Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The agricultural industry in South Africa is dominated by the use of insecticides. Producers rely heavily on chemicals that cause increased risk to health, the environment and ecology, rapid resistance development in key insect pests and pesticide residues on crops. The increased concern regarding the impact of these pest management practices on the environment and alternative pest management strategies are being investigated. Entomopathogenic nematodes (EPNs) have been identified as being promising biological control agents of key insect pests. The two EPN genera that have shown promise for use as biological control agents within an integrated pest management programme areSteinernema and Heterorhabditis. Commercialisation and the successful use of EPNs to control pests in North America, Australia, Europe and Asia have confirmed the effectiveness of these organisms as biological control agents. Unfortunately, EPNs in large enough numbers for commercial field applications are not yet available on the South African market. Large numbers of EPNs can be produced through either in vivo or in vitro culturing practices. The objective of this study was to streamline the in vivo production process by using two endemic EPN species, Heterorhabditis zealandica (SF41) and H. bacteriophora (SF351). These EPN isolates have been shown to be effective control agents of codling moth Cydia pomonella, false codling moth Thaumatotibia leucotreta, obscure mealybug Pseudococcus viburni, and the banded fruit weevil Phlyctinus callosus. A comparative study was conducted to identify suitable host insects for EPN production of local H. zealandica (SF41) and H. bacteriophora (SF351) strains. Hosts were selected according to their susceptibility to the two EPN species used, their general availability and the ease and cost of rearing. Wax moth larvae Galleria mellonella (WML) and mealworms Tenebrio molitor (MW) were selected as hosts. In order to produce nematodes of consistent quality, a continuous source of host insects reared on a standardised diet was required. WML and MW were each reared on five different diets in the dark at ±26°C. A superior diet for each host was selected according to the diet that produced, on average, the larvae with the highest body mass within a specific timeframe. The heaviest WML, at an average weight of 0.19 g per larva, were produced on a diet consisting of 118 g wheat flour, 206 g wheat bran, 118 g milk powder, 88 g brewer‟s yeast, 24 g wax powder, 175 ml honey and 175 ml glycerol. The heaviest MW larvae weighed, on average, 0.0154 g per larva, and were produced on a diet consisting of 100% wheat bran. To confirm the hypothesis that a linear relationship exists between the weight of a host and the number of nematodes produced from that host, a study was conducted to determine the number of H. zealandica and H. bacteriophora produced per g of host. WML, MW, codling moth larvae and false codling moth larvae were weighed individually and inoculated with the two nematode species respectively. In addition, nematode production in frozen MW and WML was tested. The number of nematodes harvested from each host was counted, and the average number of nematode progeny produced in each host was calculated. A significant linear correlation between the weight of WML and MW and the number of H. zealandica and H. bacteriophora respectively produced confirmed the hypothesis that nematode production within the specified host increases with an increase in host weight. WML produced the highest number of H. zealandica and H. bacteriophora per g of host (1 459 205 ± 113 670 and 1 898 512 ± 94 355), followed by MW larvae (836 690 ± 121 252 and 414 566 ± 67 017). Lower numbers of H. zealandica and H. bacteriophora per g codling moth (57 582 ± 10 026 and 39 653 ± 8 276) and per g false codling moth (192 867 ± 13 488 and 97 652 ± 23 404) were produced. Successful infection of a suitable insect host is one of the key factors in an efficient in vivo nematode production process. Three inoculation techniques were compared using H. zealandica and H. bacteriophora: inoculation with a pipette; shaking of hosts in the nematode inoculum; and immersion of hosts in the nematode suspension. With each inoculation technique, WML and MW were used as host larvae and were inoculated with nematodes at a concentration of 200 infective juveniles (IJs) / larva. The percentage mortality of insect hosts was determined after two days, and EPN infectivity, confirmed by colour change and