Biology and Ecology of Nisotra Basselae (Bryant) on Abelmoschus Manihot Medicus in Solomon Islands

Biology and ecology of Nisotra basselae (Bryant) on Abelmoschus manihot Medicus in Solomon Islands

Maclean Vaqalo

B. Sc., The University of the South Pacific

M. Sc., The University of Reading

A thesis submitted for the degree of Doctor of Philosophy at

The University of Queensland in 2014

School of Biological Sciences

Abstract

Slippery cabbage, Abelmoschus manihot Medicus, is a staple vegetable crop throughout the Pacific. The slippery cabbage flea beetle, Nisotra basselae (Bryant) (Coleoptera: Chrysomelidae), is indigenous to Papua New Guinea (PNG) and was first recorded in Solomon Islands in 1981. It is now a severe pest of the crop and is established on most of the islands in the archipelago. Soon after its discovery in the Solomon Islands, it was formally identified as Podagrica basselae Bryant (Coleoptera: Chrysomelidae) but it has since been referred to as Nisotra basselae (Bryant), causing confusion as to its true identity. To resolve this, adult beetle samples collected on Guadalcanal were compared with samples collected in PNG. Specimens from both countries were morphologically identical and characterized by two longitudinal sulci at the anterior margin of the pronotum. This characteristic definitively separates the genera Nisotra and Podagrica. All other key morphological characteristics were consistent with Bryant’s original description of material collected PNG. Thus the original decision to place the species in the genus Podagrica was an error and the current taxonomic designation of the slippery cabbage flea beetle is Nisotra basselae (Bryant) (Coleoptera: Chrysomelidae).

In the laboratory, survivorship and performance of N. basselae on excised roots was greater on A. manihot than on Hibiscus tiliaceus L. and insects could not complete development on Abelmoschus esculentus (L.) Moench. Female beetles deposited eggs below the soil surface and, at ambient temperatures (25 - 31°C), they took eight days to hatch. When reared on A. manihot roots, insects developed through three larval instars over 15 days. Pre-pupae formed cells beneath the soil surface and remained in this stage for three days, before entering a six-day pupation period. Adult beetles were long lived (>65 days) and females laid an average of 266 (59) eggs. In field studies adult N. basselae fed preferentially on leaves 3- 6, and ignored younger and older leaves.

A long-term (18-month) field study showed that the abundance of adult N. basselae was significantly reduced during wetter times of the year (November-March). Tangle-foot trapping experiments showed that female beetles moved down host plant stems at night in order to oviposit, while recently emerged beetles of both sexes moved up plant stems after eclosion in the soil. Most eggs (>95%) were laid beneath the soil surface, within 2 cm of the stem of their host plants. Similar oviposition patterns were detected in laboratory studies, which showed that eggs were not laid beyond the root zone of host plants. A positive correlation between adult beetle density in the plant canopy and egg density in the

i soil was detected in field studies. This, combined with studies showing that beetle density in the canopy declined in direct proportion to adult beetles trapped close to oviposition sites at the base of stems, indicates significant beetle fidelity to host plants. Disruption of access to oviposition sites as a cultural control tactic was investigated by comparing soil egg densities at the base of plants in which the soil immediately adjacent to the stem was covered with copra sacking, sown with clover, covered with grass mulches or kept bare to replicate typical farmer practice; copra sacking reduced oviposition >50% compared with the other practices.

In the field, more adult beetles fed on A. manihot plants growing in full-sun than on plants growing in shaded conditions. When A. manihot plants grown for three months in shaded or full-sun conditions were transplanted to shaded or full-sun conditions in A. manihot crops, more beetles were recovered from full-sun grown plants than shade grown plants in both light conditions; indicating that beetles were attracted to plants that had been grown in full–sun. Laboratory experiments confirmed the preference of N. basselae for full-sun grown foliage and similar experiments showed that urea treatment could make shade grown plants more attractive than un-treated full-sun grown plants. Semi-field experiments, which examined beetle orientation and residency on intact, mechanically damaged, N. basselae-damaged or intact plants supporting bagged male or female beetles, showed that beetles were attracted to plant damage and female conspecifics. The possible mechanisms underlying these responses are discussed.

This is the first detailed study on N. basselae biology and ecology and the improved understanding provides opportunities for cultural control based on access to and disruption of oviposition sites and manipulation of growing conditions. Approaches could improve the design of oviposition site barriers and combine these with targeted application of Tangle- foot or contact insecticides to plant stems to trap or kill ovipositing beetles. Strategic application of insecticides to oviposition sites, rather than the current ineffective practice of applications to the crop canopy, could improve control and reduce insecticide use. These approaches could significantly improve the yields of subsistence farmers who depend on A. manihot as a staple food and cash crop and reduce insecticide use by larger producers that currently supply urban markets.

Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

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Publications during candidature

No publications.

Publications included in this thesis

No publications included

Contributions by others to the thesis

No contributions by others.

Statement of parts of the thesis submitted to qualify for the award of another degree

None

Acknowledgments

First and foremost I wish to thank the Almighty God for enabling me to engage in this study project which ultimately resulted in the production of this thesis. My heartfelt thanks and gratitude must go to my Principal Supervisor, Dr. Michael Furlong, for his insightful ideas, endless support and encouragement during this project. His patience and generosity know no bounds. Immense thanks must also go to my associate supervisor, Professor Myron Zalucki, for his support and alternative approaches to problems at hand when needed. I must thank Dr. Chris Reid for providing free accommodation and meals during a four day visit at the Australian Museum in Sydney and his rendered guidance which enabled the insect in question to be morphologically reviewed and confusions on the identity of the beetle clarified. Thanks to Dr. Bronwyn Cribbs for the worthwhile critique and milestone review during the course of writing this thesis. Thanks to all my mates like Barry Ale, Fereti Atu, Dr. Paul Cunningham, Mary Finlay-Doney, Lindsay Popple, John Holland, Thomas Gorenge, Associate Professor Gimmie Walter and others unmentioned here for their friendly smiles and assistance in one way or another.

Immense thanks go to the Australian Centre for International Agricultural Research (ACIAR) for generously providing financial support for this study program.

Thanks go to all responsible officers in the Ministry of Agriculture and Livestock for the endorsement and moral support rendered during my study. Special thanks colleagues and friends from the Secretariat of the Pacific Community (especially Mr. Steve Hezalman), TerraCircle (Dr. Grahame Jackson) and Ministry of Agriculture and Livestock that participated in Improved Plant Protection for Solomon Islands project (IPPSI), especially to Mr. Edwin Valenga, Ms. Getrude Varuia, Mr. Geoffrey Oliou’ou, Mr. Graham Teakle and Ms. Helen Tsatsia; for without their invaluable hands-on assistance both in laboratory and field experiments, this study program would not have been carried out as required and ultimately successfully.

Many thanks to Mr. Graham Teakle for his assistance in collecting and sending me beetle specimens from different sites in Solomon Islands. Also, many thanks to Dr. Jacque Wright Kami and Mr. David Tanakanai for coordinating Agriculture officers in PNG to collect beetle specimens from different sites and sending them to the laboratory in the university of Queensland for taxonomic analysis.

Thanks to all friends who had rendered support and assistance during my study. These includes Ms. Heather Andersen, Mr. Ron Anderson and family, Mr. Joseph Gamatia and family, Mr. Robert Kake and family, Mr. Rodney Robertsons and family, late Reverend Lalomilo Lima and family and others that are not mentioned by name here.

Finally, my warmest and sincerest thanks to my wife (Mrs. Gillian Annie Vaqalo) and children (Mr. John Chris Vaqalo, Miss Lynn Macklyn Vaqalo, Mr. Ian Roger Vaqalo, Miss Marie Corolie Vaqalo and Miss Diana Karen Vaqalo) for your patience, interest and support during the last few years, you mean everything to me.

Keywords

Alticinae, Chrysomelidae, flea beetle, taxonomy, host-plant relationships, oviposition, light regime, shot-hole, cultural control

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060207, Population Ecology, 33.3%

ANZSRC code: 060808, Invertebrate Biology, 33.3%

ANZSRC code: 070603, Horticultural Crop protection, 33.3%

Fields of Research (FoR) Classification

FoR code: 0602, Ecology, 33.3%

FoR code: 0608, Zoology, 33.3%

FoR code: 0706, Horticultural production, 33.3%

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TABLE OF CONTENTS

ABSTRACT ...... i DECLARATION BY AUTHOR ...... iii PUBLICATIONS DURING CANDIDATURE ...... iv PUBLICATIONS INCLUDED IN THIS THESIS ...... iv CONTRIBUTIONS BY OTHERS TO THE THESIS ...... iv STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF ANOTHER DEGREE ...... iv ACKNOWLEDGEMENTS ...... v KEYWORDS ...... vii AUSTRALIAN AND NEW ZEALAND STANDARD RESEARCH CLASSIFICATIONS (ANZSRC) ...... vii FIELDS OF RESEARCH (FOR) CLASSIFICATION ...... vii TABLE OF CONTENTS ...... viii LIST OF TABLES ...... xiii LIST OF FIGURES ...... xiv LIST OF ABBREVIATIONS USED IN THE THESIS ...... xxi CHAPTER ONE: INTRODUCTION ...... 1 1.1 SOLOMON ISLANDS ...... 1 1.2 AGRICULTURE IN SOLOMON ISLANDS ...... 3 1.3 SLIPERY CABBAGE, ABELMOSCHUS MANIHOT MEDICUS (MALVALES: MALVACEAE) ...... 7 1.4 INSECT PESTS OF ABELMOSCHUS MANIHOT IN SOLOMON ISLANDS ...... 9 1.4.1 AMARASCA DEVASTANS (DIST.) (CICADELLIDAE: HEMIPTERA) ...... 12 1.4.2 HARITALODES DEROGATA FABRICIUS (= SYLEPTA DEROGATA) (PYRALIDAE: LEPIDOPTERA) ...... 13 1.4.3 EARIAS VITELLA FABRICIUS (NOCTUIDAE: LEPIDOPTERA)) ...... 14 1.4.4 NISOTRA BASSELAE (BRYANT) (CHRYSOMELIDAE: COLEOPTERA) ...... 15 1..5 THE RATIONALE FOR THE STUDY ...... 16 CHAPTER TWO: GENERAL MATERIALS AND METHODS ...... 19 2.1 INTRODUCTION ...... 19 2.2 EXPERIMENTAL SITES ...... 19 2.3 PREPARATION OF FIELD SITES ...... 20 2.4 PREPARATION OF SOIL FOR EXPERIMENTS AND CULTIVATION OF PLANTS ...... 20 2.5 HOST PLANTS ...... 21 2.5.1 ABELMOSCHUS MANIHOT ...... 21

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2.5.2 HIBISCUS TILIACEUS ...... 22 2.5.3 ABELMOSCHUS ESCULENTUS ...... 22 2.6 INSECT CULTURES ...... 23 CHAPTER THREE: TAXONOMY OF NISOTRA BASSELAE (BRYANT) ...... 24 3.1 INTRODUCTION ...... 24 3.2 MATERIALS AND METHODS ...... 26 3.2.1 SOURCE OF ADULT AND IMMATURE BEETLE SPECIMENS...... 26 3.2.2 STANDARD MEASUREMENTS AND INDICES ...... 27 3.2.3 STATISTICAL ANALYSES ...... 28 3.3 RESULTS ...... 31 3.3.1 DESCRIPTIONS ...... 31 3.3.1.1 ADULT DESCRIPTION ...... 31 3.3.1.1.1 BODY SHAPE AND MORPHOLOGY ...... 31 3.3.1.1.2 COLOUR ...... 32 3.3.1.2 DESCRIPTION OF SPECIFIC BODY PARTS ...... 36 3.3.1.2.1 HEAD ...... 36 3.3.1.2.2 PRONOTUM ...... 38 3.3.1.2.3 STERNUM ...... 39 3.3.1.2.3 LEGS ...... 41 3.3.1.2.4 WINGS ...... 42 3.3.1.2.4 ABDOMEN ...... 43 3.3.1.3 IMMATURE STAGES ...... 44 3.3.1.3.1 EGG ...... 44 3.3.1.3.2 LARVA ...... 44 3.3.1.3.3 PUPA ...... 46 3.4 CONCLUSIONS ...... 47 CHAPTER FOUR: LIFE CYCLE AND SEASONAL PHENOLOGY OF NISOTRA BASSELAE IN SOLOMON ISLANDS...... 49 4.1 INTRODUCTION ...... 49 A. 4.2 MATERIALS AND METHODS ...... 50 4.2.1 EGG VIABILITY AND INCUBATION PERIOD ...... 50 4.2.2 PERFORMANCE OF NISOTRA BASSELAE ON DIFFERENT HOST PLANTS .. 52 4.2.2.1 SURVIVAL AND DEVELOPMENT OF LARVAE AND PUPAE ...... 52 4.2.2.2 ADULT ECLOSION AND MATURITY ...... 53 4.2.2.3 EGG DEVELOPMENT: COPULATION TO OVIPOSITION ...... 54 4.2.2.4 FECUNDITY AND LONGEVITY ...... 54

4.2.3 DISTRIBUTION OF NISOTRA BASSELAE WITHIN PLANTS OF ABELMOSCHUS MANIHOT ...... 54 4.2.4 SEASONAL ABUNDANCE AND DISTRIBUTION OF NISOTRA BASSELAE IN THE FIELD ...... 55 4.2.5 STATISTICAL ANALYSES ...... 57 4.3 RESULTS ...... 57 4.3.1 EGG VIABILITY AND INCUBATION PERIOD ...... 57 4.3.2 PERFORMANCE OF NISOTRA BASSELAE ON DIFFERENT HOST PLANTS .. 59 4.3.2.1 SURVIVAL AND DEVELOPMENT OF LARVAE AND PUPAE ...... 59 4.3.2.2 ADULT ECLOSION AND MATURITY ...... 65 4.3.2.3 EGG DEVELOPMENT: COPULATION TO OVIPOSITION ...... 66 4.3.2.4 FECUNDITY AND LONGEVITY ...... 68 4.3.3 DISTRIBUTION OF NISOTRA BASSELAE WITHIN PLANTS OF ABELMOSCHUS MANIHOT ...... 72 4.3.4 SEASONAL ABUNDANCE AND DISTRIBUTION OF NISOTRA BASSELAE IN THE FIELD ...... 76 4.4 DISCUSSION ...... 82 CHAPTER FIVE: OVIPOSITION ECOLOGY OF NISOTRA BASSELAE ...... 89 5.1 INTRODUCTION ...... 89 5.2 MATERIALS AND METHODS ...... 92 5.2.1 EFFECTS OF SOIL MOISTURE ON NISOTRA BASSELAE OVIPOSITION ...... 92 5.2.2 THE EFFECTS OF MULCHES ON NISOTRA BASSELAE OVIPOSITION ...... 92 5.2.3 DISTRIBUTION OF NISOTRA BASSELAE EGGS IN THE SOIL AROUND HOST PLANTS IN THE FIELD ...... 93 5.2.4 OVIPOSITION OF NISOTRA BASSELAE IN SOIL AROUND POTTED HOST PLANTS IN THE SHADE HOUSE ...... 94 5.2.5 INTRA-PLANT MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL ...... 95 5.2.5.1 MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL ON POTTED PLANTS IN A CAGE ...... 95 5.2.5.2 MOVEMENT OF NISOTRA BASSELAE BETWEEN HOST PLANT FOLIAGE AND SOIL IN THE FIELD ...... 96 5.2.6 THE EFFECTS OF SOIL SURFACE BARRIERS ON NISOTRA BASSELAE OVIPOSITION IN THE FIELD ...... 97 5.3 RESULTS ...... 98 5.3.1 EFFECTS OF SOIL MOISTURE ON NISOTRA BASSELAE OVIPOSITION ...... 98 5.3.2 EFFECTS OF MULCHES ON NISOTRA BASSELAE OVIPOSITION ...... 99 5.3.3 DISTRIBUTION OF NISOTRA BASSELAE EGGS IN THE SOIL AROUND HOST PLANTS IN THE FIELD ...... 99

5.3.4 OVIPOSITION OF NISOTRA BASSELAE IN SOIL AROUND POTTED HOST PLANTS IN THE SHADE HOUSE ...... 101 5.3.5 INTRA PLANT MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL ...... 102 5.3.5.1 MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL ON POTTED PLANTS IN A CAGE ...... 102 5.3.5.2 MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL ON PLANTS IN THE FIELD ...... 103 5.3.6 THE EFFECTS OF SOIL SURFACE TREATMENT ON NISOTRA BASSELAE OVIPOSITION IN THE FIELD ...... 105 5.4 DISCUSSION ...... 109 CHAPTER SIX: ADULT NISOTRA BASSELAE MOVEMENT AND THE EFFECTS OF BIOTIC AND ENVIROMENTAL FACTORS ON HOST PLANT SELECTION ...... 114 6.1 INTRODUCTION ...... 114 6.2 MATERIALS AND METHODS ...... 115 6.2.1 DISPERSAL OF NISOTRA BASSELAE ...... 115 6.2.2 THE EFFECTS OF CONSPECIFIC FEEDING DAMAGE, MECHANICAL DAMAGE AND THE PRESENCE OF CONSPECIFICS ON ATTRACTION TO HOST PLANTS ...... 116 6.2.2.1 EFFECTS OF CONSPECIFIC FEEDING AND INFESTATION ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS ...... 117 6.2.2.2 EFFECTS OF MECHANICAL DAMAGE ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS ...... 117 6.2.2.3 EFFECTS OF MALE OR FEMALE CONSPECIFICS, BUT THE ABSENCE OF FEEDING DAMAGE, ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS ...... 118 6.2.3 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON NISOTRA BASSELAE DISTRIBUTION IN THE FIELD ...... 118 6.2.4 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON NISOTRA BASSELAE DISTRIBUTIONS BETWEEN POTTED SENTINEL PLANTS IN THE FIELD ...... 120 6.2.5 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON HOST PLANT SELECTION BY NISOTRA BASSELAE ...... 121 6.2.6 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME AND UREA TREATMENT ON HOST PLANT SELECTION BY NISOTRA BASSELAE ...... 122 6.3 RESULTS ...... 124 6.3.1 DISPERSAL OF NISOTRA BASSELAE ...... 124 6.3.2 THE EFFECTS OF CONSPECIFIC FEEDING DAMAGE, MECHANICAL DAMAGE AND THE PRESENCE OF CONSPECIFICS ON ATTRACTION TO HOST PLANTS ...... 127

6.3.2.1 EFFECTS OF CONSPECIFIC FEEDING AND INFESTATION ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS ...... 127 6.3.2.2 EFFECTS OF MECHANICAL DAMAGE ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS ...... 128 6.3.2.3 EFFECTS OF MALE OR FEMALE CONSPECIFICS, BUT THE ABSENCE OF FEEDING DAMAGE, ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS ...... 129 6.3.3 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON NISOTRA BASSELAE DISTRIBUTION IN THE FIELD ...... 130 6.3.4 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON NISOTRA BASSELAE DISTRIBUTIONS BETWEEN POTTED SENTINEL PLANTS IN THE FIELD ...... 132 6.3.5 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON HOST PLANT SELECTION BY NISOTRA BASSELAE ...... 138 6.3.6 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME AND UREA TREATMENT ON HOST PLANT SELECTION BY NISOTRA BASSELAE ...... 138 6.4 DISCUSSION...... 141 CHAPTER SEVEN: GENERAL DISCUSSION ...... 144 BIBLIOGRAPHY ...... 148

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LIST OF TABLES

Table 1.1 Important insect pests of A. manihot in PNG (Preston 1998) and in Solomon Islands.

Table 1.2 Minor seasonal and occasional insect pests of A. manihot in PNG (Preston 1998) that are also present in Solomon Islands.

Table 3.1 Definitions of anatomical descriptors used in the description.

Table 3.2 Mean (±SE) body lengths and widths of male and female N. basselae collected from seven sites in PNG and Solomon Islands, n = 7 beetles of each sex per site.

Table 4.1 Larval head capsule widths of N. basselae measured at different larval instars when fed with A. manihot (Broad-type), H. tiliaceus and A. esculentus.

Table 4.2 Life-tables for N. basselae fed with A. manihot, H. tiliaceus or A. esculentus.

Table 4.3 Longevity and oviposition periods of N. basselae (adult males and females) reared on A. manihot and H. tiliaceus (n = 4).

Table 4.4 The mean number of eggs laid by female N. basselae when fed two different plants. n = 4.

Table 4.5 The mean area per each leaf number of A. manihot (Broad-type) (n = 5).

Table 4.6 The index of dispersion and index of patchiness of N. basselae in the different seasons (wet season November- April; dry season May - October).

Table 5.1 The mean number of eggs laid by N. basselae on soil covered with mulches and bare soil.

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LIST OF FIGURES

Figure 1.1 Map of Solomon Islands. Source: Family Survival Protocol. Earth Watch Report 2013

Figure 1.2 a) adult Indian cotton leaf-hopper A. devastans b) Indian cotton leaf-hopper damage on a leaf of A. manihot.

Figure 1.3 a) Leaf of A. manihot rolled by cotton leaf-roller caterpillar (Haritalodes derogate); b) H. derogata caterpillar in rolled up leaf; c) H. derogata adult moth.

Figure 1.4 a) Shoot of A. manihot damaged by E. vitella caterpillars; b) Caterpillar of E. vitella feeding and tunnelling inside the stem of A. manihot; c) Adult moth of E. vitella.

Figure 1.5 a) adults of N. basselae making shot-hole damage; b) severe shot-hole damage on leaves of A. manihot.

Figure 2.1 The Guadalcanal Plain and proximity of the Tutuva experimental site. The light grey shaded areas are oil palm plantations. Source: Pacific Economic Bulletin Volume 25 number 1 © 2010 the Australian National University.

Figure 3.1 Map of Solomon Islands and Papua New Guinea; showing locations where beetle specimens were sampled from A. manihot (red stars). Islands and provinces circumscribed by the red boundary (A) area currently infested by slippery cabbage flea beetle; (B & C) areas which are not yet infested are marked in green (reported by Chief Field Officers in Solomon Islands in 2011).

Figure 3.2 Mean lengths (±SE) and mean widths (±SE) of male and female beetles.

ANOVA: Female beetles, significant longer (F1, 96 = 61.7, P < 0.05) and significantly wider

(F1, 96 = 70.1, P < 0.05) than male beetles.

Figure 3.3 Morphological characters of male and female genitalia of N. basselae. a) dissected ventral abdominal surface of female; b) dissected ventral abdominal surface of male; c) abdominal ventrites of intact female, showing the simple and evenly curved apex of ventrite V; d) abdominal ventrites of intact male, showing the truncated and bilobed apex of ventrite V; e) female genitalia featuring tergites VIII (T VIII) & IX (T IX), and sternites VIII (S VIII) & IX (S IX) (stylus); f) male gentalia, showing the drawn out aedeagus (AD) and sperm sacs (SS).

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Figure 3.4 Nisotra basselae habitus: a) dorsal view; b) ventral view; c) lateral view.

Figure 3.5 Facial view of N. basselae (vx = vertex, so = supraorbital puncture from which trichobothrial seta emerge from; ey = eye; ob = orbit; as = antennal sockets; asp = antennal space; gn = gena; cl = clypeus; lb = labrum; mb = mandible).

Figure 3.6 Mouth parts of N. basselae; a) lateral view of the head; b) labium; c) antennae; d) maxilla; e) mandible; f) labrum.

Figure 3.7 Pronotum of N. basselae showing the longitudinal grooves at the anterior margin.

Figure 3.8 Nisotra basselae prosternal and head parts; a) Ventral view of prothorax, showing closed procoxae (pc), prosternum (ps), prosternal process (psp), and postcoxal projection (pp); b) side view, showing the head parts, the transverse shape of procoxal, the overlap of prosternal process and the post coxal projection which closed the procoxal cavity c) photographic facial view

Figure 3.9 Habitus of N. basselae. Ventral and dorsal views.

Figure 3.10 Legs of N. basselae: a) Hind leg, depicting metafemoral spring mechanism; b) representation of pro- and meso-legs (these are very similar); c) ventral and dorsal views of tibia; d) tarsal segments, with 3rd segment distinctively bilobed; e) tarsal claws clearly appendiculate.

Figure 3.11 Wings of N. basselae, a) dorsal view of elytron (right hand side); b) hind wing. c. Figure 3.12 Genitalia of N. basselae: a – d, ventral view of female genitalia; A. consists of the Ventrite V, part of Sternite VIII, Tergite VIII, Tergite IX; b. Sternite VIII; c. Tergite VIII, which is pubescent at apex and showing pygidium - the longitudinal median groove; d. spermatheca (magnification = x400); e – h, show parts of male genitalia; e. shows ventral view of male Ventrite V - truncated and lobed; f., g. and h are the ventral, dorsal and side A. b. view of the aedeagus, respectively.

Figure 3.13 Nisotra basselae eggs laid in a batch. Length of an egg = 0.84mm.

Figure 3.14 Larvae of N. basselae: a) side view of first instar larva showing the position of egg bursters on meso- and meta- thoracic segments; b) shows the dorsal view of a plate

xv that all larval stages have on the last abdominal segment; c) shows the larval head capsule; d. shows pre-pupal larval stage.

Figure 3.15 Nisotra basselae pupae: a) photograph (ventral view) of fresh new pupa habitus; b) pupa habitus, dorsal view; c) frontal view of the head before eyes and mandibles are pronounced; d) ventral view of apical segments of a pupa.

Figure 4.1 A. manihot varieties compared in the N. basselae seasonal abundance study. a) Broad-type variety; b) Noodle hair-type variety.

Figure 4.2 The percentage of N. basselae eggs that hatched at different times under ambient laboratory temperatures (31.1oC ± 0.2oC to 25.7oC ± 0.1oC; relative humidity 90.2% ± 0.6% to 72.9% ± 1.5%).

Figure 4.3 Survivorship curves for N. basselae from a day old larvae to adults, showing the percentage of survival and duration at different stages within the life cycle when fed with one of three Malvaceae species, A. manihot, A. esculentus or H. tiliaceus.

Figure 4.4 Median immature development periods of N. basselae when fed with rootlets of A. manihot, H. tiliaceus or A. esculentus under ambient laboratory conditions. Within a given stage, median durations marked with a common letter are not significantly different.

Figure 4.5 Median times to sexual maturity for adult male and female of N. basselae when fed with A. manihot and H. tiliaceus. Within a given stage, median durations marked with a common letter are not significantly different.

Figure 4.6 Median times to first oviposition event when fed with A. manihot or H. tiliaceus under laboratory ambient conditions. Host plant had no significant effect on pre-oviposition period (Z = -1.5, P>0.05).

Figure 4.7 The median durations of each of the different developmental stages of the life cycle of N. basselae when fed on A. manihot under ambient laboratory temperatures (31.1oC ± 0.2oC to 25.7oC ± 0.1oC; relative humidity 90.2% ± 0.6% to 72.9% ± 1.5%).

Figure 4.8 The daily oviposition pattern of N. basselae when fed with A. manihot under ambient laboratory conditions.

Figure 4.9 The daily oviposition pattern of N. basselae when fed with H. tiliaceus under laboratory ambient conditions.

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Figure 4.10 A) mean number (±SE) of adult N. basselae per plant, and B) mean number (±SE) shot-holes per leaf of A. manihot. n = 30 for both. Columns marked with different letters are significantly different.

Figure 4.11 A) mean numbers (±SE) of adult N. basselae per cm2 of leaf and B) mean numbers (±SE) of shot-holes per cm2 of leaf. All measurement taken on A. manihot, n = 30 plants. Columns marked with different letters are significantly different (LSD, P < 0.05).

Figure 4.12 A) the mean (±SE) numbers of N. basselae per plant, and B) the mean (±SE) numbers of N. basselae per plant leaf area recorded on two A. manihot varieties (Broad-type and Noodle hair-type) between November 2007 and June 2009. Columns marked with different letters are significantly different (LSD, P < 0 .05).

Figure 4.13 The mean (±SE) numbers of N. basselae per plant recorded weekly on two varieties of A. manihot Medicus (Broad-type & Noodle hair-type) between November 2007 and June 2009. Indicate when sampling changed between plots.

