r EFFICACY OF CERTAIN TOXIC COMPOUNDS EXPOSED TO

AGRICULTURALLY IMPORTANT INSECT PEST IN RELATION TO SOME ABIOTIC FACTORS

DISSERTATION

SUBMJTTIEDIN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE A WARD OF THE DEGREE OF

f a s t e r nf ptfilnsnplig

BY

ASAF ALI MURTAZA

Under the supervision of

DR. KHOWAJA JAMAL

1 DEPARTMENT OF ZOOLOGY FACULTY OF LIFE SCIENCES

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

2011

DS4184

f e d ^^ '•^'••'-

' External: 2700920/21-3430

iBtcrnal: 3430

DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY

ALIGARH-202002 Sections: INDIA D.No ../ZD

1. Entomology Dated 2. Fishery Science & Aquaculture 3. Genetics 4. Nematology 5. Parasitology

ST. Jissistant (Profassor-Cum-^useum Curator

l^w is to certify that the research wor^presentecC in the dissertation

entitled 'efficacy of certain toxic compounds exposed to agricutturaSy

important insect pest in relation to some aSiotic factors' carried out 6y

Mr. AsafjiR 9iurtaza under my supervision. The dissertation is an ori^inaf

contriSution of the researcher and adds suSstantiaCCy to the exiting ^owledge

in the discipfine. He is permitted to su6m.it it for the partiaCfuffiUment of the

degree of Master of (Philbsoplty in Zoology, at Migarh MusCim Vniversity,

AGgarh, India. The dissertation is fit for suSmission to the ej^aminers for

evaCuation. »

It is further certified that Mr. JisafM Murtaza has fuCfiOed the

prescriSed conditions of duration and nature given in the Ordinances of the

ACigarh Muslim Vniversity, Migarh. " '

(Dr, Khowaja Jamal) Sr. Assistant Professor-Cum-Museum Curator

Supervisor

AcknomUinmnent

Tirst andforemost, I -woufdCi^ to pay my oBeisance and gratitude to "JtHah

JiCmigHty" thefaitd in wHom fieCpedme aCways.

It gives me immense pleasure to express my heartfeCt gratitude, indebtness

and dumSa regards to my esteemed teacHer and supervisor (Dr. %lwwaja JamaC

JLssistant (Professor, (Department of Zoology, JiMV, JiGgarfi. It is indeed a matter

of great pride and experience -witfiout comparison to Rave had the privilege of

•wor^ng umler his ceaseless and untiring guidance. The present wor^has cuCminated

due to his expert supervision, meticulous guidance and solves each and every proSlem

that I cropped up during the completion of this wor^ His immense knowledge and

constant help irrespective of how Busy his schedule was proved instrumental in

completion of this endeavor.

I would li^ to pay my sincere regards to (Prof. Irfan Ahmed, Chairman

(Department of Zoology, for giving me opportunity to wor^ and providing me laS

and other facilities required during my research wor^

I tender my heartfelt than^ to all my teachers especially

(Dr. Ayesha Qamar Asst. (Professor, (Department of Zoology and

(Dr. (Bachchan Ad "Kfum, for their constant help untiring encouragement, moral

support, valuaSle suggestions, inspiration and profound encouragement for

Betterment, which have Been a driving force for me to perform this wor^

I would li^ to shower my special than^ to (Dr. fHd Sadre ACam Ta^ri,

<Dr Mohd <Fannan 'Kfian, (Dr. Shamim Ahmed, (Dr. Shah Mohammad Haris, the

Budding scientists in entomology, who have Been a source of inspiration for me, too^

painsta^ng efforts during my wor^ fuelled me with his viBrant, constructive and

informative ideas and assisted me in giving the solid shape to my research wor^

m

/ tfian^ffy express my deep sense of gratitude to JAUah for giving me nice

fattier and Motfier, wHo raised me witfi a Cove of science and supported me in ad my

pursuits. What I am today is Because of my parents, their prayers, Cove, sacrifices,

sincere endeavors and sustained efforts tHat enaBCedme to acquire tfiis ^rurwCedge.

9Ay fieartfeCtfeeCings of gratitude to my grandfatRer, Cote grandmother, (Bmtfiers,

sister and ad the family numbers whose prayer and support are aCways with me and

have provided me. aCfthefaciCities whenever needed.

I aCso ta^ this opportunity to my speciaC than^ to my friends

Syed !Hassan MehntR, Jdrdesh. lOimar Sharma, SaBiur ^fiAman ^arooqi,

^ofuf Hamayun, ^ja CftoudRary, Mofid iBraAim, Q?an^j gwari, (Dr. !Mohd

Tauseef, fMofid^fi "Kfian, Mofid Inran, fW<£ Siraj Maneer, <^fyaz Ahmed Mir,

Iram "Kfian, TaSassum <Stfhmat and Tiza %fian for their support at every stage

whenever needed. My time at JACigarh MusRm University, yiCigarh was made

enjoyahCe in Carge part due to many friends that Became a part of my Cife.

Last But not the Ceast, I express my speciaCthan^ to Mrs. Tfasreen iHiissain,

Seminar Librarian, and aCf non-teaching staff of my department especially

%fiaCiq (Bhai and Zaheer (Bhai, who extended ad sorts of heCp and support to me

throughout the compCetion of this wor^

I am deepCy indeBudto Mr. A-% Azad, Mr. AhdufMannan (Munna), Mr. ShoaiS

Ahmad, Mr. Md SuhaiC dC Mr. Shafaqat-MemBers ofAH-In-One Compmmters,

AMI)., ACigarh for composing and (Designing this dissertation.

(Before concCuding, I douBt that I have forgotten to mention and

ac^owCedge many persons who heCp me in one way or the other.

(AsafAS OHurtaza)

m

CONTENTS

INTRODUCTION 1-7

REVIEW OF LITERATURE 8-20

MATERIAL AND METHODS 21 -26

RESULTS AND OBSERVATION 27-46

TABLES 47-54

GRAPHS 55-72

PHOTOGRAPHS 73-74

DISCUSSION 75-96

REFERENCES 97-123

INTRODUCTION

Cotton is an important agricultural crop commonly known as

white gold. It provides major employment in agriculture and textile

industry. The world cotton production estimated at 25.9 million

tonnes in 2007-08, 3% lower than the previous season due to

decline in area under cotton cultivation (Anonymous, 2008).

Whereas, India produced 5.3m tonnes with 11% increase

compared to the previous season because of an increase in cotton

production area by 4% touching 9.58 million ha. However, the

average productivity of cotton in India (589lint/ha) is low as

compared to other cotton producing countries (607 to 1390lint/ha).

There are two types of cotton grown in India, Desi cotton

{Gossipium arboretum L.) and American cotton (G. hirsutum L.).

The Desi cotton is grown in mountain and high rainfall areas. The

American cotton is generally grown where there is some source of

irrigation. Amongst the various constraints in cotton cultivation, the

losses caused by insect pests are of major importance. In India,

160 species of insect and non-insect pests have been reported to

damage cotton crop (Agarwal, 1978). Of these, 24 phytophagous

insect species have attained the pest status, out of which nine are

key pests, in different zones of the country. In the north zone

(Punjab), sucking pests and bollworms cause 11.6 and 49.5% loss

Introducticm

in seed cotton yield respectively and both together caused 52.1%

loss (Dhawan eta!., 1998).

The cotton strainer, Dysdercus koenigii Fabr., is an important

pest of cotton (Gossipium sp) in Punjab and Uttar Pardesh and

widely distributed throughout India. Due to feeding on half opened

cotton bolls and seeds, the oil content and seed viability is reduced

and the yellow excreta of the insect imparts a dirty colour and

spoils the cotton lint. On potted plants, the eggs were laid in cracks

and crevices of soil and covered by loose soil or dried leaves. The

average egg laying capacity of female is 288 (Chatterjeet et al.,

1985). Eggs are oval, smooth, creamy yellow in color with an

average incubation period of 6.18 days. The period of the first,

second, third, fourth and fifth instars range from 3 to 4, 3 to 4, 3 to

5, 6 to 8 and 12 to 16 days, respectively. The adults live for 9 to 25

days and the complete life cycle extends from 51 to 61 days. The

sex ratio of male to female was 50.50 to 48.50. This bug is

prevalent throughout the year and also feeds on okra, hollyhock,

eggplant and other malvaceous plants (Kamble, 1971). The feeding

habits of nymphs and adults were similar except for their

gregariousness during feeding, when food was scarce nymphs and

adults were commonly cannibalistic.

- 2 -

Iianktuction

The chemical control method of the insects is very effective

and rapid in curative action. Although the method is, in general,

ecologically unsound and has many serious limitations like

insecticide resistance, pest resurgence and pest replacement

which can occur following repeated application of these

compounds (Pedigo, 1989), it has been considered as the most

important and powerful tool in controlling insect pests both in the

field and under storage conditions for the past. If we compared with

the alternatives, the benefits of insecticides probably outweigh the

risks. But the goal of insecticide use to obtain maximum benefits

with minimum risks has not yet been achieved in most situations.

This may be achieved by proper use of pesticides and dose

selection,, to follow the rules of pesticide application. A

developmental program for chemical pesticides should involve a

thorough examination of the physical, biological and environmental

factors that can affect the pesticide toxicity. Although there is also a

need to replace dependence on environmentally disruptive

chemicals, it seems likely that the further agriculture production

system will require some new generation insecticides. The

chemical groups, mostly in use today, are organophosphates,

carbamates and synthetic pyrethroids which have broad-spectrum

activity. However, new generation pesticides like imidacloprid,

Introduction

spinosad, fipronyl are much selective, less disruptive and more

environmental benign and having new novel mode of actions,

hence likely to be more compatible with IPM.

Imidacloprid is a relatively new, systemic insecticide

chemically related to the tobacco toxin nicotine. It was first

registered for use as a pesticide in the U.S in 1994. It moves

through the plant from the place where it was applied and kills

insects when they feed. Like nicotine it acts on the nervous system.

Because of the molecular shape, size and charge, it fits into

receptor nnolecules in the nervous system that normally receive the

neurotransmitter acetylcholine. Acetylcholine transmits nerve

impulses from one nerve cell to another or from nerve cell to some

other tissues like muscles or glands. Nervous system of insects as

well as mammals has acetycholine receptors; most of the sensitive

receptors are in the nervous system of insects, but in nerves

associated with muscles in mammals (Ware, 2000). However,

insect acetylcholine receptors are more sensitive to imidacloprid

than are mammalian receptors (Zwart etal. 1994). Imidacloprid has

high affinity to insect nicotinic acetylcholine receptors (nAchR). It

interacts with nAchR, resulting in excitation and paralysis followed

by death of the insect. Their selectivity results from a higher affinity

to the insect nAchR as compared to that of vertebrates, in contrast

Intfoductum

to the original nicotine compounds (Tomizawa et al., 1995). Hence,

it can be suggested that imidacloprid and related compounds be

called as nicotinoids (Yamento et al., 1995). The nicotinoids act

especially on sucking pests and have mild or no effect on

parasitoids and predators and as such fit well in various IPM

programs.

Spinosad (Dow Agrosciences), a mixture of spinosyns A and

D that are tetracyclic macrocyclic compounds produced by an

actinomycete Saccharopolyspora spinosa. Mertz and Yao isolated

from a jamican soil sample (sparks et al., 1998). As these products

are created biosynthetically during fermentation of S. spinosa,

spinosad has been classified as a bio insecticide (Cropping and

Menn, 2000). It is primarily a stomach poision with some contact

activity and is particularly active against Lepidoptera and Diptera. It

is a neurotoxin with a novel mode of action involving the nicotinic

acetylcholine receptor and apparently the GABA receptors as well

(Salgado, 1997, 1998). Exposure of spinosad results in cessation

of feeding followed by paralysis and death. Conventional toxicity

tests indicate that spinosad has virtually no toxicity to birds and

mammals, with contact LC50 value of 200ppm. Spinosad has also

been reported to be practically non-toxic to natural enemies of

insects(Schoonover and Larson, 1995; Bretefa/., 1997).

- 5 -

Introduction

Furthermore, insecticide use is generally the preferred

strategy for its high efficacy during a pest outbreak. In some

environments such as greenhouses, storehouses and quarantine

facilities, with their relatively closed conditions, high temperatures

can be used to control insect pests. In previous studies

(Alexandresu, 1986; Turnbull and Harris, 1986; Horn, 1988; Morytz,

et ai, 1997; Cho et ai, 1999; Rahman et al. 1999) the effects of

temperature on toxicity of some insecticides were investigated.

Generally, the toxicity of chlonnated hydrocarbons,

organophosphates and carbamate compounds increases with

temperature i. e. exhibit positive temperature coefficient, while

toxicity of some pyrethroids has negatively correlated with

increasing temperature (Hinks, 1985; Grafius, 1986; Thaung and

Collins 1986; Horn 1988; Johnson 1990; Arthur 1999; Wadleigh et

al., 1991). The degradation rates of most organophosphate

insecticides generally increase rapidily with increase in temperature

and either grain moisture content or relative humidity (Snelson,

1987; Arthur et al 1992). Although pyrethroid insecticides are

generally' more stable than organophosphates, increased

temperatures will also enhance pyrethroid degradation (Noble et al.

1982). The realized efficacy of an insecticide is a function of the

inter-relationship between vaporization and cuticular penetration.

Introduction

Residual activity of an insecticide is usually correlated with

temperature. Generally, an insecticide will remain active for longer

periods when temperatures are lower, due mostly to reduced

vaporization (Felsot 1989). The influence of temperature on the

efficacy of insecticides in the field has usually agreed with results of

bioassays in the laboratory (Ayad and Alyousef, 1984; Chandler, et

al., 1991) but very few studies have dealt with the association

between insecticides and abiotic factors. Therefore, the present

study aim's to investigate the effects of temperature, photoperiod

and constant relative humidity on the toxicity of different classes of

insecticides to Dysdercus koenigii (Fabr.) in laboratory.

- 7 -

REVIEW OF LITERATURE

There are certain factors that affect the growth and reproduction

of insects. These factors are endogenous as well as exogenous.

Several author studied the effect of various exogenous factors such as

temperature, humidity, light, food, chemicals etc on various

physiological processes including mortality, longevity, fecundity, fertility,

and malformation.

Abdullah et al., (2002) tested the efficacy of two insecticides

imidacloprid and temik against whitefly and jassid and reported that

both insecticides were good in controlling the jassid and whitefly

population.

