CONTROL OF Dermestes maculatus (COLEOPTERA:

DERMESTIDAE) IN AN INTERIOR STORAGE SITUATION WITH

NEEM, Azadirachta indica.

BY

Cory M. Keeler

Department of Natural Resource Sciences

McGiil University, Montreai, Quebec

March, 1999

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulf llment

of the requirements for the degree of Master of Science

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Controi of an interior storage beetle pest with Neem. Control of Dermtcs macuiatu (Coleoptera: Dermestidae) in an interior storage

situation with neem, Azuduachto indica.

M. Sc. Cory M. Keeler Department of Natural Resource Sciences

Neem, Azadirachtu indica, products were tested for toxic, growth regulating, primasr anti feedant, and secondaxy anti feedant effects on Dermestes maculatus, under conditions approximating those found in storage facilities. Toxic and growth regulating effects were uivestigated using topid application of minera1 oil, neem oil, purifted azadhchtin/methanol solution. and 10% neem seed kernet extract/methanol solution. Al1 neem treatments exhibited higher mortality than the mineral oil treatment 5, 10, and 14 days afier the application of the treatments; larvae treated with neem products ofien failed to pupate and never emerged as adults. Pnmary antifeedant effects of azadirachtin (1 -5 g/L and 5 g/L) were investigated with an original no-choice feeding bioassay. Significant primary antifeedant e ffects were observed which were persistent for up to 13 weeks for adults and 1 7 weeks for larvae. Significant secondary antifeedant effects were also demonstrated afier topical application of azadirachtin (. 125 g/L, .25 g/L and -5g/L) to the larvae. Contrôle de Dermestes maculutus (Colcoptera: Dermestidae) avec du neem,

Azadirachta india, dans une situation d'emmagasinage intérieur.

M. Sc. Cory M. Keeler Department des Sciences Resources NaturaI.

Les produits du neem, Azudirachta indica, ont été testés pour la toxicité, le contrôle

de la croissance et les effects anti appetant primaires et secondaires sur Dermesres maculatus,

sous conditions similaires à celles retrouvées en situation d'emmagasinage intérieur. La

toxicité et les effets sur la croissance ont été testés par l'application topique d'huile minerale,

d'huile de neem. d'une solution purifie d'azadirachtine/méthan01, et d' une solution d'extrait

de grain* de neem (1 O%)/methanol. Tout les traitements ont démontré un plus haut taux de

mortalité que le traitement d'huile minérale, 5, 10, et 14 jours après l'application des traitements. Les larves traitées avec les produits de neem ont souvent echoué la pupaison, et aucune n'a réussi à émerger comme adulte. Les effets anti-appétant primaires de iàzadirachtine (1.5 g/L and 5 gL)ont été testés à l'aide d'un essai sans choix de noumture.

Des effets anti-appétant primaires significatifs ont été observés et ont persistés jusquoà 13 semaines pour les adultes et 17 semaines pour les larves. Des effets anti-appétant secondaires ont aussi été demontrés chez les larves après l'application topique d'azadirachtine (. 125 g/L,

.25 g/L and .5 g/L). Acknowledgments

There are large number of people without whose help and input 1 would not have been able to complete this thesis. 1 would like to thank my CO-supervisorsDr. David Lewis and Dr. Roger Stewart both of whom were quick to help and never seemed to muid my unannounced visits or the large numbers of favors 1 asked of them. 1 would like to thank Dr.

Raymond Manuel who spent much of his fiee time giving me advice and looking over my work, not to mention listening patientiy while 1 argued with him. For help with my statistics,

1 would like to thank Dr. Pierre Dutillieul and for advice on my experiments and for supplying me with samples of azadirachtin free of charge I would like to thank Dr. Murray

Isrnan from the University of British Colombia. Most of dl 1 would like to thank my supervisor. Dr. C. C. Hsiung. Dr. Hsiung helped me in every way possible with my research and with anything else 1 needed. provided me with hanciai suppon and the opportunity to work and gain experience at the Lyman Museum... unfortunately there is not room to list everyhng here. Without his help, patience, and friendship this work would never have been completed.

Thank you to everyone at the Lyman Museum for making every day interesting and for helping to create an aunosphere that was enjoyable to work in. Thank you also to al1 the people who have been such great fiends throughout rny time here at Mac. Unfortunately. I have been here so long that there is no room to Iist everyone.

Without my farnily. 1 would never have been able to finish this thesis. My parents provided me not only with suppon and advice, but they have set an example that 1 hope to

a.- 111 be able to follow throughout my life. They were also very patient and put up with many crazed phone calls and anxiety attacks. Thanks to Lei& Lori and everyone else for their support and inspiration. I would W

Abstract ...... i .. Résumé ...... ii ... Acknowledgments ...... iii

Tableofcontents ...... v

.* . List of Figures ...... viii

List of Tables ...... xii .... List of Appendices ...... xi111

1.Introduction ...... 1

2 . Literature Review ...... -4

2.1 The Neem Tree ...... 4

2.2 Chemistry of Neem Products ...... -5

2.3 Neern Products Available for the Control of Insect Pests ...... 7

2.4 Anti-Insect Effects of Neem ...... 9

2.4.1 Anti feedant Effects of Neem Products ...... 9

2.4.2 Growth Regulating and Toxic Effects ...... 12

2.5 Effects of kemProducts on Beetle Pests ...... 14

2.6 Effects of Neem Products on Beetle Pests of Stored Products ...... 15

2.7 Dermestid Beetles ...... 16

3 . Materials and Methods ...... 18

3.1 Dermesres maculatus Cultures ...... 18 3.1.1 Selection of Test Insects ...... 20

3 -2 Preparation of Neem Extracts and Neem Products ...... -20

3 -3 Topical Application Experiments ...... -21

3.3.1 Relative Efficacy of Mineral Oil, and

Neem Products ...... -21

3 -3.2Effects of Mineral Oil and Neem Products on

Molting. Pupation and Adult Emergence ...... -22

3.4 Antifeedant Experiments ...... -23

3.4.1 Primary htifèedant Effects ...... -23

3.4.2 Secondary Antifeedant Effects ...... 25

4.Results ...... 26

4.1 Topical Application Experiments ...... 26

4.1.1 Relative Efficacy of Mineral Oil and

Neem Products ...... 26

4.1 -2 Effects of Mineral Oil and Neem Products on

Molting, Pupation and Adult Emergence ...... -27

4.2 Antifeedant Experiments ...... -29

4.2.1 Primary Antifeedant Effects of Azadirachtin

onLarvae ...... 29

4.2.2 Primary Antifeedant Effects of hdirachtin

onAdults ...... 30

4.2 -3 Secondary Anti feedant Effects of Azadirachtin onLarvae ...... 30

5.Discussion ...... 51

5.1 Topicai Application Experiments ...... -51

5.2 Primary Antifecdant Effects of Azadirachtin ...... -58

5.3 Secondary Antifeedant Effects of Azadirachtin ...... 66

6. Conclusion ...... 69

7.References ...... 71

vii List of Figures

re:

Figure 1. Effect of treatments on cumulative mortality in larvae of

D. rnaculatlls 5, 10, and 14 days afier application ...... -32

A. Cumulative mortality 5 days afier application

oftreatments ...... 33

B. Cumulative mortality 1 0 days afler application

oftreatrnents ...... 33

C. Cumulative rnortality 14 days afier application

oftreatments ...... 34

Figure 2. Effects of topical application of minera1 oil,

neem oil, azadirachtin/methanol solution, and 10% neem

seed kemel extract on molting. pupation, maturation to

the adult stage. and final mortalit). of D. maculatus larvae ...... 36

viii B. Mean number of larvae to reach the

pupalstage ...... 38

C. Mean number of Iarvae to become

adults ...... 38

Figure 3: Effect of azadirachtin on consumption of fish

foodchipsbylarvae ...... 41

A. Arnount of food consumed by the larvae &er

fish food chips were stored in the climate control

chamber for 1 week ...... -42

B. Arnount of food consurned by the larvae afier

fish food chips were stored in the climate control

chamber for 3 weeks ...... 42

C. Amount of food consurned by the larvae afier

fish food chips were stored in the clirnate control

chamber for 5 weeks ...... 43 D. Amount of food consumed by the iarvae afier

fish food chips were stored in the climate control

chamber for 9 weeks ...... -43

E. Arnount of food consumed by the larvae afier

fish food chips were stored in the climate control

chamber for 13 weeks ...... -44

F. Amount of food consumed by the larvae fier

fish food chips were stored in the climate control

chamber for 17 weeks ...... 44

Figure 4. Effectsof time on consurnption of azadirachtin treated fish

food chips by D.maculatus iarvae ...... -45

A. Fish food chip treated with 1 .S g/L

azadirachtin/methanoI solution ...... 46

B. Fish food chip treated with 5.0 g/L

azadirachtin/rnethanolsolution ...... 46 Figure 5 : Effect of azadirachtin on consumption

of fish food chips by adult D. maculatus ...... -47

A. Amount of food consumed hy the adults

after fish food chips were stored

in the climate control charnber for 1 week...... -48

B. Arnount of food consumed by the larvae

afier fish food chips were stored

in the climate control chamber for 13 weeks ...... 48

Figure 6. Effects on food consumption of increasing

concentrations of azadirachtin topically applied to

the D. macularus larvae ...... -50 List of Tables

Table:

Table 1. Mortaiity of the larvae 5, 10, and 14 days after topical

application of minera1 oil, neem oil, 20 g/L and 1 0 g/L .

azadirachtidmethanol solution and IO% NSK/methanol solution ...... -31

Table 2. Molting, pupation, and adult emergence

in larvae of D. macularus îreated topically with mineral oil,

neem oil, 20 g/L and 5 g/l azadirachtin/methad solution, and

10% neem seed kernel extracthethano1 solution ...... 35

Table 3. Mean (+/- standard error) consumption (mg) by

larvae of D. macuiarus of fish food chips treated with O g/L,

1.5 g/L, and 5 g/L azadirachtin/methanoI solution subsequent

to storage at 25°C and 80% r. h. for 1,3,5,9, 13, and 17 weeks ...... -39

Table 4. Mean (+/- standard error) consurnption (mg) by

adults of D.macularus of fish food chips treated with

O g/L, 1.5 g/L, and 5 e/L subsequent to storage at 25°C and

80%r.h.for 1 and 13weeks ...... 40

xii Table 5. Consumption of cat food (mg)by D. rnacuIafus

larvae after topical application of azadirachtin and methanol ...... 49 List of Appendices Paee:

Appendix 1. Analysis of Cat Food and Fish Food ...... -79

xiv 1. Introduction

Many insects, including many beetle species are senous pests of non-food intenor

stored products. Beetle pests might destmy .non-food stored products such as fabrics.

leathers, wood. and even synthetic and minerai fibers such as rayon and asbestos (Bennett

et al., 1988). They are ais0 known to be pests of naturai history and insect museurn collections, and pet food factories (Hinton, 1945). Currentiy these beetle pests are controlled with a range of synthetic chemicai insecticides. In agriculture there has ken a widespread movement towards ecologically based pest management as a result of the obvious dangers of chemicai pesticides. These methods have ken largely overlooked when considering the control of household and industrial intenor stored product insect pests. The destructiveness of such pests and the current trend away fiom the total reliance on dangerous synthetic chemicals, indicate that there may be a need for ecologically based Pest controls.

