Optimization of formulated artificial diets with the addition of sterols and cryoprotectants for effective rearing and fitness of Eldana saccharina Walker (Lepidoptera: Pyralidae)

Thesis (MScConsEcol)--Stellenbosch University, 2021.

ENGLISH ABSTRACT: Eldana saccharina is amongst the most economically damaging pests in South African sugarcane. Many attempts have been made to control the pest. The Sterile Insect Technique (SIT) as part of an Integrated Pest Management (IPM) program against E. saccharina, offers great potential to reduce pest damage to below the economic injury level. Since the success of SIT depends upon the production of high quality, competitive sterile male insects for field releases, it follows that mass- rearing of E. saccharina on artificial diet is a principal step in the process. To this end, three separate trials were conducted to study the nutritional requirements and formulate better and more economical diets for mass rearing E. saccharina. The diet currently used to rear this species was developed from a previously published diet for Ostrinia nubilalis, which, even though it was much more efficient and cost effective than previous diets developed for E. saccharina, it specifically did not take into account the actual nutrient requirements needed for optimal development of E. saccharina. Four artificial diets, based on the following were formulated using the carcass milling technique: the first, formulated according to the minimum specification of a summary of literature diets proven to be effective at rearing E. saccharina (MS); and the other three based on the ideal amino acid composition and profile (IAAP) of the second (IAAP2), third/fourth (IAAP3/4) and fifth/sixth (IAAP5/6) instar larvae. The current diet used at SASRI was used as the control diet (ECBMOD). Survival was significantly high on all diet formulations with more than 92 % of inoculated neonate E. saccharina life stages surviving at day 20 and more than 95 % surviving up till harvest (Day 27). The life stages developed fastest in the IAAP3/4 and MS diets (25 % and 17 % prepupae and pupae produced at day 20, respectively) compared to life stages from the remaining carcass milling diets and the control diet. Within dietary formulations female pupae were significantly heavier (0.1908 g) than male pupae (0.1138 g). Male and female pupal weights were not significantly influenced by the carcass milling diets, as the control diet produced heavier male and female pupae (0.1204 g and 0.2085 g, respectively) compared to them. Adult emergence from pupae was significantly highest (98 %) for the MS diet, followed by the IAAP3/4 (97 %) and control diet (96 %). The sex ratio of adults emerging from pupae harvested from the different diet formulations was close to 1. There were no significant differences observed in male chill coma recovery time (recovery time: 267.60 s) but females from the IAAP5/6 diet recovered the fastest from chill coma treatment. Males from the IAAP5/6 diet mated with significantly more females (6 different females) than those of the remaining diets, who mated with an average of 4 different females. Although not significantly different from the control diet, females from the MS diet mated with more males (3 different males) than those of the remaining carcass milling diets, who mated with an average of 1 male. Females from all diet formulations produced more than 870 eggs that were more than 90 % fertile. The physical properties (pH: 4.79, moisture content: 81.43 % and water activity: 0.92 aw) of the diets were not significantly different, and maintained the quality and stability of the diets produced, ensuring optimal growth and development of E. saccharina throughout the trial duration. Due to the faster larval development of E. saccharina reared on the MS diet, further improvements to this diet were investigated through the inclusion of sterols (cholesterol (C) and stigmasterol (S)), which have been shown to be essential to insect growth and have improved insect performance when fed to them in artificial diets. The larval development period was significantly shortened in the sterol diets, compared to the MS diet without sterols added, irrespective of the type and concentration of sterol added. Larvae that developed fastest, determined by highest percentage of pupae 20 days after diet inoculation, was recorded on the MS (1.0gS) diet (72 %) followed by the MS (0.2gC: 0.2gS) diet (70 %). Pupal weight was increased on females that fed on the MS (0.1gC), MS (0.1gS) and MS (0.2gC:0.2gS) (0.2143 g, 0.2271 g and 0.2252 g, respectively) compared to those of the MS diet without sterols added (0.1864 g). To improve the cold tolerance of this insect to make it more fit for the environmental conditions into which it would be field released, the inclusion of cryoprotectants (i.e. L-proline (P) and trehalose (T)) into the MS diet was also investigated. Pupal weight increased in males (0.1295 g) that fed on the MS (0.2gP:0.2gT) diet and the chill coma recovery time of male and female moths (204.00 s and 259.20 s, respectively) was reduced on E. saccharina reared on this same diet, compared to that of the MS diet without cryoprotectants added (253.20 s and 306.60 s, respectively). The addition of cryoprotectants into the MS diet did not improve fertility of chill coma exposed female moths, but instead it severely reduced fertility to less than 44 %, compared to females not exposed to the chilling treatment whose eggs were on average 84 % fertile. The carcass milling technique proved to be effective at developing superior diets (i.e. the MS and IAAP3/4 diets) than the Ostrinia based diet, and their qualities as a good food source were even more improved when sterols and cryoprotectants were added (particularly in the MS diet) as supplements. The findings of this study demonstrated that the MS diet incorporated with the lower concentration of the sterol mix (0.2gC:0.2gS) and the cryoprotectant mix (0.2gP:0.2gT) can result in a positive impact on E. saccharina’s life history traits, indicating that this species can be effectively mass reared with a significant reduction in rearing time and resultant costs for the SIT program. The MS diet formulation including the sterol mix (0.2gC:0.2gS) is the preferred choice to replace the current diet used to rear E. saccharina at SASRI, as it reduced the larval growth period dramatically by 60 % compared to the other diets in this study, including those incorporating cryoprotectants, without having any negative effects on key quality parameters of E. saccharina.

