Optimization And Evaluation Of The Artificial Diet Of Coccinella Septempunctata L.

Coccinella septempunctata L.(Coleoptera: Coccinellidae),is a natural enemy of insect pests.It has received increasing attention as a bio-control agent,due to its strong predatory ability against many important pests,including aphids,whiteflies,spider mites,leafhoppers,and psyllids.However,due to the affect of climate and farming operations,it is often difficult to maintain sufficient C.septempunctata in the field and can't control pests effectively.Therefore,it is necessary to artificially rear them as supplement predator.Currently,lady beetles are reared mainly using aphids as food.This method is costly and time-consuming for the mass rearing of both aphids and lady beetles.Therefore,successful and continuous mass rearing of ladybug at a commercial level is important for this purpose,which requires the availability of artificial diets.In this study,an orthogonal test was used to optimize an insect-free artificial diet for adult C.septempunctata.The nutritional ingredients of the artificial diet and the natural prey species Aphis craccivora were compared.We also studied the effect of food on development of C.septempunctata and its predation on A.craccivora,which was continuously rearing for 8 generations on different food types.Moreover,the transcriptomes of C.septempunctata reared on A.craccivora and on the artificial diet were sequenced.The results were as followings.1.Screening and optimization of the artificial diet for C.septempunctataA 16-factor,2-level orthogonal test was used to optimize an insect-free artificial diet formula for C.septempunctata.We found that the 5 parameters: weight gain,survival rate,pre-oviposition,fecundity and hatch rate of beetles feeding artificial improved diet were 167.46,114.75,96.71,228.93 and 102.78% compared with the averaged values of orthogonal test.The weight gain and egg laying capacity of C.septempunctata feeding on artificial diet were 87.46% and 62.70% of that feeding on A.craccivoraa,respectively.However,there was no significant difference in the survival and hatching rates between C.septempunctata rearing on either food type.C.septempunctata feeding on the artificial diet entered into the pre-oviposition period significantly earlier than C.septempunctata fed on A.craccivora.2.Nutritional comparision of the artificial diet and A.craccivoraThe contents of crude protein,crude fat,total sugar,zinc,iron,calcium and manganese were 18.60 g/100 g,1.26 g/kg,27.80 g/100 g,58.00 mg/kg,200.00 mg/kg,1084.00 mg/kg and 6.10 mg/kg in the artificial diet respectively,while they were 14.50 g/100 g,3.35 g/kg,31.70 g/100 g,52.00 mg/kg,62.00 mg/kg,118.00mg/kg and 7.20 mg/kg in the A.craccivora.Nutritional comparision indicated that the crude protein content in the artificial diet was higher than that in A.craccivora.Similarly,contents of both total and essential amino acids were significantly higher in the artificial diet than in A.craccivora.Crude fat was significantly lower in the artificial diet compared to A.craccivora.Fatty acids,myristic acid,palmitic acid and arachidic acid were lower in the artificial diet than in A.craccivora.The total sugar content in the artificial diet was lower than that in A.craccivora.Minerals including zinc,iron,and calcium in the artificial diet were higher than in A.craccivora,but there was only a significant difference in iron and calcium.3.An artificial diet and A.craccivora for continuous maintenance of C.septempunctataThe optimized artificial diet was developed for continuous maintenance adult C.septempunctata,and A.craccivora was developed for continuous maintenance larvae C.septempunctata for 8 generations.At all 8 generations,the total number of eggs laid by C.septempunctata feeding on the artificial diet were lower than feeding on A.craccivora.The C.septempunctata eggs feeding on the artificial diet had a similar weight as those of C.septempunctata feeding on A.craccivora.However,the weight gain rate of larvae of C.septempunctata increased as a result of supplementing A.craccivora prey to meet nutritional demands during the larval period.There were no significant differences on weight in fourth instar larval and pupal stages of C.septempunctata,or in the first four-generation female adults.After feeding C.septempunctata with the artificial diet to the 8th generation,the sex ratio(?:?)was 57.5%,which was higher than that in C.septempunctata fed on A.craccivora(54.2%).4.Predatory ability of C.septempunctata feeding on different nutrient sourcesThe predatory capacity was compared between C.septempunctata fed on the artificial diet,or fed on A.craccivora.Both response curves of predatory capacity fit Holling II equation.The estimated maximum of aphids predated by 1st to 4th instar larvae and the adult females and males of C.septempunctata feeding on diet per day were 20.9,36.0,100.2,403.2,490.9 and 395.3,respectively.The estimated maximum of aphids predated by 1st to 4th instar larvae,the adult females and males of C.septempunctata feeding on aphids per day were 20.0,28.9,102.1,476.2,472.2 and 396.4,respectively.At different age stages,there were no significant differences in the theoretical maximal daily predatory capacity between C.septempunctata feeding on either food type,which indicated that C.septempunctata fed on the artificial diet also has robust control over A.craccivora.5.Transcriptome sequencing of C.septempunctata feeding on different nutrient sourcesTranscriptome sequencing of C.septempunctata feeding on the artificial diet and on A.craccivora revealed that the expression of 38,315 genes was affected by the artificial diet,1182 of these genes had a significant change in the expression levels.These DEGs(Differentially Expressed Genes)were likely associated with the decreased egg laying capacity,hatching rate,longevity,and increased sex ratio(?:?)of adult C.septempunctata feeding on the artificial diet.The DEGs of five hemolymph juvenile hormone binding proteins,and of two male sterility proteins,were down-regulated.The DEGs of six cytochrome P450 and three MRJPs(Major Royal Jelly Protein)were up-regulated in female adult C.septempunctata fed on the artificial diet.Furthermore,in the DEGs metabolic pathways for C.septempunctata feeding on the artificial diet were accumulated 8 amino acids metabolic pathways,and more DEGs were down-regulated.In seven lipid metabolic pathways,most of the DEGs that belonging to fat digestion and absorption pathways were up-regulated.Conversely,starch and glucose metabolism were accumulated two pathways,and most of the DEGs were down-regulated.We found some differentially expressed nutrient-controlled genes and metabolic pathways are related to nutrition,from which a more informative feedback for diet formulation was obtained and the artificial diet could be more efficiently optimized.