Fumigation, residual sprays. dusts and vapor boards (Su and Scheffiahn, 1990) are methods currently used for the control of interior stored product beetle pests as are synthetic chemicals such as paradichiorobenezene, naphthalene, chloropyrifos. dichlorovos and malathion (Bennett et al., 1988; Jubb and Perkins, 1985). While these chemical conuol methods are usuaily effective. there are senous problems associated with chemical pesticides. in fact, synthetic chemical pesticides have in some cases caused more problems that they have solved. Problems associated with the use of chemical pesticides, described by Ascher

(1 993) include: poisoning of mammals. fish. birds and other beneficial organisms: episodes of widespread human poisoning. especially in developing countries; poisonings of the environment (soil, air, and water); resurgence of pest populations; development of resistance to insecticides by pest populations; and the destruction of nanual bio-control agents as a result of non-selectivity. Wesome of these effects are of le= concem in interior storage situations, it is obvious that an alternative to synthetic chemicai pesticides for the control of non-food interior storage beetle pests should be found.

Botanical insecticides are one alternative to synthetic chernical pesticides since these compounds are biodegradable and less persistent in the environrnent. Plants are a rich source of insecticidai compounds and the effectiveness of these compounds has been demonmted against many stored product insects (Xie et ai., 1995). Extracts of the neem tree, Azudirachfa zndica, have yielded botanical insecticides which have been tested extensive1y in agricultural situations. Shce neem products have ken shown to be effective against many insect pests, including many beetle pests, main1y agriculturai and almost exclusively phytophagous, they might also be a safe alternative to synthetic chemicals for the control of keratinophagous interior storage beetle pests such as Dermesies rnacufatlrs(Coleoptera: Dermestidae) if it can be proven that these beetles are sensitive to the effects of neem products.

While generally considered Iess harmfùl to the environment than synthetic chemicals, botanical insecticides are not completely without danger to human health and the environment. Pyrethroids, nicotine. and rotenone are exarnples of well know botanical insecticides. These insecticides are considered toxins and while Iess dangerous and persistent in the environrnent than many synthetic chemicals (DDT), they are poisonous to a variety of organisms other than insects (Ware. 199 1 ). Neem products are generally considered to be safe when compared to synthetic chernical pesticides; they are considered to be non-toxic to mammals, birds and fish (Ascher, 1993) and cause less disturbance to ecosystems than chernical insecticides (Sundaram, 1996). There has been no evidence of the development of resistance to crude neem products to date, although there has been some evidence of the developrnent of resistance to purified azadirachtin (Jilani et al., 1990; Ascher, 1990). In outdoor agricultural situations, neem products have been shown to have low persistence and to degrade rapidly in the presence of moisture, high temperatures and ultraviolet radiation

(Mohapatra et al., 1995). The effects of these factors are reduced in intenor storage situations, therefore neern products might be more effective as a result of increased persistence, than they are in outdoor situations.

There has ken very little. or no research examining the effects of neem products on keratinophagous interior storage beetle pests, such as Dermestes maculatus, or on the persistence of neem products in intenor storage situations. The objectives of this study are to

1 ) examine the potential of Neem products for controlling a keratinophagous intenor

stored product beetie pest.

2) deterrnine if anti feedant. poulh regulating, and toxic effects, or a combination of

these effects. are exhibiied by beetle larvae treated with a variety of neem products.

3) deterrnine if the persistence of neem is increased in an interior storage

environment, and to detemine if neem chernicals are effective over a pe60d long

enough to be usefiil in interior storage situations. 2. Literature Review

2.1 The Neem Tree

The neem tree, Amdirachta Mica, is a member of the mahogany (Meliaceae) family.

It is indigenous to arid regions of southeni and southeastem Asia, but is now widespread in

adjacent counûies including Bangladesh, Pakistan, Sn Lanka, indonesia, Malaysia, Thailand,

and Burma During the last 150 years the tree has been introduced into Afiica and is now

concentrated in a belt stretching across the African continent fiom Somaiia to Mauritania;

and in semiarid areas between the 1 Oh and 14' degree of latitude north of the equator. It is

also propagated on islands in the South Pacific, in Australia, in seved countries in the

Caribbean, adin Centrai and South Arnerica (Ascher, 1993). The tree is a fast growing plant

which may reach a height of 25 rn: it is best grown under tropical conditions and can tolerate an extended dry season. It can tolerate severe droughts and poor shailow soils (Koul et al.

1990; Schmutterer, 1990), but is susceptible to excessive frost (Koul et al., 1990). It is propagated easily by seed and 9 to 12 .month old seedlings transplant well. The fruits, the main source of pesticide products. are produced on drooping panicles, usually one a year. although two fniiting penods have been known in some areas (Ascher, 1993). Seed is nonnally produced afier about five years and afier this time 37 to 50 kilograms of seed per tree are produced every year (Koul et al.. 1990).

The neem tree has an arnazing variety of uses and this is one of the main reasons that it has been planted in so many pans of the world. Products fiom the neem tree have ken used for centuries in traditional medicine and agriculture. Neem products have been used as mouthwashes and toothpastes, and the tme is also known to posses contraceptive properties

which are currenly king investigated (Koul et al., 1990). Neem oil, extracted fiom the

seeds, was used in traditional Indian medicine to treat leprosy, skin diseases, and malaria,

(Baladrin et al., 1988) although the efficacy of these treatments has not been documented.

Leaves fiom the neem tree have been used as feed for cattle and sheep, as organic manure.

and as a soi1 conditioner. Cnide neem extracts, mainly fiom the seeds and leaves, have been

shown 10 exhibit antibactwial, antifùngal, and antiwal properties, as well as a range of anti-

insect properties. in some localities, neem trees have been planted for shade, for reforestation

and for erosion control. The quality of the wood makes it an excellent tree for timber and in

some cases it is used for fûel (Koul et ai., 1990; Schmutterer, 1990; Isman, 1997).

It is obvious that the neem tree is an extremely usefirl tree, but it has gained the most

attention from scientists in recent years as a result of its anti-insect properties.

2.2 Chemistry of Neem Products

Over 300 compounds have been isolated fiom neem seeds, one third of these being

tetranortritepenoids (limnoids) (Kumar et al., 1996). These limnoids include the most active,

azadirachtin-A (Schroeder and Nakanishi, 1987), and eight other highly active isomers,

azadirachtin B-1, moderately active nim bandiol, saiannin, and the less active gedunin.

vi lasinin, azadirone and azadiradione (Sundaram, 1996). Although the other azadirachtin

isomea are found at lower concentrations than azadirachtin-A, they are considered to have the same order of biological activity (Govindachari et al., 1996).

The compound azadirachtin, the most polar and highly oxygenated of these limnoids (Johnson et al., 1996), has received the most attention because of its importance as an

environmentally Wendly insect control agent. Schmutterer (1988) points out that the

structure of azadirachtin closely resembles the structure of ecdysone, an insect hormone

which stimulates epidermal molting. It is generally believed that the bio-activity of neem

preparations is mainiy the resuit of their a.riidirachtin content (including al1 the azadirachtin

isomers) (Govindachari et al ., 1996; Isman, 1997). Salannin has antifeedant and growth

regulating properties (although lesthan azadirachtin), and is present in neem oil at 3-4 times

the concentration of Azadirachtin-A. Nimbin, deacetylnimbin, azadiradione and epoxy-

azadiradione (other limnoids isolated fiom neem products), are also present in considerable

quantities. Therefore, while azadirachtin is an important antifeedant, the effects of the other

limnoids should not be forgotten (Govindachari et al.. 1996). It is believed that al1 the neem triterpenoids are derived fiom the parent tetra cyclic triterpenoid tirucallol (Ascher, 1993;

Mordue and Blackwell, 1993).

Azadirachtin is usually found in neem seeds at levels of 0.2-0.6% (Yakkundi et al..

1995). but the exact content in the seeds and other parts of the tree varies with the locality in which the tree was grown, and the length of time the neem products have been stored.

Yakkundi et al. (1 995) studied the azadirachtin content of seeds stored in the dark, and found that afier one month the azadirachtin content fell by 5%. and by 35% afier four rnonths.

Ascher (1993), indicates that seeds fiom indian neem trees might contain 0.1 ta 0.3% azadirachtin, while neem trees fiom the Afncan continent rnight have an azadirachtin content of 0.5 to 0.6?40and in extreme cases 0.9%. The highest concentrations of azadirachtin are found in the seeds, and at decreasing concentrations in the leaves, bark, roots and stems (Sundaram, 1996). During the course of the development of the neem seed, Johnson et al.

(1 996) indicated that while the proportions of active chemicals remained constant throughout the development of the seed, the quantity of these active chemicals increased during the unripe stages of the early green seed. Since neem seeds might be the major source of compounds which are biologically active against kcts, it is important to know the optimal time to collect the seeds in order to maximite the concentration of the bio-active compounds fiom this study it was concluded that the seeds should be harvened as soon as possible after the seed becomes ripe.

in addition to the limnoids, other natural products such as lipids, carbohydrates, fatty acids, hydrocarbons. and volatile sulphur compounds have also been isolated (Kou1 et al.

1990; Sundaram, 1996).

2.3 Neem Products Available for Control of Insect Pests

A large variety of both crude and refined neem products are available for use in controlling iwect pests. Neem leaves have been combined with stored grain products to repel insects in traditioiial apiculture (Schmutterer. 1990; Ascher, 1993); dry powders of neem seeds and ieaves might atso be used for the same purpose. Cnide extracts. extracted with different solvents from the seeds and leaves. are used as sprays to control insect pests in field conditions, and in many cases. are the bais of commercial formulations (Isman, 1997).

Examples of solvents used inciude water (Karel. 1989; Mohapatra et al., 1995). ethyl alcohol

(Reed et al., 1982; Karel, 1989; Mohapatra et al., 1995), methanol (Meisner et al. 1983;

Kaethner, 1992; Mohapam et al., 1995), petroleum ether and chloroform (Jaglan et al., 1997). Extracts rnight be obtained from virtuaily any part of the neem tree, but are most

comrnody taken fiom the seed and leaves as these are the plant parts with the highest

concentrations of active chemicals (Ascher, 1990). Extracts might also be taken from neem

cake; neem cake is the product rernaining after crushed neem seed has been de-oiled. The oil

extracted fiom the seeds is cornmonly used for insect Pest control, although there are

problems with its large droplet size and oily nature. Various extraction techniques have been

used to remove the biologically active chemicals, which are often highly concentrated in the neem oii compared to other neem products (Prakash and Rao, 1997).

