Influence of different diets and oviposition substrates on Lygus rugulipennis biology ( Heteroptera : Miridae )

The aim of this study was to evaluate the effects of different diets on the development and reproduction of Lygus rugulipennis Poppius (Heteroptera: Miridae). Using 2 laboratory generations (F1 and F2) obtained from field-collected L. rugulipennis, the following diets were tested: beans, beans plus Tenebrio molitor (L.) (Coleoptera: Tenebrionidae) pupae, and a commercial artificial diet, which was developed for mass rearing of Lygus hesperus Knight. As oviposition substrates, beans and agar/parafilm rolls were used. Our data show that both the artificial diet and the artificial oviposition substrate were ineffective substitutes for beans for both laboratory generations. Stage-dependent and total survival rates clearly indicated that F1 Lygus bugs survive significantly longer when they are reared on vegetable substrates i.e., beans and beans plus pupae. The differential effects of the diets were more pronounced in the F2 generation, in which the embryonic development was longer for eggs from females reared on the artificial diet than on beans, and in which the second instar nymphs did not survive on the artificial diet. Both the total duration of post-embryonic development and the longevity of F1 males were shorter on the artificial diet than on beans. Female fecundity was affected by diet in terms of total duration of the oviposition period and mean number of eggs laid/female, since these parameters were lower on the artificial substrate, compared with those obtained on the bean substrate. However, the diet did not affect the morphological parameters, as there were no significant variations in weight, width of cephalic capsule, and tibia and hemelytra length. Since L. rugulipennis cannot be reared on the commercially available artificial diet, we discuss the necessity to improve both the artificial diet and oviposition substrate so that this Lygus bug and its specific egg parasitod Anaphes fuscipennis Haliday (Hymenoptera: Mymaridae) can be mass reared.

Over the last few decades there has been an increasing interest in Northern America in research focused on mass production of Lygus spp. in laboratory (Debolt, 1982;Patana, 1982;Debolt & Patana, 1985;Cohen, 2000;Habibi et al., 2001;Zeng & Cohen, 2001), in order to develop on effective method of producing the specific biological control agent Anaphes iole (Girault) (Hymenoptera: Mymaridae).In this respect, the objectives were to develop and improve the rearing methodology so that these insects can be produced continuously.Improvement of diets regarded not only the use of natural substrates, such as beans, cotton and alfalfa plants (Beards & Leigh, 1960), or beans supplemented with animal material such as heat-killed beet armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) pupae, but also artificial diets.Artificial diets for Lygus spp.were developed assuming that they only can ingest materials that are already liquid prior to the onset of feeding.However, the presence of specific salivary enzymes indicate that they can feed on non liquid food (Cohen, 2000).Based on this premise Debolt (1982) developed the first artificial diet for L. hesperus Knight, which was subsequently improved by Patana (1982), and used to produce feeding packets.These packets were further improved for this same species by Cohen (2000).
Therefore, in the present work, the artificial diet developed for the American species was tested for the first time on L. rugulipennis.In particular, the effect of different diets, i.e., fresh green beans, beans plus Tenebrio molitor (L.) (Coleoptera: Tenebrionidae) pupae, and artificial diet (Debolt & Patana, 1985) on L. rugulipennis development and reproduction, were determined.Pupae of T. molitor were chosen as a protein source, since in preliminary tests it was observed that these were readily preyed by L. rugulipennis.