Figure 4.14 A) the mean (±SE) numbers of N. basselae per unit area of leaf recorded weekly on two varieties of Abelmoschus manihot (Broad-type and Noodle hair-type) over a year and a half (November 2007 to June 2009). (B) total monthly rainfall and mean temperature taken at Henderson Meteorological Station which was the closest (≈6 km) available weather recording station to Tutuva Experiment site where the beetles were recorded.

Figure 4.15 Frequencies of N. basselae recorded per A. manihot plant (Broad-type and Noodle hair-type combined from the population seasonal abundance data set).

Figure 5.1 Oviposition chamber to investigate N. basselae oviposition in different soil zones around a potted A. manihot plant in the laboratory.

Figure 5.2 Mean (±SE) number of eggs laid by female N. basselae in moist and dry soil, No eggs laid in dry soil. Different letters marked significant difference between the two treatments.

Figure 5.3 Mean (±SE) number of eggs laid by female in mulched and bare soil

(ANOVA: F1, 16 = 25.31; P < 0.05). Points marked with different letters are significantly different.

xvii

Figure 5.4 The mean (±SE) number of eggs recovered from the concentric zones around the base of A. manihot (Broad-type variety) in the field (n = 10). Columns marked with a different letter are significantly different (ANOVA: F4, 45 = 34.75; P < 0.05; LSD P < 0.05).

Figure 5.5 Relationship between the total number of eggs of N. basselae within a radius of 15 cm at the depth of 2 cm at the base of host plants and the number of adult N. 2 basselae on foliage. (Linear regression: F1, 9 = 39.83; P < 0.05; r = 0.83).

Figure 5.6 The mean (±SE) numbers of eggs laid by adult female N. basselae in different concentric zones around the base of A. manihot (Broad-type variety) plants in an oviposition cage. Columns which are marked with a different letter are significantly different

(ANOVA: F2, 33 = 8.22, P < 0.05; LSD, P < 0.05).

Figure 5.7 Adult male and female N. basselae trapped on the upper and lower margins of Tangle-foot traps 0 - 24, 24 - 48 and 48 - 72 hours after traps were set. Within time intervals, columns marked with a different letter are significantly different (ANOVA: F2, 33 = 8.22, P < 0.05; LSD, P < 0.05).

Figure 5.8 The mean numbers of N. basselae recorded 24 h prior to trapping and at 24 h intervals after the setup of Tangle-foot traps (ANOVA: F1, 45 = 11.34; P < 0.05). Points marked with different letters are significantly different

Figure 5.9 Mean (±SE) number of N. basselae eggs collected from soil within a 5 cm radius (2 cm depth) of experimental plants Columns marked with different letters are significantly different (ANOVA: F3, 60 = 3.86; P < 0.05; LSD, P < 0.05).

Figure 5.10 Linear relationships between the numbers of adult N. basselae per plant and the number of eggs deposited within 0 – 5 cm of the base of the plant. Linear regressions of egg density against adult beetle density were significant for all treatments. Mulches (F1, 2 2 15 = 57.61; P < 0.05; r = 0.805); Copra sack (F1, 15 = 14.51; P <0.05; r = 0.509), Clover (F1, 2 2 15 = 16.65; P < 0.05; r = 0.543) and bare soil (F1, 15 = 12.16; P < 0.05; r = 0.465).

Figure 6.1 Plan of A. manihot plot used to assess the distribution of adult N. basselae and feeding damage between plants grown in the shade of rain trees and plants grown in full-sun.

xviii

Figure 6.2 Diagrammatic representation (not to scale) of the experiment to investigate effects of full-sun and shade on the distribution of N. basselae between A. manihot plants in the field. Grey plots represent areas under the shade of rain-trees; white plots represent areas in full-sun. Space between adjacent subplots was 0.5 m apart.

Figure 6.3 Percentage of N. basselae recovered (n = 700) on pot plants of A. manihot and yellow sticky traps at 12 h intervals after release.

Figure 6.4 Proportion of N. basselae recovered on plants positioned at different orientations from a central release point. Black squares represent the proportion of the beetles that were released (n= 700) and recovered 72 hours later.

Figure 6.5 The proportion of adult N. basselae recaptured at 12 h intervals for 72 hours on A. manihot (Broad-type variety) at different distances from the central release point (n=700).

Figure 6.6 Mean (±SE) proportion of released N. basselae on infested plants and mean (±SE) proportion of released N. basselae on intact plants 12 h and 48 h after release.

Figure 6.7 Mean (±SE) proportion of released N. basselae recaptured on mechanically damaged plants and mean (±SE) proportion of released N. basselae on intact plants at 12 h intervals for 48 h after release.

Figure 6.8 Mean (±SE) proportion of released N. basselae on plants supporting caged female N. basselae and mean (±SE) proportion of released N. basselae on plants supporting male N. basselae at 12 h intervals for 48 h after release.

Figure 6.9 Nisotra basselae and shot-hole densities on plants grown under shaded and full-sun light regimes in the field at Tatuva village in January 2007. A) mean (±SE) number of N. basselae adults plant-1; significantly more adult N. basselae on full-sun grown plants than on shade grown plants (F1, 28 = 15.94; P < 0.05). B) mean (±SE) number of shot-holes plant-1; significantly more shot-holes on full-sun grown plants than on shade grown plants

(F1, 28 = 43.30; P < 0.05).

Figure 6.10 Nisotra basselae shot-hole mean (±SE) densities on plants grown under shaded and full-sun light regimes in the field at Tutuva village in January 2007, mean

(±SE) number (F2, 24 = 4.31, P < 0.05 of shot-holes/plant; significantly more shot-holes block 1and 3 than on shade grown plant block.

xix

Figure 6.11 Nisotra basselae and shot-hole densities on field grown plants raised under shaded and full-sun light regimes in the field at Tutuva village in October 2008 A) mean

(±SE) number of beetles on plants grown in full-sun and plants grown in the shade (F1, 44 = 1.09; P > 0.05). B) Mean (±SE) number of shot-holes on plants grown in full-sun and plants grown in the shade (F1, 44 = 0.27; P > 0.05). Columns marked by a different letter are significantly different (LSD, P < 0.05).

Figure 6.12 A) mean number (±SE) of N. basselae and B) mean number (±SE) of shot- holes on potted-plants that had been reared in full-sun or shaded conditions in the nursery and then transplanted among field plants grown in full-sun or shade at Site 1. Within a given time interval (24 h and 48 h), columns marked with a different letter are significantly different (LSD, P < 0.05).

Figure 6.13 A) mean (±SE) numbers of N. basselae and B) mean number of shot-holes on potted-plants that had been reared in full-sun or shaded conditions in the nursery and then transplanted among field plants grown in full-sun or shade at Site 2. Within a given time interval (24 h and 48 h), columns marked with a different letter are significantly different (LSD, P < 0.05).

Figure 6.14 A) mean (±SE) number of N. basselae and B) mean (±SE) number of shot- holes on plants previously reared in full-sun and shade (n = 15) when tested in cage choice tests. For a given time interval, columns marked with a different letter are significantly different (LSD, P < 0.05).

Figure 6.15 A) mean (±SE) number of N. basselae and B) mean (±SE) number of shot- holes on plants previously reared in full-sun or shade (n = 15) treated with urea 12 h after exposing beetles to treated plants, columns marked with a different letter are significantly different (LSD, P < 0.05).

LIST OF ABBREVIATIONS USED IN THE THESIS

ACIAR: Australian Centre for international Agriculture Research

BMNH: British Museum of Natural History

DPI: Department of Primary Industry

GPPOL: Guadalcanal Plantation Limited

MAL : Ministry of Agriculture and Livestock

NPK: Nitrogen Phosphate and Potash

PNG : Papua New Guinea

SBD: Solomon Bokolo Dollar

SIPL: Solomon Islands Plantation Limited

SPC: Secretariat of the Pacific Community

xxi CHAPTER ONE: INTRODUCTION

1.1 SOLOMON ISLANDS

Solomon Islands is an independent sovereign nation in the southwest Pacific basin. It is comprised of around 990 islands that range over a distance of approximately 1400 km, between 5° 10’ S 155° 30’ E and 12° 45’ S 170° 30’ E. The eastern outer islands are located close to the northwest border of Vanuatu while the western group of islands are located close to the southeast border of Bougainville Island, Papua New Guinea (Fig 1.1). The islands are naturally diverse, ranging from high mountains to low-lying coral atolls. Most of the bigger and mountainous islands are intersected by deep, narrow valleys and are mostly covered with tropical rainforest vegetation.

The total land area of Solomon Islands is approximately 28,000 km2. However, its exclusive economic zone is about 46 times greater than its total land area. The islands are separated by vast oceanic space and the nation is endowed with abundant land and marine-based natural resources, which are pivotal to the cultures and livelihoods of its people (Whitmore 1969).

Before becoming an independent state on July 7th 1978, Solomon Islands was a protectorate of Great Britain for 85 years. Its population is currently estimated at 553,935, with an annual growth rate of 2.3% (Solomon Islands National Statistics Office 2012). The population is 94.5% Melanesian, 3% Polynesian, and 1.2% Micronesian with a small proportion of Chinese, Europeans and other migrants (CIA Fact Book 2012). About 85% of the population resides in the rural areas and depends on subsistence agriculture and fishing (Jansen 2006). In many cases subsistence food production is the sole source of livelihood (National Agriculture and Livestock Sector Policy 2010).

Solomon Islands has a narrow economic base that is largely dependent on natural resources, especially round logs and timber, minerals, tuna, palm oil, copra and coconut and cocoa (CBSI 2011). The production and export receipts of these main commodities are as follows: log harvest 1.9 million cubic metres, valued at Solomon Bokolo Dollar (SBD) $1,457 million; mineral production 39,429 ounces, SBD $518 million; tuna 19,300 tons, SBD $341 million; palm oil and kernel 31,592 tons and 3,537 tons respectively, combined value SBD $326 million; copra and coconut oil production 24,939 tons, SBD

$216 million; cocoa 4,553 tons, SBD $119 million. Currently, the country depends heavily on overseas development assistance.

The climate in Solomon Islands is hot and humid, with high, but seasonal, annual rainfall and frequent storms and tropical cyclones; especially on Guadalcanal, Makira, Rennell and Bellona and the eastern outer islands. Daily temperatures typically fluctuate between 25°C and 32°C throughout the year, with a mean diurnal temperature of 27°C. Annual rainfall is greatest in the mountainous regions of the larger islands (up to 5500 mm per annum), but it is also considerable in the coastal regions (3,000 mm per annum). The dry season usually occurs between April and October and the wet season between November and March (Solomon Islands Meteorological Services 2013).

The vegetation in Solomon Islands consists of lowland rainforest, hill forest, montane forest, freshwater swamp and riverine forest, saline swamp forest, grassland and other non-forest areas. The first floral diversity documentation was carried out about five decades ago, at which time 1931 species were recorded (Whitmore 1966). More recent work recorded 3210 plant species; one sixth of these are species exotic to the Solomon Islands; they are represented mainly by ornamental and weed species and a few species of forestry importance (Henderson and Hancock 1988)

The field experiments for this study were undertaken on the northwest coast of the Guadalcanal Plain of Guadalcanal Island. Guadalcanal Island is part of one of the southern chains of the Solomon archipelago that lies 9° 15’- 10°S and 159° 3’- 160° 45’ E. It is the largest of all the islands in Solomon Islands, with land area of 5,302 km2. Honiara is the capital city of Solomon Islands and it is situated 9° 25’ - 9° 26’ S and 159° 55’ E and 160° 2’ on Guadalcanal Island. Most vegetable crops are produced by subsistence farmers on the Guadalcanal Plain to supply urban markets. The plain, which covers an area of 450 km2, is the only alluvial plain of this size in the country and stretches from the Lungga River in the west to Koaka Bay in the east (Hansel and Walls 1979) (Figure 1.1). About 6,000 ha of the Guadalcanal Plain is currently occupied by oil palm plantation estates belonging to the Guadalcanal Plains Palm Oil Limited (GPPOL) (Fraenkel et al. 2010) (Figure 2.1).

The climate around Guadalcanal Island is typically tropical, characterized by uniformly high temperature, humidity and rainfall with an average rainfall between 3,000 and 5,000mm per year (Solomon Islands Meteorological Service 2009). Rainfall generally increases in areas where elevations exceed 600m. Topographical characteristics of higher mountains

2 and steep slopes are closer to the southern side of the island than to the northern side, where the Guadalcanal Plain stretches further inland. It is for these reasons that the south coast usually experiences wet and adverse weather conditions and it is referred to as the ‘weather coast’ of Guadalcanal. By contrast, the north coast, which includes the Guadalcanal Plain, is a much drier region of the island, and one of the driest parts of Solomon Islands (MEFC 1995).

1.2 AGRICULTURE IN SOLOMON ISLANDS

About eighty five per cent of people in Solomon Islands live in rural areas, and derive their livelihoods from a combination of subsistence agriculture and small-scale income- generating activities, particularly export cash cropping and marketing of fresh produce (Jansen et. al 2006; CBSI 2012). The other twenty five per cent of people live in urban areas and depend on import goods and fresh market products supplied by rural farmers. The scope of employment opportunities in these rural areas is informal, and driven mostly by agricultural production and marketing. In fact, it is the rural farmers that are the backbone of Solomon Islands’ overall economy, through the production of exportable commodities in sectors such as agriculture, forestry, fisheries and mining (CBSI 2011). Apart from palm oil and kernels, which are produced by a commercial company with major contributions by out-grower commercial farmers, rural farmers also produce other agricultural commodities, particularly copra and cocoa.

Most of the food consumed in rural villages in Solomon Islands is grown in local subsistence gardens. Other important food sources, which are not grown in these gardens, include coconuts, fish, other marine resources such as shellfish and mangroves, wild ferns, and fruit and nuts obtained from trees in the forest. Important starch food crops that are widely grown in Solomon Islands include sweet potato (Ipomoea batatas (L.) Lam), cassava (Manihot esculenta Crantz), banana (Musa sp.), kongkong taro (Xanthosoma sagittifolium (L.) Schott), taro (Colocasia esculenta (L.) Schott), coconut (Cocos nucifera L.), pana (Dioscorea esculenta L.) and yam (Dioscorea alata L.). Although, sweet potato is still by far the most important locally grown starch staple in the villages of Solomon Islands, imported rice has become preferred to sweet potato, even in rural villages of Solomon Islands (Taki et. al 2010). Apart from rice, there are also other important food sources such as flour, Solomon Blue tuna, corned beef, noodles, biscuits, sugar, tea and

3 salt that are sold in retailing shops in the villages that supplement locally grown food to rural villagers.

Abelmoschus manihot Medicus is by far the most widely grown and eaten leafy vegetable in both rural villages and urban centres in Solomon Islands. Other important leafy vegetable crops include brassica, taro leaves, pumpkin leaf tops and cassava leaves. Villagers also collect wild ferns and other leaves of a variety of plants from the forest to supplement to their diets.

Most farmers in Solomon Islands still rely on simple tools such as bush knives, axes and digging mattocks and spades to produce food for their families and markets. A traditional farming practice that is still prevalent in Solomon Islands is known as ‘shifting cultivation’, where a plot of land is initially slashed and burned from either a virgin forest or one that has been under fallow for a period of time. Shifting cultivation has been practiced by farmers for many years, not only in Solomon Islands but also in other parts of the world, and is a traditional method of rejuvenating soil fertility (Aweto 2013). The slash and burn method of clearing and cleaning the land before planting can be viewed as an effective way of controlling pest populations in subsistence gardens. Usually, shifting cultivation also follows a sequential pattern, with one end of a garden area progressively extending into recently cleared bush, while the other end of the garden is harvested and then left to revert to bush fallow for a period of years. Quite often, a number of families of a clan would work in one area on their individual garden plots for some years and then abandon the whole area for a longer fallow period and move on to another part of their tribal land (Jansen et. al 2006).

Historically, fallow periods under shifting cultivation typically took 15 – 25 years. However, nowadays fallow periods have been typically reduced to just a year, or even less, due to increased demand for agricultural land following the rapid rise in the human population (Jansen et. al 2006). As fallow periods become shortened, farmers are forced to extend cropping periods on the same piece of land for longer, leading to reduced crop yields due to declining soil fertility and high pest populations. This is now becoming a major problem in some locations in Solomon Islands. Potential improvement of food production under the current system of farming may be achieved through planting of early bearing and higher yielding varieties of important food crops. The slash and mulch systems as practiced by farmers in Guadalcanal have shown promising results with sustained high yields provided that fallow periods are 4 – 5 years (Jansen et. al 2006).

Most of the farmers living close to urban centres, for example, Honiara and Auki, are driven by market demands to produce more from their farms. A majority of these producers are subsistence farmers, and the fruit and vegetable produce that is sold in urban centres represents the collective production from this sector. In recent years, increased settlement and subsequent population rises in peri-urban villages, coupled with customary land tenure systems, have led to land shortages for crop production for urban markets. Fallow periods have shortened and soil fertilities have declined, lessening crop yields and vigour and making them more susceptible to pest attack. Also, since farmers have used their land excessively, with little crop rotation, continuous pest problems have developed on farms. Farmers have resorted to relentless use of insecticides to control pests in these farms, without consideration for personal safety, environmental contamination or the impacts of excessive insecticide use on consumers. A recent survey on some urban centres in Solomon Islands showed that many subsistence farmers use insecticides to control insect pests on their farms, but that many of these were either not registered, were not intended for use against agricultural pests (e.g. Icon (lamda cyhalothrin) which is registered only for indoor mosquito control) or were defunct insecticides acquired from obsolete stock (e.g. DDT (dichlorodiphenyltrichloroethane) remaining from failed rice growing enterprises of the 1980s (Teakle and Bakale 2007).

Figure 1.1: Map of Solomon Islands. (Source: Family Survival Protocol. Earth Watch Report 2013)

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1.3 SLIPERY CABBAGE, ABELMOSCHUS MANIHOT MEDICUS (MALVALES: MALVACEAE)

The slippery cabbage, Abelmoschus manihot Medicus (Malvales: Malvaceae), is a perennial shrub which is eaten as a leafy vegetable in many Pacific Island countries and territories. The archaic synonym for slippery cabbage was Hibiscus manihot L. (Linnaeus 1753, Medicus 1787) and two subspecies, A. manihot manihot Medicus and A. manihot tetraphyllus Medicus, are now recognized (Van Borssum Waalkes 1966). Abelmoschus manihot tetraphyllus Medicus. consists of the wild forms with prickly and hairy stems, pedicels and leaf veins while A. manihot manihot Medicus consists of the cultivated forms which are devoid of prickly hairs. The cultivated form is commonly referred to as Abelmoscus manihot Medicus. Within the cultivated forms of A. manihot differences in shape, size and colour of leaves, leaf stalks and stems give rise to distinctions between varieties. The stems of A. manihot varieties are typically light or dark green, brown, purple or red. Leaf shape varies from pinnate to cordate, flowers usually have distinctive sulfur yellow petals and a purple base, fruits are conical and seeds are typically about 3 mm in diameter when mature (Sowei 1993).

The world’s greatest diversity of cultivated A. manihot cultivars is found in the southwest Pacific region, particularly in New Guinea, Solomon Islands and Vanuatu. In these countries, it is quite common to find more than ten cultivars or varieties together grown in any single garden (Preston 1998). The presence of a high number of cultivated A. manihot varieties in this region has led to suggestions that the plant is of Melanesian origin. However, similar forms and genetic relatives grow in the wild in Asia, particularly in rocky areas, grassy wasteland, near streams and margins of farmland (Phillips 1998; Manandhar 2002), which may also suggest the plant to be of Asian origin. It has been suggested that A. manihot was introduced to Melanesia 3,000 years ago with the influx of migrants from Southeast Asia settling in the Pacific region (Barrau 1958; Bellwood 1978; 1982; Williams 1979; Bourke 1981; Sowei and Osilis 1995).

Despite its presumed Asiatic in origin, A. manihot is not a popular or important vegetable crop in Asia as compared to its status in the Pacific, especially among

Melanesians. Also, in Melanesia the crop has been given many different local names owing to the richness in local dialects (Lewis 2009; Henderson and Hancock 1988; Preston 1998). Although known by so many names in different dialects, the leafy vegetable crop is commonly known as slippery cabbage in Solomon Islands, aibika in PNG, bele in Fiji and as pele in most Polynesian countries and territories.

Perhaps one of the main reasons for the crop being so popular in Melanesia is the fact that it is a relatively easy crop to grow by both subsistence and commercial farmers (Kimoto et al. 1984, Bourke 1981, Sowei and Osilis 1995, Jansen 2006.). There is a high diversity of varieties in Melanesian countries; for example, 114 A. manihot accessions were once kept at Laloki Research Station in PNG (Kambuou et al. 2003, Kambuou 2005) and in Solomon Islands, 45 A. manihot accessions collected from farmers’ fields were once held at Dodo Creek Station (Eldridge and Wells 1999). Based on this diversity, the crop has been referred to as the ‘true traditional vegetable of Melanesia’ (Barrau 1958). This diversity of A. manihot is likely to have arisen through natural insect mediated cross-pollination and also the continuous selection of new varieties by farmers over time (Preston 1998).

Abelmoschus manihot is a perennial shrub that can grow up to between two and six metres tall, with soft, mostly smooth, stems that are entire or hollow (Sowei 1993). The plant is sexually hermaphrodite and pollination is usually facilitated by insects (Preston 1998). Although seed propagation is possible, farmers prefer to propagate it by woody stem cuttings when cultivating the crop, as this is the fastest, and easiest, method of propagation (Sowei et al. 1991). The period from planting to first crop harvest may take about 3 months, depending on the variety, soil fertility, temperature and light intensity under which the crop is grown.

Abelmoschus manihot can grow easily in most soil types and requires minimal agronomical knowledge, or care, to produce; thus making it a popular crop among subsistence farmers. However, for better production, the crop requires well-drained clay loam soils with pH between 5 and 7 and plenty of moisture and sunshine (Huxley 1992; Sowei 1993). A. manihot responds well to application of inorganic fertilizers with high nitrogen content and to animal manure (Kurika 1988). Growing A. manihot crops under moderate tree shade is a common practice in Solomon Islands

and there are anecdotal reports that planting under shade enhances plant growth and deters pests.

Abelmoschus manihot is usually harvested by plucking off the upper seven leaf internodes of the tender shoot tops. Crops are continually harvested from regenerating shoots when the shoot tops produce seven leaves (Preston 1998; Sowei et al. 1991; Sowei 1993; Sutherland1962).

1.4 INSECT PESTS OF ABELMOSCHUS MANIHOT IN SOLOMON ISLANDS

A wide range of insect pests attack A. manihot and they variously cause damage to leaves, shoot-tips, stems and roots (Sowei 1993). Two lists, compiled by Preston (1998), document 22 common insect pest species from five orders and a mite species, and 34 occasional insect pest species, from four orders, in PNG (Tables 1.1 and 1.2). Most of the important pests listed in PNG are also present in Solomon Islands.

The most serious insect pests of A. manihot in Pacific Island countries and territories are the slippery cabbage flea beetle, Nisotra basselae (Bryant), slippery cabbage leaf roller moth, Sylepta derogata Fabricius. (Lepidoptera: Pyralidae), slippery cabbage shoot-tip borer, Earias vitella Fabricius. (Lepidoptera: Noctuidae), and slippery cabbage green jassid, Amarasca devastans (Dist.) (Hemiptera: Cicadellidae) (Sutherland 1982, Preston 1998).

Table 1.1 Important insect pests of A. manihot in PNG (Preston 1998) and in Solomon Islands

Pest species Common name Feeding guild Damage severity

Nisotra basselae (Bryant) Coleoptera: Chrysomelidae Slippery cabbage flea beetle Leaf chewer *** Earias vittella Fab Lepidoptera: Noctuidae Slippery cabbage tip bore Tip borer *** Sylepta derogate Fab. Lepidoptera: Pyralidae Slippery cabbage leaf roller Leaf chewer *** Amrasca devastans Dist. Hemiptera: Cicadellidae Slippery cabbage jassid (1) Sap sucker *** Amblypelta lutescens papuensis Brown Hemiptera: Coreidae Banana-spotting bug Sap sucker *** Tetranychus sp. Acarina: Tetranychidae Spider mite Sap sucker *** Anomis flava Fab. Lepidoptera: Noctuidae Slippery cabbage semilooper caterpillar Leaf chewer *** Xanthodes transversa Guer. Lepidoptera: Noctuidae Slippery cabbage hairy caterpillar Leaf chewer *** Spodoptera litura Fabricius Lepidoptera: Noctuidae Cluster caterpillar Leaf chewer *** Oribius cruciatus Faust Coleoptera: Curculionidae Oribius beetles Leaf chewer ** Oribius improvidus Marshallt Coleoptera: Curculionidae Oribius beetles Leaf chewer ** Oribius spp. Coleoptera: Curculionidae Oribius beetles Leaf chewer ** Colgar chlorospilum Walker Hemiptera: Flatidae Flatid Sap sucker ** Unaspis citri Comstock Hemiptera: Diaspididae White scales Sap sucker ** Pseudaulacaspis pentagona Targ Tozz Hemiptera: Diaspididae White peach scale Sap sucker ** Parasaisstia nigra (Nietner) Hemiptera: Coccidae Black scale Sap sucker ** Thrips sp. Thysanoptera: Thripidae Thrips Sap feeder ** Valenga sp, Orthoptera, Acrididae Grasshopper Leaf chewer ** Monolepta sp. Coleoptera: Chrysomelidae Leaf beetle Leaf chewer ** Cassena intermedia Jac. Coleoptera: Chrysomelidae Short – hole beetle Leaf chewer nm Cassena sp. Coleoptera: Chrysomelidae Short – hole beetle Leaf chewer nm Empoasca quadripunctata Evans Hemiptera: Cicadellidae Slippery cabbage jassid (2) Sap sucker nm Pulvinaria psidii Maskel Hemiptera: Coccidae White louse scale Sap sucker nm

Pest status: *** = serious, **= not serious but of some importance, * = minor, nm=not mentioned

Table 1.2 Minor seasonal and occasional insect pests of A. manihot in PNG (Preston 1998) that are also present in Solomon Islands

Insect species Order Family Acrocercops cathedrae Mey Lepidoptera Gracillariidae Acrocercops sp. Lepidoptera Gracillariidae Aleurodicus dispersus Russ. Hemiptera Aphididae Anadastus albertisi Harold Coleoptera Languridae Aphis craccivora Koch Hemiptera Aphididae Aphis gossypii Glov. Hemiptera Aphididae Apirocalus ebrius Faust Coleoptera Curculionidae Aspidiomorpha australiasiae Bdv. Coleoptera Curculionidae Caridophthlmus sp. Hemiptera Pentatomidae Cicadella sp Hemiptera Cicadellidae Cleoporous hisci Gressitt. Coleoptera Chrysomelidae Colgar missior Medler Hemiptera Pentatomidae Cosmophila flava Fabricius Lepidoptera Noctuidae Cosmophila lyona Swinhoe Lepidoptera Noctuidae Dysdercus sp. Hemiptera Pyrrhocoridae Elytrurus griseus (Guerin-Meneville) Coleoptera Curculionidae Epilachna signatipennis Bdv. Coleoptera Coccinellidae Euproctis sp. Lepidoptera Lymantridae Glenea aluensis Gahan Coleoptera Cerabycidae Lampromicra sp. Hemiptera Scutelleridae Leptoglossus australis Faust Hemiptera Coreidae Nazara viridula L Hemiptera Pentatomidae Nipaecoccus viridis Newstead Hemiptera Pseucoccidae Nysius sp. Hemiptera Lygaeidae Oribius inimicus Coleoptera Curculionidae Oxycarensus bicolor Fieb. Hemiptera Lygaeidae Phaenacantha sp. Hemiptera Colobathristidae Phanaeroptera brevis Audient-Serville Orthoptera Tetigoniidae Platyacus ruralis Faust Coleoptera Curculionidae Rhyparidella sobrina Bryant Coleoptera Chrysomelidae Saissetia coffea Walker Hemiptera Coccidae Seiza sp. Hemiptera Flatidae Tectoris diophthalmus (Thunberg) Hemiptera Pentatomidae