Wang et al., (2008) determined the efficacy of sub lethal

concentrations (LC50) of six insecticides (imidacloprid, rotenone,

fanvalerate, abamectin, pericarb and azadirachtin) on fecundity and

wing dimorphism of green peach aphid Mysius persicae under

laboratory and green house conditions separately. In laboratory

condition, after being exposed to sub lethal doses of imidacloprid and

fanvalerate, the proportion of alate progeny in aphid progeny were

significantly higher than that of the control while in green house, the

percentages of alate offsprings from the mother aphid treated with

imidacloprid and fanvalerate and abamectin increased pronouncedly

compared with controls. Mortality rates of offsprings in the nymphal

^^fviewofLiumture

stages from adults treated with insecticide revealed no significant

differences between laboratory and green houses.

Richman et ai, (1999) investigated the toxicity of imidacloprid to

cat fleas on glass at 20, 26, 30, and 35°C and found that it is the most

toxic to adult cat fleas at 35°C and to larvae at 20°C. Further he

observed that piperonyl butoxide (PBO), a synergist, increased the

relative potency of imidacloprid (1:5 imidacloprid:PBO) 16-fold at 26°C

against adults, but had no effect at 35°C. No synergism occurred in

larvae at 20°C, but addition of PBO (1:5 imidacloprid: PBO) doubled

toxicity at 26°C. PBO (1:5 imidacloprid: PBO) could possibly be used to

synergize imidacloprid premise treatments (20-30°C), but it is not likely

to be effective in pet treatments because no synergism occurred in adult

fleas at 35°C (average fur temperature of tested cats and dog).

De Cock et ai, (1996) investigated the susceptibility of the

predatory bug Podisus maculiventris (Say) to imidacloprid in the

laboratory through direct topical exposure, residual contact and

ingestion and observed that imidacloprid toxicity decreases in order of

topical exposure> ingestion > residual contact.

Nishi and Takashashi (2002) reported that the pre-oviposition as

well as egg incubation period of Amphibolus venator decreased with

increasing temperature, whereas, the average number of eggs laid per

-9-

^fviewofLiUmtttn

ovipositing female increased with increasing temperature, however, the

hatchability remain unaffected.

Ali and Qureshi (1982) observed that virgin females of Dysdercus

cingulatus lay no eggs, however the topical application of the juvenile

hormone analogue methoprene (Z15) on the thorax resulted in some

oviposition by virgins. Further he found that the number of eggs laid

increased with increasing doses, but it was never as great as the

number laid by mated females. Treatment of both mated and newly

emerged females with increasing concentrations of methoprene

resulted \n progressive reductions both in numbers of eggs laid and in

the time required for the completion of vitellogenesis.

Yasmeen et ai, (1984) assesed the iox\c\iy of juvenile hormone

analogue methoprene against 5' instar nymphs of Dysdercus koenigii in

the laboratory and reported that the infection of LD50 dose i.e.

45|jg/nymph caused abnormalities in the 5th-instar, whereas, the topical

application of LD50 i.e. 28|jg/nymph prolonged the mstar duration. Chitin

synthesis and moulting were also inhibited.

Ansari et al., (2008) studied the total effect of imidacloprid on

development of Plutella xylostella and reported that feeding of 3" instar

larvae on cauliflower leaves impregnated with 0.002% of imidacloprid

resulted in decrease of net reproductive rate (RQ) of a surviving adult

from 10.99 to 8.47 females/female. The potential fecundity (Pf) was

-10-

^ItfitnewofLftemhtre

significantly decreased to 166.5, wliereas, intrinsic rate of increase (r^)

was 0.073 females/female/day as compared to 0.09 in untreated

control.

Rathod et ai, (2003) conducted an experiment in India during

1997-2000 to determine the efficacy of imidacloprid against jassids

{Amrasca biguttula), aphids {Aphis gossypii), and thrips {Thrips tabaci)

infesting cotton. The treatments comprised imidacloprid at 5.0, 7.5, and

10 g/kg; diafenthiuron 300 and 400 g/ha; and dimethoate at 1.25

litres/ha. The lowest mean population of jassids (0.99), aphids (4.41),

and thrips (1.73) per 3 leaves were obtained with 10 g imidacloprid/kg,

300 g diafenthiuron/ha. and 5 g imidacloprid/kg, respectively. The

highest cotton yield (826 kg/ha) was obtained with 5 g imidacloprid/kg.

Hu and Prokopy (1998) tested imidacloprid in the laboratory as

well as in the field against Rhagoleta pomonella flies to determine

ingestion/ contact or contact alone toxicity and reported that

imidacloprid 10-12 times more toxic and acted more rapidly by oral

ingestion than by surface contact. Affected flies were observed to cease

feeding and then regurgitate. Mortality stabilized 4 days after treatment.

Compared with control flies, females exposed to imidacloprid showed

reduced fecundity regardless of whether exposure was by oral or

surface contact. In field experiments, spray applications of imidacloprid

to foliage at the manufacturer's recommended rate resulted in no

-11-

^fview of Literature

significant mortality of flies, either among flies released immediately

after treatment or 24 h later.

Imidacloprid residues upto 2ppm in soil did not affect egg viability

or days to hatch of Japanese beetle Popillia Japonica, but they killed

neonates soon after eclosion (George et al., 2007).

Bao et al., 2009 tested four insecticides against brown plant

hopper and reported that imidacloprid and dinotefuran reduced the

fecundity of BPH to 68.8% and 52.4% in macropterous families, and

57.9% and 43.1% in brachypterous families, as compared with the

untreated controls, by contrast, triazophos and fenvalerate increased

the fecundity.

Christopper et al., (2009) observed that when Green peach aphid,

Myzus persicae (Sulzer) were exposed to potato leaf discs dipped in

sublethal concentrations of insecticide, almost all measured endpoints-

adult longevity, F1 production, F1 survival and F2 production-were

affected, and a statistically significant (P < 0.05) stimulatory response

was recorded for F2 production following exposure to imidacloprid.

Boina et al., (2009) studied the effects of sublethal concentrations

of imidacloprid on life stages of Asian citrus psyllid (ACP), Diaphorina

citri and reported that feeding by ACP adults and nymphs on plants

treated daily with a sublethal concentration (0.1 microg mL" ) of

imidacloprid significantly decreased adult longevity (8 days), fecundity

-12-

^fviewofLiUrature

(33%) and fertility (6%), as well as nymph survival (12%) and

developmental rate compared with untreated controls. The magnitude of

these negative effects was directly related to exposure duration and

concentration. Furthermore, ACP adults that fed on citrus leaves treated

systemically with lethal and sublethal concentrations of imidacloprid

excreted significantly less honeydew (7-94%) compared with controls in

a concentration-dependent manner suggesting antifeedant activity of

imidacloprid. It has been concluded from the experiment that sublethal

concentrations of imidacloprid negatively affect development,

reproduction, survival and longevity of ACP, which likely contributes to

population reductions over time. Also, reduced feeding by ACP adults

on plants treated with sublethal concentrations of imidacloprid may

potentially decrease the capacity of ACP to successfully acquire and

transmit the HLB causal pathogen.

Banerjee et al., (2001) studied the growth regulatory effect of

naturally occurring quinines and their derivatives against D. koenigii

and reported that all the test compounds disrupted the normal growth at

sublethal doses while 2,6-dimethylbenzoquinone and menadione at low

doses. Baised on LD50 values, the following increasing order of toxicity

could be established: plumbagin < 2, 6-dimethylhydroquinone < 2,3,6-

trimethylbenzoquinone<juglone<menadione<2,6dimethylbenzoquinone.

-13-

^^gviewofLiUrahtn

Amarasekare and Edelson (2004) evaluated the efficacy of

diflubenzuron, azadirachtin, Beauveria bassiana, spinosad, endosulfan,

esfenvalerate, and naled to third instars of differential grasshopper,

Melanoplus differentialis (Thomas), at temperatures ranging from 10 to

35°C. In the laboratory, treatment with esfenvalerate resulted in 100%

mortality at temperatures of 10 to 35X, and efficacy was not

temperature dependent. Treatment with spinosad resulted in similar

mortality as with esfenvalerate at all temperatures except lOX. The

activity of B. bassiana was greatest at 25°C and was adversely affected

by high and low temperatures.

Gregory et al., (2005) studied the persistence and efficacy of

spinosad against Rhyzopertha dominica (F.) in wheat stored for 9

months at 30°C temperature and 55 and 70% relative humidity, and

reported that spinosad applied at 0.5 or 1 mg kg" was completely

effective for 9 months, with 100% adult mortality after 14 days of

exposure and no live Fi adults produced. Adult mortality was <100% in

some samples of wheat treated with 0.1 mg kg" of spinosad, and live

progeny were produced in all samples treated at this level. Further, they

concluded that spinosad is likely to be an effective grain protectant

against R. dominica in wheat stored in warm climates.

Behnam Amiri-Besheli (2009) determine the efficacy of Tracer

(Spinosyn), Insecticidal Gel (Palizin), Insecticidal Emulsion (Sirinol),

-14-

^^fviewofLittmiun

Runner (Methoxifenozide) and Tondoxir with and without mineral oils

(MO) for the control of citrus leaf-miner (CLM), Phylocnistis citrella

Stainton (Lepidoptera: Gracillariidae) in laboratory condition at 24, 48,

72 and 96 h post treatments. Leaves of citrus with second and third

instars of leaf-miner larvae were used in all tests and the larval mortality

were monitored. Analysis of variance showed that there were significant

differences between treatments and control, and also significant

differences were found among treatments. Tukay-Test among above

treatments has shown that Tracer + oil, Runner and Runner + oil and

Tondxir + oil with 98 ± 3. 2, 98 ± 8.1 and 93 ± 5% mortality were more

effective than MO, Tondxir and Sirinol + oil with 85 ± 8.3 and 81 ±7.2 and

78.25 ±8.2% mortality and significant different with control (P< %1),

respectively. Tukay-Test among 96 and 72h post treatments with 77 ±

3.82 and 70.65 ± 7.5% of total mortality are more effective than 48 and

24 h post treatments with 52.27 ± 4 and 35.90 ± 3.8% of total mortality

respectively

Bill et al., (2009) assessed the combining effects of spinosad at

0.01, 0.1 and 0.5 ppm, with the diatomaceous earth (DE) formulation

SilicoSec at rates of 150, 300 and 600 ppm against larvae and adults of

three different populations of Tribolium confusum du Val (Coleoptera:

Tenebrionidae), originating from different European countries (Greece,

Portugal and Denmark). Tests were conducted on wheat and maize at

•15-

^tfviewafLiUmtttre

25 and 30°C and survival of larvae was assessed after 7 d exposure

and survival of adults was assessed after 7 d and 14 d of exposure. At

each dose of spinosad, survival of T. confusum individuals decreased

as the rate of DE increased. As temperature increased, the efficacy of

spinosad and Silicosec alone or in combination also increased. The

efficacy of spinosad alone was slightly higher on maize than wheat,

while the reverse was noted for all the tested combinations of spinosad

with DE as well as in the case of the application of DE alone. The strain

from Portugal was always the least susceptible of the three tested.

Xian-Hui et ai, (2008) investigated the effects of sublethal

concentrations of spinosad applied for 48 h at LC25 (0.12 mg L"^) and

LC50 (0.28 mgL"^) on the biological characteristics i.e. development

time, pupation rate, pupal weight, fecundity, fertility, larval weight and

survival rate in the parent and offspring, of the diamondback moth,

Plutella xylostella (Lepidoptera: Yponomeutidae) and reported that the

pupation rate and pupal weight were significantly lower in treated

groups in which the third instars were treated with spinosad at LC25 or

LC50 than in the control group. The fecundity, egg size and reproductive

effort (fecundityxegg size) were strongly reduced, and the hatchability of

smaller eggs tended to be lower in treated groups. The survival rate of

the offspring at immature stages was lower in the LC25 and LC50

treatment groups than in the control group, and the development time

-16-

^^fviewofLiUmture

tended to be prolonged in the treatment groups. The mean values of the

intrinsic rate of increase (rm), finite rate of increase (D), gross

reproductive rate (GRR) and net reproductive rate (Ro) were

significantly lower in the treatment groups than in the untreated group.

Generally, the effects on biological characteristics were greater in the

LC50 treatment group, and the effects on the offspring were inuch

smaller than those on the parent generation.

Khowaja et al., (1994) studied the effects of sub-lethal

concentrations (10, 15, 20, 25 and 30 ppm) of monocrotophos applied

topically on 5th-instar nymphs of Dysdercus cingulatus Fabr. in the

laboratory at controlled conditions (29 ± VC and 70-80% R.H.) and

reported that the longevity and weight of the treated nymphs increased

linearly and showed positive correlation. On the other hand, fecundity

and fertility of the adults emerging from the treated nymphs decreased

linearly and showed negative correlation with the increasing sub-lethal

concentrations. The LD 50 value representing a very small percentage

of monocrotophos (30 ppm/nymph) increased 5th-instar nymphal

longevity by 43.14%, whereas, the longevity of the adult males and

females enhanced by 31.48 and 26.50%, respectively as compared to

control. Similarly, the weight of the 5* instar treated nymphs increased

by 15.9%; but adult males and females gained 7.9 and 11.8% more

weight as compared to control. On the contrary, fecundity and fertility of

-17-

^ftfiewofLitemtun

the females emerging from the surviving nymphs declined by 18.1 and

22.4%, respectively as compared to controls.

Khowaja et al., (1996) observed that the fecundity and fertility of

the emerging females of Dysdercus cingulatus following the topical

application of 6, 4, 2, 1, and 0.5 Dg doses of two analogues of D-

ecdysone (22, 25-dideoxyecdysone and 2, 22, 25-tndeoxy-5

D-hydroxy ecdysone) decreased linearly and showed negative

correlation with the increasing dose levels. After the application of the

highest dose (6 Dg/nymph) of 22,25-dideoxyecdysone, the maximum

reduction in the egg production and egg hatching of the females that

emerged from the treated nymphs was 30.85 and 35.28%, respectively

as compared to control. But, the same dose of 2,22,25-trideoxy-5n-

hydroxyecdysone resulted in 27.55 and 29.18% reduction in fecundity

and fertility of the females which was 3.3 and 6.1% less when compared

with 22,25-dideoxyecdysone, respectively.

Khowaja and Qamar (2003) observed by topical application of

sublethal concentrations (10, 15, 20, 25 and 30 ppm) of monocrotpphos

on the individual 4th instar nymphs of D. cingulatus resulted in

concentration-based changes. The regression between concentration

strength and average longevity showed a positive correlation. The life

span of the treated 4th instar nymphs as well as those of the next instar

and adult males and females increased linearly, whereas, the weight of

-18-

^StfviewofCiUmtun

the treated nymphs as well as those of ensuing stages did not change

significantly compared to the control. However, the egg production of

the females emerged from the treated nymphs and egg hatching

decreased linearly and showed a negative correlation with increasing

concentrations. Furthermore, the anatomical structure of gonads of the

insects was also affected. The application of higher concentrations, i.e.

