THE LIFE HISTORY AND COMPARATIVE INFESTATIONS

OF SPINULOSA (BURMEISTER)

ON NORMAL AND RIBOFLAVIN-DEFICIENT RATS

dissertation :

Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy In the Graduate School of The Ohio State University

3y DEFIELD TROLLINGER HOLMES,, B. Sc., M; Sc.

The Ohio State University 1958

Approved by

BriLd: Adviser Department of Zoology and Entomology ACKNOWLEDGMENTS

X would like to make special acknowledgment to my adviser, Dr. C. E. Venard, Department of Zoology and

Entomology, The Ohio State University, for his understanding encouragement and oontlnued assistance and stimulation throughout this research. Thanks are also due to Dr. D. M.

DeLong and Dr. F. W. Fisk, also of this department, for the contribution of materials when needed; to Mr. Fhelton

Simmons of the Columbus Health Department for kindly contributing rats from various areas In Columbus; to

Mr. William W. Barnes and Mr. Roger Meola for technical assistance. Finally, I wish to express sincere appreciation to my wife, Ophelia, for her patience and enoouragement throughout this research and for her aid In the preparation and typing of this material.

11 TABLE OF CONTENTS

Pag©

Introduction ...... 1

Historical Review and ...... 6

Materials and Methods ...... 14

Life History ...... 22

Incubation Period of the E g g ...... 23

First Stage Nymph ...... 26

Second Stage Nymph ...... 2 8

Third Stage Nymph ...... 30

Adult Female ...... 33

Adult Male ...... 35

Moulting ...... 37

Comparative Infestations on Normal and Riboflavin-deficient R a t s ...... 38

D i s c u s s i o n ...... 41

Summary . . 46

Literature Cited ...... 48

ill List of Figures

Figure Page

1 - Normal and Rlboflavln-deflolent R a t s ...... 15

2 - Rlboflavln-deflclent Rat (Mild Symptoms) . . . 20

3 - Egg ...... 25 4 - First Xnstar Nymph ...... 27

5 - Seoond Instar Nymph ...... 29

6 - Third Instar Nymph ...... 32

7 - Adult Female ...... 34

8 - Adult Male ...... 36

Text Illustrations

Table Page

1 - Mode of Infestation ...... 18

2 - Time Required for Hatching of E g g B ...... 24

3 - Life Cycle of Lice on Normal and Deficient R a t s ...... 31

4 - Areas of Infestation on Ho s t s ...... 39

lv INTRODUCTION

It has been noticed by various Investigators that

laboratory rats varied widely In the number of rat lloe

Polyplax snlnulosa (Burmelster) which they harbored. It

was found that the more heavily infested rats had been maintained on vitamln-deflolent diets.

Gyorgy (1938) noticed that heavy infestations of lloe

became apparent In about 20 per oent of the that were

kept for a sufficient length of time (more than eight to ten

weeks) on rlboflavln-deflolent diet. Searls and Snyder

(1939) found that rats most heavily Infested had been

maintained on a vitamin A deficient diet. Kartman (1942)

also noted that rats maintained on a vitamin A deficient

diet showed heavy Infestations, but he stated that diets

deficient In riboflavin also Increase the size of

Infestations and that no particular vitamin plays the

singular role, or even a limiting role, In determining

the resistance of rats to their lice.

The relation of host nutrition to other species of

ectoparasites has also been studied, especially by

De Melllon and co-workers (1946-47) In South Africa. These

workers reported that egg laying of Clmex leotularlus was

drastically reduced and that a large proportion of the eggs

were sterile when the bedbugs were fed on thlamln-deflcient

rats. When the tick Ornlthodorus moubata was fed on thiamin- deficient rata, there was a decrease In the rate of growth

and size of nymphs, a prolongation of the interval between

eaoh moult and an additional moult before maturity. When these two speoles of parasites were fed on rlboflavln-

deflolent rats, normal growth and reproduction took place In all cases.

An experiment with cattle lice was reported by

Matthysse (1946) In which he suggested that vitamins A and D

In the diet of the host apparently had no relation to the

economy of these parasites. Barger and Card (1943)

theorized that when fowls are either qualitatively or

quantitatively undernourished, they become more susceptible

to the attacks of lloe and mites than under conditions of

optimal nutrition. Life histories of the lice of mammals have been

determined for only a few species. Some aspects of the

biology of Fedloulus humanus have been worked out by

Nuttall (1917) and Buxton (1940).

According to Weber (1939), our main knowledge of the

biology of lice, sucking and biting, Is largely confined to

ovlposltlon, hatching, and the fact that there are three

nymphal stages which resemble the adults in habits and

general appearance. It has been ascertained that the eggs

are fastened to hair or feathers, and It has been observed

that In most Instances the eggs will not hatch If kept at a temperature much below the body temperature of the host.

2 The body of man la an exoeptlon to the rule In that the eggs are usually attached to seams of the clothing worn next to the skin. The duration of the egg-stage and pre­ adult life is known only for a few species, and it varies with the temperature of the host as well as with the species of the louse. Hopkins (1950), In a comprehensive study on the host-assoclations of the lice of mammals, suggests a general life history of the lice, biting and sucking, as follows: egg stage, one to three weeks; the nymphal life, about a week to 14 days; and the pre-ovlpoBltlon period, from one to three days, thus giving the average period from egg to egg at about three to four weeks, and the number of generations a year about 12 to 14.

Florence (1921) observed that the life history of the hog louse Haematoplnus suls was 29 to 33 days from egg to egg, which Is summarized as follows:

Time from laying to hatching of egg..13 to 15 days First moult occurred after 5 to 6 days Second moult ocourred after ...... 4 days Third moult occurred a f t e r ...... 4 to 5 days Sexual maturity occurred after ...... 3 days

There Is also little Information on the longevity of the adults, but for several species It is shown that this period Is not great. Unfortunately, much of this Information has been obtained by keeping the lice under unnatural con­ ditions and therefore Is of little value. Watts (1918) gives the duration of a generation of the hog louse Haematoplnus as 30 to 40 days, of which half represents the adult stage.

