Development of an Artificial Diet for Rearing Conogethes punctiferalis (Lepidoptera: Crambidae)
Conogethes punctiferalis (Guenée) (Lepidoptera: Crambidae) is one of the most damaging Lepidoptera, attacking fruit in the temperate and tropical regions of Asia, Australia, and Papua New Guinea. Effective methods of control will likely remain limited until further physiological and ecological studies can be conducted, many of which will require effective means of rearing of the insect in the laboratory. To that end, this study was undertaken to develop and assess four meridic diets for rearing C. punctiferalis. The four diets differed in the amounts of chestnut meal, corn meal, and soybean meal. The diet containing 30 g chestnut meal, 70 g corn meal, and 70 g soybean meal per 700-ml diet yielded a larval survival rate of 94.5%, a generation developmental time of 42.4 d, mean pupal weights of 73.6 mg for males and 77.3 mg for females, and an adult fecundity rate of 97.9 eggs/female. Performance on this diet compared favorably with rearing on fresh corn.Abstract
Conogethes punctiferalis (Guenée) (Lepidoptera: Crambidae), commonly known as the yellow peach moth in its native range in Asia, is one of the most damaging Lepidoptera attacking fruit grown in the temperate and tropical regions of South and East Asia, Australia, and Papua New Guinea (CABI International 2011). Larvae of C. punctiferalis are highly polyphagous and feed on a broad range of hosts, including peach, chestnut, durian, citrus, papaya, cardamom, ginger, eggplant, and maize (Sekiguchi 1974, Waterhouse 1993). Infested fruits usually are stunted and/or scorched, and may drop from the tree before maturation (CAB International 2011).
Because of its broad host range and its potential to be spread long distances, C. punctiferalis is expected to negatively impact many susceptible plant hosts. In China, for example, C. punctiferalis was reported to damage more than 100 species of trees, crops, and vegetables (Lu et al. 2010) and was identified as one of the most destructive pests of peach. Various methods, including application of insecticides, use of sex pheromones, biological control with Bacillus thuringiensis Berliner, and the use of cultural practices (e.g., bagging fruits) have been proposed for the management of C. punctiferalis (CAB International 2011, Du et al. 2014). However, C. punctiferalis larvae bore into and feed within host fruits for the entire larval stage, making most traditional management strategies, especially those directed to the larval state, largely ineffective. Development of effective management strategies for C. punctiferalis will likely involve intensive studies on the biology, ecology, physiology, and toxicology of the insect (Du et al. 2012). The nature of many of these studies will be facilitated by or require readily available specimens of eggs, larvae, pupae, and adults for testing. Thus, a meridic diet for rearing C. punctiferalis in laboratory is needed.
Over the past several decades, artificial diets have been widely used for mass rearing of insects in both commercial insectaries and scientific laboratories (Cohen 2004, Vanderzant 1974). Only slight differences in composition of a diet can impact rearing quality and efficiency, which is usually translated as higher survival rates, greater larval and pupal weight, shorter developmental time, and higher fecundity rates. Honda et al. (1979) and Utsumi et al. (1990) proposed meridic diets for rearing C. punctiferalis, but colonies fed on those diets had a lower larval survival rate and a larger variation in development duration than colonies fed on natural host plant materials (Utsumi et al. 1990). Therefore, efforts to define a meridic diet for rearing C. punctiferalis must continue.
Previous reports provide a basis for continued development of an artificial diet for C. punctiferalis. For example, larvae fed on chestnuts developed faster and had a lower mortality rate than those fed on peach, persimmon, or cypress (Choi et al. 2006, Honda et al. 1979), suggesting that chestnut could enhance performance on a meridic diet. Li et al. (2014) reported C. punctiferalis larvae fed on fresh corn and chestnut performed better than those fed on apple, pear, or plum. Our own preliminary testing showed that C. punctiferalis larvae were repelled by diets containing formaldehyde and sorbic acid as microbial inhibitors, whereas methyl paraben was not repellent. Furthermore, Wang et al. (2011) developed a diet to successfully rear the Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), which has feeding habits and hosts similar to those of C. punctiferalis. Our objective, therefore, was to compare four meridic diets, each based on the diet recipe of Wang et al. (2011) but each with different ratios of chestnut meal, corn meal, and soybean meal, in successfully rearing C. punctiferalis in the laboratory.
Materials and Methods
Insects
Insects used in these studies were from a laboratory colony of C. punctiferalis, originally established from larvae collected on 9 October 2009 from cornfields at the Agricultural Experiment Station of Beijing University of Agriculture and maintained on fresh corn for >20 generations. The stock colony was held in an environmental incubator (RTOP-B, Zhejiang Top Instrument Co., Ltd., Hangzhou, China) maintained at 23 ± 1°C, 75 ± 2% relative humidity, and on 16:8-h light:dark photoperiod with 3,500 lux light intensity. All experiments for this study were conducted therein.