25 and 30 ppm, resulted in the gradual degeneration of oocytes, which

ended up in total dissolution of the ovarian follicles. Eventually, all the

oocytes degenerated completely. The lower concentrations of

monocrotophos did not change the morphological and cellular structure

of the gonads, which remain more or less unchanged.

Khowaja et al., (2001) examined the sublethal levels of cythion

(malathion) at the concentrations of 10, 20, 40, 60 and 80 ppm/4th

instar nymph of Dysdercus koenigii resulted in concentration-based

changes. The nymphal loss and survival duration of the treated insects

were directly proportional and increased linearly with the increase in

concentration. On the other hand, the fecundity and fertility of the

adults, emerged from the treated nymphs were inversely proportional,

decreased linearly and showed negative correlation with concentrations.

The initial mortality following the topical application of higher

concentrations (i.e. 80, 60 and 40 ppm) was very high within 24 hours.

However,, the subsequent mortality up to adult emergence was

-19-

^^fmwofLitemture

insignificant. Out of the total adults that emerged from the treated

nymphs, few were with malformed wings and legs. The lower

concentrations (i.e. 20 and 10 ppm) had negligible effect and were well

tolerated.

-20-

MATERIAL AND METHODS

Breeding and maintenance of stock culture of Dysdercus koenigii

Adults of Dysdercus koenigii were collected from the cotton field

during the kharief period and were kept in glass jars measuring

20x15cm, containing 2-3cm thick layer of moist and loose sterilized

sand at the bottom. These jar were kept at two different temperatures

with photoperiod regime, viz. 25±1°C / 12L: 12D and 30 ± 1°C / 14L:

10D in different B.O.D incubators where as the relative humidity was

kept constant in both incubators i.e. 60-70%R.H. These insects were

fed on water soaked healthy cotton seeds, which were changed daily.

After egg laying, the adult insects were transferred to fresh rearing jars

to avoid overcrowding. On hatching, the nymphs were daily provided

with soaked healthy cotton seeds.

Sampling of experimental insects

Newly moulted one day old 4** instar nymphs of Dysdercus

koenigii were used for the experiments. The samples of freshly moulted

3 ^ instar nymphs between 11:00am and 12:00 noon each day were

isolated and maintained age wise. The experiments were run under

controlled laboratory conditions at two different temperature and

photoperiod i.e. 25±1°C / 12L: 12D and 30 ± 1°C / 14L: 10D where the

relative humidity was kept constant in both cases. Subsequently, fresh

MaUmCand9ietfu)ds

moulted 4* instar nymphs of Dysdercus koenigii were treated with

different concentrations of selected insecticides for observation as

described below.

Insecticides Used

1. Confidor (Imidacloprid 17.80% SL)

2. Tracer (Spinosad 45%SC)

These chemicals were purchased commercially from the authorized

dealer.

Dilution and application of insecticides

The following formula was proved helpful to make the stock

solution as

X = AxB/C

Whereas

X = Volume of the solution in hand to be diluted to desired volume

A = Concentration desired

B = Volume of solution desired

C = Concentration of solution in hand.

The stock solution of each insecticides were further diluted to sub

lethal concentration of each insecticide on the basis of LD 30 value as,

Imidacloprid: 0.008%, 0.006%, 0.004%, 0.002%, 0. 001%

Spinosad: 0.1%, 0.08%, 0.06%, 0.04%, 0.02%.

-22-

Material and Methods

These sub lethal concentrations of each insecticide were used for

topical application on the 4* instar stage of Dysdercus koenigii. The

effect of these chemicals was observed on mortality, malformation,

fecundity, fertility and longevity by using five-sub lethal concentrations

of each insecticide at their respective temperature and relative humidity.

For each concentration of diluted insecticide only 1|j| was applied

topically on the thoracic pleuron of individual IV** instar of Dysdercus

koenigii by 26-gauge needle attached to a tuberculin syringe fitted into a

manually operated micro applicator. At the time of application of

insecticides the age of the experimental insect was one day. For each

concentration and temperature with their photoperiod 30 individual IV'

instar nymphs were used. Two controls at each temperature,

photoperiod and humidity consisting of same number of nymphs of

same age were also run parallel to each series of experiment. One

control was treated with acetone (solvent) and served as control-l while

untreated nymphs of the same age served as control-l I. The controlled

groups are maintained at their respective controlled conditions. When

females subsequently emerged from the treated nymphs they were

paired with the males emerged from the untreated nymphs of

corresponding age from control II (Magdum et al., 2001). The data on

mortality, malformation, fecundity, fertility and longevity were recorded

on both treated and control insects.

-23-

Material aiuf94etfukb

Method of observation

Following the application of each concentration of insecticide on

24-hour-olcl 4'^ instar nymphs of D. koenigii. Observations were made

on mortality, malformation, longevity as well as fecundity and fertility of

females emerged from the treated nymphs. The data was compared

with acetone treated control (control-1). The selection of concentrations

was made on the basis of LD30 value. For determining LD30 value of

insecticides different concentration was prepared and applied topically

by micro applicator. For observations on the fecundity and fertility pf the

females, the adult males emerged from the survived treated nymphs

were paired with the affected females of the corresponding age and

stage and each pair was kept in separate rearing jar at controlled

conditions. The number of eggs laid by each female and fertility were

recorded. The unhatched eggs were also counted. Similar observations

were also made in controls consisting of the males and females

emerged from acetone-treated as well as untreated nymphs.

Interpretation of data

The data on mortality, malformation, fecundity, fertility and

longevity of Dysdercus koenigii affected by different concentrations of

various insecticides were recorded for each replicate and mean value of

four such replicates was calculated. The determination of any change

was not only ascertained arithmetically but appreciated by statistically

-24-

MaUfiaC and Methods

analysis also. The standard deviation (S.D) was calculated by the

following formula

S.D.= V^Z)'/«-l

Where,

S.D = Standard deviation

D = Sum of squares of the difference of the mean value.

n = Number of observation

On the basis of S.D (Standard deviation), Standard error was

calculated by the formula as

5£.= S.DJ 4n

Where,

SB = Standard error

S.D = Standard deviation

n • = Number of observation

It was necessary to appreciate the difference between mean

values of different sets of samples. Arithmetically it was uncertain;

therefore we tested the mean value statistically for this purpose the

statistical technique of variation significance (t-Test) was applied

(Bailey-1959) by using the formula

SD, m L 4- 1 «,

-25-

MaUmCan^Metfukb

Where,

t = Significant value

mi = Mean value of first set of observation

m2 = Mean value of second set of obsen/ation

SDi = Standard deviation of first set of observation

SD2 = Standard deviation of second set of observation

ni = Number of observations of the first set

n2 = Number of observations of the second set

The calculated 't' was compared with tabulated 't' (Bailey- 1959)

at 5% level. If the calculated value remained higher than the tabulated 1'

value, the data were regarded significant otherwise insignificant. The

tabulated value oft' at 5% level is 2.447.

Further, to measure the intensity of association between mortality,

malformation, fecundity, fertility and concentration regression line was

fitted to the data, followed by co-relation analysis (Sokal and Rohlf,

1981) using the formula

Y= A+Bx

-26-

RESULTS AND OBSERVATION

Efficacy of Imidacloprid at 30±rc temperature, 14L: 10D

photoperiod and 60-70%RH

For control-1 treatment, 4*' instar nymphs of same age were

sorted out from the culture and treated topically with 1pl acetone while,

for control-!! nymphs of the corresponding age remained untreated. No

significant effects were observed on comparing acetone treated nymphs

{control-1) with untreated nymphs (control-ll). Mortality was not seen

upto the adult emergence during the course of control experiments

(Table 1). The female emerged from acetone treated nymphs, when

mated with normal males, laid almost equal number of eggs to those

emerged from the untreated nymphs. Similarly, there was no significant

effect on the fecundity (t = 1.180, P > 0.05) and fertility (t = 2.130, P >

0.05). The longevity recorded during 4* and 5* nymphal instars as well

as adult male and adult female life stages was 5.25± 0.24, 6.75± 0.24,

20.25 ± 0.24 and 16.75± 0.24 days respectively.

Different sublethal concentration of imidacloprid i.e. 0.001%,

0.002%, 0.004%, 0.006%, and 0.008% were applied topically on 24-

hour-old 4** instar nymphs of Dysdercus koenigii. The number of insects

taken for each treatment was thirty and the experiment was repeated

three times. Each insect was treated topically with 1 | j | of these sublethal

concentrations of insecticide by 26-guage needle attached to a

^l(psu&s and^OSs&vation

tuberculin syringe fitted into a manually operated micro applicator. The

insecticide was applied on the thoracic pleuron of individual 4* instar

nymphs. Observations were made on various aspect of biology like

mortality, malformation, fecundity, fertility and longevity.

Effect on Mortality

The mortality was recorded at two stages, initial mortality upto 24

hour and the total mortality upto adult emergence. The initial nymphal

mortality upto 24 hours at five different sublethal concentration viz.,

0.001%, 0.002%, 0.004%, 0.006% and 0.008% were recorded as 3.33 ±

0.33 (t = 4.186, P > 0.05), 4.00 ± 0.57(t =3.464, P > 0.05), 6.66 ± 0.33(t

= 5.920, P > 0.05), 8.66 ± 0.33(t =6.751, P > 0.05) and 9.00 ± 0.57 (t =

5.196, P> 0.05) respectively. The results were found to be statistically

significant when compared with the acetone treated (Control-I). The

total nymphal mortality upto adult emergence following the topical

application of above said sublethal concentration were recorded as 3.66

± 0.33 {t = 4.388, P > 0.05), 6.00 ± 0.57{t = 4.242, P > 0.05), 10.66 ±

0.33 (t = 7.490, P > 0.05), 12.66 ± 0.33 (t = 8.162, P > 0.05) and 14.00 ±

0.57 (t = 6.480, P > 0.05) respectively, which were found to be

statistically significant as compared with control-1. The initial mortality

upto 24 hours and total mortality upto adult emergence of the treated

nymphs increased linearly with increasing sublethal concentration of

imidacloprid at 3 0 ± r c temperature, 14L:10D photoperiod and 60-

-28-

^^§su&s aiufOSservation

70%RH (y = 1004.4X + 1.910, R = 0.878, P > 0.001 and y = 1624.7x +

2.387, R- = 0.918, P > 0.001 respectively) which shows positive

correlation coefficient (Graph land 2).

Effect on Malformation

Malformations in the nymphs and adults emerged from treated 4*

instar nymphs of D. koenigii were of variable degree in the whole body,

wings and legs. A number of adults were found having folded, twisted

and deformed wings as well as sunken abdomen. In controlled sets, the

malformations were not observed. The malformation either in wings or

legs caused by these sublethal concentrations viz., 0.001%, 0.002%,

0.004%, 0.006% and 0.008% were recorded as 2.66 ± 0.33 (t = 3,741,

P > 0.05), 3.33 ± 0.33 (t = 4.186 P > 0.05) 3.33 ± 0.33 (t = 4.186 P >

0.05), 3.66 ± 0.33 (t = 4.186, P > 0.05) and 5.00 ± 00 (t = 6.388, P >

0.05) respectively (Plate-1). These results were statistically significant

when compared with the acetone treated nymphs or untreated nymphs.

The malformation caused due to treatment of nymphs increased linearly

with increasing sublethal concentration of imidacloprid (y = 417.14x +

1.599, R = 0.665) showing positive correlation coefficient (Graph 3).

The malformations were much pronounced in higher concentrations.

The abnormal adults formed were found to have shorter life span.

-29-

^Stpsu&s andOSservation

Effect on Fecundity and Fertility

The Fecundity and Fertility were affected by sublethal

concentration of insecticides viz., 0.001%, 0.002%, 0.004%, 0.006%,

and 0.008% and fall in fecundity were recorded as 179.75 ± 1.49 (t =

4.418, P > 0.05), 144.25 ± 1.49 (t = 5.853, P > 0.05), 110.75 ± 0.85 (t =

7.454, P > 0.05), 92.00 ± 1.29 (t = 8.092, P > 0.05) and 83.25 ± 0.85 (t =

8.291, P > 0.05) at respective concentrations as compared to control

whereas, the fertility were recorded as 154.50 ± 1.04 (t = 6.902, P >

0.05), 122.25 ± 1.24 (t = 5.853, P > 0.05), 86.75 ± 0.85 (t = 10.725, P >

0.05), 66.00 ± 0.91 (t = 11.441, P > 0.05) and 52.25 ± 1.24 (t = 11.200,

P > 0.05) respectively. Both the results were statistically significant

when compared with control-1. The results showed reduction in

fecundity and fertility on increasing the concentration of imidacloprid. At

lowest selected concentrations the fecundity dropped to 20.72% while

the fertility / egg hatching were reduced to 26.42%. Similarly, at highest

selected concentration the fecundity dropped to 63.28% and fertility

reduced to 75.11%. The average egg production of the females that

emerged from treated 4*'' instar nymphs decreased linearly with

increasing sublethal concentration (y = -15864x + 192.6, R = 0.855)

showing negative correlation coefficient (Graph 4) while the fertility of

the eggs laid by the affected females also decreased linearly (y = -

-30-

^^fsu&s awCOBsefwition

17018X + 172.3, R = 0.858) and showed negative correlation

coefficient (Graph 5).

Effect on Longevity

The effect of topical application of imidacloprid on longevity has

been recorded during nymphal instar (4* and S*" ) and adult (male and

female) stage of Dysdercus koenigii. The longevity for 4 ^ nymphal instar

at sublethal concentrations viz., 0.001%, 0.002%, 0.004%, 0.006% and

0.008% were 5.50 ± 0.28 (t = 0.975, P > 0.05), 5.75 ± 0.24 (t = 1.428, P

> 0.05), 6.00 ± 00 (t = 2.474, P > 0.05), 6.50 ± 0.28 (t = 5.182, P > 0.05)

and 6.75 ± 0.24 (t = 2.474, P > 0.05) days respectively. The result at

lower concentration (0.001% and 0.002%) showed statistically

insignificant increase in nymphal duration whereas, at higher

concentration (0.004%, 0.006% and 0.008%) gave significant when

compared with the control-1. Similarly, the 5* nymphal instar span were

7.25 ± 0.24 (t = 1.428, P > 0.05), 7.75 ± 0.24 (t = 2.020, P > 0.05), 8.00

± 00 (t = 3.194, P > 0.05), 8.50 ± 0.28 (t = 2.581, P > 0.05) and 8.75 ±

0.24 (t = 2.857, P > 0.05) days at above mentioned concentrations

respectively. The results at lower concentrations i.e. 0.001% and

0.002% gave insignificant increase whereas higher concentration i.e.