Puri fied extracts of azadirachtin and other active chemicals are available commercially, although they are expensive (as a result of the extraction process, usually by high pressure liquid chromatography) and not easily obtained. Commercial formulations of crude neem extracts and neem oil fractions, in some cases enriched with purified azadirachtin are also available. These neem products might also be combined with extracts fiom other plants for greater efficacy (Prakash and Rao. 1997).

In general. it is easier and less expensive to obtain either neem products such as seeds. neem cake, oil, and leaves or commercially produced crude extracts of these neem products. as there is a large supply of these available, than to obtain the purified active chemicals. Currently. extraction of active chemicals in quantities large enough for commerciaI use is expensive and time consuming (Xie et al., 1995). The structure of the azadirachtin molecule is extremely complicated, and to date it has not been produced synthetically. Other techniques, such as the production of azadirachtin in in-vifro tissue culture (Aerts and Mordue, 1997) have not produced quantities large enough for commercial

8 use. As a result, naturai products and cade extracts will be the most important source of products for insect control.

2.4 Anti-Insect Effects of Neem Products .

Neem has received much attention from entomologists as a result of its anti-insect properties. Previous studies have shown that neem products are effective against over 300 difierent insect species, or 90 percent of those tested (Isman, 1997). The extensive attention given to the ad-insect properties of neem is not surprising, since mixing neem leaves with stored grain products to protect the grain fiom insect pests (Xie et al., 1995); and using neem oit and neem seed kernel extracts to protect crop plants (Govindachari et al.. 1996) are ancient traditional practices in India.

There are two main categories of effects of neem products on insects: an antifeedant effect. and a growth regulatory effect. Other effects of neem products on insects. although less important. and in sorne cases open to debate. include repellency, oviposition repellency. intenupted settling behavior. reduced fecundity and sterility in aduits, and egg sterility

(Ascher. 1993): neem products might produce more than one of these effects on a particuiar species of insect (Isman. 1993). contrïbuting to their overall effectiveness.

2.4.1 Antifeedant Effects of Neem Products

One of the most important and most studied effects of neem products on insects is the antifeedant effect. Antifeedant behavior is ofien confwd with repellency. Schoonhoven

(1 982) states that a product is an antifeedant when it is contacted and interrupts feedinp behavior; he aiso States that the term repellency should not be used in this context as it involves an oriented rnovement away hmthe source of the stimulus, and more ofien is the resuit of olfactory cues fiom a volatile substance. Originally, it was thought that azadirachtin might produce a repellent effect in some species of insects; it has since ken show that the repekncy was caused by voIatile sulphur compounds in crude neem products, or in the case of the oils, by their oily texture (Baladrin et al., 1988).

Neem products have been shown to produce antifeedant behavior in ail feeding stages of insect growth (duits, nyrnphs and larvae) and in a wide range of unrelated species. There are differences in the susceptibility of different insect groups (even at the species level) to neem, and the effects are often dificult to predict, although in general oligophagous insects are considered more susceptible than polyphagous insects (Ascher, 1993).

Neem products are knowri to produce two distinct antifeedant effects: a primary antifeedant effect (gustatory effect) and a secondary antifeedant effect (non-gustatory)

(Schmutterer. 1990). The pnmary antifeedant effect is defmed as gustatory since antifeedancy, and rejection of the food, is usually the result of sensory orgms in the mouthparts contacting active chemicals in or on the food. Secondary antifeedant effects are a reduction in food conswnption observed afier topical application, injection or ingestion of neem's active chemicals into the insect (Ascher 1993; Schmutterer, 1990) and therefore are not a result of such sensitivity of the sensory organs in the insects mouthparts to the active chemicals. The primary antifeedant effect has received more attention than the secondary antifeedant effect, probably because it produces a more complete protection of food crops.

Azadirachtin is largely responsible for the primary antifeedant effect of neem producis although other triterpenoids have kenshown to have an effect (Ismanet al. 1990).

When tested against Spadoptera lâtura (Lepidoptera: Noctuidae) by Govindacharï et al.

(1996),azadirachtin was found to be the most potent antifeedant, and was twice as active as salannin. Nimbin and deacetyinimbin were also found to have significant, although lesser, antifeedant effects. Govindachari et al. (1996), found al1 the triterpenoids tested to be equal ly effective against Oxya jiuscovittatu (Orthoptera: Acndidae).

Electrophysical experiments indicate that azadirachtin molecules interact with specific deterrent chemoreceptors in the insects mouthparts to produce the primary antifeedant effect. Therefore, feeding detemence occurs on contact with the azadirachtin molecule (Mordue and Blackwell, 1993; Isman, 1997). Sirnrnonds et al. (1 999, state that the antifeedant activity of azadirachtin might be a result of its ability to stimulate a neuron in both the lateral and mediai maxillary styloconica sensilla in both lepidopteran larvae and locusts. This neuron has been described as the deterrent neuron by Simmonds et al. (1 995) because increased antifeedancy is found with and increased response from the newon.

The primary antifeedant effect is so strong in many cases that the insect will starve rather than eating. In this way. azadirachtin and other neem chemicals indirectly affect growth and fitness of nymphs and larvae. and fitness, reproduction and survival of adults

(Xie et al. 1995). Other insects have been shown to become desensitized to the antifeedant effects of azadirachtin (Bomford and Isman, 1996), and it is assumed that if an insect is able to overcome the antifeedant effects. consuming the neem products would probably result in toxic and growth regulating efiects (Mordue and Blackwell, 1993). Bomford and Isman

(1 996) were able to show that insects could become desensitized to purified azadirachtin, but not to cmde neem extracts containing the sarne absolute concentration of azadirachtin. This is probably due to the combined action of al1 the lirnnoids with antifeedant effects. These multiple modes of action on a single insect species contribute to the overall effectiveness of neem products.

The secondary antifeedant effect is more cornplex, and reduced feeding might be the result of changes in the insects physiology. It is thought that reduced feeding rnight be the result of a feedback mechanism, regulated by the central nemous system (Isman. 1997). a reduction in gut motility (Schmutterer, 1990), or changes to the neuro-endocrine system. A combination of these effects and a general reduction in fitness of the insect afier application of neem chemicals might also cause the reduction in food consurnption (Mordue and

Blackwell, 1993). Less attention has been given to the saiondary antifeedant effects; regardless, it is still an important effect of the application of neem products to pest insects.

2.4.2 Growth Regulating and Toxic Effects

The growth regulating and toxic effects of neem on insects have been investigated extensively and are still the subject of much debate. Neem products are extremely effective ai producing a large range of growth regulating and toxic effects on a large nurnber of unrelated insect groups. Unlike the anti feedant effects, the growth regulating and toxic effects of neem products are more consistent between these unrelated insect groups (Mordue and Blackwell, 1993).

Delays in the development of the immature stages of insects have been observed afier treatment of feeding and non-feeding stages or instars ( Mordue and Blackwell, 1993; Ascher, 1993). This delay in development rnight be partidy the result of a reduction in food

intake caused by primary and seconâary antifeedant effects (Sieber and Rembold, 1983;

Ascher, 1993). Other observed growth regulating effects include high mortality between

molts, lethal disturbance of ecdysis, incomplete ecdysis, the occurrence of larval-pupal,

prepupal-pupal, nymphal-adult, and pupae-adult intermediate forms, and other general

disturbances of molting, pupation, and adult emergence (Ascher, 1993, Mordue and

BIackwell, 1993; Schmutterer, 1990); these effects usually result in mortality. Mordue and

Blackwell (1993) have documented the development of wingless adults or adults with

crippied or deformed wings, and the death of treated insects soon afier application of neem

products to the larvae. They also described reductions in adult fecundity, although it is not

clear if the adults were treated with neem products as adults or if they developed from neem

treated immature stages.

It is believed that azadirachtin and other active chemicals inhibit ecdysis by

disrupting the ecdysteroid titre or by disrupting neuro-endocrine events; more speci fically,

by blocking the release of prothoracicotropic and allotropie hormones which control the

levels of juvenile hoxmone (JH) and ecdysone (molting hoxmone), respectively (Banken and

Stark. 1997; Ascher. 1993: Mordue and Blackwell, 1993; Schmutterer, 1988). In spite of

strong evidence pointing to this expianation (Bamby and Klocke, 1990; Kou1 et al.. 1986),

Mordue and BiackwelI (1993) argue that al1 the toxic effects of azadirachtin do not result

from changes to the neuro-endocrine system: they suggest that neem has a variety of effects on a range of tissues and organs. which implies a number of different modes of action or a direct toxicity to the cells of insects. Sieber and Rembold (1983) found evidence that azadirachtin might also interfére with the building and reluise sites of barsicon and eclosion hormones in the brain, supporthg the hypothesis that there are multiple sites of action for azadirachtin. in this case, inhibition of ecdysis is caused by a gradua1 and lasting decrease in ecdysteroid titres which prevents eclosion honnone hmking released at the appropriate the. Barnby and Klocke ( 1990), indicate that the prothoracic glands are not as receptive to prothoracicotropic hormone atier an insect is treated with azadirachtin; the prothoracic glands are therefore not stimulated to produce moiting hormone (ecdysone) and the levels of this honnone are reduced in the hemoiymph.

There is no recognized optimal time period for the appiication of neem products to increase the sensitivity of larval or nymphal insects to the growth regulating and toxic effects of neem products; although it is known that if the next molt is due within 48 hours, there is no irnmediate effect, and disturbances in ecdysis usually occur in the next molt (Ascher,

1993).

2.5 Effects of Neem Products on Beetle Pests

Neem products have ken show to be effective against a large nurnber of beetie pest species. and have ken tested extensively as an alternative to synthetic chemicals for the control of many agricu1tural beetle pests.

Larvae of the Colorado potato beetle, Leplinotama decemlineata (Chrysomelidae), showed highly increased mortality when feeding on neem treated foliage or when treated directly with neem products in the laboratory (Kaethner, 1992); neem products were highl y effective against al1 lard instars. Results of laboratory studies by Zirnmerman et al. (1 995)

14 indicated significant mortality to eh leaf beetle larvae, Xanthogalemca lureola

(Chrysomelidae); antifeedant efTects were observed in al1 larvai instars. Another

chrysomelid, the foliar beetle, Ootheca bennigseni, was shown by Karel(1989) to be affected

by neem products; aqueous extracts of neem products reduced feeding of the beetle in field

and laboratory trials. Azadirachtin can be transloçated in Iodgepole pines and was

demonstrated by Naurnann et al. (1994) to reduce the numkrs of mountain pine beetle,

Dendroctonrrsponderosae (Scolytidae), attacking the ûee. While the systemic action of neem

products would not be important in an interior storage situation, the reduction in numbers of

the mountain pine beetle fiom increased mortality as a result of neem applications is.