1.4.1 AMARASCA DEVASTANS (DIST.) (CICADELLIDAE: HEMIPTERA)

Amarasca devastans (Dist.) (Cicadellidae: Hemiptera) is commonly known as the Indian cotton leafhopper, and is a common pest of cotton and other cultivated crops of the family of Malvaceae (Ullah et al. 2012; Selvaraj et al. 2011). It is a serious pest of A. manihot in Solomon Islands and attacks okra as well as peanuts, beans, and other leguminous crops. Adult leafhoppers and nymphs feed on the underside of leaves of A. manihot and can cause chlorosis and characteristic “hopper-burn”. The presence of more than three adult leafhoppers per leaf can cause severe damage, which can result in complete defoliation and death of plants, especially under dry conditions (Preston 1998). Eggs (which usually take 8 -10 days to hatch) are laid on the leaves and leaf stalks. The yellow-green nymphs develop through four instars and look similar to adults, but they are wingless and smaller than adults, which are 2 - 3 mm long. The life cycle of the leafhopper is completed in 25 to 28 days (Preston 1998). Populations are negatively affected by rainfall, which causes significant mortality of both nymphs and adults.

a) b)

Figure 1.2 a) adult Indian cotton leaf-hopper A. devastans b) Indian cotton leaf- hopper damage on a leaf of A. manihot

1.4.2 HARITALODES DEROGATA FABRICIUS (= SYLEPTA DEROGATA) (PYRALIDAE: LEPIDOPTERA)

Haritalodes derogata Fabricius (Pyralidae: Lepidoptera) is commonly known as the “cotton leaf-roller”. It is a polyphagous insect pest of many agricultural and forestry plants in Solomon Islands but associates more with A. manihot than other crops. The main host plants belong to the family Malvaceae (Zhang 1994). Adult female moths, which are yellow, lay eggs on the leaves, and larvae feed gregariously under a rolled leaf. Out-breaks can result in complete defoliation of the plants, leaving a skeleton of branches. When they pupate they usually drop into the soil or litter to complete development. The life cycle of this moth takes approximately 34 days, depending on environmental conditions (Anioke 2008). Picking caterpillars off leaves and killing them and crop rotations or extended periods between crops can be an effective way of controlling the pest.

a) b)

Figure 1.3 a) Leaf of A. manihot rolled by cotton leaf-roller caterpillar (Haritalodes derogata); b) H. derogata caterpillar in rolled up leaf; c) H. derogata adult moth

1.4.3 EARIAS VITELLA FABRICIUS (NOCTUIDAE: LEPIDOPTERA)

Earias vitella Fabricius (Noctuidae: Lepidoptera) is commonly known in Solomon Islands as slippery cabbage stem-borer. Adult moths are wedge shaped, about 12 mm long and wings are very pale brown and green. Adult female moths usually lay eggs singly and in small scattered groups on the shoot tips or under surface of leaves close to the shoot tips at night. The eggs are small, spherical in shape, bluish green in colour, with parallel longitudinal ridges projecting upward. The larvae are dark purple with white and orange marks. Egg takes 3 - 7 days to hatch. After hatching from the egg the larva crawls to the soft tip of the shoot and chews downwards, inside the stem. The larval period lasts 10 - 16 days. When closer to pupation the larva exits the stem and drops onto the soil where it pupates for 8 - 14 days (Al-Mehmmady 2000). A complete life cycle of E. vitella usually takes 22 - 35 days (French 2006).

a) b)

Figure 1.4 a) Shoot of A. manihot damaged by E. vitella caterpillars; b) Caterpillar of E. vitella feeding and tunnelling inside the stem of A. manihot; c) Adult moth of E. vitella

1.4.4 NISOTRA BASSELAE (BRYANT) (CHRYSOMELIDAE: COLEOPTERA)

The slippery cabbage flea beetle, Nisotra basselae (Bryant) (Chrysomelidae: Coleoptera) is a tiny orange and black flea beetle that is approximately 4 mm long and 2 mm wide (Table 3.1; Figure 3.3). It is the most serious pest of A. manihot in Solomon Islands, with adults causing direct damage by chewing on leaves (Figure 1.5) and larvae causing indirect damage to the harvested parts of plants by feeding on the roots. This pest was first recorded from the foliage of a Basella species (Basellaceae) and subsequently sent to the British Museum of Natural History (BMNH) where it was identified as Podagrica basselae Bryant (Bryant 1941). It was first recorded in Solomon Islands in 1982 and is presumed to have invaded from PNG (Macfarlane 1986, Henderson and Hancock 1988). a) b)

Figure 1.5 a) adults of N. basselae making shot-hole damage; b) severe shot-hole damage on leaves of A. manihot.

To date, N. basselae is only known to be present in PNG and Solomon Islands (Preston 1998; Macfarlane 1986). Another beetle in the same genus, Nisotra obliterata Jacoby, is only present on the New Guinea mainland and it causes the same type of damage to Malvaceae hosts (Jacoby 1885; Kimoto et al.1984). However, while the distribution of N. basselae in PNG (including its distribution on New Guinea Island) is not fully understood, it is believed to be widely established on many islands of the country, including Bougainville Island in the far southeast, which

is very close to Choiseul in the north of Solomon Islands. Like most flea beetles, this beetle is generally a poor flyer (Matthew and Reid 2002). For this reason, the spread of the beetle is believed to be facilitated mainly by people taking infested planting materials from infested locations to places that are free from the pest.

The first record of the beetle in Solomon Islands was in 1982 at the village of Binu in the Guadalcanal plain, on Guadalcanal Island (Macfarlane pers. Comm.). There was some confusion when the beetle was first reported in the area. Local farmers were mistaken and complained that their A. manihot crops had been attacked by an oil palm pollinating weevil, Elaedobius kamerunicus Faust (Cucurlionidae: Coleoptera) that was introduced from Malaysia to oil palm plantations in the Guadalcanal plain between 1981 and 1982. The introduction of the beneficial weevil was carried out in a co-operative effort by the Ministry of Agriculture and Lands (MAL), the Solomon Islands Plantation Limited (SIPL) and the PNG Department of Primary Industries (PNG DPI) (Caudwell et al. 2003, Greathead 1983, Macfarlane 1984, 1985, 2009 (pers. comm.). Subsequent to its initial discovery, the slippery cabbage flea beetle was collected and sent to the BMNH in London for identification. The beetle was identified as Podagrica basselae Bryant; the species previously identified in PNG (Bryant 1941). Following anecdotal reports that the true identity of the pest was Nisotra basselae (Bryant) (for example, Preston 1998; Waterhouse 1998) a second sample of beetle specimens was sent from Solomon Islands to BMNH, these again were identified as Podagrica basselae Bryant.

1.5 THE RATIONALE FOR THE STUDY

Abelmoschus manihot is an important leafy vegetable in many Pacific Island countries and territories, particularly Solomon Islands and Papua New Guinea (PNG) where most subsistence farmers cultivate it extensively (Preston 1998; Morris 2004; Fa’anunu 2009). The consumption of A. manihot has declined in many areas in recent years due to pest problems, particularly damage by the slippery cabbage flea beetle (Jansen 2006). However, the demand for A. manihot in suburban and urban areas has escalated as the population in these areas continues to increase. In trying to meet the high urban demand for A. manihot farmers supplying urban markets have

been pressured to provide marketable A. manihot despite the significant pest problems that they face. In addition to high local demand, A. manihot has potential for export to niche markets in Australia and New Zealand where Pacific Island communities exist (Fa’anunu 2009).

The main insect pest of A. manihot that farmers are having problems with is the slippery cabbage flea beetle. No previous detailed study has been carried out to investigate its biology and ecology; such a study is essential in order to develop effective management approaches for the pest. As a result, most farmers that produce for urban markets have relentlessly applied insecticides in their attempts to control the pest and meet market demands for the crop. Scores of farmers have complained that the insecticides that they have been using for some time are no longer effective and expensive (Jansen 2006). Therefore, this study investigated some basic aspects of the biology and ecology of N. basselae that can be used to help farmers and researchers manage the pest in a sustainable manner.

The focus of the study was to: a) determine the taxonomic status of the chrysomelid beetle that is commonly known as “slippery cabbage flea beetle” in Solomon Islands, b) investigate the life cycle of slippery cabbage flea beetle on different species of Malvaceae host plants, c) study the seasonal abundance of slippery cabbage flea beetle in the field in Solomon Islands, d) investigate host plant relationships between slippery cabbage flea beetle and its plant host, A. manihot, e) study the oviposition ecology of slippery cabbage flea beetle, f) Investigate the distribution of slippery cabbage flea beetle between plants grown in full-sun and shaded conditions and between herbivore infested and undamaged host plants.

Slippery cabbage flea beetle is known to be present in most of the provinces of Solomon Islands, except Temotu and Renbel on the far southeast of the archipelago (Figure 3.1). The pest poses both national and international quarantine threats to localities that are still free from infestation. Understanding the pest identity, biology and ecology status will help to develop a sustainable management plan and provide essential information that might help managers prevent its further incursion within Solomon Islands and its invasion of neighbouring countries.

CHAPTER TWO: GENERAL MATERIALS AND METHODS

2.1 INTRODUCTION

This chapter presents the materials and methods that were common among experiments reported in several chapters of the thesis. These include descriptions of study sites, techniques for field preparation, laboratory insect rearing methods, soil preparation for nursery and experimental purposes, nursery practices and host plant materials and rearing facilities.

2.2 EXPERIMENTAL SITES

Field experiments in this study were undertaken on Guadalcanal Island at the village of Tutuva (9 26’ S; 160 6’E), situated 20 km east of Honiara (9 25’S; 160 2’E) on the northwest coast of the Guadalcanal plain. Guadalcanal Island is part of the southern chain of the Solomon archipelago and is the largest of all the islands in Solomon Islands, with land area of 5,302 km2. It is about 160 km long and 45 km wide at the centre.

Rainfall and temperature records were not available for Tutuva, but those routinely collected at nearby Henderson Airport Meteorological Station were representative of the weather conditions experienced at the experimental sites.

Laboratory and shade house experiments were conducted at the MAL precinct in Honiara.

Tutuva experimental site

Lungga R

Figure 2.1: The Guadalcanal Plain and location of the Tutuva experimental site. The light grey shaded areas are oil palm plantations. Source: Pacific Economic Bulletin Volume 25 number 1 © 2010 the Australian National University.

2.3 PREPARATION OF FIELD SITES

Field sites were prepared manually and involved clearance of vegetation (grasses, shrubs and secondary forest trees) with machetes and axes. All tree stumps were uprooted and slashed plant material was collected and heaped at the edges of plots, leaving the field fairly free of vegetation before A. manihot cuttings were planted.

2.4 PREPARATION OF SOIL FOR EXPERIMENTS AND CULTIVATION OF PLANTS

The soil media used in all experiments carried out in the laboratory and for raising plants in the nursery was collected from beneath rain-trees at the banks of the Lungga River, on the eastern fringe of Honiara. This soil is a mixture of dark alluvial topsoil and decomposed humus. The soil was heated over an open fire and then stored in cool and dry conditions before use in experiments or in the nursery. Prior to use in experiments soil was broken up and sieved through a fine mosquito proof

stainless steel mesh (1 mm x 1 mm). Soil used for raising plants in the nursery constituted coarse soil particles that were passed through a courser steel wire mesh (6 mm x 6 mm).

2.5 HOST PLANTS

All the plants used in both indoor and outdoor experiments were grown in a specially constructed nursery (5 m x 20 m), which had two equal sized compartments. In one compartment, a wooden framed roof covered by strips of green nylon shade-cloth (Site Shade Company, Australia) blocked out 70% of incident light while in a second compartment there was no roof and seedlings were exposed to incident sunlight. Plants were raised in poly-bags (15 cm diameter and 20 cm tall) on 1 m raised benches under conditions of shade or full natural sunlight as required for experiments.

2.5.1 ABELMOSCHUS MANIHOT

Two varieties of Slippery cabbage were used in the various experiments. The varieties were the “Noodle-type” and “Broad-type”. The Broad-type variety has distinctive broad shaped leaves, while the Noodle-type variety has finger like slender leaves. The leaf area of the Broad-type variety was approximately four times that of the Noodle-type variety.

Propagated cuttings of both varieties were purchased from farmers on the Guadalcanal Plain and were approximately 2.0 - 2.5 cm diameter and 30 cm long when planted. The cuttings were taken from the top end of plant stems, or branches, after the growing tips were cut approximately 10 cm below the tip. A single cutting was planted into a hole made in the soil of a poly-bag and then pushed down to a depth of 15 cm. Hence, for each of the 30 cm cuttings, half (15 cm) was buried beneath the soil in poly bags, while the other half was exposed to the air and subsequently produced new shoots. Several shoots typically sprouted on each cutting, but only one was allowed to remain on each potted plant; the remainder were

removed manually. Fertilizer (NPK 5:7:4, 250 kg ha-1) was administered to all pot plants at planting and every three months thereafter. Urea (75 kg ha-1) was applied one month after planting and at monthly intervals thereafter. Plants were irrigated by hand twice daily, in the morning (08.00 - 09.00) and in the afternoon (15.00 – 16.00).

When cuttings were planted in the field, plant spacing distances were 50 cm x 50 cm. Holes were made in the soil using a sharp stick and two cuttings were planted at a depth of 15 cm into each hole, leaving 15 cm of the cuttings exposed to the air to sprout. One of the cuttings planted in each hole was removed about three weeks after planting, leaving only one plant, which was pruned to leave one shoot. Fertilizer (NPK 5:7:4, 250 kg ha-1) was administered to all plants at planting and every three months thereafter. Urea (75 kg ha-1) was applied one month after planting and at monthly intervals thereafter. Plants were manually irrigated by a casual labourer on site in the morning between 08.00 and 09.00 and in the afternoon between15.00 and 16.00. Weeding in the field was carried out fortnightly throughout the planting period.

2.5.2 HIBISCUS TILIACEUS

The cuttings of H. tiliaceus that were propagated and used in the experiments were all from scion branches of a wild plant that was grown beside the Mataniko River in central Honiara. Each of the cuttings was 2.0 – 2.5 cm diameter and 30 cm length. Single cuttings were planted in prepared soil and fertilized and irrigated in the same manner as described for A. manihot.

2.5.3 ABELMOSCHUS ESCULENTUS

Plants of an unknown variety of Okra (A. esculentus) were grown from seeds bought from a retail shop in Honiara. Single seeds were buried 2 cm deep in prepared soil in polythene potting bags and fertilized and irrigated in the same manner as described for A. manihot.

2.6 INSECT CULTURES

Laboratory insect colonies were established as required during the course of the study. Laboratory colonies consisted of approximately 1000 adult N. basselae, which were collected from insecticide-free A. manihot gardens in and around Tutuva village. Insects were collected from plants using an aspirator and then transferred into a nylon mesh cage (40 cm x 40 cm x 40 cm). Colonies were supplied with fresh nursery grown A. manihot shoots daily; foliage was kept fresh by placing the plant stem in a glass bottle (length 20 cm x diameter 3 cm) filled with rain-water. A piece of cloth was placed in the mouth of the bottle to hold the stem in place and to prevent the insects from entering and drowning. The N. basselae population collected and maintained in 2006 was fed with fresh leaves of the Broad-type variety of A. manihot. Humidity in the cages was maintained by applying a fine mist of rainwater using a hand held sprayer.

The laboratory was well ventilated with large louvre windows covered with mosquito proof screen nets. Natural light in the laboratory was supplemented by two banks of double florescent light bulbs, which were kept on a L: D 12: 12 cycle. During the course of the study, average maximum and minimum temperatures were 31.1 ± 0.2oC and 25.7 ± 0.1oC respectively while average maximum and minimum relative humidities were 90.2 ± 0.6% and 72.9 ± 1.5%.

CHAPTER THREE: TAXONOMY OF NISOTRA BASSELAE (BRYANT)

3.1 INTRODUCTION

The slippery cabbage flea beetle is a member of the order Coleoptera and family Chrysomelidae. Members of this family feed on a wide range of plants (Jolivet and Hawkeswood 1995). Within this family is the subfamily Galerucinae, which contains the tribe Alticini and all other tribes that are commonly known as the flea beetles (Matthews and Reid 2002). Beetles of this tribe are called ‘flea beetles’ because of their ability as adults to jump into the air in a repulsive movement using a special metafemoral spring mechanism (Furth 1982).

Among the flea beetles are two closely related genera, Podagrica and Nisotra, which feed mainly on plants within the Malvaceae (Matthews and Reid 2002). Adults of these beetles feed on the leaves and leave behind trails of characteristic ‘shot-hole’ damage in the leaves, while their larvae feed on the roots of the host plants (Matthews and Reid 2002).

The flea beetle analysed in this chapter is commonly known in Solomon Islands as the “slippery cabbage flea beetle”. The beetle is native to Papua New Guinea (PNG) (Bryant 1941, Macfarlane 1986, Henderson and Hancock 1988), where it is commonly known as the Aibika flea beetle (Kimoto et al. 1984).

The Aibika flea beetle in PNG was first recorded as Podagrica basselae Bryant (Bryant 1941). Its distribution there covered the Papuan mainland and spread throughout the country, as far as Bougainville Island in the far south east, close to the border with Solomon Islands (Kimoto et al. 1984).

In 1981 a flea beetle that resembled P. basselae in PNG was reported feeding on A. manihot in the Guadalcanal Plain on Guadalcanal Island of Solomon Islands (Macfarlane 1986, Henderson and Hancock 1988). This was the first time that such a beetle damaging the crop was recorded in Solomon Islands and so sample specimens of the beetle were rapidly collected and sent to the British Natural History Museum (BNHM) in London for identification. The identity of the beetle was

confirmed by BNHM as Podagrica basselae Bryant, as already recorded in PNG (Macfarlane 1986, Macfarlane pers. Comm.).

Since its introduction to Solomon Islands, the beetle has spread through most of the islands, except those in Temotu and Renbel provinces which are situated on the east-south fringe of Solomon Islands according (pers. Comms. Chief Field Officers of Ministry of Agriculture based in the provinces of Solomon Islands) (Figure 3.1).

Before this taxonomic study of the beetle was undertaken, the identity of slippery cabbage flea beetle in Solomon Islands had been a subject of much contention and confusion. The name Nisotra basselae (Bryant) began to be used for the insect that had been officially described as Podagrica basselae in both 1941 and 1981 (see Preston 1998 and Waterhouse 1998). Therefore, a second sample of specimens was collected from the Guadalcanal Plain (Guadalcanal of Solomon Islands) in 2005 and resent to the BNHM in London for the second time; again the identity was confirmed as Podgarica basselae Bryant.

Nisotra and Podagrica are two very similar genera within the tribe Alticini (Foudras 1859; Baly 1864). The genus Nisotra, with the type species: Haltica gemella Erichson, 1834, was first collected from Sumatra in Indonesia (Baly 1864). This first collection is currently archived in the Museum of Berlin in Germany. Comparisons of morphological characters of these two genera have been described (Scherer 1969; Konstantinov and Vandenberg 2000). Apart from other morphological characters, which they shared with other members in the family and tribe, these two genera have the following morphological characters in common: 9 - 11 antennal segments; hind femur void of apical spines; hind tibia with simple apical spines; claw segment of hind tarsus weakly dilated; procoxal cavities closed behind; mid and hind tibia with a subapical excavation; and pronotum with two longitudinal sulci at the basal margin of the pronotum (Scherer 1969).

An important feature that definitively separates Nisotra from Podagrica is the presence of two longitudinal sulci at the anterior margin of the pronotum in Nisotra; these are absent in Podagrica (Foudras 1859; Baly 1864; Scherer 1969). Other differences include a round-ovate body shape in Nisotra but a longitudinal-ovate body shape in Podagrica, and elytra confusedly punctured or in narrow more or less

correct rows or double rows in Nisotra while punctures in the elytra tend to form exact single or double rows in Podagrica (Scherer 1969).

This chapter describes work that was undertaken to determine the identity of the slippery cabbage flea beetle in Solomon Islands through examination of morphological characters. The approach taken to identify the beetle involved the comparison of beetle specimens from Solomon Islands and PNG and comparison of Solomon Island collected material with established taxonomic keys for Nisotra and Podagrica.

3.2 MATERIALS AND METHODS

3.2.1 SOURCE OF ADULT AND IMMATURE BEETLE SPECIMENS

Adult beetle specimens (50 - 100 per site) were collected from slippery cabbage plants (A. manihot) at three sites in PNG and at four sites in Solomon Islands in 2006 (Figure 3.1). Specimen samples from PNG were collected at the following locations on the dates detailed: (i) Mis village (Madang Province), collected on 22nd August 2006; (ii) Amanab village (Western Province), collected on 7th September 2006; and (iii) Lido (Vanimo) village (Western Province), collected on 9th September 2006. Specimen samples from Solomon Islands were collected at the following locations on the dates detailed: (i) Uefi Resort - Marovo (Western Province), collected on 19th November 2006; (ii) Auki town (Malaita Province), collected on 12th November 2006; (iii) Tutuva village (Guadalcanal Province), collected on 10th November 2006; and (iv) Ngasukana village (Choiseul Province), collected 9th December 2006. All beetle specimens collected were preserved in 100% ethanol and shipped to the University of Queensland for laboratory examination.

Initial examination of the collected samples involved a detailed study of seven male and seven female specimens collected from each of the three PNG and four Solomon Islands collection sites. Specimens were examined particularly for the following characters: (1) presence or absence of two additional longitudinal sulci at the anterior margin of the pronotum; (2) whether the beetle body shape was round- ovate or longitudinally ovate; and (3) whether elytra were confusedly punctured or

whether punctures formed exact single or double rows. The sizes of beetles at different sites were then determined from subsamples of seven males and seven females of each site. Adult beetle specimens were measured to determine the average overall body sizes. Length and width of measurements of each sampled beetle were obtained using a binocular microscope (x10) with an inbuilt graticule to determine the average body size of males and females. A further subsample of seven males and seven females (a sexed couple representing each site) were examined for the morphological description of beetles.

Immature beetle specimens (including eggs, larvae and pupae) were obtained from laboratory cultures reared in Honiara in 2006 (see Chapter 4 for details of the rearing methods). Eggs were collected directly from females that laid them in the soil in laboratory cages while larvae were fed and reared on A. manihot roots and then sacrificed by drowning in ethanol (100%). Five specimens of each larval instar (1st, 2nd and 3rd) and five pupae were collected and examined for morphological description.

3.2.2 STANDARD MEASUREMENTS AND INDICES

It is important to note that the description of slippery cabbage flea beetle, in terms of integument sculpturing, coloration, and genitalia characters used in this chapter is based on a descriptive standard by Samuelson (1973). The terminologies of beetle morphological characters used in the description analysis are taken from Lawrence et al. (2000).

Measurements are a crucial part of the description of the beetle. Measurements in the study were obtained from a microscope that was mounted with ocular linear scales with 100 micron units. These measurements were taken initially as ocular linear scale readings and then subsequently converted to metric units by calibration against a linear scale (0.5 mm graduation) viewed under each magnification of the inbuilt ocular-mounted linear scale. Length and width measurements were taken on various parts of the head, pronotum, elytra, and segments of sternum, leg parts and the abdomen ventrites. Indices were calculated from microscope measurements without conversion to metric units.

3.2.3 STATISTICAL ANALYSES

Statistical comparisons of different measurements were made by ANOVA or t-tests as appropriate. All statistical tests were performed in Statview 5.0 (SAS Institute (1999).

PNG

SolomonSolomon IslandsIslands

C B

Figure 3.1 Map of Solomon Islands and PNG, showing locations where beetle specimens were sampled from A. manihot (marked with red stars). Islands and provinces circumscribed by the red boundary (A) area currently infested by N. basselae; (B and C) areas which are not yet infested are marked in green (reported by Chief Field Officers in Solomon Islands in 2011)

Table 3.1 Definitions of anatomical descriptors used in the description.

Anatomical descriptor Definition body length the maximum linear dimension of the insect body, measured from the anterior-most part of head to the posterior-most part of elytron body breadth the maximum transverse dimension of the insect body, which is the broadest part of the insect elytra head breadth the maximum transverse dimension of the head, measured between the lateral-most part of each eye antennal socket the transverse diameter of socket; measurements are taken across the aperture and do not include the margins of the antennal socket interantennal space the narrowest transverse space between inner margins of antennal sockets eye the maximum diameter of an eye; measuring between margins across the greatest diameter orbit the narrowest transverse dimension between the lateral margin of antennal socket and the inner margin of the eye interocular space the narrowest transverse dimension between eyes eye breadth the transverse diameter of an eye interocular index the product of interocular space x 100/eye pronotal length maximum linear dimension of thorax; measured along the imaginary mesal line of the pronotum pronotal index pronotal length x 100/pronotal breadth elytral length maximum linear dimension between elytra base at scutellum and elytral apex

3.3 RESULTS

3.3.1 DESCRIPTIONS

Based on morphological analysis, it was confirmed that all the flea beetles sampled from the foliage of A. manihot both in Solomon Islands and PNG were from the same genus. All the specimens examined had two additional longitudinal sulci on the anterior margin of the pronota, round-ovate body shapes and elytra with confused punctures, indicating that the beetle specimens examined were all from the genus Nisotra.

The specimens examined in this study were compared against archived duplicates of specimen samples that were sent from Solomon Islands to BNHM in 2005 and which were identified as Podagrica basselae Bryant. These duplicate specimen samples were archived in the Entomology Laboratory in Honiara, Solomon Islands. No morphological differences between the specimens were found, confirming that the previous identification, which placed the slippery cabbage flea beetle in the genus Podagrica, was erroneous.

Since the genus has been confirmed to be Nisotra, this study from now onwards refers to the slippery cabbage flea beetle as Nisotra basselae (Bryant).

3.3.1.1 ADULT DESCRIPTION

3.3.1.1.1 BODY SHAPE AND MORPHOLOGY

The body length of adult N. basselae, irrespective of sex, was twice the size of the body width, resulting in convexed ovate-rounded body. (Figure 3.4 a, b and c; also see Table 3.2). Differences in sexual dimorphism between male and female beetles were identified. Adult females (n = 7) were significantly longer (mean (±SE), (3.99 ± 0.04 mm) than males (2.56 ± 0.04 mm); F1, 96 = 78.05; P < 0.05). Adult females (n = 7) were also significantly broader (mean (±SE), (3.61 ± 0.03 mm) than adult males (2.19 ± 0.02 mm);

F1, 96 = 61.70; P < 0.05) (Table 3.2). Furthermore, there was a difference between sexes on the 5th abdominal ventrite, where the female ventrite was simple, even and smooth

curved at the apex (Figure 3.3 a), c), e)), in male specimens it was truncated and lobed at the apex (Figure 3.3 b), d) and f)).

3.3.1.1.2 COLOUR

Differences in coloration of adult beetles are sometimes used for differentiating the sexes of a species (Peterson et al. 2007). However, for N. basselae, there was no colour difference between sexes in adults. The colour of head, pronota, legs, antennae and abdomen of both sexes were distinctly yellowish brown and elytra of both sexes were metallic black.

Table 3.2 Mean (±S.E) body lengths and widths of male and female N. basselae collected from seven sites in PNG and Solomon Islands, n = 7 beetles of each sex per site. There were no significant size differences among beetles collected from the different sites (length (F6, 91 = 0.333, P > 0.05); width (F6, 91 = 0.212; P > 0.05).

Female Male Locality length (mm) width (mm) length (mm) width (mm) Mis village (PNG) 3.94 (±0.05) 2.61 (±0.06) 3.52 (±0.05) 2.00 (±0.04)

Amanab village (PNG) 3.99 (±0.10) 2.47 (±0.13) 3.73 (±0.06) 2.36 (±0.06)

Lido village (PNG) 3.92 (±0.13) 2.67 (±0.11) 3.64 (±0.06) 2.21(±0.05)

Uefi Resort (SI) 4.03 (±0.06) 2.48 (±0.09) 3.64 (±0.06) 2.21 (±0.05)

Ngasukana village (SI) 3.97 (±0.13) 2.54 (±0.03) 3.61 (±0.05) 2.23 (±0.06)

Tutuva village (SI) 4.02 (±0.07) 2.56 (±0.11) 3.56 (±0.09) 2.10 (±0.05)

Auki village (SI) 4.09 (±0.09) 2.60 (±0.13) 3.60 (±0.09) 2.22 (±0.07)

5 Adult female beetle Adult male beetle 4.5 a 4 b

3.5 (mm)

2.5 N.baselae c c 2

1.5

1 Measurement of adultofMeasurement 0.5

0 Adult beetle body length Adult beetle body width

Figure 3.2 Mean lengths (±SE) and mean widths (±SE) of male and female beetles.