0.004%, 0.006%, and 0.008% gave statistically significant enhancement

when compared with control-1. The adult male longevity were recorded

at these concentrations as 21.75 ± 0.24 (t = 2.474, P > 0.05), 22.00 ± 00

-31-

^^fisufts andOSsetvation

(t = 3.779, P > 0.05), 22.75 ± 0.24 (t = 3.194, P > 0.05), 23.50 ± 0.28 (t

= 3.761, P > 0.05) and 24.00 ± 0.28 (t = 5.532, P > 0.05) days

respectively. The results were found to be significant when compared

with control-1. The adult female longevity at these concentrations were

recorded as 17.00 ± 00(t = 1.428 P > 0.05), 17.25 ± 0.47 (t = 1.428, P >

0.05), 18.50 ± 0.28 (t = 2.569, P > 0.05), 20.00 ± 00 (t = 5.150, P >

0.05) and 20.50 ± 0.28 (t = 3.761, P > 0.05) days respectively. The

results were found to be significant at higher concentration (0.008%,

0.006%, and 0.004%) but were insignificant at lower concentrations

(0.001% and 0.002%). The longevity of treated 4"" instar nymphs

increased linearly with increasing sublethal concentrations and same

affects were observed in rest of the life stages as 4'* instar (y = 176.3x +

5.3677, R2 =0.976), 5* instar (y = 223.5x + 7.084, R = 0.929), adult

male (y = 387.9x + 21.07, R =0.872) and adult female (y = 487.9x +

16.69, R? = 0.9676) shows positive correlation with increasing

concentrations (Graph 6-9).

Efficacy of Imidacloprid at 25±1*C temperature, 12L: 12D

photoperiod and 60-70%RH

For control set I, when the 4* instar nymph was treated topically

with 1 1 acetone solution no mortality was observed upto adult

emergence (Table 2). For control set II the initial mortality upto 24

hours, total mortality upto adult emergence, malformation, fecundity,

-32-

^§su&s andOBsewation

fertility and longevity were compared with the acetone treated control I.

The ecdysed 5' ^ instar nymphs and as well as emerged adult male and

female were normal in appearance showing no malformation. The adult

females emerged from the acetone treated 4th instar nymphs niated

with the untreated normal males (control- II), laid almost equal number

of eggs as compared to females emerged from the untreated 4 * instar

nymphs (Table 3), showing no significant difference in fecundity (t =

1.052, P > 0.05) and fertility (t = 1.973, P >0.05). The longevity recorded

during 4* and 5* nymphal instar as well as adult males and adult

female stage were 6.50 ± 0.28, 6.50 ± 0.28, 22.25 ± 0.24 and 17.50 ±

0.28 days respectively. The survival duration of all the stages in both

control I and control II remain almost same indicating no statistical

difference.

Effect on Mortality

The initial mortality upto 24 hours and total mortality upto adult

emergence were recorded with treated 4* instar nymphs of D. koenigii.

The 4'^ instar nymphs were treated with different sublethal

concentrations as, 0.001%, 0.002%, 0.004%, 0.006% and 0.008% of

imidacloprid to observe the initial nymphal mortality upto 24 hours and

total mortality upto adult emergence. The initial mortality upto 24 hours

were recorded at these sublethal concentrations as, 2.33 ± 0.33(t =

3.501, P > 0.05), 3.00 ± 0.57 (t = 3.000, P > 0.05), 5.00 ± 0.57 (t =

-33-

^S^fsufts cmdOSsmmtion

3.872, P > 0.05), 5.33 ± 0.33 (t = 5.292, P > 0.05) and 7.00 ± 0.57 (t =

4.542, P > 0.05) respectively. The results were found to be significant

when compared with control-1. The total nymphal mortality upto adult

emergence were found to be 3.33 ± 0.33 (t = 4.186, P > 0.05), 6.00 ±

0.57 (t = 4.242, P > 0.05), 9.00 ± 0.57 (t = 5.196, P > 0.05), 11.00 ± 0.57

(t = 5.744, P > 0.05) and 12.00 ± 0.57 (t = 6.000, P > 0.05) respectively.

The results came to be statistically significant when compared with

control-1. The initial mortality of nymphs upto 24 hours and total

mortality upto adult emergence at this temperature and relative humidity

increased linearly with increasing sublethal concentrations (y = 727.1x +

1.340, R = 0.891 and y = 1364.5x + 2.3171, R = 0.904, P > 0.01

respectively) showing positive correlation coefficient (Graph 10 and 11).

Effect on Malformation

The affects on malformations of variable degree were noticed in

the wings, legs and body of adults that emerged from the treated 4*

instar nymphs. The malformation formed at sublethal concentration viz.,

0.001%, 0.002%, 0.004%, 0.006% and 0.008% were found to be 0.67 ±

0.33 {t = 1.877, P > 0.05), 2.33 ± 0.33 (t = 3.501, P > 0.05), 3.00 ± 00 (t

= «°, P > 0.05), 3.33 ± 0.33 (t = 4.186, P > 0.05), and 4.33 ± 0.33 (t =

4.773, P > 0.05) respectively {Plate-2). There was no significant effect

on malformation at 0.001% and 0.004% concentration while treatment

with 0.008%, 0.006% and 0.002% indicate statistically significant results

-34-

^^^ and observation

as compared to control-1. The proportion of malformation increased on

increasing tiie concentration of insecticide (y = 485.7x + 0.649, R =

0.912). Thus malformation showed positive correlation with increasing

sublethal concentration at 25±1°C/ 12L: 12D temperature, photoperiod

and constant relative humidity {Graph 12).

Effect on Fecundity and Fertility

The fecundity were recorded at sublethal concentrations viz.,

0.001%, 0.002%, 0.004%, 0.006% and 0.008% as 157.75 ± 1.54 (t =

3.762, P > 0.05), 128.50 ± 1.70{t = 4.687, P > 0.05), 103.75 ± 1.88 (t =

5.300, P > 0.05), 70.75 ± 0.62 {t = 6.692, P > 0.05) and 65.25 ± 0.62 {t =

6.824, P > 0.05) eggs respectively. While, the egg hatching at these

sublethal concentration were recorded as 132.50 ± 1.70 {t = 6.049, P >

0.05), 105. 50 ± 0.28 (t = 9.396, P > 0.05), 76.50 ± 1.49 (t = 8.954, P >

0.05), 46.25 ± 0.62 (t = 12.492, P > 0.05) and 40.00 ± 0.91 (t = 11.854,

P > 0.05) respectively. The results were statistically significant when

compared with the control-l. The fecundity decreased linearly with

increasing sublethal concentrations (y = -15623x + 174.5, R = 0.868)

showing negative correlation coefficient (Graph 13), similarly the fertility

also reduced linearly (y = -15827x + 150.6, R = 0.863) with increasing

sublethal concentrations and shows negative correlation coefficient

(Graph 14).

-35-

^fstdts andOBsermtion

Effect on Longevity

The longevity was reported at three life stages such as, 4*^ S**"

nymphal instars as well as adult male and female. The longevity of 4*

instar nymphs at sublethal concentrations viz., 0.001%, 0.002%,

0.004%, 0.006% and 0.008% were recorded as 6.75 ± 0.24 (t = 0.971,

P > 0.05), 7.00 ± 00 (t = 1.873, P > 0.05), 7.50 ± 0.28 (t = 1.873, P >

0.05), 8.00 ± 00 (t = 3.244, P > 0.05) and 8.25 ± 0.24 (t = 2.564, P >

0.05) days respectively. The results at lower concentrations i.e. 0.001%,

0.002% and 0.004% were statistically insignificant while at higher

concentrations i.e. 0.006% and 0.008% these were statistically

significant. The longevity were recorded for 5* nymphal instar at these

concentrations were 7.00 ± 00 (t = 1.873, P > 0.05), 7.75 ± 0.28 (t =

1.428, P > 0.05), 8.50 ± 0.28 (t = 2.649, P > 0.05), 8.75 ± 0.24 (t =

2.913, P > 0.05) and 9.50 ± 0.28 (t = 3.244, P > 0.05) days respectively.

These results were statistically insignificant at 0.001% and 0.002%

while higher concentrations i.e. 0.004%, 0.006% and 0.008% gave

statistically significant results. The adult male longevity were recorded

as 23.50 ± 0.28 (t = 2.171, P > 0.05), 24.25 ± 0.24 (t = 2.857, P > 0.05),

25.00 ± 00 (t = 4.738, P > 0.05), 25.75 ± 0.24 (t = 3.779, P > 0.05) and

26.50 ± 0.28 (t = 4.004, P >0.05) days respectively. The results were

statistically insignificant at 0.001% while at 0.002%, 0.004%, 0.006%

and 0.008% showed statistically significant. The adult female longevity

-36-

^fsulis amfOSsewation

was recorded as 17.75 ± 0.24 (t = 0.971, P > 0.05), 18.00 ± 00 (t =

1.873, P > 0.05), 18.50 ± 0.28 (t = 1.873, P > 0.05), 19.00 ± 00 (t =

3.244, P > 0.05) and 19.50 ± 0.28 (t = 2.649, P > 0.05) days

respectively. These gave insignificant result at 0.001%, 0.002% and

0.004% whereas, at higher concentrations i.e. 0.006% and 0.008% it

was significant. The longevity of treated 4 ^ instar (y = 214.Ox + 6.616,

R > 0.981), 5* instar (y = 339.8x + 6.861, R = 0.954), adult male (y =

456.8X + 23.011, R =0.921) and adult female (y = 236.1x +17.58, R =

0.989) increased linearly with increasing sublethal concentrations of

imidacloprid showing positive correlation coefficient (Graph 15-18)

Efficacy of Spinosad at 30±1*'C temperature, 14L: 10D photoperiod

and 60-70%RH

Different aspects of biology as mortality upto 24 hours, mortality

upto adult emergence, malformation, fecundity and fertility and longevity

were recorded. No significant effects were found while comparing the

acetone treated control-1with untreated control-ll. The initial mortality

upto 24 hours and total mortality upto adult emergence and

malformation were not found during different stages of life cycle. The

fecundity and fertility were recorded as 226.75 ± 3.32 (t = 1.180, P >

0.05) and 210.00 ± 1.29 (t = 2.130, P > 0.05) respectively. The

untreated females laid 231.25 ± 3.06 numbers of eggs and the fertility

was recorded as 218.25 ± 2.35. The longevity of treated 4**" instar and

-37-

^ifisu&s otuCOSsmHition

5' instar nymphs, adult male and adult female were recorded as 5.25 ±

0.24, 6.75 ± 0.24, 20.25 ± 0.24 and 16.75 ± 0.24 days respectively and

no effect was found while comparing acetone treated nymphs (control-l)

with untreated ones (control-ll).

Effect on Mortality

The initial mortality of nymphs upto 24 hours at sublethal

concentrations viz., 0.02%, 0.04%, 0.06%, 0.08% and 0.1% were

recorded as 0.66 ± 0.33 (t = 3.473, P > 0.05), 2.33 ± 0.57 (t = 3.501, P >

0.05), 5.00 ± 0.57 (t = 3.872, P > 0.05), 7.33 ± 0.33 (t = 6.211, P >

0.05), 8.33 ± 0.33 (t = 6.621) respectively. The results were statistically

significant when compared with control-1. The total mortality upto.adult

emergence was also recorded as 1.66 ± 0.33 (t = 2.955, P > 0.05), 7.00

± 0.57 (t = 4.582, P > 0.05), 10.66 ± 0.33 (t = 7.490, P > 0.05), 13.33 ±

0.33 (t = 8.376, P > 0.05), and 17.00 ± 0.57 (t = 7.141, P > 0.05)

nymphs respectively. The results were statistically significant when

compared with control-1. The initial mortality upto 24 hrs and total

mortality upto emergence of treated 4*'' instar nymphs increased linearly

with increasing sublethal concentration (y = 91.9x - 0.653, R = 0.973

and y = •176.6x - 0.558, R = 0.987 respectively) showing positive

correlation coefficient (Graph 19 and 20).

-38-

^^fisu&s ondOSserMtim

Effect on Malformation

The malformation were recorded at sub lethal concentrations of

spinosad viz., 0.02%, 0.04%, 0.06%, 0.08% and 0.1% as 6.00 ± 0.57 (t

= 4.242, P > 0.05), 5.00 ± 00 (t = 3.501, P > 0.05), 5.66 ± 0.33 (t =

5.457, P > 0.05), 4.33 ± 0.33 (t = 4.773, P > 0.05), 5.33 ± 0.33 (t =

5.296) respectively (Plate-3). The results came to be statistically

significant when compared with control-1. The malformation during their

life stages increased linearly with increasing sublethal concentration (y

= 31.85X .+ 2.793, R = 0.287) showing positive correlation coefficient

(Graph 21)

Effect on Fecundity and Fertility

The number of eggs (aid by the affected females at sublethal

concentration viz., 0.02%, 0.04%, 0.06%, 0.04% and 0.1% were 161.50

± 1.32 (t = 5.300, P > 0.05), 138.75 ± 1.24 (t = 6.202, P > 0.05), 130.00

± 1.95 (t = 6.053, P > 0.05), 118.75 ± 2.24 (t = 6.224, P > 0.05) and

103.75 ± 1.88 (t = 6.878, P > 0.05) respectively. The egg hatching was

also recorded on these concentrations as 142.50 ± 0.64(t = 7.942, P >

0.05), 117.25 ± 0.85(t = 9.302, P > 0.05), 106.25 ± 0.85(t = 9.830, P >

0.05), 90.75 ± 0.33 (t = 9.979, P > 0.05), and 72.50 ± 1.04 (t = 10.863, P

> 0.05) respectively. These results came to be statistically significant

when compared with acetone treated control-1. The average fecundity

of the females that emerged from the treated 4*'' instar nymphs

-39-

§tpsu&s andOSservatUm

decreased linearly with increasing sublethal concentration (y = -1074.3x

+ 200.3, R = 0.842) showing negative correlation coefficient while the

fertility of the egg laid by these affected females also decreased linearly

(y = -1219.x + 184.1, R = 0.878) and showed negative correlation

coefficient (Graph 22 and 23).