dissection, after seven days. The highest percentage EPN infection was obtained using pipetting for both nematode isolates and hosts. Nematode infection rates for all nematode-host combinations obtained with pipetting were above 90%, with the exception of MW inoculation with H. bacteriophora, where the percentage of infection obtained was 76%. The current study conclusively demonstrated that variations in infection levels occur, depending on the inoculation technique used. In an additional effort to enhance infectivity during inoculation, H. zealandica, H. bacteriophora and MW were subjected to host-stressor regimes and to nematode- infectivity-enhancing additives. Three treatments, plus a control treatment, were compared. Exposing MW to 70°C tap water prior to inoculation did not increase infection levels. On the contrary, reduced infection levels were observed with host immersion in 70°C tap water followed by inoculation with H. bacteriophora, compared to the control. Only 12% infection was obtained compared to the 48% infection achieved in the control. Infection obtained using H. zealandica was 21%. Treating H. zealandica and H. bacteriophora IJs withMn2+SO4.H20 in a suspension, prior to inoculating MW, did not significantly enhance nematode virulence. Inoculation of MW with treated H. zealandica IJs led to an infection rate of 81%, compared to the control, with which 80% infection rate was obtained. Heterorhabditis bacteriophora caused 47% MW infection, compared to the control, which was subject to 48% infection. A combination of the two above-mentioned treatments did not enhance the infection levels either. Immersing MW into 70°C tap water prior to inoculation with nematodes treated with Mn2+SO4.H20 led to infection levels of 13% and 9% respectively when H. bacteriophora and H. zealandica were used. Future research is required to optimise the protocol used in this study of subjecting MW and local nematode isolates to stressor regimes. The ability of two formulations to maintain biological activity and virulence of H. zealandica was investigated. A quality standard control measure was used to measure the percentage survival and virulence of formulated H. zealandica over a period of 21 days. IJs were formulated into Pesta granules and coconut fibres, while nematodes stored in tap water served as the control. The numbers of live H. zealandica in Pesta granules and coconut fibres decreased drastically after seven days of storage. The survival of nematodes in Pesta granules dropped to 9.79% after 21 days compared to the control, where the survival rate was 79.79%. Nematode survival in coconut fibres was even lower, at 25.84% after seven days and 2.25% after 21 days. After 21 days in storage, 100%+of nematodes survived in the control for coconut fibres. The application of the standard quality control measure, which was used to determine the virulence of formulated H. zealandica, proved to be ineffective. Higher MW mortality rates were obtained in the control where no nematodes were added to larvae, compared to where nematodes were added in varying dosages. However, adjusting certain aspects in the protocol of this quality control measure specifically to accommodate local conditions could possibly make it a more effective tool for measuring endemic nematode virulence. / AFRIKAANSE OPSOMMING: Die landboubedryf in Suid-Afrika word oorheers deur die gebruik van insekdoders. Vervaardigers steun swaar op chemikalieë wat toenemend gesondheids-, omgewings- en ekologiese risiko's, asook die snelle ontwikkeling van weerstand in sleutelinsekteplae veroorsaak, en wat reste van plaagdoders op gewasse laat. Na aanleiding van toenemende besorgdheid oor die impak van hierdie plaagbestuurspraktyke op die omgewing, word alternatiewe plaagbestuurstrategieë ondersoek. Entomopatogeniese nematodes (EPNs) is geïdentifiseer as belowende biologiese beheeragente van sleutelinsekteplae. Die twee EPN genera wat belofte inhou vir gebruik as biologiese beheeragente binne 'n geïntegreerde plaagbestuursprogram is Steinernema en Heterorhabditis. Kommersialisering en die geslaagde gebruik van EPNs om insekplae te beheer in Noord-Amerika, Australië, Europa en Asië, het die doeltreffendheid van hierdie organismes as biologiese beheeragente bevestig. Ongelukkig is EPNs in groot genoeg getalle vir kommersiële aanwending in die veld nog nie op die Suid-Afrikaanse mark beskikbaar nie. Groot getalle EPNs kan deur in vivo en in vitro teling verkry word. Die doelwit van hierdie studie was om die in vivo produksieproses te stroomlyn deur die gebruik van twee endemiese EPN spesies, Heterorhabditis zealandica (SF41) en H. bacteriophora (SF351). Hierdie EPN isolate is deur navorsing bewys om doeltreffende beheeragente van kodlingmot Cydia pomonella, vals kodlingmot Thaumatotibia leucotreta, ligrooswitluis Pseudococcus viburni, en gebande vrugtekalanders Phlyctinus callosus te wees. 