seal, 6 % AN M8 ot a * Oe DIOR OBE > BANE Pe re HAN TA ais Ben T87 pc aks Historic, archived document Do not assume content reflects current scientific knowledge, policies, or practices. ‘UNITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 780 : Contribution from the Bureau of Entomology - L, O. HOWARD, Chief Washington, D.C. | PROFESSIONAL PAPER ‘June 12, 1919 NOSEMA-DISEASE | G,. F. WHITE, ue in fee Disease CONTENTS © Page Page Introduction . . . . - - « - 1 | Resistanceof Nosema apis toPutrefaction 35 Name of Disease ...... . « 3 | Resistance cf Nosema apis to Direct _ Digestive Tract of Aduit Bees. . . . _4 Sunlight so. we ew te ww. OF Cause of Nosema-Disease . . . . . 7 | Period Nosema apis Remains Virulent . A Three-Year Study of Nosema Infec- — Infectiousness of Brood-Combs from tioninan Apiary. . . . . .. - Nosema-Infected Colonies . . . .-: Symptoms of Noseima-Disease. . . . Resistance of Nosema apis to Carbolic Metkods Employed in Experimental SS GACH 6) ape ain er) on er eee ee ren es Pe EON a St onan hee ee est Effect of Drugs on Nosema-Disease. . Effect of Nosema Infection on the Colony Modes of Transmission of Nosema-Dis- and on the Apiary. . ..... WEBS er coe 6 RL a aH ve Resistance of Nosema apisto Heating . Diagnosis of Nosema-Disease. . . Resistance of Nosema apis te Drying . Prognosis in Nosema-Disease. . . Resistance of Nosema apis to Permenta- Summary and Conclusions. . . . MNO Aer le ede cy ere eo he alae Literature Citedis oo. Geet eet | WASHINGTON GOVERNMENT PRINTING OFFICE 1919 - rive ¢ | ae Per peatye . 1 et eae aS er pe ae 8 werk a AN ee ie UNITED STATES DEPARTMENT OF AGRICULTURE Contribution from the Bureau of Entomology L. O. HOWARD, Chief Washington, D. C. PROFESSIONAL PAPER June 12, 1919 NOSEMA-DISEASE. By G. F. WHITE, Specialist in Insect Diseases. CONTENTS. Page. Page. HT MOGUCHONE eseoo tae wcneoh wee eee el oe. 1 | Resistance of Nosema apis to putrefaction... 35 INaimevondisea semen aa nace eas sire a 3 | Resistance of Nosema apis to direct sunlight. . 37 Digestive tract of adult bees................- 4 | Period Nosema apis remains virulent........ 39 Cause of Nosema-disease.................---- 7 | Infectiousness of brood-combs from Nosema- A three-year study of Nosema infection in an Imfectedicolonie se ey anu auedgnnya |i sees Un 43 PIMA Sse SSea eet NSA Aa Gee AG iol Sie peices 13 | Resistance of Nosema apis to carbolic acid... - 44 Symptoms of Nosema-disease...............- 21 | Effect of drugs on Nosema-disease-........-- 44 Methods employed in experimental studies. - 22 | Modes of transmission of Nosema-disease.... - 46 Effect of Nosema infection on the colony and Diagnosis of Nosema-disease................. 48 OMCNEA DIARY A ee eae cee eee se IS 23 | Prognosisin Nosema-disease..-............-- 53 Resistance of Nosema apis to heating..--...- 29 | Summary and conclusions................--- 56 Resistance of Nosema apis to drying.......-- Sie eelGi tera buUrerei Ged aya cy ape eee ee 58 Resistance of Nosema apis to fermentation. .. 33 INTRODUCTION. Nosema-disease is an infectious disease of adult honeybees. It causes the death of many individual bees, tending thereby to weaken the colonies.infected. Many colonies die of the disease, but the per- centage of deaths is comparatively small and entire apiaries are rarely, if ever, destroyed by it. It is not to be considered, therefore, as a particularly serious disorder. This is shown by the results recorded throughout the present paper. It is to be thought of rather as a disease the losses from which are less to the infected apiary than the losses from either of the foulbroods, although greater than those 103789°—19—Bull. 780 —1 2 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. from sacbrood. ‘The disease is one, however, of considerable eco- nomic importance. The fact that Nosema-disease is not a new disease deserves em- phasis. The knowledge of the disease and its name only are of recent origin. Nosema-disease, like the brood diseases, has probably existed among bees longer than history records the keeping of bees by man. Since the disease is not a new one, fear regarding additional losses from it would not be justified. On the other hand, as we know of the disorder, we may entertain the hope that the losses due to it may now be lessened. Until 1909 the existence of Nosema infection among bees was not generally known to beekeepers, although it had been studied some- what by Dénhoff (1857) about a half century earlier. Zander began his studies a decade ago and since the appearance of his first paper (1909) a number of investigators have made studies on the disorder. In the papers which have been written concerning the infection, widely differing views regarding certain points have been expressed. To discuss these different views would be to go beyond the scope of the present bulletin. The writer began the study of Nosema infection in 1910 following the demonstration by him that the disorder exists in the United States. In pursuing these investigations the object has been not to devise a treatment for the disease, but rather to ascertain such facts concerning the disorder that the beekeepers might be able to devise methods for its treatment with the assurance that they would be not only efficient but also economical. While there is yet much to be learned about the disease, this object has been fairly well attained. Relations which the results obtained bear to practical apiculture should be borne in mind, therefore, in reading the paper. During the studies the effect of the disease on colonies and on aplaries, the transmission of the disease, the resistance of the infecting germ to heat, drying, sunlight, fermentation, putrefaction, and dis- infectants, and the effect of drugs on the disease are among the problems which have been considered. An earlier paper (White, 1914) refers briefly to the nature of the results obtained from these studies. The present bulletin gives all the results obtained from them which are believed to be of direct practical value to the beekeeper or otherwise of particular interest to him. The nature of the bulletin is similar, therefore, to the one on sacbrood (White, 1917) recently published.t The semitechnical points which could not well be omitted are briefly explained in the sacbrood paper. Unless the reader is familiar with the nature of such investigations, the sacbrood bulletin will probably be found helpful in following the present one, NOSEMA-DISEASE. 3 NAME OF DISEASE. About 60 years ago Dénhoff (1857, March) discovered small oval bodies upon examining microscopically the stomachs from adult bees which he supposed had died of exposure. He sent some of the bees to Leuckart, who after an examination of them expressed the belief that the oval bodies were the spores of a fungus (‘‘Pilz’’). The dis- order was referred to by Dénhoff (1857, August) by the term “ Pilz- sucht” (fungous disease). These observations apparently had been practically forgotten at the time Zander (1909) reported his studies on a disease of adult bees in which he found small oval bodies in the walls of stomachs taken from affected bees. These were in fact the parasites that cause the disease. To the germ Zander (1909) gave the name Nosema apis and for the disease he (1911) used the name ‘‘ Nosema- seuche.”’ The disorder studied by Dénhoff and the one studied by Zander are almost without question one and the same condition. It will be noted that each of these men in referring to the disorder used a term containing a reference to the parasite considered by each, respectively, as being its cause. The term ‘‘ Nosema-disease,”’ + which the writer (1914) has suggested as the common name? for the disease, is not a new one, it will be observed, but simply an English translation of the term ‘‘Nosema-seuche’”’ used by Zander. In Switzerland ‘“‘Nosemakrankheit’’ (Nosema-disease) (Nussbau- mer, 1912; Angst, 1913) is the term commonly used in referring to the disease. In Denmark Bahr (1915) used the term ‘‘Nosema- sygdommen”’ (Nosema-disease). The name “‘Nosema-disease’”’ possesses certain features which com- mend it: (1) It is definite, as it can refer only to the disease caused by Nosema apis; (2) it suggests the nature of the disease by referring to its cause; (3) it is readily understood; and (4) it is not long. Care should be observed that Nosema-disease is not confused with dysentery. Leuckart (1857, March) early raised the question regard- ing its relation to dysentery. The question was soon afterwards 1Jt will be observed that there are two parts to the name and that the name of the disorder is not “Nosema,’? but ‘‘Nosema-disease.”? It is suggested, therefore, that the name be written, for the present at least, as a compound word. By so doing the difficulty which has been experienced by some will be avoided. 2 While working on a disorder which had received the common name ‘‘Isle of Wight disease,’? Fantham and Porter (1911), in England, encountered a protozoan parasite belonging to the group Microsporidia which they identified as being Nosema apis. In selecting a technical name for the disorder caused by the parasite they chose the term “‘ Microsporidiosis,”’ derived, as will be observed, from the group name Micro- sporidia, under which the parasite is classified. The name is, therefore, an appropriate one. The term has received some criticism on account of its length and possibly on account of its not being readily understood. As the parasite is now believed to belong to the genus ‘‘ Nosema,’’ the writer begs to suggest that as a technical name for the disorder the term ‘‘nosemosis’’ would have some arguments in its favor, This is not to be interpreted as proposing a substitute for the earlier term “‘Microsporidiosis.”” It is meant, rather, as an explanation of it. 4 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. taken up by Brotbeck (1857). Zander (1909) in his first paper re- ferred to Nosema infection as a (malignant) dysentery. Other discus- sions have appeared from time to time in regard to such relationship (Maassen and Nithack, 1910; Beuhne, 1911; Maassen, 1911). In fact the two disorders are very different and should be con- | sidered, for the present at least, as having no direct relation to each other. As both conditions are widely distributed and occur most frequently in the spring of the year, it is to be expected that not infrequently both of them may be encountered together in the same colony. Efforts have been made to determine the name by which Nosema- disease has been known to beekeepers in the past. In these studies it was found (p. 16) that the highest percentage of Nosema-infected bees occurred in weak colonies. Consequently in asking beekeepers for samples bees from weak colonies were requested. In response to the request made approximately 150 samples were received. Fully half of these contained Nosema apis. Nine representative beekeepers located in different sections of the country that sent Nosema-infected bees were asked concerning the name by which the colonies showing the weakened condition were known. Three replied spring dwindling; two, not spring dwindluig; two, weak colonies; one, bad queen; and one, ‘‘Don’t know.” None suggested paralysis and none dysentery. In reply to requests for bees from colonies showing spring dwindling 38 samples were received from 14 beekeepers located in different sections of the country. Out of the 38 samples 15 upon examination revealed the presence of Nosema apis. From these 15 samples 314 bees were examined, of which 70 were found to be Nosema-infected. Samples have been received from five beekeepers who diagnosed the condition in the colonies from which the bees were taken as paralysis. Nosema apis was not found in any of them. The facts indicate, it would seem, that beekeepers had not learned to recognize the disease produced by Nosema apis by any one name. DIGESTIVE TRACT OF ADULT BEES. In Nosema infection the parasite Nosema apis enters, infects, and leaves the bee by way of the digestive tract. It is well, therefore, to know something of the location, arrangement, appearance, and structure of the organs of the alimentary canal of the healthy adult bee in order that the disease when encountered may be recognized and more fully understood. The following description is an abbreviation of a general survey of the alimentary tract by Snodgrass (1910). The part of the alimentary canal (fig. 1) immediately following the mouth forms an enlargement called the pharynx (Phy). Succeeding this is the esophagus (CZ), NOSEMA-DISEASE. 