Effect on .Longevity

The longevity at different life stages has been investigated with

sublethal concentration of spinosad viz., 0.02%, 0.04%, 0.06%, 0.08%

and 0.1%. The survival duration of 4** instar nymphs was recorded as

5.50 ± 0.28 (t = 0.975, P > 0.05), 6.00 ± 00 (t = 2.400, P > 0.05), 6.50 ±

0.28 (t = 2.182, P > 0.05), 7.00 ± 00 (t = 3.818, P > 0.05) and 7.25 ±

0.24 (t = 2.857, P > 0.05) days respectively. Here the results at 0.02%,

0.04% and 0.06% sublethal concentrations came to be statistically

insignificant while at higher sublethal concentrations at 0.1% and 0.08%

results came to be significant. The 5** instar nymphal longevity was

recorded as 7.25 ± 0.24 (t = 1.428, P > 0.05), 7.75± 0.24 (t = 1.020, P >

0.05), 8.25 ± 0.24 (t = 2.474, P > 0.05), 8.75 ± 0.24 (t = 2.857, P > 0.05)

and 9.50 ± 0.28 (t = 3.236, P > 0.05) days respectively. The results at

lower concentration i.e. 0.02%, 0.04% came to be insignificant while at

higher concentrations i.e. 0.06%, 0.08% and 0.1% results came "to be

statistically significant. The longevity of adult males were 21.50 ± 0.28 (t

= 2.182, P > 0.05), 22.25 ± 0.24 (t = 2.857, P > 0.05), 23.00 ± 00 (t =

-40-

^fsuCts atutOBsermtum

4.787, P > 0.05), 23.75 ± 0.24 (t = 3.780, P > 0.05) and 24.50 ± 0.28 (t =

4.023, P > 0.05) days respectively. The results were statistically

significant at 0.1%, 0.08%, 0.06% and 0.04% while at 0.02% remain

insignificant. The longevity of adult females were also recorded at

aforesaid sublethal concentrations as 17.50 ± 0.28 (t = 1.690, P > 0.05),

18.25 ± 0.24 (t = 2.474, P > 0.05), 19.00 ± 00 (t = 4.330, P > 0.05),

19.75 ± 0.24 (t = 3.500, P > 0.05) and 20.50 ± 0.28 (t = 3.780, P > 0.05)

days respectively. The results at 0.02% came to be insignificant while at

higher concentrations i.e. 0.04%, 0.06%, 0.08% and 0.1% results came

to be statistically significant. The longevity of 4** instar (y = 21.42x +

5.178, R2'= 0.989), 5* instar (y = 26.78x + 6.702, R = 0.994), adult

male (y = 41.07x + 20.48, R = 0.99) and adult female (y =37.5x +

16.75, R = 0.99) of the treated 4* instar nymphs increased linearly with

increasing sublethal concentration. Thus the result showed positive

correlation coefficient (Graph 24-27).

Efficacy of spinosad at 25±1''C temperature, 12L: 12D photoperiod

and 60-70%RH

The initial nymphal mortality upto 24 hours, mortality upto adult

emergence, malformation, fecundity, fertility and longevity were

recorded at 25±1°C temperature, 12L:12D photoperiod and 60-70%

relative humidity. The initial mortality of nymphs upto 24 hours and total

mortality upto adult emergence and malformation were not found during

-41-

^fsu^ muCOBservatum

different stages of life cycle at 25±1°C temperature, 12L:12D

photoperiod and 60-70% relative humidity. The fecundity of females,

which emerged from acetone treated nymphs and untreated nymphs

were found to be as 207.50 ± 5.48 and 213.00 ± 4.45 (t = 1.052, P >

0.05) respectively. The fertility of females, which emerged from acetone

treated nymphs and untreated nymphs were recorded as 184.75 ±1.18

and 195.25 ± 4.21 (t = 1.973, P >0.05) respectively. No statistically

significant effects were shown. The longevity of different life stage i.e.

4*^ 5*^ adult male and adult female were as 6.50 ± 0.28, 6.50 ± 0.28,

22.25 ± 0.24 and 17.50 ± 0.28 respectively. The longevity of untreated

nymphs remains unchanged when compared with untreated nymphs

(control-ll).

Effect on Mortality

The initial mortality of nymphs recorded from the treated 4** instar

at sublethal concentrations viz., 0.02%, 0.04%, 0.06%, 0.08% and 0.1%

were as, 0.33 ± 0.33 (t = 1.736, P > 0.05), 0.67 ± 0.33 (t = 1.877, P >

0.05), 1.33 ± 0.33 (t = 2.645, P > 0.05), 3.00 ± 00 (t = «>, P > 0.05) and

4.33 ± 0.33 (t = 4.773, P > 0.05) respectively. The results at lower

concentrations i.e., 0.02% and 0.04% were statistically insignificant

whereas at higher concentrations i.e. 0.06%, 0.08% and 0.1% found to

be statistically significant. The mortality upto adult emergence were also

recorded at sublethal concentrations as, 0.66 ± 0.33 (t = 1.878, P >

-42-

^^psu&s amCOSsavation

0.05), 3.00 ± 0.57 (t = 3.000, P > 0.05), 4.33 ± 0.33 (t = 4.773, P >

0.05), 7.00 ± 0.57 (t = 4.582, P > 0.05) and 9.33 ± 0.33 (t = 7.007, P >

0.05) respectively. The results were Insignificant at 0.02% while at

higher concentrations results were significant as compared with control-

1. The Initial mortality upto 24 hours and total mortality upto adult

emergence increased linearly with increasing sublethal concentration (y

= 43.31X - 0.5557, R^ = 0.904, y = 95.71x - 0.732, R^ = 0.976

respectively) showing positive correlation coefficient. (Graph 28 and

29).

Effect on Malformation

The malformation in treated 4** instar nymphs at sublethal

concentrations viz., 0.02%, 0.04%, 0.06%, 0.08% and 0.1% were 4.66 ±

0.33 (t = 4.952, P > 0.05), 5.33 ± 0.33 (t = 3.000, P > 0.05), 5.66 ± 0.33

(t = 3.872, P > 0.05), 6.33 ± 0.33 (t = 5.771, P > 0.05) and 6.33 ± 0.66 (t

= 4.063, P > 0.05) respectively (Plate-4). The result shows significant

result as compared to control. The malformation increased linearly with

Increasing sublethal concentrations (y = 52.84x + 2.076, R^ = 0.680)

showing positive correlation coefficient (Graph 30).

Effect on Fecund/ty and Fertility

The fecundity of females, which emerged from the treated 4*

instar nymphs at sublethal concentrations of spinosad viz., 0.02%,

-43-

J^^'Wrf-Qfen^t^, ' "''"'''' I'll'' " ' " , u.uQVo and n 10/ . —

^•°^^. - 0.05) .espec..e,v r.e . s ' « ' ' ' " ' " ' • ' ' ^ ^'^ , .

=°ncen,rat,on were recorded ' " " ' ' ' ^"'"^•^a' recorded as, 140.75 + Ofl"; »

' ' ^ • = 0 - . a 4 ( , = a . 8 4 , P > 0 0 5 ) S S 7 / " ' ' ^ ' ' ^ ° ° ^ ' '

^ '-^-—- J : :;;:::: - °- '= - ^ compared wl,h contro, , JU ' "''"'""'"' * " « "

' ' -eased „near,v mu i n c r e ' " " ' ' ' " " " * ' ° ' '^^ ^ - ^ ' -

"^•3. R = 0.978) Showing neaat,v» "

(^-P-^ 31), White the fertility of tH " ' " " ' ° " ~ ^ ' " * " ' 3 , . , . , _ ^ ' - e . , . , d . , t h e 3 . e c , e d f e . a i e s

-native correlation coefficient (Graph 32, " " ' ' ' ^ ='°^^"^

Effect on Longewty

' " ^ ^ ' " ' ° " '°"9-^'V were investigated t>v . "ymP' s Of Oy . .e . . s fee.,, , , „ . , ' ' ' ^ ' " * « ^'^ - t a r

°----.oe.,ooarr"""""°"^^"' *"-^ ' ^-^o/o, and 0 1% r,f

""nation Of treated 4%nstarn. K ' " " ° ' ' * " ^ ="™Val '"Star nymphs werp « 7c

°0^X 7.25 . 0.24 (t - , eao P n ' " ' ^ ' = "'^^^^ ^ > V / .oyu, P > 0 0 5 ) 7 7C

°°5) ,8 .00t0O(t = 3 2 7 3 p ^ ' ' ' * "'^^ « = 2-182, P >

^•273, P> 0.05) and 8 .50.0 28 rt , - "ZS (t = 2.672, P > -44-

^S^fSuO^ atufOBservatum

0.05) days respectively. The results were significant only at two higher

concentration i. e. 0.1% and 0.08% while, rest of sublethal

concentration shows statistically insignificant effects. The longevity of 5*

instar nymphs recorded as 7.25 ± 0.24 (t = 1.690, P > 0.05), 8.00 ± 00 {t

= 3.273, P >0.05), 8.50 ± 0.28 (t = 2.672, P > 0.05), 9.00± 00 (t = 4.226,

P > 0.05) and 9.25 ± 0.24 (t = 3.237, P > 0.05) days respectively. The

results gave statistically significant at higher concentration

{0.1%,0.08%, 0.06%, 0.04%) while at 0.02%. results was insignificant

effects. The male longevity recorded as 23.50 ± 0.28 (t = 2.182, P >

0.05), 24.00 ± 00 (t = 3.818, P > 0.05), 24.75 ± 0.24 (t = 3.194, P >

0.05), 25.25 ± 0.24 (t = 3.499, P > 0.05) and 26.25 ± 0.24 (t = 4.040, P >

0.05) days respectively. The results are insignificant only at 0.02% while

rest of all higher sublethal concentration remain significant. The female

longevity recorded as 17.75 ± 0.24 (t = 0.976, P > 0.05), 18.25 ± 0.24 (t

= 1.690, P > 0.05), 19.00 ± 00 (t = 3.273, P >0.05), 19.75 ± 0.24 (t =

2.927, P > 0.05) and 20.25 ± 0.24 (t = 3.236, P > 0.05) days

respectively. Here, the results at higher concentrations (0.1%, 0.08%,

0.06% is significant while rest of sublethal concentrations remained

insignificant. The affected longevity of 4* instar nymphs (y

=20.35x+6.440, R = 0.991), 5* instar nymphs (y =27.85x+6.690,. R =

0.972), adult males (y =37.14x+22.47, R = 0.982) and adult females

-45-

^^fisu&s (md^OSservatmi

(y =29.28x+17.28, R = 0.980) increased linearly with increasing

concentration of spinosad (Graph 33-36).

-46-

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y=1004.4x+1.9103

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^ ^ • 30±1X/14L:10D

^ ^ ^ ^ Linear (30+1X/14L:10D)

1 ! [ !

0.002 0.004 0.006 0.008 0.01

Concentration %

Graph: 1. Fitted linear regression line for mortality upto 24 hours of treated 4* instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 30±1°C/14L:10D temperature and photoperlod.

Showing total mortality upto adult emergence at

4>

g 18 o) 16

^ 14 ^

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/ ^ Linear {30±1°C/14L:10D)

.,_ , ,j , , , .

0.002 0,004 0.006 0,008 0.01

Concentration %

Graph: 2. Fitted linear regression line for mortality upto adult emergence of treated 4* ^ instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 30±1 "0/141:100 temperature and photoperlod. ,-.^-

Showing malformation at30±1*C/14L:10D

Vt i

o ^ 4 \

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V= 417.14x+1.5992 R = 0.6657

• 30±1''C/14L:10D

- Linear (30±1''C/14L:10D)

0.002 0.004 0.006 0.008 0.01

Concentration %

Graph: 3. Fitted linear regression line for malformation of treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 30±1°C/14L:10D temperature and photoperiod.

Showing fecundity at 30±1X/14L:10D

y=-15864x4 192,6

R = 0.8551

• 30±1"C/14L:10D

Linear (30±1X/14L:10D)

0.002 0,004 0.006 0,008 0.01

Concentration %

Graph: 4. Fitted linear regression line for fecundity of adult female, which emerged from treated 4* ^ instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 30±1°C/14L:10D temperature and photoperiod.

-56-

Showing fertility at 30±rc/14L:10D

250

200

w

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I 150 f

Ig 100 a>

• 30±1''C/14L:10D

Linear (30±1°C/14L:10D)

0 0.002 0.004 0.006 0,008 0.01

Concentration %

Graph: 5. Fitted linear regression line for fertility of adult female, which emerged from treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 30±1°C/14L:10D temperature and photoperiod.

Showing longevity of treated IV**' instar at 30±rc/14L:10D

(0

I c o

8

7

6

5

4

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R = 0,9764 Linear (30±1°C/14L:10D)

0 0.002 0.004 0.006 0.008 0.01

Concentration %

Graph: 6. Fitted linear regression line for longevity of treated 4"" instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 30±1°C/14L:10D temperature and photoperiod.

-57-

Showing longevity of V**' Instar nymph which emerged from treated IVth Instar at

30±rC/14L:10D

> OJ c o

• 30+1'C/14L:100

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(30±1X/14L:100)

0.002 0.004 0.006 0.008

Concentration %

0.01

Graph: 7. Fitted linear regression line for longevity of V** instar which emerged from treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 30±rC/14L;10D temperature and photoperiod.

>> (0

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Showing longevity of adult male which <

24.5 24

23.5 23

22.5 22

21.5 21

20,5

20

emerged from treatedIV*''instarnymphs at30±l''C/14L:10D

i H J

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0.002 0,004 0.006 0.008 0.01

Concentration %

• 30±1"C/14L:10D

Linear (30±1"C/14L:10D)

Graph: 8. Fitted linear regression line for longevity of adult male which emerged from treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 30±rC/14L:10D temperature and photoperiod.

-58-

Showing longevity of adult female which emerged from treated IV'^instar nymphs at

30±rC/14L:10D 25

> (D O) c o

10 y=487.0xt 16.699

R = 0.9676 • Linear (30±1"C/14L:10D)

0.002 0.004 0.006 0.008 0.01

Concentration %

Graph: 9. Fitted linear regression line for longevity of adult female which emerged from treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 30±1°C/14L:10D temperature and photoperiod.

(A

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r—

0.002

25±rC/12L:12D

V= 727.14x4 1 . 3 4 M , ^

R = o.soy^ ^ ^ ^

^ ^ • 25+lX/12L:12D

Linear (25+rC/12L:12D)

J , .—-r~----- — ;

0.004 0.006 0.008 0.01

Concentration %

Graph: 10. Fitted linear regression line for mortality upto 24 hours of treated 4* instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 25±1°C/12L:12D temperature and photoperiod.