3 Orauford and Benson (1941) stated that for the cattle louse

Haematoplnus eurvsternus the maximum longevity of the adults was 16 days for the female and ten days for the male. In

Pedloulus humanus and Phthlrlua nubls the average life of the adult is about one month. According to Bacot (1917) and

Buxton (1940), In Pedloulus the difference In length of life between tbe sexeB 1b small. Matthysse (1944) found the maximum life of the female oattle-bltlng louse Damallnla bovIs to be 42 days.

The number of eggs laid has been determined for a few species and varies according to species. Lamson (1917) states that the cattle louse Haematoplnus eurvsternus lays from 35 to 50 eggs. Parthenogenesis has not been reported for most species, but Matthysse (1944) found parthenogenesis to be normal in Damallnla bovls. Hopkins (1950) states that in theory a single fertilized female could give rise to an enormous infestation within a very short period of time, but he suggests that this Is unlikely to occur. Elohler (1940) records the rise of an artificial Infestation of chewing lice from 200 to 14,000 In 80 days. However, he stated that because of biological checks such a great lnorease does not generally occur. Buxton (1940) has shown that a biological check in the case of Pedloulus humanus is the Injury to females which Is Inflicted by the males while making numerous attempts to mate. This occurs in populations where males exceed females.

4 No complete life history of the Incubation period of the egg and the longevity of the nytnpha.1 stages and the adult stage was found for Polyplax aplnulosa. Thus the purpose of this writer's Investigation was to work out the complete life history of the rat louse Polvplax aplnulosa and to oompare the degree of Infestation on rats kept on rlboflavln-deflolent diet and on normal diet.

ft HISTORICAL REVIEW AND TAXONOMY

The origin of the sucking lloe Is not very clear, but It Is generally accepted that lice are descended from

Psocid-llke ancestors, and they have In the past been placed In the superorder Psoooldes, a position which appears to express their relationship with the other better than any other which has been suggested. The relation of lice to the Psooldae was perhaps first suggested by Packard

(1887)- Kellogg (1896 and 1902) came to the same conclusion. Lice belong to the primitive hemlmetabolous

Insects, In which group the newly hatched Insects resemble the adult In most respects. They have formerly been divided into two orders: the , or ohewlng lice, and the Anoplura, or sucking lice. The ordinal name to be applied to the lice Is In dispute.

Leach (1815) gave the name Anoplura to the whole group, and

Nltzsch (1818) named the chewing lice Mallophaga, The latter, together with Ryncophthlrlna (Ferris, 1931) for the elephant-louse, has never been seriously disputed, but the discovery that the chewing and sucking lice are closely related and should be regarded as suborders has created ohaos as to the use of the name Anoplura. A few authors use this name for the whole group (applying the name

Slphunculata to the sucking lice), while the great majority continue to use It for the sucking lice alone, as was

6 universal until a few years ago. Weber (1939) Included all lice in the order Phthiraptera of the superorder

Psoooidea. Hopkins (1950) oontlnues to use Anoplura as a suborder for the sucking lice and has adopted Weber*s name

Phthiraptera for the whole order, giving the arrangement of the order as follows:

Order; Phthiraptera

Suborders: .... biting lice Isohnocera .... biting lice Rynchophthlrlna. . biting lice Anoplura .... sucking lice

The first three suborders are considered by many as super- families of the suborder Mallophaga. Dalla Torre (1908), in his synopsis of the Anoplura, recognized four families, and these have been generally recognized by others. They are the Pediculidae, for those lice with eyes and which Infest primates; the Haematomyzldae, for those with tubular heads

and occurring on elephants; the Echinophthirlldae, for the

short-splned lice of marine mammals; and the Haematoplnidae,

for the remaining blind forms that are found chiefly on

rodents and ungulates.

Ferris (1951) employs a system in whioh he uses the

order Anoplura for the sucking lice and divides it into five

families; the Echinophthirlldae, Haematoplnidae,

Hoplopleurldae, Linognathldae, and Pediculidae.

Enderleln (1904) divided the family into three sub­ families: Haematopinae, Trlohaullnae, and Euchaematopinlnae.

7 Ferris (1922) accepted these three divisions, but he reduced

the genus Trlohaulus to a synonym of Linognathus, thus

ohanging the subfamily Triehaullnae to Llnognathlnae.

The Hoplopleurldae in whloh Ferris (1951) places the

genus Polvplax is divided into four subfamilies, with the genus Polvplax in the subfamily Polyplaclnae. Thus, the

classification of Polvplax splnulosa as employed by Ferris

(1951) is as follows:

Phylum: Arthropoda Class: Hexapoda

Order: Anoplura

Family: Hoplopleurldae

Subfamily: Polyplaclnae

Genus: Polyplax

Species: splnulosa

The family Hoplopleurldae to which the rat lice belong

is by far the largest of those in the order Anoplura. In

it are included 29 genera and more than half of the

approximately 250 species of sucking lice. These lice are

confined largely to rodents, inseotlvores, and ungulates.

The genus Polyplax belongs to that group of genera

of the subfamily Polyplaclnae in which there are not more

than two transverse rows of setae to a single abdominal

setment. There are sternal and tergal plates present in

both sexes, but the antennae are different in the two sexes.

The third antenna! segment of the male is either modified

8 or Is provided with one or more short stout spines. The

Common of Rats, Polyplax splnulosa feurmelster) oocurs on all of the domestic rats and la almost worldwide In distribution. The Old World Mouse Louse, Polvplax serrata (Burmeister), has been found In the

United States only on laboratory animals. It Is a common parasite of the house mouse In Europe.

One other species of rat louse Hoplopleura oenomydla has been found commonly on domestlo rats (Ra.ttus rattus and

Rattus norveKloua) in the United States. Polvplax has been known to occur on rats for over a oentury, while the tropical rat louse Hoplopleura oenomydls (Ferris), also of the family

Hoplopleurldae, has been positively determined from this country only since 1937 and 1945 (Roudabush 1939, and Pritchard 1947).

The adults and immature stages of Polvplax splnulosa have been described and figured In detail by Cummings (1915),.