5 a slender tube traversing the entire thorax. In the anterior part of the abdomen the cesophagus expands into a large thin-walled sac which is known as the honey stomach (HS); next is the short neck- like portion, the proventriculus (Pvent); then comes the large U- shaped portion, the stomach or ventriculus (Vent), an organ with thick walls and many annular constrictions. Following the stomach is the short, narrow and coiled, small in- testine (SJ/nt) having a circle of about one hundred long, greatly coiled, blind, thread- like tubes opening into its anterior end. These tubes are the Malpighian tubules (Mal). Following the small intestine is the large intestine or rec- tum (Rect). When bees have been con- fined for some time this latter portion of the canal is found dis- tended with material to be voided. Since the stomach is always invaded by the parasite in Nosema- disease, and the Mal- pighian tubules occa- sionally are, a further description of the structure of these or- gans seems warranted. The stomach (fig. 1, Vent) is a relatively Fig. 1.—Alimentary canal of worker bee: Pharynx (Phy), oesophagus : (@), honey stomach (HS), proventriculus (Pvent), stomach or thick-walled 0 s S out ventriculus (Vent), small intestine (S/nt), and large intestine or lying U-shaped within rectum (Rect), rectal glands (RGI), Malpighian tubules (Mal), the abdomen When Salivary glands of head (2Gl) and thorax (3Gl), and pharyngeal : glands (1G1) arealsoshown. (Snodgrass.) iy i removed and straight- ened it is seen to be in general cylindrical but somewhat spindle-shaped inform. (Pl.I.)} Circular constrictions present give to it asegmented appearance. The number and distinctness of these transverse mark- ings vary somewhat. The size of the organ and its color vary also. The color varies within wide limits, being usually some shade of 6 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. brown. It may be quite light, approaching a yellow, or it may be dark, approaching the red observed in the flesh of the ox. Stomachs of the lighter shades especially are translucent. : The rather thick walls of the stomach (fig. 2) consist of an inner epithelial and an outer muscular portion. Between these is the ass SEEN SEA NTI) SI ne = yO as Of an a Bee AZ EFRON» In I> © < Ss ais ZA3 Se w= ab bee g TMcl; «eqs EAS ecu 2 Zs 2 SFE i eg LENS) Ray nO yn My r bane d ei mara «fjrteeantin OOO EA) CaN Sear K { DML Ah eS 0) { Ne 4 Y BY) OS &é ind & Ale ox Ep eee) > Ges Ee 2 oar =e, a | : Se) ee, heya Boy: 2a aS = aS ws R= 7 € ye ue ot Ler ak Fic. 2.—Microscopic anatomy of alimentary canal of worker bee: A, cross Section of stomach showing peritrophic membranes (Pmb); B, wall of stomach, more highly magnified, showing epithelial layer (Epth), basement membrane (BM), and muscular portion; C, section of Malpighian tubule showing epithelium ( Epth) and basement membrane (BM); D, crosssection of smallintestine. This portion of the canal, the rectum, and the esophagus havea heavily chitinizedintima. (Snodgrass.) basement membrane. Both surfaces of the epithelial layer are irregular. This consists of epithelial cells (Zpth) varying in size and outline. Closely associated with the outer surface of the epithelial layer is the basement membrane (BM). In connection with its inner surface is the more or less indefinite intima (Jnt) which possibly NOSEMA-DISEASE. q bears some relation to the peritrophic membranes (Pmb). Outside the basement membrane is the muscular portion of the stomach wall consisting of three (White, 1918) muscular layers (Pl. II, D; and PI. III, L). The outer and inner ones are made up of longitudinal and the middle one of circular fibers (fig. 3). Each layer is made up of a single layer of branched fibers. Digestion and absorption, comparable to some extent to those obtaining in the human stomach, are functions which have been attributed to the stomach of the bee. The Malpighian tubules (fig. 2, G Mal) empty into the alimentary tract at or very near the juncture of the stomach and small intestine. Microscopically their structure is seen to consist of a single layer of Fig. 3.—Longitudinal section of stomach of honeybee showing infection with Nosema apis: ep, Epithelial portion, containing the spores of the parasite stained black. (The younger parasites, not differentiated so easily by staining, are not shown; they are found toward the base of the cells reaching the basement membrane (BM), but do not extend beyondit. Younger spores sometimes show an unstained area at one end and occasionally at both ends.). m, muscular portion of stomach wall showing an outer and an inner longitudinal muscular layer and a middle circular one. (Author’s illustration.) epithelial cells (fig. 2, C, Epth) and a basement membrane (B M), but no pronounced intima. The function attributed to these tubules is one comparable in a measure to that of the kidneys of the vertebrates. CAUSE OF NOSEMA-DISEASE. THE EXCITING CAUSE. On December 4, 1856, Dénhoff (1857, August) inoculated a colony of bees with the oval bodies he had found in the stomachs of adult bees. The inoculation was made by feeding the colony the crushed stomachs of the infected bees in a honey suspension diluted with water. Upon examining stomachs from adult bees taken from the inoculated colony in eight days following the inoculation no spores were observed. In 11 days, however, they were found to be teeming with the parasites. A second colony was then similarly fed on Decem- @ 8 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. ber 16. On the twenty-ninth of the same month all of the bees examined from the colony were found to be infected. The results of these experiments strongly indicated that the disorder in which the oval bodies were found was an infectious one and that the bodies © were parasites which bore a causal relation to the disease. Other studies made by Dénhoff (1857, September) indicated that the parasite was quite prevalent in Germany but that there were colonies apparently free from infection. ; About 50 years later Zander (1909) inoculated colonies experimen- tally by feeding material containing the oval bodies he had encoun- tered in his studies. In bees from the colonies inoculated he demon- strated that the oval bodies were in the walls of the stomach. This fact showed still more conclusively that there was an infectious disease of adult bees in which the oval bodies were parasites bearing a causal relationship to the disease. The oval bodies studied by Zander and those studied by Dénhoff in all probability are the same. To Zander, however, is due the credit for having determined their true nature. Together with Déflein he (1909) classified the germ as a protozoan (a one-celled animal parasite) belonging to the group Microsporidia and to the genus Nosema. Zander gave the name Nosema apis to the species he found in the honeybee. The parasite Nosema apis grows and multiplies for the most part in the epithelium of the stomach (fig. 3; Pls. IZ and ID) of the adult bee. Occasionally, but rarely, it is found within the epithelial cells of the Malpighian tubules (Pls. II and III). When Nosema apis is encountered in making an examination for the parasite it is the spore form (fig. 4; Pl. III, G, H) that is most often encountered and most readily recognized. Viewed microscopically the spore in unstained preparations is seen to be a small, refractile, more or less oval body varying somewhat in size but measuring about 2/10,000 of an inch in length and about 1/10,000 of an inch in width. Its width seems, however, to be slightly greater than one-half its length The spore is surrounded by a somewhat resistant coat which tends to maintain for it a constant form. It is not, however, a rigid structure, since, when studied in fresh preparations, it will be seen to bend to and fro as it is carried along by a current under the cover glass. The manner in which a bee becomes infected with Nosema apis is in general as follows: Spores which have left the body of an infected bee with the excrement are ingested by the healthy adult bee. The environment within the stomach of the bee is favorable for the 1 Measurements were made of spores in smears stained with iron hematoxylin and of others in prepara- tions made by an India-ink method. In making the latter preparations thin smears of the spore containing material were made and allowed to dry, and over these smears a thin film of undiluted India ink was spread, The average length of the spores measured in the stained preparations was 4.15 » and the average breadth 2.06 u; the average length in the India ink preparations was 4.46 u and the average breadth 2.44 u. & (*[CULsT10 ) "41 SUT 1d ouo Oj UB} JovUOT poliod & JOy po ut AJOATPOdSoI Yoro (9g) pur ‘(g¢) ‘(F) puv ‘potsod JNSMO, & IOF po Ul oUO (g) ‘poJIOJUT-vUIESO NY AT} TO euo (Zz) ‘youurojs AY[Coy @ (TL) FUSI 07 YJo[ WI “WMOYS ov Os[B YovuIo}s AoUOY OY ‘OOWw4SUT OU TI ‘PUB ‘OUI4SOJUT[[VUIS OY} “OUTISOJUT OBIV] OY} “WoWOpg’ oY} Jo dry ouL "NOILVNINWX> YOS GHAOWSY SAAQ YANYOM AO SHOVWOLS 9 a 4 € G 0) : =] ed = ro) cc op P<@ ye [o) ra] Qa oO ja) ”) =) roy [0 0) mB iS oa Bul. 780, U. S. Dept. of Agriculture. PLATE II. PHOTOGRAPHS OF SECTIONS OF THE STOMACH OF THE HONEYBEE AS SEEN THROUGH THE MISCROSCOPE. A, entire cross section of stomach (queen) and Malpighian tubules, showing infection of these organs with Nosema apis; B, a portion of A more highly magnified; C, a small portion of a longitudinal section of a stomach from a healthy bee; D, similar to C, but from a Nosema-infected bee; £, infected epithelium highly magnified, the disease as seen in America; fF’, similar to E, but from a preparation made by Zander in Germany. (Original.) Bul. 780, U. S. Dept. of Agriculture. PLATE III. af = = ang MES a vir A : team . wy FURTHER STUDIES ON NOSEMA APIS AS REVEALED BY THE MICROSCOPE. G, Nosema apis as seen in a stained smear preparation; H, a stained smear preparation showing within the groups how closely the cells are packed with parasites (note the nucleus of an epithelial cell below and to the right of the center); J, smear showing young forms (note the paired appearance); J, por- tions of epithelial cells are shed into the lumen of the stomach, carrying with them the contained parasites, accounting for the groups in this photograph; K, cross sections of Malpighian tubules highly magnified (the epithelial cells of the one to the left are not infected, some of those of the one above contain parasites, while all of those of the one to the right are heavily infected); L, tangen- tial section of stomach wall showing the three muscular layers, the fiber of all of them being branched and striated. The inner and outer layers are made up of longitudinal fibers while the middle one consists of circular ones. (Original.) NOSEMA-DISEASE. 