-59-

u c 4)

Showing total mortality upto adult emergence at 25±rC/12L:12D

y=1364.5x+ 2.3171 R = 0.9048

• 25+lX/12L:12D

• Linear (25±rC/12L:12D)

0.002 0.004 0.006 0.008

Concentration %

0.01

Graph: 11. Fitted linear regression line for mortality upto adult emergence of treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 25±rC/12L:12D temperature and photoperiod.

Showing malformation at 25±1*C /12L: i2D

v> o Z c o m E

m

• 25±1'C/12L:12D

V= 485.73x4 0.6495 R = 0.9127 - Linear

(25±1°C/12L:12D)

0.002 0.004 0.006 0.008 0.01

Concentration %

Graph: 12. Fitted linear regression line for malformation of treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 25±1°C/12L:12D temperature and photoperiod.

-60-

Showing fecundity of females at 25±1 "0/121:12D

250

y=-15623x+ 174.59 R2-0.8681

• ZStlX/lZL-.lZD

Linear (25+l*C/12L:12D)

0 0.002 0.004 0.006 0.008 0.01

Concentration %

Graph: 13. Fitted linear regression line for fecundity of adult female, which emerged from treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 25±1°C/12L:12D temperature and photoperiod.

1 2' 1 41

200 180

160 140

120 100 80

60

40

20

0

Showing fertility of female at 25±1

t - i V ,

1

( i

J

0

• ^

0.002

y=-15827x+150.6 R - 0.8638

• \ ^

1 1 1

0.004 0.006 0.008

Concentration %

0.01

"C/12L:12D

• 25±1'C/12L:12D

Linear (25±1''C/12L:12D)

Graph: 14. Fitted linear regression line for fertility of adult female, which emerged from treated 4*" instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 25±1°C/12L:12D temperature and photoperiod.

-61-

Showing longevity of IVth Instar nymph at 25±rC/12L:12D

(A >» (0

> a> o> c o

y=214.05x+6.6163 R = 0.9817

• 25±rC/12L:12D

Linear

(25±1°C/12L:12D)

0.002 0.004 0.006 0.008 0.01

Concentration %

Graph: 15. Fitted linear regression line for longevity of treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 25±1°C/12L:12D temperature and photoperiod.

Showing longevity o f Vth instar at 25±1°C/12L:12D

• 25±l''C/12L:12D

V-339.82X+5.8616 R2 = 0.9542 Linear

(25+rC/12L:12D)

0 4

0 0.002 0.004 0.006 0.008

Concentration %

0.01

Graph: 16. Fitted linear regression line for longevity of 5^^ instar which emerged from treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 25±1°C/12L:12D temperature and photoperiod.

-62-

Showing longevity of adult male at 25±1°C/12L:12D

27 -

• 25+l'-C/12L:12D

y=456.81x t 23.011 R = 0.9216 - Linear

{25±1°C/12L:12D)

0.002 0.004 0.006 0.008 0.01

Concentration %

Graph: 17. Fitted linear regression line for longevity of adult male which emerged from treated 4** instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 25±rC/12L.12D temperature and photoperiod.

in >. (0

t>

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18.5 -

18

17.5 i

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Showing longevity of adult female at 25±rc/12L:12D

0.002

^4f • 25±1''C/12L:12D

y=236.12x» 17.584 Lmp^^

R' = 0-9852 (25±rC/12L:12D)

1 \ i n

0.004 0.006 0.008 0.01

Concentration %

Graph: 18. Fitted linear regression line for longevity of adult female which emerged from treated 4'^ instar nymphs of D. koenigii with various sublethal concentrations of imidacloprid at 25±rC/12L:12D temperature and photoperiod.

-63-

in o

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8

6

4

2

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Showing Initial mortality upto 24 hour at 30±rC/14L:10D

y=91.9x-0.6S33 R2 = 0.973

• 30+l'C/14L:10D

• Linear (SO+l-C/lAL-.lOD)

0.02 0.04 0.06 0.08

Concentration %

0.1

Graph: 19. Fitted linear regression line for mortality upto 24 hours of treated 4'^ instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 30±1°C/14L:10D temperature and photoperiod.

4> O

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1 2 0 ^

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Showing mortality upto adult emergence at 30±rC/14L:10D

^y^ y^ y = 176.67X - 0,55*6 30±rC/14L-.10D

y ^ R - 0.9876

y^ Linear y ^ (30+l"C/14L:10D)

0.02 0.04 0.06 0.08 0.1

Concentration %

Graph: 20. Fitted linear regression line for mortality upto adult emergence of treated 4'" instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 30±rC/14L:10D temperature and photoperiod.

-64-

Showing malformation at 30±1'C/14L:10D

• 30±l''C/14L:10D

y=31.857x+2.7938 R = 0.2872 • Linear

(30±1"C/14L:10D)

0.02 0.04 0.06 0.08 0.1

Concentration %

Graph: 21. Fitted linear regression line for malformation of treated 4*^ instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 30±1 "0/141:100 temperature and photoperiod

250

Showing fecundity of adult female which emerged from treated IV** instar nymph at

30±rC/14L:10D y -1074.3X t 200.3

o Z >% •5 c O 4)

200

150 -

100

50

0 H

c

>-., . ,^^^ R' = 0.842

1 1 ! '•

) 0.02 0.04 0.06 0.08

Concentration %

- ^

0.1

• 30±1'"C/14L:10D

Linear (30±1'C/14L:10D)

Graph: 22. Fitted linear regression line for fecundity of adult female, which emerged from treated 4*" instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 30±1°C/14L;10D temperature and photoperiod.

-65-

Showing fertility of female emerged from treated IV i lnstar nymph at 30±1°C/14L:10D

250

_ 200 V=-1219.5xf 184.19 R = 0.8785

• 30±1'"C/14L:10D

Linear (30+l"C/14L:10D)

0.02 0.04 0.06 0.08

Concentration %

0.1

Graph: 23. Fitted linear regression line for fertility of adult female, which emerged from treated 4** instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 30±1°C/14L:10D temperature and photoperlod.

Showing longevity of treated IV*" instar at 30±rC/14L:10D

m

I c o

V= 21.429x4 5.1786 R = 0.989

• 30±1''C/14L:10D

• Linear {30±1°C/14L:10D)

0.02 0.04 0.06 0.08 0.1

Concentration %

Graph: 24. Fitted linear regression line for longevity of treated 4" instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 30±rC/14L:10D temperature and photoperiod

-66-

Showing longevity of V*'' instar emerged from treated IV*" instar at 30±1°C/14L:10D

• 30±1"C/14L:10D y^ 26.786x4 6.7024

R'= 0.9941 2 J Linear

(30±1'-C/14L:10D)

0.02 0.04 0.06 0.08

Concentration %

0.1

Graph: 25. Fitted linear regression line for longevity of 5*' instar which emerged from treated 4* instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 30±1°C/14L:10D temperature and photoperiod

Showing longevity of adult male emerged from trated IV** instar nymphs at 30±1°C/14L:10D

a •D

> O) c o

30 1

25 J

20

15 i

10 ;

0 4

• 30+rC/14L:10D

y^41.071x+20.488 R = 0.99 • Linear

(30±1''C/14L:10D)

0.02 0.04 0.06 0.08 0.1

Concentration %

Graph: 26. Fitted linear regression line for longevity of adult male which emerged from treated 4* " instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 30±rC/14L:10D temperature and photoperiod.

-67-

Showing longevity of adult female emerged fron treated IV*' instar nymph at 30±1°C/14L:10D

V=37,5x > 16.75

R ^ - 1

• 30±1X/14L:10D

• Linear (30±1''C/14L:10D)

0 ^

0 0.02 0.04 0.06 0.08 0.1

Concentration %

Graph: 27. Fitted linear regression line for longevity of adult female which emerged from treated 4** instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 30±1°C/14L:10D temperature and photoperiod.

>" in O Z

3 o XL

O Q. 3 >» 15 •c £

.2 c

5

4

3

2

1

0

-1

I

1 i

1

, i

' i !

• • 1 ,

• -

Showing initial mortality upto 24 hours at

0.02

^ ^

0.04

25±rC/12L:12D

•

^ ^ ^ • 25±l''C/12L:12D ^ ^ y-43,314x-0.5557

^ ^ ^ ?} -~ 0.9043 • Linear

(25+rC/12L:12D)

0.06 0.08 0.1

Concentration %

Graph: 28. Fitted linear regression line for mortality upto 24 hours of treated 4** instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 25±1°C/12L:12D temperature and photoperiod.

-68-

Showing total mortality upto adult emergence at 25±rC/12L:12D

• 25±1'C/12L:12D

y^95.714x-0.7324 R = 0.9767

0.02 0.04 0.06 0.08 0.1

Concentration %

Linear (25±1X/12L:12D)

Graph: 29. Fitted linear regression line for mortality upto adult emergence of treated 4** instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 25±1°C/12L:12D temperature and photoperiod.

I I a

• 25±1''C/12L:12D

Linear (25+lX/12L:12D)

Concentration %

Graph: 30. Fitted linear regression line for malformation of treated 4' instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 25±1 "0/121:120 temperature and photoperiod.

-69-

w o z >> *•* c 3 u

250

200

150

100

50

n

1

4 ( i J 1 j

0

Showing fecundity of adult female at 25±1°C/12L:12D

0.02

y=-1245.7x+197.37 R' = 0.9781

0.04 0.06 0.08 0.1

Concentration %

• 25±l'-C/12L:12D

Linear (25±1''C/12L:12D)

Graph: 31. Fitted linear regression line for fecundity of adult female, which emerged from treated 4" instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 25±1°C/12L:12D temperature and photoperiod.

200

- r 150

o Z ^ 100 -•E <v u. 50

0

(

Showing fertility of adult female at 25±rC/12L:12D

--v^,^^ V=-1288.6x+172.93 ^•"^^-^.^^^ R = 0.9723

^ " ~ \ , . ^ ^ • 25±1*C/12L:12D

^ ^ ^ Linear {25+l"C/12L:12D)

1 1 ! • • r 1

) 0.02 0.04 0.06 0.08 0.1

Concentration %

Graph: 32. Fitted linear regression line for fertility of adult female, which emerged from treated 4'* instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 25±1°C/12L:12D temperature and photoperiod.

-70-

n

I at c o

Showing longevity of IV** instar nymphs at 25±rC/12L:12D

y= 20.357x4 6.4405 R = 0.9911

• 25±1'C/12L:12D

Linear (25±1°C/12L:12D)

0.02 0.04 0.06 0.08

Concentration %

0.1

th ;. Graph: 33. Fitted linear regression line for longevity of treated 4 instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 25±1°C/12L:12D temperature and photoperiod.

(0 n •a

l c o

Showing longevity of V** Instar nymphs at 25±rC/12L:12D

10

8

6 j

4 ] I

2 H

0 +-

y=27.857x+6.6905 R = 0.9729

0.02 0.04 0.06 0.08

Concentration %

0.1

• 25+l'C/12L:12D

Linear (25±1"C/12L:12D)

Graph: 34. Fitted linear regression line for longevity of 5^*" instar which emerged from treated 4* ^ instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 25±rC/12L:12D temperature and photoperiod.

-71-

Showing longevity of adult male at 25±rC/12L:12D

o-i ,„o -n All 25±rC/12L:12D y = 37.143x+22.4/6 R = 0.9821

Linear (25±1"C/12L:12D)

0.02 0.04 0.06 0.08 0.1

Concentration %

Graph: 35. Fitted linear regression line for longevity of adult male which emerged from treated 4* instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 25±1°C/12L:12D temperature and photoperiod.

Showing longevity of adult female at 25±rC/12L:12D

• 25±1X/12L:12D

y= 29.286x4 17.286 Lj^ear R = 0.9802 (25±rC/12L:12D)

0.02 0.04 0.06 0.08 0.1

Concentration %

Graph: 36. Fitted linear regression line for longevity of adult female which emerged from treated 4'*" instar nymphs of D. koenigii with various sublethal concentrations of spinosad at 25±rC/12L:12D temperature and photoperiod.

-72-

Plate 1: Malformation In adults emerged from 4* instar treated nymphs following topical application of imidacloprid at 30±rc/14L:10D temperature and photoperiod.

Plate 2: Malformation in adults by treated 4* instar treated nymphsJoll^^g topical application of imidacloprid at 25±rC/12L:12D temperature awtJphotoperi J

. /

2Ww L//1??]

M

Plate 3: Malformation in adults emerged from 4**" instar treated nymphs following topical application of spinosad at 30±1°C/14L:10D temperature and photoperiod.

Plate 4: Malformation in adults emerged from 4'^ instar treated nymphs following topical application of spinosad at 25±1°C/12L:12D temperature and photoperiod.

- 7 4 -

m M

^iieajuuefi

r [f-

DISCUSSION

Application of chemical insecticides is still one of the most

powerful measures against insect pests. Insecticides are often the only

effective remedy for quickly and inexpensively reducing pest

populations to below economic injury levels. The use of insecticides is

generally the preferred strategy for their high efficacy during a. pest

outbreak. However, there are many negative side effects associated

with the frequent spraying of chemical compounds, especially to human

and environment. There have been efforts, during the past few

decades, to develop biorational insecticides and systemic

neonicotinoids affecting the target pest with minimum effect on natural

enemies and the environment. This approach has led to the

development of botanicals (such as citrus oil derivatives, neem-

azadirechtin, garlic oils, hot pepper oils etc), soaps and oils, microbial

insecticides (like Bacillus thuringiensis, Beauveria, Steinernema),

growth regulators, pheromones, non-organic natural products,

fermentation products (like spinosads and avermectins) and

neonicotinoids (like acetamiprid, clothianidin, dinotefuran, imidacloprid,

nitenpyram, thiacloprid, thiamethoxam etc), which are able to efficiently

control agricultural pest species with minimum effects on natural

enemies. Spinosad is a mixture of spinosyns A and D, which are

fermentation products of the soil actinomycete Saccharopolyspora

(Oiscussion

spinosa (Mertz and Yae 1990, and Thompson et al., 1997). It is a

broad-spectrum natural bio-insecticide offered a new mode of action

which is relatively safe on natural enemies (Temerak 2003). This

compound has two unique modes of action, acting primarily on the

insects nervous system at the nicotinic acetylcholine receptor, and

exhibiting activity at GABA receptor (Salgado 1997, Watson 2001).