Female beetles, significant longer (F1, 96 = 61.7, P < 0.05) and significantly wider (F1, 96 = 70.1, P < 0.05) than male beetles.

2.0 mm

a) b)

Simple apex

Truncated apex c) d)

T VIII

T IX

S VIII AD S IX

e) f)

Figure 3.3 Morphological characters of male and female genitalia of N. basselae. a) dissected ventral abdominal surface of female; b) dissected ventral abdominal surface of male; c) abdominal ventrites of intact female, showing the simple and evenly curved apex of ventrite V; d) abdominal ventrites of intact male, showing the truncated and bilobed apex of ventrite V; e) female genitalia featuring tergites VIII (T VIII) and IX (T IX), and sternites VIII (S VIII) and IX (S IX) (stylus); f) male gentalia, showing the drawn out aedeagus (AD) and sperm sacs (SS).

1.0mm

Figure 3.4 Nisotra basselae habitus: a) dorsal view; b) ventral view; c) lateral view.

3.3.1.2 DESCRIPTION OF SPECIFIC BODY PARTS

3.3.1.2.1 HEAD

The head of N. basselae (Figure 3.5): is prominently large, convex, strongly protuberant with entire eyes (ey) located on the sides. Eyes iridescently metallic black; distance between eyes equivalent to two and a half times the breadth of the eye and equivalent to two times the length of first antennal segment. Antennae positioned between and below the eyes (refer to position of antennal socket (as) (Figure 3.5). Face of the insect inclined forward (less than 45°), head not abruptly constricted at posterior (Figure 3.6 a). Vertex (vx) smooth, shiny, broadly convex and impunctate. Trichobothrial setae on supraorbital punctures (so) between posterior margin of eyes and frontal ridge and vertex intersection; vertically above antennal insertion. Interantennal space (asp) about two and a half times as broad as transverse diameter of antennal sockets; space flat with central ridge and frons moderately narrow, cut-off dorsally by postantennal calli, forming a triangular shape ridge with clypeus (cl). The orbit (ob) as broad as antennal socket. Depth of gena (gn) half that of eye. Clypeus slightly convex with anterior margin sinuated. Antenna with eleven segments (Figure 3.6 c); the first five segments fulvous and sparsely pubescent, the remaining six segments dark and pubescent. First antennal segment thickened and twice as long as second segment; second segment shorter but thicker than third, fourth and fifth, all of which more or less similar. Antennal segments from sixth to tenth similar, apices twice as broad as bases. Eleventh segment measured one and a half times longer than the 6th – 10th segments, with mesal broadened and pointed at apex. Length of the antennae half-length of entire insect body, stretching to approximately a third elytra length. Antenna length approximately twice as long as head length. Fronto-clypeus suture distinctly impressed. Antennal insertion located anterior to line joining eye anterior edge. Labrum (lm) slightly transverse, with anterior margin convexed and sinuated. Mandibles (mb) (Figure 3.6 e)) with apices bidentated. Distal labial palp (Figure 3.6 b)) conically pointed at apex; mentum transverse, mesally broadened, about 2 x submentum length. See maxilla in Figure 3.6 d).

1.0 mm

1mm 2mm

a) b) c)

2mm 2mm 2mm

d) e) f)

Figure 3.6 Mouth parts of N. basselae; a) lateral view of the head; b) labium; c) antennae; d) maxilla; e) mandible; f) labrum

3.3.1.2.2 PRONOTUM

Pronotum of N. basselae (Figure 3.7) is transverse, approximately half as long as wide (ratio of pronotal length to pronotal width = 2.1 : 4.1). It widens at middle, hence making lateral margins slightly rounded. Its length measures a quarter of insect entire body ength (mean pronothal length and mean body length ratio = 1: 4). Its widest width slightly narrower than elytral widest width (mean ratio of pronotal base to elytra base = 1 : 1.4). It extends to ventral portion of prothorax on both sides as hypomera (Figure 3.9 a), and lateral to prosternum. Hypomeron continued to extend behind procoxae (Figure 3.9 a). Base of pronotum with a pair of longitudinal sulci, extending anteriorly about a quarter of length of pronotum; anterior margin of pronotum with a pair of longitudinal sulci. Longitudinal sulci contain four small fine pits (punctures), extending posteriorly a third of pronotal length; further fine pits extending alongside pronotal anterior margin (5th and 6th) towards anterolateral angulated corners and continue alongside lateral pronotal carina or margin (7th to 14th) towards base; all anterolateral and basalateral angle corners of pronotum with trichobothrial setae. Scutellum small, smooth and triagularly shaped.

Longitudinal groove at anterior margin

0.68mm 1.0 mm

Figure 3.7 Pronotum of N. basselae showing the longitudinal grooves at the anterior margin.

3.3.1.2.3 STERNUM

The sternum of N. basselae comprises of pro-, meso- and meta-sternum; all fulvous and with sparsely recumbent pubescence. Prosternum infront of coxa even and moderately convexed. Procoxal cavities externally closed from behind by postcoxal projections which connect by overlap to lateral apex of prosternal process. Procoxal cavities slightly transverse (Figure 3.8).

psp ps pc

a) 1 mm b)

pc c)

pp psp

Figure 3.8 Nisotra basselae prosternal and head parts; a) is the front view b) is the caudo ventral view of prothorax, showing closed procoxae (pc), prosternum (ps), prosternal process (psp), and postcoxal projection (pp); c) is the lateral view of head and anterior part of prothorax - showing the head parts, the transverse shape of procoxal projection, the overlap of prosternal process and the postcoxal projection which closed the procoxal cavity.

Meso-ventrite transverse with anterior margin slightly concaved and sinuate (Figure 3.9 a). Meso-ventral process (posterior projection of ventrite extended and connecting anterior projection of meta-ventrite) concaved (Figure 3.9 a)). Meso-coxal cavities laterally opened (meso-ventrite (=mesosternum) but not connecting metaventrite (=metasternum) laterally); meso-metaventral junction convexed and overlapping metaventral process; metacoxae narrowly separated; mesepisterna distinctly separated at midline; mesocoxal cavities not partly closed by metepisterna; mesocoxal cavities not oblique; mesocoxal cavities slightly transversed; mesosternum with sparse recumbent pubescence.

Metacoxae narrowly separated by less than longest coxal diameter. Metacoxae not extending laterally to meet elytra or sides of the body; metacoxial plate well developed messally, weak laterally; metaventral discrimen moderately long (terminated halfway); metaventrite slightly shorter than 1st abdominal ventrite; metendosternal laminae well developed; apical portion of metendosternite (only slightly emarginated; broadly, deeply and emarginated); metasternum with sparsely recumbent pubescence.

1.0 mm

a) 1mm b) a) 1.0 mm b)

Figure 3.9 Habitus of N. basselae. Ventral and dorsal views.

3.3.1.2.3 LEGS

The legs of N. basselae fulvous. Meta-femur much broader (evenly curved anteriorly) and slightly longer than meso- and pro- femors. Endo-sclerite meta-femoral spring mechanism translucent on metafemoral surface. Length of femora slightly longer than tibia in all legs; a ratio of 1.03 to 1.00. Ratio of meta-femora length to width = 2.55 : 1.00. Apex of tibia (pro-, meso- and meta- inclusive) fringed with dense stiff setae with a spur produced ventrally. Third tarsal segments of all legs (pro-, meso-, and meta-) deeply bilobed and pubescent. First segment of male anterior tarsus widened and more pubescent. Mid and hind tibia with subapical excavation.

a) b)

1.0 mm 6.8mm

c) d) e)

41 c.

3.3.1.2.4 WINGS

The elytra of N. basselae are metallic black and covered with numerous fine impressions, confusedly arranged (Fig 3.11 a). Base of elytra slightly broader than base of prothorax; not overlapping each other. Hind-wings translucent and well developed. Hind wings with only one anal cell present (See Figure 3.11 b), and ephipleuron stretched through to elytra apex.

a) 1.0 mm

. b)

Figure 3.11 Wing of N. basselae: a) dorsal view of elytron (right hand side); b) hind wing.

3.3.1.2.4 ABDOMEN

The abdomen of N. basselae has five subglaborous ventrites. Ventrite I much longer than ventrite II. Ventrite V simple in female (Figure 3.10 a) but truncated and lobed in male (Figure 3.10 e). Abdominal tergites covered by elytra apex; tergite IX in male emarginated; anterior edge of sternite VIII in male with medial strut.

a) b) c) d)

1.01mm mm

e) f) g) h)

Figure 3.12 Genitalia of N. basselae: a – d, ventral view of female genitalia; A. consists of the Ventrite V, part of Sternite VIII, Tergite VIII, Tergite IX; b. Sternite VIII; c. Tergite VIII, which is pubescent at apex and showing pygidium - the longitudinal median groove; d. spermatheca (magnification = x400); e – h, show parts of male genitalia; e. shows ventral view of male Ventrite V - truncated and lobed; f., g. and h are the ventral, A. b. dorsal and side view of the aedeagus, respectively.

3.3.1.3 IMMATURE STAGES

3.3.1.3.1 EGG

Nisotra basselae egg is elongate-ovate and cylindrical; length and width of 0.84 mm and 0.23 mm, respectively (Figure 3.13). Fresh egg appears golden-yellowish but gradually turns light yellowish and then to deep orange before hatching.

Figure 3.13 Nisotra basselae eggs laid in a batch. Average length of an egg = 0.84 mm.

3.3.1.3.2 LARVA

Nisotra basselae larvae develop through three larval instars. All larval stages exhibited three pairs of thoracic legs and a head capsule from which a pair of minute papillae-like single segmented antennae emanated. In all instars the abdomen was devoid of legs with a flattened rounded plate and minute setae covered the body and head. First larval instar head capsule width measured 0.25 mm and there was a pair of egg bursters on the mesonotum and metanotum. Second larval instar head capsule width measured 0.35 mm; however, egg bursters were no longer present and body colour dull yellowish. Third larval instar head capsule width measured 0.45 mm; body of the larva larger in size and dull light yellowish, milky and translucent. By comparison, the mandibles of the third instar larva were blunter than in first and second instars, which had sharp, serrated mandibular teeth. Pre-pupation is defined as the immature stage when the larva stops

feeding, burrows into the soil, makes its soil chamber and rests until it pupates. At this stage the insect possessed all the characteristics of a third larval instar, but was generally larger in size, turgid and with a glassy dull whitish brown colour. Also at this stage, it was common to find the insect constricting the ventral mid-section of its

abdominal segments and forming a “u” shape in the pupation chamber. 25.0 µm

2.0 mm

45.0 µm 2.0 mm

Figure 3.14 Larvae of N. basselae: a) Lateral view of first instar larva showing the position of egg bursters on meso- and meta- thoracic segments; b) shows the dorsal view of a plate that all larval stages have on the last abdominal segment,; c) shows the larval head capsule; d. shows pre-pupal larval stage.

3.3.1.3.3 PUPA

Nisotra basselae has exarate pupae (see Figure 3.13 a-d) covered with scattered hairs. Anal segment tipped with two spines. Pupa appeared wholly creamy white in colour at

the onset of pupation (Figure 3.13 a). Eyes, mandibles, elytra and hind wings became visibly pronounced at late stage.

20.0mm 2.0 mm a) b)

c) d)

3.4 CONCLUSIONS

Analysis of the insect specimens collected from foliage of A. manihot in both Solomon Islands and PNG confirmed that all specimens had the same morphological characteristics (see Section 3.3). By systematically comparing and matching the established descriptions of Podagrica (Foudras 1859) and Nisotra (Baly 1864) along with the latest taxonomic keys of Alticini (Scherer 1969) to the beetle specimens collected in this study, there is sufficient evidence to confirm that the beetle commonly referred to as the Slippery cabbage flea beetle and Aibika flea beetle in Solomon Islands and PNG, respectively, is the same species belonging to the genus Nisotra.

Below are the descriptions that explicitly separate the two genera (Scherer 1969 P. 22). Beetle specimens collected from both Solomon Islands and PNG possessed characters of the genus Nisotra as described below:

82 (87). Pronotum with short longitudinal impressions on each side of middle on anterior or basal margin, or on both of them.

83 (84). These longitudinal impressions on anterior and basal margin (the latter can be much reduced); body round-ovate. Elytra confusedly punctured or in narrow more or less correct rows or double-rows………...... Nisotra”

84 (83). Longitudinal impressions only on base of pronotum; body longitudinal ovate.

85 (86). Clypeus broad; antennal calli absent; hind femur distinctly thickened. Punctuation of elytron with tendency to form exact single or double rows...... ”Podagrica”

The following extract is taken directly from the original description of the beetle in question by Bryant in 1941 (Bryant 1941, p. 100):

”the posterior margin having on either side near the hind angle a deep short perpendicular impression, while behind the eyes on the anterior margin a very short perpendicular impression. Elytra metallic blue, slightly broader than the base of the

prothorax, nearly parallel-sided, gradually rounded to the apex, finely punctuate-striate, in irregular double rows, the intervals between the striae at the side slightly costate”

Information from literature above confirmed that the original description of the flea beetle in question conducted by Bryant (1941) also matches all the characteristics of the genus Nisotra as described by Foudras (1859) and Scherer (1969). It is therefore concluded that the placement of the flea beetle into the genus Podagrica (Bryant 1941) was an error. The change in usage of the genus to describe the flea beetle in recent years (i.e. adoption of Nisotra basselae (Bryant)) (Waterhouse 1998; Preston 1998) is likely due to a realization that this error had been made but there is no recorded study that explicitly identifies this mistake before the current study. Results of morphological analysis in this study categorically confirm that the slippery cabbage flea beetle is a species of the genus Nisotra. The species, “basselae”, was mentioned for the first time in the original description without further systematic analysis with other congeneric species. It is therefore an arbitrary given name to the beetle, perhaps in respect of the plant, Bassela species (Basellaceae), from which the type specimens were collected (Bryant 1941).

This study presents systematic morphological analysis of the beetle that usually causes severe damage to A. manihot in both PNG and Solomon Islands and confirmed the beetle in both countries to be the same species, Nisotra basselae (Bryant) and not Podagrica basselae Bryant as previously asserted (Bryant 1941).

CHAPTER FOUR: LIFE CYCLE AND SEASONAL PHENOLOGY OF NISOTRA

BASSELAE IN SOLOMON ISLANDS

4.1 INTRODUCTION

Many species of the genus Nisotra associate with plants of the family Malvaceae, some of which are important agricultural crops (Jarvis 1919; Rawat and Singh 1971; Macfarlane 1986; Preston 1998; Oke and Odebiyi 2008). Adults of this genus usually feed on plant foliage, while their larvae feed on the roots of the same type of host plants (Jolivet and Hawkeswood 1995). Adults feed on leaves by making shot-holes which can result in complete loss of an entire crop, particularly when no form of control measure is applied. Larvae feeding on roots can also have an impact on the health of the plants, but this is difficult to quantify economically, and no such study has yet been performed.

Life cycles of some Nisotra species have been studied in different places and at different times in the past. An early study was conducted in Queensland (Australia) on N. breweri Baly when it was reared on rosella (Hibiscus sabdifera L.) under an unmonitored ambient temperature regime (Jarvis 1919). Since then, another life cycle study was carried out in the Philippines, also under unspecified environmental conditions, when N. gemella (Erichson) was reared on okra (A. esculentus) and rosella (H. sabdifera) (Reveche 1922). The life cycle of N. orbiculata (Motschulsky) was determined on okra in Sehore (Madhya Pradesh, India) at temperatures between 28 – 30oC (Rawat and Singh 1971), and on mesta (Hibiscus cannabinus L.) in the laboratory in West Bengal (India) at temperatures between 31.3oC and 32.0oC and a relative humidity of 82% (Pandit and Chatterji 1978). Another life cycle study on the genus was carried out on N. sjostedti (Jac.) in Nigeria. These insects were reared on A. esculentus at temperature between 24oC and 28oC and 79% and 92% relative humidity (Adenuga 1970). Apart from these studies, there are no other studies documenting life cycles in this genus.

Females of Nisotra species typically deposit yellow elongated eggs, either singly or in

clusters of between two and eleven, in loose moist soil (6 - 11 mm below the surface) very close to the base of host plants (Jarvis 1919; Reveche 1922; Rawat and Singh 1971). These eggs hatch into larvae that develop through three larval instars (Jarvis 1919; Pandit and Chaterji 1987; Oke and Odebiyi 2008). When N. orbiculata was reared on H. cannabinus in the laboratory (31 - 32°C), the first and second larval instars each took 3 - 4 days to complete, the third instar took 4-6 days, pre-pupation took 1 - 2 days, and pupation took 7 - 9 days (Pandit and Chatterji 1987).

This chapter describes the first detailed study of the life cycle of N. basselae in Solomon Islands and also investigates basic relationships between N. basselae and its host plants. The laboratory study, which was conducted at measured fluctuating ambient temperatures, investigated the development time for each life stage of the beetle on different host plants and examined the effect of host plant on immature survival and adult performance. In field studies, the distribution of adult beetles, and the feeding damage that they caused, within the canopy of two different varieties of A. manihot was investigated and the seasonal abundance and distribution of the pest between plants was investigated in the field over a continuous 18 month period.

4.2 MATERIALS AND METHODS

4.2.1 EGG VIABILITY AND INCUBATION PERIOD

Eggs for experiments were produced by adult N. basselae collected from two villages, Gavaga and Tutuva, in the Guadalcanal plain. Eggs from the Gavaga village population were used in experiments in October 2006 and eggs from the Tutuva village population were used in October 2008. In each experiment eggs were collected from about 1000 adult beetles that were collected from the respective field population and placed in a nylon net cage (1 m x 1 m x 1 m) in the laboratory a week previously. The eggs were collected from 10 oviposition dishes (Petri dishes, diameter 9.0 cm) that contained moist soil (1:7 v/v soil : water) that was partially covered by patches of mulch (dry leaves of A.

manihot). The oviposition dishes were placed on the floor of the cage. The origin and initial preparation of the soil was as described previously (Chapter 2).

In order to collect cohorts of eggs of uniform age, oviposition dishes were introduced to the cage at 08:00 and then removed 6 hours later (14:00 that afternoon). Immediately after removal from the cage, the soil in the oviposition dishes was examined under a dissecting microscope (x40) and the eggs isolated using a fine camel hair brush. Isolated eggs were placed together on a moistened filter paper ready for use in experiments.

One egg from each of the 10 oviposition dishes was collected and then carefully placed on the surface of a moist filter paper in a Petri dish (9 cm diameter); thirty filter papers, each supporting 10 eggs, were prepared in this manner. The exterior surfaces of the Petri dishes were covered with black sticky tape to provide a dark environment, similar to that that eggs would experience in soil. The Petri dishes were then placed on top of a raised ant-proof bench in the laboratory, and incubated at ambient temperature (mean maximum and minimum daily temperatures = 31.1 ± 0.2°C and 25.7 ± 0.1°C respectively) and humidity (mean maximum and minimum daily RH = 90.2 ± 0.6% and 72.9 ± 1.5% respectively). To prevent desiccation of eggs, a very fine water mist was applied to the surface of eggs on the filter paper in each dish during the daily morning assessments. Eggs were examined under a dissecting microscope (x10 magnification) twice daily until they hatched; the first examination took place between 06:00 and 08:00 and the second between 16:00 and 18:00 on the day after they were laid. Hatched eggs were identified by the presence of larval exit slits along the chorion. Examination of eggs continued for a period of 5 days after the hatching of the last egg, at which time the experiment was terminated.

4.2.2 PERFORMANCE OF NISOTRA BASSELAE ON DIFFERENT HOST PLANTS

4.2.2.1 SURVIVAL AND DEVELOPMENT OF LARVAE AND PUPAE

The survival and development of N. basselae larvae was tested on three different species of Malvaceae: A. manihot (slippery cabbage, Broad-type variety), A. esculentus (okra) and H. tiliaceus (native tiliaceus).

Egg incubation period for N. basselae was determined by counting the number of hatched eggs in oviposition dishes. Cohorts of eggs of the same age were collected from the soil in 10 oviposition dishes previously placed in the cage as described in section 4.2.1. Soil in the dishes was not disturbed after they were removed from the cage and the precise number of eggs in each dish was not determined. The Petri dishes were then covered with black tape and incubated as previously described in section 4.2.1 for six days. At 06:00 on the sixth day after oviposition, five succulent rootlets of A. manihot (Broad-type variety) (<1.0mm diameter and approximately 40 mm in length) were placed on the soil surface in each Petri dish in order to collect neonate larvae following egg hatch. The rootlets were then removed at 18.00 on the same day. The number of larvae collected was recorded and they were removed, together with the A. manihot roots, from the Petri dish. Daily placement and removal of roots and, the counting and removal of larvae from the Petri dishes continued until no more larvae were collected.

Ninety Petri dishes (9 cm diameter) of moistened soil were prepared as described previously; 30 Petri dishes were then furnished with 5 portions (≈ 40 mm in length) of succulent A. manihot rootlets, 30 Petri dishes were furnished with 5 portions (≈ 40 mm in length) of succulent A. esculentus rootlets and 30 Petri dishes were furnished with 5 portions (≈ 40 mm in length) of succulent H. tiliaceus rootlets. At 4.00 pm that day (day 8 after oviposition), nine neonate larvae were collected from each dish and these were then uniformly allocated to Petri dishes containing soil and roots of each of the three host plants. All 90 dishes were supplied with a single larva. Once larvae had been introduced to the dishes they were labelled and incubated in the dark as previously described (section 4.2.1). Each day, 5 fresh succulent rootlets (≈ 40 mm in length) of the

appropriate host plant were placed on top of the soil surface of each Petri dish and old rootlets were removed; this was repeated daily until larvae died or pupated.

Each larva was examined twice daily (06:00 - 08:00 and 16:00 - 18:00) and the width of the broadest portion of the head capsule of each larva was determined using a graticule under a dissecting microscope (x40 magnification). Measurements were taken each day until larvae ceased feeding and burrowed into the soil to excavate a pupal chamber. Pre-pupae that burrowed into the soil tended to settle at the very bottom of the Petri dish and thus were visible if the dish was turned upside down. As for the larval stages, pre-pupae were monitored twice daily (06.00 – 08.00 and again 16:00 – 18:00) under the dissecting microscope and pupae were monitored in the same way until adults eclosed.

4.2.2.2 ADULT ECLOSION AND MATURITY

Cohorts of pupae were monitored at intervals of 3 hours as they eclosed. The eclosion process was carefully monitored by turning the Petri dishes upside down, and then viewing pupae from the bottom of the Petri dishes under the dissecting microscope.

Upon emergence from pupal chambers, adult beetles were individually transferred to clear Petri dishes (9 cm diameter) and examined from the underside using a dissecting microscope, to determine their sex by the differences on the last abdominal ventrite (refer to Figure 3.3). After being sexed, each beetle was put together with a mature partner of the opposite sex. Paired beetles were then supplied with intact fresh young leaves of the host plant on which they had been reared as larvae. All insects paired in this manner were then monitored every hour to record the first feeding and mating events. A copulation event was recorded when the male genitalia connected to the female genitalia (viewed under a dissecting microscope – 10x magnification).

4.2.2.3 EGG DEVELOPMENT: COPULATION TO OVIPOSITION

The period from the first copulation of a female adult N. basselae to the time of first oviposition event was recorded for newly emerged adults under ambient conditions in the laboratory. After copulation, each female beetle was monitored twice daily (morning between 08:00 – 09:00 and evening between 16:00 – 17:00) to record when eggs were first laid.

4.2.2.4 FECUNDITY AND LONGEVITY

Following copulation, four pairs of male and female N. basselae that had developed on A. manihot and four pairs of beetles that had developed on H. tiliaceus remained paired in separate Petri dishes; each pair was supplied with fresh young leaves of the host plant on which they were reared as larvae daily. Using a dissecting microscope, the number of eggs laid by each female beetle was recorded twice daily (08:00 – 09:00 and 16:00 – 17:00) and eggs were then removed from the dish. Daily egg production was recorded until the beetles died.

4.2.3 DISTRIBUTION OF NISOTRA BASSELAE WITHIN PLANTS OF ABELMOSCHUS MANIHOT

This study was conducted at the Tutuva field site in January 2007. The garden (30 m x 30 m) was owned by a subsistence farmer and contained plants of the A. manihot (Broad-type variety). Land preparation and planting of this patch was conducted by the farmer. At the time of experimentation, plants were approximately 3 months old, of uniform size and were spaced approximately 50 cm apart. Most plants had a single stem with twelve leaves. The farmer did not use any agrochemicals on the crop.

Thirty plants were selected randomly and marked by tying coloured plastic tape to the stems prior to assessment. Plant assessments were conducted between 06:00 and 08:00 in order to minimize the effects of full-sun or shade on beetle distributions within

plants. Leaves within a stem were numbered from the apical meristem down to the ninth leaf on the stem. Plants were examined with the minimum amount of disturbance possible. Beginning at the first leaf, and taking great care not to disturb beetles on other leaves within the stem, the number of adult beetles on both the abaxial and adaxial surface of each of the nine leaves of the selected stem was recorded. Once beetle numbers had been recorded, the number of shot-holes on each leaf was also recorded. This procedure was repeated for each of the selected stems from each of the 30 selected plants.

In order to assess the area of foliage within stems, five plants within the field were randomly selected, with each plant stem bearing nine leaves. All nine leaves from a single stem were then labelled by a permanent marking pen, before being removed from the stem and placed into a plastic bag which was then sealed. In the laboratory the area of each leaf was estimated by placing it on a sheet of squared graph paper (2 mm x 2 mm) and then tracing around the margin of the leaf with a pencil. The area of each leaf was then determined by counting the number of squares within the outline.

4.2.4 SEASONAL ABUNDANCE AND DISTRIBUTION OF NISOTRA BASSELAE IN THE FIELD

The experiment was carried out in a field plot (30 m x 30 m) at Tutuva village; the area was open and cleared from trees. Land preparation, field planting, irrigation, control of other pest and fertilization were carried out in the same way as described in Chapter 2. The N. basselae populations on two varieties of A. manihot, Broad-type and Noodle hair-type were monitored over a period of 18 months (October 2007- April 2009).

The experiment was carried out in a complete randomized block design with two varietal treatments. Each treatment had four replicate plots, with each plot having plants arranged in a 4 x 4 grid. Adjacent plants were spaced 0.5 m apart. Adjacent plots were spaced 2 m apart and separated by bare soil.

The experiment was carried out over a period of 18 months in two successive plantings of both varieties. The first planting occurred in October 2007, while the second planting occurred 9 months later in adjacent plots to the south of the first ones planted.

Monitoring activity involved weekly visual sampling and recording of the numbers of N. basselae on 4 plants in each of the 4 plots of each plant variety. Sampling was conducted on the same day each week (08:00 and 09:00) for the duration of the study. Plants were carefully searched, branch by branch and leaf by leaf, with minimal disturbance. The first recordings on plants in the first planting commenced a month after planting and were conducted weekly for a year. Recordings on the second planting commenced a week after the last recording of the first planting was taken, at this time plants in the second planting had been in the field for three months and had reached an average height of about 1 m and stems contained 13 leaves. The Broad-type and Noodle hair-type A. manihot varieties have very different leaf sizes and shapes (see Figure 4.1). In order to compare beetle densities relative to the foliage available in the two varieties studied, the leaf area available on a single stem of each variety was measured. Five plants of each variety that had suffered little leaf damage were selected. A single stem on each plant that contained 9 leaves was then randomly selected from each of the 5 plants of each variety. Each leaf was then labeled and placed into a plastic bag that was then sealed and stored in a refrigerator. The area of each leaf was then measured by tracing its outline on graph paper as previously described.