9 growth and multiplication of the parasite. The digestive fluids are believed to assist in removing the spore coat. The liberated young parasite finds its way to the walls of the stomach and invades the epithelial cells. Within this epithelial tissue it grows and multiplies with great rapidity, giving rise finally to numerous spores. The cells of the epithelium at times seem to become virtually filled with the _ parasites (fig. 3; Pls. Il and III). That portion of an epithelial cell that is normally shed into the lumen of the stomach in case of infection bears with it many spores. These are liberated gradually from the fragments, become mixed with the partially digested food of the stomach, and are carried onward first into the small and then into the large intestine and finally pass out of the alimentary tract with the excrement. Other bees ingesting these spores become infected. This in brief is the life cycle! through which the parasite passes. ; a0 @ Nosema apis reaches the tissues of O10 0 O 2 ae: Bizser the bee by way of the alimentary 06 e CES eeG tract. In infecting the stomach the Oo Oe a parasite reaches the basement mem- |O_ o ue Die brane but does not penetrate it (Pls. Co DO & Oe II and III). The muscular part of = Goa 0 me O we the organ is therefore uninvolved (fig. 3). Likewise when the infec- tion is found in the Malpighian tubules the soe does not proceed Fig. 4.—Spores of Nosema apisas seen in a fresh beyond the basement membrane _ preparation, indicating their general oval form. (Pls. II and III). Furthermore the (nsinal.) germ does not infect (fig. 1) the pharynx (Phy), the cesophagus (@), the honey sac (ZS), the proventriculus (Pvent), thesmall intestine, or the large intestine (fect)—organs which possess a pronounced chitin- ized intima. Infection with the parasite seems, therefore, to be con- fined to the epithelium of the stomach and of the Malpighian tubules. So far the writer has not encountered the germ in the blood, muscu- lature, or any of the other tissues of the body. _ Nosema apis has not been cultivated in pure cultures by artificial methods. The nature of the organism makes the accomplishment of such a task at the present time especially difficult. Direct proof ob- tained by the inoculation of bees with cultures of the parasite has not, therefore, been obtained. Fortunately such direct proof is not 0 OQ 1 Fantham and Porter (1911 and 1912) encountered a parasite in bees taken from colonies affected with Isle of Wight disease which they have identified as Nosema apis. Their studies on the morphology of the parasite are interesting. The morphology of Nosema apis and of Nosema bombycis are apparently quite similar and studies made by Stempell (1909) on the latter parasite may be referred to with profit in studying Nosema apis. 103789—19—Bull. 780 2 10 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. always necessary to establish the causal relationship between the germ and the disease. Because of the absence of any of the higher animal parasites and of fungi in bees suffering from Nosema-disease these groups of para- sites naturally can be eliminated as possible causal factors. Malden (1912, 1913) studied the bacteriology of Nosema-infected bees. He found that the number of bacteria in the diseased bees was much greater than in normal ones, the proportion being as 12 to 1. He found, however, no evidence of a direct etiological relation existing between these bacteria and the disease. Whether they play a secondary role is a question which admits of much discussion but one which is somewhat foreign to the present paper. Some preliminary experiments were made by the writer in regard to the possibility of the presence of a filtrable virus in Nosema- disease. The results obtained indicate that no such virus is present. By thus eliminating, at least tentatively, the higher animal para- sites, the fungi, the bacteria, and the filtrable viruses—groups of parasites which cause diseases in animals—there remains another group, the protozoa. Of this group there is only one species, Nosema apis, that is constantly present in Nosema-disease. Other protozoa are occasionally encountered in adult bees, but when found are present usually in small numbers only. The conclusion is naturally reached, therefore, that Nosema apis is the cause of Nosema-disease. Such a conclusion is in harmony with views generally accepted at the present time in regard to proof necessary to establish the causal re- lation of such a germ to the disease. PREDISPOSING CAUSES. AGE. Experimental inoculations have shown that in general adult bees of all ages are susceptible to Nosema infection. In nature it is found that the youngest bees are always free from infection and that the old shiny bees usually are. The absence of Nosema apis in the younger ones may be attributed simply to the fact that they have not yet been infected through the taking of food containing the germ. In the case of the shiny bees it seems probable that they have escaped infection, although it is possible that some of them might have been infected at one time and later recovered. The brood does not seem to be at all susceptible to infection with Nosema apis. In heavily infected colonies the larve and pupe appar- ently remain healthy. In these studies larve were inoculated more or less directly by means of a pipette and examinations * were made daily following the inoculation. The spores were found mixed with 1 The examinations were made through fixing and sectioning inoculated larve, 4 NOSEMA-DISEASE. il the food within the stomach for from 1 to 3 days after the inoculation, but there was no evidence that the parasite had increased in numbers or that it had invaded the tissues. 4 SEX. Nosema infection is encountered most frequently in workers, al- though drones and queens are susceptible. In nature it is not un- usual to find from 10 to 20 per cent of the workers of diseased colonies infected. Frequently a much higher percentage is encountered. In no instance has the writer found Nosema infection in drones taken from colonies in which the disease occurred in nature. In a few in- stances only were the queens that were examined from such colonies found to be infected. As a result of artificial inoculation practically 100 per cent of the workers of the experimental colony become infected. If drones are present a very large percentage of them also become infected. Queens in experimental colonies may or may not be found infected. To obtain data relative to queens a number of inoculations were made. Table I summarizes the experiments together with the results obtained. TaBLE I.—Nosema infection in queens in experimental colonies. Period before | Workers examina-| infected. tion. Date of inoculation. Results of inoculation. Maree Ors eee eet he tens tue kA Cee EEL Moher 8 100 | Queen not infected. July 12, ORB cas oc ae Aaah See RIS GE re ete ino 13 100 Do. DD SABO SEU OH Ei 0 & CBS eT a ee ena 16 100 Do, TEES She TUN ENS eS a Cos ah a gn PR gn ep 19 100 Do. Wc inioreten ee: Bieta AN er ee 22 40 Do. OCS HOS he ae Bean SS ees Se OS ate Ce 23 50 Do, Oct. 2; HOT Paes pee Re ae PEE 48 100 Do. SOEOUOSS SOS SE Sa OS eS Eo a Ee Sar eh aren 53 100 Do. Feb. rs Oise eee AGEL MIS LC Hes: icy Fae PBs Seg yee. 2a Queen Nosema infected Senile wlOlmeemm esta Sse 49 100 Do, INOW 20 SIGN 2R Ve eee ee Fo 3S Se EB 48 100 Do Oct. 29, 1912 SECO SO OD Oe es CRIS Cet aE a Ree RCS er anon 53 100 Do Aug. 6, MIG ete seeds EMIT ce 162 100 Do It will be seen from the foregoing table that out of the 13 experi- mental colonies 9 of the queens upon examination were found to be free from infection while the other 5 were infected. Infection in the queen occurs less frequently, apparently, when the inoculations are made in the spring and summer than when made in the autumn or winter. Queens in colonies inoculated and kept at room tempera- ture were found infected in some instances and not in others although practically 100 per cent of the workers in all of them became in- fected. 12 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. RACE. In experiments recorded in the present paper the bees used have been largely hybrids, being for the most part grade Italians. Two each of tested Carniolans and Caucasians and a few common blacks have been among the colonies used. The bees were found to be sus- ceptible to Nosema infection in all instances. It is not unlikely that future studies will show a difference among the races as to their relative immunity to the disease, but sufficient data are yet wanting to justify a definite statement in regard to the point. CLIMATE. Nosema infection has been reported from Australia (Price and Beuhne, 1910), Brazil (Zander, 1911), Canada (White, 1914), Eng- land (Fantham and Porter, 1911), Germany (Zander, 1909), and Switzerland (Nussbaumer, 1912). Studies have not yet been made in Denmark on the disease (Bahr, 1916). The writer (1914) has found it in samples of bees received from 27 different States of the United States. Out of 120 samples examined 40 contained the parasite. Samples showing infection were recetved from the coast plains and mountains of the East, from the plains of the Mississippi Valley, from the plateaus and plains of the West, and from the South and the North. The infection was found in bees received from Florida and southern California, but in 15 samples received from Texas it was not found. The data thus far obtained indicate that less infection occurs in the southern portion of the United States than farther north. Whether it is found in the Tropics or in the coldest climate in which bees are kept is not yet known. Laidlow (1911) reports that heavier infection was encountered in some parts of Australia than in others. Nussbaumer (1912) reported the infection from 14 of the cantons of Switzerland. The practical import of these observations in connection with the climate, to the beekeepers of the United States at least, is that the presence of the disease in a region can not be attributed entirely to the climatic conditions present. It is possible, however, that the climate of a particular region may affect somewhat the occurrence and the course of the disease in that locality. SEASON. Infection in apiaries has been found to occur at all seasons of the year, but is greatest during the spring. In the studies reported in the present paper (p. 20) infection was greatest in April and May, being greater in these months than in March. Very little of a definite character is known of the infection as it occurs in nature during the winter. Experimentally it has been found that bees are susceptible to infection with Nosema apis at all seasons of the year. , | NOSEMA-DISEASE. 