2.6 Effects of Neem Products on Beetle Pests of Stored Products

Toxic and growtb regulating effects of neem products have been demonstrated

against the red flour beetle, Tribolium custaneum (Tenebrionidae) by Jilani et al. (1988). and

toxic effects to this beetle were also demonstrated by Xie et al. (1995). Two other beetle

pests of stored gmin products. the nisty grain beetle, Cryptolestesjerrugineus (Cucujidae),

and the rice weevil. Siiophih oryzae (Curculionidae), also exhibited toxic effects (Xie et

al.. 1995). Al1 three beetles reduced feeding when exposed to neem products including

azadirachtin. Jilani and Saxena ( 1990)demonstrated an antifeedant effect of neem products

to the lesser grain borer. Rhyroperrh dominica (Bostrychidae).

Since neem products produce toxic. growth regulating and antifeedant effects againsi many families of phytophagous agricultd beetle pests and beetle pests of stored grains, then they might be effective against keratinophagous intenor storage pests of the family Dennestidae, specificaily, the hide beetle, Dermesres maculatus. in fact, another dermestid beetle, Anthrenocetus ausrnalis, was found to reduce its feeding on woolen cloth treated with azadirachtin; no growth regulating or toxic effects were demonstrated for this species

(Gerard and Ruf, 1994).

2.7 Dermestid Beetles:

Severai species of dermestid beetie may damage non-agricultural intenor stored products. Descriptions of several of the more cornmon are provided by Bennett et al. (1 988), including the varied carpet beetle, Anrhrenus verbasci, and the furniture carpet beetle,

Anhenus flmipes. Dennestid beetles have a complete metamorphosis, with the larvae king the only stage which causes serious damage to fibers; the adults consume less food and therefore cause less damage. Adults rnay be found indoors or outdoors and are frequently found on flowers where they feed on pollen. Carpet beetles are considered scavengers; they are a common pesi of museurn collections and ofien destroy vaiuable specimens. They feed on a variety of animal products such .awoolens, carpets, hides, feathers, as well as plant products and are very hardy and difficult to control. The fumiture carpet beetle, as its name implies, often damages upholstered himinire and, like the varied carpet beetle, feeds on hair, feathers. homs, silk. and other keratin containing materiais; they will also feed on fibers such as rayon, Iinen, Cotton and jute.

There are several species of the pnus Dermestes that are of econornic importance in

Canada and worldwide. These beetles can be found in tanneries and warehouses which process hides and skin; they can also be found in the home attacking furs, animal skins, feathers and meats or cheeses. The three most econornically important hide beetles are D. lardius (larder beetie), D. ater (biack larder beetle), and D. maculatus. D. maculatus is known in Canada as the hide or leather beetle -ton, 1945; Bennett et al. 1988); it is considered cosmopolitan, but is thought to have originated in Eurasia (Hinton, 1945).

These beetles are important economically because the larvae, and in some cases the adults, feed on a wide variety of stored products, mostly animal proteins. Their habit of boring into solid materials to pupate also considerable economic loss (Hinton, 1945;

Bennett et al. 1988). Larvae have been known to feed on hom and feathers, fur. brides and glue of bnishes, cottons and linen, cacao beans and al1 stages of silk woms. These beetles have been known to be vectors of pebrine, and because they consume carcasses of dead mimals, they may also spread anthrax (Hinton, 1945). 3. Materials and Methods

3.1 Bermestes maculatus Cultures

D.macufatus was chosen as the subject of this study for several reasons. This species

is larger than other species of Dermestid beetle, and is therefore easier to work with. It has a shorter life cycle so larger numbers of beetles might be obtained in a shorter period of the.

This species is a common pst, and is representative of other derrnestid beetle species.

Cultures of D. maculatus were rnaintained in order to provide a sufficient population fiom which to select healthy and uniform test individuals. Adults and larvae for the preparation of the initial cultures were obtained fiom Ward's Biologicd Science. Adults and larvae were seIected fiom these cultures for al1 experirnents.

The optimum temperature range for development is between 18-20°C and adult beetles will copulate above temperatures of 16- 18 OC. Femaies may copulate one or more times during the egg laying period and the pre-oviposition period varies fiom 1 O to 15 days; eggs are laid in batches of 3-20. The number of eggs laid by the females, which may live up to 90 days, varies considerably. but to lay the maximum number of eggs the females mut have access to water and food. The incubation period of the eggs varies with temperature. but development appears to be fastes1 at 28°C. The larvae normally molt six times before pupating but they may molt anywhere from seven to eleven times. The total duration of the larval period is dependent on temperature. relative humidity, available moisture in the food, and the type of food available (Hinton, 1945).

Larvae are very active and strongly negatively phototrophic. When they are full grown they leave their food in order to find a suitable place to pupate. Holes are bored into any suitable available compact material, including oak, cork, books, and cotton and iinen.

Pupation takes place in the last lwdskin with the pupal period usuaily lasting fiom 5 to 14 days. The duration of the entire life cycle is about 42-46 days at 28 OC. and approxirnately

55 days at 23 OC vinton, 1945).

The culture jars were kept inside aquaria, with sealed screen tops to prevent escape of the Iarvae and adults. The aquaria and the jars were kept inside a walk-in, environment controI chamber in the Lyman Entomological Museum and Research Laboratory, McGill

University. The temperature inside the chamber was maintained at 25 OC with a 12h: 12h

1ight:dark photo-period, and a relative humidity of 80%. Although the beedes are able to obtain water through atmospheric humidity, they appeared to be healthier and maintained higher populations when water was readily available. Therefore, a piece of tightly folded wet paper towel was piaced into the culture jars on top of the culture media every second day afier the previous paper towel was rernoved. Ail experiments were performed inside the same environment control chamber to ensure uniform experimental conditions.

The culture medium used in this study consisted of pine wood shavings used for pet litter; small shavings for use in the cultures were separated from larger shavings using a screen. Approxirnately 500 ml of dried cat food and 500 ml of dried goldfish flakes were added to 3.5 liters of small pine shavings: beef was dried in a food dehydrator and small pieces were added to the culture for additional nutrition as required. The entire mixture was pIaced in a 4.0 liter jar with a fine screen top.

Afier approximately three months, or when accumulated shed larvd skins became numerous, the cultures were discarded and new ones started. New cultures were stted by

piacing a nwnber of young adults and/or pupae from previous cultures into a kshculture

medium. Whenever possible, adults fiom different culture jars were mixed in order to reduce

inbreeding. Larvae reached a size useful for experirnentation afier approximately 6 to 8

weeks. 12- 15 culture jars were maintained at al1 times so that adequate numbers of beetles

rnight be obtained. Larvae and adults were separated fiom the cultures by spreading out the

medium in smail quantities in a porcelain pan and removing them with soft tweezers.

3.1.1 Selection of Test lnsects:

Larvae were removed fiom the culture medium as required. individuals of

approximately the same size (6-7 mm) and the sarne stage of growth (5"-6. instar) were selected. Larvae which appeared unhealthy, which were too large or too srnall, or which had just recently molted were discarded. Adults were selected in a similar manner, although they were selected only on the basis of size.

3.2 Preparation of Neem Extracts and Neem Products

Neem products (seed, cake and oil) were obtained fkom the Fundacion Agricultura y Medio Ambiente, San Cristobal. Dominican Republic. A previous analysis of the neem seeds and oil prepared by Dr. Maus Ermel, of the same institution, gave the following results:

9.4% humidity and 0.9 mg/g of azadirachtin-A in the seeds and 0.08% azadirachtin-A in the neem oil. Purified neem extract powders, 36.4%, 16.4%, 10- 1% and 7.0% azadirachtin-A, were provided by Dr. Murray Isman, Department of Plant Science, University of British

20 Columbia, Vancouver, British Columbia In the topical application experiments, solutio.?s

were prepared using the 10.1% extract, the 16.4% extract was used in primary antifeedant

experiments and the 36.4% extract was used to prepire the solutions for the secondary

an ti feedant experiment .

The neem seed extracts were prepared as follows: 400 g of crushed neem seed kernels

were mixed with 1 L of methanol; the mixture was vigorously shaken for 1 minute, every

hour, for eight hours and was then let stand for a further 48 hours. Mer this period, the

mixture was shaken one last time for several seconds. To remove large particles the mixture

was immediately poured through a coarse screen and the remaining methanol solution was

filtered through a fluted Whaunan #1 filter. The methanol was evaporated in a shallow

aluminum pan until no visible liquid remained and the resulting residue was dried funher .

under blowing air for 12 hours; approximately 15 gram of a sticky yellow extract was

produced. This extract was redissolved in methanol as required to produce a 10% neem seed

kernel extract/methanol solution. Neem extracts were prepared at the tirne of the experiment

and were used immediately to prevent variation as a result of reduction in active chernicals

in the neem seeds over tirne. The sarne extract was used for al1 treatments in each experiment.

Purified neem exvacts were also redissolved in methanol to produce the appropriate

concentrations.

3.3 Topical Application Expenments

3.3.1 Relative Effkacy of Mineral Oil and Neem Products Comparative mortality and efficacy of minera1 oil, neem oil, purified azadirachtin/rnethanolsolution (at a concentration of IO g/L and 20 g/L), and 1û% neem seed kemel extract/methanol solution were compared in this expriment. 2 pl of each treatment was applied to the anterior dorsal section of each lama using a micro-pipette. 6 replicates of

20 larvae per replicate were tested in a completely randomized design. Treated larvae were placed in a petri dish, fish food was supplied ad libitum to prevent starvation. and a moist paper towel was provided for drinking water; the papa towel was replaced every second &y.

For each treatment the number of dead larvae were counted and then removed daily, and the total number of dead lmae, after 5,10 and 14 days, was recorded. The &ta were square root transformed and analyzed using one-way ANOVA. The means of each treatment were compared using Duncan's multiple range test (P=0.05).

3.3.2 Effects of Mineral Oil and Neem Products on Moltiog, Pupatioo, and Adult

Emcrgence

Five treatments, mineral oil, neern oil; 5 g/L azadirachtin/methanol solurion and 20 g/L azadirachtin/rnethanol solution; and a 1 0% neem seed extract/methanol solution, were tested. As in the previous experiment. 2 pl of each treatment was applied to the anterior dorsal portion of each iarva. Treated larvae were placed in a petri dish and given goldfish food ad libitum. A moist paper towel was placed in the petri dish to provide drinking water and was replaced every second day when the old one was removed. The treated larvae were examined daily until al1 were dead or had reached the adult stage; the total number of larvae to pupate, and the total nurnber of larvae to reach the adult stage were recorded. The number of molts per group were counted daily by recording the number of shed lamal skins in each

petri dish. 4 replicates of 25 larvae per replicate were tested in a randomized complete block

design. The larvae were blocked by the &y on which the treatments were applied. The data

for each treatment were square root tramformed be fore anal ysis of variance. Di fferences

between treatments were tested using Duncan's multiple range test (P=0.05).

3.4 Antifeedant Expcrimcnts

3.4.1 Primary Antifeedant Effects of Azadirachtin

Xie et al- (1 995) used the following methods to test the primary antifeedant effects

of neem products on pest insects of stored grains. A flour disk was prepared by mixing an aqueous solution containing the appropriate concentrations of test materials with flour. The

suspension was then thoroughiy mixed and quantities were placed in a plastic petri dish for drying. Afier stabilization in a humidity controlled chamber. individually weighed flour chips were transfened to another petri dish for bioassay.