'n Vergelykende studie is gedoen om geskikte gasheerinsekte vir EPN produksie van plaaslike H. zealandica (SF41) en H. bacteriophora (SF351) isolate te vind. Gashere is geselekteer op grond van vatbaarheid vir die EPN spesie wat gebruik word, en algemene beskikbaarheid en gemak en koste van teling. Wasmotlarwes Galleria mellonella (WML) en meelwurms Tenebrio molitor (MW) is as gashere gekies. Ten einde nematodes van konsekwente kwaliteit te teel, word 'n deurlopende bron van gasheerinsekte benodig wat op 'n gestandaardiseerde dieet voed. WML en MW is onderskeidelik op vyf verskillende diëte geteel by ±26°C in die donker. Die beste dieet vir elke gasheer is gekies op grond van die dieet wat, gemiddeld, die swaarste larwes binne 'n spesifieke tydsraamwerk opgelewer het. Die swaarste WML, teen 'n gemiddelde massa van 0.19 g per larwe, is geteel op 'n dieet wat bestaan het uit 118 g koringmeel, 206 g semels, 118 g melkpoeier, 88 g brouersgis, 24 g verpoeierde was, 175 ml heuning en 175 ml gliserol. Die swaarste MW larwes het gemiddeld 0.0154 g per larwe geweeg en is geteel op 'n dieet van 100% semels. Ten einde die hipotese te bevestig dat 'n lineêre verwantskap bestaan tussen die massa van 'n insekgasheer en die aantal nematodes wat deur daardie gasheer geproduseer word, is 'n studie gedoen om die aantal H. zealandica en H. bacteriophora per gasheergram te bepaal. WML, MW, kodlingmotlarwes en vals kodlingmotlarwes is individueel geweeg en met infektiewe larwes van die twee onderskeidelike EPN spesies geïnokuleer. Daarbenewens is die vermeerdering van nematodes in bevrore MW en WML ook getoets. Die aantal nematodes wat in elke gasheer geoes is, is getel, en die gemiddelde nematode-afstammelinge in elke gasheer bereken. 'n Beduidende lineêre korrelasie tussen die massa van WML en MW en die aantal H. zealandica en H. bacteriophora wat onderskeidelik geproduseer is, het die hipotese bevestig dat nematode-vermeerdering binne hierdie gashere toeneem namate die gasheermassa toeneem. WML het die meeste H. zealandica en H. bacteriophera per gasheergram opgelewer (1 459 205± 113 670 en 1 898 512± 94 355 onderskeidelik), gevolg deur MW larwes (836 690± 121 252 en 414 566± 67 017 onderskeidelik). Laer getalle H. zealandica and H. bacteriophora per gram kodlingmot (57 582 ± 10 026 en 39 653 ± 8 276) en per gram vals kodlingmot (192 867 ± 13 488 en 97 652 ± 23 404) is egter geproduseer. Een van die sleutelfaktore vir die doeltreffendheid van die in vivo vermeerdering van nematodes is geslaagde gasheerinfeksie. Drie inokulasietegnieke is dus geëvalueer en vergelyk deur H. zealandica en H. bacteriophora te gebruik: inokulasie met 'n pipet, skud van gashere in 'n nematode-inokulum, en gasheerindompeling in 'n nematode-suspensie. WML en MW is as gashere gebruik vir elke inokulasietegniek, en is geïnokuleer met nematodes wat uit 'n konsentrasie van 200 infektiewe larwes (ILs) / insek larwe bestaan het. Die persentasie dooie insekgashere is na twee dae bepaal, en infeksie soos bevestig deur kleurverandering en disseksie, na sewe dae. Die hoogste persentasie infeksie deur sowel nematode-isolate as gashere te gebruik, was met die pipet-tegniek. Die infeksiekoerse vir alle nematode-gasheerkombinasies met die pipet-tegniek was hoër as 90%, met die uitsondering van MW-inokulasie met H. bacteriophora, waar die infeksie 76% was. Hierdie studie toon dat afwykings voorkom in die mate van gasheerinfeksie, na gelang van die inokulasietegniek wat gebruik is. In 'n bykomende poging om infeksie na inokulasie te verhoog, is H. zealandica, H. bacteriophora en MW onderwerp aan stressors en bymiddels om nematode-infeksie te bevorder. Drie behandelings, asook 'n kontrole-behandeling, is vergelyk. Infeksievlakke het nie verhoog deur MW voor inokulasie aan kraanwater van 70°C bloot te stel nie. Inteendeel, laer infeksievlakke is opgemerk waar gashere in kraanwater van 70°C gedompel is en daarna met H. bacteriophora geïnokuleer is, vergelyke met die kontrole. Gasheerinfeksie van slegs 12% is verkry, vergelyke met 48% in die kontrole. Infeksie van 21% is met H. zealandica verkry. Die virulensie van nematodes het nie beduidend toegeneem deur H. zealandica en H. bacteriophora IL in 'n suspensie met Mn2+SO4H20 te behandel voor MW geïnokuleer is nie. Inokulasie van MW met behandelde H. zealandica IL het tot 'n infeksie van 81% gelei, vergelyke met die kontrole waar 'n infeksie van 80% behaal is. H. bacteriophora het 'n MW-infeksie van 47% veroorsaak, vergelyke met die kontrole se infeksiekoers van 48%. 