AFRIKAANSE OPSOMMING: Eldana saccharina is een van die mees skadelikste plae in suikerriet in Suid-Afrika. Daar is baie pogings aangewend om die plaag te bestry. Die Steriele Insek Tegniek (SIT) as onderdeel van 'n geïntegreerde plaagbeheer (IPM) teen E. saccharina, bied 'n groot potensiaal om plaagskade tot onder die ekonomiese beseringsvlak te verminder. Aangesien die sukses van SIT afhang van die produksie van kompeterende steriele manlike insekte van hoë gehalte vir veldvrystelling, volg dit dat die grootmaak van E. saccharina op kunsmatige dieët 'n belangrike stap in die proses is. Vir hierdie doel is drie afsonderlike proewe gedoen om die voedingsbehoeftes te bestudeer en beter en meer ekonomiese diëte vir die grootmaak van E. saccharina te formuleer. Die dieët wat tans gebruik word om hierdie spesie groot te maak, is ontwikkel uit 'n voorheen gepubliseerde dieët vir Ostrinia nubilalis, wat, hoewel dit baie meer doeltreffend en koste-effektief was as vorige diëte wat vir E. saccharina ontwikkel is, het dit nie spesifiek die werklike voedingsstowwe in ag geneem wat benodig word vir optimale ontwikkeling van E. saccharina. Vier kunsmatige diëte, gebaseer op die volgende, is geformuleer met behulp van die karkasmaaltegniek: die eerste, geformuleer volgens die minimum spesifikasie van 'n opsomming van literatuur diëte wat bewys word dat dit effektief is om E. saccharina (MS) groot te maak; en die ander drie gebaseer op die ideale aminosuursamestelling en -profiel (IAAP) van die tweede (IAAP2), derde/vierde (IAAP3/4) en vyfde/sesde (IAAP5/6) stadium larwes. Die huidige dieët wat by SASRI gebruik is, is as die beheerdieët (ECBMOD) gebruik. Die oorlewing was beduidend hoog op alle dieëtformulerings, met meer as 92 % geëntde pasgebore E. saccharina-lewensfases wat op dag 20 oorleef het en meer as 95 % tot die oes oorleef (Dag 27). Die lewensfases het die vinnigste ontwikkel in die IAAP3/4- en MS-diëte (25 % en 17 % prepupae en papies wat onderskeidelik op dag 20 geproduseer is) in vergelyking met lewensfases van die oorblywende karkasmaal-diëte en die kontroledieët. Binne dieëtformulerings was vroulike papies aansienlik swaarder (0.1908 g) as manlike papies (0.1138 g). Die gewig van die manlike en die vroulike papie is nie beduidend beïnvloed deur die karkasmaal-diëte nie, aangesien die kontroledieët swaarder manlike en vroulike papies (onderskeidelik 0.1204 g en 0.2085 g) opgelewer het. Volwassenes se opkoms van papies was beduidend die hoogste (98 %) vir die MS-dieët, gevolg deur die IAAP3/4 (97 %) en kontrole-dieët (96 %). Die geslagsverhouding van volwassenes wat voortspruit uit papies wat uit die verskillende dieëtformulerings geoes is, was ongeveer 1. Daar is geen beduidende verskille waargeneem in hersteltyd vir koue koma (hersteltyd: 267,60 s) nie, maar vroue uit die IAAP5/6-dieët het die vinnigste herstel koue koma behandeling. Mans van die IAAP5/6- dieët het gepaar met aansienlik meer wyfies (6 verskillende wyfies) as dié van die oorblywende diëte, wat gemiddeld met 4 verskillende wyfies gepaar het. Alhoewel dit nie beduidend van die kontrole- dieët verskil nie, het vrouens uit die MS-dieët gepaar met meer mans (3 verskillende mans) as dié van die oorblywende karkas-maal-diëte, wat gemiddeld met 1 mannetjie gepaar is. Wyfies uit alle dieëtformulerings het meer as 870 eiers geproduseer wat meer as 90 % vrugbaar was. Die