Artificial diets.
Four artificial diets, designated AD-I, AD-II, AD-III, and AD-IV, were prepared for feeding C. punctiferalis larvae. Casein, cholesterol, glucose, citric acid, l-ascorbic acid, methyl paraben (a microbial growth inhibitor), brewer's yeast, and water were present in equal amounts in all four diets (Table 1). The amount of agar differed in only the AD-II diet. The total amount of corn meal, chestnut meal, and soybean meal combined in the AD-I, AD-III, and AD-IV diets was 170 g/L, whereas the amount of those combined ingredients in the AD-II diet totaled 165 g/L. The amount of agar added to AD-II diet was increased by 5 g/L to compensate for the smaller amount of those combined ingredients in the AD-II diet. The diets differed in the amounts of chestnut meal, corn meal, and soybean meal incorporated into each (Table 1).
Briefly, each diet was prepared by combining the chestnut meal, corn meal, soybean meal, and brewer's yeast powder, to which 300 ml distilled water was added. This mixture was then simmered over heat for approximately 30 min. The agar was suspended in the remaining 400 ml water and heated to boiling point (100°C), after which glucose and citric acid were added and mixed. This mixture was cooled to approximately 60°C, and l-ascorbic acid, casein, methyl paraben, and cholesterol were added and mixed. The two mixtures were then combined and thoroughly mixed. Each diet was allowed to cool to ambient temperature and was then stored at 4°C until used.
Rearing of insects.
Forty neonate larvae obtained from the stock colony were transferred into individual glass containers (12 × 8 cm) containing four blocks of a specific diet, each measuring 25 × 50 × 50 mm. The container was covered with a transparent plastic lid with small holes for ventilation and placed in the environmental chamber. Diet was replenished every 3 days, and diet residues and wastes were removed as needed. Larval survival and development were checked daily. As larvae reached the penultimate stage, cheesecloth (150 × 100 mm) was placed over the diet blocks to provide pupation sites. After pupation, the newly molted pupae were collected, weighed, numbered, and individually placed into 50-ml tubes with ventilated lids and placed in the chamber. Pupal survival, duration, and adult emergence were monitored daily. Each diet treatment was replicated five times with a total of 200 larvae per diet treatment.
For each diet treatment, adults (1:1 ratio of females:males) were placed in a plastic cage (350 × 270 × 250 mm). Moths were provided with a honey solution (8%, v/v) soaked in cotton in a 2.5-ml vial. The honey solution was replenished each day. Three days after placement of adults in the cages, an apple wrapped in a wet piece of cheesecloth was placed in each cage as an oviposition site for the females. The cheesecloth was replaced each day, and the numbers of eggs deposited on the cloth were counted. Collection of eggs was continued until no living females were available in the cage. Adult survival also was recorded. Cloths with eggs were dated, numbered, and placed into individual glass containers (12 × 8 cm). The number of hatched F1 larvae was counted daily.
For this phase of the study, each diet treatment was replicated four times. A total of 40 moths (20 males, 20 females) was used for the AD-I diet treatment, while totals of only 10, 30, and 30 moths were used for the AD-II, AD-III, and AD-IV diet treatments, respectively, because of insufficient numbers of moths available for each treatment.
Fresh corn collected from a cornfield in Beijing University of Agriculture was used as a control diet treatment. Rearing conditions and experimental procedures were the same as that of the artificial diet treatments.
Data analyses.
Response variables of duration of development (time required to complete specified periods in the development of the insect), preoviposition duration (time between the adult emergence and initial oviposition), generation duration (time of development from egg through moth preoviposition), pupation rate (ratio of pupae to larvae), and adult emergence rate (ratio of adults to larvae or the ratio of adults to pupae) were compared among the diet treatments using analysis of variance and the Bonferroni test with SPSS 16.0 statistical software. Selected life table parameters were calculated for each treatment according to methods of Ding (1994) and Liu et al. (2009). These also were subjected to analysis of variance and the Bonferroni test.
Results
Statistically significant (P < 0.05) differences among the treatments were observed for duration of the entire generation, with AD-I = corn check < AD-IV < AD-III < AD-II. These durations ranged from 42.4 d for AD-I to 63.3 d for AD-II. This same pattern of differences was reflected in the duration of the larval stages, with AD-I = corn check < AD-IV = AD-III < AD-II (Fig. 1). Larval development ranged from 20.3 d for AD-I to 41.8 d for AD-II.