'n Kombinasie van die twee bogenoemde behandelings het eweneens nie gasheerinfeksievlakke verhoog nie. Die indompeling van meelwurms in kraanwater van 70°C voor inokulasie met nematodes wat met Mn2+SO4H20 behandel is, het tot gasheerinfeksie van 13% en 9% onderskeidelik gelei wanneer H. bacteriophora en H. zealandica gebruik is. Toekomstige navorsing is nodig om die protokol te verbeter wat in hierdie studie gebruik is om MW en plaaslike nematode-isolate aan stressors te onderwerp. 'n Ondersoek is gedoen na die vermoë van twee formulasies om biologiese aktiwiteit en virulensie van H. zealandica te onderhou. 'n Kwaliteitsstandaardtegniekis gebruik om weekliks die persentasie oorlewing en virulensie van geformuleerde H. zealandica oor 'n tydperk van 21 dae te meet. IL is in Pesta korrels en klappervesel geformuleer, terwyl nematodes in kraanwater gedien het as kontrole. Die aantal lewende H. zealandica in Pesta korrels en klappervesel het drasties verminder na sewe dae in die formulasie. Oorlewing van nematodes in Pesta korrels het gedaal tot 9.79% na 21 dae vergyleke met die kontrole, waar 79.79% oorleef het. Nog minder nematodes - 25.84% - het na sewe dae in die klappervesel oorleef, en slegs 2.25% na 21 dae. Na 21 dae van berging het 100%+ van nematodes oorleef in die kontrole vir klappervesel. Die toepassing van die kwaliteitsstandaardtegniek om die virulensie van geformuleerde H. zealandica te bepaal, het ondoeltreffend geblyk. Verhoogde MW sterftesyfers is verkry in die kontrole waar geen nematodes by die inseklarwes gevoeg is nie, vergelyke met die byvoeging van hoër dosisse nematodes. Nietemin, die aanpassing van sekere aspekte in die protokol van hierdie kwaliteitsbeheermeting om spesifiek plaaslike toestande in ag te neem, sou dit moontlik 'n meer doeltreffende middel kon maak om die virulensie van endemiese nematodes te bepaal.

Thesis (MSc (Conservation Ecology and Entomology))--Stellenbosch University, 2009. / ENGLISH ABSTRACT: An understanding of the interactions of herbivores, plant nutrients and soil properties is crucial for grazing management. Of particular interest are plant palatability and herbivore grazing preferences. These aspects, the focus of this thesis, were studied in a Swartland Shale Renosterveld and Swartland Alluvium Fynbos mosaic in the Western Cape of South Africa. This vegetation is classified as critically endangered due to the transformation of its landscape in favour of agricultural production and urban settlement. Pre-colonial herbivore vertebrates were hunted out by 1700, leading to a sparse knowledge of the early dynamics of the vegetation. However, currently game farmers and landowners are re-introducing selected species into the area. Thus, managers require knowledge on how to implement practical guidelines for best-practice grazing management. This study investigates, firstly, the relationship between plants and animals. This included seasonal assessment of seed germination from dung of bontebok and eland; the effect of stocking rates on plant palatability (crude protein, crude fibre, crude fat, moisture, tannin and mineral) of herbage was studied with the aim to understand how palatability influences herbivore forage decisions. Secondly, this study looked at how stocking rates influence soil nutrients (nitrogen, phosphorus), and others soil properties such as pH and soil moisture. The distribution of alien grasses was facilitated by grazers. About 58 % of grasses germinated from the dung of eland and Bontebok were alien species. Additionally, there was significant variation in palatability related to grazing pressure and plant maturity between sites and over seasons. Grasses on the high grazing site had higher carbohydrate (3%-5% higher) and protein content (1%-5% higher) than at the least grazed site. Mature grasses contained less water (10%-20%), but no obvious variation in tannin concentration than immature grasses. However, species such as Ficinia sp. showed variations with grazing pressure and maturity. Ficinia sp. on the high grazing site had higher tannin concentration (5mg/ml-22mg/ml) and crude protein (4%-9%) than at the least grazed site. When the plant ages, tannin concentration increase (ranging from 15% to19% higher). Two types of plant