150.0 mm

Figure 4.1 A. manihot varieties compared during the N. basselae seasonal abundance study. a) leaf of Broad-type variety; b) leaf of Noodle hair-type variety.

4.2.5 STATISTICAL ANALYSES

Where appropriate non-parametric data were analysed using Kruskal-Wallis and Mann- Whitney tests. Other data were analysed by ANOVA as indicated. All statistical tests were performed in Statview 5.0 (SAS Institute (1999).

4.3 RESULTS

4.3.1 EGG VIABILITY AND INCUBATION PERIOD

Freshly laid eggs of N. basselae were bright yellowish in colour and they remained so for a day after being laid. They then became light yellowish and by day 6 they were

transparent and the milky- yellow bodies and dark mandibles of developing larvae were quite visible.

Eggs hatched between days 7 and 11 after oviposition, but most eggs hatched on day 8 (Figure 4.2). Across both studies, 20% of the eggs did not hatch. There was a significant difference between the number of eggs that hatched on successive days after day 7 (H = 17.43; P < 0.05) and the median incubation period was 8 days (Figure 4.2).

At the time of hatching the larva was observed to rapidly exit from a slit at one end of the egg chorion. Exiting the egg was assisted egg bursters on the mesonotum and metanotum segments of the larvae (refer to the description of larvae in Chapter 3). Egg hatching occurred very rapidly and took between 30 and 80 seconds. Most empty egg- shells left behind by neonate larvae could be easily recognized by their grey-brown colour, distortedness and presence of slits on one end.

35 Gavaga beetle population 30 Tutuva beetle population

eggs hatchedeggs 25

15 N. N. bassela

Percentage ofPercentage 0 < 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12

Incubation time (days)

Figure 4.2 The percentage score of N. basselae eggs that hatched at different times under ambient laboratory temperatures (31.1oC ± 0.2oC to 25.7oC ± 0.1oC; relative humidity 90.2% ± 0.6% to 72.9% ± 1.5%).

4.3.2 PERFORMANCE OF NISOTRA BASSELAE ON DIFFERENT HOST PLANTS

4.3.2.1 SURVIVAL AND DEVELOPMENT OF LARVAE AND PUPAE

The larva of N. basselae has three instars (Table 4.1). The first instar larva has a pair of egg-bursters on the mesonotum and metanotum segments (refer to the description of larvae in Chapter 3). The colour of the first instar larva was bright yellowish for the first 24 h but thereafter it turned pale and yellowish brown as it aged. Larvae crawled swiftly on the soil immediately upon hatching but movement ceased as soon as the beetle found a root and started to feed. The width of the head capsule of first instars was 0.25 mm (Table 4.1).

The microscope graticule used to measure larval head capsule was limited at 0.05 mm and thus no errors for the absolute measurements presented in Table 4.1 could be determined. Durations of first larval instars of N. basselae were not significantly different (H = 2.23, P > 0.05) when fed with the three host plants (A. manihot (Broad-type), H. tiliaceus and A. esculentus. Larvae fed with A. esculentus only developed to the second larval instar before they died. Durations of second and third larval instars were both significantly different when fed with A. manihot and H. tiliaceus host plants (for second instars, Z = -3.16, P < 0.05; and for third instars Z = -2.49, P < 0.05); that is, second and third instar larvae fed with H. tiliaceus took longer to moult than when fed with A. manihot (Figure 4.3). No significant differences were observed in the pupal duration when larvae were reared on A. manihot and or on H. tiliaceus (Z = -1.50; P > 0.05 and Z = -1.67; P > 0.05 respectively). Overall, the median of total development period from first instar to adult was not significantly different between insects reared on A. manihot and H. tiliaceus (Z = -0.75; P > 0.05) (Figure 3).

The egg-bursters, which were clearly visible in first larval instar, were not present in the second instar, but otherwise characteristics of the second instar larvae were very similar to those of the first instar. Larval head capsule width at second larval instar was 0.35 mm (Table 4.1). Third larval instar head capsule width was 0.45 mm (Table 4.1) and at this stage of development larvae were turgid and turned creamy white and translucent.

Significant mortality occurred in the first larval stage on all host plants: 30% in A. manihot, 43% in H. tiliaceus, and 93% in A. esculentus (Table 4.2). Mortality was first recorded on day three after hatching (Figure 4.3). Larvae could not survive on A. esculentus roots; 93% died within the first instar and the remaining larvae died early in the second instar. When larvae were reared on A. manihot or H. tiliaceus, mortalities in the second and third instar were considerably lower than in the first instar and 50% and 47% respectively successfully completed development to adulthood (Table 4.2).

At the onset of the pre-pupal period, larvae ceased to feed, burrowed into the soil and excavated a pupal chamber. The larvae then moulted, turned into an exarate pupa (see description in Chapter 3) and occupied the soil chamber throughout its pupation period. The change from pre-pupae to pupae was subtle and occurred within hours. Freshly formed pupae were snowy and creamy white and a pair of spines projected from the posterior apex of the abdomen. Two initially faint black spots on the facial area (eyes) gradually became clearer and prominent, as the pupa grew older. The colour of pupae gradually turned light yellowish brown as the insects matured.

The pre-pupation period of insects reared on A. manihot was significantly shorter than that of the insects reared on H. tiliaceus (H = 6.62, P < 0.05). However, there was no significant difference in the development periods of pupae reared on either host plant (H = 2.20, P > 0.05) (Figure 4.4).

Based on these measurements, under the conditions in which the life-cycle was studied, N. basselae can complete development from egg to adult in 29 days (Figure 4.7) and, after a period of 6 days to reach sexual maturity and a further 3 day pre-oviposition period, female beetles can lay their first eggs 9 days after eclosion (Figures, 4.5, 4.6 and 4.7).

Table 4.1 Larval head capsule widths of N. basselae measured at different larval instars when fed with A. manihot (Broad-type), H. tiliaceus and A. esculentus. SE is 0 for all of them.

Larval head capsule measurements (mm)*

Larval Instars A. manihot H. tiliaceus A. esculentus

I 0.25 (n = 30) 0.25 (n = 30) 0.25 (n = 30)

II 0.35 (n = 21) 0.35 (n = 17) 0.35 (n = 2)

III 0.45 (n = 18) 0.45 (n = 15) -

* The microscope graticule scale resolution errors were fixed at 0.05 mm for all the readings.SE is 0 for all of them.

Table 4.2 Life-tables for N. basselae fed with A. manihot, H. tiliaceus or A. esculentus.

N. basselae fed with A. manihot N. basselae fed with H. tiliaceus N. basselae fed with A. esculentus (Broad-type) Mortality Marginal Mortality Marginal Mortality Marginal Stage lx dx Stage lx dx Stage lx dx factor mortality factor mortality factor mortality L1 30 L1 30 L1 30

unknown 9 0.30 unknown 0.43 unknown 28 0.93

L2 21 L2 17 L2 2

unknown 3 0.14 Unknown 2 0.12 unknown 2 -

L3 18 L3 15 L3 -

unknown 0.17 unknown 1 0.06 - - -

Pupa 15 Pupa - Pupa -

- 0 0 - 0 0 - - -

Adult 15 Adult - Adult -

Pupation a a A. esculentus H.tiliaceus A. manihot Prepupation a b

3rd larval instar a b

a 2nd larval instar b

a Ist larval instar a a

a Egg incubation a a

0 2 4 6 8 10

Time (days)

4.3.2.2 ADULT ECLOSION AND MATURITY

Adult N. basselae eclosion was a gradual process in which the beetle’s elytra turned greyish and creamy white, bluish metallic silver and then eventually black. Other parts appeared light yellowish and fulvous. Newly hatched adults remained inside individual soil pupal chambers for between one and two days before emerging to the soil surface. There was no significant difference in the time to emergence between insects fed with A. manihot and those fed with H. tiliaceus (H = 0.14, P > 0.05). Once emerged from the soil surface, the new adults spent most of their early hours crawling, remaining stationary and jumping a little before taking their first flights. Most newly emerged adults started to chew on the first leaves that they came into contact within the first hour.

The time to sexual maturity of an adult beetle was measured from the time of the beetle’s eclosion to its first mating event. Adult female N. basselae matured significantly earlier than its adult male (H = 25.55), P < 0.05 (Figure 4.5). Overall, adult N. basselae fed with A. manihot matured significantly earlier than those fed with H. tiliaceus (H = 15.75; P < 0.05).

H. tiliaceus b A. manihot Adult female beetle b

a Adult male beetle a

0 2 4 6 8 Time (days)

Figure 4.5 Median times from eclosion to first mating of adult male and female of N. basselae when fed with A. manihot and H. tiliaceus. Median durations marked with a common letter are not significantly different.

4.3.2.3 EGG DEVELOPMENT: COPULATION TO OVIPOSITION

There was no significant difference in the pre-oviposition period between newly emerged adult females of N. basselae when fed with A. manihot or fed with H. tiliaceus (Z = -1.5, P > 0.05 (Figure 4.6).

H. tiliaceus a

A. manihot a

0 1 2 3 4 5

Time (days)

Life Cycle of Nisotra basselae (Bryant)

Male – 6.0 days 3.0 days Female – 3.0 days

Sexual Maturity

Adult Egg

6.0 days 8.0 days 1 Generation = 38.0 days

Larva Pupa

3.0 days 15.0 days

Pre-pupa

4.3.2.4 FECUNDITY AND LONGEVITY

Longevities of adult males and females of N. basselae when fed with A. manihot were significantly higher than when fed with H. tiliaceus (adult males: F1, 6 = 271.81, P < 0.05; adult females: F1, 6 = 100, P < 0.05) (Table 4.3). The oviposition period of adult female beetles fed on A. manihot was longer than that of female beetles fed on H. tiliaceus (F1,

6 = 10.94, P < 0.05) (Table 4.3). The average number of eggs laid by female beetles fed with A. manihot was significantly higher than those laid by females beetles fed with H. tiliaceus (F1, 6 = 9.11, P < 0.05) (Table 4.4).

Eggs were laid singly or in clusters. The number of eggs laid by individual females, irrespective of host plants, fluctuated daily and large oviposition events were typically followed by comparatively low numbers of eggs being laid in the next 24 h, before an increase in egg laying in the flowing 24 h period; this pattern was repeated serially over the life of study insects until oviposition ceased (Figures 4.8 and 4.9).

Table 4.3 Longevity and oviposition periods of N. basselae (adult males and females) reared on A. manihot and H. tiliaceus (n = 4)

Mean time (days) (±SE)

A. manihot H. tiliaceus P - value

Oviposition period (days between 43.25 (±8.28) 13.25 (±3.71) < 0.05 eclosion and last oviposition)

Adult female longevity (days between eclosion and death) 68.00 (±3.74) 18.75 (±3.17) < 0.05

Adult male longevity (days 65.00 (±1.73) 16.00 (±2.42) < 0.05 between eclosion and death)

Table 4.4 Fecundity as a function of host plants. When fed different host plants the mean number of eggs laid by female N. basselae (n = 4) was significantly different between treatments (F1, 6 = 9.1, P < 0.05).

Plants reared on: Mean number of eggs laid*

A. manihot 266 ± 59

H. tiliaceus 55 ± 37

30 Host plant: A. manihot 300

25 250 eggs laideggs

20 200

eggs laideggs daily Eggs laid daily N. basselae N. 15 Cumulative eggs laid 150

N. N. basselae 10 100 Cumulativenumber Number ofNumber 5 50

0 0

Longevity (days)

Figure 4.8 The daily oviposition pattern of N. basselae when fed with A. manihot under ambient laboratory conditions

18 Host plant: H. tiliaceus 60

16 50 14 Eggs laid daily

Cumulative eggs laid laideggs

12 40 eggs laideggs daily 10

30 basselae N.

8 N. basselae 6 20

Number ofNumber 10 Cumulativenumber 2

0 0 0* 4 8 12 16 20 24

Longevity (days)

Figure 4.9 The daily oviposition pattern of N. basselae when fed with H. tiliaceus under laboratory ambient conditions

4.3.3 DISTRIBUTION OF NISOTRA BASSELAE WITHIN PLANTS OF ABELMOSCHUS MANIHOT

There were significant differences in the mean numbers of both N. basselae (F8, 261 =

3.02; P < 0.05) and shot-holes (F8, 261 = 7.84; P < 0.05) between the different leaves within a plant (Figure 4.9). No beetles were recorded on leaf-1 or leaf-2. Beetles were recorded on leaf-3 and above; the mean number per leaf increased from leaf-3 to leaves 5 and 6 but thereafter the mean number of beetles per leaf declined incrementally to the oldest leaf. The mean numbers of beetles on leaves 4, 5 and 6 were not significantly different from each other (LSDs, P > 0.05). The mean number of beetles on leaf 5 was significantly higher than the mean number of beetles on leaf- 3 (LSD, P < 0.05), leaf-7 (LSD, P < 0.05), leaf-8 (LSD, P < 0.05) and leaf-9 (LSD, P < 0.05) (Fig 4.9). The mean number of beetles on leaf-4 was significantly higher than the mean numbers of beetles on leaf-8 (LSD P < 0.05) and leaf-9 (LSD P < 0.05). Also, the mean number of beetles on leaf-6 was significantly higher than the mean numbers of beetles on leaf-8 (LSD, P < 0.05) and leaf-9 (LSD, P < 0.05).

There was no shot-hole damage on leaf-1 or leaf-2 but leaf-3 and all subsequent leaves suffered shot-hole damage. There was no significant difference between the level of damage between leaf-3 and leaf-4 (LSD, P > 0.05) but these leaves were less damaged than leaf-5 and all the older leaves (Figure 4.10).

When analysed in terms of beetles and shot-holes per unit leaf area, significant differences in both beetle and shot-hole numbers were detected (F8, 261 = 3.22; P <

0.05 and F8, 261 = 7.52; P < 0.05, respectively) (Figure 4.10). There was no significant difference between the beetle densities on leaf-3 and leaf-4 (LSD, P > 0.05). The density of beetles on leaf-3 was significantly higher than the densities of beetles on leaves 5, 6, 7, 8 and 9 (LSD, P < 0.05). Similarly the density of beetles on leaf-4 was not significantly different to the density of beetles on leaf-5 (LSD, P > 0.05) but it was significantly greater than the density of beetles on leaves 6 to 9 (LSD, P < 0.05). The highest density of shot-holes was recorded on leaf-3 and leaf-4, but there was no significant difference in the density of shot holes on these leaves (LSD, P > 0.05). The density of shot-holes on leaves 3 and 4 was significantly higher than the densities on leaves 5 to 9 (LSD, P < 0.05) (Figure 4.11).

A) 9 a ab 8

leaf ab 7

N. basselae/ N. 5

4 b

3 b

SE) numberSE) ± 2 bc bc

Mean ( Mean 1 d d 0

B) 800 a ab 700 ab ab

600

holes/leaf - 500 b 400

300 c

SE) number of shotnumberSE)of 200 ±

100

c Mean ( Mean d d 0 1 2 3 4 5 6 7 8 9 Leaf number

Figure 4.10 A) mean number (±SE) of adult N. basselae per plant, and B) mean number (±SE) shot-holes per leaf of A. manihot; n = 30 for both. Columns marked with different letters are significantly different.

Table 4.5 The mean area per each leaf number of A. manihot (Broad-type) (n = 5)

Leaf Number Leaf area (mm2 ; mean ± SE)

1 20 ± 10

2 130 ± 30

3 1589 ± 375

4 5937 ± 842

5 14267 ± 2390

6 19188 ± 3611

7 16380 ± 2764

8 19187 ± 3611

9 16380 ± 2763

A) 0.7

a ) 2 0.6

0.5

leaf(cm area 0.4 a 0.3

N. basselae/N. 0.2 SE) SE) ± 0.1 d e e d d d d

Mean( 0

B) a ) 2 a 10

holes/leaf(cm area 6 -

SE) shot SE) ± 2 b b Mean( b b b c c 0 1 2 3 4 5 6 7 8 9

Leaf number

4.3.4 SEASONAL ABUNDANCE AND DISTRIBUTION OF NISOTRA BASSELAE IN THE FIELD

The mean (±SE) numbers of beetles recorded per plant over the entire one and a half year monitoring period was significantly different between the two A. manihot varieties (F1, 132 = 54.25; P < 0.05) (Figure 4.12 A)). The Broad-type variety had more beetles per plant than Noodle hair-type variety.

The mean number of N. basselae per plant recorded in the dry season (May to October) was significantly greater than the number recorded in the wet season

(November to April) (F1, 132 = 159.80; P < 0.05) (Figure 4.12 A)). The interaction between season and plant variety was also significantly different (F1, 132 = 11.69; P < 0.05).

The mean number (±SE) of beetles recorded per unit leaf area over the entire one and a half year monitoring period showed a significant difference between the two varieties of A. manihot (F1, 132 = 14.54; P < 0.05). Noodle hair-type variety had significantly more beetles per plant leaf unit area than the Broad-type variety (Figure 4.12 B)).

Season had a significant effect on the mean number of beetles per unit area of leaf,

F1, 132 = 166.2; P < 0.05. The average number of beetles per unit leaf area recorded in the dry season months (May to October) was significantly greater than those recorded in the wet season (November to April) (LSD, P < 0.05) (Figure 4.12 B)). The number of beetles did not differ significantly between varieties during the wet season but did differ during the dry season, when numbers were higher on the Noodle hair-type variety Broad-type (LSD, P < 0.05) (Figure 4.12 B)).

The distribution of N. basselae within the experimental plots was aggregated (Figure 4.15; Table 4.6), beetles were more aggregated in the wet season, when beetles were fewer, than in dry season when beetles were more abundant (see index of patchiness in Table 4.6).

14 A) Noodle hair-type variety a Broad-type variety

12 plant 10

N.bassela/ 8 b

SE) no. of no.SE) ±

4 c Mean ( Mean

2 d

0 25

a 20

plant leaf unitplantleafarea 15 b

N. basselae/ 10 SE) no. ofno.SE) ± c

5 d Mean ( Mean

0 Dry season Wet season

30 Wet Dry Wet Dry

25 Broad-type variety

Noodle-type variety

(Bryant) per plant (Bryant) per 20

15 N. N. basselae

SE) number of adultnumberofSE) ±

0 Mean ( Mean

Monitoring dates

Figure 4.13 The mean (±SE) numbers of N. basselae per plant recorded weekly on two varieties of A. manihot (Broad-type and Noodle hair-type) between November 2007 and June 2009. Indicate when sampling changed between plots.

50 Wet season Dry season Wet season Dry season 45

40 Broad type variety /unit leaf area area /unitleaf 35 A Noodle type variety 30 25

N. N. basselae 20 15

10 SE) adultSE)

± 5

0 Mean ( Mean

700 B 33 Average total rainfall 600 32.5

Average temperature C)

32 o 500 31.5 400 31 300 30.5 200

Total rainfall (mm) Total 30 Mean temperature ( temperature Mean 100 29.5 0 29

Figure 4.14 (A) the mean (±SE) numbers of N. basselae per unit area of leaf recorded weekly on two varieties of A manihot (Broad-type and Noodle hair-type) over a year and a half (November 2007 to June 2009). (B) total monthly rainfall and mean temperature taken at Henderson Meteorological Stati on which was the closest (≈6 km) available weather recording station to Tutuva Experiment site where the beetles were recorded.

a) 400 November 2007-April 2008 b) 250 May 2008- October 2008 350 200

300

plants plants 250 150 200 100

150 A. manihot A. A. manihot 100 50

50 Number ofNumber Number ofNumber 0 0

Number of N. basselae per plant Number of N. basselae per plant

c) 700 November 2008- Aprill 2009 d) 80 May 2009- June 2009 600 70 60

500 plants plants 50 400 40 300

A. A. manihot 30 A. A. manihot 200 20

100 10 Number ofNumber Number ofNumber 0 0

Number of N. basselae per plant Number of N. basselae per plant

Figure 4.15 Frequencies of N. basselae recorded per A. manihot plant (Broad-type and Noodle hair-type combined from the population seasonal abundance data set).

Table 4.6 The index of dispersion and index of patchiness of N. basselae in the different seasons (wet season November- April; dry season May- October)

Index of Index of Period Mean Variance Dispersion(V/M) Implication Patchiness Implication

Nov 07 - Apr 08 2.34 16.79 7.17 Aggregated 3.64 Aggregated

May 08 - Oct 08 7.72 58.22 7.54 Aggregated 1.84 Aggregated

Nov 08 - Apr 09 2.10 14.75 7.02 Aggregated 3.87 Aggregated

May 09 - Jun 09 10.45 115.56 11.06 Aggregated 1.96 Aggregated

4.4 DISCUSSION

Like other chrysomelid beetles, Nisotra larvae and adults both feed on the same host plant (Matthews and Reid 2002). Adults feed on foliage while their larvae feed on the roots. Species of the Nisotra genus primarily feed on Malvaceae but some members are occasionally found feeding on host plants from other families (Matthew and Reid 2002). The first record of N. basselae was collected from a Basella species (Basellaceae) (Bryant 1941). From general field observations, N. basselae usually feeds and associates more with A. manihot (Malvaceae) than any other plant species in the field.

The full extent of the host range of N. basselae is still unknown. This study investigated whether or not the pest could also feed and breed on a native hibiscus, H. tiliaceus (Malvaceae), a plant which is quite common in coastal areas where A. manihot is cultivated. Results from laboratory experiments showed that larvae could complete their development by feeding on the roots of H. tiliaceus but in laboratory studies N. basselae performed better (higher immature survival rates, longer adult longevity and greater egg production when reared on A. manihot (Tables 4.2 - 4.4; Figures 4.4 - 4.8) Inspections for adult feeding damage on leaves of H. tiliaceus and the presence of larvae in the soil near roots of H. tiliaceus which grew adjacent to A. manihot plots that were severely infested with N. basselae found no traces of leaf damage or larval presence (unpublished data). This suggests that H. tiliaceus is not an important host of N. basselae in the field on Guadalcanal.

This study showed that under ambient conditions adult N. basselae reach sexual maturity between 3 and 6 days after eclosion. From general observation, the beetles were observed mating throughout the day, but more mating occurred early in the morning and late afternoon. Such observations agree with an assertion by Jarvis (1919), where he observed adult males and females of N. breweri mated on foliage of host plants regularly and multiple times. Rawat and Singh reported that mating of N. orbiculata was observed to have occurred at night and that actual mating typically occurred for 11 minutes. However, males of N. basselae mounting on the backs of females of N. basselae can be misleading since such mountings might represent courtship and not copulation pairing.

Gravid females of N. basselae typically laid eggs 3 days after their first copulation. Eggs were laid either singly or in clusters in the soil or on the soil surface beneath leaf litter close to the base of the host plant (see more detailed information in Chapter 5). This oviposition behaviour of adult female beetles is similar to that of related species. For example, it was found that Nisotra breweri Weise laid eggs singly and clusters and close to the base of host plants in loose moist soil at a depth of 7-14mm (Jarvis 1919). Reveche (1922) reported eggs of N. gemella were laid in groups of 5 to 11 around the base of the plant and a majority of them were laid 2 to 3 mm under the soil surface.

Egg incubation period is an embryonic period of development between egg fertilization and egg hatching (Gordh and Headrick 2001). The lengths of such a period are usually regulated and determined by temperature of the environments that the eggs are exposed to (Gilbert and Raworth 1996; Nahrung and Merritt 1999). Thus, egg incubation period is in fact a function of accumulative heat energy. It is typically shortened in hotter tropical climates and lengthened in cooler temperate regions (Santiago-Blay 1994; Gilbert and Raworth 1996; Charnov and Gillooly 2003). Previous research has shown that egg incubation periods of Nisotra species can range between four and thirteen days (Rawat and Singh 1971; Pandit and Chatterji 1978. This study recorded N. basselae incubation period to be eight days. Eggs were observed to hatch very quickly, in a matter of seconds. This observation supports that of Pandit and Chatterji (1978) who observed that eggs of N. orbiculata hatched rapidly, between 30 and 80 seconds, once larvae began to leave the chorion.

Immediately after hatching, the neonate larvae of N. basselae moved swiftly through debris and soil particles. Reveche (1922) observed a similar phenomenon and concluded that larvae were very active on the surface of the soil straight after hatching as they looked for roots on where to feed at the base of the host plant.

The larva of N. basselae has three larval instars that fed on the new and tender roots of host plants. When reared on A. manihot, larval development lasted 15 days (1st instar = 4 days, 2nd instar = 5 days and 3rd instar = 6 days) at temperatures between 31.1 ± 0.2oC and 25.7 ± 0.1oC (Figure 4.3). This developmental period was very similar to a related species, N. orbiculata, in which larval development was completed in 13 - 15 days when reared on Hibiscus cannabinus, H. sabdariffa or

Urena lobata at temperature between 31.3oC and 32oC (Pandit and Chatterji 1978). Similarly the developmental periods of each instar also corresponded to those of N. orbiculata (1st instar 3 - 4 days; 2nd instar 3 - 4 days; the 3rd instar 4 - 6 days; the pre- pupa 1 - 2 days; and pupation 7 - 9 days; Pandit and Chatterji 1978).

Shorter development duration and low mortality on a host plant usually imply better performance of an insect population (Dank 2006). The development period of first instar N. basselae was the same when reared on A. manihot, H. tiliaceus and H. esculentus. However, the different host plants did significantly affect the survival of larvae. Thirty, 43 and 93 % mortality was recorded in larvae fed with A. manihot, H. tiliaceus and A. esculentus, respectively (Table 4.2). More beetles died during the first instar than in the second and third instar in all three host plants tested (Table 4.2). Second and third instar larvae feeding on H. tiliaceus took longer to develop than larvae feeding on A. manihot (Figure 4.6). Larvae feeding on A. esculentus only developed to the second larval instar, whether this was due to an intrinsic property of A. esculentus roots or whether the particular roots used were not of sufficient quality is uncertain and further experiments should be conducted to investigate the suitability of A. esculentus as food for larvae of N. basselae.

Newly hatched adults of N. basselae were observed to remain inside individual soil pupal chambers for one to two days before emerging on the soil surface. Similarly Reveche (1922) reported that young N. gemella adults remained in the pupal chamber for 2 to 3 days after eclosion.

The life cycle of N. basselae took 38 days to complete at ambient temperature (mean maximum and minimum daily temperatures = 31.1 ± 0.2°C and 25.7 ± 0.1°C respectively) and humidity (mean maximum and minimum daily RH = 90.2 ± 0.6% and 72.9 ± 1.5% respectively) (Figure 4.7). By comparison, this result was similar to that for other Nisotra species. Rawat and Singh (1971) found the life cycle of N. orbiculata to be between 22 and 25 days at temperatures between 28.4 and 29.5oC. Pandit and Chatterjii (1978) also determined the life cycle of N. orbiculata to be between 27 and 32 days when the average temperature was 29.5oC.

In terms of fecundity of N. basselae, A. manihot was a better host than H. tiliaceus, as more eggs (mean (SE) = 266 (± 59) eggs) were laid by female N. basselae reared

through from larvae to adults on A. manihot than by female N. basselae reared through from larva to adults on H. tiliaceus (mean (SE) = 55 (± 37) eggs) (Table 4.4). Similarly, beetles fed with A. manihot lived significantly longer (65 – 68 days) than those fed with H. tiliaceus (16 – 18 days) (Table 4.3). The results of this study are similar to those reported by Jarvis (1919) where N. breweri female adults feeding on Rosella, Hibiscus sabdifera, lived for 68 days and laid a total of 271 eggs. Pandit and Chatterji (1978) observed newly emerged adults when fed on green leaves of H. cannabinus, H. sabdifera and U. lobata, lived 36 – 44, 30 - 32 and 40 – 45 days, respectively.

The life cycle of N. basselae can be summarized as follows. Adults feed and mate on the foliage of host plants. Gravid females lay eggs three days after copulation (pre- oviposition period). The eggs are laid singly as well as in clusters. Most eggs are laid in the soil near the base of host plants.

Larvae hatch from eggs after an average of eight days. Neonate larvae feed on the succulent roots of the host plants and develop through three larval instars over 15 days before pre-pupating in soil chambers for four days and subsequently spending about 7 days in the pupal stage. Newly emerged females and males mature in 3 and 6 days, respectively.