13 FOOD. As is pointed out under the heading ‘‘Climate,’’ Nosema-disease occurs in a wide range of localities. The food and water obtained in these localities naturally differ as to quality and quantity. Infection is found in colonies having an abundance of stores and in others having a scarcity. The disease is produced readily by ex‘perimental inoculations in colonies with much and in colonies with little stores. From these observations the conclusion seems to be justified that the réle played by food in the causation of Nosema-disease is slight, if indeed it contributes at all appreciably to it. A THREE-YEAR STUDY OF NOSEMA INFECTION IN AN APIARY. The presence of Nosema infection among bees in the apiary of the Bureau of Entomology was discovered in May, 1910 (White, 1914) In April, 1912, a more or less systematic study was begun on the prev- alence and persistence of the infection in the apiary and was con- tinued until June, 1915, As the apiary was being used for other purposes than these studies, it was not possible to follow all of the colonies throughout this entire period. In Table II are summarized observations made during the first year of the study. a0) ef |°"" "| d6 TS Lae a) dM | MJs0T|°°" 7} OT)" "} AA} aT peaks || aes CD) ee a POSE IPS ll (N)-|/2029) sete SO eae ot LT | LT | O€ | 8Z | TZ | 8T | OT *I0q WN | ano00q *1090190 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. €16r 14 Fire io() a | eet | cee ee eee OO ae > | aoe dOE|R ame PETE HCY Nya | nce pesca (ere aaa Coat (cog OM fiat Vette Sra aa Hey are Gia je bos oods foie Sucre () ial meer |eoee, lec ale se 20 |°°° "°°" *] a Sd LC OCT Ea BO CCH Hp e0 Cn bo eat) Vy (YR cil een Lae CL OVo-| Pat Gynct | Stee] Weems! Uiteiecsoh| jamin eae hICY 0 Ye] ht esl = Sa fags Rae C0 )0) bec cell (a dQ ‘TON queun | *4u980 -tod | 19g “Xi "roquieydeg |-ysnsny ZI6L 8% “Ayn “oun & | 8% | £ | OL of ‘ON AtOlOD ‘hundo up ur uoroafur puasoyy fo ipnys p ur ‘aaasnjowa ‘er6r ‘youopy 07 ‘erEr ‘udp wosf pourwjiqo synsay— JI AAV, 398 aac 2 108 = 0G en 4 ea9G Beets GG Le Elke 15 NOSEMA-DISEASE. So}COrpul acts) *POAOUIAI SVM 4T 48} ;,‘ -e 04 pesn oe soyep oyeullxoiddy s--+] oT ---+| 99 ----| 49 ----| 99 --++] 97 Cg ed i ne es Gee ed ed ee mead I Bad oe gg | 900 | -- Ses) needs, os ef | ag |" ” Terese | lea ily | Ga are SON INN tnd Gin eae Oya) |e comt C1 Ya eens ae CYA CVG Won Pe OO ile ge ha ge 6 Cy ca a: 6 20 | M]| 30 [>> 7} eG | FAG) PE poser Resch ales | eae leet |e gear = ele eae ef | of | d6 - "yeom ATOATIC[OI SBA 4191 ,.‘ AA ,, PUB ‘4ISUOI]s Ul VAINIpeuT SV IEC ,,“W,, ‘ZuoTs APAAMeToI sem ATOTOO oy 4eq} UW,, ‘peep sem ATIO[OO 04} 184} SMOYS ,,_,, 10}}9[ OY, “SOULVIJ POOIG Ct} UIOIJ Uae} O1OM Ady) YVY} ,,‘J,, pue {107eM JO AoMOY JUIAIIVO O10M Aoyy 4eY} ,, Y,, Moye} Moy A[lreurtid Uejod sutA11¥e9 919M pouluexs soeq oY} Ye} ,,“d,, ‘eATY 94} JO doue1}U OT} NOG seoq ou} SUOWIe TIO1J Maye} SVM O[dures 9} 184) ZUIMOYS «<2 yy 1040] OY} -Taye} SUM o[dures B YOY U1OIJ 90INOS oY} SojvoIpUl JoquiInu v suTAUeduI0N08 10340] OUT, “SoTep 1OexKe oy} JO S Vp OM} UIGIIM o1e LOG} OINIVSy °9[qe} oY} 97eTAIIq “@PeUl 919M SHOT{VUIUILX OY} YOIYM WO oyep oyeultxoidde oy} Aq pepeoy SMuINIOo UI UaAIs o1e SyNsei OYJ, “WuIN[Od sIy OY Ul Jequinu $71 Aq TMOYS SI Uoxe} SUM o[dures oy} YOIyM wo] AUO[OD ey, “pep1000d SI peutuIexd Soeq OT Jo e[dures Yous UI puNoJ seeq po_daJUI-eUIESON Jo JoquInU oY ,.— JI 219D,,, 40f wouvunjiday ee ee ee ee PB. Vise acl oc cae o oe Wag) |°°77]---5 MA | 48 | af |o77 W|°"*"| a0tl"*7" HN valerate ral sce los oot pOGee lets a/E eal Nwode Se cae | Sa te goa te |e Ee COTW OIG on alts dZ | dQ |" PETE |e SS al a Slane il gars (a | ear, dg Halecaillelctercite CZ eatira lieve ces |Pealeltens sie mee Silt a aera Neues e Wal pete mace | Gol bees |e CUD Bt dses eit} Seacaage ey | Re So 1 aa Gut |OOSS eee ee ¢ Tre COA sa) Of mali oe oats Ties ee Pee tie ame a (stoph|tereave che OI a Pane call eusvere' lteter aielltererere (OS feria Bait rae) Sia | ae lar ay Bes mated (ner ema Bo ee aed | oe ee 290 | eB fT] og | | i “aN (ated Nea ee OL | aQl ine ctln as 0G SE SAIL ABS ieee eO) [eats Stems oc aG | yf | 00 Fons epi hela Meare te |e ae Boas OW Weat6a le = SS cfs 7g amen | cea ipa Ns | gana OL Hea |e nape enn) oO Be ie eee | (eee oat BQ) | es | ae Eo Gea a ae a [se way Femme opal | Oper | Wes et | acca a0 |] 4%)" |} 7 | of Gie'ctallleislaveUstarere lloiatete leteie s|| ie aiate aa loess ecicve a lee 16 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. From Table II it will be noted that in April there were 24 colonies in the apiary. Out of 240 bees examined from them during the month, 72 (30 per cent) were Nosema infected. The number of bees out of each sample of 10 was found to vary from 0 to 10. During May, out of 410 bees examined 96 (23 per cent) were found to be Nosema infected. During June, out of 130 bees taken from 13 colonies 19 (15 per cent) were found to be Nosema infected. During July, out of 130 bees examined 21 (16 per cent) were found infected. During September, out of 170 bees examined 14 (8 per cent) proved to be Nosema infected. Out of a total of 1,140 bees examined in 1912, from April to Sep- tember, inclusive, 236 (20 per cent) Nosema infected bees were found. The number of infected bees found in the different colonies varied from 5 to 100 per cent.? Five of the 24 colonies died. These were dead by the end of May. It was found that the number of infected bees present in them varied from 50 to 100 per cent. The number of infected bees in the colo- nies that lived varied from 5 to 33 per cent. All of the colonies that died were weak when first examined in the spring and dwindled until they disappeared. The colonies that lived gained in strength and behaved as healthy ones. The colonies that died had sufficient stores. The queen in each of them was apparently in good condition and brood was being reared. At times, indeed, the brood was in excess of the amount that could properly be cared for by the diminishing number of bees present. These and other facts which have been observed justify the belief that the immediate cause of death in each of the five colo- nies that died was the Nosema infection that was present. These colonies, therefore, may be said to have died of Nosema-disease. The number of colonies in the spring was increased during the bee season through swarming and by division. In September an experiment was begun in the apiary in which 10 colonies were inoculated with Nosema apis. The results of these inoculations will be referred to later under experiment No. 1 (p. 23). Examinations were made in 1913 for the prevalence and persist- ence of Nosema infection in the apiary studied in 1912. Naturally the colonies present were not altogether the same as those of the previous year. Some of them had been lost and some represented the increase. The results obtained are summarized in Table III. 1 Fractions are omitted in this paper, as a rule. 2 As the younger bees and the older ones were avoided in selecting samples for examination, the results recorded in this paper show a higher percentage of Nosema-infected bees in the colonies than actually existed. - NOSEMA-DISEASE. 17 Taste III.—Results obtained in 1913 from a study of Nosema infection in an apiary.' Sep- Ex- | March. | May.| June. July. Ex. | August. ces October. peri- eri- er. Per Colony No. | ment ent | cent. No. 1. No. 2. IP PAS | PA) 3. | 18. | 14. | 16. | 19. | 22. OEE Zounee2oceultloon| alos ipoce 3 ee vet | eg eae an ba Mra (ONS Fo ses eee Fe cg Fs Peni (rte aed as Me el fon NS Ue Ee Se Pata (eed Teron be ie rae seat en a Pies hel Nes pt aS (| (ea (Von laneene le |. OD [hee se | Sees teases Qos RX eeer 2 Lies TLS ROS er Es a eee eee Oers | ae Sia bevel ODM eee ees a ee 8 1p 5 es rel we | em cvereval epee Oeni aero: 2 153) Uys igh 2 1 ge L a | ol | a ll) | 2P 10 Boe Sires OS Aes eer eee On Seeaee Qe 1 Lhe se |e eed ae 3 1 PAS i See De PASe Ris AY as ala ASP | ae Lee Ue [ogee ne [oaths ta] ee 60 AG ie 5 Ba eR ed | eee el ene Wee ee oee SIO Sic [sees lt Sie | raecey fp al ees oe Oerle2en|ess ee 5 SOM eee emcee -| le SE pees ees topeeseal Pepe ce rues eh ewes Le Doo" Bie hese seal priya ees NP cles | ee alr of tes en tS nae | ge a |pae DS or lt esse (ee ese el hnean| Lee 2h Ka or (earn | penn SMe | ars Sa ey VF | Pare |e ot Peete BO ee ees AS | RO eh eae. Op | 3h |. ive) | esse anaes Mer erates OeaitOene esse 16 BOE serene eee Hetiers se. Sool MoO oale Sole ase XE a es ea ot tes 6P Oe 20 CBee ei a ed te Lies | ears Op ENC) Ny | eeant | Paros | cr aes mee le 1p 6 AOR ROSS 5 oa 7 | Rye ae Set [ol HUNG) Wee ee EF Oe epee Eee Oe ale as |e ek ee er |iseer QDiA Paste | ee. 8 DOD e stesewteclloaee oo Myer || OS rs sce ete ls OS fereeen pu Ley reeves. cee 1 Bile (OYO 1) |e DA Shs bes Aare Nid ES tas 2 aa 7 Gt, 5 SOS ere res Gp |sOeriees: P33 Sel ae al aeeh [cst reg yds vl 1 eR Pa re aes ace) en fa [nee ec Fee (ee all eee fe ee es Hose AES eal SeSU Se ns ee nee Sees [feces Fetal RRS aga [ati Earce (Oral {eae oe OOM Peas 30 (tg Se ee eee (Ne ae ees Der FORD | ORF et tet pa ae aoa ea ACIS MOK) | Feiee see el eee 6 (GO nc SSR a oe | Eine ea igen Eee eer Ds Ree SILO [fers ar cer ID lA | aoe PADI is Ae ee XS 12 Oe oS Scersees Sabo! fee DOA MO TSS SSO Se alias PAN Pe ALOIS OKI Eos aie Ss te es eae 4 GO GE CSE CRS Sale Sane a heise ey | Pa ODE Siow [ees erase E | Oe 3P | Oe 10 CQ ees See BER eels Ogres | ene | (0) 20351 eg EC eee ae incre) ies At B15) | epee eee 5 LOSER 10 | Oe | 2e 4e OP | Oe By li 3 LE | (eae i BBCI Fg es a ae a en a Py 11 UES EACLE ee Samet ie ee Oem eee a ae le | 0e a ie yar ae OSs O eri reser eee 10 CO ee ee es) eae (0) 4 | eee ee tees (OY CIE IE es |S Sh i ee el eee ee ODE Aes 0 DS epccie ae SaeH EBC ae eee Cer esses (OY NY HN ee a te | a Bs Sa eer ote Bead Pea 0 CO eta FN pe ek corel Sy, ahh a beslaaey Oe |. OSS Seed |S arerel eevee NOs Saae (iscod tacos 6 Siete Soke it, se. Cera teen [ica Stee Res el ok Pia ul ana rea ea on ACO ag Es Shae ce ee Da ea aed (STS (SE taeda Ni Be Oer ee temo: (zee lees Oe 3 5 GOS GORE ee Se lca Ie [prs a Op |..- CO al ie (8 ee le | Oe PA sot Sat a ee ssc 6 Dea SBOE BOCES SEGA eG see | Sea] RORRer tein OpE Gee (OP) Ft he Bets 339 1s oa|lesce 10 Osses sn sh Gsscad Soe S| Sie le St RS Ee FRE Te ae) (en ee Steed Nec eet Speier Renan LOK | eee iy Lrg Vs SET Da tg Gos ACESS EEL OS Ss Are eV ee (aU tyes | tel ocean em eee Oe feo aes wees 20 QasogSotG CoE OSE BGA es ered eae ol eens De ee FR aa oe tl De [oD | eee eee 25 1 Where the number of bees examined is small, the rate indicating the percentage frequently is not given. Explanation for Table III.—The method of recording results is the same asin Table II. Colonies exam- ined in 1913 that were examined in 1912 bear the same numbersin Table III asin Table II. Colonies repre- senting the increase in the spring are designated by the letters ‘‘a’’ to ‘‘e,”’ inclusive. Colonies in experi- ment No. 1 areindicated by numbers; colonies in experiment No. 2, by capital letters. From Table III it will be observed that in March, 1913, out of 270 bees examined from the 25 colonies then in the apiary 28 (10 per cent) were found to be Nosema infected. During June bees were examined from 21 colonies, and out of 220 bees 8 (4 per cent) were found to be infected. During July 21 colonies were examined and out of 260 bees 23 (9 per cent) were found to be infected. During August bees from 18 colonies were examined and out of 240 bees 11 (5 per cent) were found to be infected. During September, out of 170 bees from 17 colonies 43 (25 per cent) were found to be infected. During October bees were examined from 6 colonies only, and out of 60 bees 1 (2 per cent) was found to be infected. Out of a total of 1,270 bees examined during the year 1913, 121 (10 per cent) were infected, being less than the percentage found in 1912, which was 20 per cent. The spring infection was very much less in 1913 than in 1912. 