A variation of the methods used by Xie et al. (1995) were used in this antifeedant bioassay; a solid material with an even distribution of azadirachtin throughout, which could be easily weighed to determine the amount of food consurned by the beetle larvae was produced. As a control, 5 g of goldfish flakes were mixed with 30 ml of water and 5 ml of methanol. 5 ml of 1.5 g/l azadirachtinlmethano1 solution was substituted for the 5 ml of pure methanol to prepare the first treatment, and 5 ml of 5 g/L azadirachWmethano1 solution was used for the second treatment. in al1 cases, 5 g of goldfish flakes were combined with 35 ml of liquid. The amounts of liquid and fish food were chosem as a result of previous mals to

detennine proportions which would provide rapid drying and still allow for easy spreading

of the mixture.

Al1 the ingredients were mashed and thoroughly mixed with a mortar and pestle until

a paste with an even consistency was produceci. The paste was poured into 150 mm diarneter

disposable plastic petri dish and spread to a uniform thickness. The paste was then dned

using gentiy blowing wam air until it became hard and began to lifi fiom the surface of the

petri dish. The dried fish paste circles were then cut into squares with 15 mm sides. These

pieces were dried at room temperature for 48 hours, and then placed in the climate control

chamber at 25 OC and 80% r.h. until they reached arnbient temperature and hurnidity.

Imrnediately pnor to the stari of the experiments the fish food chips were weighed.

One chip per treatrnent was placed in a 150 mm diameter plastic petri dish. 10 similar sized

larvae were selected per ment;5 larvae were placed in each petri dish dong with the fish

food chip, and 5 larvae were reserved to replace larvae which died during the experiment.

The iarvae were permitted to feed for 120 hours and then removed. The chips were then re-

weighed to determine the arnount of the food consumed. The experiment was performed afler

the fish food chips had ken stored in the climate control charnber for 1,3. 5, 9, 13, and 17

weeks. Each treatrnent was replicated 3 times per time period. In preliminary experiments.

it appeared that there might be a negative effect on feeding if beetles fed on fish food chips

that had been fed on previously by other beetles. For this reason, fiesh chips were used in

each experiment. All the chips were prepared at the sarne time fiom the same stock solution of azadirachtin and methanol to reduce variation. Anaiysis of variance was performed for each tirne period; and ciifferences between treatments in each time period were tested using

Duncan's multiple range test. A Iinear regression was perfomied to test for the effect of time

on the persistence of the antifeedant effects of azadirachtin, at 1-5 gR and 5 g/L.

This experiment was repeated for only two tirne periods for adult Dermestes

maculatus because of a shortage of adults; rnethods used were the same as for the larvae.

3.4.2 Secondary Antifeedant Effects of Azadirachtin

Larvae were treated with -5 g/L, .25 g/L, and -125 g/L methanoVazadirachtin solution

and a methanol control. 9 larvae were randomly assigned to each treatrnent and starved for

24 hours afier the treatrnent was applied. 4 were then placed in a glass jar with o piece of cat

food which was weighed before the experiment, and 5 were placed in a separate glass reserve jar, also with a piece of cat food. The larvae were checked twice daity, and those which appeared unhealthy or which had died were removed and replaced with larvae from the reserve jar. The larvae were allowed to feed on the piece of cat food for 120 hours and then were removed. The piece of cat food was re-weighed to determine the amount of food eaten by the larvae. The experiment was replicated 12 times in a randomized complete block design; larvae were blocked by they day on which the treatment was applied. A linear regression was performed to test for the effects of concentration on food consumption. 4. Results

4.1 Topical Application Experirnents

4.1.1 Relative Effect of Minera1 oü, Neem oi), 20 g/L and 10 g/L hadirachtinlMethanol

Solution, 10% Neem Seed Kernel Extract/Methanol Solution Larval Mortality.

There was a significant difference in larval mortality (average number of dead larvae per replicate) resulting fiom the treatments (F= 10.9 1; df%,25; p=.000 1 )(Table 1 ) five days afier application. Neem oil caused a significantly higher mortality than dl the other treatments (Figure 1A); the 20 g5azadirachtin/methanol solution and the 10% neem seed kemel extracthnethano1 (NSWrnethanol) solution caused significanly higher rnortality than the minera1 oil treatment (Figure 1A). The 10 g/L azadirachtin/methanoI solution appeared to have a higher mortality than the mineral oil treatment, but difference was not significant

(Figure 1 A).

Ten days after the application of the treatments, the difference between the total mortalities was significant (F=24.47; de4.25; p=.OOOl)(Tabie 1); ail neem treatments exhibited a significantly higher mortality than the mineral oil treatment, with neem oi1 exhibiting the highest mortality (Figure 1 B). There was no significant difference between the other neem treatments, but the 20 g/L azadirachtidmethand solution appeared to produce the next highest mortality, followed by the 10 g/L azadirachtin/methanol solution, and the

10% NSWmethanol solution (Figure 1B).

Founeen days afier the application, none of the treatments caused 100% mortality (Figure 1 C)(Table 1). There was a significant différence between the total monalities caused by the treatments (F=50.08; df-4,25; p=.0001) and al1 neem treated lamae exhibited significantly higher mortalities than the mineral oil tieatment (Figure 1C). The highest rnortality appears to be caused by neem oil. followed by 20 g/L, azadirachtidmethanol solution and the 1O gR. azadirachtin/methanol solution but there was no significant differehe between these treatments (Figure 1C). There was no significant difference between the 10 gR. azadirachtin methanoi solution and the 10% NSWmetbanol solution (Figure 1C).

Beyond 14 days it was observed that none of the neem treated larvae reached the adult stage, whereas a large portion of those treated with mineral oil survived to pupate and reached the adult stage. This data was not recorded during this experiment since the experimental methods were not designed to examine these effects.

4.1.2 Effects of Mineral Oil, Neem Oil, 5 g/L and 20 g/L Azadirachtin/Methanol sotution and 10% Neem Seed Kernel Extract/Methanol solution on Molting, Pupation, and

Maturation to the Adult Stage

There were significant differences in the number of molts recorded in each group of treated larvae (F=9.62; df-4.12; p4.05 1 )(Table 2); the group of larvae treated with minera1 oil molted significantly more times than the poup of larvae in the other treatments except the group of larvae treated with the 20 g/L azadirachtin/methanoI solution. Of the groups treated with neem products. the group of larvae in the 20 g/l azadirachtidmethanol soIution treaunent molted more times than ihe other neem groups, but the difference is not significant

(Figure 2A). The nwnber of larvae to reach the pupal stage was significantly different between the treatments (F= 1 1.39; df4,12; p=O.OOOS)(Table 2); significantly more larvae

reached the pupal stage when aeated with mineral oil, than larvae treated with neem

pr0du~t.S(Figure ZB), but there was no sigdicant diffmnce between the pups treated with

neem products. Larvae survived to the adult stage only in the mineral oil treatrnent (Figure

2C). 4.2.1 Primary Antiieedant Effccts of Azadiracbtin on Larvae

There was a significant difference in the amount of fish food chip consumed by the

larvae derthe chips were stored in the climate control chamber at 25 " C and 80% r-h. for 1

week (F=32.24; df=2,8, p=0.0006). There were also si@cant differences in al1 tirne periods

after this; afier 3 weeks (F=159.47; df--2,8; p=O.Oûûl), after 5 weeks (F49.62; d&2,8;

p=0.0023), afier 9 weeks (F=44.48; d+2,8; p=0.0003), after 13 weeks (F=282.47; dF2,8;

p=0.0001), and afier 17 weeks (F43.92; d+2,8; p=0.0063) (Figure 3, B-F)(Table 3). In al1

time periods there was significantly more food consumed when the larvae fed on the control

chip containhg no azadirachtin (Figure 3, A-D). It appeared that there was more food

consumed by the larvae feeding on the fish food chips with the iower concentration of

azadirachtin in al1 of the tirne penods, but this difference was only signi ficant after 3 weeks

(Figure 3B) and 13 weeks in the climate control chamber (Figure 3E).

The amount of the fish food chip with the lowest concentration of azadirachtin (1 -5 g/L) consumed by the larvae appeared to decrease slightly as the storage time increased

(r=0.06; dope= -0.229) (Figure 4A). At the highest concentration of azadirachtin (5 g/L). the arnowit of food consumed by the larvae appeared to increase slightly as the storage tirne increased (?=O. 19; slope=0.296) (Figure 4B). 4.2.2 Primary Antifdant Effects of Azadirachtin on Adults

There was a significant difference in the arnount of food consumed by the adults in each treatment afier the fish food chips were stored in the climate control chamber at 25 OC and 80% r.h. for 1 week (F=22.14; &-2,8; @.O01 7) (Figure 5A)flable 4), and after the fish food chips were stored in the climate control chamber for 13 weeks (F=5.59;d+2,8; p=0.0426) (Figure SB)(Table 4). In the first time period, there were significant differences in the amount of fish food chip consumed by the adults between al1 treatments (Figure 5A).

After 13 weeks in norage, there was still a significant difference between the control group and the group treated with the highest concentration (5 g/L) of azadirachtin (Figure 5B). In al1 time penods none of the fish food chip treated with the highest concentration of azadirachtin (5 g/L) was consurned.

42.3 Secondary Antifeedant Effects

Linear regression analysis indicates a slight trend (F =0.17; siope = -18.22) of decreasing food consurnption as the concentration of azadirachtin applied increased

(Figure 6). Table 1. Mortality of the larvae 5, 10, and 14 days afier topical application of mineral oil,

neem oil, 20 g/Land 1 0 g/L amdirachtinlrnethanol solution and 1 OO/o NSWmethanol solution.

Days After Treatment Standard Application

1 Mineral Oil 0.50 0.223 1 Neem Oil

1 Neern Oil

1 Mineral Oil 1 Neem Oil

'. Average of 6 replications. 20 larvae treated per replication b. The total number of dead larvae per group of 20 treated larvae Figure 1. Effect of treatments on cumulative mortaiity in larvae of D. maculatus 5. 10 and

14 days after application. Data are presented as the mean of 6 replicates. Bars in the figures having the same letter are not significantly different at the 0.05 level of significance according to Duncan's multiple range test.

A. Cumulative mortality 5 days afier application of treatments.

B. Cumulative mortality 10 days afier application of treatments.

C.Cumulative mortality 14 days afier application of treatments. Figure 1A.

Figure 1 B. Figure 1C. Table 2. Molting, pupation, and adult emergence in larvae of D. mucuIutus treated topically with mineral oil, neem oil. 20 g/L and 5 gll azadirachtirdmethanoi solution, and 10./o neem seed kemel extract/methanol solution.