Ferris (1923), Ferris and Stojanovlch (1951), and Pratt and

Lane (1951). The adults and Immature stages of Hoplopleura oenomydls have been described and figured In detail by

Ferris (1921 and 1932), Ferris and Stojanovlch (1951), and

Pratt and Lane (1951). The adults of Polyplax splnulosa appear somewhat more slender than the adults of Hoplopleura. Specimens of

Polvplax in alcohol show more conspicuous Indentation of the lateral margins of the abdomen than do those of

9 Hoplopleura. All of the Immature stages of Hoplopleura oenomydls have a characteristically heart-shaped abdomen but laok spiracles and submedian lines of setae on the abdomen. All of the Immature stages of Polyplax have a longer, more slender abdomen with spiracles and submedian lines of setae. Seoond and third nymphs have lateral plates as well as spiracles. According to Ferris (1951) and Pratt and Karp (1953), the Important characteristics used In recognizing the stages of Polyplax splnulosa may be summarized as follows:

Sgg: The egg is about 500-525 microns long by 175-

200 microns wide, with polygonal, honeycomb pattern on the exochorlon. The cap has seven or eight air pores In the center. The base of the egg is evenly rounded.

First stage nymph: The length is about 0.4 mm. The abdomen is elongated with two submedian rows of spines dorsally but not ventrally. The spiracles are not surrounded by lateral plates. The posterior part of the abdomen has four long setae.

Seoond stage nymph: The length Is shout 0.6 mm. The abdomen is elongated with two submedian rows of spines dorsally and ventrally. The spiracles are surrounded by small lateral plates. The seventh and eighth abdominal segments have a pair of long setae on either side, the tip of the abdomen thus having eight long setae.

Third stage nymph: The length Is about 0.7“1»1 mm. The

10 abdomen ts elongated with two submedian rows of spines dorsally and ventrally. The spiracles are surrounded by small lateral plates. The sixth abdominal segment has one long and one short setae on either side, the tip of the abdomen thus having ten long setae.

Females The length is about 1.5 mm. Antennae are stout, five-segmented, the seoond segment being about as long as it

Is wide. The head Is slightly narrowed anteriorly. The sternal plate Is approximately five-sided, the anterior margin being truncated to somewhat pointed. Abdominal segments— four to six each— have two tergal and two sternal plates, the anterior plate being longer than the posterior plate, with each plate typically bearing five to seven setae. The lateral plates on segments three to six are small, triangular; the posterior margin is slightly convex with two short subequal setae; the setae on lateral plates seven and eight are very long. The posterior margin of the abdomen is slightly concave.

Male: The length is about 1.1 mm. The male Is similar

In general appearance to the female, but Its abdomen has one tergal and one sternal plate each on segments four to six.

The third antennal segment has a projecting spur. The posterior margin of the abdomen Is somewhat pointed. The male termlna.Ha with the pseudopenis Is stout, wedge-shaped, and strongly curved dorso-ventrally, appearing markedly hook- llke In the lateral view.

11 The Important characteristics used In recognizing the stages of Hoplopleura oenomydls were described by Ferris

(1951) and Pratt and Karp (1953) and may be summarized as follows %

Egg: The egg, exclusive of the holdfast, Is about

550-600 microns long by about 225-275 microns wide. The cap has nine or ten air pores near the periphery. There Is a definite projection at the base of the egg. The exochorlon has a reticular appearance In oertaln lights.

First stage nvmph: The length Is about 0.4-0.6 mm.

The abdomen Is rather heart-shaped, with a double row of six plates dorsally and two long, slender setae at the posterior end of the abdomen. Spiracles are absent.

Seoond stage nvmph: The length Is about 0.6-1.2 mm.

The abdomen Is heart-shaped, finely granular, entirely with­ out pistes, spiracles, or long hairs. There are six minute setae at the posterior end of the abdomen dorsally, which can be seen with high magnification of the compound microscope (50 power or above).

Third Btage nvmph: The length Is about 0.6-1.2 mm.

The abdomen Is heart-shaped, finely granular, entirely with­ out plates, splraoles, or long hairs. There are six minute setae at the posterior end of the abdomen dorsally and a pair of slightly larger setae on either side just anterior to them. These can be seen with magnification of the compound microscope (50 power or above).

12 Female: The length Is about 1.2-2 mm. The antennae are stout and five-segmented, the segments being longer than they are wide, with a slightly emerglnated area between segments four and five. The head Is more narrow anteriorly than In

Polvplax. The sternal plate Is rather elongated and egg- shaped. The third sternlte has stout spines on each end and three or more slender setae In between. Abdominal segments four to six each have three short tergal plates, each plate typically bearing six to eight rather slender setae. Lateral plates on segments four to six have spiracles, with a broad shoulder and a deep, wide emarglnatlon on the posterior margin, one large and one minute setae in the emarglnatlon.

The lateral plates on segments seven and eight are much smaller, each bearing two long setae. The posterior margin of the abdomen Is slightly concave. Male: The length Is about 1-2 mm. The male is similar to the female In general appearance except that each of abdominal segments four to six has only one tergal plate and two sternal plates. The posterior margin of the abdomen Is somewhat pointed. The male termlnalia with basal plate Is slightly larger than the parameres; the arms of the pseudopenis are slightly serrulate.

13 MATERIALS AND METHODS

Sixteen white laboratory rats, two per cage, were plaoed In the basement of the Botany and Zoology Green­ house in observation cages especially designed for rats kept on vitamin-deficiency diets. These cages were purchased from Kauffman-Lattlmer Company, Columbus, Ohio.

Ten rats were plaoed on a basal synthetic diet minus riboflavin, while six were used as controls and were kept on a normal basal synthetic diet. Both of these diets were obtained from Nutritional Blochemicals Corporation,

Cleveland, Ohio.

The ten rats were continued, on the deficient diet until they showed definite symptoms of riboflavin deficiency

(Fig. l), these symptoms being non-specific dermatitis on the ears, neck, legs, and paws. A marked reduction In weight was also noticeable. When the rats were In this condition, attempts were made to Infest them with lice from domestic rats. All of the laboratory rats were examined thoroughly prior to the beginning of the experiment, and none was found to have any lice. In order to get an Infestation, the lice

Polyplax had to be obtained from the domestic rats and transferred to the experimental rats.