103789°—19—Bull. 780-3 18 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. The percentage of infected bees found during the spring and sum- mer remained quite constant, increasing unexpectedly in September. The reason for the increase can not be assigned at present. Out of the 25 colonies in the apiary in March, 1913, 1 (No. 12) died. As this colony contained a high percentage of Nosema-infected bees, and as it dwindled until it disappeared, it may be assumed that Nosema-disease was the immediate cause of its death. As in the preceding year all of the colonies that lived behaved much as do uninfected ones. In this year another experiment was begun in the apiary. This one is described as experiment No. 2 (p. 25). Studies similar to these made in 1912 and 1913 were continued throughout 1914 and until June, 1915. While in the main the col- onies of the apiary were those of the previous years, naturally there had been some changes. The results obtained are summarized in Table IV. TaBLE IV.—Results obtained from May, 1914, to June, 1915, from a study of Nosema infection in an apiary. 1914 | 1915 | Sep- | No- Colony No. May. June. July.| tem- | vem- March.| April. | May. ber. ber DNS AS 254 Sula Salt AF) tet Or 23 5 2 SD 126 Aight Py kei /Qe|ip}.....0p|1p|0r]5>|0r| Op! Or] or] oe Ge.0e.1-De. Ord eah Ta Sa ll te a | Qe | Qp 1p | 1h | Op OP Op | OP | Oe FY =m (net Ie ar eer S| | see eatin |i «Wt (Fe OM eee. ots eee a | ee ee 3P | 2h} Ip 1p OP OR Qe teks) mn - |leme is Le pS ciate a Same pe hand es eed a Antares atone uta le | Ip | Ip | Qh | Op Ope Op | Oe QOS acca eee ee eee | ee ee jae Lee Pewee a2 on Oe | 1p 1p | 2h | Ob 1p Op | OP | Oe Oe Oe | Oe} OP | Ip | Ob Tes eer eee Oe | OP Ip} 2h | Op }. Op QD QR ee ae ore | a red Te ere el eee ee [is cee Ferny ecg le | 2p Op {| 2h | 1h]. 19 fp OPy | OPppel@ Qet|> cod eee g: fo] eka le eal leprae Rae ee ey ee Oe | OP | 2p 1h | Oe Op Op | OP | Oe Oe Sa sidyom pa itis a5 gee NaS ere" Bea 1 | OP Op | 4h | Oh | Ip | Ip Opy|Op ye . alesis] a eb ed eee le fesse OMe prc ere ae oe | OP | 2e Qe | 1h | Op | 2P | Op Gp Op [eas ence ses | este meee ea ae ahi | baa sa See ge ees i} je} Ip Op | 1h | Oh Op Op |rOP ee lee seo chee Sette ss leg eealeeret it aes 11 ee ie eae ee WA 2e | 2e | 2e | Oe | le Op OD | OR Ye cal Ges ccle eee eee ee ee ee ees ts eee aie oe le | 1p 2p | 2h | Qh Op OP: Peet fe Feces eee heb e a ee | ee ee ee ele 1 Ges Qe | 2p Qp | 1h! Oh | Ip} Op De see wae aise deta Qe. (Ferre cea peas 1 7/3 enna ae ee 2 2e | 4p ip | 4h | 0h | Ip | Op ApaQpale. | eres a! le | Oe Ip | Qh AR ee oe ee aa eur Oe | Ip Ip | 3h | 1h} 3p} Ip OD hee lee eee seer lees Ges hee ase e eee Tit ee Ba Re a ahs Oe | OP 1p | 2h | 2h | 2p | Op OPwy eek els. re) s. £2 ae BS pe eee Sows Oe | Op | Ob Qh | ep Ape heme Seva le | Ip 2p | 1h | 1h | 3p | Op ey ea ea eet ee Oe | Op | Op Qh TAL eS ae ee Bk eH) le | 3p Ope Qh ||pAby yt h oc Opa ee pee oe iets So setae Qe] 1p] Op] Ob AS Ae a hs Oe | 2p 74 RS 3k | Ta JT bn Pe Pe ee Be ooe ood petedoos ees (Se Sos SE ae AAD tak! Oe | 2p 2p | 1h | Op | ip | ip OP Qerltes=se Syuleass. 4s Oe] Oe sce SRY IE ed el ee 3 (eee ae 2p | 3h | Ob | 3p | ip Op seers ee eae Gilet ocak WYSE) fal Ges ea Che | (ee ee cage SOF 4e | 4p |__..| 3p | 2h | Ip | 3P | Op Opi Opis: sipeseee Myeres bee 6e | Qe | Ip Oe SOG G Cee Seed lan De PD ey EO ee cee OW HSE Ly) Pea SR, Paral RSs ie =" (epee RS Oe | Oe | Oe Oe SEGR 220 Ree ne so Oe | 2p |__.-] Op | 35 | OP | Op | Op OP Hees les alee ee Wil; Saher e Neale: cree S34 feo ot 5s aie eh 7e | 5e|....] 4p | 6h | Ip | 1p | 3p OPS eee da] PRS ee 0e"|o 22 jkece lees LOR ODIEE EET PREECE Ped 3p ig2s/ ip yes-2pObri2pr | itp Opa erent et ee yi lseeede Oe | 9e}9r]} le 7a Misael see: Oe} le | 3P | Oe | Explanation of Table IV.—The colonies reported in Table IV for 1914 do not bear the same numbers that were assigned to them for 1913 in Table III except those designated by numbers in quotation marks. The first 9 colonies aes in the table for 1915 bear the same numbers they did in 1914. The identity of col- onies numbered by letters “‘s”’ to ‘‘z,”’ inclusive, had been lost through changes made in the apiary. oe | uw cad NOSEMA-DISEASE. 19 Table IV shows that out of 1,050 bees examined during May, 1914, 166 (16 per cent) were Nosema infected. In June, out of 700 bees examined 60 (9 per cent) were found infected. In July, out of 240 bees examined 2 (1 per cent) were infected. In September, 220 bees were examined and no Nosema-infected . one was found. In November, 60 bees were examined and none was found infected. Out of 2,270 bees examined during the summer of 1914, 218 (10 per cent) were found infected. It will be noted that during the early months of the active bee season of 1914 there was a higher percentage of Nosema-infected bees in the apiary than during a similar period of 1913. Two colonies were so weak in May that they were disposed of. In one of these at least (No. 13) the weakness was most probably due to Nosema infection. During the first week in July the apiary was moved to a new loca- tion. It is interesting to note that the amount of Nosema infection after removal was reduced to practically nothing. This is not defi- nitely accounted for by the results obtained by these investigations. ! Examinations were made of a portion of the apiary in 1915. In March, out of 50 bees taken from 5 colonies, 6 (12 per cent) were found to be Nosema infected. In April, out of 280 bees taken from 17 colonies 24 (9 per cent) were found infected. In May, out of 200 bees taken from 10 colonies 16 (8 per cent) were infected. Out of 530 bees examined from the apiary during the spring of 1915, 46 (9 per cent) infected ones were found. Among the colonies that were examined during the spring of 1915 two (Nos. 8 and 18) died by the end of April. Both of these contained a rather high percentage of Nosema-infected bees. Two others containing an equal or greater number of infected bees lived throughout May and had recovered apparently by June. In case of these 4 colonies it can properly be said that the two colonies that died died of Nosema disease, whereas the two that lived recovered from it. In Table V is given a summary of the results obtained in the study of the apiary from April, 1912, to June, 1915. 1 That the immediate environment of the apiary determines, to some extent, the presence or absence of Nosema-disease and its transmission seems quite likely. In searching for the cause for such a difference the water supply of the bees, if near by, must not be overlooked (p. 46). In this connection, it may be pointed out that in the experimental apiary (Pl. IV) Nosema infection at no time exceeded 1 per cent, excepting naturally in inoculated colonies, although the source from which these colonies were obtained had been largely the apiary which, it will be seen from Tables II and III, showed Nosema infection in from 10to 20 percentofthebees. Herethere wasnoslowly moving body of water used by the bees as the source of their water supply. 20 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. TaBLeE V.—Summary of results from a study of Nosema infection in an apiary. March. April. May. June July sear . {so Ae =| Sed 2 | 3 s/s] 1313 33 18] | 8/3 Year. A lo Alo a © & 2 Eel wc Be [Ova oloe beod OFS. el oleae 323i 32/2 ~ a = 3 rat Sleie(S5 18/8] 8 1/8 |e] 2 ale) eiala 212/41 8/1/2/5| 81/8 ]21 8 /8le/8/8le OH SilusxidyootS lave oa so 8 4-65 1..34 2s AIA eats em Pa tae ey ee | ey ee ea; SS ge ee Se oe (CRE [erst 240 | 72| 30] 410] 96] 23] 130/19] 15| 130] 21| 16 19192 2M BESS AV 270)! B84) 10-45. 27 aE 50| 7 2 8 260 9 ROTA ere ef SS eet ho Ete ce eat oul ee lee | AND 1,050 | 166} 16 | 700 | 60 AAO} QA ok TIS TG eS ae eae eae 50} 6| 12} 280 | 24 200° | AG ITs PELLLLCIE ipa tbe ie pa be Ba Potales eee es 320 | 34 | 11 | 520 | 96 | 18 |1,910 | 285 | 17 |1,050 a7 | 8 | 630 | 46| 7 August. September. October. , 3 Bale 31 31% 313 g | > 3 Year a & q 3 g Ss 5 g H |e | .. | 8 | 2 | .. (ee peal ombesaie ae 3 » 3 3 ~~ 3 » 7] = Sg (igide Sai gih So] Sieg) duis oleae Bt dcile lb Selenite ala Bocledee ce a nee ® S By) >) & 5 ro) 5 B iS) iS io) ea) Z a ea) a a aa) Z a & & | A 1912 Tee ea oe G07 §4.).22.28 170} 14 i} | Neral 4 Ne Be a APs 1,140 | 236 | 20 TRE {evan 5 rel cd afin 240} il BI 701" 4341> 25 168 1 ooREe ,270 | 228] 10 TEV Sie eS Co ES SRS el geen | Bt PARA Hea B= Yel eee MeN een alte fetes cI ae OI ue 2,210 | 121} 10 1 AY ts Sale delete Mie lahe | eerie benched ae tecan thee ila h(t ea Lk Me (EN Te Ce 530] 46] 9 From Table V it will be observed that the number of infected bees found at different periods of the year varied considerably. April and May furnished the highest percentage, being 18 and 17 per cent respectively. In March, June, July, August, and Sep- tember the number of Nosema-infected bees among those examined was 11, 8, 7, 5, and 10 per cent respectively. Out of 5,150 bees taken from the apiary from April, 1912, to June, 1915, and exam- ined, 631 (12 per cent) were Nosema infected. Laidlow (1911) reports that out of somewhat more than 1,500 bees received from various parts of Australia, 17 per cent were found to be Nosema infected. From an examination of the foregoing tables it is seen that Nosema infection was found to be present in practically every colony of the apiary. Had further examinations been made of the few colonies in which Nosema apis was not found, one could well expect, from what is known of the disease, that these, too, would have revealed the presence of the infection. It is seen also that the infection persisted throughout all seasons of the year, and that it was heaviest 1 While this three-year study was being made the apiary served for other work. It is likely that the attending manipulations were accompanied from time to time by a certain amount of robbing. From the nature of the disease, however, it is not belioved that this fact affected materially the results obtained. PLATE IV. s ("NOILVYLSNT1] YOHLNY) “GALONGNOD 3yY4aM GI6I 40 YAWWNS AHL ONIYNG Savi] SLNSAWIYSdXy SSVSSIG-VWASON SHL HOIHM NI AYVIdW TIWLINAWIYSdxX3 NOSEMA-DISEASE. 