Biological parameters of Duponchelia fovealis (Lepidoptera: Crambidae) reared in the laboratory on two diets

Duponchelia fovealis Zeller is a polyphagous insect that has been recently reported attacking strawberry plants (Fragaria x ananassa Duchesne). Despite its economic importance there are few studies on this pest because it is diffi cult to rear it in the laboratory. With a constant supply of insects, studies on alternative methods of pest control can be conducted. This study aimed at developing an artifi cial diet for rearing D. fovealis with biological characteristics similar to those reared on their natural diet. This study was carried out in a climate-controlled room (25°C ± 2°C, RH 70% ± 10%, and 14L : 10D). The natural diet consisted of ‘San Andreas’ strawberry leaves (D1), while the artifi cial diet (D2) was developed in which beans, casein, soy protein, yeast and wheat germ are used as sources of protein. Five instars were identifi ed. D. fovealis completed its life cycle in 38 and 40 days when fed D1 and D2, respectively. Survival was highest for the larvae fed the artifi cial diet. Females fed D1 lay a mean of 300.2 ± 62.3 eggs, while those fed D2, 220.3 ± 41.8 eggs. The artifi cial diet is suitable for the continuous rearing of D. fovealis in the laboratory.

Given the recent history of D. fovealis as a nonnative pest and the signifi cant damage it causes (Zawadneak et al., 2016), lack of pest control can have serious economic implications.Thus, biological control may provide an alternative way of managing D. fovealis (Zawadneak et al., 2016).However, in the development of a biological control program of any agricultural pest, the fi rst step is to obtain a good understanding of the basic biology of the target species and how it can be effi ciently reared in the laboratory (Sørensen et al., 2012).The rearing of a pest could be optimized by developing an artifi cial diet (Cohen, 2005;Parra, 2012;Sørensen et al., 2012).The main advantage of using artifi cial diets instead of natural foods is the continuous supply of laboratory-reared insects of known quality (Sing, 1983).
The objective of this study was to develop an artifi cial diet for producing D. fovealis with biological characteristics similar to those reared on a natural diet.
Kolmogorov and Bartlett tests and the results transformed when necessary.The results were then analyzed using Student's t-test at P ≥ 0.05.Analyses were carried out using statistical software ASSISTAT, 7.6 beta version (Silva & Azevedo, 2002).