At the beginning of the experiment, for over a period of a year, domestic rats were collected from many areas of metropolitan Columbus, Including the city dump, granaries,

14 A. NORMAL RAT

B. RIBOFLAViN-DEFICIENT RAT supermarkets, homes, restaurants, and The Ohio State Univer­ sity farm. All domestic rats collected were examined for lice. Fifty-three rats were oolleoted, of which 23 were found to have no lice; 20 were found to have as few lice as an average of 8.2 per rat; five were moderately infested, with an average of 23 per rat; and five were considered heavily Infested, with an average of 1,125 per rat.

Early attempts were made to collect the lice from the domestic rats by placing the freshly killed rats In cello­ phane bags, with the idea that the lice would desert the rats when the rats became cold and the lice could be collected from the bags. After several attempts only a few lloe were obtained In this way, as the rats collected were generally not very lousy. It was observed that the few lice which deserted the rats were adults and that most all of the lice will remain on the host even when a strong light

Is kept on the dead host in the Berlese apparatus.

Another method employed was to rub thoroughly pyrethrum dust onto the fur of the host and then comb or brush off the lice with the dust, but a good number of lice were killed In this way, and unless the host was heavily Infested, this method was not practicable.

Since few lice were obtained In the above fashion and these few failed to become established on the laboratory rats,

It was deemed necessary to resort to the searching technique, which Is slow, but the lice may be picked off with forceps

16 soon after the host has been killed, the lice are still active, and the number, sex, and stages can be selected.

Several attempts were made In order to get an Infestation of lice on the laboratory rats. For the first effort the number of active lice used was few and consisted of adults.

Twenty adult lloe, ten per rat, were placed on two deflelent- dlet rats, and 20 adult lice, ten per rat, on two oontrol rats. After one week, no lice were found on the normal rats; only three were on the deflolent-dlet rats, and few eggs were discovered. After a period of four weeks no lice were found. This Information supports the theory of Hopkins

(1950) that lice whloh spend their entire life on the body of the host develop a very high degree of host-speclflclty and that there Is a brief period In which they can survive off their host In natural conditions. The permanent establish­ ment of a species of louse on different host species does not

readily occur, although a few oases have been noted. One reason advanced for the fact that lloe normally can not

flourish on an abnormal species of host is that the chemical

constitution of blood, skin, fur, or plumage of the unnatural host Is such that Its body does not provide an attractive

souroe of food and shelter but may possibly provide actual

lethal conditions for the parasite.

At this time It was discovered that a mite population had built up tremendously and had to be controlled before additional louse Infestations could be made. To rid the

17 rats of the mites, the hosts and the cages were dusted with

the mltlcide Neotran applied with a hand sieve. One week

later no mites were found. It has been observed that

domestlo rats with few or no lice were usually heavily

Infested with mites, and those with heavy louse Infestations

had few, if any, mites.

Upon the collection of rats with heavy louse populations, the laboratory rats were Infested again, but as follows:

Table 1

Mode of Infestation

Number of rats Number of lloe Stape of lice

Rats on rlboflavln-deflolent diet

2 50 Eggs 2 50 Nymphs 2 50 Adults

Oheok rats (normal)

1 50 Eggs 1 50 Nymphs 1 50 Adults

Pour weeks after this Infestation the louse population was observed to be Increasing, and eggs and nymphs were

found on all rats Infested.

The Incubation period of the egg was determined by

placing only adult lloe on a rat and examining the rat each

day for eggs. On the day that the eggs were laid, the area

18 containing the eggs were marked. The eggs were considered to be no older than 12 hours. One hundred eggs were marked and observed on two rats on the normal diet; also 100 eggs were marked and observed on two rats on the rlboflavln- deflolent diet. The relative number of eggs laid on normal host and on deficient host were compared.

Then, the longevity of the instars was determined.

Forty newly hatched nymphs were transferred to four rats, two deflclent-dlet rats and two normal rats. Eaoh rat received ten nymphs. Each louse, while being transferred, was spotted with a phosphorescent enamel In order to facilitate the Identification of the stages, although the stages can be recognized with a good hand lens. The nymphs were observed every day, and the length of time required for the life cyole to be oompleted was compared on the normal and the deficient rats.

During the course of the experiment It was observed that the rats which were in the poorest condition developed alopeola and died within a short time unless riboflavin was added to the diet. It was also noted that those rats which developed alopecia lost their lice quickly and that few eggs were deposited on rats In this oondltlon. Thus, It was necessary to relnfest some of the deficient rats. The rats

showing mild symptoms of riboflavin deficiency (Fig. 2) were more easily infested. The longevity of the males and females was determined by

19 RIBOFLAVIN-DEFICIENT RAT (MILD SYMPTOMS)

FIG. 2

2 0 marking them after the last moult with a phosphorescent enamel and noting the number of days they lived.

The total number of lice on each rat was determined by killing the rats and Immediately freezing and maintaining refrigeration of the freshly killed rats for 24 hours. This rapid chilling, it was found, caused external parasites to release the firm hold on the hair or body of the host.

Skinning was not successful because deficient rats having severe dermatitis could not be skinned. Each rat killed was placed immediately in individual cellophane bags in order to prevent contamination. After 24 hours each rat was examined for the number of lice on the various areas of the host; afterwards, each rat was soaked and washed for 30 minutes in detergent water contained in separate vessels. The detergent water was then strained through a fine mesh sieve, the smallest eggs or parasites being unable to pass through. The sieve was then placed over a large petri dish, and the parasites were washed off with a mild stream of water from a wash bottle. All of the stages of lloe could be picked up, using a narrow-mouth pipette, and counted.

21 LIF.fi HISTORY

The rat louse Polyplax splnulosa Is an obligatory parasite, and it has traveled with its host, the domestic rat, until it is now found all over the world.

The louse passes through five stages in its life history: the egg, three nymphal stages, and the adult.

The lmmatures moult three times before reaching the adult stage.