2 in the spring. Some colonies died as a result of the disease, while a greater number recovered from the infection, increased in strength, and behaved in all respects as healthy colonies. The total of all the spring counts, during the period from 1912 to 1915, inclusive, of the apiary under study, was 94 colonies. , Out of this number at least 12 (13 per cent) died more or less directly as the result of Nosema disease. An equal or greater loss to the apiary than this colony loss probably is the aggregate loss in strength sustained by colonies weakened by the infection but which recover from the disease. Naturally it is particularly unfortunate from an economic point of view that the highest percentage of infected bees, and consequently the heaviest loss in strength sustained by colonies from Nosema infection, occurs in the spring. Beuhne (1916) has reported investigations made on colonies from his own apiary which are similar in nature to the foregoing studies. The results he obtained indicate that Nosema infection in Australia is similar to the infection as it occurs in America. SYMPTOMS OF NOSEMA-DISEASE. Nosema-disease presents only a few symptoms. In describing them the colony rather than individual bees should be considered as the unit, since it is the colony as a whole that is of primary interest to beekeepers. Weakness is a colony symptom which invariably will be manifest if a sufficiently large percentage of the bees of the colony are Nosema in- fected and if the infection persists for a sufficient period. When only a small percentage of the bees are infected the weakness resulting may never be apparent. ‘The loss in strength may be gradual or sudden. The behavior of a Nosema-infected colony is similar to that of a healthy one. The stores are sufficient. The queen does her work well. As the colony dwindles the queen usually is among the last handful of bees. The brood in general is normal in appearance, but in colonies weakened by the disease not infrequently it is seemingly in excess of the amount that can be properly cared for by the adult bees present. In Nosema-disease the workers especially suffer from the infection. An infected bee manifests no outward symptoms of the disease when seen among the other bees of the colony and it performs functions similar to those performed by healthy ones. - When the stomach of an infected bee is removed it may show marked changes which are characteristic of Nosema-disease. The organ pales as a result of infection. The brownish yellow or dark reddish hue of the normal stomach is gradually lost as the disease advances. The organ (PI. I) is often increased in size, the circular 22, BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. constrictions are less marked, and the transparency is diminished. In late stages of the disease, however, the stomach approaches the normal in size and the constrictions are again well marked. The organ is then white and opaque and the tissues are friable and easily crushed. When crushed the mass presents a milky appearance. Upon microscopic examination Nosema apis is found in very large numbers in.the crushed tissues. The presence. of the parasite is almost invariably recognized by its spore form. The presence of Nosema-infected bees in a colony is the one constant colony symptom of the disease. METHODS EMPLOYED IN EXPERIMENTAL STUDIES. As Nosema apis has not been grown in the laboratory by artificial methods, in carrying out these investigations it has been necessary to inoculate a large number of colonies of bees. The use of a few bees in cages was found to = be inadequate for experimental purposes. A 4 to 6 frame nu- cleus in a 10-frame hive body (fig. 5) serves well the purposes of an experimental colony. The experimental apiary (PI. IV), consisting usually of about 50 colonies, was the same one that was used in the sacbrood studies. During the bee sea- son the colonies were inocu- lated and kept in the apiary in the open under conditions PS Bopanna nie, tev fos Homer similar to. those occurring in entrance nearly closed with wire cloth, and the open- nature. Precautions similar ing on the side of the hive body occupied by the to those observed in the sac- Shae seme brood studies were followed in the present studies. During the winter colonies to be inoculated were removed to and kept in the laboratory. The top of the hive body was screened and the bees given free opportunity for flight through a hole in the window. The manner of obtaining the parasite Nosema apis from diseased bees for use in the inoculations is described under ‘‘ Diagnosis” (p. 48). The stomachs of from 5 to 10 infected bees are amply sufh- cient for each inoculation. After their removal from the bees they are crushed, suspended in sirup, and fed to a colony free or practically free from Nosema infection. The methods throughout are similar €5 NOSEMA-DISEASE. 23 in general to those employed and described in the sacbrood studies. It should be stated in addition that no watering place for the bees was provided at the time of these experiments and none with sluggish water was near by. The results of an experiment usually can be determined during the second week following the inoculation. The diagnosis 1s made as described later in the present paper (p. 48). Usually one examina- tion of 10 bees is sufficient for the determination of results. It is advisable sometimes, however, to make others. As a rule experimental colonies inoculated during the summer recover from the infection and can be used again. The period which must elapse, however, before they can be used for a second experiment varies. An examination of the field bees should show no infection among them or only an occasional infected bee before another inocu- lation is made. A colony used in the laboratory is good for one inoculation only if by it Nosema-disease is produced. Should the results be negative following an inoculation, however, the colony may be used in a subsequent experiment. It is not necessary to disinfect a hive which has housed a Nosema- infected colony. The experimental colony may or may not have a queen. If one is present no concern need be felt in regard to whether or not she is infected. No fear need be entertained that drones from infected colonies in the apiary will transmit the infection to the experimental colony. EFFECT OF NOSEMA INFECTION ON THE COLONY AND ON THE APIARY. To determine the effect which Nosema infection in a colony pro- duces on the colony, and on the apiary of which the colony is a part, is a problem in the study of Nosema disease which is of vital interest to the beekeeper. Some observations have been made bearing directly upon this point. EXPERIMENT NO. 1. On September 13, 14, 15, and 18 ten colonies were fed a sirup suspension of the crushed intestines of Nosema-infected bees. These colonies (Table II, Nos. 6a, 12, 25, 35, 41, 55, 65, 66,67, and 70) were in the apiary mentioned on page 13. Those selected for inoculation were not especially strong, the bees being easily accommodated on six or seven brood frames and being about an average for the apiary. Examinations show that about 10 per cent of the pollen-carrying bees of these colonies were Nosema infected at the time of the inoculation. The 32 uninoculated colonies in the apiary served as checks. It will be seen from Table II that after inoculation 50 to 100 per cent of the pollen-carrying bees in the inoculated colonies were Nosema infected. Out of the 100 bees examined from these colonies 24 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. during the period from October 5 to October 16, inclusive, 132 © (70 per cent) were found infected. These colonies when examined on October 28 showed that, out of 100 bees examined, 78 (78 per cent) were infected. It will be noted, therefore, that following the feeding inoculations there was a marked increase in the percentage of Nosema-infected bees in each of the 10 colonies inoculated. In the experiment sufficient precautions were not taken to prevent robbing at the time the inoculations were made. This resulted in an increase also of Nosema-infected bees in some of the uninoculated colonies (Table II) of the apiary—the checks. The increase in the number of infected bees disappeared more readily from the check colonies, however, than from the moculated ones, suggesting that probably a comparatively small amount of the contaminated sirup was obtained by the robbing bees. On December 17, out of 100 bees taken from the 10 inoculated colonies 49 (49 per cent) were found to be Nosema infected, showing that the percentage of infected bees had decreased. From comparison of the moculated colonies in October and in December, it was observed that their strength had decreased and that they were relatively weaker than the checks. Toward the last of December one of the 10 inoculated colonies died. During the last week of the year the remaining 9 were packed for the winter as were also the check colonies. Some of the weaker check colonies were united, giving them a slight advantage in strength over the inoculated ones. The winter 1912-13 being a favorable one for bees, the winter losses were low. In March, 1913, when the first examination of the apiary was made, 4 of the 10 colonies that had been inoculated had died out. Four of the six inoculated colonies that were still alive showed 4, 6, 2, and 2 Nosema-infected bees respectively in samples of 10 bees examined. Neither of the other two inoculated ones showed at the time the presence of Nosema infection. All of the 19 uninoculated colonies packed in December were alive in March, 1913. Out of 190 bees caught from the entrance of these check colonies during March only 6 (3 per cent) were Nosema infected. By the middle of May another of the moculated colonies (No. 12) had died, making 5inall. Of the 10 colonies that had been inoculated in September, 1912, the 5 that lived through the winter and the following spring continued to gain in strength during the summer of 1913 and by autumn were apparently as strong and healthy as any in the apiary. By experiment No. 1 it is shown that when colonies are inoculated with Nosema apis a high percentage of adult bees of each colony becomes Nosema infected—results which confirm similar ones previously obtained by Donhoff (1857), Zander (1909), and others. — i fu NOSEMA-DISEASE. DNs Such results, together with facts which are recorded on the foregoing pages, are sufficient to demonstrate that Nosema-disease is an infec- tious disease of adult bees. It is shown also by the results of this experiment that there is a tendency for the infected colonies to become weakened. It is further shown that when inoculated in September colonies do not die out readily as a result of the inoculation. Furthermore the results indicate that the infection is not readily transmitted from the infected to the healthy colonies of the apiary. It is further shown that colonies inoculated in September may die as a result of the infection during the winter that follows, or they may survive the winter, gain in strength during the brood-rearing season, and by the following autumn present the appearance of healthy colonies. EXPERIMENT NO. 2. Beekeepers are always desirous of knowing whether combs from diseased colonies can be used in healthy ones without causing a spread of the infection. To obtain data relative to this point experi- ment No. 2 was begun in July, 1913 (Table III). In the experiment, brood combs from diseased colonies were inserted into colonies comparatively free from Nosema infection and kept under observa- tion for more than a year afterwards. Combs from the 5 colonies of experiment No. 2, which died during the winter and spring following their inoculation with Nosema apis in September, 1912, were inserted into the 6 colonies (Nos. 36, 50, 61, 66, 68, and 82, numbered by capital letters ‘‘A” te ‘‘F,”’ respectively) used in the present experiment, each colony receiving _ from two tofour combs. The colonies from which the inserted frames were obtained had been dead for from seven weeks to five months before they were given to the colonies. None