Mineral Oil 9

Total Number of Moltsb

Mineral Oil 1 2 O 1 O 1 NIA Total Number of Larvae Reaching the Pupal Stage

Nam Oil 11 0 Total Number of Larvae Reaching the Adult Stage

'. Mean of 4 repliates. 25 treated larvae per repliate. b. Obtained by counting the nurnber of shed larval skins for each group of 25 larvae, until al1 larvae died or reached the pupal stage. Figure 2. Effects of topical application of mineral oil, neem oil, azadirachtidmethanol solution, and 10% neem seed kemel extract on molting, pupation, maturation to the adult stage, and final mortality of D. maculatus larvae. Data are presented as the meam of 4 replicates. Bars having the same letter are not significantly different at the 0.05 level of significance according to Duncan's multiple range test.

A. Mean number of molts per 25 treated Iarvae.

B. Mean nurnber of larvae to reach the pupal stage of 25 treated iarvae.

C. Mean number of larvae to become adults of 25 treated larvae. Figure 2A.

Figure 2B. Figure SC. Table 3. Mean' (+/- standard enor) consumption (mg)by larvae of D- maculatus of fish food chips treated with O &, 1.5 g/L, and 5 g& subsequent to storage at 2S°C and 80% r. h. for

1,3,5,9, 13, and 17 weeks.

- . - . - - - 1 Consurnption (mg) of Azadirachti Number of Weeks in Food Chip Storage

' . Average of 3 replications. Arnounc of food consumed by 4 larvae per replication over a period of 120 hours. Table 4. Meana (+/- standard error) consurnption (mg)by adults of D. wzuculatus of fish food chips treated with O g/L, 1.5 g/L,and 5 g/L subsequent to storage at 25 OC and 80% r. h. for

1 and 13 weeks.

. Average of 3 replications. Amount of food consumed by 4 adults per replication over a period of 120 hours. Figure 3 : Effect of azadirachtin on consumption of £kh food chips by larvae. Data are presented as the mean of three replicates per treatment, during each the period. Bars in each figure having the same letter are not significantly different at a 0.05 level of significance according to Duncan's multiple range test.

A. Arnount of food consumed by the larvae der fish food chips were stored in the climate control chamber for 1 week

B. Amount of food consumed by the larvae afler fish food chips were stored in the climate control chamber for 3 weeks.

C. Amount of food consumed by the larvae after fish food chips were stored in the climate control chamber for 5 weeks.

D. Arnount of food consumed by the larvae after fish food chips were stored in the climate control chamber for 9 weeks.

E. Amount of food consumed by the larvae after fish food chips were stored in the ciimate control chamber for 13 weeks.

F. Amount of food consumed by the larvae after fish food chips were stored in the climate control chamber for 17 weeks. Figure 3B. Figure 3C.

Figure 3D. Figure 3E.

Figure 3F. Figure 4. Effects of tirne on consurnption of azadkchtui treated fish food chips by D. maculatus larvae. Data are presentcd as the meam of t&reereplicaîes per time period. The regression equations are:

A. Fish food chip treated with 1.5 g/L A7rrdirachtin/methanol solution. Y=0.229X + 13 -89; ?= 0.06

B. Fish food chip treated with 5.0 g/L. azadirachtin/methanol solution. Y=0.296X + 1 -02;?= 0.19 Figure 4A.

Figure 4B. Figure 5: Effect of ehtinon consumption of fish food chips by aduit D. mculatus.

Data are presented as the mean of thme replicates per treatment during each time period. Bars in each figure having the same letter are not significantly different at a 0.05 level of significance according to Duncan's multiple range test.

A. Amount of food consumed by the adults after fish food chips were stored in the climate control chamber for 1 week.

B. Amount of food consumed by the larvae after fish food chips were stored in the climate control chamber for 13 weeks. Figure SA.

Figure SB. Table 5. Consumption of cat food (mg) by D. macuIatus larvae afier topicai application of azadu.achtin and methanol.

Consumption (mg) of Fish Food by I Larvae

Mean of 12 replications. Amount of cat food consumed by 4 treated larvae per replication over 120 hours. Figure 6. Effects on food consumption of increasing concentrations of azadirachtin topically appIied to the D. macufo~uslarvae. Data are presented as the mean (quantity of food consurned by 4 larvae over 120 hours) of 12 replications. nie regression equation is:

Y = -1 8.229X+24.05; f 4.17 Figure 6.

Mean Quantity of Cat Food Eaten by Larvae

, 1 I 0.0 0.1 0.2 0.3 O -4 0.5 O .6 Concentration of Azadirachtin (g/L) 5. Discussion

5.1 Topical Application EspcrimenQ

Topical application of neem oil to D. mcuIatus larvae produces the highest initial

mortality in this experiment, or most rapid lethal effcct; it is also the most effective bio-

insecticide tested. Five days abr application to the larvae, neem oil produced the highest

overall mortality. The other neem products do not act as rapidly as neem oil, but produce

equally high mortalities afker 14 days. There are several factors which might account for the rapid efficacy of neem oil, and the high moaality after 14 days. The oily properties of neem oil might have increased mortdity by blocking the intake of oxygen or by other negative

factors affecthg lamal health resulting fiom the larval cuticle becoming coated with the oil. .

If this was the case, high mortalities should also have been observed in the mineral oil treatment, which has a similar consistency to neem oil. The minerai oil treatrnent had very low mortaiity even afier 14 days, therefon, the high mortaiity caused by neern oil compared to the minerai oil treatment and the other neem treatments, must be caused by sorne other factor or combination of factors. The most likely explanation for the greater activity of neem oil as opposed to mineral oil and the other neem treatments is the higher concentration and greater number of bio-active chemicais. The higher mortality of the neem oil compared to the azadirachtin/methanoI solutions might be because the oily nature of the neem oil eased the spread of the active chernicals ovcr the land body and increased the persistence of the active chemicals.

The results fiom this topical application experiment also indicate that, the 20 g/L azadirachtin/methauol solution produced a higher morîaiity than the 10 g/L azadkach?.in/rnethanoI solution. Since these are purified extracts, the ody explanation for the higher mortality of the 20 g/L azachchtiiamethanol solution when cornpared to the 10 g& azadirachtin/rnethanol solution is the increased concentration of atrrdirachtin; this agrees with - obsewations by Isman et al. (IWO), who found that the bio-activities of neem products were proportional to their azadirachtin content.

The increased mortality of the neem oil treatment compared to the other neem treatments rnight in part be due to higher concentrations of amdirachth. Isman et al. (1990) reported that neem oil is obtained hm crushed neem seeds, which have the highcst concentration of azadirachtin in the neem tree. It is also possible that the Iipophilic properties of the neem oil facilitate the rapid absorption of the active chemicals into the insect, through the larvai cuticle, or that the oil produces better contact thaa the other solvents because it did not evaporate, allowing more time for the active chemicals to be absorbed.

Five days derapplication, the rnethanoheem seed kernel extract (NSK) solution produced higher mortalities than the methanol solutions of purified azadirachtin, As with the neem oil treatment, this high mortality rnight be the result of a greater number of bio-active chemicals extracteci from the fksh neem seeds, which act together to produce the initial high mortality. Unlike the neem oil treatment, the NSK extract did not cause as high a mortaiity after 14 days. This may be because methanol is a very polar solvent, and it extracts many materials other than the active chemicals including sugar and tannins. These materials are reported to dilute the active chemicals (Jaglan et ai., 1997) and this may make the NSK extract les effective over time. The redts of îhese topical application experiments to compare the effects of minera1

oil and neem products on overail mortality indiate that ail nccm products produce higher

mortality than the mineral oil matment when topically applied to Dermestes maculatus

Iarvae. This incrcastd mortality after topicd application of neem products has also been

observed in other insects. Schmutterer (1990), observed increased mortality in late instar

nyrnphs of Blatta orientalis (Orthoptera: Blattidae) when Margosan-O, a neem based

insecticide, was applied topically, and in experiments similar to those reported here for D.

maculatus, Banken aud Stark (1998) demonstrated that al1 CoccineIZa septempuncfatca

(Coleoptera: Coccinellidae) larvae treated directly with, not ingesting, a commercial neexn

pesticide died within 10 &YS of exposure.

Ladd et al. (1984), used injection methods to test the effects of azadirachtin on the

Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae); azadirachtin was dissolved in

ethyl alcohol and the solutions (3.5 PL) were injected into the latero-ventral thoracic regions of the larvae. Larvae were returned to a growth chamber and dead and deforrned larvae were recorded every 5 to 7 days after treatment; none of the larvae treated wmpleted development.

In the experiments used for this study, solutions of neem products were not injected into the larvae, but the observed increase in mortality and morphological defects were similar to those described by Ladd et al (1984). injection of neem products into the lmal hemolyrnph is suitable for detennining and quantifying the effects of neem prducts on immature insects, but is less accurate for approxirnating applied situations than topical applications. It is likely that lower or delayed mortalities would be found afier topical application of the neem products than wouid be found after injection. Injection also rtquires more expensive and complicated equipment.

There has ken very litde investigation of the toxïcity of topicdy applied neem products to beetle larvae and none with respect to bcetle larvae which are pests of non- agricultural interior stod pducts. Topical application experiments have been used extensively for testing of synthetic chemical pesticides and other botanical pest control agents

(Perry et al., 1998; Hoskins and Craig, 1%2), but only in a few cases have these methodsbeen used to test the effects of neem pmducts on coleopteran pest species. Aem and Mordue

(1997) used topid applications of azdhchtin dissolved in acetone (1 PL) 10 investigate the toxic effects on Spodoptera littorafis (Lepidoptera: Noctuidae), Schistocerca gregaria

(Orthoptera: Acrididae), and 0ncopltu.sfaciatw (Hemiptera: Lygaeidae). Toxic and growth regulating effects of azadirachtin were demonstrated for al1 these insécts. Toxic and growth regulating effects of neem products were aiso demonstrated with topicai application to fourth instar larvae of severai moths of the family Noctuidae (Lepidoptera) (Isman, 1993).

Teswig the toxicity of an insecticide using topical application has several advantages, as described by Redfern (1985). Large numbers of tests can be perfonned in a short penod of tirne, and small numbers of test organisms are needed (5 -20 per replicate), a high degree of precision can be obtained. vexy srnail amounts of the test chemical are required, and the experiments have a high degree of repeatability. Topical application, because of these advantages, was therefore an ideal method for testing and comparing the effectiveness of neem products on an interior storage pst such as D. maculatus.

