this time both stocks
appeared perfectly healthy and were working normally, except that the
parent stock had only a small number of bees, and drones were unusually
numerous. Thus it will be seen that in the case of the parent stock a full
year had elapsed since the bees took possession of a hive and combs with a
recent history of Isle of Wight disease.

On the 26th of June “crawling” symptoms were developed in the bees
of the swarm, and these were very marked when the stocks were visited on
28th June. The garden in front of the skep for a considerable distance was
covered with crawling bees, with characteristic grouping very noticeable.
The internal appearance of the crawling bees was quite characteristic of
Isle of Wight disease, and we satisfied ourselves that this disease was
present. It should be added here that both in 1914 and 1915 the whole
surrounding district had been swept by the disease, and there were very few
stocks surviving in it. Examination of the combs showed that both queens
were laying, and there was a good deal of sealed brood in the skep.

The crawling symptoms above noted became less noticeable from day to
day. There were, however, fluctuations, till on 23rd August both stock and

60 Proceedings of the Royal Physical Society.

swarm appeared to be free of the disease and were working normally. The
skep being overcrowded had been provided with an “eke.” On the
11th September, both stocks were found apparently in perfect health.
The bees in the skep had extended their combs down into the eke.

All this time there had been no symptoms of Isle of Wight disease in the
parent stock.

On 6th October, the parent stock in the bar-frame hive was found rather
weak in numbers, and the bees in the skep were driven and united with it.
The queen of the skep was removed and introduced to a stock in Desswood
apiary. The combs of the skep were cut out and piled over the frames in
the wooden hive.

On 29th December, the bottom of the hive was found to be thickly
covered with dead bees, but there were bees still alive amongst the piled
combs just under the quilt.

It may be noted that to date bees have been living in this hive on the
old “Isle of Wight” combs for over 18 months without any symptoms of
the disease appearing, and the conclusion is legitimate that the appearance
of Isle of Wight disease in the swarm is to be traced to a separate source.

There has also come under our observation the case of a bee-keeper near
Perth, who had lost several stocks from the disease, but had several remaining
which were apparently in excellent condition, although he had fed them on
the stores left by the diseased stocks. One in particular, through an accident,
had been soaked with water during last winter (1914-15), and by spring the
bees had been reduced to a handful. We examined this stock in October
1915, and found it in fine condition. The bees were black, but the owner
declared they were Italians because they had had an Italian queen three
years previously. Some of the surviving stocks displayed yellow bands, and
the bee-keeper attributed their resistance to the admixture of Italian blood.

Of direct interest in connection with our failure to produce Isle of Wight
disease by means of presumably infected hives and combs is the experience of
Beuhne, of Victoria, as given in The Australian Bee-keeper, 15th August
1915. He says: “When a very heavy mortality occurred amongst the bees
in the Grampians in 1909-10, and microscopic examination showed the
parasite in the bees of the apiaries concerned, the question arose whether
bee-keepers should follow the directions of the British Board of Agriculture,
and destroy all the remaining bees as well as all the combs, or give the
survivors a chance, and take a risk with the combs of the defunct colonies.

“My personal experience with this kind of mortality was in 1900-01, when

Puate I.

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“Tste oF Wicut” Ber DIsEase.

Isle of Wight Disease in Hive Bees. 61

nothing was known of Nosema, and therefore I was unaware of any risk, and
used the combs from which the bees had disappeared, when restocking the
apiary with purchased bees, and ‘nothing happened.’”

Beuhne is of opinion that “under ordinary conditions, that is, conditions

favourable to bees, the parasite is merely a casual inhabitant of the intestine
of the bee.”

GENERAL CONCLUSION REGARDING INFECTIVITY.

The general conclusion to which the foregoing facts point is that Isle of
Wight disease, although probably an infectious disease, is one which requires
the coincidence of other and presently unknown external factors (besides a
specific organism) before the disease develops. The disease is not necessarily
conveyed by mere contact with contaminated hives or combs, or by feeding
upon contaminated stores.

EXPLANATION OF PLATE.

Fic. 1. Section of the chyle-stomach of a bee displaying symptoms of Isle of Wight disease.
Spores of Nosema are not present, and no young forms were found in this outbreak,
x 180 (Iron-hematoxylin),

Fic. 2. Section of the chyle-stomach of a bee infected with Nosema, but not suffering from
Isle of Wight disease. The stained spore contents came out as black dots in the
photograph. x 180 (Iron-hematoxylin).

Fic. 3. Portion of the teased chyle-stomach of a bee, showing detached cells, some contain-
ing spores of Nosema. Fresh preparation. x 180.

Fic. 4. Fresh unstained preparation, showing Nosema spores lying free in the gut. x 180.

Fig. 5. This shows the behaviour of a stock (E14) suffering from Isle of Wight disease.
Note the bees, incapable of flight, clustering on the hive front. Large numbers on the
grass cannot be seen, but some can be observed climbing up the leg of the hive.

cs. Chyle-stomach of bee.

e, Epithelium lining chyle-stomach,
sp, Spores and epithelium of chyle-stomach.

(Issued separately, 14th September 1916.)

as, ell University Library

bservations and experiments bearing on

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