Citation: Journal of Entomological Science 50, 2; 10.18474/JES14-29.1
Survival rates also were significantly impacted by diet. Percentage of survival of larvae from egg hatch to pupation was 94.5% on AD-I, 80.0% on the fresh corn control, 26.5% on AD-III, 25.6% on AD-IV, and 18.0% on AD-II (Table 2). Percentage of survival from egg hatch to adult emergence was 78.5% on AD-I, 71.4% on the fresh corn control, 20.9% on AD-III, 20.6% on AD-IV, and 8.0% on AD-II (Table 2). The hatch rate of eggs from those adults was highest for the AD-I diet (67.7%) and lowest for the AD-III (29.8%) and AD-II (26.9%) diets.
Pupal weight, adult longevity, and adult fecundity also were significantly affected by larval diet treatments. Pupal weights on the AD-I diet were greatest among all treatments (73.6 mg per male, 77.3 mg per female), did not differ significantly from weights obtained when fed on fresh corn, and were significantly greater than those observed on remaining three diets (Table 3). Adult longevity appeared to have no relationship to fecundity, with no significant differences among females developing from larvae fed on the fresh corn control, the AD-IV diet, and the AD-I diet (Table 3).
Discussion
Cohen (2004) states that relative amounts of components of artificial insect diets impact performance and fitness of the insects. We demonstrated in a previous study that the survival and reproduction of C. punctiferalis were impacted by the choice of host plant material incorporated in meridic diets (Li et al. 2014). In this current study, the amount of chestnut meal as an ingredient in the artificial diet was a key factor in survival, weight gain, generation time, and fecundity of C. punctiferalis. The AD-I diet, containing the highest concentration of chestnut meal of the diets tested, resulted in enhanced survival rate, shortened developmental duration, increased pupal weight, and increased numbers of eggs produced by females.
Furthermore, the AD-I diet compared favorably with the control diet using fresh corn as the larval food source. Larval survival and total survival rates on the AD-I diet and the fresh corn control were statistically similar, whereas survival rates on the remaining three artificial diets were much lower and not acceptable in terms of the diets sustaining population growth under artificial conditions. Pupal weights and numbers of eggs produced per female showed the same relative responses. Generation times (T) for the AD-I diet and the fresh corn control were statistically equal; both were statistically shorter than T values of the AD-II, AD-III, and AD-IV diets (Table 4).
The calculated natural (or intrinsic) rate of increase (rm) for the insect cohorts fed on the different diets also demonstrated that C. punctiferalis performed best on the fresh corn control and the AD-I diet (Table 4). The intrinsic rate of increase is a theoretical maximum that may be reached in a given environment if the population is not resource-limited and represents the potential of the increase of a population in specified environmental conditions (Southwood and Henderson 2000). The rm is not only dependent upon birth and mortality rates, but also on age assembly, fecundity, and developmental rate (Li et al. 2012, Niu et al. 2008). The rm value of 0.073 for the cohort fed on fresh corn was likely due to a higher fecundity rate and survival rate, whereas the rm value of 0.074 for the cohort fed on the AD-I diet likely resulted from high development and survival rates. The rm value is an indicator of fitness, with a higher value indicating a higher level of fitness. In this study, the rm values for C. punctiferalis larvae fed on the AD-I diet (0.074) and fresh corn (0.073) were higher than those observed with the AD-IV (0.037), the AD-III (0.008), and the AD-II (−0.031) diets. Cohorts fed on fresh corn or the AD-I diet thus had a higher level of fitness than cohorts fed on the other artificial diets.
In summary, these results demonstrate that the AD-I meridic diet as outlined herein is conducive to successfully rearing C. punctiferalis under laboratory or other artificial conditions. We believe that chestnut meal is a key component for the success of this diet in enhancing C. punctiferalis performance. We have thus adopted this as the meridic diet to be used for production of our laboratory colonies.
The AD-III, AD-IV, and especially the AD-II diets will not satisfactorily support performance of C. punctiferalis. All three of these diets have equivalent or higher concentrations of corn meal and soybean meal than the AD-I diet. However, the AD-I diet contains a higher concentration of chestnut meal than any of the other three diets. Furthermore, the AD-II diet contains a higher concentration of agar than the other three. Agar is often used in artificial diets for insects and plays important roles as a solidifying agent and in increasing water-holding capacity. A higher concentration of agar might impede the boring activity and thus the survival of C. punctiferalis neonates.
Developmental duration of Conogethes punctiferalis reared on meridic diets and fresh corn. Bars represent means ± SE; significant differences among six host plants are indicated by letters over each bar (Bonferroni test, P < 0.05).
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