of the 6 colonies were strong, the bees being easily accommodated on from four to six brood frames, a strength representing about an average for the apiary. Out of 110 bees examined from the 6 colonies of the experiment prior to the insertion of the combs 10 (9 per cent) were found to be infected; and out of 170 bees examined after they were inserted 26 (15 per cent) were found to be infected. This increase in Nosema- infected bees can not be attributed to the introduction of the combs, since a similar increase is noted in the other colonies of the apiary® serving as checks. All of the colonies of the experiment lived through the winter and spring except one (No. 61). This colony was dead when examined in May, 1914. Dead bees taken from the bottom board of the hive showed a high percentage of Nosema-infected bees. The 5 colonies that survived gained in strength, behaved as healthy colonies, and contained a percentage of Nosema-infected bees approximating that 113789°—19—Bull. 730-4 26 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. of the other colonies of the apiary (Table IV, colonies numbered 36, 50, 66, 68, and 82). The results obtained indicate, therefore, that by inserting combs from Nosema-infected colonies, as was done in experiment No. 2, the infection is not transmitted appreciably. An explanation for this is easily seen from results recorded throughout the present paper. Further experiments on the point are summarized in Table XXVI. EXPERIMENT NO. 3. In this experiment 7 colonies free from Nosema infection were inoculated by feeding them sirup to which Nosema apis had been added. The bees from which the parasites were obtained for this experiment were from various sources (p. 12). They had been dead and drying in the laboratory at room temperature for at least three months. All of the 7 colonies received the first inoculation feeding on October 8. On each succeeding day for four days the feeding was repeated. Each of the inoculated colonies of the experiment was examined from time to time, but no Nosema-infected bees were found. The final examination in connection with this experiment was made on October 28. Out of 70 bees examined from the 7 colonies only one Nosema-infected bee was found. The infection in this instance probably did not result from the inoculations. In this experiment it is shown that Nosema apis drying in the abdomen of bees at room temperature for three months does not produce infection when fed to healthy bees. This result suggested the interesting fact that the parasite of the bee resisted drying for a comparatively short time only (see other experiments, p. 40). EXPERIMENT NO. 4. In experiment No. 4, four of the colonies used in experiment No. 3 were inoculated on October 29, 1912, with Nosema apis taken from infected bees recently killed. Nine days after the inoculation sam- ples of bees were examined from each of the four colonies inoculated. Nosema infection was found in nearly all of the bees examined. Two weeks after inoculation 50 bees were examined from each of the 4 colonies. All of the 200 bees were found to be infected. At the end of three weeks a similar condition prevailed. On Decem- *ber 16, 48 days after inoculation, all of the 4 inoculated colonies were alive. A large number of bees were now found on the bottom board of the hive. By this time the colonies had become very much weakened. The bees were uneasy, the cluster being easily disturbed. During the following week 1 of the colonies died out completely. The remaining 3 were chloroformed. Another colony inoculated in November gave like results, and died in January, 1913. Each of the 5 colonies of the experiment were four-framed nuclei. As the inoculations were made late in the autumn there were no NOSEMA-DISEASE. 97 young bees emerging. All of the bees of the colonies were exposed, therefore, to infection by the inoculation. It is seen from this experiment that during the autumn workers infected with Nosema apis live, as a rule, for more than one month, but that most of them die during the second month after infection. These results led to the conclusion that heavy infection in a colony when no brood is being reared will destroy the colony, but that it may live for two or three months following the infection. | Although 100 per cent of the workers in each of the 5 colonies were infected, the queens from 3 of them were free from infection at the death of their respective colonies. The other 2 were found to be infected. NOSEMA INFECTION WEAKENS THE COLONY. There is good evidence at hand indicating that Nosema infection weakens the colony. The fact that the epithelial layer of the stomach is filled with parasites (fig. 3; Pls. I and II) at once suggests that the functions of the organ, digestion and absorption at least, would be decidedly impaired thereby. Likewise, when the Malpighian tubules are invaded (Pls. II and III), it is to be expected that the bee suffers impaired functions. The abnormal condition argues strongly that such a bee is less efficient as a member of a colony than an uninfected one. Further evidence that infection weakens a colony is seen in the fact that in nature the heaviest infection is encountered in the weaker colonies. Still further evidence is seen in the results obtained in experiments Nos. 1, 3, and 4, just recited, and from inoculations made in 1913, 1914, 1915, and 1916, now to be referred to. On June 4, 1913, a colony was inoculated by feeding it Nosema apis in a sirup suspension. On the 13th it was found to be heavily infected. At this time the inoculation was repeated. When exam- ined on July 12 the colony had not increased in strength as the unin- oculated ones had done. On this date it was reinoculated. By the middle of August it had not gained in strength. No reason could be assigned for the failure of the colony to become strong other than the presence in it of Nosema infection resulting from the inoculation. On June 9, 1914, a colony was inoculated with Nosema apis. On the 22d it was found to be heavily infected. On July 8 it was reinoculated, at which time it was weaker than the check colonies. On August 6 the colony was still relatively weak and was reinocu- lated. On the 17th it was still weak. The failure on the part. of the colony to become stronger is attributed to the Nosema infection. On August 6, 1914, a colony was inoculated with Nosema apis. It became heavily infected and on September 9 it seemed to, be weakened as a result of the infection. It was reinoculated on this date. On December 1 it was found to be heavily infected and on January 15, 1915, it was dead. 28 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. On March 30, 1915,,a colony was inoculated, resulting in heavy infection with Nosema apis. On June 17 the inoculation was repeated. Later a swarm was cast. Inoculations were repeated on July 3, 9, 17, 24, 31, and August 13. The colony became much weakened and later in the autumn died. . Beginning on March 22, 1916, a colony was inoculated at irregular intervals thereafter until September. Much brood was being reared in it throughout the season, but its strength in September was about equal to its strength in March. The evidence obtained, it will be observed, is sufficient to justify the conclusion that the Nosema infection in a colony tends to weaken it. The weakness resulting does not occur immediately following the infection, however. During the active brood-rearing season the young bees reared may exceed the loss from disease and the colony will then actually gain in strength. On comparison of colonies that are infected with those that are not, however, it will be seen that the infected ones are the weaker. An experimental colony receiving repeated inoculations increases in strength, as a rule, during the first two weeks following the initial feeding through the emergence of young bees, but comparatively little, if any, after the first month. The question arises as to whether the weakness is the result of infection in workers, drones, or the queen, or in a combination of these different members of the colony. Brood apparently does not become affected with Nosema apis (p. 10). The weakness in a colony can not be attributed, therefore, to infection of the brood. Infection among drones is rare (p. 11). Loss in strength, therefore, could not be expected to result from infection in the drones. The queen in an infected colony is more often free from the infection than not (p. 11). Weakness from Nosema infection can result, therefore,when the queen is free from infection. By elimination in this way the conclusion is reached that the weakness produced by Nosema infection in a colony is due primarily to infection among the adult workers. Other observations made point to the same conclusion. Workers taken from colonies in which Nosema infection had reached a rather advanced stage were confined in the McIndoo wire-screen cages * and kept at room temperature. Healthy ones were similarly caged and kept under observation. The relative length of time that the infected and uninfected bees lived under these conditions was noted. On December 8, 1914, in each of four cages were placed from 15 to 30 bees taken from colonies heavily infected with Nosema apis. By the end of one week, out of 79 bees confined 62 (78 per cent) had died. On the same date bees from another infected colony were simi- larly confined. At the end of a week out of 119 bees confined 108 (91 per cent) had died. On December 15, 1914, the experiments were 1 Small triangular cages devised by McIndoo (1917, p. 4) in his studies on the honeybee, Star & NOSEMA-DISEASE. 29 repeated. Out of 138 bees in one set of four cages 125 (91 per cent) were dead at the end of one week. In the other set of four cages out of 136 bees confined 98 (72 per cent) were dead at the end of a week. On December 8 a check experiment was begun. In each of two cages bees taken from healthy colonies were confined and kept at room temperature. At the end of one week out of 59 bees confined 5 (8 per cent) had died. Out of a total of 472 diseased bees confined 393 (83 per cent) were dead at the end of one week, while out of a total of 59 healthy bees kept under similar conditions only 5 (8 per cent) were dead at the end of aweek. Although such experiments are subject to great variation and should be repeated many times for definite results, yet the difference between 83 per cent of loss in the case of infected bees and 8 per cent of loss in the case of healthy ones is sufficiently great to justify the conclusion that the heavily infected bees under the con- ditions of the experiment possessed less endurance than the healthy ones. These results indicate that weakness in a colony may result directly from infection among the workers. - Throughout the investigations which have been made on the dis- ease, therefore, evidence has been obtained indicating that weakness results not from the infection of the queen, drones, or brood, but of the workers. RESISTANCE OF NOSEMA APIS TO HEATING. NOSEMA APIS SUSPENDED IN WATER. Preliminary results indicating the minimum amount of heating that is necessary to destroy Nosema apis were given in an earlier paper (White, 1914). Other experiments have been performed. In conducting the experiments a suspension was made in water of the crushed stomachs and intestines of Nosema-infected bees. This suspension was distributed in test tubes in such a dilution that the amount in each tube contained the infective material of from 5 to 10 bees. The tubes were stoppered and heated at different degrees of temperature by immersing them in water. Colonies free from infec- tion were inoculated with the heated material and the results noted. Table VI summarizes some of the experiments made with the results obtained. 