were recoreded according to their chemical response following grazing pressure. These types are 1) plants that are grazing tolerant and which produce more carbohydrate when grazed and 2) plants that are grazing intolerant under high grazing pressure and which produce chemical defence compounds such as tannin to deter herbivores attacks. The results indicate that in fynbos and renosterveld, the relationship between preference and plant chemical compounds is not consistent since no IV patterns emerged to explain what compounds drive preference. A combination of chemical compounds may be the reason for the selection by the grazers; alternatively, other compounds not included in the study may influence the forage selection by an herbivore. The second part of the study showed that stocking rates appeared to have a significant effect on soil properties investigated. Soil moisture was significantly affected by the stocking rate in autumn. The high grazing intensity site had the lowest soil moisture especially in autumn (10% lower than the control site), likely due to heavy trampling and soil compaction. pH was the lowest at the high grazing intensity site in all seasons compared to the control site. Likely reasons were the high deposition of nitrogen through dung and urine deposition, high removal of basic cations and animal exportation through hunting activities. Lastly, grazing maintains inorganic nitrogen at stable levels regardless of seasonal changes and increases the concentration of phosphorus especially under high grazing pressure compare to less grazed sites. The grazing dynamics of Swartland Shale Renosterveld - Swartland Alluvium Fynbos mosaic system are complex and multifaceted. Farmers need to pay attention to the nutritional status of plant species grazed to know whether they meet the nutritional requirements of the game in the area. Overstocking should be avoided in this system as it triggers the production of tanniferous compounds that could decrease the survivorship of herbivores. Moderate grazing 0.09 LAU/ha pressure provides the best stocking rates for effective grazing management. / AFRIKAANSE OPSOMMING: Vir suksesvolle weidingsbestuur is dit noodsaaklik om die interaksie tussen herbivore, plantvoedingstowwe en grondeienskappe te verstaan. Van besondere belang is plantsmaaklikheid en herbivore se weidingsvoorkeure. Hierdie aspekte, wat die fokus van hierdie tesis is, is bestudeer in ‘n Swartland Shale Renosterveld en Swartland Alluvium Fynbos-mosaïek in die Wes-Kaap Provinsie van Suid-Afrika. Hierdie plantegroei word as kritiek bedreigd geklassifiseer weens die transformering van sy landskap ten gunste van landbou-aktiwiteite en stedelike vestiging. Pre-koloniale herbivoor vertebrata is teen 1700 deur jagters uitgeroei, wat gelei het tot min kennis oor die vroeë dinamiek van die plantegroei. Wildsboere en grondeienaars is egter besig om geselekteerde spesies in die area te hervestig. Bestuurders moet dus weet hoe om praktiese riglyne vir beste-praktyk weidingsbestuur te implementeer. Hierdie studie ondersoek eerstens die verhouding tussen plante en diere. Dit sluit seisoenale evaluering van saadontkieming uit die mis van bontebokke en elande in; die effek van veegetalle op plantsmaaklikheid (ruproteïen, ruvesel, ru-vet, vogpeil, tannien en minerale) van die weiveld is bestudeer om vas te stel hoe plantsmaaklikheid herbivore se weidingsbesluite beïnvloed. Tweedens het die studie die invloed van veegetalle op grondvoedingstowwe (stikstof, fosfor) ondersoek, asook ander grondeienskappe soos pH en grondvogpeile. Die verspreiding van uitheemse grasse is deur weidende diere gefassiliteer. Ongeveer 58% van alle grasse wat uit die mis van elande en bontebokke ontkiem het, was uitheemse spesies. Verder was daar beduidende variasie in plantsmaaklikheid verwant aan beweidingsdruk en plantvolwassenheid tussen verskillende persele en oor seisoene. Grasse op die hoogs beweide persele het 3%-5% hoër koolhidraatinhoude en 1%-5% hoër proteïeninhoude gehad as die minder beweide persele. Volwasse grasse het 10%-20% minder water bevat as onvolwasse grasse, maar het nie in terme van tannienkonsentrasie van die onvolwasse grasse verskil nie. Spesies soos Ficinia sp. het variasies getoon met beweidingsdruk en volwassenheid. Ficinia sp. het op die hoog-beweide perseel ‘n hoër tannienkonsentrasie (5mg/ml-22mg/ml) en meer ruproteïen (4%-9%) gehad as op die minste beweide perseel. Wanneer die plant verouder, verhoog die toename in tannienkonsentrasie (met tussen 15% en 19%). Twee tipes plante is aangeteken