The change in seasons can affect fecundity of the beetles. For example, oviposition of N. orbiculata reached its peak during late July but subsequently declined to zero by the end of September, by which time the insects contained mostly immature ovaries (Rawat and Singh 1971). A similar phenomenon might explain the fluctuations in seasonal abundance reported during the wet and dry seasons (see Fig 4.3) or the heavy rainfall might cause adult mortality due to the impact of rain on exposed insects or larval mortality in waterlogged soil; this requires further investigation.

Adult N. basselae tended to feed more on younger A. manihot leaves (leaves 3 to 5) than on older leaves (leaves 7 to 9) (Figures 4.9 and 4.10). Several previous studies have established that young and growing tissues of plants, including leaves, are particularly rich in nutrients because they require more resources to support their rapid development and therefore are most attractive and vulnerable to herbivorous

insect damage (e.g. Mattson 1980, Fortin and Mauffette, 2001, 2002; Yamasaki and Kikuzawa, 2003) as they require these highly nutritious plant tissues for optimal growth and performance (Mattson 1980; Chen et al 2008a; Chen and Poland 2009).

Studies have shown that the distribution of nitrogen in the canopy is in proportion to the distribution of absorbed light, with the result that leaves exposed to high levels of light have high nitrogen concentration and contribute the bulk of canopy carbon fixation (Leuning et al., 1995; dePury and Farquhar, 1997). Young leaves of A. manihot are small in size (Leaf 3 - 5) (Table 4.5) and are normally positioned nearer the top of the canopy than the older leaves, thus allowing them more time exposure to solar radiation. Leaf-1 and 2 are not exposed to direct sunlight thus photosynthesis may be limited in these leaves. Although older leaves are broader than young leaves, the elevation of the sun during the day may reduce their diurnal exposure to light compared to the younger leaves.

The overall extent of damage on each leaf of A. manihot at a given space and time is usually a result of cumulative shot-holes chewed by adult N. basselae. This is evident in the Figure 4.9, where it can be seen that most of the damage on leaves 6 to 9 was accumulated when leaves were younger; particularly when they were at leaf stages 3 and 5 (Figure 4.9 A and Figure 4.9 B). Correspondingly low numbers of beetles were observed settling and feeding on the older leaves (Figure 4.9 A and Figure 4.9 B A). The toughness of senescing leaves may contribute to them being less damaged than younger leaves. King et al. (1997) reported that the larvae of willow leaf beetle, Plagiodera versicolora Laicharting feed profusely on the younger leaves but feed less on older leaves to avoid their mandibles wearing out.

The understanding of the life cycle of N. basselae in the laboratory is an important step but it is vital to understand its life cycle in relation with its seasonal cycles in the field in order to develop effective management strategies (Pedigo 2002). In this study the life cycle of N. basselae under ambient conditions was 38 days. Theoretically, the expected number of generations per year is approximately eight. In reality these numbers of generations may either be higher or lower depending on prevailing weather conditions. The general weather pattern in Solomon Islands (Solomon Islands Meteorology Service 2013) and a weather station closest to where

N. basselae population seasonal abundance was monitored both showed an obviously distinctive weather pattern throughout the year (Figure 4.12 and 4.11).

The results of weekly monitoring of beetle populations in two A. manihot varieties clearly showed significant higher N. basselae population during dry season than during wet season (Figure 4.11 A and B; and 4.12 A and B). Whether population difference was due to prevailing weather conditions or biotic factors, such as predators and parasites was unknown. High rainfall during wet seasons reduced beetle population as eggs, larvae and pupae in the soil were likely to be washed away by the physical impact of rainfall or possibly killed by prolonged waterlog on the soil surface. The hypothesis which suggested that low rainfall could lead to high water stresses on host plants, leading to increased insect populations (Thatcher 1981) could possibly explain the seasonal abundance pattern for N. basselae too. The relative effects of high rainfall depressing populations and of times of low rainfall allowing populations to increase require further study to understand the mechanism that are operating so that the underlying factors driving N. basselae population dynamics can be better understood. The long term weather-beetle density relationship suggests that in order to avoid severe damage on A. manihot crops it is advisable to plant the crop in the field during wet season.

The number of beetles on a single plant usually correlates positively with feeding damage on that plant damage. Only the numbers of beetles per plant but not the damage per plant were recorded in the seasonal abundance monitoring. The Broad- type variety has leaves three times surface area of the Noodle hair-type variety and had more beetles per plant. However, in terms of beetles per leaf unit area, there were more beetles on the Noodle hair-type variety than on the Broad-type variety. It is common to find more than ten varieties of A. manihot in a farmer’s garden (Preston 1998). At this stage, it is not clear what actually caused the differences in the distribution of beetles in the different varieties. Leaf size or preference due to palatability or nutritional value could be among the reasons. Broad-type variety was found to sustain more beetle damage than Noodle hair-type variety (Figure 4.12 B).

The numbers of N. basselae per leaf area in both varieties were not significantly different during wet seasons but were significantly different during the dry seasons,

as the number of beetles recorded on Noodle hair-type variety were significantly greater than those recorded on Broad-type variety.

The population of N. basselae started to increase at the onset of dry seasons and continued to do so until reaching maximum levels in the months between June and July, at this time populations declined rapidly before the wet seasons. It is possible that this sudden drop in numbers was due to significant damage to the crop foliage resulting in limited resources that could not support the large population that had developed (Pollard 1981).

The distribution of N. basselae recorded throughout the seasons showed that the population was aggregated (Figure 4.15; Table 4.6). It was observed that the beetles were more aggregated in the wet season, when the overall beetle number in the field was low, than in the dry season when population were higher (see Table 4.6). Aggregation during wet season was probably due beetles drawing to themselves together to feed and to mate when not many beetles were around compared to the dry seasons. The effect of the saturation level of damage plant volatiles and beetle sex pheromones (chapter 6) might also contribute to the factors determining this distribution.

In conclusion, this study has determined the basic life cycle of N. basselae on A. manihot, A. esculentus and H. tiliaceus. Of the three host plants tested, A. manihot supported better performance N. basselae in terms of shorter life cycle duration and lower mortality. The study also determined longevity and fecundity of N. basselae on A. manihot and H. tiliaceus and again found A. manihot to be the better host for beetle performance.

CHAPTER FIVE: OVIPOSITION ECOLOGY OF NISOTRA BASSELAE

5.1 INTRODUCTION

Oviposition site selection can have far-reaching consequences for the survival and development of a female insect’s progeny (Prokopy et al 1982; Quiring and McNeil 1987; Tschanz et al. 2005). Insects usually lay their eggs either directly on host plants or in proximity to them, so neonate larvae can easily reach feeding sites soon after hatching (Courtney and Kibota 1990; Selman 1994). Many soil dwelling insects usually lay their eggs in the soil, on the soil surface or in cracks and crevices near the base of the host plant or within its root zone (Chamberlin and Tenhit 1924, Pandit and Chatterj 1978; Martin and Herzog 1987, Degeo et al. 1993).

Soil dwelling species of Chrysomelidae typically lay their eggs close to the base of host plants at depths of up to 30 cm (Gray et al. 1992). Most of these beetles lay eggs singly or in clusters of up to a dozen at depths of approximately 5 mm below the soil surface (Swaine and Ironside 1983, Parker 1910). Nisotra species also typically lay a single egg or small cluster in soil very close to the base of their host plants, thereby providing newly hatched larvae easy access to roots upon which they feed (Jarvis 1919). Unlike other chrysomelid larvae that feed on aerial parts of host plants, and where the larvae hatch from eggs that are usually laid directly on the surface of feeding sites, neonate larvae of Nisotra species must search for root feeding sites after hatching. Jarvis (1919) reported that immediately after hatching, neonate Nisotra breweri Baly larvae crawled swiftly between soil particles in search of host plant rootlets upon which to feed. Plant volatiles that are emitted from feeding sites as olfactory stimuli are believed to assist neonate larvae to find their feeding sites (de Jong and Stradler 1999; Hilker and Meiners 2002).

Gravid insects usually follow a series of searching, orientation and encountering, landing and surface evaluation behaviours before choosing oviposition sites (Renwick and Chew 1994; Courtney and Kibota 1990; Selman 1994). These behaviours are mediated by plant stimuli, particularly, chemical and mechanical stimuli, from the host plants on which to lay eggs (Prokopy 1986; Visser 1986; Quicke 1997; Dudareva et al. 2006). The final decision of whether an insect accepts

or rejects a host plant may be based on a combination of visual and chemical cues which are detected after landing (Renwick and Chew 1994).

Quality of host plants is a factor that drives oviposition site preference in insects. Some insects prefer to lay eggs on plants that are healthy and vigorous (Howlett et al 2001; Forister 2004). There are other insects that prefer plants that are under stress (Courtney and Kibota 1990, Larsson and Ekbom 1995). Plants that are under stress are possibly less well equipped with defensive chemicals than those grown in high resource environments (Rhoades 1979; White 1984; Mattson and Haack 1987). It also possible that plants under stress are more likely to avoid natural enemies, which are often, associated with high resource environments (Singer and Stireman 2003).

Physical conditions of soil, especially moisture and texture, also often influence the behaviour of adult female insects in their choice of where to deposit eggs (Pandit 1998). Most gravid females prefer moist conditions for oviposition, as demonstrated by Diabrotica longicornis barberi Smith and Lawrence which laid significantly more eggs within corn rows in irrigated fields than in dry land fields (Weiss et al. 1983). Work by Brust and House (1990) underscored the importance of soil moisture by demonstrating significantly higher survival and more rapid development of larvae of southern corn root worm, D. undecimpunctata howardi Barber, in moist soil than in drier soil.

Cultural practices that are applied for crop production also can affect insect oviposition (Steffey et al. 1999). Mode of land preparation for crops often has an impact on insect populations, particularly when oviposition and pupation sites are disturbed or obstructed. For example, the southern corn rootworm, D. undecimpunctata howardi Barber, has been recorded to lay 20% less eggs in tilled soil compared to a non-tilled soil (Brust and House 1990). Irrigation and rainfall can have negative effect on oviposition and development of juvenile insects in the soil (Jackson et al. 2002). Artificial irrigation and natural rainfall often saturate the soil with water which can displace, suffocate or drown eggs, larvae and pupae. In India, continuous monitoring of populations of Nisotra orbiculata (Mots.) on mesta (Hibiscus cannabinus L.), rosella (H. Sabdariffa L.), congo-jute (Urena lobata L.), and malachra (Malachra capitata L.) over a period of three years showed that high rainfall reduced insect numbers in all crops (Pandit 1998).

Application of mulches and other forms of protective covers over the soil and around plants is a common cultural practice among home gardeners and commercial vegetable growers, especially in warmer periods and areas in general (Waggoner et al 1960, Ashworth and Harrison 1983). Mulches have various benefits which include retention of soil moisture, reduction of soil erosion, suppression of weeds and the provision of nutrients after the mulch materials are decomposed. Materials used as mulches vary from organic residues (e.g. compost, grasses, leaves, hay, straw, kitchen scraps, saw dust, newspapers) to manufactured mulches (e.g. rubber and plastic mulches) and living mulches or ground covers (Louise 1996). The choice of the type of mulches used may depend on factors such as the availability of mulch materials, their costs, the effects that they have on the soil and how durable they are when applied.

Depending on the type and thickness of materials, mulches can either impede or promote insect development. For instance, the cucumber beetle, Diabrotica sp., was deterred from laying eggs in the ground near the base of plants that was mulched (Diver and Hinman 2008). In addition, Stone (1996) reported a significant reduction in the numbers of eggs and larvae of Leptinotarsa decemlineata (Say) on the leaves of plants in fields that had been mulched compared with plants that had not been mulched.

In this chapter I report on my investigation of N. basselae oviposition in field and laboratory experiments. In the laboratory I investigated N. basselae oviposition under different soil moisture and mulching regimes. In follow up field experiments I investigated the relationship between oviposition site and proximity to the host plant and these were complemented by laboratory experiments to study precise relationships between oviposition sites and host plant root distributions in the soil. In another set of experiments, which were conducted in both the field and laboratory, I investigated the movement of adult beetles between foliar feeding sites and soil oviposition sites. The final experiment investigated the impact of different cultural practices on oviposition of N. basselae at the base of host plants and its impact on adult feeding on plant foliage.

5.2 MATERIALS AND METHODS

5.2.1 EFFECTS OF SOIL MOISTURE ON NISOTRA BASSELAE OVIPOSITION

Dry soil for this experiment was collected and prepared as described in Chapter 2, section 2.3. Soil was then placed into two separate 2L beakers. One beaker of soil was treated with rainwater (1: 7; volume: volume; water: soil) and then thoroughly mixed by hand. The other beaker of soil was not treated with water. Four Petri dishes (9 cm diameter) were then filled to the brim with dry soil, while another four Petri dishes were filled to the brim with soil that had been moistened as described above. The eight dishes were arranged in a grid (2 x 4) on the floor of an oviposition cage (40 cm x 40 cm x 70 cm) which contained approximately 1000 field collected adult N. basselae. The beetles were collected from the field five days before the experiment was carried out (see Chapter 2 for details). The placement of the dishes within the grid was done in a complete randomized design with each Petri dish positioned 1 cm away from adjacent Petri dishes. The Petri dishes were left in the cage for three days, during which time the wet soil was kept moist by spraying with a fine mist of rainwater every 24 h, before they were removed and the number of eggs in each Petri dish was determined. Soil from each Petri dish was emptied into a large plastic tray (20 cm x 30 cm) and then carefully spread out. Using a binocular microscope (x10 magnification) the soil was meticulously searched (a small wooden pointer was used to move soil particles) and all eggs recorded. The experiment was repeated two times using the same cage of beetles.

5.2.2 THE EFFECTS OF MULCHES ON NISOTRA BASSELAE OVIPOSITION

Eight Petri dishes (9 cm diameter) were filled to the brim with moist soil as described previously. The soil surfaces in four Petri dishes were covered with a single uniform layer of dry A. manihot (Broad-type variety) leaves while soil surfaces in the other four dishes were left bare. The eight dishes were randomly arranged in a grid (2 x 4; adjacent dishes positioned 1 cm apart) on the floor of an oviposition cage (40 cm x 40 cm x 70 cm) which contained approximately 1000 adult N. basselae that were collected from the field 5 days before the experiment was set up (see Chapter 2 for details). Dishes were left in the cage for 24 h before they were removed and the

number of eggs laid in the soil in each dish was determined by examination of soil under a binocular microscope (x10 magnification) as described previously. This was a single experiment which was replicated four times using the same cage and beetles.

5.2.3 DISTRIBUTION OF NISOTRA BASSELAE EGGS IN THE SOIL AROUND HOST PLANTS IN THE FIELD

The relationship between oviposition site and distance from the base of the stem of A. manihot plants was investigated in the field at the Tutuva village site between 16th July and 7th August 2008. The land was cleared and prepared as described previously (Chapter 2). Sixteen plots (plot size = 2 m x 2 m) were prepared in a 4 x 4 grid. Sixteen A. manihot (Broad-type variety) cuttings were planted in a 4 x 4 grid within each plot, plants were spaced 0.5 m apart and adjacent plots were separated by 2 m of bare soil.

Once plants had reached the 11 leaf stage, approximately 3 months after planting, a single plant from the central four plants in the middle of 10 randomly selected plots was selected for sampling. In order to sample the number of N. basselae eggs in a unit volume of soil around the base of each of the ten randomly selected plants a fine string was tied to the base of the plant and a small wooden pointer tied to the other end. This was then used to mark a circle of 5 cm radius from the stem in the soil. The soil within this circle was then carefully removed by hand to a depth of 2 cm and transferred to a labelled plastic bag that was then sealed. This method of marking the soil surface and then collecting the soil sample to a depth of 2 cm was repeated for successive concentric circles of radius 10 cm, 15 cm, 20 cm and 25 cm from the base of each of the ten selected plants. Soil samples were taken to the laboratory and processed immediately to ensure that eggs were counted before they hatched. In the laboratory, each soil sample was carefully poured into a white plastic tray (20 cm x 30 cm). The soil was then carefully sifted and examined under a dissecting microscope (x10 magnification) and the number of N. basselae eggs in each sample was recorded. In order to ensure that samples could be processed as soon after collection as possible a maximum of 3 plants were sampled on any given day, the ten

plants were sampled over an 8 day period (16 July – 7 August, 2008).

5.2.4 OVIPOSITION OF NISOTRA BASSELAE IN SOIL AROUND POTTED HOST PLANTS IN THE SHADE HOUSE

Abelmoschus manihot (Broad-type variety) plants used in the experiments were grown in polythene potting bags (15 cm diameter, 20 cm deep) in the shade-house as described previously (Chapter 2). Plants were at the 11 - leaf stage and were 3 months old when used in the experiments.

A plant was placed in the centre of a large plastic bowl (60 cm diameter x 25 cm deep) and the bowl was then filled to the brim with soil. The upper margin of the polythene bag surrounding the roots of the plant was then trimmed to ensure that the surface of the soil within the bag and the surface of the soil in the bowl was level (Figure 5.1). The soil surface in each bowl was then carefully moistened by spraying with rainwater held in a hand sprayer. Five plants prepared in this way were then each introduced into individual oviposition cages (70 cm x 70 cm x 100 cm) covered by ant-proof white cotton screen-netting. Twenty recently eclosed adult N. basselae (10 females and 10 males) were introduced into each cage. These beetles were collected from the field at Tutuva (Guadalcanal Plain) and were placed inside a cage in the laboratory for a week. Plants were exposed to beetles in the oviposition cage for four days before they were removed.

A similar method to that used to determine egg numbers in field collected soil samples was used to determine the egg numbers in the different zones of soil around each potted plant in each cage. A length of fine string was tied to the base of each plant and a circle 2 cm in radius from the stem was marked on the soil surface by a small wooden pointer tied to the other end of the string. The soil within this circle was then carefully removed to a depth of 2 cm and transferred to a labelled plastic bag that was then sealed. This process was repeated for the soil remaining within a radius of 7.5 cm from the plant stem and finally for the soil remaining within a 15 cm radius from the plant stem; the soil in the latter sample was external to the polythene potting bag and thus outside of the plant’s root zone. Once all soil samples from each of the five plants had been collected, they were then taken to the laboratory and

the number of eggs in each sample was determined using a binocular microscope (x10 magnification) as previously described.

Plant

0-2.0cm zone

2.0 -7.5cm zone (root zone)

7.5-15.0 cm zone (beyond root zone)

Nylon cage

Poly-bag potting

Plastic bowl

Figure 5.1 Oviposition chamber to investigate N. basselae oviposition in different soil zones around a potted A. manihot plant in the laboratory.

5.2.5 INTRA-PLANT MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL

5.2.5.1 MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL ON POTTED PLANTS IN A CAGE

A cage experiment was setup to investigate the intra-plant movement of N. basselae between foliage and soil. Six A. manihot plants (Broad-type variety) potted in polythene bags (15 cm diameter, 20 cm deep) were grown for 3 months (11-leaf

stage) as previously described. They were then placed inside a net cage (70 cm x 70 cm x 100 cm) and a 2 mm thick by 10 cm long layer of Tangle-foot was applied around the stem of each plant, starting at 2 cm above soil level. Sixteen male and 16 female N. basselae were randomly selected from the laboratory population from Tutuva village and released on the foliage of each plant at 08:00, immediately after application of Tangle-foot to the base of plants. The Tangle-foot traps on each plant were examined the next morning between 08:00 and 09:00. All beetles trapped in the upper margin of Tangle-foot on a given plant were collected, counted and transferred to a labelled glass vial before the beetles trapped on the lower margin were collected, counted and transferred to a separate labelled vial. The beetles were then examined and dissected under a dissecting microscope and the sex of each (based on the presence or absence of male aedeagus) was then determined. This procedure was repeated 48 h and then 72 h after the release of beetles on the foliage.

5.2.5.2 MOVEMENT OF NISOTRA BASSELAE BETWEEN HOST PLANT FOLIAGE AND SOIL IN THE FIELD

Sixteen A. manihot plants (Broad-type variety) grown in the field at Tutuva village that were approximately six months old and were severely infested with N. basselae were used to investigate the movement of beetles between foliage and soil in the field.

Four plants, spaced 0.5 m apart, in the centre of four 4 x 4 plots were used in the experiment. On the first day of the experiment, at approximately 09:00, the total number of adult N. basselae on each selected plant was carefully counted and recorded without disturbing the beetles. Immediately after the count on a given plant a 2 mm thick layer of Tangle-foot was pasted around the stem of each plant, starting 2 cm above the soil surface and covering the next 10 cm portion of the stem. The following morning, the number of adult N. basselae on the foliage and the number and sex of those trapped on the upper and lower margins of Tangle-foot traps were recorded as previously described. This procedure was repeated 48h and then again 72 h after the setup of the Tangle-foot traps.

5.2.6 THE EFFECTS OF SOIL SURFACE BARRIERS ON NISOTRA BASSELAE OVIPOSITION IN THE FIELD

An area of 30 m x 30 m adjacent to the southern end of the A. manihot plot used to monitor the seasonal abundance of N. basselae (see Chapter 4) was planted with A. manihot (Broad-type variety) in late March 2009. Sixteen plots, each consisting of a 4 x 4 grid of plants spaced 0.5 m apart and separated by 2 m of bare soil, were prepared. At the time of planting one of four soil surface treatments was applied to each of the plots. Each treatment was replicated four times and treatments were arranged in a Latin square design. The soil surface treatments tested were applied to the entire surface of each plot. In the bare soil surface treatment, the soil surface of plots was thoroughly cleared of any plant debris at the time of planting and plant debris and weeds were routinely removed from the plot by hand throughout the experiment. In the mulched soil surface treatment, a 10 cm-thick layer of grass straw mulch (Imperata cylindrica (L.)) was applied to the soil surface of the plot at planting and maintained throughout the experiment by addition of extra mulch as required. In the living clover cover treatment, clover plants (Trifolium repens L.) were transplanted (four blocks of grafted clover 10 cm x 10 cm) around the base of each A. manihot plant at the time that experimental plants were transplanted. In the copra sack treatment, copra sack material from new knitted fabric bags (60 cm x 60 cm x 100 cm) was cut into strips and laid over the surface of soil in the plots at the time of planting. Adjacent sheets of sacking overlapped by 20 mm to ensure that no soil was exposed in the designated plots.

The number of adult N. basselae feeding on the foliage and the number of eggs laid within a 5 cm radius of the base of each plant were determined and compared in the different treatments. The first assessment was conducted in late June 2009 (87 days after planting) while the second assessment was carried out two weeks later in early July 2009 (101 days after planting). The four plants at the centre of the 4 x 4 grid of plants in each plot were assessed; two were randomly selected for the first assessment and the remaining two plants were then examined for the second assessment. During each assessment, the number of adult beetles on each plant was recorded followed by the removal of a 2 cm deep soil sample (5 cm radius) from the base of the plant as previously described. The mulch, copra sack and clover

were carefully removed from the soil surface in these treatments before the soil for collection was marked. Once the soil samples had been collected they were placed into labelled bags as previously described and taken to the laboratory where they were stored in a fridge for up to 3 days to prevent egg hatch before assessment. The number of eggs in each soil sample was then determined by carefully examining the soil on a white tray (20 cm x 30 cm) under a binocular microscope (x10 magnification) as previously described. It took approximately one hour to examine the soil from each plant and it took three days to complete examination all of the samples.

5.3 RESULTS

5.3.1 EFFECTS OF SOIL MOISTURE ON NISOTRA BASSELAE OVIPOSITION

Adult N. basselae females preferred to lay eggs in moist soil rather than in dry soil

(F1, 9 = 20.0; P < 0.5) and moist mulched soil rather than in moist but bare soil, (F1, 16 = 25.31; P < 0.05) (Figure 5.2). In fact no eggs were recovered from the dry soil in any of the experiments.

35 a

30 eggs laid laid eggs

20 N. basselae basselae N. 15

SE) no.SE) of ± 5 b

Mean Mean ( 0 Moist soil Dry soil

Figure 5.2 Mean (±SE) number of eggs laid by female N. basselae in moist and dry soil, No eggs laid in dry soil. Different letters marked significant difference between the two treatments.

5.3.2 EFFECTS OF MULCHES ON NISOTRA BASSELAE OVIPOSITION

Adult female N. basselae deposited significantly more eggs in Petri dishes with soil covered with mulch than in Petri dishes with bare soil (F1, 16 = 25.31, P < 0.05; Figure 5.3). Eggs were laid singly and in clusters of 2 to 22 eggs. Most of the eggs were deposited beneath the soil surface but some were laid on the soil surface directly under the mulch layer.

50 a 45

40 eggs laid eggs 35

N. basselae N. 25

15 SE) number ofSE)number

± 10

Mean ( Mean b 0 Mulched soil Bare soil

Figure 5.3 Mean (±SE) number of eggs laid by female in mulched and bare soil (F1, 16 = 25.31; P < 0.05). Points marked with different letters are significantly different.

5.3.3 DISTRIBUTION OF NISOTRA BASSELAE EGGS IN THE SOIL AROUND HOST PLANTS IN THE FIELD

Adult female N. basselae deposited greater numbers of eggs close to the base of A. manihot plants than in soil further away. The numbers of eggs in the different concentric zones around the plant were significantly different (F4, 45 = 34.75; P < 0.05), and eggs decreased in number with increased distance from the base of plant

stem; Figure 5.4). Approximately 92%, of the total eggs recovered were laid within 0 - 5 cm of the base of the host plant, while only 7% and 1% of eggs respectively were recovered from the zones 5 - 10 and 10 - 15 cm away from the base of the plant. No eggs were recovered from the soil samples collected more than 15 cm from the base of the plant (Figure 5.6).

There was a significant linear relationship between the total number of N. basselae eggs recovered from soil within a 15 cm radius of a plant and the total number of 2 adults on the foliage of that plant (F1, 9 = 39.83, P < 0.05; r = 0.83) (Figure 5.5).

of soil soil of 3

cm 20

15 N. basselae/N.

SE) no.of eggs ofeggs no.of SE) ± 5 b

Mean ( Mean c d 0 0 - 5.0 cm 5.0 - 10.0 cm 10.0 - 15.0 cm 15.0 - 20.0 cm

Distance from base of the stem of A. manihot

100

60 soil

3 y = 1.2454x - 1.8205

50 R² = 0.8328 /cm

40 N. basselae N. 30

10 Number of eggs ofeggs of Number

0 0 10 20 30 40 50

Number of adult N. basselae /plant

Figure 5.5 Relationship between the total number of eggs of N. basselae within a radius of 15 cm at the depth of 2 cm at the base of host plants and the number of 2 adult N. basselae on foliage. (Linear regression: F1, 9 = 39.83; P < 0.05; r = 0.83).

5.3.4 OVIPOSITION OF NISOTRA BASSELAE IN SOIL AROUND POTTED HOST PLANTS IN THE SHADE HOUSE

The number of eggs laid in each of the designated zones around plants was significantly different (F2, 33 = 8.22, P < 0.05; Figure 5.4). Over 99.9% of all eggs recovered was collected from soil within a 7.5 cm radius of the stem and 74% of these eggs were recovered from soil collected 0 - 2 cm from the base of the stem (LSD; P < 0.05; Figure 5.6).

101

Root zone Non root zone

25 a

3 20

SE) eggs per per eggsSE) cm 10 ± b

5 Mean ( Mean

c 0 0.0 - 2.0 cm 2.0 - 7.5 cm 7.5 - 15.0cm Distance from plant stem

5.3.5 INTRA PLANT MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL

5.3.5.1 MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL ON POTTED PLANTS IN A CAGE

Only female beetles were caught on Tangle-foot on the stems of A. manihot. Significantly more N. basselae were trapped on the upper margin than on the lower margin of the Tangle-foot placed around the stems of A. manihot (F2, 33 = 8.22, P < 0.05; LSD, P < 0.05) (Figure 5.7).

102

80 a 70

Female beetles trapped 60 Male beetles

50 b

N. basselae N. 40 a a

30 a

a SE) no. of no. SE)

± 20 c 10 c

Mean( b b b b 0 Upper Lower Upper Lower Upper Lower 24 Hours 48 Hours 72 Hours

(ANOVA: F2, 33 = 8.22, P < 0.05; LSD, P < 0.05).

