Studies of the reproductive biology and of the structure, composition and physiology of the egg of Graphognathus leocoloma Boheman (Coleoptera : Curculionidae): a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Zoology at Massey University

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private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

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Studies of

t he R eproductive

Biology and of

the Structure, C omposi ti on,

and

Physiology of the Egg of

Grapho!:J"'">Wthus leucolorna Boheman

(Co l eopte r a : Curc ulio ni dae )

A thesis presented in partial fulfil m ent of th e requirements for the degree of

Mas t er of Science in Zoology at Massey University

David George Holdom

1977

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A. Graphognathus leucoloma Boheman, adult.

B. Graphognathus leucolornu bofiemun, ebgs.

•

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· 5mm

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To the late Stan and Blanche BaGon, 1,hose enthusi.:isr.i and love oJ nature

were u ~ajar influence on my interest in biolo~

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ABSTRACT

The published information rec;ardinG the oiology c:md pest .status of Graphognathus leucoloma Boheman .i.s summarized.

A study was made of ecc devclop~e~t at temperatures ranginG from

4

to

37 . 8°c

&nd relative hwnidities ranging from ¹!-U to 100';~. Egg hatch

was frequently very low and showed consideraole variability. ~:edian duration of development rani;ed fro• about ¹¹^-1 ^daysat 31.5 0 ^C to 95-97 days at

15°c,

with the developmental-hatchinp; threshold between 12.1

~

50

and 1~. C. Sub-threshold temperatures were lethal. Virtually no

hatching occurred below 1CO'ib RH but the eGgs could withstand considerable dessication and would hatch when moistened. Possible effects of humidity on the duration of develop1:1cnt and of parental ar;e on egg viability are also discus::;ed. Some reasons for the vet~y low egg viability nre

suggested, along with modifications to the experimental design to elim- inate them.

Studies were made of adult and pupal size, and of longevity, pre- oviposition period and rep::-oductive output. Soi~e reasons for unexpect- edly low fecundity ^a.nd lon₆ pre-cviposition i,eriod are discusced.

The structure of the egg cnvelope.s ,.rnd the cement in which the

esgs are laid was studied, using scanning and t~ansmission electron microscopy, and :;o:narsk:i. diffcre11t1.al intcrfcrcJ,lCC microscopy. A histochemical study of these structures, supple~entcd by some simple chemic&l tests, 1,.:as also r:iade, to elucidate :i.n par-: their composition. The

chorion is L;-9µ..".:! thic~t and compo.::;ed entirely of protein; some 2_50 ,000

aeropyles ure scattered over the surface, and its structure is such that it could probably act as a plastron when the egg is submerged. There is no micropyle. Disulphide linkages are probably important in

the structure of the chorion, as in many other species, but unlike other beetles so far studied, no crystalline proteins were found. The vitelline membrane was found to be a three-layered structure

o.3-0.5µm

thick composed of protein and acid mucopolysaccharides, and to be highly

resistant to chemical attack. Waterproofing is probably provided by a layer of lipid on the outer surface of the vitelline membrane. The cement was found to be a complex, variable and heterogeneous mixture of protein and up to five acid mucopolysaccharides, which is unlike that of any other species reported. Some possible functions of the cement and the significance of its composition are suggested.

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A note on the citation of publications, and the referral to species and their systematic status.

Any publication by three joint authors is cited in full the first time it is referred to and thereafter is abbreviated to the for•: senior author at al.

are cited in the form: including the first.

Publications with more than three joint authors senior author~ al. each time they are cited, The specific name of any organism is given in full only on the first time it is referred to, and the names of insect species and e;enera and their taxonomic status are listed in Appendix Five.

V

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ACKNOWLED.'.iE}'IENT S

I would like to thank Nr. I.A.N. Stringer, who supervised the major part of this work, for his constant advice and helpful suggestions, and Dr. J. Crofton for his supervision in its early stages.

Thanks are also due to Dr. M.J. Merrilees, who offered helpful advice during the early stages and on histochemistry; to Mr. M.