30 BULLETIN 780, U..S. DEPARTMENT OF AGRICULTURE. Taste V1.—Experiments to determine the heat required to destroy Nosema apis suspended in water.) Temperature Period of Date of inoculation. employed. heating, Results of inoculations. =C: 2708 Minutes. ars 918 ic 2 Oey 2 a hg & i ee 50 122 20 | Nosema infection produced. Jan. 8, 1913 ihe Suck as ees cere mem eee Eee ae 55 131 10 Do. Oct. 4 1A TG Ea eae | ae eas ek eae tl ee 56 133 10 Do Oct. 15, OTS Se Oo ae eee ce ne 56 133 10 Do. May 21, BOT se Pee Ey Seley pe 90 58 136 10 Do. Oct. 15, LOTS See eae Cee ACR ae ee eae ce ty eee 57 135 10 | No anfection produced. Feb. 8, 1913 DES ee eye ae ee eas Stee A Se 58 136 10 Oct. 4 TR TTS Sas, ig a 8 aes Sy thats i Bn Oe es ee 58 136 10 De Aug. 28, SLO ae eee hl Se le ie ak ay paar: | 59 138 10 Do. Nov. “ie RET EP ae Re AI re ed eae eS 60 140 =~ 10 Do. Nov. 20, IQUQ Si eed oe = A gi aie ys he ee a 60 140 10 Do. May 21, "1915 Be eee At, Cee aE een Bates 60 140 10 Do. Aug. 28, ON eee Oey nh ete tp due bes 61 142 10 Do. Nov. 12, LIE) 1 RS ei SIRS Pe At NOR RNS 65 149 10 Do. Jan, 8, DIS hy a chee eh ene a 65 149 10 Do. Oct. 29, LAUD a aay eee his Id os ra ee Be 80 176 20 Do Nov. 12, QI Dek eaten le sr epee es eg FS 100 212 5 Do 1 In omitting fractions of degrees the nearest whole number is given. From Table VI it will be observed that Nosema apis in a water sus- pension was destroyed in 10 minutes at a temperature somewhere between 135° F. (57° C.) and 138° F. (59° C.). NOSEMA APIS SUSPENDED IN HONEY. From preliminary experiments it was learned that the amount of heating that is required to destroy Nosema apis suspended in glycerin is approximately equal to that required to destroy it when suspended in water. It was anticipated, therefore, that the minimum amount of heating that would destroy the germ suspended in honey would approximate that required to destroy it when suspended in water. Experiments were made to determine the approximate thermal death point of Nosema apis when it is suspended in honey. In making the experiments the technique used was similar in the main to that of the preceding group of experiments wherein suspensions in water were heated. In Table VII are summarized the experiments performed, together with the results obtained. TaBLe VII.—Experiments to determine the heat required to destroy Nosema apis suspended in honey. Fl | Temperature Period of Date of inoculation. Results of inoculations. employed. heating. =C: oes Minutes. ALIS 25 AOlorenee nen ens fee ee eee ences 58 136 10 | Nosema infection produced. Aug. 27, MOLD eb ee Sas ck secetewe cee 59 138 10 Do. June 9, MOUR SS cere ee eck Bie Pe tee 59 138 10 | No infection produced. May 21, LOL ee eee ee ae ae Be a 60 140 10 Do. June 8, 1915 AGS aj Se MAES fay SE eR Ss RR 61 142 10 Do. Aug. 28, OTS ee en ee As RE Eee 61 142 10 Do. June 9, 1915 Pi a irate ak Scene ha pe Fe kN ee re ta 62 144 10 Do. June 8, IO oe ee dks ee ata ee a 63 145 10 Do. Maya OTb tart ee a ol este ee 65 149 10 Do. May 21, 1} | PR ae SR ee ee et ed Sk 70 158 10 Do. Rint y AAU ol eee ee abet 80 176 10 Do. NOSEMA-DISEASE. 81 Table VII shows that Nosema apis in a honey suspension was destroyed by heating for 10 minutes at a temperature between 136° F. (58° C.) and 140° F. (60° C.), the death point being about 138° F. (597°C:). RESISTANCE OF NOSEMA APIS TO DRYING. In experiments relative to the effect of drying on Nosema apis, stomachs from Nosema-infected bees were crushed, and the crushed tissues were smeared on slides to the extent of a thin layer. The slides were placed in incubator, room, outdoor, and refrigerator tem- peratures, respectively. At different intervals after the preparation of the smears an aqueous suspension was made, germs from two slides representing the material from 5 to 20 bees being used. This was added to sirup and fed to a healthy colony. Whether or not the parasite had been destroyed was determined by the presence or absence of Nosema-infection in the colony following the inoculation with the sirup. NOSEMA APIS DRYING AT INCUBATOR TEMPERATURE. In Table VIII are summarized the experiments, together with the results obtained, in which the Nosema material was allowed to dry at incubator temperature. TABLE VIII.—Resistance of Nosema apis to drying at incubator temperature. Date of inoculation. Period of drying. Results of inoculation. Months.| Days. MND A SOR OL om ee ae ccictieclas ajo dieie oaticnc ceieee cs eedsswece 0 10 | Nosema infection produced. Nalyslet loom ane Ln OR UNNLN SO a UA a 0 13 Do. Dias Bs TNC A Ie Ma 0 14 Do. Umi Os TO ST a a ee a 0 18 Do. ul ya29 1 91 Gans ee a ne eee Rd 0 15 | Noinfection produced. Sead Os ree AR ee aa ie ie seen gene ay Lae 0 21 Do. Nov. 2, TORIC OS ce, Ta a a a ee 0 30 Do. Sept. 29, NOUS Spiked aged lacie Aneta aie ei, ean an ff) 40 Do. Oct IGN OIAR ans aan eee ee a Se 0 56 Do. Dilys QeplOl neti eee een am Me ae Seki Oe Ee Tee | 2 15 Do. Jb Mee PELs SITUS a ea Nn he ee eA A eS 7 27 Do. CTU Es TT) a ee ie ek le a a NUL I 9 19 Do. From Table VIII it will be seen that Nosema apis drying at incu- bator temperature was destroyed in from 15 to 21 days, that is, re the third week. NOSEMA APIS DRYING AT ROOM TEMPERATURE. In Table IX are summarized experiments in which the drying of Nosema apis took place at room temperature. 32 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. TaBLe IX.—Resistance of Nosema apis to drying at room temperature. Date of inoculation. ee, Results of inoculation. Days. uily226 1 91G eee anceee eee Biel Re Ms et er 4 sete Stor aise 18 | Nosema infection produced. Seppe O14 Seon ease Race cet tele eat. SN ee eee 21 Do. JA TVS UU LD) CER ae Men etecs el gegen Semin 5 tik anes hee 35 Do. Oct 2, 1914 sere se4 TC Nea a Se A RGAE ae ai cd 3 Rees Sem: 42 Do. DOPE. MONO ees ee eae oe saat a be ea eon 43 Do. PALE 20, pA OG Ae. os BEAR Aye PR AN EE ARE Leena SES eee 50 Do. OCERIGS AQT 42 pay Ss 89th 0 2S i ge ane ok ee ee 56 Do. Mary 245 AQNGE ets. SSS ISS Ge TE A 60 | No infection produced. Sie} O16 GAIUS) Us fame ey ee ar. epee ol Oe Ae CNET he 21S, pine envy 1s EE Re 61 Do. ume}27) TOG Moe see LL te FP EERE DEG | GEG 95 Do. From results recorded in Table IX it will be observed that Nosema apis drying at room temperature remained virulent for from 56 to 60 days, that is, about 2 months. NOSEMA APIS DRYING AT OUTDOOR TEMPERATURE. Table X summarizes experiments in which Nosema apis was allowed to dry at outdoor temperature. TABLE X.—Resistance of Nosema apis to drying at outdoor temperature. Date of inoculation. Period of drying. Results of inoculation. Months. | Days. SOPcep hl Ole rr crete dah Oh ee sen mem Ser it iets 0 21 | Nosema infection produced. Octo MIO TAR ie ins ae Ne ca Lic eee as Rm 0 42 Do. AMIS HM ONAL a wots ene cee eens Seite Me Se See ee 0 46 Do. WCiMIG RIOT tek eee UE ay See Sua ae 0 56 Do. VUITTON eel OU Gia eyes ce eee ee res eee eaters atte eto 0 60 Do. Sep taza Gsia ee see oieveene tes Mie ere san soi mee 0 60 | No infection produced. JITLyAO POLS Esa c Nee eeee oe eee ee eee oe eee eee 0 75 Do. SUITS F271 OI Gi ahs stirs me ae te ee eee oe Stee lea oe 9 80 Do. ANT C25 FN TOY SS eer eee ares el rors sey een tas Sete ee meget IE 0 85 Do. UTR sl fe gh UG choy ete Oey Soe He Ree a ee eee eae 0 100 Do. May 20, HOV Gis Secs sete ce a tee en aetna ee oe epee ge 9 0 Do. ib Re [7a RO Ie es aa ee ey te a ne ae Cen aes a 10 10 Do. ULL yAde aL ONG ase ests yl ere ern Ne AR tetra eye 11 0 Do. The results recorded in Table X show that Nosema apis ceased to be virulent after 2 months of drying at outdoor temperature. NOSEMA APIS DRYING AT REFRIGERATOR TEMPERATURE. Table XI summarizes experiments in which Nosema apis was allowed to remain dry in the refrigerator. TaBLe XI.—Resistance of Nosema apis to drying at refrigerator temperature.} Period of Date of inoculation. drying Results of inoculation. Months. Dec. 2, 105s ee een cee Wen scat tee Ba eae aah peepee a 3 | Nosema infection produced. Vans! OG Bee eens vice salsa atseninsieek oes aeeene eben camer + Dol (Mares, UO1G otek aoe stare eel rntatetacnets Sle isiats ae orate eee ore 6 Do. TAZ 1 916: Bi le A ee ee I ek OR ee ee | 7 Do. WAT 22S OL Ge Beet a Seb Neen Re SN AI eerie eae eieree 74 | No infection produced. Madyas 1916S) ee eis cate ead cle yep eee as ewe ee outed engage 8 Do. Lute Re Gee aS aC oes SSeS ROMA koh Aan 4 AChE ee arc AR CODGAGO DAE 10 Do. 1A few times aunine. the experiments in which the refrigerator temperature was used, the ice became exhausted, allowing the temperature to approach and possibly to reach that oftheroom. This higher temperature, when present, however, at no time prevailed for more than a day. NOSEMA-DISEASE. 83 It is learned from the results recorded in Table XI that Nosema apis drying at refrigerator temperature remained virulent for seven months but that no disease was produced following inoculation with the material after seven and one-half months drying. From the results obtained in the experiments relative to the resist- ance of Nosema apis to drying, given in Tables VITI—XI, it will be observed that the period the parasite remained alive, or at least virulent, varied, depending upon the environment of the germ. ‘The shortest period for the destruction of spores was obtained under incubator conditions, while the longest period occurred under refrig- erator conditions. The death probably was not due to the drying alone but to a combination of factors of which drying was an impor- tant one. RESISTANCE OF NOSEMA APIS TO FERMENTATION. Experiments have been made to obtain data relative to the resist- ance of Nosema apis to fermentative processes. In conducting the experiments suspensions of the crushed stomachs from Nosema- infected bees were made in a 10 per cent sugar (saccharose) solution and in a 20 per cent honey solution. These solutions were distributed in test tubes. Each tube contained infectious material equal to that present in the stomachs of from 5 to 10 infected bees. To each sus- pension was added a bit of soil to inoculate it further. Suspensions were allowed to ferment at incubator, room, outdoor, and refrigerator temperatures, respectively. At intervals reckoned in days the fer- menting suspension from a single tube was transferred to about one- half pint of sugar sirup and fed to a colony free from the infection. The results were then noted. FERMENTATION AT INCUBATOR TEMPERATURE. In Table XIT are summarized some of the results that were obtained when a suspension of Nosema apis in a 20 per cent aqueous solution of honey was allowed to ferment at incubator temperature. TABLE XII.—Resistance of Nosema apis to fermentation in a honey solution. Period of Date of inoculation. fermen- Results of inoculation. tation. Days Munlive2 Ol Ol Geena ene re WO Ee SR UNS oe Sac 1 | Nosema infection produced. elyg 2ONL ONG Meee aire LRN Sa OM ue 2 Do. Diu yt 2a OIG Bree eemael a Cy es Be ee ee oe 3 | Noinfection produced. SUVA SER Omer epee ee