The results of this expcriment illustraie a problem associated with the testing of botanical pest control agents using topical application. Except for neem oil, the other neem products did not have an immediaîe effkct on mortality. With neem products and other crude

botanical insecticides there is no immcdiate knockdown effect as is obscrved with synthetic

chernical insecticides. In fact, the lwae which are not killed after application of neem

products may live in an extended larval stage, but neither pupate nor reach the acîuit stage before they die of old age. Schmutterer (1990) indicates that these extended larval periods rnay be a problem as the treated larvae are ohobserved to continue feeding after treatment with neem products; but reductions in fitness and secondary antifeedant effects might be a factor in reducing this feeding afier treatment with neern. in fact, the results of these experiments, which are discussed in more detail later, indicate a secondary antifeedant effect of azadûachtin which might reduce the feeding of D. rnucu2a1~1~after treatment with azadirachtin. Extended immature stages were observed by Isman (1997) in the grasshopper

Mefanoplus sanguinipes (Orthoptera: Acrididae), which faiied to undergo its final nymphal molt to reach the adult stage afier application of adirachth to the immature stages; nymphs continued to live for 60 days and died without ever becoming addîs. Unless the larvae are observed over a longer period of time, it is likely that the effects of these insecticides might be underestimated. in this experiment, the larvae were observed for only 14 days, at which time even neem oil had not killed al1 the treated larvae. Further unquantified obsewations subsequent to day 14 revealed that many of the larvae treated with mineral oil survived to pupate, while those treated with the neem products, even the NSK extract which had the lowest monality afkr 14 days, failed to pupate and eventuaily died. 'lheir deaths in most cases were not immediate, and pnor to death a variety of morphological defects of the larvae and pupae were observed. In order to determine the eff~tsof topifal application of neem products on molting,

pupation and addt emergence, the second topical application expriment was performed. The

results of this topical application experiment indicate a reduction in the nurnbers of molu in

the groups of larvae treated with nccm products, although the reduction was not statistically

sigaificant denthe Iamae were treated with the 20 g/L azadhchtin methanol solution. Few

larvae treated with neem products survived to pupate, and none of those that did pupate

emerged as healthy abults. A large majority of the neem ~atedlarvae died while attempting

the fhmolt to the pupal stage. In some cases, the lwae that died appdto sbrivel up and

turn black, or the pupae appeared to be making the transition to the adult stage, but were

unsuccessfùl and died with a combination of pupal and adult characters. In al1 cases, none of

the deformities associated with the neem treatments were obsemed in the groups of larvae

treated with mineral oil, possibly because of the absence of bio-active chemicals.

In the first topical application experirnent, there appeared to be a slight concentration

effect on mortality afier 14 days. in this experiment, concentmions of 20 g/L and 5 g/L

azadirachtin/methanoI were used in order to make this concentration effect more apparent.

The results were unexpected. More molts were recorded in the group treated with the highest

concentration (20 gk), where less molts were expected because of the higher concentration

of azadirachtin. The nurnber of larme to pupate was not significantly different and no larvae

reached the adult stage at either concentration. This suggests that the number of molts does

not accurately reflect the growîh regulating properties of the neem chemicals on D. macula~ur

larvae. The growth regulating effects of neem products on D. maculatus seem to manifest themselves during the final molt to the pupal stage. It is at this point that morphological deformities and problems with ecdysis wmmoa evident. The growth regulating effects of

neem products on D. muCUIutu larvae might be more effectively compared by cxarnining the

number of larvae to pupate and mach the adult stage. -

These topical application experiments have demonstrated that neem products have

toxic and growth regulating effects on D. rnuculatus larvae, and that neem oil is the most effective neem product since it produces a more immeâiate effect on the larme. As a result of its oily consistency and yeiiow staining color, there are few indusaial situations were pure neem oii rnight be applied dkdyto a storeci product to prevent destruction by insects. There is a greater possibility that purified adirachth and an appropriate solvent which would have reduced visuai effects might be used for this purpose.

5.2 Primary Antifeedant Effects of Azadirachtin

Interior stored products might also be protected from destruction by insect pests as a result of the primary antifeedant properties of azadirachtin and other neem extractS. There has been littie research on the response of insects pests of non-agricultural interior stored products, and even less on the response of beetle pests of non-agriculturai interior stored products to the prirnary antifeedant effecu of neem; therefore, it was necessary to develop original methods for testing for these antifeedant eff- on keratinophagous beetie pests. The methods used in this study, a variation of those used by Xie et al. (1996) were designed specificaliy to test for primary antifcadant effects of neem products on Dermestes macuIatus larvae and adults, and to accurately test the peaistence of azadirachtin in an interior storage environment. Dermesres muctlIatus and many other denncstid beetles dcstroy intenor stored products made of animal protein, thmfore, fish faa matcrial with a high protein content

(Appendix l), was substiMed for flour, which was used in the antifeedant experiments perfomed by Xie et al. (1996) . The chips used for this expriment were made of fish food for several reasons. The fish food was part of the culture mix, so it was certain that the beetles would feed on it. Whni the goldfish flakes were combinecl with water, a was produced which was easily rnixed and reformeci; this ensuted an evcn distribution of azadirachtin throughout the test material and a uniformity in size and shape of the finai chips. It was important to keep the chips in a climate control chamber since there wem some weight changes as a result of fluctuations in humidity.

A method had to be found where dirachtin could be incorporated into the beetles food evenly, and the arnount of food before and derexposure to the beetles couid be weighed accurately. Xie et al. (1 996) descnbed îhe advantages of their flour disk bioassay; they found that the flour chips were simple and quick to prepare, and that the chips produced were uniform if the test solution was thoroughly mixed. When spraying wheat kemels for use in antifeedant bioassays, it is difficuit to get even coverage of the kemels with the test chernical, and it is even more dificuit to detennine the exact amount of food consumed. Similar problems would be encountered when testing for primary antifeedant activity of chernicals on other stored products. The destruction of stored product materials such as leather rnight be visually assessed, as in bioassays designed to quanti@ the destruction of 1- plant parts (see

Karel et al. 1989). but this method is less accurate and difficult to repeat becaw of biased observations. in cornparison, weighmg the arnount of food consumed by the test insect is easily repeatable and less subject to error. The advantages of the flour disk bioassay were

duplicated in this experiment with the fish food chips, and several other advanîages are

described. This experiment was also extended to include a test of the persistence of the

primary antifeedant effects caused by azadirachtin.

Since the beetles are in constant contact with their food, and since the weight change

due to feeding was weighed in milligrams, it was important that the food not become

contaminated with feces or shed larval skins which might affect the weight of the fish food

chips. The fish food chips produced in this experiment were hard, and wuld be removed hm

the test petri dishes without feces or larval skins attachecl. The chips maintained theù integrity

and withstood significant feeding by the beetle larvae and adults. These characteristics

enabled easy weighmg and accurate measurement of the food consumed by the lawae.

The resuits of this experiment demonstrated a strong primary antifeedant effect of neem products on D. macuIatw larvae and adults; less food is consumed with higher concentrations of azadirachtin and in some cases the antifeedant effects of the azadirachtin were so strong that at the higher concentrations none of the neem treated food was consumed.

More food was consumed by the larvae than the adults; this observation suggests that the majority of damage done to stored products by D. macuIatus is a result of feeding by the larval stage and agrees with observations by Hinton (1945). In one of the few experirnents on the effects of neem on a non-phytophagous interior storage beetle pest, Gerard and Ruf (1 994) tested a neem seed cxtract on the Australian carpet beetle, Anthrenocerus australis

(Dennestidae). Wool was treated with various concentrations of neem seed extract and late instar larvae were allowed to feed on the treated wool for 14 days. The wool was weighed before and after fceding to cietennine the total amount comed; it was found that the larvae

reduced feeding on the ncem treated wool and that there was lesfecding as the concentration

of azachcbtin increased. This Mehas similar food and habits to D. maculutus, and since

prirnary antifeedaat effects of aauhchtin are found for both species, it is likely that there

will be effécts on other dermestid Mesand other non-phytophagous beetle pests of interior

stored products.

In this experiment it was observeci that although there was a reduction in the amount

of food consumed as a result of the application of aadirachtin, some food was still ingested.

Another experiment might investigate the growth regulating and toxic effects of neem

products after ingestion by D. rnacufatus larvae. in some insect species, neem chernicals do

not produce prirnary antifeedant effects. These insects will feed on products mted with neem chernicals and usually exhibit reductions in health and fecundity, morphological defonnities and death as a result of neem's growth regulating and toxic properties at a later tirne (Ascher,

1993). Schistocerca gregaria (Orthoptera: Acrididae) were show to be extremely sensitive to azadirachtin with alrnost 100Yo reduction in feeding at concentrations of 0.001 ppm.

Locwta rnigratoria (Orthoptera: Acrididae) were found to much less sensitive to azadkchtin

(ED, 10 ppm) and ingested signifiant amounts of the chernical (Mordue et al., 1996). If D. maculatus larvae were insensitive to azadirachtin, the larvae would have ingested the neem chemicais in the fish food chips; there wouid have been no primary antifeedant effect of the azadirachtin incorporated in the fish food chips, but growth regulating and toxic effects might have been observed.

At the highest concentration of azadirachtin, the larvae consumed some of the fish food chips, but the adults did not consume any; at the lowest concentrations of azadirachtin

the Iarvae drastically reddthtu consumption of the fish food chips while it was observed

that the adults consumed amounts of food sirnilar to the amounts consumed in the control.

This suggests that adult D. madafus adults are not as sensitive to the Iower concentrations

of a7aditachti.n used in this experiment, but will drastically reduce feeding at an undetermhed

threshold concentration somewhere between the highest and the lowest concentrations. This

has interesthg implications for the use of azacbchtin for conml of Mepests of intenor

stored products. In order to control beetle pests of non-agicultural interior stored products, . a bait control strategy might be useful. Aradirachth could be incorporated into the bait material at concentrations below the threshold required to produce primary antifeedant effects.

Beetles consuming these baits wouid experience mortality, providing short tem control, a reduction in feeding as a result of physiological changes (secondary antifeeàant effects) and later reduced fecundity and longevity, decreasing the size of fume generations. This bait strategy would be usefûl where long term control is required. It is known that some adult beetles will ingest neem products at certain concentrations but when feeding occurs in anti feedant trials, signi ficant mortaiity ohen occurs to the adult insects. Lepinotorsa decemlineatcl (Coleopima: Chrysomelidae), the Colorado potato beetle, showed 73% mortaiity after feeding on potato foliage ntated with neem seed kemel extract (Zehnder and Warthen,

1988). It could be assumed that with a general reduction in fimess, reproduction and egg production would also be reduced (Schmutterer, 1990).

Even though the= was significantly les neem treated fish food chip consumed by the

D. maculafus larvae compared to the control fish food chips, some food was conswned, mainly at the Iowa concentrations of azadirafhtin. It is not likely that simply tasting the neem

treated food wouid bave resulted in the arnount of food loss recordecl, so there must have been

some consumption of the fi& food chips. It is possible that due to starvation, the D. muczùahr~

larvae became unresponsive a the antiféedant effiof adirachth and began to feed on the

fish food chips at the end of the experiment. Schistocerca greguria (Orthoptera: Acrididae) has been documented to become habituated to azadhchtin Bfter a pcriod of 4 days to concentrations which initiaJiy gave cornpiete protection (Schoonhoven, 1982). Bomford and

Isman (1996), tested the effects of hunger on desensitization and habituation of Spodoptera lirura (Lepidoptera: Noctuidae) to azadirachtin and neem; afkr repeated exposures, iarvae becarne desensitized to purified azadirachtin but not to neem products containing the same absolute arnount of azadirachtin. They also determined that hunger was responsible for approxMately one third of the desensitization response in the no-choice bioassays. Since the fish food chips in this study were presented to the laivae in a nochoice bioassay, it is possible that the antifeedant effects of azadirachtin on the D. maculatus larvae were underestirnated.