5.3.5.2 MOVEMENT OF NISOTRA BASSELAE BETWEEN FOLIAGE AND SOIL ON PLANTS IN THE FIELD

There was a significant difference between mean number of N. basselae on the foliage at successive monitoring times (F1, 45 = 11.34; P < 0.05) (Figure 5.8). The mean number of beetles at 0 h was significantly higher than the mean number of beetles at 24 h and 48 h (LSD, P < 0.05) but there was no difference between the mean number of beetles on foliage 24 h and 48 h after trapping began (LSD, P > 0.05) (Figure 5.8).

Overall, there was a significant difference in the number of N. basselae trapped per plant at the different time intervals (F1, 45 = 21.8; P < 0.05) (Figure 5.8). The mean

103

number of beetles trapped per plant at 24 h was significantly higher than the number trapped at 48 h and 72 h (LSD, P < 0.05). The mean number of N. basselae trapped at 48 h was also significantly higher than the mean number trapped at 72 h (LSD, P < 0.05).

At 24 h the mean number of beetles caught on the upper margin of the Tangle-foot trap was significantly greater than the mean number of beetles caught on the lower margin (F1, 60 = 6.78; P < 0.05; Figure 5.8). Also, there were significantly more female beetles trapped than male beetles (F1, 60 = 64.65; P < 0.05; Figure 5.8).

At 48 h there was no significant difference between the numbers of beetles caught at the upper and lower margins of the Tangle-foot traps (F1, 60 = 0.01; P > 0.05).

However, there were more female beetles caught than male beetles (F1, 60 = 61.55; P

< 0.05; Figure 5.8). A similar pattern was observed after 72 h (F1, 60 = 0.02; P > 0.05), but the overall number of beetles caught at that time was reduced by approximately 40%. Again, there were significantly more female beetles than male beetles caught at both the upper and lower margins of the Tangle-foot paste trap (F1, 60 = 57.08; P < 0.05; Figure 5.8) and there was no significant difference between the overall numbers of beetles caught at the upper and lower margins of the Tangle-foot paste traps, (F1, 60 = 0.016; P > 0.05).

104

35 a Adult beetles on foliage

/plant 30 Adult beetles trapped in tangle-foot

25 b N. N. basselae 20 c

15 a1

SE) number of SE) number ± 10

b1 Mean ( Mean c1 5

0 0 hours 24 hours 48 hours 72 hours

Monitoring time intervals

5.3.6 THE EFFECTS OF SOIL SURFACE TREATMENT ON NISOTRA BASSELAE OVIPOSITION IN THE FIELD

There was no significant difference between the number of eggs recorded in the first and second assessments (F1, 60 = 1.541, P > 0.05); nor was there a significant interaction between assessment time and soil surface treatments (F3, 60 = 0.31; P > 0.05). Consequently, data were pooled together for analysis (Figure 5.9). Overall, soil surface treatment had a significant effect on the mean number of N. basselae eggs laid at the base of plants (F3, 60 = 3.86; P < 0.05; Figure 5.9). Fewer eggs were recovered from the base of plants covered with copra sack than in any of the other treatments (LSD, P < 0.05; Figure 5.9). Plants covered with copra sack reduced the average number of eggs recovered by 53%, 51% and 45% when compared to

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numbers recovered from plants treated with mulch, clover and bare soil, respectively. There were no significant differences between the mean numbers of eggs collected around plants in soil that was left bare, covered with grass mulch or sown clover (LSD, P > 0.05; Fig 5.9).

There was a significant linear relationship between the number of beetles on the foliage of a plant and the number of eggs deposited at the base of that plant (within 0 2 – 5 cm of the stem) in all treatments (mulch treatment (F1, 15 = 57.61; P < 0.05; r = 2 0.81); Copra sack treatment (F1, 15 = 14.51; P < 0.05; r = 0.51), Clover treatment (F1, 2 2 15 = 16.65; P < 0.05; r = 0.54) and bare soil treatment (F1, 15 = 12.16; P < 0.05; r = 0.47), Figure 5.10). There was no significant difference between the slopes of these regressions in any of the treatments (F3, 56 = 1.28; P > 0.05) and a pooled slope of 1.16 could be fitted the data. However, the y-axis intercepts of the regressions were significantly different (F3, 59 = 7.49; P < 0.05) (Figure 5.8).

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12 a

ofsoil 3

10 a /cm

8 N. basselaeN.

6 SE) no. ofofeggs no. SE)

± 4 Mean(

0 Mulches Copra Sack Clover Bare Soil

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35 35 a) Mulch b) Copra sacking 30 30 25 25 20 20 15 15 10 10

5 y = 1.449x + 1.4372 5 y = 1.3611x - 3.8039 Eggs withinstem Eggsof 5cm R² = 0.8045 withinstem Eggsof 5cm R² = 0.509 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Adult beetles per plant Adult beetles per plant

35 35 c) Clover d) Bare soil 30 30 25 25 20 20 15 15 10 10

5 y = 0.9427x + 5.5301 5 y = 0.8896x + 2.0752 Eggs withinstem Eggsof 5cm Eggs withinstem Eggsof 5cm R² = 0.5432 R² = 0.4648 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Adult beetles per plant Adult beetles per plant

Figure 5.10 Linear relationships between the numbers of adult N. basselae per plant and the number of eggs deposited within 0 - 5 cm of the base of the plant. Linear regressions of egg density against adult beetle density were significant for all 2 treatments. Mulches (F1, 15 = 57.61; P < 0.05; r = 0.81); Copra sack (F1, 15 = 14.51; P 2 2 < 0.05; r = 0.51), Clover (F1, 15 = 16.65; P < 0.05; r = 0.54) and bare soil (F1, 15 = 12.16; P < 0.05; r2 = 0.47).

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5.4 DISCUSSION

According to laboratory experiment results, most N. basselae females preferred to lay eggs in moist or mulched soils, rather than in either dry or bare soil. One of the properties of mulches is their ability to retain moisture in the soil (Louise 1996). There are advantages and disadvantages when using mulches around A. manihot plants. Although mulches can promote plant growth by maintaining high soil moisture levels, they may also provide favourable environments for N. basselae oviposition. Eggs can rapidly lose viability when laid in dry environments (Jarvis 1919) and the moist environments that are provided by soil surface mulches can increase the egg viability and lead to increased pest populations (Pandit and Chatterji 1978).

Materials used as mulches can vary from organic residues (for example compost, grasses, leaves, hay, straw, kitchen scraps, saw dust, newspapers) to manufactured mulches (e.g. rubber and plastic mulches) and living mulches or ground covers (Louise 1996). The type and volume of mulch material can determine whether mulch is either impeding oviposition or promoting oviposition and overall insect development (Diver and Hinman 2008).

In the laboratory experiment which measured the number of eggs that female N. basselae laid, only a thin layer of grass straw mulch was tested and so it could not be determined if the higher number of eggs laid was due to the provision of a cryptic oviposition site under the mulches, higher soil moisture levels or a combination of both factors. Choice of oviposition site is widely considered to be driven by selection of environments where eggs and larvae will have greatest chance of survival through provision of favourable abiotic conditions and the provision of food (Futuyma 1983; Thompson 1988a; Jaenike 1990; Janz et al. 2001).

Other factors that require consideration when dealing with mulches relate to their ability to suppress pests, especially weeds (Diver and Hinman 2008). Mulches that are very rich in organic materials provide micro-environments suitable for possible biological control agents such as mole crickets, lizards, ground slaters, spiders and ants that may feed on eggs, larvae, pupae and adult beetles (Brust, 1994; Chang et. al 2004).

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Mulches (typically debris of slashed shrubs and grasses) are often applied to the surface of the soil around A. manihot plants to retain soil moisture, prevent weeds and to supplement soil nutrients when the mulches eventually decompose. In the Guadalcanal plain, where much of A. manihot is cultivated for sale in Honiara markets and where the weather is often drier than elsewhere, mulching of plants is typical in A. manihot cultivation. However, while mulches can be beneficial to plants by retaining moisture they can provide ideal conditions ideal for N. basselae oviposition and increase survival rates of eggs and larvae. Striking a balance to overcome the problem of creating the ideal environment for N. basselae oviposition but at the same time promoting soil moisture retention for A. manihot is important and yet to be explored.

Mulches underneath the plants can create warm, buffered microclimates which could promote oviposition and the survival of immature insects (Bristow 1988, Nahrung and Merritt 1999, Weston and Diaz 2005). Both the field and laboratory experiments conclusively showed that female N. basselae preferred to deposit more eggs both beneath the soil surface close to the base of the host plant stem. The precise depth at which eggs were laid was not determined in the experiments but most of the eggs observed were buried beneath the soil surface. Such oviposition patterns are common for flea beetles (Parker 1910; Chamberlin and Tenhit 1924; Hanson 1933; Gray et al. 1992; Johnson and Gregory 2006). Eggs of N. basselae were only recovered within the root zone and they were particularly concentrated within 2 cm of the base of the plant stem (Figure 5.2 and 5.4).

The results from the Tangle-foot trap experiment (Figure 5.5) clearly showed that gravid female N. basselae crawled from the A. manihot foliage, down through to the base of the stem and finally to the soil where they laid their eggs. The fact that in both field and laboratory experiments significantly more female beetles than male beetles were trapped on Tangle-foot traps (Figures 5.5 and 5.6 ) suggests that gravid female beetles crawl from the host plant foliage to the soil to lay eggs. The female beetles were trapped on the upper margin of Tangle-foot traps were probably gravid female beetles moving towards oviposition sites (Figure 5.6). Beetles that were caught on the lower side of the Tangle-foot traps included males and females, presumably representing returning adult females after a transient period of

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oviposition in the soil and newly emerged adult males and females. The period that a single gravid female N. basselae would normally spend in the soil during oviposition was not observed and is therefore unknown; but, according to Jarvis (1919), this can take several days. Daily Tangle-foot trapping of beetles from single plants decreased the number of beetles on the foliage of experimental plant in proportion to the number of beetles trapped, suggesting high fidelity to a given plant and limited movement between plants within a crop.

The final experiment was designed to investigate the possibility of using physical barriers to oviposition on the soil surface to reduce N. basselae densities within a crop. Covering the soil surface with copra sack reduced egg densities in the soil immediately adjacent to the plant stem by 45%, 51% and 52% when compared with bare soil surface, clover surface and mulches respectively (Figure 5.7). The beetles that did lay eggs probably crawled down the stems of host plant through the hem of copra sack and laid eggs in the soil.

Significant positive linear relationships between the total number of adult N. basselae on the foliage of plants and the number of eggs deposited within the defined soil zones at the base of the plant under different soil surface treatments were recorded (Figure 5.2 and Figure 5.6). The results indicate that an increase in the density of adult beetles on the foliage of each plant had a corresponding effect on the egg density in the soil around the stem of that plant.

In all the soil surface treatments there was a significant positive linear relationship between the number of adult beetles in the crown of the plant and the number the number of eggs laid in the soil at the plant base (Fig 5.8). In the copra sack treatment the regression line had a negative y-axis intercept value, indicating that at low adult beetle densities in the crop canopy no eggs were laid in the soil (Fig 5.8). This attests to the effectiveness of its physical barrier and indicates that it has potential for development as a cultural control strategy.

Making recommendations based on the results from this study, especially the soil surface practices tested, at this stage would be too early as subsequent survival experiments have not yet been done. However, the promising results, and the confirmed results by others as discussed earlier (Stoner 1996; Jackson et al. 2002;

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Johnson et al 2004, Ciobanu et al. 2009), suggest that cultural control practices that manipulate the soil surface to inhibit oviposition site access may have great potential for the management of this pest. Specifically, copra sack barriers resulted in fewer eggs being laid at oviposition sites and the adoption of these, or similar, physical barriers could be an option for subsistence farmers attempting to control N. basselae in A. manihot crops. However, the cost of sacks can be high and they are not always available.

The clover ground cover was included as a treatment based on anecdotal reports that it reduced N. basselae populations in the field. Past studies have shown that clover can reduce pest abundances and improve crop quality (Theunissen et al. 1994). A similar result, in which egg and larval densities were reduced significantly but spider density increased, was found in plots of broccoli (Brassica oleracea L.) intercropped with clovers (Hooks et al. 2007). The reduction of the number of eggs laid and larval densities could be in part due to natural enemies promoted by mulches (Hooks et al. 2007). Johnson et al (2004) also reported a similar effect by straw mulches which increase the number of adult L. decemlineata but reduced larval numbers.

In conclusion, it is clear that N. basselae, like other soil inhabiting Chrysomelidae, prefer to lay eggs in the soil within the root zone, especially close to the base of their host plant stem (Parker 1910; Jarvis 1919; Chamberlin and Tenhit 1924; Hanson 1933; Gray et al. 1992; Johnson and Gregory 2006). Although, not determined, it is speculated, with support from previous studies (Jones and Coaker 1978; Singh and Mullick 2002), that volatiles and contact chemicals either from the roots or base of host plant stem, or both, played the role of attracting gravid female beetles to lay eggs on the sites. It was evident from the Tangle-foot trap experiment that beetles, particularly females, actively move between the foliage and soil along the stem of the host plant. Beetles caught moving up the stem could include newly emerged individuals, those that had dropped off from the foliage, and returning females after periods of oviposition in or on the soil. Male beetles caught trying to move down the stem were possibly attracted to female beetles that were already caught in the traps.

The previous lack of knowledge of the ecology of N. basselae has made control of this pest difficult in Solomon Islands. Farmers that were informally interviewed while

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carrying out this study expressed concern about the seriousness of this pest and consider that if it is not controlled it can destroy whole crops. While some subsistence farmers often have no choice but to tolerate the pest, others, especially those that live close to urban centres and markets, rely heavily on insecticides for control. A survey carried out in villages close to urban markets on Malaita and Guadalcanal (Teakle and Bakale, 2007), showed that as many as 54% of farmers have used insecticides (Orthene, Carbaryl, Target, Maldison, Applaud, Permethrin, Lannate, DDT) on their A. manihot crops. The use of insecticides by subsistence farmers to control N. basselae and other pests in the peri-urban villages is a concern, as these chemicals are expensive and associated with adverse effects on the health of consumers and the labourers who apply them as well as causing environmental contamination.

Farmers apply insecticides only to crop foliage as they target the visible and damaging adult stages of the pest. The effect of larval root feeding on crop yield and quality is currently unknown but it warrants future study. The distribution of larvae feeding on the roots of plants is not known but eggs are concentrated in a restricted area within soil immediately beneath the base of hosts. This represents a possible target for the application of insecticides. If successful, such an approach would have the advantages that insecticide would not be applied to the foliage that is eventually consumed by humans and precise small areas (immediately around the base of plants) could be targeted; thereby reducing insecticide inputs and destruction of egg and larval stages would reduce adult populations that damage crop foliage.

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CHAPTER SIX: ADULT NISOTRA BASSELAE MOVEMENT AND THE EFFECTS OF BIOTIC AND ENVIROMENTAL FACTORS ON HOST PLANT SELECTION

6.1 INTRODUCTION

Light drives the process of photosynthesis in plants, affecting the production of simple sugars and secondary metabolites (Kitaya et al. 1998; Dieln et al. 2004). Therefore, light greatly influences insects’ behaviour in terms of their distribution and feeding habits (Close and McArthur 2002). Light is a dynamic commodity that varies in quantity, as in instantaneous dose accumulated over time and day length, as well as in quality, as in spectral or wavelength balance (Bjorn 2002). Thus, the attributes of light emitted from the sun in terms of quality and quantity and captured by plants on earth depend on factors such as the elevation of the sun in the sky during the diurnal cycle, season and latitude. Other factors that attribute to light variation include greenhouse effect, cloud cover, slope elevation, and density of plant canopies (Fitter and Hay 2002).

The distribution of herbivorous insects and corresponding damage inflicted on host plants are clear evidences of their feeding site preferences. Nutritional status of host plant is very much a factor that determines such feeding site preference that also shapes the overall distribution of insects. Usually such preference and distribution pattern vary with plant age, attributes of light exposed by the plant, availability of water, nutrient elements and other resources in the soil that plants exposed to and so on (Mattson 1980, Slansky 1993; Harbone 1993; Goverde and Erhardt 2003). Also, usually, young seedlings, new leaf flushes and re-growth tissue are much preferred due to the higher composition of nitrogen that they exhibit as compared to aging plants and older plant parts which tend to have high carbon-based secondary compounds.

The first experiments in this chapter investigated the distribution of N. basselae (Bryant) between A. manihot plants grown in full-sun and shaded conditions in the field. Laboratory experiments then examined the effects of full-sun and shaded

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growing conditions and the application of nitrogen fertilizer on preferences on host selection the preferences of adult beetles.

6.2 MATERIALS AND METHODS

6.2.1 DISPERSAL OF NISOTRA BASSELAE

Seven hundred adult N. basselae were collected from A. manihot (Broad-type variety) plants in a garden at Tutuva village. The insects were then reared on potted A. manihot (Broad-type variety) plants in the laboratory, as previously described (Chapter 2). Prior to use in the experiment, beetles were transferred to collecting vials (dia. 3 cm x 12 cm) with a pooter and then divided into 35 equal batches of 20 beetles that were held in separate Petri dishes (9 cm diameter) ready for field release.

The experiment was conducted on a recently mowed flat lawn area (45 m x 70 m) that was situated in the middle of Honiara. The surrounding buildings and trees were far enough away so that no shadows encroached on the study site during experiments. On the 7th October 2008, nursery plants at the 11 leaf stage (reared as described in Chapter 2, Section 2.4.1) were transported to the experiment site by a truck. Plants were then arranged in a series of concentric circles, 5 m, 10 m, 15 m and 20 m from a central point. A compass was used to position plants on the perimeter of each concentric circle, the plants were positioned as follows: (1) on the perimeter of a circle of 5 m radius, individual plants were placed at the N, E, W and S cardinal points; (2) on the perimeter of a circle of 10 m radius, individual plants were placed at the cardinal points and at the NE, SE, SW and NW points between these and (3) on the perimeter of circles of 15 m and 20m radius, on each circle, individual plants were placed at the following points, N, NNE, NE, EES, E, SEE, SE, SSE, S, SSW, SW, SWW, W, NWW, NW and NNW. Single yellow sticky card traps (standard whitefly traps, 7.5 cm x 12.5 cm held at 0.5 m above the ground on wooden stakes) were positioned at approximately 2 m intervals between the plants within each ring. When all plants and sticky traps were in position, 700 beetles (equal sex ratio) were released at the central point of the concentric circles at 18:00 on 7th October 2008 by placing all 35 Petri dishes on the ground and gently removing the lids.

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The numbers of N. basselae on the plants and the numbers trapped on the sticky traps were recorded every 12 h after the release until 72 h after release. The first assessment took place at 06:00 on 8th July, the next day after the release. Assessments were carried out by visual observation of beetles on each leaf of each plant while taking care not to disturb beetles.

6.2.2 THE EFFECTS OF CONSPECIFIC FEEDING DAMAGE, MECHANICAL DAMAGE AND THE PRESENCE OF CONSPECIFICS ON ATTRACTION TO HOST PLANTS

Potted plants used in these experiments were reared to the 11-leaf stage as previously described (Chapter 2, Section 2.4.1). Nisotra basselae used in the experiments were collected from A. manihot (Broad-type variety) at Tutuva and reared as previously described (Chapter 2, Section 2.5) before use in the experiments. All experiments were conducted at the field site used to investigate the flight and directional movement of N. basselae (see Section 6.2.1). The same basic procedure was used to test the effect of N. basselae infestation and feeding, mechanical damage and the presence of male or female beetles (but no feeding damage) on the attraction of beetles to treated plants relative to intact control plants. In each experiment, eight individual potted plants were placed at 2 m intervals on the circumference of a 5 m diameter circle; plants were arranged at each of the four cardinal points and then at points between these, such that a plant was positioned in the following directions from the centre of the circle, N, NE, E, SE, S, SW, W and NW points. For each experiment six rings spaced 10 m apart were set up. In the first, second and third rings of plants, plants at the cardinal points underwent the requisite treatment, while the plants on either side remained untouched, and in the second, fourth and six rings of plants, plants at the cardinal points were untouched while the plants on either side underwent the requisite treatment. This ensured that any directional bias due to external factors was minimised. All six replicates for a given study were conducted simultaneously. The three different experiments were conducted one week apart from each other.

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6.2.2.1 EFFECTS OF CONSPECIFIC FEEDING AND INFESTATION ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS

This experiment investigated the effect of N. basselae infestation on host plant selection by conspecific beetles. Treated plants were infested by releasing ten N. basselae (five males and five females) into 24 cylindrical vinyl insect-proof screen net cages (diameter 30 cm x height of 70 cm) and placing these over the selected plants. Intact plants received no treatment. Beetles were allowed to feed on selected plants for 12 h. Infested and intact plants were then positioned within six rings as previously described and the cages removed from infested plants. One hundred N. basselae (50 males and 50 females) were then released at the centre of each ring of plants. The number of N. basselae on each plant was recorded every 12 h after the release for 72 h.

6.2.2.2 EFFECTS OF MECHANICAL DAMAGE ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS

The effect of mechanical damage on host plant selection by N. basselae was investigated in a similar manner. In treated plants 10 simulated shot-holes were punched in leaves 4 to 11 using a 5 mm diameter hole-puncher. The holes on the leaves were made about 30 minutes before the beetles were released in the centre of the circles. Untreated plants remained intact. Treated plants and intact plants were then arranged around the perimeters of six 5 m diameter rings as previously described. One hundred N. basselae (50 males and 50 females) were then released at the centre of each ring of plants. The number of N. basselae on each plant was recorded every 12 h after the release for 72 h.

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6.2.2.3 EFFECTS OF MALE OR FEMALE CONSPECIFICS, BUT THE ABSENCE OF FEEDING DAMAGE, ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS

A final experiment investigated the responses of beetles to plants supporting caged conspecific male or female adult N. basselae. Forty-eight nylon net sleeves were prepared, 10 adult female beetles were carefully introduced to 24 of these while 10 male beetles were introduced to the remaining 24 sleeves. The sleeves were then carefully attached to plants so that treatments in each ring alternated between male and female treated plants. One hundred N. basselae (50 males and 50 females) were then released at the centre of each ring of plants. The number of N. basselae on each plant was recorded every 12 h after the release of the beetles for 72 h.

6.2.3 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON NISOTRA BASSELAE DISTRIBUTION IN THE FIELD

This study was conducted in an isolated commercial A. manihot crop (30 m x 30 m) surrounded by grassland (south and east) and secondary forest (north and west) at Tutuva village in January 2007. Prior to planting, the plot was prepared in the usual manner by slashing and collecting cleared vegetation, which was then heaped at the side of the plot (Chapter 2). Within the plot, remnants of primary forest trees (mainly rain-trees, Samanea saman Merrill (Fabaceae)) were left to provide shade for the crop; a common practice when growing A. manihot in Solomon Islands.

Within the plot a mixture of 12 cultivars of A. manihot was planted following typical subsistence standard farming practice (slash and burn land preparation). Some plants were grown under shade provided by the rain trees, while others were grown in open areas and were more exposed to full sun. The cultivar selected for the study was the Broad-type variety since it was the most common cultivar, accounting for approximately 80% of all plants in the plot. Plants had grown in the field for approximately 4 months and an initial harvest had been taken one month before the study. At the time of the study, plants contained at least nine leaf nodes and they were due for a second harvest. The plot was divided into three blocks, each block was clearly marked by wooden stakes and each contained plants grown in full-sun

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and those grown in shaded conditions under trees. Five plants grown in full-sun and 5 plants grown in shade were selected at random from each of the three blocks (Figure 6.1) and marked by plastic tape tied to their stems. The number of adult beetles feeding on the foliage of all marked plants was counted and recorded as previously described (Chapter 4, Section 4.2.3).

The number of adult beetles and the number of shot-hole feeding sites on leaves 3, 4 and 5 of a randomly selected stem of each of the 10 selected plants in each of the 3 blocks was systematically determined between 06:00 and 08:00. Leaves 3, 4 and 5 are preferred feeding sites for adult beetles (Chapter 4, Section 4.3.3).

30m

B 2

30m

B 3

Tree shade Plant in full-sun Plant in shade

Figure 6.1: Plan of A. manihot plot used to assess the distribution of adult N. basselae and feeding damage between plants grown in the shade of rain trees and plants grown in full-sun.

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6.2.4 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON NISOTRA BASSELAE DISTRIBUTIONS BETWEEN POTTED SENTINEL PLANTS IN THE FIELD

In late June 2008, 60 cuttings of A. manihot (Broad-type variety) were planted into soil in poly-bags as previously described (Chapter 2). The plants were then divided into two groups, one group of 30 plants was grown in the nursery under green nylon shade-cloth which allowed 70% of incident full-sun to penetrate (Site Shade Company, estimate) and the remaining 30 plants were grown in the nursery exposed to full-sun. No measurements of light intensity or the radiation spectrum were taken in either of the light growing regimes. The plants in each of the light regimes were irrigated with approximately 1 L of water per plant two times a day and treated with fertilizer as previously described (Chapter 2). The plants grew at a similar rate in each environment and were raised to the 11 - leaf stage before they were used in the field experiment, approximately three months later.

The experiment was conducted in plots of A. manihot (Broad-type variety) at Tutuva village in the Guadalcanal plain. It was replicated at two sites; Site 1 and Site 2. These sites were located 30 m apart (Figure 6.2). The plants at each site were planted in late June 2008, at the same time as those planted in the nursery, and were grown under the agronomic practices previously described in Chapter 2. Each plot of plants contained two sub-plots of 30 plant that were arranged in a 6 x 5 grid; plants within each grid were spaced 0.5 m apart (Figure 6.2). One subplot was located in the shade under paper mulberry trees (Broussonetia papyrifera (L.) (Moraceae family)), and the other was located 3 m away, it was not shaded but in full-sun (Figure 6.2).

The number of adult N. basselae on 6 randomly selected plants in each of the shaded and full-sun subplots was recorded. The number of shot holes on leaves 3, 4 and 5 on each of the selected plants were counted and recorded. Care was taken not to disturb the adult beetles on these plants during assessments. Before dawn on the following day, the nursery grown full-sun and shade A. manihot plants were transported to the Tutuva trial field sites. Great care was taken not to damage plants during transport and handling. Five of the plants that were previously reared under shade and five of the plants previously reared in full-sun were carefully positioned at

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alternate points on a 2 x 5 grid among plants in each of the field subplots (Figure 6.2). Adjacent potted plants were placed 0.5 m apart and were located 0.25 m from the nearest field grown plant (Figure 6.2). All plants were put in position between 06:00- 07:00.

Assessments, systematic recording of the number of adult N. basselae and shot- holes on every leaf of selected plants, were carried out 24 h and 48 h after the potted plants were positioned in the respective subplots in the field.

6.2.5 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON HOST PLANT SELECTION BY NISOTRA BASSELAE

This experiment was similar to the above but set up in cages in full-sun environment. The experiment had 5 replication cages and involved exposing Broad-type variety of A. manihot that were previously reared in full-sun and shade in the nursery. Plants used in the experiment were 12 weeks old in the nursery when used (as previously described in 6.2.4 above).

Five wooden-framed, nylon net (ant proof) cages (70 cm x 70 cm x 100 cm) were placed on top of a bench (100 cm width x 600 cm length and 100 cm height) in full- sun. Two labelled nursery grown plants, one previously reared in the full-sun and one previously reared in the shade, were placed inside each cage; within a cage plants were spaced 25 cm apart, and 20 cm and 35 cm from the cage walls. At 18:00 that evening 20 adult N. basselae (10 males and 10 females) from the laboratory colony were released on the floor, equidistant between the two plants, of each of the cages. The number of beetles on each of the plants inside the cages was recorded every 12 h for 36 h following beetle release.

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6.2.6 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME AND UREA TREATMENT ON HOST PLANT SELECTION BY NISOTRA BASSELAE

The experiment was set up in a similar manner to that described in sections 6.2.3 to 6.2.5 above. A total of 20 plants that were previously reared under different light regimes in the nursery were used. Five plants previously reared in the full-sun and treated with urea; five plants previously reared in the full-sun but not treated with urea; five plants previously reared in the shade and treated with urea; five plants previously reared in the shade but not treated with urea. In urea treatments urea was applied to the surface of the soil close to the base of the plants at a rate of 2.5g/plant. Two basal applications were made, one a month after planting and the other a month later.