22 Ineubatlon Period of the Egg

The egg (Pig. 3) emerges within a few seconds as the female remains quiet for several minutes until the egg is cemented to a hair. It Is grayish In appearance and remains this color until hatohlng. The free end of the egg Is provided with an operculum containing seven or eight air pores in the center. It breaks away to permit the exit of the larva. It was observed that each egg was oriented with this end away from the base of the hair upon which the egg was attached. The egg Is generally glued to a single hair about 1.5 mm. from the skin of the host. The majority of the eggs were looated on the hair of the mid-body region, dorsally and ventrally.

The Incubation period averaged the same on both the normal and deficient rats. For the majority of the eggs to hatch, five days were required. Very few eggs hatched after the

five-day period. The Incubation period was observed on two normal and two deflolent rats having a total of 200 eggs attached and marked. Of the 200 eggs, 157 hatched within five days; ten hatched on the sixth day; and 35 failed to hatch.

23 The results obtained are summarized In the following table:

Table 2

Time Required for Hatohlng of Eggs

Number of e«fcs Number of davs required for epcpcb to hatch 1 2 3. 4 5 6 7 8 9 10 Normal rats

50 0 0 3 8 36 3 0 0 0 0 50 0 0 0 5 34 2 0 0 0 0 Deficient rats

50 0 0 1 7 28 5 0 0 0 0 50 0 0 5 3 27 0 0 0 0 0

24 FIG. 3, EGG

25 First Stage Nymph

The newly hatohed nymph (Fig. 4) Is whitish in appearance before the Ingestion of blood. After a blood meal and several hours, the nymph turns gray. The contents of the digestive traot are clearly visible and this makes the nymph easy to see once the observer knows what to look for.

Also, the newly hatched nymph is very active and Is attracted by pieces of string or hair. It Is easily recognized by Its having only four long setae on the posterior part of the abdomen. At room temperature It does not live over two hours when removed from the host. The distribution of the nymphs over the body area of the host varied on the normal and deficient rats. On the deficient rats there appeared to be a greater number of nymphs about the mid-body. On the normal rats the nymphs were more concentrated about the rump and hlnd-body. It was also noted that the activity of the deficient rats decreased rapidly as the symptoms of the deficiency appeared. According to Kartman (194-9), the hosts having great activity could apparently rid themselves of a large number of lice, while the deficient rats became less active and did not remove any of their lice. The length of time required for the first nymphal ln- stars to moult on the deficient rats and on the normal rats was five days.

26 FIRST IN STAR NYMPH DIVIDED DORSA--VENTRAL DRAWING Second Stage Nymph

The aeoond stage nymph (Fig. 5) Is a light tan shortly after ecdysis and is easily recognized by its having eight long setae; the seventh and eighth abdominal segments have a pair of long setae on either side.

The second stage nymphs are not as active as the first stage nymphs and they remain pretty much in the same place.

On the normal rats the greatest number of second stage nymphs were also found on the rump and hind-body region, whereas on the deficient rats they were more concentrated about the mid-body, and occasionally they were found pretty well scattered over the trunk. The second instars begin feeding soon after ecdysis and grow very rapidly. At this stage the sex of the nymphs can not yet be determined. The length of time required for the average number of second stage nymphs to moult on the deficient rats was six days and on the normal rats it was seven days.

28 SECOND INSTAR NYMPH

DIVIDED DORSAL-VENTRAL DRAWING

FIG.

2 9 Third Stage Nymph

Closely paralleling the color of the adult, the third stage nymph (Fig. 6) Is light brown in color and can be differentiated from the first and second nymphal Instars by

Its having an Increased number of setae on the abdomen. The sixth abdominal segment of the third nymphal Instar has a long and a short setae on either side; the seventh and eighth abdominal segments have a pair of long setae on either side, thus giving a total of ten long setae on the tip of the abdomen.

The distribution of the third stage nymphs on the normal and the deficient rats was similar to that of the second stage nymphs. On the normal rats more nymphs were found on the mid-body and rump and hind-body areas, while on the deficient rats the majority of them were found on the mid-body region.

The length of time required for the third stage In­ star to moult to the adult stage on the deficient rats was four days; on the normal rats, seven days.

In summarizing the stages, It was observed that the cycle from egg to egg on deficient rats was 22 days and on normal rats, 26 days. This oould give about 17 generations per year on deficient rats and 14 generations per year on the normal rats.

30 Table 3 summarizes the Ilf© cycle of Polvolax aplnulosa on normal and deficient rata.

Table 3

Life Cycle of Lice on Normal and Deficient Rats

Eggs First Second Third Sex (Incubation Instar Instar Instar Maturity Period) Number of days Total

Normal Rats 5 5 7 7 2 26 Deficient Rats 5 5 6 4 2 22

31 THIRD INSTAR NYMPH

DIVIDED d o r s a l- v e n t r a l DRAWING

FIG. 6

32 Adult Female

The female (Fig. 7 ) is slightly longer than the male, averaging 1.5 mm. In length. The antenna Is stout and five segmented, and the head is slightly narrowed anteriorly. Each abdominal segment has two sclerites, dorsally and ventrally. The posterior margin of the female Is slightly concave and Is broader than th8t of the male. The females are quite aotive and copulate 24 hours after moulting and lay eggs 48 hours after moulting. Egg- laying has been observed to occur on hosts where there were no males; however, none of the eggs hatched.

The longevity of 20 marked females was found to be

28 days. The aversge female lays two eggs per day, thus giving a total of approximately 56 eggs during her life time.

33 ANTENNA

STERNAL PLATE

LATERAL PLATES

f t

ADULT FEMALE

DIVIDED DORSAL-VENTRAL DRAWING

FIG. 7

3 4 Adult Male

The male (Fig. 8 ), averaging 1.2 mm. In length, Is similar In general appearance to the female but Is usually smaller. It has only one sternal and one tergal plate each on segments four to six. The third antennal segment has a projecting spur. The posterior margin of the abdomen Is pointed. The termlnalla. with a pseudopenis Is stout, wedge- shaped, and curved dorso-ventrally, appearing hook-llke In the lateral view.

The males are attracted to the females, and they copulate within 24 hours after moulting. The writer observed the aot to last from five to eight minutes, with the male on the dorsum of the female, both remaining rather lnaotlve. After copulation, the male seeks other females.

The males did not have as great longevity as the females, 25 days being the length of time determined on the basis of 20 marked males.