Mannering and Dr. J. Riddle for advice regarding histological methods;

to Dr. W.M. Kain who made a number of helpful suggestions during the planning stages of both season.:s I work; and to Mr. il .J. Esson for his constant advice.

I am indebted to hr. H.J. E.sson and Dr. W.M. lain for the loan of equipment without which this work would not have been possible; to Professor Bacon for the use of a constant temperature room; to l-lr. E.

Roberts for the loan of a refrigerator; and to Mr. '-i/. 'I'homas for con- str\~c:ing r.iuch cf the other equ.ipmento I am also grateful to l·lr. P.

Couchman for supplying a aaciple of basic fuchsin that worked; to Viessrs. C. Fletcher and R. Hanson for r:ian:; of the chemicals used in

the histocher:iistry, and to Dr. ~1.J. Merrilees for a sample of cetyl- pyridinu;n chloride. The ever helpful and patient staff of Chemistry

'

Stores have also earned my gratitude.

Thanks to Mr. D. Hopcroft who prepared the material for electron microscopy and for operating the micro::,copes, and, together with Mr. I. Simpson, for processing the electron micro£raphs.

Credit for the frontispiece and F.igs. 3.1, 3.2 and

3.3

must go to the staff of the Central Photographic Unit, who also processed the light micrographs. Miss A. Turner drew Figs. 2.1 to 2.6.

I wish to thank Dr. P. Elliott, Vir. ~ .• J. Esson, Mr. P.G. Fenemore and Hr. N. Pomeroy who read parts of the manuscript and who made many useful suggestion.s; Mr. C.G. Arnold and Dr. B.S. Weir, who advised me on statistical methods; and Di Machin for assistance with the final pre- paration and for a translation from the French text. Thanks are due also to Glenis Hobberley for typing references on to cards; to my sister, Diana Cloud, for sending several references from Canada and for her constant encouragement; and Mrs. J. Parry for typing the final manuscript.

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xi xiv

1 1 2 4

5

8 3 ,, 0

8 11

12 12 12 12 14 17 17 19

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2.3.2.1 Measurement and representation of the duration 19 of the egg stage

2.3.2.2 Some possible effects of humidity on the 22 duration of the egg stage

2.3.2.3 Some effects of temperature on the duration of 23 the egg stage

2.3.3 Second Season

26

2.3.3.1 AssessMent of survival 26

2.3.3.2 Some effects of temperature on survival 27 2.3.3.3 Effects of ter:iperature on the duration of the 31

egg stage

2.3.3.4 Effect of parental age on eGG viability

34

2.3.4 The Shadehouse Experiment

34

2.3.5 Effect of

T.

uutrescentiae on

G.

leucoloma eggs

34

2.4 General C0nclusions

36

CHAPI'E.-=t THREE s·rUDIES OF VARIABILI'rY AND REPRODUCTD!E 38 CAPACITY IN ADUL'l'S AJJD PUPAE

3.2 3.2.1 3.2.2

3.3

3.3.3

3.4

CHAPrER FOUR

4.1

4.2

4.2.1 4.2.2 4.2.3

4.2.3.1 4.2.3.2

Introduction

haterials a.nd Hethods

Collection an.ct haintenance of Weevils Shadehouse Experiment

Results

Size Characteristics of Adults and Pupae LonGevity, Pre--oviposition Period and Reproductive Capacity

Shadehouse Experiment Discussion

STUDIES ON THE STRUCTURE AND COMPOSITION OF THE EGG SHELL AND CEMENT OF G. leucoloma

Introduction

Materials and Methods

Transmission Electron Microscopy (T.EM) Scanning Electron Microscopy (S:EM) Light Microscopy