volgens hul chemiese response ná beweidingsdruk. Hierdie tipes is 1) plante wat beweidingstolerant is en wat meer koolhidrate produseer wanneer hulle as weiding dien en 2) plante wat onder hoë beweidingsdruk beweidingsintolerant is en wat chemiese verbindings VI soos tannien produseer om herbivooraanvalle af te weer. Die resultate dui aan dat die verhouding tussen voorkeur en plante se chemiese verbindings nie konstant bly in fynbos en renosterveld nie, aangesien geen patrone na vore getree het wat kon verklaar watter verbindings bepalend is vir voorkeur nie. ‘n Kombinasie van chemiese verbindings mag die rede wees waarom die herbivore sekere plante verkies; alternatiewelik mag ‘n herbivoor se plantseleksie beïnvloed word deur chemiese verbindings anders as dié wat in hierdie studie ondersoek is. Die tweede deel van die studie het aangetoon dat veegetalle blyk ‘n beduidende invloed te hê op die grondeienskappe wat ondersoek is. Grondvogpeile is in die herfs beduidend beïnvloed deur die veegetalle. Die perseel met die hoë beweidingsintensiteit het die laagste grondvogpeil gehad, veral in die winter (10% laer as dié van die kontrole-perseel), waarskynlik weens erge getrappel en grondkompaktering. Vergeleke met die kontrole-perseel was pH die laagste op die perseel met die hoë beweidingsintensiteit, in alle seisoene. Waarskynlike redes hiervoor is die hoë neeerslag van stikstof deur mis- en uriene-neerslae, hoë verwydering van basiese katione en dierevervoer wat deur jagaktiwiteite teweeg gebring word. Laatstens behou weiding anorganiese stikstof teen stabiele vlakke ongeag van seisoenale veranderinge en toenames in die konsentrasie van fosfor, veral onder hoë beweidingsdruk vergeleke met minder beweide persele. Die weidingsdinamiek van die Swartland Shale Renosterveld - Swartland Alluvium Fynbosmosaïeksisteem is kompleks en veelvlakkig. Boere behoort aandag te skenk aan die voedingstofstatus van die plantspesies wat as weiding dien, sodat hulle kan weet of die voedingsbehoeftes van die wild in die omgewing bevredig word. Die aanhou van te veel vee in hierdie sisteem behoort vermy te word aangesien dit as sneller dien vir die produksie van tannienbevattende verbindings wat die oorlewingsvermoë van herbivore kan verlaag. Matige beweidingsdruk (0.09 LAU/ha) bied vir effektiewe weidingsbestuur die beste veegetalle.

<p>North American <i>Speyeria </i>butterflies are a group whose species hypotheses are confounded by shared wing color patterns between sympatric populations of closely related recognized species due to a putatively recent origin in evolutionary time. Previous studies of this group and the closely related Palearctic genus <i>Argynnis</i>, suggest that <i>Speyeria </i>is monophyletic but derived from within <i>Argynnis</i>. Sampling in these studies has either involved few basal <i>Speyeria</i> species, or too few <i>Argynnis</i> species (Simonsen 2006, Simonsen <i>et al. </i>2006). Thus, no comprehensive phylogenetic analysis exists for all members that answers the question of monophyly of <i>Speyeria</i>, or other subgeneric taxa,and their relationship to <i>Argynnis </i>species. A phylogenetic analysis was completed of all North American <i>Speyeria </i>species and nearly all species within <i>Argynnis</i>, using one mitochondrial (CO1) and four nuclear genes (EF1?, WG, GAPDH, and RPS5). The results indicate that North American <i>Speyeria</i> is a monophyletic group, but that Palearctic <i>Argynnis</i> is paraphyletic. Three major lineages are identified within <i>Argynnis sensu lato</i>: two Palearctic and one containing both Palearctic and Nearctic species. <i>Argynnis</i> species representing the subgenera <i>Argyreus</i>, <i>Argyronome</i>, <i>Childrena</i>, <i>Damora</i>,<i> Pandoriana, </i>and <i>Nephargynnis</i>, belong to a well-supported lineage that split early in the evolution of the group and is comprised of species with long branches. <i>Fabriciana</i> and <i>Mesoacidalia</i> were both recovered as strongly supported lineages, except for <i>A. clara</i> which was recovered as sister to <i>Speyeria</i>. In summary, the phylogenetic analyses suggest the need for reorganization into three genera: <i>Argynnis</i>, <i>Fabriciana</i>, and <i>Speyeria</i>. The results have implications for the conservation of these butterflies across the temperate zone by providing a framework for understanding potential gene flow between sympatric species complexes, proper taxonomic validity, and the natural history of threatened populations of <i>Speyeria </i>and <i>Argynnis </i>butterflies.