Imidacloprid which belongs to the class neonicotinoid group is similar to

the natural insecticide nicotine and acts on the central nervous system.

It blocks a specific neuron pathway that is more abundant in insects

than warm-blooded animals, hence this insecticide is more selectively

toxic to insects than mammals. In animals and humans, imidacloprid is

quickly and almost completely absorbed from gastrointestinal tract, and

eliminated via urine and feces within 48 hrs.

The newer insecticides are not immune to the development of

resistance in the insect pests, and since their registration, several cases

of resistance have been reported (Horowitz et al., 2002, 2004, 2007).

However, the availability of compounds with diverse modes of action

has assisted in managing resistance to insect pests. As most of the new

biorational insecticides and neonicotinoids have shown effectiveness

against insect strains having resistance to conventional insecticides with

no evidence of cross resistance, they can play an important role in IPM

programs.

-76-

(Discussiott

It is a well known fact that the efficacy of insecticides is greatly

influenced by several biotic and abiotic factors such as target species,

temperature, humidity, photoperiod, type of surface that insecticide is

applied to. The effect of temperature on the efficacy can either be

positive or negative. The response relationship between temperature

and efficacy has been found to vary depending on the mode of action of

the insecticide, target species, method of application and quantity of

insecticide ingested or contacted (Johnson, 1990). Stability,

vaporization, penetration, activity and degradation of insecticides are all

partly dependent on physical and biochemical processes that proceed

at characteristic rates at different temperatures. Positive correlations of

temperature with effectiveness for organophosphorus insecticide have

been noted in the laboratory tests by Zwick (1962) using malathion at

21 and 25.5°C against Sitophilus granarius. Also using malathion,

Teotia and Pandey (1967) reported the positive correlation against 7.

casteneum at 27°C and 35°C. O'Donnell (1980) found positive

correlations between temperature and effectiveness of insecticide in T.

confusum with malathion, jodfenpho, pirimiphosmethyl and fenitrothion.

The temperature may synergize activity of an insecticide, or an

insecticide may enhance the impact of high temperature. Ebeling (1990)

demonstrated this by exposing confused flour beetles, T. confusum, and

German cockroaches, Blattella germanica to bo r i c ^ i t f ^ ^ ^^ f t a t 46°C;

-77-

Oiscussian

the mortality due to heat and boric acid together was greater than the

due to either factor alone. Fang et al., 2002a; Subramanyam et al.,

2003; Toews and Subramanyam 2003; Toews et al., 2003; Nayak et al.,

2005; Daglish and Nayak 2006; Getchell 2006; Subramanyam 2006;

Subramanyam et al., 2007 also observed that the efficacy of spinosad is

affected by several biotic or abiotic factors, such as the target species,

the type of commodity, the exposure interval and the type of surface

that spinosad is applied to. These parameters are crucial for the

performance of the currently used grain protectants, since they

determine their efficacy (Noble e^ al., 1982; Johnson 1990; Braness et

al., 1998; Arthur 1996, 1999). For example, pyrethroids are generally

less effective at high temperatures, while the reverse is true for OPs

(Johnson 1990). Musser and Shelton (2005) found that the increase in

temperature decreased the efficacy of spinosad against Ostrinia

nubilalis (Hubner) (Lepidoptera: Crambidae). However, there is still

inadequate information on the effect of temperature and humidity on the

insecticidal effect of spinosad and imidacloprid against hemipterous

insect pests. Combining or integrating temperature with other control

strategies would be beneficial for modern pest management

approaches for controlling insect pest (Arthur and Dowdy 2003). In the

present work, we examined the efficacy of imidacloprid and spinosad

against Dysdercus koenigii, (Hemiptera: Pyrrhororidae) at two different

-78-

(Discussion

temperatures and photoperiods i.e. 30±1°C/14L:10D and

25±1 °C/12L: 12D with constant 60-70% relative humidity.

Imidacloprid

In the present investigation topical application of sublethal

concentrations of imidacloprid viz., 0.008%, 0.006%, 0.004%, 0.002%,

and 0.001% on 4* Instar nymphs of D. koenigii at tvi/o different

temperature and photoperiod (i.e. 30+1 °C/ 14L:10D and

25±1°C/12L:12D) with constant relative humidity (60-70%) caused

immediate mortality within 24 hrs and successive mortality upto adult

emergence. This initial mortality within 24 hour as well as mortality upto

adult emergence increased with increasing sublethal concentrations at

both the temperature and photoperiod. The results are similar to those

reported by Eger e^ a/., (1998) in which the mortality of Franklinella

species showed concentration dependent response. Tows and

Subramanyum (2003) also reported the mortality of T. casteneum that

increased with increasing dosage of insecticide. The mortality within 24

hour as well as total nymphal death upto adult emergence was

comparatively higher at 30±rC/14L: 10D as compared to 26±1°C/12L:

12D temperature and photoperiod with constant (60-70%) relative

humidity. The total nymphal loss within 24 hour after the topical

application of highest selected sublethal concentration of imidacloprid

(i.e. 0.008%) at 30±rc/14L: 10D was 30%, whereas, it was 6.67 %

-79-

(Discussion

less when the treatment was made at 25±rC/12L: 12D. However, the

topical application of the lowest selected sublethal concentration (i.e.

0.001%) resulted in 11.1% and 7.76% nymphal death at 30±1°C/

14L:10D and 25±1°C/12L:12D temperature/photoperiod respectively as

compared to control. The total nymphal loss upto adult emergences

following the topical application of 0.008% imidacloprid at 30±1°C/ 14L:

10D and 25±1°C/ 12L: 12D temperature and photoperiod was 46.66 and

40.00% respectively. Agarwal and Tilak (2006) studied the efficacy of

imidacloprid gel bait (2.15%) upto 8 weeks against German cockroach

and reported that there was 96.8 to 98.8% reduction in the population

as compared to control. Pruthi and Jerath (1960) also determined the

effectiveness of endrin against Dysdercus cingulatus and Chrotogonous

trachypterus adults at different temperature and found that the toxicity is

accelerated with rise in temperature. Uddin and Ara (2006) studied the

toxicity of six insecticide on Trilobium casteneum (Herbst) at the

temperatures of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32°C and

observed that there was a marked decrease in the effectiveness of all

insecticide tested with a lowering of temperature. The order of toxicity of

insecticide was chloropyrifos > lamda-cyhalothrin > Malathion >

cypermethrin > carbosulfan > dicofol. Ming and Qiang (2002) studied

the selective toxicity of six different insecticides against Myzus persicae

and receded the order of toxicity as imidacloprid + SV1 > imidacloprid >

-80-

(Discussion

endosulfan > methomyl > fanvalerate > dimethoate. Ateyyat et. al.,

(2009) compared the toxicity of aqueous extracts of nine plants with

imidacloprid against sweet potato whitefly, Bemissia tabaci and found to

killed 71% of early stage nymphs, which was not significantly different

from mortality caused by imidacloprid. Khowaja et. al., (1995) reported

that the topical application of 10, 08, and 06 ppm of temik (aldicar) to

the 4'* instar nymphs of Dysdercus cingulatus caused 50, 41, and 27%

more nymphal loss than that of control 2% respectively. The initial

mortality following the topical application of higher sublethal

concentrations (i.e. 80 and 60 ppm) of cythion on 4^^ and 5" instar

nymphs of D. Cingulatus was very high within 24 hrs, however, the

subsequent mortalities up to adult emergence were insignificant. The

total nymphal loss and survival duration of the treated insects were

directly proportional and increased linearly with the increase in

concentration, whereas, the fecundity & fertility of the adults were

inversely proportional and decreased linearly with increase in

concentrations (Khowaja et. al., 2001 and Khowaja and Qamar, 2005).

The malformation, moult inhibition and emergence of abnormal/

sub-abnormal individual increased with increasing sublethal

concentration of imidacloprid at these two temperatures and

photoperiods (Plate-1 & 2). The percentage of malformation at the

highest selected sublethal concentration, i.e. 0.008%, was 16.67 and

-81-

Oiscussion

14.43% respectively, whereas, at lowest concentration (i.e. 0.001%) the

percent malformation was 8.86 and 2.23% respectively. Tiwari et. al.,

(2006) noted that the topical application of neem based insecticides on

Dysdercus koenigii caused ecdysial stasis and development of

adultoids and imagoes with varied degree of deformalities. Topical

application of azadiractin through contact method evoked various

specific and non-specific effects during the course of development in

various stages of red cotton bugs (Koul, 1984). RD-9 Replin at higher

concentration prolonged the nymphal period and affected the

emergence of adults of Dysdercus koenigii (Gupta et. al., 1997).

Khowaja et. al., (1995) reported that the topical application of temik

(aldicarb) to the 4* and S**" instar nymphs of D. Cingulatus at 10, 08, and

06 ppm dose produced 7.1, 6.3, 4.8% and 9.6, 8.2, 7.0% adults with

malformed wings respectively. In contrary to the earlier reports some

insecticide, such as imidacloprid and fanvalerate, increased the

percentage of alate offspring of aphids. Field observations of the cotton

aphid, Aphis gossypii, by Kring and Conway (2004) also indicated an

increase in the percentage of winged aphids in cotton fields after an

application of imidacloprid. Many factors such as crowding (Kawada

1989; Matsumura 1996), host plant quality (Lees 1966; Lu and Chen

1993), photoperiod (Joknson 1966; Uddin and Baqui 1993),

temperature (Liu and Wu 1994; Joknson 1966), hormones (Zera and

-82-

discussion

Tobe 1990; Fairbairn 1994) and seasonal changes (Muraji et. al., 1989)

could affect aphid wing development.

The fecundity and fertility of D. koenigii following the topical

application of different sublethal concentrations of imidacloprid at

30±1°C/14L: 10D and 25±1°C/12L: 12D temperature/photoperiod with

constant relative humidity were also adversely affected. The reduction

in egg production and egg hatching of the affected females emerged

from the 4* instar treated nymphs were concentration based and

increased with increasing sublethal concentration. On comparing the fall

in egg production, it was more pronounced at 25±1°C/12L: 12D as

compared to 30±1°C/14L: 10D. Following the topical application of the

highest selected sublethal concentration (i.e. 0.008%) at 30±1°C/14L:

10D, the fall in average egg production was 63.28%, whereas, this loss

was enhanced to 5.27% when the treatment was made at 25±1°C/12L:

12D. At lowest selected sublethal concentration, the reduction in

fecundity was 20.72 and 23.97% respectively as compared to control.

Reduced fecundity of D. koenigii affected with sublethal concentrations

of imidacloprid is consistent with previous studies investigating other

hemipterous insects. The fecundities of two aphid species {Myzus

persicae and /W. nicotiana Blackman), two green leafhopper species

{Neptioteitix virescens, Distant and N. cincticeps Uhler) and a brown

planthopper species {Nilaparvata lugens Stal) were reduced by more

-83-

(Discussum

than 50 % after feeding on plants/leaves treated with sublethal

concentrations of imidacloprid (Divine et. al., 1996; Widiarta et. al.

2001; Bao et. al., 2009). Similarly, Lashkari e^ a/., 2007 reported that

the oviposition rate, longevity and growth of cabbage aphid, Brevicoryne

brassicae L., were negatively affected by sublethal concentration of

imidacloprid. Nishi and Takahashi, (2002) observed that the average

numbers of egg laid per ovipositing female of Amphibolous venator

increased with increasing temperature (i.e. 25°C, 27.5°C, 30°C 32.5°C

and 35°C/ 60-70%RH and 16L-8D of photo regime), however

differences were not significant. RH 5849, an analogue of halofenozide,

was shown to inhibit ovariole development in Lepidoptera and some

Coleoptera (Wing e . al., 1988). Monthean and Potter (1992) found that

feeding on foliage treated with RH 5849 (1,2-dibenzoyl-1-

tertbutylhydrazine) reduce the fecundity of Japanese beetles by 66-

74%, but did not reduced the percentage of hatch. Aller and Ramsay

(1988) also observed that ingestion of RH 5849 by newly emerged

adults of several phytophagous beetle species prevented oviposition

due to the absence of developing oocytes, whereas, the ingestion of RH

5849 by older female caused cessation of oviposition due to the

resorption of the oocytes. Destruction of egg chamber after treatment

with tepa, metapa, apholate, hempa, methlmethane sulphonate and

hydroxy urea has been reported by Morgan and La Brecque, (1964);

-84-

Oisotssian

Morgan, (1967); Kissam et. al., (1967). The effects on division of

oogonia in the germahum, the inhibition of growth of oocytes and

trophocytes, degeneration of follicular epithelium and the resorption of

contents of egg chamber has been reported by Rezabova and Landa,

(1967); Landa and Matol, (1971); Khowaja et. al., (2001) and Khowaja

and Qamar, (2005). The application of 06, 04, and 02 pg of 22, 25-

dideoxyecdysone to the 4** instar nymphs of Dysdercus cingulatus

reduced the egg production of the emerged females by 30.9, 18.5, and

11.0% respectively as compared to control, however, the application of

the corresponding doses of 2,22,25 trideoxy-5phydroxyecdysone

decrease the fecundity by 27.6, 21.4, and 15.4% respectively as

compared to control (Khowaja et. al., 1996). The fertility of the females

emerged from the IVth instar treated nymphs following the treatment of

0.008% imidacloprid at 30±1°C/14L: 10D and 25±1°C/12L: 12D reduced

by 75.11 and 78.34% respectively, however, at the lowest concentration

the reduction was 26.42 and 28.28% respectively. It has been noted

that the reduction in fertility was more at 25±1°C/12L: 12D than

30±1°C/14L: 10D temperature and photoperiod. Boina et. al., 2009 also

observed that the fecundity and fertility of the Asian citrus psyllid,

Diaphorina citri, were reduced significantly following the long term

exposure of imidacloprid through systemic method. The topical

application of various sublethal concentrations of monocrotophos to 4 *"

-85-

OiscMSsion

and 5* instar nymphs of D. cingulatus resulted in linear fall in egg

production and egg hatching and showed a negative correlation with the

increasing concentrations (Khowaja et. al., 1994 and 2002). Likewise,

the average fertility of the eggs laid by the affected females of D.