Histological methods Histochemistry

39

41 44

44

55

59

62

62 62 62

62

63

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4.2 . 3.2.1

Carbohydrates

4.2.3.2.2

Proteins

ix

64 4. 2.4

Chemical Tests Performed on Fresh Whole or

65

Dechorionated Eggs

4.3

Results

and

Discussion

66

4.3.1

Structure and Composition of the Chorion

66

4. 3. 2

The Vitelline Membrane 80

4.3.3

The Cement

83

4.3.3. 1

Component one

83

4.3.3. 2

Component two 84

4. 3.L1-

The Yolk 93

4. 4

Summary and Conclusions

91-

APPENDIX ONE MISCEI...LAN:EOUS OBSERVATIONS 96

A1 .1

Resistance of Larvae to Dessication

96

J\'l .2 Observations on the Helationship Between Larval 96

Populations and Crop Damage

A1.3 Jur.tpin;; hy Newly Hatched Larvae

96

A1.4 Cannibalism by Larvae

96

A1 .5

Duration of the Pupal Stage 97

A1 .G Drinking of v/ater by Adults 97

A1.7 Deformed Adults in the Field 97

A1.8 Bacteria in the Eggs 97

APPENDIX 'l'WO ARTIFICIAL DIEr FOR THE ADUL'l' WIHrE-FRINGED 99 WEEVILS

APPENDIX THREE APPENDIX FOUR APPENDIX FIVE

References

HISTOLOGICAL AND HISTOCHEMICAL MEI'HODS 100

A LIST OF REAGENTS USED IN THE PRESENT STUDY 109

A LIST OF INSECT SPECIES REFERRED TO IN THE TEXT 111

AND THEIR TAXONOMIC POSITION

113

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Table

;!-I 2-II

LIST OF TABLES

Facilities for maj.ntenance of constant temperatures

Allocation of eggs to test temperatures through allocation of adults

Page 9 13

2-III Survival of G. leucoloma eggs after exposure to different 18 temperatures and humidities

2-IV 2-V

2-VI

3-I

3-II

Hatching of G. leu.coloma eggs during first season Effects of temperature on survival and duration of development of G. leucoloma eggs - 2nd season

Viability of samples collected at different times and incubated at 25.4°C

\\Teight characteristics of pupae and adults from different collection sites

Size cruu-acteri::.;t.ics of adults used in the experiments ,rnd of the pupae from vJhich they emerged

3-III Longevity, pre-oviposition period and reproductive capacity of G. leucoloma

28

32

34

45

49

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Figure

A

B 2.1

2.2

2.4

2.6

2.7

2. 8

2. 9

2.10

2.11

3.1

3.2 3. 3

3.4

LIST OF FIGURES

Graphognathus leucoloma Boheman, adult Graphognathus leucoloma Bo.heman, eggs

Metal oviposition cage with alwninium foil floor used for housing adults during the first season

Jar used for holding eggs at constant humidity during the first season

Jar used to maintain eggs in laboratory during the second season

Jar

used to maintain eggs at high humidity in the shadehouse

Tube used to contain eggs exposed to ambient conditions in the shade.house

Maintenance of eggs at a~bient conditions in the shade.house

Cumulative hatch curves of samples held at 10..,"'fo RH and specified temperature (first season)

(a) Median duration and (b) rate of egg development in relation to teQperature (first season)

Cumulative hatch curves at specified temperatures (second season)

Survival of eggs in relation to temperature (second season)

(a) Nedian duration and (b) rate of egg development in relation to temperature (second season)

Container used to house adults singly during the second season (natural size)

The shadehouse

The shelter beneath which the adults were kept, with the cover removed, as seen from the left-hand door of the shade.house

Distribution of pupal weight at the a) Massey and b) Longburn collection sites

xi

ii ii 10

10

16

21

29

30

40

43

46

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Figure

3.6 3.7

3.8 3.9 4.1

Distribution of adult weight at the a) Massey and b) Longburn collection sites

Longevity of weevils in lots 3 - 9

Pre-oviposition period of weevils in lots 3 - 9

Fecundity of weevils in lots 3 - 9

Mean size of egg-batches laid by weevils in lots 3 - 9

Scanning electron micrograph of the egg, showing the aeropyles distributed densely over the surface