<p> Eastern Equine Encephalitis virus is a vector-borne virus amplified by the <i>Culiseta melanura</i> mosquito in an enzootic avian cycle, causing high morbidity and mortality to horses and humans when contracted as incidental hosts. The virus is distributed across most of the eastern United States, Canada, and Gulf coast, and has been expanding in geographic range and season of activity over time. Spatial-temporal trends in <i>Cs. melanura </i> abundance were correlated with available meteorological (temperature and precipitation) and remotely sensed environmental data for the period of 1997-2012 in Connecticut. The effects of inter-annual changes in precipitation, temperature, and groundwater levels on <i>Cs. melanura</i> abundances using time-series linear regression and cross-correlation analyses were inconclusive. Habitat modeling using logistic regression and landscape-based predictive variables demonstrated strong efficiency (46.2%) and acceptable sensitivity and specificity (65.6 and 78.6%, respectively) using NDVI difference and distance from palustrine areas as predictive factors. Remotely sensed data can improve the understanding of vector abundance patterns, helping to forecast future outbreaks and regional expansions by guiding surveillance efforts. </p>

Grasshoppers are able to spontaneously drop, or autotomize, their large metathoracic legs, at the joint between the second and third leg segments. Previous work demonstrated that in the grasshopper, Barytettix psolus, autotomy transneuronally induces atrophy in a group of six metathoracic muscles on the ipsilateral side. These muscles are neither damaged nor denervated by the loss of the leg. The experiments presented in this dissertation support and extend the earlier work by including twelve additional metathoracic muscles, one of which was discovered as a result of these studies. These experiments demonstrated that atrophy varied both between muscles, as well as between fibers within individual muscles. Some muscles degenerated uniformly, whereas others contained persisting fibers long after neighboring fibers degenerated. Extent of atrophy in each muscle was quantified by measurement of total protein content. Histochemical staining of myofibrillar ATPase activity in a subset of muscles indicated that slow type fibers comprised the majority of those persisting following autotomy. Immunohistochemical experiments suggested that persistance of muscle fibers was correlated with innervation both by motor neurons (MNs) containing proctolin-like immunoreactivity (PLI), and by an inhibitory MN containing gamma-aminobutyric acid (GABA)-like immunoreactivity (GLI). The distributions of PLI and GLI indicated that these two types of MNs innervate muscles in parallel. In an attempt to identify the proctolin-like substance, muscle extracts of fourteen muscles were purified by HPLC and subjected to bioassay for proctolin-like bioactivity (PLB). Nine muscles with PLI also contained PLB. These experiments support the presence of proctolin in muscles with PLI. Four out of the five muscles lacking PLI also contained PLB. It is likely that these muscles contain at least one bioactive peptide that is biochemically similar to proctolin. These experiments are the first to suggest the association of proctolin-like substances with the majority of leg muscles in any insect. My immunohistochemical data combined with the results of previous experiments on the functions of proctolinergic and GABAergic innervation suggest that proctolin or proctolin-like substances are commonly used to maintain postural tonus in metathoracic leg muscles during standing and locomotory activities.

<p>Ecological risk assessment (ERA) is a framework for monitoring risks of exposure and adverse effects of environmental stressors to populations or communities of interest. One tool of ERA is the biomarker, which is a characteristic of an organism that reliably indicates exposure to or effects of a stressor like chemical pollution. Traditional biomarkers which rely on characteristics at the tissue level and higher often detect only acute exposures to stressors. Sensitive molecular biomarkers may detect lower stressor levels than traditional biomarkers, which helps inform risk mitigation and restoration efforts before populations and communities are irreversibly affected. In this study I developed gene expression-based molecular biomarkers of exposure to metals and insecticides in the model toxicological freshwater amphipod Hyalella azteca. My goals were to not only create sensitive molecular biomarkers for these chemicals, but also to show the utility and versatility of H. azteca in molecular studies for toxicology and risk assessment. I sequenced and assembled the H. azteca transcriptome to identify reference and stress-response gene transcripts suitable for expression monitoring. I exposed H. azteca to sub-lethal concentrations of metals (cadmium and copper) and insecticides (DDT, permethrin, and imidacloprid). Reference genes used to create normalization factors were determined for each exposure using the programs BestKeeper, GeNorm, and NormFinder. Both metals increased expression of a nuclear transcription factor (Cnc), an ABC transporter (Mrp4), and a heat shock protein (Hsp90), giving evidence of general metal exposure signature. Cadmium uniquely increased expression of a DNA repair protein (Rad51) and increased Mrp4 expression more than copper (7-fold increase compared to 2-fold increase). Together these may be unique biomarkers distinguishing cadmium and copper exposures. DDT increased expression of Hsp90, Mrp4, and the immune response gene Lgbp. Permethrin increased expression of a cytochrome P450 (Cyp2j2) and decreased expression of the immune response gene Lectin-1. Imidacloprid did not affect gene expression. Unique biomarkers were seen for DDT and permethrin, but the genes studied were not sensitive enough to detect imidacloprid at the levels used here. I demonstrated that gene expression in H. azteca detects specific chemical exposures at sub-lethal concentrations, making expression monitoring using this amphipod a useful and sensitive biomarker for risk assessment of chemical exposure.