Cingulatus, after the topical application of higher selected doses (06,

04, 02 pg of 22, 25-dideoxyecdysone) on 4'^ instar nymphs, decreased

by 35.3, 30.1, and 26.0% respectively as compared to control, however,

with corresponding dose of 2,22,25trideoxy-53hydroxyecdysone, the

hatchability of the eggs was dropped by 29.2, 20.3, and 13.8%

respectively as compared to control (Khowaja et. al., 1996). Inhibition in

fecundity and fertility after the application of natural ecdysones or their

analogues have also been reported by Jalaja et. at., (1976), Ahmad &

Khan (1985), Ahmad (1983), and Khan et. al., (1990). Wright et. al.,

(1970) observed that p-ecdysone prevented lipid synthesis which is

necessary for vitellogenesis. On the contrary, Zeng and Wang (2010)

reported that the exposure of sublethal dose of imidacloprid to

Tetranychus cinnabarinus led to a significant increase in the hatch rate

of eggs. James and Price (2002) also reported that the twospotted

spider mites, Tetranychus urticae, either directly exposed to spray

formulations or fed on discs cut from a systemically treated bean plant

produced 10-26% more eggs during the 1'* 12day of adult life and 19-

23% more during adult hood compared with a water only treatment.

-86-

"-—««»».,, r:*"'-"*--*«

0008%) , , "^""centratjon im/rt , ^°' ^ ' 30±l.c/i4L- 10D ° " ""'^3<:'0Prid (i.e.

tempera,..e/pho,ope.oc< on 4%n., " " ' ' * ' ° ^ ' ' ^ ^ - ^20

J^'t/b/oancf 3 84% r^. *=" me surv/Va/

^ ' ^ - - n V ' ^ P . s , emerge. . 0 . . " " ' ^ ^ ^ ^ ' ^ ' — • T.e

'^•^2 and 46. ^5% „,,, 3, „ '^^ated nyn^p^s,

^ ° " ^ ^ " " " ° " « - - O a „ . . J •"''^^''^---=°" ' - ' - ^"e ,.e span of ,,e s . ' " ' " " " ' > ' ^^ - -Pared ,0

'"'-^^---.ooa.Ir^^"^^"--"--v - - ^ » / . . . p . . . , , . ^ ^ : : - - - a 3 , . 0 . 0 0 , . . . . . , :

P^ ed to contro/. L/kew/se fh ,

-87-

° ^ ^ ^ - enhance. . , , , 33 ^^^ ^ ^ " " ^ °^ '^^^^^^^^^

— a « o n (,e. o.oo. / , , , J " " ' ' " ' " ^ ' ^ ' - ' ' - a s , a, ,owes, /oy 11 Was onlv 1 AO

^ - ' - ^ results are in agree.e , ' " ' ' " " ' '^^P-'-ely

'°P'=al appncation of highest , , ' ' ' " ' ' " ^ ' '"^ •"ynest selected QI.M *..

'"°"ocro.ophos on 4' instar <=°"oen.ration of mstar nymphs resuiten • .

"V -PM ...a«on, Whereas ,he , ''°''° ' "=^^ - ^^ " «'eas, the longevity of V" ; .

- - pa red focomroi. ^ ' "^"^"^^ ' - ^ 50.9% , , , , ^^

^eng and Wang ^2010^ r.

="«^"V P-onge. an. increasef ^ ^ " ^ ~ ° " -

' ^ - " ' - ' - - - - o f s i ; : : : ^ " ' ' ^ - - - -- - ' ^ e n , e a n , e n g . o f g e n J " " ^ ^ " ^ - ^ - - • ( - 0 ,

° ' ""^'elle .ylostells .as „ , , " " " ' ' ° " " ' ' ' ^ ^ " - - " ^ '™e " 35 prolonged to ?«on

- P - V e M . e . , h e , a h o r a , o ^ _ ^ ' - ^ ^ ^ - ^^-^^ ^ays

- 'oe . 0 0 . , a,so reporfe. . a f l ' " " " " ^ ^ ^ ^

- • • - " . c . . . . . _ ^ ^ ^^^^^^^^^^^^

° - - ^^e .eve,op.en,a, . .af ion f l " ^ ' ^ " '^^ ^ - - d ----o.aofapp„oa::: ^^^^^^ a/., 2003). ^^°'°"9-^ significanfiy (Wang e,

-88-

th

Oiscussum

Spinosad

The topical application of different sublethal concentration of

spinosad viz., 0.1%, 0.08%, 0.06%, 0.04% and 0.02% at two different

temperature/photoperiod and constant relative humidity (60-70%) on 4

instar nymphs of Dysdercus koenigii causes instant mortality as well as

mortality upto adult emergence. The initial mortalities within 24 hour and

upto adult emergence increased with increasing sublethal

concentrations. The initial mortality within 24 hour and the mortality

upto adult emergence was comparatively higher at 30±1°C/14L: 10D as

compared to 25±1°C/12L: 12D temperature and photoperiod. The

topical application of the highest selected sublethal concentration i.e.

0.1% of spinosad at 30±1°C/14L:10D temperature caused 27.76% initial

nymphal mortality within 24 hour, however, at 25±1°C/12L:12D the initial

nymphal loss within 24 hrs was 13.33% less as compared to higher

temperature/ photoperiod. The topical application of the lowest selected

sublethal concentration (i.e. 0.02%) of spinosad at 30±1°C/14L: 10D

produced 2.2% initial nymphal loss, whereas, at 25±1°C/12L. 12D

temperature/ photoperiod it was only 1.1%. However, the total nymphal

death uptp adult emergence following the topical application of 0.1% of

spinosad at 30±1°C/14L: 10D and 25±1°C/12L: 12D

temperature/photoperiod goes to 56.67 and 31.10% respectively as

compared to control, whereas, topical application of the lowest selected

-89-

(Discussion

sublethal concentration (0.02%) at the same condition produced only

5.53 and -2.20% loss respectively. Athanassiou et. al,. (2008) studied

the influence of temperature and humidity on the efficacy of spinosad

against four stored-grain beetle species viz., lesser grain borer,

Rhyzopertha dominica (F.), rice weevil, Sitophilus oryzae (L.), confused

flour beetle, Tribolium confusum Jacquelin du Val and Prostephanus

truncates (Horn) and concluded that there was a significant impact of

temperature and relative humidity on spinosad efficacy against all

species tested. The influence of these two parameters is complex since

it varied according to the dose rate and the exposure interval. Kowalska

(2008) studied the activity of three different concentrations of spinosad

viz., 0.2, 0.1 and 0.05% against the larvae and adults of colerado potato

beetle Leptinotarsa decemlineata at three different temperature viz., 15,

20 and 25°C in laboratory and concluded that temperature play an

important role on the efficacy of spinosad and all the concentrations

tested caused mortality, which increased as concentration of spinosad

increased. Fang and Subramanyam (2003) found that 0.1 ppm of

spinosad on wheat caused 100% mortality of R. dominica adults and

notably suppressed progeny production. Nayak et. al., 2005 also tested

several R. dominica strains with various susceptibility levels to some of

the most commonly used grain protectant and found that 0.1 ppm of

spinosad provided 93-99% adult mortality after 7 days of exposure,

-90-

(Discussum

regardless of the resistance strain to other pesticide of the beetle

tested. Irani and Asghar, 2007 used a commercial formulation of

spinosad, Tracer® against 7-14 days old adults of rice weevil Sitophilus

oiyzae and red flour beetle Tribolium castaneum at three different

temperature (i.e. 24, 28 and 32°C and 65±5% RH) and observed that

there was a dose-dependent response and mortality increased, with

increasing the dosages in all temperatures in both species. Eger et al.,

(1998) also reported the same results in which mortality of Frankliniella

sp. showed a concentration dependent response. Likewise, Toews and

Subramanyam (2003) also recorded the same trend and reported that

the mortality of T. Castaneum increased with increasing dosage of

insecticide. A biochemical reason for this similarity is imposing of the

same enzymes with spinosad in these experiments.

The exposure of spinosad to the 4* instar nymphs of O. Koenigii

resulted in the emergence of malformed adults (Plate-3 & 4). This

percentage of malformation increased with increasing sublethal

concentration at both 30±1°C/14L: 10D and 25±1°C/12L: 12D

temperature/photoperiod with constant relative humidity. The

percentage of malformation at the highest selected sublethal

concentration was 17.76 and 21.1% with respect to 30±1°C/14L: 10D

and 25±1°C/12L: 12D temperature/photoperiod, whereas, at the lowest

selected dose it was only 20% and 15.53% respectively. El-Barkey et.

-91-

Q)iscusswn

al., (2009) studied the biological effects of Radiant and Hexaflumuron

against the eggs of Pectinophora gossypiella (Saunders) and reported

that both compound caused malformation in larvae, pupae and adults

emerged from treated eggs. Sublethal effects of insecticide on the

malformation and reproductive capacity reduction observed in the

survivors could have a negative impact on insect population dynamics

(Trisyono and Chippendale, 1998; Lopez and Latheef, 1999; Gobbi et.

al., 2000 Knight, 2000; Pineda et. al., 2004).

The average egg production as well as average egg hatching of

D. Koenigii were also adversely affected following the topical application

of sublethal concentrations of spinosad at both 30±1°C/14L:10D and

25±1°C/12L:12D temperature/photoperiod. The loss in fecundity and

fertility was also concentration based and increased with increasing

concentration. The reduction in average egg production of the females

following the topical application of the highest selected sublethal

concentration at two above said temperatures/photoperiods was 54.24

and 61.68%, whereas, at the lowest sublethal concentration the loss in

fecundity was 28.77 and 19.75% respectively. Similarly, a significant

reduction of fecundity was observed when Ceratitis capitata (Diptera;

tephritidae) adults ingested O.lmg/liter spinosad (Adan et. al., 1996).

Temerak, 2007 also found that Radiant at 5.76 gram active/ HA showed

100% mortality of entire hatched egg masses of Spodoptera littorilis

-92-

Q>iscussitm

after the spray in the field. The fertility of the female which emerged

from the 4 ^ instar nymphs treated with 0.1% of spinosad at both

30±1°C/14L:10D and 25±1°C/12L:12D temperature/photoperiod was

reduced by 65.47 and 72.12%, whereas, at lowest concentration, the

drop in fertility was 32.14 and 23.81% respectively as compared to

control. Similarly Yin et. al., (2008) recorded that the adult longevity,

population growth and fecundity of Plutella xylostella was strongly

reduced when the larvae were treated with spinosad. Amer (2004)

recorded that spinosad caused reduction in adult longevity, fecundity

and fertility of P. gossypiella. Also, Liu and Trumble (2005) and

Zaiiznaik and Nugegod (2006) reported that spinosad high affected on

fertility and fecundity of Bactehcerca cockerelli. Egg fertility was

reduced in Helicoverpa zea (Boddie) when females were fed with

concentrations <1 mg (Al)/liter and then paired with untreated males

(Lopez and Latheef 1999).

Besides the acute toxicity produced by the present chemicals, the

biology and physiology of the survived insects were also adversely

affected by their sublethal dosages. Some earlier reports suggest that

the treatment with chemicals may enhance the survival duration of the

affected insects (Knutson, 1955; Katiyar & Lemonde, 1972; Abo-Elghar

et. al., 1972; Parker et. al., 1976; Khowaja et. al., 1991, '92; '93; '94;

2001, '02, '05), whereas, other workers have the opinion that treatment

-93-

Qiscussion

with chemicals reduced the longevity of the survived insects (Haynes,

1988; Mackenzie & Winston, 1989; Abd-Elghafar and Appel, 1992). The

present observations on D. koenigii also prove that the topical

application of sublethal concentrations of spinosad at both 30±1°C714L:

10D and 25±1°C/12L: 12D temperature/photoperiod enhance the

longevity of all the stages of survived treated insects. The topical

application of the highest selected sublethal concentration i.e. 0.1%

spinosad on 4* instar nymphs at both 30±1°C/14L: 10D and

25±1°C/12L: 12D temperature/photoperiod showed average

enhancement of nymphal duration by 38.09 and 30.76% respectively as

compared to their control, while, at the lowest concentration (i.e. 0.02%)

the prolongation of nymphal duration was 4.76 and 3.84% respectively.

El-Barkey et. al., (2009) revealed that the exposure of Pectinophora

gossypiella to radiant and hexaflumur significantly prolonged the

duration of the larval stage than that of untreated check. The longevity

of the 5* instar nymphs which emerged from the 4* instar treated

survived nymphs following the application of 0.1% spinosad was also

enhanced by 40.74 and 42.30% respectively as compared to control,

while at the lowest concentration, the survival duration was increased

by 7.40 and 11.53% respectively as compared with control. Yin et. al.,

(2008) reported that prolonged in the immature stage and the survival

rate of Plutella xylostella was lower in the LC25 and LC50 for spinosad.

-94-

"•'""'—* ..«„«:!:, 7""-»«~ wnicn emerged frnm •»,

'"oreasedby 4.47 and , 420/ re ' " ' " ° ' '^"^^'^^ *-=

•^^' ^P-sad a, s.5,e,.a, o o n c e n r r ' ' ' " ' " ' ' ' ^ ' ° ° ' ' ^ ^ ° - ' '

°^ - • - ,a.ae a . . . . ^ ^ " " ^ " ^ ^ - ' - ^ « -

' - - ' - ° - . . a . a M . e , o p . e n , o " " ' ° " " " ^ • ^ - -

- ^ - - - . a s c o n e e . a . . . p . ^ ^ ^ " ^ ^ " ^ " ' ^ ^ ^ • ^ ^

<=y» ion (malathion) was 33 90/. " ' " " " ' " * ° ""^-^^

•• 1991). Acord,ng ,0 Conney et a/ nsee^ •

'"^*«ed hy*oxy,atlon process of so.e , ' ' " ----0---.one, j : ; : r ' ° '--"-- - K a « y a r a n d . e ™ n d e ( , 9 7 2 , o " ' * " ^ " ' ^ " ' " " -

(1972) oPse^ed .onoc.o.ophos, ga.dona

-95-

Q>iscussum

and aldicarb to act as antiacetylchoiinestrase agents resulting in greater

prolongation of the larval period of T. confusum. Perhaps, the increase

in longevity of D. koenigii may be attributed to the

antiacetylchoiinestrase activity when spinosad was applied on the

nymphs.

The present finding of these biological parameters can be helpful

in manipulating some of the chemical methods to keep the pest

population at the minimum. The recommended doses should be

regarded temperature/photoperiod specific and some consideration

must be given to decline in effectiveness with lowehng of temperature. It

may well not prove feasible to compensate for this decline by increasing

the dose applied since this could put spray operator at greater risk, lead

to residue hazards or prove uneconomic. Certainly the less effective

insecticides should not be used at all where temperatures are very low.

In some situation, it may be possible to raise the temperature of the

treated area sufficiently to activate the insects so that they may pick up

a lethal dose.

-96-

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