Five wooden-framed, nylon net (ant proof) cages (70 cm x 70 cm x 100 cm) were placed on top of a bench (100 cm width x 600 cm length and 100 cm height) in full- sun. A single plant from each of the light rearing regime/urea treatment combinations was placed at the base of each cage such that the four plants in each cage were positioned about 25 cm apart from each other and 20 cm from the cage walls. At 18:00 that evening 20 adult N. basselae (10 males and 10 females) from the laboratory colony were released on the floor, equidistant between the four plants, of each of the cages. The number of beetles on each of the plants inside the cages was recorded 24 h later.

122 N

. Space for other

experiments

Seasonal

Cocoa plantation Cocoa

Secondary forests Secondary

Population experiment Abundance plots

30m Field plants Potted plant reared in shade Potted plants reared in full sun Field monitored field plant

Figure 6.2 Diagram representation (not to scale) of the site of the experiment that investigated effects of full-sun and shade on the distribution of N. basselae on A. manihot plants in the field. Grey plots represent areas under the shade of rain-trees; white plots represent areas in full-sun. Space between adjacent subplots was 0.5 m apart.

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6.3 RESULTS

6.3.1 DISPERSAL OF NISOTRA BASSELAE

Assuming that there was no influx of N. basselae into the experimental field, almost 40% of the N. basselae released were recovered on the potted plants (Figure 6.3). At 12 h, 24 h, 36 h, 48 h, 60 h and 72 h after release 3, 26, 31, 36, 38 and 37% of released insects respectively were recovered. Only 2% of released insects were caught on the yellow sticky traps. Nisotra basselae moved in all directions from the release point, but most were recovered in the N (12%) and NE (22%) directions from the release point (Figure 6.4).

No N. basselae remained in the Petri dishes when the first check was carried out 12 h after the beetles were released. Nisotra basselae were recovered as far as 20 m away from the release point; but this took 12- 24 h (Figure 6.5). After 48 h, there was little change in the average numbers of beetles on plants at the different distances from the release point (Figure 6.3); a drop in cumulative recaptures, from 38% to 37% over the last observation period probably due to beetles leaving the plants for some unknown reason.

30 recaptured

20 Recaptures on plants

N. basselaeN. Recaptures on yellow sticky traps 10

Percentage 0 12h 24h 36h 48h 60h 72h Time (Hours)

Figure 6.3 Percentage of N. basselae recovered (n = 700) on pot plants of A. manihot and yellow sticky traps at 12 h intervals after release.

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N NNW 0.25 NNE 0.2 NW NE 0.15 NWW 0.1 NEE 0.05 W 0 E

SWW SEE

SW SE

SSW SSE S

125

0.2 72 hours 0.18 12 hours 24 hours 36 hours 48 hours 60 hours 0.16

0.14

0.12

(Bryant) recaptured 0.1

0.08

0.06 N. basselaeN. 0.04

0.02

Proportion 0 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20

Distances (m)

Figure 6.5 The proportion of adult N. basselae recaptured at 12 h intervals for 72 hours on A. manihot (Broad-type variety) at different distances from the central release point (n = 700).

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6.3.2 THE EFFECTS OF CONSPECIFIC FEEDING DAMAGE, MECHANICAL DAMAGE AND THE PRESENCE OF CONSPECIFICS ON ATTRACTION TO HOST PLANTS

6.3.2.1 EFFECTS OF CONSPECIFIC FEEDING AND INFESTATION ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS

The mean proportion of released N. basselae on damaged plants was significantly higher than the mean proportion of N. basselae on intact plants (F1, 183 = 216.66; P < 0.05) (Figure 6.6). There was no significant difference in the mean proportion of N. basselae on plants at the different time intervals (F3, 183 = 2.16; P > 0.05).

0.16

Beetles recaptured on beetles infested plants

0.14 Beetle recaptured on intact plant a a a 0.12 a

0.1 recaptured/plant

0.08 N. basselae N. b b 0.06 bc

c 0.04

0.02

SE) proportion of no.ofproportion ofSE) ±

Mean( 0 0 h 12h 24h 36h 48h

Time

Figure 6.6 Mean (±SE) proportion of released N. basselae on infested plants and mean (±SE) proportion of released N. basselae on intact plants at 12 h interval for 48 h after release.

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6.3.2.2 EFFECTS OF MECHANICAL DAMAGE ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS

The mean proportion of released N. basselae on mechanically damaged plants was significantly higher than that on intact plants (F1, 183 = 53.25; P < 0.05) (Figure 6.7). There was a significant difference in the mean proportion captured at different time intervals (F3, 183 = 24.52; P < 0.05); significantly fewer N. basselae were recaptured in the first 12 h than in the following time intervals (LSD; P < 0.05)

0.1 Beetles recaptured on mechanically damaged plants

0.09 Beetles recaptured on intact plants a 0.08 a

0.07 recaptured/plant a

0.06

N. basselae N. basselae 0.05 b b 0.04 b 0.03

0.02

SE) proportion no. of no. SE) proportion ± 0.01

c Mean ( Mean 0 0h 12h 24h 36h 48h

Time

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6.3.2.3 EFFECTS OF MALE OR FEMALE CONSPECIFICS, BUT THE ABSENCE OF FEEDING DAMAGE, ON THE ATTRACTION OF NISOTRA BASSELAE TO ABELMOSCHUS MANIHOT PLANTS

The mean proportion of released N. basselae on plants supporting caged female N. basselae was significantly higher than on plants supporting caged male N. basselae

(F1, 120 = 220.76; P < 0.05) (Figure 6.8). There was a significant difference in the mean proportion of N. basselae on plants at different time intervals (F1, 120 = 36.61; P < 0.05); significantly fewer N. basselae were recaptured in the first 12 h than in the following time intervals (LSD; P < 0.05) and significantly more N. basselae were recaptured after 36 h and 48 h than after 24 h (LSDs; both with P < 0.05)

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0.2 Beetles on plants with female beetles

0.18 Beetles recaptured on plants with male beetles a 0.16 a

recaptured/plant 0.14 b 0.12

N. basselae N. basselae 0.1

0.08

0.06 c c c c 0.04

SE) proportion of SE) of no. proportion d ± 0.02

Mean ( Mean 0 0h 12h 24h 36h 48h

Time

6.3.3 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON NISOTRA BASSELAE DISTRIBUTION IN THE FIELD

Significantly more beetles were recorded on plants grown in full-sun than those grown in shade (F1, 28 = 15.94; P < 0.05) (Figure 6.9). Similarly, plants that grew in full-sun suffered significantly more shot-hole damage than plants grown in the shade

(F1, 28 = 43.30, P < 0.05) (Figure 6.9). Beetle numbers did not vary significantly

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between blocks (F2, 24 = 2.32; P > 0.05) but there was a significant block effect on the number of shot-holes recorded on plants (F2, 24 = 4.31, P < 0.05). Plants in block 1 and 3 suffered more shot-hole damage than plants in blocks 2 (LSDs, both with P < 0.05) (Figure 6.10)

10 a A) 9

plant 8

6 N. N. basselae/ 5

4 SE) no. SE) of no.

± 3

2 Mean ( Mean 1 b

0 1200 B) a 1000

holes/plant holes/plant 800 -

600

400

SE) no. of no. shot SE) ±

200 Mean ( Mean b 0 Full-sun Shade

Figure 6.9 Nisotra basselae and shot-hole densities on plants grown under shaded and full-sun light regimes in the field at Tatuva village in January 2007. A) mean (±SE) number of N. basselae adults /plant; significantly more adult N. basselae on full-sun grown plants than on shade grown plants (F1, 28 = 15.94; P < 0.05). B) mean (±SE) number of shot-holes/plant; significantly more shot-holes on full-sun grown plants than on shade grown plants (F1, 28 = 43.30; P < 0.05).

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70 a 60 a

50 holes holes plant per - 40 b 30

SE) no. of shot SE) of no. ±

10 Mean ( Mean 0 1 2 3

Block numbers

Figure 6.10 Nisotra basselae shot-hole mean (±SE) densities on plants grown under shaded and full-sun light regimes in the field at Tutuva village in January 2007, mean (±SE) number (F2, 24 = 4.31, P < 0.05 of shot-holes/plant; significantly more shot-holes block 1and 3 than on shade grown plants block.

6.3.4 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON NISOTRA BASSELAE DISTRIBUTIONS BETWEEN POTTED SENTINEL PLANTS IN THE FIELD

There was no significant difference between the number of N. basselae per field grown plant (F1, 44 = 1.09; P > 0.05) nor the number of shot-holes per field grown plant (F1, 44 = 0.27; P > 0.05) between site 1 and site 2. Significantly more N. basselae (F1, 44 = 88.64; P < 0.05) and shot-holes (F1, 44 = 35.73; P < 0.05) were recorded on plants that had grown in full-sun than in shade at both field sites (Figure 6.11).

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At Site - 1, 24 h after potted plants were placed in the field, there were significantly more N. basselae on potted plants in full-sun plots than on potted plants in the shaded plots (F1, 36 = 24.49; P < 0.05; Figure 6.12). There were also significantly more N. basselae on potted plants that were previously reared in full-sun than those reared in shade (F1, 36 = 12.22; P < 0.05; Figure 6.11). However, there was no significant interaction between the light conditions in the field and the light regimes under which plants were previously reared in the nursery (F1, 36 = 1.73; P > 0.05).

At Site - 2, 24 h after potted plants were placed in the field significantly more N. basselae were recorded on potted plants in the full-sun plots than on potted plants in the shaded plots (F1, 36 = 86.98; P < 0.05; Figure 6.12). There were also significantly more N. basselae on plants that were previously reared in full-sun than on plants previously reared in shade (F1, 36 = 21.75; P < 0.05; Figure 6.1). However, there was no significant interaction between the light conditions in the field and the light regime under which plants were previously reared in the nursery (F1, 36 = 0.60; P > 0.05).

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A) FullDirect-sun sunlight Field field Shade Field 18

16 a a

b SE) number of beetles per plant per numberofSE)beetles

± 4

2 Mean( b 0

B) 300 a a 250

200

holes per plant holesper -

150

100

SE) numberofSE)shot ± 50

Mean( b 0 Site 1 Site 2

Figure 6.11 Nisotra basselae and shot-hole densities on field grown plants raised under shaded and full-sun light regimes in the field at Tutuva village in October 2008 A) mean (±SE) number of beetles on plants grown in full-sun and plants grown in the shade (F1, 44 = 1.09; P > 0.05). B) mean (±SE) number of shot-holes on plants grown in full-sun and plants grown in the shade (F1, 44 = 0.27; P > 0.05). Columns marked by a different letter are significantly different (LSD, P < 0.05).

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Figure 6.12 A) mean number (±SE) of N. basselae and B) mean number (±SE) of shot-holes on potted-plants that had been reared in full-sun or shaded conditions in the nursery and then transplanted among field plants grown in full-sun or shade at Site 1. Within a given time interval (24 h and 48 h), columns marked with a different letter are significantly different (LSD, P < 0.05).

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12 Full-sun reared A) SITE 2 Shade reared

10 plant

N. basselae/ N. 6 a

4 a

SE) no. ofSE)no. ±

2 Mean ( Mean b b c c 0

180 B) SITE 2

160

140 a a

120 b holes/plant - 100 80 60

40 SE) no. ofshotSE)no. ± 20 b b c c 0 Mean ( Mean Full-sun field Shade field Full-sun field Shade field 24 hours 48 hours

Figure 6.13 A) mean (±SE) numbers of N. basselae and B) mean number of shot- holes on potted-plants that had been reared in full-sun or shaded conditions in the nursery and then transplanted among field plants grown in full-sun or shade at Site 2. Within a given time interval (24 h and 48 h), columns marked with a different letter are significantly different (LSD, P < 0.05).

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At Site 1, 24 h after potted plants were placed in the field, there was no significant difference in the numbers of shot holes on plants in full-sun plots and plants in shaded plots (F1, 36 = 3.84; P > 0.05; Figure 6.12). However, plants previously reared in full-sun had significantly more shot-holes than plants previously reared in shade

(F1, 36 = 4.60; P < 0.05; Figure 6.12). Again, there was no interaction between field plots and where plants were reared previously (F1, 36 = 0.39; P > 0.05).

At Site 2, 24h after potted plants were placed in the field, there were significantly more shot-holes on plants in full-sun plots than on plants in shade plots (F1, 6 = 13.84; P < 0.05; Figure 6.13). However, the number of shot-holes on plants previously reared in full-sun was not significantly different from the number of shot-holes on plants previously reared in shade after 24h (F1, 36 = 0.002; P > 0.05; Figure 6.12). There was no interaction effect between field plots and previous rearing conditions

(F1, 36 = 0.002; P > 0.05).

At Site 1, 48h after placing plants in the field, the number of shot-holes on plants in full-sun plots was significantly higher than the number of shot-holes on plants in shade plots (F1, 36 = 27.16; P < 0.05; Figure 6.12). The number of shot-holes on plants previously reared in full-sun was significantly higher than the number of shot- holes on plants previously reared in the shade (F1, 36 = 10.69; P < 0.05; Figure 6.12).

There was no interaction between field plots and previous rearing conditions (F1, 36 = 0.26; P > 0.05).

At Site 2, 48 h after placing plants in the field, the number of shot-holes on plants in full-sun plots was significantly higher than the number of shot-holes on plants in shade plots (F1, 36 = 86.64; P < 0.05; Figure 6.13). The number of shot-holes on plants previously reared in full-sun was not significantly different from shot-holes on plants previously reared in the shade (F1, 36 = 2.83; P > 0.05; Figure 6.13). There was no interaction between field plots and previous rearing conditions (F1, 36 = 2.83; P > 0.05).

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6.3.5 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME ON HOST PLANT SELECTION BY NISOTRA BASSELAE

The number of adult N. basselae on plants reared in full-sun was significantly higher than the number of adult N. basselae on plants reared in shade at 12 h (F1, 18 =

8.65; P < 0.05), 24 h (F1, 18 = 6.97; P < 0.05) and 36h (F1, 18 = 22.71; P < 0.05) respectively (Figure 6.14 A)). Beetles created only a small number of shot-holes during the first 12h which resulted in no significant difference between the mean number on full-sun reared plants and shade-reared plants (F1, 18 = 2.945; P > 0.05)

(Figure 6.14 B)). However, after 24 h (F1, 18 = 15.46; P < 0.05) and 36h (F1, 18 = 10.02; P < 0.05) there were significantly more shot-holes on plants reared in full-sun than on plants reared in the shade (Fig 6.14 B)).

6.3.6 EFFECTS OF ABELMOSCHUS MANIHOT LIGHT REARING REGIME AND UREA TREATMENT ON HOST PLANT SELECTION BY NISOTRA BASSELAE

After 24 h the number of adult N. basselae on plants reared in full-sun was significantly higher than the number on plants reared in shade (F1, 8 = 9.184; P < 0.05; Figure 6.15 A)). Similarly after 24 h the number of N. basselae on plants treated with urea was greater than the number of N. basselae on plants not treated with urea (F1, 8 = 9.184; P < 0.05) (Figure 6.15 A)). There was no significant interaction between light regime and urea treatment (F1, 8 = 2.00; P > 0.05). After 24 h, the mean number of shot-holes on plants reared in full-sun was significantly higher than the number of shot-holes on plants reared in shade (F1, 8 = 6.677; P < 0.05) (Figure 6.15 B)). There was no significant interaction between full-sun or shade and urea treatment (F1, 8 = 1.715; P > 0.05) (Figure 6.15 B)).

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14 a A)

12 a /plant 10 b 8

b N. basselae N. 6

4 a SE) no. ofno. SE) ± 2 b

Mean ( Mean 0

140 a B) 120

holes/plant 100 - b 80 a

60 b

SE) no. of shotofno. SE) ± 20

Mean ( Mean a a 0 Full-sun Shade Full-sun Shade Full-sun Shade 12 hours 24 hours 36 hours

Figure 6.14 A) mean (±SE) number of N. basselae and B) mean (±SE) number of shot-holes on plants previously reared in full-sun and shade (n = 15) when tested in cage choice tests. For a given time interval, columns marked with a different letter are significantly different (LSD, P < 0.05).

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18 a A) Full-sun reared plants 16 Shade reared plants

14 per per plant 12

N. N. basselae b b 8

SE) of SE) b ± 6

4 Mean number( Mean 2

160 B) a 140

120

holes per plant holesper 100 - b b

80 SE) of shotof SE)

± 60 c 40

Mean number(Mean 20

0 Urea fertilizer applied No Urea fertilizer applied

Figure 6.15 A) mean (±SE) number of N. basselae and, B) mean (±SE) number of shot-holes on plants previously reared in full-sun or shade (n = 15) treated with urea 12 h after exposing beetles to treated plants, columns marked with a different letter are significantly different (LSD, P < 0.05).

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6.4 DISCUSSION

Light quality and quantity affects the distribution of N. basselae (Bryant) between host plants. In a series of studies reported in this chapter, different light intensities were generated by making use of full sun, tree-canopy shade and shade-cloth material (70% block-out light penetration). The measure of light penetration in the field under tree canopies was not determined but shade was fairly uniform throughout the fields. In natural tree shade situations, the density of foliage, leaf thickness, leaf texture, leaf colour, amount of solar radiation and cloud cover are all factors that contribute and create shade under plants (Lin and Lin 2010).

This chapter reports the results of experiments that explore the influence of full sun and shade on behaviour of N. basselae adults. It should be noted that light intensity and quality from the full sun is variable, depending on atmospheric conditions and sun angle. The initial study in this chapter investigated the distribution of N. basselae on A. manihot (Broad-type variety) crops in full-sun and shade. Shade is a product of reduced light intensity and is commonly known to be responsible for lowering soluble carbohydrate level in plants thus contributing to decline in herbage dry matter digestibility (Wilson 1984).

In all the trials that compared full-sun against shade in this chapter, it is clear that N. basselae preferred to occupy and feed on A. manihot grown in full-sun than in shade. These results supported a study by Larrsson et al. (1986) and which explained that the relative availability of carbohydrates obtained through photosynthesis, apparently in full-sun situation has resulted in the susceptibility of plants to herbivorous insects. On the contrary, beetles probably did not like plants grown in shade which promoted production of carbon-based secondary compounds such as phenols and terpenes can render food indigestible, therefore reducing plant nutritional values for insects (Coley 1983, Hargerman and Butler 1991; Chacon and Armesto 2005).

It was further observed in a trial in the series of studies reported in this chapter that plants previously exposed to full-sun were preferred by beetles more than plants previously reared in shade preferred full-sun plants than plant in the shade. It occurred in the field experiments (Figure 6.5 and 6.6) and similarly in the laboratory experiments (Figure 6.7) plants previous reared in shade condition but placed in full-

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sun plots were as susceptible as plants previously reared in full-sun but reared in shaded plots. This result clearly indicates that changes in the light regime or growing condition exposed by the plant also affects the chemistry of the plant in terms of carbon-nutrient balance (Larsson et al.1986; Hauck et al 2007). The nature of light regime determines the distribution of the population in this case thus resulted in more beetles in full-sun environment than in shade. The reasons of beetles’ preference toward full-sun environment than shade were merely speculated and unknown. Evidently the high beetle presence had a strong correlation to high shot-hole damage thus high number of beetles presence in full-sun plots correlated with high beetles feeding in that same light regime environment.

Addition of urea both in plant grown in full-sun and shade affected the selection of plants by adult N. basselae (Bryant) and that urea made shade grown plants as attractive and preferred as a plant grown in full-sun but data does not provide an unequivocal explanation.

In conclusion, both laboratory and field experiments clearly demonstrated shade reducing number of N. basselae (Bryant) shot-hole damage on A. manihot Medicus as host plant. In the absence of data on nutrient and secondary metabolites balance, it was not possible to explicitly explain the chemistry that had taken place that caused such poor preference of shade environment and plants grown in shade. In terms of pest management, shades under canopies are variable and optimal shade for plant growth may vary from plant species to plant species and so reduction of the pest may subject to the nature of canopy used. Shade underneath tree canopies usually provides micro-climatic environments that are different from adjacent unshaded open space. The effects of such micro-climatic environments can also be among factors that can influence herbivorous insect distribution and populations. Lin and Lin (2010) measured air and soil-surface temperatures of nearby full-sun space and under canopies and found both air and surface-soil temperatures in shade were both lower than in full-sun space (between 0.64 and 2.52°C and 3.28 and 8.07°C, respectively). The effect of low temperature was inferred to be responsible for reducing plant cell wall concentrations and therefore increasing nutrient concentrations lower rate of plant maturity, which in turn can prolong damage on the plant (Kephart and Buxton 1993; Lin and Lin 2010). It becomes obvious from results

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obtained in the series of studies here that shade plays a vital role in reducing N. basselae (Bryant) pest. However, there is a need to strike a balance on the knowledge of optimal full-sun under shade that provides the lowest economic injury level but at the same time favourable for slippery cabbage production in terms of light intensity level that will still provide the necessary agronomic requirements for the crop.

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CHAPTER SEVEN: GENERAL DISCUSSION

Slippery cabbage (Abelmoschus manihot Medicus (Malvaceae)) is an important leafy vegetable in many Pacific Island nations, particularly Solomon Islands and Papua New Guinea (PNG), where it is extensively cultivated by subsistence farmers (Preston 1998; Morris 2004; Fa’anunu 2009). In Solomon Islands, consumption of the crop has declined in many areas due to damage by the slippery cabbage flea beetle, Nisotra basselae (Bryant) (Jansen 2006). However, the demand for slippery cabbage in urban areas has escalated as the population in these areas continues to increase. Consequently, production has intensified and inappropriate insecticide use is commonplace on farms supplying urban centres (Teakle and Bakale 2007). Hence, the subsistence sector is confronted with difficult management problems while the reaction of the commercial sector is the adoption of unsustainable and environmentally damaging insecticide based control strategies. The goal of this study was to establish a better understanding of the biology and ecology of the pest to lay down the foundation for more sustainable pest management practices.

When attempting to control a pest organism, it is essential that its identity is established so that relevant information can be gathered (Pedigo and Rice, 2008). Prior to this study, there was considerable confusion surrounding the identity of the slippery cabbage flea beetle. This was directly addressed in Chapter 3: Taxonomy of Nisotra basselae (Bryant). Based on morphological characteristics, specifically the presence of two longitudinal sulci at the anterior margin of the pronotum (Foudras 1859; Baly 1864; Scherer 1969), the study confirmed that the beetle infesting slippery cabbage crops in Solomon Islands is Nisotra basselae (Bryant), and that its previous classification as Podagrica basselae Bryant (Bryant 1941) was a mistake. Further morphological studies corroborated the original description by Bryant (1941), which matches all other characteristics of the genus Nisotra described by Foudras (1859) and Scherer (1969). Currently, N. basseale is only present in PNG and Solomon Islands, where it is established in all provinces except Temotu and Rennel in the far southeast of the archipelago (Figure 3.1). Thus, the pest poses both national and international quarantine threats to localities that are still free from infestation.

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The first recorded specimens of N. basselae were collected from a Basella sp. (Basellaceae) host plant (Bryant 1941). However, in Solomon Islands N. basselae is found associated with A. manihot more frequently than with any other plant species in the field. The complete host range of N. basselae still has to be established but, in Chapter 4: Life cycle and seasonal phenology of Nisotra basselae (Bryant) in Solomon Islands, the life cycle and bionomics of N. basselae on A. manihot and two other members of the Malvaceae, Hibiscus tiliaceus L. and Abelmoschus esculentus (L.) Moench, were established. As is common in this genus (Rawat and Singh 1971; Pandit and Chatterji 1987), N. basselae fed on foliage as adults and on rootlets as larvae. Of the three host plant species investigated, A. manihot was shown to be the most suitable for both immature and adult insects. Larvae were unable to complete their development on A. esculentus and, although N. basselae could complete development on H. tiliaceus, adults were less fecund and did not live as long as those reared on A. manihot. Despite the demonstrated potential of H. tiliaceus as a host for N. basselae, when the roots and foliage of H. tiliaceus growing next to heavily infested by N. basselae plots of A. manihot were sampled, no beetles were found and no feeding damage was recorded on foliage. This suggests that H. tiliaceus is not an important host and an unlikely reservoir of insects in the field in Solomon Islands.

Long term monitoring of adult N. basselae populations showed that the pest attacked crops throughout the year, but that adult populations were depressed in the wet season. Whether this is due to the effects of rainfall on adult populations (through the direct impact) or larval populations (through waterlogged soils) is unknown and warrants further study. In the laboratory N. basselae took 38 days to complete a generation when feeding on A. manihot, and adults typically lived for approximately two months, thus field populations are likely to consist of multiple overlapping generations.

In Chapter 5: Oviposition ecology of Nisotra basselae (Bryant), it was established that female N. basselae preferred to lay single or clusters of eggs in moist soil within the root zone of host plants, and the vast majority of these were laid within close proximity (2 cm) to the base of the stem. In field studies a strong positive correlation between adult density on foliage and egg density on roots was detected

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and it was established that beetles moved between foliar feeding sites and soil oviposition sites by crawling up and down host plant stems. Trapping of insects in Tangle-foot at the base of plant stems lead to a decline in adult densities on the foliage of these plants, suggesting a high degree of fidelity to individual host plants. In experiments that tested mechanical disruption of female access to oviposition sites, it was shown that copra sack barriers strategically placed around the stems of plants could significantly reduce egg densities on roots and adult densities on foliage when compared to standard practice (bare soil), the application of mulch or under- sowing with clover. The efficacy of copra sack mechanical barriers and Tangle-foot traps for reducing eggs and adult densities in the soil and on the foliage respectively are important applied findings that have considerable potential to form the basis of cultural control based IPM strategies for N. basselae on A. manihot in Solomon Islands.

Host plant relationships and the movement of adult beetles between plants were investigated further in Chapter 6: Adult Nisotra basselae (Bryant) movement and the effects of biotic and environmental factors on host plant selection. Dispersal studies indicated that beetles aggregated on host plants and that they tended not to move between plants following establishment on a given host. Host plant choice tests conducted in the field showed that mechanical damage and N. basselae herbivory increased the attractiveness of host plants to adult beetles and there was also evidence that beetles of both sexes aggregated in response to female conspecifics, the later possibly indicating the production of an aggregation pheromone. The chemical basis for these responses were not investigated but this warrants further research as understanding the cues that mediate these responses could lead to the development of attractants for improved sampling and/or trapping of adult beetles. In Solomon Islands A. manihot crops are often planted under the shade of trees. Laboratory and field experiments showed that adult N. basselae preferred to occupy and feed on A. manihot grown in full sun over plants grown in the shade. Transplant experiments showed that plants grown in full sun, but transferred to shaded sites in the field, were preferred over plants grown in the shade and placed in the shade and over shade grown plants placed in full-sun. These effects were rapidly lost, suggesting that the mechanism could have a physiological/ chemical basis. Investigating this further was beyond the resources of this study but, as for the

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studies on the effects of herbivory and the possible aggregation pheromone production by female beetles, it warrants further study. Interestingly, application of urea to plants grown in full-sun or shaded environments increased the attractiveness of plants, suggesting that mobilization of nitrogen might play a role in the attraction.

This work clearly shows that practices for growing slippery cabbage in Solomon Islands could be improved by adopting cultural controls to mitigate damage caused by the major pest, N. basselae. Preliminary work on the chemical ecology of N. basselae- A. manihot interactions suggests that chemical cues are very impart and that their development could add significantly to an integrated management program. Pending these developments, farmers in Solomon Islands can take advantage of the fact that N. basselae tends not to move great distances and as a consequence it is slow to invade new areas; ensuring that planting material and soil are free from infestation are basic practices that could aid crop production significantly.

In places where the beetle is already present, and where management is the only option available, farmers are advised to make sound informed decisions on what form of control to take and based on some of the fundamental findings in this thesis. The type of control measures applied will be different between resource poor subsistence farmers and commercial producers but both groups can benefit significantly by gaining a better understanding of the ecology of N. basselae and applying this when they make management decisions.

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