We report on susceptibility to insecticides of whiteflies (Bemisia tabaci) collected from cotton, melons and ornamental plants during the 2004 season. No major problems with field performance of insecticides against whiteflies were observed or reported in 2004 in Arizona cotton, vegetables, or melons. However, monitoring revealed further statewide reduction in susceptibility to pyriproxyfen (Knack®) and showed that whiteflies possessing pyriproxyfen resistance could be detected in all low desert areas of the state. Susceptibility to buprofezin (Applaud®/Courier®) has not changed significantly since 1997. Mean susceptibility to synergized pyrethroids (e.g., Danitol® + Orthene®) has increased strikingly on a statewide basis since 1995 though highly resistant whiteflies were detected in some collections from cotton, melons and ornamentals. Whiteflies from throughout Arizona continued to be highly susceptible to imidacloprid (Admire®/Provado®). However, susceptibility to the related neonicotinoid insecticide, acetamiprid (Intruder®) varied widely and was lowest in collections from melons and greenhouse plants. Whiteflies from cotton that were least susceptibile to acetamiprid were significantly less susceptible to a second neonicotinoid, thiamethoxam (Actara®/Centric®/Platinum®). The most worrisome findings of our 2004 studies stemmed from detection of a strain of B. tabaci, at a retail nursery, that was essentially unaffected by pyriproxyfen in egg bioassays. It also possessed strikingly reduced susceptibility to acetamiprid, buprofezin, mixtures of fenpropathrin and acephate, imidacloprid, and thiamethoxam. This strain was found to be a biotype of B. tabaci previously undescribed in the US, the Q biotype. We cannot predict with accuracy the timecourse of future resistance problems or the spread and impact of this new whitefly biotype. However, our findings point to the need to formulate contingency plans for management of resistance, in order to insure that Arizona agriculture does not revisit the severe whitefly control problems experienced in the past.

The follicle cell-specific (FCS) gene, a 3,023 bp gene specific to follicle cells of the mosquito, Aedes aegypti, was characterized at the nucleotide level. Genomic Southern blots demonstrated that there was only one copy of this gene in the A. aegypti genome. An ovary-specific cDNA library was constructed from female mosquitoes 24 hours post blood meal. Then a cDNA clone containing the complete coding region was identified, and its nucleotide sequence was determined. The deduced protein contained unusually high levels of alanine and proline. Search of a protein data base revealed that the FCS gene was similar to Drosophila vitelline membrane protein genes, with 47.5% similarity in nucleotide sequence and 46.7% similarity in amino acid sequence. The conserved hydrophobic regions from several vitelline membrane proteins were compared. Another cDNA clone, 1D, was isolated from the cDNA library screened with the FCS gene. These two mosquito genes shared a 60% similarity at the nucleic acid level and a 79.8% similarity at the amino acid level.

Antisera against leucokinin IV were used to test for the presence of leucokinin-like peptides in the central nervous systems of Nauphoeta cinerea, Acheta domesticus, Schistocerca americana, Aedes aegypti, Manduca sexta and Apis mellifera. Leucokinin immunoreactive neurosecretory cells were found in the pars intercerebralis and pars lateralis of the brains of N. cinerea, A. domesticus, A. aegypti, but not in the brains of S. americana, M. sexta and A. mellifera. The neurohemal release sites were also very different among species. In contrast, the distribution patterns of leucokinin immunoreactive neurosecretory cells were very similar among abdominal ganglia of all six species. The identity of the leucokinin immunoreactive material in the brain, corpora cardiaca and abdominal ganglia of N. cinerea was shown by HPLC combined with ELISA. In N. cinerea and A. domesticus, leucokinin and vasopressin were found to co-localize in the same neurosecretory cells. In M. sexta, leucokinin and diuretic hormone co-localized in the same neurosecretory cells in abdominal ganglia, but not in the brain.

In Diptera, small-field retinotopic neurons supply achromatic motion sensitive neurons in the lobula plate, a region thought to control stabilization of yaw, pitch, and roll during flight. If different body shapes, and the flight performance associated with them, place specific demands on the design of control pathways, it might be expected that flies with long bodies and high aerodynamic pitch stability would show specific differences in the organization of such magnocellular achromatic networks. Striking differences emerge in the comparison of 9 species. Tipulids and culicids possess small non-stratified lobula plates, containing only 2-3 large diameter neurons. In syrphids, all VS cells have wide diameters and overlapping fields covering 2-3 times more of the mosaic than those of calliphorids. In asilids (robber flies) vertical cells cannot be identified. Despite these differences, we observe a conserved relationship between terminals of lamina afferents with neurons in the medulla.