35 ANTENNA

LATERAL ASPECT

MALE GENITALIA

ADULT MALE DIVIDED DORSAL-VENTRAL DRAWING

DORSAL-VENTRAL FIG. 8 ASPECT

36 Moulting

Moulting has been observed several times, and the process has been found to be similar In each case. At the beginning of the moult, the louse, which Is already attached to a hair, begins to arch dorsally In such a way that the front legs and posterior tip of the abdomen come close together and the head Is bent downward. The first rupture oocurs along the dorsal median line of the thorax and gradually extends caudad to about the third abdominal segment and cephalad to the frons where a large oval opening ocours, extending to the base of the eyes. The body becomes

Inflated and begins to emerge through the dorsal rupture.

The body straightens, and the head and thorax are withdrawn, followed by the legs, which enables the to free the abdomen, and It walks away, leaving the exuvlum still attached to the hair, with an extended diamond-shaped opening.

The moulting process Is similar to that of Haematoplnus suls as described by Florence (1921), but the latter leaves a T- shaped rupture.

37 Comparative Infestations on Normal & R15oflavln-aei'icrentr hats"

The number of lloe in the different stages— eggs, nymphal instars, and adults— on deficient and normal rats was compared. The final numbers represent the averages taken from a total of eight rats, four of which were on a deficient diet and four were on a normal diet. Because the deficient hosts' hides were so ulcerated that skinning was not practical In most cases, the locations of the lice on the hosts were estimated without employing the dissolving technique, which Involves dissolving various areas of the host's skin but leaving any lice present In the solution.

Instead of the dissolving teohnlque, a quick freezing method was devised. The quick freezing of a host Immediately after death keeps migration of the lice at a minimum, and counts made on different areas of the host are quite accurate.

It was also observed that the quick freezing caused the

Insects to release partially their hold on the hairs, thus making the lice easily obtained by washing the rats in detergent water. After a thorough washing In detergent water, the rats were examined carefully, and only a very few lloe remained. Table 4 summarizes the proportion of lice of all stages found on the different areas of the bodies of the deficient and normal rats after 14 weeks of Infestation.

38 Table 4

Areas of Infestation on Hosts

A. Rats Kept on Rlboflavln-deflcient Diet

Area Eggs First Second Third Adult (Unhatched) Instar Instar Instar Male Female

Head and neck 17 170 98 67 42 86 Shoulders and fore-body 53 264 195 133 58 88

Front legs 9 25 11 22 7 18

Mid-body 380 645 478 456 85 164 Rump and hlnd-body 96 84 76 43 28 67

Hind legs 7 18 11 18 6' 21

Total 562 1206 869 739 226 444

B. Rats Kept on Normal Basal Synthetic Diet

Area Eggs First Seoond Third Adult (Unhatched) Instar Instar Instar Male Female

Head and neck 7 9 13 11 9 16 Shoulders and fore-body 35 28 19 13 6 16

Front legs 0 4 7 0 0 0

Mid-body 37 28 19 20 12 27 Rump and hlnd-body 15 63 38 24 5 27 Hind legs 0 0 2 0 0 0

Total 94 132 98 68 32 86 39 The Infestation on the deficient rats was considerably heavier than that on the normal rats. The Infestations on the deficient rats ranged from 500 lloe on one rat to a high of 1,300 on a rat deemed heavily Infested. These

Infestations were built up In 14 weeks from an Initial Infestation of 50 nymphal lice.

The Infestation on the normal rats was comparatively small, and it stayed at a minimum. Only 50 lice were counted on one rat, and the highest number found on a normal rat was 175. These Infestations were also started from 50 nymphal lloe and lasted 14 weeks. The life cycle of the lice on the normal host was 26 days, as compared to

22 days on the deficient host.

It was noted that the deflolent rats with severe pedioulosls when fed riboflavin lost at least 90 per cent of their lice as the rats approached normal health and activity.

This was also observed by Searls and Snyder (1939) and

Kartman (1949)• Some rats were fed additional amounts of riboflavin In their normal diet and they showed no Increased resistance to lousiness.

On the normal and the deficient rats, the females out­ numbered the males approximately two to one.

40 DISCUSSION

The Information found In the literature concerning the life history of the louse. Polyplax splnulosa dealt with the number of stages and a description of these stages. It had been ascertained that the louse passes through five stages in Its life history* the egg, three nymphal stages, and the adult stage. However, the length of time spent In each stage and the total length of time required for a generation on the host were not found.

The life history of Polyplax splnulosa was determined on two groups of laboratory rats, one group kept on normal diet and the other group kept on rlboflavln-deflclent diet; the two life histories were then compared.

It was observed that the length of time required for the eggs to hatch on normal and deficient rats was five days; very few eggs on normal or deficient rats hatched on the fourth or sixth day.

The first nymphal instar was found to moult five days after hatching on the normal and the deficient rats. The longevity of the second nymphal Instar was not the same on the normal and deficient rats. On the deficient rats the longevity was six days; on the normal rats the longevity was seven days. The longevity of the third nymphal instar also differed on the rats on normal and deficient diets. The duration of the third Instar on the deficient rats was four

41 days, whereas on the normal rats It was seven days. The

reason for these differences In the second and third lnstars might be speculated to be due to the differences In the

resistance of the hosts kept on normal and deficient diets.

The rats kept on deficient diet were weak, did little

scratching, and showed marked cutaneous fragility and apparent changes In the composition of the hairs, making these hosts more susceptible to continuous feeding.

Twenty-six days were required for one generation on the normal rats, while 22 days were required for one generation on the deficient rats.

The female was found to copulate 24 hours after moulting and to lay eggs 48 hours after moulting. The female lays an average of two eggs per day, thus giving her a maximum of 56 eggs laid during her life time. Parthenogenesis was not observed to occur.

The longevity of the females and males was not the same; however, the host’s nutrition did not affect the longevity

Inasmuch as on the two groups of hosts the longevity of the females was 28 days and that of the males was 25 days.

At the beginning of the research, it was difficult to get an Infestation of lice started on rats kept on normal and deficient diets when using only adult lice, but this was finally accomplished with the use of great numbers of adults.