fisiese eienskappe (pH: 4,79, voginhoud: 81,43 % en wateraktiwiteit: 0,92 aw) van die diëte was nie beduidend verskillend nie en het die kwaliteit en stabiliteit van die geproduseerde diëte gehandhaaf, wat die optimale groei en ontwikkeling van E. saccharina gedurende die proeftydperk verseker het. As gevolg van die vinniger larwale ontwikkeling van E. saccharina wat op die MS-dieët geteel is, is verdere verbeterings aan hierdie dieët ondersoek deur die insluiting van sterole (cholesterol (C) en stigmasterol (S)), wat bewys is dat dit noodsaaklik is vir die groei van insekte, en het insekprestasie verbeter as dit in kunsmatige diëte aan hulle gevoer word. Die ontwikkelingsperiode vir larwes is in die steroldiëte aansienlik verkort, vergelyke met die MS-dieët sonder dat sterole bygevoeg is, ongeag die tipe en konsentrasie sterol wat bygevoeg is. Larwes wat die vinnigste ontwikkel het, bepaal deur die hoogste persentasie papies 20 dae na dieëtinenting, is aangeteken op die MS (1.0gS) dieët (72 %), gevolg deur die MS (0.2gC: 0.2gS) dieët (70 %). Die gewig van die papies is verhoog by vroue wat grootgemaak is op MS (0.1gC), MS (0.1gS) en MS (0.2gC: 0.2gS) (onderskeidelik 0.2143 g, 0.2271 g en 0.2252 g) vergelyk met dié van die MS-dieët sonder sterole bygevoeg (0.1864 g). Die insluiting van kriobeskermingsmiddels (dws L-proline (P) en trehalose (T)) in die MS-dieët is ook ondersoek om die kouetoleransie van hierdie insek meer geskik te maak vir die omgewingstoestande waarin dit in die veld vrygestel word. Die gewig van die papies het toegeneem by mans (0.1295 g) wat die MS-dieët (0.2gP: 0.2gT) gevoer is.Die hersteltyd van koue koma van manlike en vroulike motte (onderskeidelik 204,00 en 259,20 s) is verminder op E. saccharina wat grootgemaak is op dieselfde dieët, in vergelyking met die van die MS-dieët sonder dat kriobeskermingsmiddels bygevoeg is (onderskeidelik 253,20 en 306,60 s). Die toevoeging van kriobeskermingsmiddels in die MS-dieët het die vrugbaarheid van vroulike motte wat aan koue koma blootgestel is, nie verbeter nie, maar dit verminder vrugbaarheid tot minder as 44 %, vergeleke met wyfies wat nie blootgestel is aan die koue behandeling nie, waarvan die eiers gemiddeld 84 % vrugbaar was. Die karkasmaaltegniek blyk effektief te wees vir die ontwikkeling van superieure diëte (dws die MS- en IAAP3/4-diëte) as die Ostrinia-dieët, en die eienskappe daarvan as 'n goeie voedselbron is nog beter as sterole en kriobeskermingsmiddels bygevoeg word (veral in die MS-dieët) as aanvullings. Die bevindinge van hierdie studie het getoon dat die MS-dieët wat saam met die laer konsentrasie van die sterolmengsel (0.2gC: 0.2gS) en die kriobeskermingsmengsel (0.2gP: 0.2gT), 'n positiewe uitwerking op E. saccharina se lewensgeskiedenis kan hê, wat aandui dat hierdie spesie effektief grootgemaak kan word met 'n aansienlike vermindering van die opvoedingstyd en gevolglike koste vir die SIT-program. Die MS-dieëtformulering, insluitend die sterolmengsel (0.2gC: 0.2gS), is die voorkeur keuse om die huidige dieët wat gebruik word om E. saccharina by SASRI te verhoog, te vervang, aangesien dit die larwagroeiperiode dramaties verminder het met 60 % in vergelyking met die ander diëte in hierdie studie, insluitend diegene wat kriobeskermingsmiddels bevat, sonder om negatiewe effekte op die sleutel kwaliteits parameters van E. saccharina te hê.

Masters