50

51 52

53

67

4~2

S~anning electron micrograph showing detail of the surface

67

of the chorion

4.3

Scanning electron micrograph showing pores exposed through

68

gaps in the cement

4.4

Transmission electron. micrograph of the chorion and cement

69 4.5

TransmisGion electron micrograph of the chorion and cement

69 4.6

Scanning electron microgTaph of a freeze fractured egg 70

showing the broken edge of the chorion

4.7 4. 8

Unstained section photographed using Nomarski optics 70

Transmission electron micro~raphs of the vitelline membrarie 71 and inner region of the chorion

4. 9

Micrograph of an unstained section

75

4.10

Mixed anhydride reaction for side-chain carbox:yl groups

75

(glutamic and aspartic acids)

4.11

The DDD reaction for disulphide groups (cystine) 76

4.12

The DDD reaction for sulphydryl groups (cysteine) 76

4.13

The diazotization-coupling method for tyrosine

77 4.14

The Sakaguchi-dichloronaphthol method for arginine

77 4.15

^The U1AB-nitrite method for tryptophan

78

4.16

The PAS technique

85

4.17

The modified PAS method for uronic acid-containing

86

glycoasminoglycans

4.18

Alcian Blue staining at pH 2.5

87

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Figure 4.19 4.20

4.21

A1.1

Alcian Blue staining at pH 1.0 The PAD technique

A) ,liter 7 hrs staining B) After 21+ hrs staining

The carbodiimide reaction for carbohydrate-bound carboxyl groups

Transmission electron micrographs of the yolk showing the rod-shaped bacteria which were observed in the eggs

xiii Page

88

89

90

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oc

p R."9:

<

aer B C cem G

ic ilin ivm lae mvm oc

ovm

trl vtm

ABBREVIATIONS USED IN THE TEXT

degrees celcius Probability

relative humidity approximately equal to less than

aeropyle bacteria chorion cement

ABBREVIATIONS USED IN THE FIGURES

glycogen granules inner layer of chorion inner lining of chorion

inner layer of vitelline membrane lining of aeropyle

middle layer of vitelline membrane outer re₆ion of chorion

outer layer of vitelline membrane trabecular layer

vitelline membrane

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1 CiiJ\,'l':.:;.: ,i,,

'l'hc 1::1itc-i'rin_;cd '.:ecvil, Gra.J>o n.:::.t:t~·s lc·icolo.:a .3ohc:;a~, (Colco₁)tcra, Curculionidae, Brn.c:1yderinne), i::; n rc;,ti vc o;· 2,cJtrt /\;::crica, Hlwrc it occurs in J,r,;entina, Er.::i.zil, C,,ilc, }er~, o.,.rl Ur11·;uaJ (t3crr:-/, 1t;11.7; Youn~

.,_ 7 ~c-o)

~ ~ •, I;-:; • Its 1::,0\m ran ;e 110·.1 i::-Kl't,:es ... a~l/ socit',-eastern st.:J.tes of the U.S.A. as far .:rnrth as :.aryland (Anor.:·:.ou:..;, 1;·:·:;), .::i.11 ~i-✓ e :.o.ir:land Austral ian states (Cliach-,ic.:, 1S170), Sout~1 .\i'ric,:;_ (Jou::icrt , 1S!:,i1) o.::-;d

Nc1v iealand. Iu :;e·.-; Zcalanci., G. le:uco.l.o!. ¹,m.s found in ;;ort!l.l.:mci in 1941;., , .. ;here it 11as believed to ho.ve bee!: i,i'c..:.·od.;ccd 1·1it:1 A.:1crican , .. :;:,.r equip::1ent about 1940 (Cottier, 1~L)2), u'Jt it :,0¹.1 occur::; L:1rou i101~t tile iiorL. I.sla.nd and llas been ::.·ou;:1d i11 iiel::;on (:i.\c'rrot L, 1 ✓:-,11.) .::i.:-.d ;,s.:.Jurto1, (. ,-1.j', 1 //'.'3) in

. ' . .

,

^..

o::. ·.n~lC!l "lVC

occu1· in t:.c

u

.S .1\. C:1lj G. 2.e:ucoJ.o .:t ^{~ • J} ~·e:co1·ciccl out.:;iJc :;orL1 crnC.:.

publi0:1ed.