However, it was found that the nymphal stages became established In a short time, especially on the deficient rats,

42 though with more difficulty on the normal rata; this supports

Hopkln*s theory of host-speclflclty.

Rat lloe were found to be highly host-speolflc, and very few lice were seen to abandon the host after death, as has been reported for many species (Hopkins, 1950). Infestation was not accomplished by placing a killed rat having lice In the cage with a laboratory rat, wherein the quarters were so close that eontaot was unavoidable. In order to ensure

Infestation, the lice were picked manually from the domestic rats and placed on the laboratory rats. In this way, It was oertaln that the stage of lice desired was being transferred and that the lice being used were active.

When the subsequent louse Infestation of the laboratory rats was assured, the results on rlboflavln-deflclent rats and on normal rats were noted. It was observed that the normal rats showed great activity, such as scratching, and that they were highly resistant to the Infestation; but the rats that had advanced symptoms of riboflavin deficiency showed very little activity and more susceptibility to Infestation.

In order to obtain the lloe from the dead hosts, the quick freezing method was devised. This method Involves the immediate freezing and maintained refrigeration of the freshly killed rat for 24 hours. This rapid chilling caused the lice to release the firm hold on the hair or body of the host. The lice were then readily obtained In a detergent

43 wash. The deficient hosts' hides were so ulcerated that skinning was not practical In most cases.

Among those rats deficient In riboflavin, pediculosis was found to range from mild to severe. After 14 weeks of

Infestation, 3,484 lice were taken from four rats on the rlboflavln-deflolent diet. The lowest number from one host was 500, and the highest number was 1,300. The majority of the lice were found about the mid-body where the greatest number of eggs were attached; the next area of heavy Infes­ tation was the shoulder and fore-body, and then the neck region. Few lice were found on the legs. More first stage nymphs were found than any other.

On the other hand, the rats kept on normal diet showed very light Infestation after 14 weeks. Few eggs were found, and there seemed to be a tendency to lose the newly hatched nymphs. A total of 416 lice was taken from four normal rats.

Greater numbers were found about the mid-body and then the shoulder and fore-body; few were found about the neck, and none on the legs. As few as 50 lice were taken from one rat, and the highest number taken was 175. Deficient rats having severe pediculosis lost as high as

90 per cent of the lice when given riboflavin for two weeks, and In this period some of the rats recovered their health completely. Normal rats, when given additional amounts of riboflavin, showed no increased resistance to pedloulosls. It was observed that those rats with severe dermatitis

44 ‘ • and In very poor health began to lose their lice rapidly and had to be fed riboflavin for a few days In order to be kept alive and maintain the Infestation. Those rats which showed mild dermatitis were the most heavily Infested and lost very few of the lice.

During the oourse of the research, some Interesting side observations were made. An early observation was that only one species of lice was found on the 53 domestic rats collected In Columbus and the Franklin County area: Polyplax splnulosa (Burmelster). It was also noticed that there was a definite relationship between the number of lice on rats and the rats' diet. The domestic rats caught around the Columbus area showed that there was a. relationship between the number of lice they harbored and the amount of food available.

Those rats collected In the areas of granaries, barns, and grocery stores had fewer lice than those rats collected about the city dump and river banks.

Further, It was observed that among domestic rats the mite population was not abundant when there was an abundance of lice, and vice versa. Before the laboratory rats were

Infested with lice, they were found to have an abundance of mites, but after treatment with the mltlclde Neotran and subsequent louse Infestation, the mite population decreased.

45 SUMMARY

The life history of the louse Polyplax splnulosa was determined on two groups of white laboratory rats. One group was kept on a normal basal synthetic diet and the other on a basal synthetic diet minus riboflavin. The length of time required for the completion of a generation on the normal rats was 26 days; on the rlboflavln-deflolent rats it was 22 days.

The complete life cycle from the egg to egg stage on the two groups of rats may be summarized as follows:

With hosts on normal diet

Time from laying to hatching of eggs . . . 5 days First moult occurred after ...... 5 days Second moult occurred after ...... 7 days Third moult occurred a f t e r 7 days Sexual maturity occurred after ...... 2 days T o t a l 26 days

With hosts on rlboflavln-deflcient diet

Time from laying to hatching of eggs . . . 5 days First moult occurred after ...... 5 days Second moult occurred after ...... 6 days Third moult occurred after ...... 4 days Sexual maturity occurred after...... 2 days T o t a l ...... 22 days

The number of generations per year that could occur on the deficient rats could be about 17, and for the normal rats, about 14. The Important difference in the life cycle of the lice on the normal and deficient rats occurred In the length of life of the second and third nymphal lnstars. On the normal rats there is a difference of four days. The longevity of the adult females was greater than that.

46 of the adult males on hoth the normal and deficient rats, the females living 28 days a.nd the males, 25 days. This may account for the fact that there were found In most cases more females than males, although the differences were not very great.

The average female lays two eggs per day, thus giving a total of approximately 56 eggs during her life time.

Those rats maintained on a rlboflavln-deficlent diet could not rid themselves of the Infestation of lice; thus by

14 weeks after the Initial Infestation, they had a high degree of pediculosis.

The rats maintained on normal diet showed high resistance to pediculosis and manifested very light Infestation after

14 weeks from the Initial Infestation.

The quick freezing method was devised to facilitate the collection of lice from the host. This method involves the freezing of the freshly killed host for 24 hours and the subsequent washing of the host In detergent water.

It may be concluded that the host's nutrition and activity, such as scratching, appear to have some effect on the life history of the rat louse Polyplax splnulosa. which

Is an obligate and blood-sucking parasite throughout Its life cycle. Those rats maintained on an abundance of riboflavin remained resistant, while rats deprived of riboflavin became highly susceptible to louse infestation.

47 i LITERATURE CITED

Bacot, A. 1917- A Contribution to the Blonomlos of Pedloulua humanus (veatlmenta) and Pedloulua caoltla. Parasitology 9* 228-258. Barger, E. H. and L. E. Card. 194-3. Dlseaaea and Paraaltea of Poultry. War Dept. Educ. Manual, EM879.