Bomford and Iman (1996) stated that in no-choice experimentai designs where desensitization might occur, experiments in which the insects are exposed to the antifeedant substance for longer durations will be less sensitive to initial feeding deterrence than experiments of a shorter duration. It was also stated that paired choice assays might be more sensitive to antifeedant effects than no choice bioassays because the effects of hunger would be removed. If the test D. mucuiat~~lawae had the choice of feadiag on some 0th material, as they might have in an interior storage situation, then less of the test matenal might have been consumed. It would be interesting to examine if the food was consumed at the beginning

63 of the test, or at the end, which would indicatt wcther the larvae became less sensitive to azadirachtin as theu hunger ind.

One problem with testing the primary antifeedant effects of azadirachtin on 13.

~~~cuiaiularvae is the difficutty of weighing the srnail amount of food consumed in a short

Mieperiod because oftheu srnail size. To compensate for tbis in the experimenîs used in tbis study, the larvae were left in contact with the fish food chips for a longer penod of time to increase the amount of food consumeâ. Instead of leaving the larvae in contact with the test material for a longer tirne, more larvae might have been used over a shorter period of time.

This would result in the consumption of an amount of food which is easily weighed and decreases the risk of desensitization to the neem products which might occur in a longer bioassay. In this case, larger numbers of larvae would have to be available at the tirne of the experiment.

Even though neem products have proven veiy efféctive in reducing insect feeding on many agricultural plants, there are problems associated with the use of neem products outdoors. Neem products have been shown to be extremely unstable when exposed to the weather. Azadirachtin and other triterpenoides found in neem products degrade rapidly when exposed to air, heat. moisture and ultraviolet light (Mohapatra et al., 1995; Isman, 1997); in fact, neem products arc known to have a rcsidual life of only 4-8 chys in outdoor conditions

(Ascher, 1993). In an intenor storage situation the effects of the environment on neem's active chemicals are greatly reduced. Temperature and moisture are usually kept low in order to preserve the stored products, and ultra-violet light is much lower than in an outdoor agricdtural situation. With these tnvironmental effects reduced, neem chemicals should be more persistent in an indoor storage situation and therefore more eEative than when applied

to agriculhual insect pests in an outdoor situation. There are isolated reports of longer

persistence of neem chernicals (Ascher, 1993); if this increased persistence could be proven,

then neem products might be considered suitable for use in an interior storage situation.

The results of this experiment indicate that neem products have a strong prirnary

antifeedant effect on D. maculatus larvae and adults; but, if these products are to be used to

protect stored goods fiom destruction by the feeding activities of beetle pests, these antifeedant effects must persist for a relatively long period of the. Larval feeding was reduced on azadirachtin treated fish food chips even after the treated fish food chips were stored in the climate control chamber for 17 weeks. The amounts of the control fish food chips eaten varied considerably over the six time periods tested, but in al1 time periods the amount of fish food consurned by the larvae feeding on the control fish food chips was significantiy more than the amount of food eaten in any of the fish food chips treated with azadirachtin. This variation in food consumption on the control fish food chips can be attributed to variation in the groups of larvae tested since a different group had to be tested for each time period. Sirnilar rpsuits weni observed for the adult beetles; adult feeding on the azadirachtin ûeated fish food chips was reduced, even after the treated fish food chips had ken in storage for 13 weeks. ûver this time there is very little change, if any, in the amount of protection aorded by azadirachtin, at either concentration. It might be assurned that the prirnary antifeedant effaon both adults and larvae could have lasted much longer than the duration of the experiment.

Neem products have ken show to have fairly constant growth reguiating and toxic effects beîween spcies, but the primary antifecdant eff~of neem tend to Vary, with some species afSected more than othen (Aerts and Mordue, 1997). It has been hypothesized that many insects are not sensitive to botanical antifeedant compounds because they have never corne in contact with the substances during the course of their evolution (Schoonhoven,

1982). In the case of neem it has been suggested that the primary antifeedant effects are an adaptation of the insect to protect itself fiom the toxic and pwthregulating properties of neem chernicals (Schoonhoven, 1982). If this is the case, why would an insect such as D. maculatus which feeds almost exclusively on animal proteins, be sensitive to a compound which evolved to protect a plant? The evolution of susceptibility to the antifeedant effects of neem products is beyond the scope of this study; it is important to note however, that this hypothesis rnay be one on of the rasons there has not been more research on the effects of botanical insecticides on non-phytophagous beetle pests of inteIior stored products. This study has show that a keratinophagous insect pest of non-agricultural stored products is susceptible to the primary antifeedant effects of neem products, and that a substance which evolved to protect a plant fiom phytophagous insects is also effective against a non-phytophagous insect.

It is possible then, that other non-phytophagous beetle pests of stored products might be susceptible to the pnmary antifeedant effects of neem.

5.3 Secoodary Antifeedant Effects of Azadirachtin:

One of the major disadvantages of neem products compared to synthetic chemical pesticides is their slow cffect on mortality (Schrnutterer, 1990; Ascher, 1993, Isman, 1997).

Since the insects do not die immediately afkr treatment, it is likely that they will continue to feed on, and cause damage to, stod proâucts. In the previous topical application experiments the neem products did not kill the D. macu1atm larvae immediately aertreatment. In fact, it was observed that a portion of the treated larvae were alive after 14 &YS. It was also observed, but not quatltified, that the neem treated larvae appeared to consume less food than those treated with mineral oil. It could not be determined however, wether there achiaily had been secondary antifeeâant effects of neem, or if a reduction in the numbers of beetles resuited in the consumption of less fd.

To determine if th- was a reduction in feeding as a resdt of topical application of neem products to the D. maculatus larvae another expcriment was neeâed. in this experiment, lower concentrations of azadirachtin were tested than in the previous topical application experiments in order to prevent mortality of the larvae during the cokeof the experiment.

The larvae were fed on cat food pieces; the cat food pieces were the same as those used in the culture matenal so it was known that the larvae would use them for food. Like the fish food chips used in the primary antifeedant experirnent, the cat food pieces were solid and could be easily weighed at the beginning and at the end of the experiment without fear of contamination by shed lamal skins or feces, to determine the amount of food consumed by the beetle larvae. Fish food chips were not used in this case since the chemicals were king applied to the larvae, not to the food they were consurning.

The results of this experiment showed that less food was consumed as the concentration of topically applied azadirachtin increased, indicating a mild secondary anti feedant effect which increaxs as the concentration of azadirachtin applied to the larvae incrûases. This effect can be defined as a secondary antifeedant effect because food was not rejected as a dtof contact of azadirachtin with mouthparts of the larvae. Since azadiracbtin

was applied topically, the secondary antifeedant effect bad to be the resuit of the absorption

of azadirachtin through the lamal cuticle. This expcriment was no

physiological changes which dtedin reduced fecding by the beetle larvae; but, it is Iikely that the secondary antifcedant effect was a result of a general loss in fitness and appetite due to the multitude of negative effets caused by neem products on immature insects. It is possible that if higher concentrations of azadirachh were used, such as at the levels tested in the other topical application experiments, an even stronger antifeedant effect would be exhibited.

The secondary antifeedant effect demonstrateci in this experiment is important ifneem chernicals are to be used for the control of interior storage pests. If necm products are to be applied directly to the pest insect species, a minimal knockdown efféct is to be expected. The secondary antifeedant effects might afford additional protection to the stored products as reduced feeding will result in less destruction by the swiving insects. 6. Conclusion

The dtsof these experiments demonsûate tbat neem pducts produce signifiant

growth regulating, toxic, primary antifeodant and secondary antifeedant effects on D.

macuIahc~,a beetie pst of non-agricultural interior stored products. These eEects singly, or

çombined, might be used to protect non-agricdtural storeci products fkom a variety of beetle pests. More importantiy, it was demonstrateci that the primary antifeedant effects of azadirachtin are persistent for a period of at least 17 weeks with little or no reduction in escacy.

Cnide neem extracts, neem oil, and purified azadirachtin solutions might be used as an alternative in situations where spraying is the main method of insect control. In such situations neem products contacting the immature stages of a beetle pest species, would lead to mortality, reduced fitness, possible secondary antifeedant effects, and adverse effects on the next generation. Ln addition, pest beetles not contacted directiy by the spray may exhibit a primary antifeedant effect to droplets of neem product remaining in the environment Neem residues would thus provide extended protection for the stored products. If neem products do not provide adequate control, they could be used in conjunction with culturai methods such as heat and cold. The combined growth regulating, toxic, primary antifeedant, and secondary antifeedant effects of neem products might provide adequate protection to stored products and reduce the use of dangerous synthetic chernical pesticides.

Cnide neem products and purified azadïrachtin were tested and found to be effective against D. macuIafus.but these neem products are in a fomi that could only be applied to a limited number of situations. There are severai problems associated with neem oil and crude neem extracts which limit theu uschiness in stored product situations. Neem oil alone is

ditncult to apply because of its consîstency; and both nemi oil and deneem ex- might

stain certain materials, especially fabrics. Ail neem products are extremely bitter, which might

affect their application where food products are concemed. Nevertheless it is important that

research be conducted on the applied use of neem chernicals in non-agriculturaj interior

storage situations. If effective methods for the application of neem products are found they

might prove to be a useful alternative to synîhetic chemical pesticides.

Since the neem products testcd are effective against D. maculutus, a representative

species of Dermestidae because it has similar lifecycle, ecology and feeding habits, it is likely

that they might also be effective in controllhg 0th- beetle pests of this farnily. The methods

used in this study, specifically those used to test the primary and secondary antifeedant effects,

could be applied to other beetle pests of non-agricultural interior store products. This research

demonstrated the effcctiveness of neem products under laboratory conditions approximating

an environment which might be found in storage facilities. A logicai extension of this work

would be to test the efficacy of neem products in storage facilities.

The dangers of synthetic chemical pesticides used in agriculture have prompted a

movernent towards the use of alternative pcst control methods. Synthetic chemicd pesticides

used in interior storage situations arc no less dangerou and are associated with many hurnan health problems. In the sarch for safer control methods for pests of intenor stored products, more attention should be paid to the potential of neem products and other botanical insecticides. Botanical pesticides might a part of the solution as they have been in agriculture. 7. References

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Analysis of FWh Food and Cat Food Used in Erperiments:

Fish Food: Wardley Goldfish Flakes

Cnide Protein: 30%

Cnide Fat: 4%

Cnide Fibre: 7%

Moisture: 9%

Cat Food: Purina Original Cat Chow

Cnide Protein: 70%

Crude Fat: 8%

Cnide Fibre: 4.5%

Moisture: 12%