RESUL TS AND DISCUSSION
D. fovealis underwent fi ve larval instars on both D1 and D2 diets (Table 1).Knowledge of the number of instars is essential for both basic and applied research, such as studies on the biology of this insect, construction of life tables and development of control strategies (McClellan & Logan, 1994;Logan et al., 1998).
No signifi cant differences were found in head capsule width attributable to diet, except for the 1 st (t = 10.83;P ≤ 0.001) and 5 th instars (t = 6.67;P < 0.05) (Table 1).Regarding growth rate, means followed a geometric progression, as reported by Dyar (1890).The mean growth rates (K) were 1.50 (D1) and 1.52 (D2), which are within the range of variation expected according to Dyar's rule of 1.10 to 1.90, and similar to the mean ratio of 1.52 reported by Cole (1980) for holometabolous insects.Measurements of head capsule are a good way of determining the growth of insects with homogeneous growth rates (Schmidt et al., 1977).
The mean larval development time recorded for D. fovealis (25-28 days) reared on the two diets under controlled laboratory conditions was similar to that reported by Romeijn (1996) and CABI (2016) in the fi eld (28 days).The pupal stage (approximately eight days) was shorter than that reported by Romeijn (1996) (7 to 14 days).D. fovealis adults reared in the laboratory survived approximately 15 days, whereas Romeijn (1996) reported a longevity of approximately 7-14 days and CABI (2016), 14-21 days.This variation indicates that apart from nutrition, environmental conditions may also infl uence the development of D. fovealis.distilled water (1,000 mL), to which V8 ® vegetable juice (50 mL) was added as a phagostimulant.Following preparation, according to Parra (2001), 5 mL of the diet was placed in sterile Petri dishes (6 cm diameter).After cooling, the Petri dishes were kept at 5°C.
Newly emerged adults were placed in polypropylene cages (17 × 12 cm) that were each topped with a screen, lined with paper towels and kept under controlled conditions (25°C ± 2°C, 70% ± 10% RH, and 14L : 10D).Paper towels were changed daily and eggs were kept in a Petri dish with moistened paper until the larvae hatched.Larvae were transferred to clear plastic boxes (11 × 11 × 3.5 cm) with either diet D1 or D2.Approximately 100 larvae were placed in each box.Leaves (D1) were replaced every two days and the artifi cial diet (D2) when it began to dry out.Pupae were sexed and kept in Petri dishes.Newly emerged adults were transferred to cages lined with paper towels to collect eggs of the fi rst generation reared on D1 and D2 and for use in experiments.
The greatest width (mm) of the head capsule of ten newly hatched larvae in each treatment were measured.These measurements were made daily, always at the same time, using a stereomicroscope Zeiss ® fi tted with an ocular micrometer.
For treatments D1 and D2, 210 and 215 eggs were used, respectively.Each egg was kept individually in a Petri dish with moistened paper (6 cm of diameter) and monitored daily.For determining the number of instars and their duration larvae after hatching were kept separately and fed one of the diets (D1 or D2).The prepupal stage was characterized based on morphological (shortening and wrinkling of the body) and behavioral changes (webbing for the construction of the cocoon and inactivity).Pupae up to 24 h of age were sexed (Genc, 2005), and then placed individually in Petri dishes lined with moistened fi lter paper until adult emergence.Development time (days) and survival (%) were determined for each of the stages: egg, larva, prepupa, pupa and egg to adult.
Using the adults that were reared in the laboratory on the two diets, pairs of individuals of the same age were formed (n = 10).One pair was placed in each cage, as previously described, in order to monitor reproduction and longevity.Each pair of moths was considered a replicate.Paper towels were changed daily and eggs were counted and kept in a Petri dish lined with moistened paper until the larvae emerged.Fecundity was determined based on the daily and total number of eggs produced per female in each cage, until the death of both male and female.
Larvae from two cages per diet used in the previous experiment were individually placed in clear plastic cups (80 mL) and fed their corresponding diet until pupation.Twenty-four hours after pupation, pupae were sexed, and weighed (mg) on an analytical balance.The sex ratio, defi ned as the number of males (m) / number of females (f): SR = m / f, was then calculated.
The percentage survival of the developmental stages of D. fovealis were highest on the artifi cial diet, except in the 4 th instar, when there was no difference between diets (P > 0.05), and in the prepupal stage, when the percentage survival was higher for individuals fed the natural diet (Table 2).Mortality was highest for 1 st instar larvae fed D1.In this developmental stage, larvae measure approximately 1.5 mm in length (Zawadneak et al., 2015) and are diffi cult to detect and remove from strawberry leaves due to the hairs.Thus, the lower survival recorded on D1 may not be due to nutritional inadequacy, as indicated by the results on the biology of this pest.Rather, this difference in survival may be due to the handling of larvae during the replacement of food (leaves) in D1.Also, upon entering the prepupal stages, larvae fed D1 use remains of leaves provided as food to construct the cocoon, similar to that observed in nature (Zawadneak, 2011(Zawadneak, , 2015)), while larvae fed D2 do not have access to this substrate and more energy might have to be used to produce the cocoon using only webbing, consequently contributing to the lower viability of prepupae reared on D2.
From the larvae that hatched from the eggs collected from the two cages for each diet used to determine fecundity, 600 (D1) and 500 pupae (D2) were obtained.Female pupae from the larvae fed the artifi cial diet were heavier (Table 3), while no differences were recorded for males.This is normal in lepidopteran females, since they are heavier than males, feed for longer and need more nutrients for reproduction (Moreau et al., 2006).The mean percentage of deformed pupae was low, 5.4% in D1 and 7.2% in D2.Deformities in pupae resulted in abnormal metamorphosis and adults with deformed or swollen wings.The sex ratio (SR) was 1.1 : 1 for D1 and 0.8 : 1 for D2.A sex ratio of approximately 1 : 1 is appropriate for insects (Hamilton, 1967) and according to Parra (2001), in populations of insects with sexual reproduction, one male to one female is supposedly adequate to ensure mating and production of offspring.
Although viability over the whole life cycle (egg to adult) on diets is not above 75% (Parra, 2012), the diet developed by Greene used in this study to rear D. fovealis met the requirements proposed by Sing (1983).The biological parameters of this insect reared on the diet are similar to those recorded on natural diets.The viability and duration of larvae and adults reared on diets should be similar to that observed in nature, and according to Parra (2001), be suitable for more than one species.This is the fi rst study on biological parameters of D. fovealis under controlled conditions.Our results indicate that the artifi cial diet allows the continuous rearing of D. fovealis in the laboratory and that their biological characteristics are similar to those of insects reared on a natural diet.Also, the artifi cial diet is more effective for the mass rearing of this species, as it is easier to prepare and does not need to be replaced daily, and consequently the insect is subject to less handling.Stable laboratory cultures of this insect are essential for future studies on the development of management strategies for D. fovealis.
Table 3. Mean (± SE) longevity of females and males, pre-oviposition and oviposition time (days), daily fecundity (mean daily number of eggs), mean total fecundity (mean total number of eggs), mass of pupae (mg) of Duponchelia fovealis fed a natural diet consisting of 'San Andreas' strawberry leaves (D1) and the artifi cial diet (D2).Means followed by different letters in a row differ based on Student's t-test at P < 0.05.Pupa n = 600 (D1); n = 550 (D2).

Table 1 .
Head capsule widths (mean ± SE) (mm) and growth ratio (K) of Duponchelia fovealis larvae fed a natural diet consisting of 'San Andreas' strawberry leaves (D1) and the artifi cial diet (D2) (n = 10) (Means followed by different letters in a row differ based on Student's t-test at P < 0.05).