Buxton, P. A. 1940. The Biology of the (Pedloulua humanus corporis: Anoplura) III Parasitology 32:: 296-302. Crauford-Benaon, H. J. 1941. The Cattle Lice of Great Britain. Parasitology 33: 331-342, 343-358.

Cummings, B. F. 1915. On Two New Species of Polyplax Anoplura) from Egypt. Proc* Zcol. Soc. London. Part 2 ? 245-272.

Dalla Torre, K. W. 1908. Anoplura, Genera Inaectorum. Faso. 81: 22 pp. 1 pi. De Melllon, B. and L. Goldberg. 1946. Nutritional Studies on Blood-sucking . Nature 158: 269.

______. 1947. Development of OrnIthodorus moubata on Normal and Thlamln-deflclent Rats, Nature 159: 171.

De Melllon, B., J. M. Thorp and F. Hardy. 1947. The Relation­ ship Between Ectoparasite and Host I. The Development of Clmex lectularlus and Ornlthodorua moubata on Rlboflavln-deficlent Rats. South Afr. J. Med. Scl. 12: 111-116. Elohler, W. 1940. Die Wlrtschaftllche Bedeutung Der Mallophagen (Haarllnge und Federllnge) Anz. Scbfi.dllngsk 16: 32-36. Enderlein, G. 1904. L&use-Studlen Zool. Anz. Bd. 28 Nr. 4, pp. 121-147, Figs. 1-15. Ferris, G. F. 1921. Contributions Toward a Monograph of the Sucking Lice. Part II. Stanford Univ. Pub. Univ. Ser., Biol. Sol. 2 : 53-133. ______. 1922. Concerning Lice. J. Mammology 3: 16-18. Perris, G. F. 1922. A summary of the Sucking Lice. Ent. News 45s 70-74.

______. 1923. Contributions Toward a Monograph of the Sucking Lloe. Part IV Stanford Univ. Pub. Univ. Ser., Biol. Sol. 2 : 179-270.

______. 1931. The Louse of Elephants Haematomvzus elephantls. Parasitology 23s 112-127.

______. 1932. Ectoparasites of Marquesan Rats. Vernlce P. Bishop Museum Bull. 98s 117-127.

Perris, G. F. and C. J. Stojanovlch. 1951. The Sucking Lice. Pacific Coast Ent. Soc. Mem. Is 1-320.

Florence, L. 1921. The Hog Louse, Haematoplnus aula Llnnes Its Biology, Anatomy, and Histology. Mem. Cornell Grlc. Exp. Sta. 51s 635-743. Gyttrgy, P. 1938. Pediculosis In Rats Kept on a Rlboflavln- deficlent Diet. Proo. Soo. Exp. Biol. Med. 38s 383-385. Hopkins, G. H. E. 1950. Host-Assoclatlons of the Lice of Mammals. Proc. Zool. Soc. Lond. 119s: 387-604.

Kartman, L. 1942. A Note on Vitamins In Relations to Ectoparasite Resistance. J. Paraslt. 28s 170-171.

Kellogg, V. L. 1896. New Mallophaga. II Proc. Calif. Acad. Sol. (2 ), 6 s 431-548.

______. 1902. Are the Mallophaga Degenerate Psoclds? Psyche, Camb., Mass. 9s 339-343.

Lamson, G. H. 1917. The Llfe-Hlstorles of the Cattle Lice. J. Econ. Ent. 10s 446-447.

Leach, W. E. 1815. Entomology In Brewster’s Edinburgh Encyclopedia. 9s 77.

Matthysse, J. G. 1944. Biology of the Cattle Biting Louse and Notes on Cattle Suoklng Lice. J. Econ. Ent. 37s 436-442. ______. 1946. Cattle Lice, Their Biology and Control. Cornell U., Agrle. Exp. Sta., Bull. 832.

49 Nuttall, Gr. H. F. 1917. The Biology of Pedlcalua humanus. Parasitology 10: 80-185.

Packard, A. S. 1887. On the Systematlo Position of the Mallophaga. Proc. Amer. Phil. Soc. 24: 264-272.

Pratt, H. D. and H. Karp. 1953. Notes on the Rat Lice Polyplax snlnul08a (Burmelster) and Hoplopleura oenomvdls Ferris. J. Paraslt. 39: 495-504.

Pratt, H. D. and J. E. Lane. 1951. Hoplopleura orvzomvdls New Species, with Notes on Other United States Species of Hoplopleura (Anoplura: Haematoplnldae). J. Paraslt. 3 7 s I2^ ^ .

Prltohard, A. E. 1947. Hoplopleura oenomydls Ferris, A Louse Found on Doraestlo Rats In the United States. J. Paraslt. 33: 374-375.

Roudabush, R. L. 1939. Survival of the Troploal Rat Flea In the United States. Sol. 88: 79-80.

Searls, E. M. and F. M. Snyder. 1939. A Study of the Relation of Vitamin A to Louse Resistance In Rats. J. Paraslt. 25: 425-430. Watts, H. R. 1918. The Hog Louse. Univ. Tenn. Agric. Exp. Sta. Bull. 120, 16 pp.

Weber, H. 1939. Zur Elablage und Entwlcklung Der Elephantenlaus Haematomyzus. Nebst Verglelchenden Betrachtungen uber Die Lage des Embryos lm El und Das Auskrlechen. Biol. Zbl. Leipzig 59: 397-409.

50 AUTOBIOGRAPHY

I, DeField Trollinger Holmes, -was born In Frazier,

Pennsylvania, November 23, 1921. I attended grade and high school at Graham, North Carolina, graduating from high school In 1939. My college education, which was begun at Hampton

Institute, Hampton, Virginia, In September, 1940, was disrupted for service In the Army from November, 1942, to

January, 1946. Returning to Hampton Institute In June, 1946,

I received the Bachelor of Science degree In biological soience In August, 1948.

From 1948 to 1951, I served as an Instructor of biological science at Graham High School. To further my education, in 1951, I matriculated In the Graduate School of

Zoology at The Ohio State University, completing the requirements for the Master of Soience degree In 1953. From

1953 to 1955, I taught biological science at Shaw Unlver<y,

Raleigh, North Carolina. In 1956, I returned to The Ohio

State University to work toward the Doctorate of Philosophy degree In entomology.

51