MATERIAL AND METHODS
Adults of L. rugulipennis were collected from alfalfa fields in central Italy and kept in a controlled-environment growth chamber (25 ± 1°C, 50-60% r.h. and photoperiod 14L : 10D) in plastic cages (180 × 105 × 100 mm; ~ 50 adults/cage) containing strips of paper to increase the surface area for walking.The adults were fed with fresh green beans, sunflower seeds and supplied continuously with water through a glass tube, inserted in a hole and plugged with moistened cotton.The food was replaced every day and beans containing eggs, which were counted using a stereo microscope, were transferred (~ 100 eggs) to different plastic cages (120 × 85 × 80 mm) containing strips of paper to improve air circulation.Egg development was observed daily.Upon hatching, single nymphs of the 1 st laboratory generation (F1) were gently transferred, using a thin brush, to single plastic pots (Ø = 40 mm, h = 65 mm) kept under the same controlled environmental conditions, and fed on one of three different diets: (1) beans (n = 147), (2) beans + T. molitor pupae (n = 137) or (3) artificial diet (n = 221).The bean diet consisted of a portion (3 cm) of fresh green beans, while the bean + pupae diet consisted of a similar portion of fresh green beans and one pupa of T. molitor (Esche Grifo, Perugia, Italy), to provide a source of animal proteins.In both cases the food source was changed every day.For the artificial diet, a meridic diet developed for L. hesperus (Patana & Debolt, 1985) purchased from BIO-SERV ® was fed to the bugs in a plastic cap (Ø = 11 mm, h = 8 mm) covered with tight parafilm, and changed every two days.
In the case of beans and beans + pupae, the green beans served as a source of food and as an oviposition substrate.In the case of the artificial diet the oviposition substrate was an agar cylinder (Ø = 10 mm, l = 10 mm) (1.5 g agar / 100 ml of water), totally covered with parafilm, which was slightly stretched in order to favour ovipositor insertion.This substrate was changed every day.
The entire Lygus development, through 5 nymphal instars, was followed daily and recorded until adult emergence.The adults were weighed 2 days after emergence at the same time of the day (11-12 am) and then males and females of approximately the same age (~ 4 days), separated according to diet, were allowed to mate by placing a couple in a container (Ø = 40 mm, h = 65 mm) in the same environmental conditions.
Daily observations were made to evaluate the life cycle of both males and females and the number of eggs laid in each oviposition substrate, i.e., green beans or agar.The eggs laid daily by each couple were counted and placed in plastic cages (120 × 85 × 80 mm), which were separated according to diet.Because a large number of eggs were laid on beans by females of the F1 generation, only a representative sample was taken, equal to twice the number of F1 adults.In the case of eggs embedded in the artificial oviposition substrate, the parafilm was removed, stretched and the eggs were placed on filter paper soaked with distilled water.This procedure is a modified version of that described for L. hesperus by Debolt & Patana (1985).Eggs remaining inside the agar were drawn out gently using tweezers to avoid damaging.
The rearing of the F2 generation was done using the same technique and on the same diet as the F1 generation, and the nymphal development recorded as previously described.
The initial number of replicates per type of diet tested varied from 137 to 221 in the F1 and from 15 to 282 in the 2 nd generation (F2), because fewer eggs were produced by the females reared on the artificial diet.Each insect was treated as a replicate.
The following biological parameters were evaluated for each diet and generation (F1 duration of post-embryonic development; adult longevity; durations of the pre-oviposition, oviposition and post-oviposition periods; and number of eggs/female.Additionally, the following morphological parameters were also recorded, of both males and females: weight, using a high precision balance; width of cephalic capsule, length of one fore tibia and one hemelytron per bug, using a micrometric optic device (Leitz Wetzlar Germany 16×) fitted to a stereo microscope.
The stage specific and total survival of F1 and F2 were analysed using the Karl Pearson χ 2 test and, for the internal comparisons, the Goodman's post hoc procedure (Marascuilo & Serlin, 1988) was applied.The total duration and stage dependent duration of post-embryonic development of F1 and F2, the adult longevity in both generations and the morphological parameters were submitted to 2-way ANOVA.The duration of embryonic development of the F2 generation was analyzed with 1-way ANOVA.All internal comparisons were submitted to unequal N HSD Tukey test (Statistica 6.0, Statsoft Inc., 2001).Data were tested for normality (Shapiro-Wilks W-test) and heteroscedasticity (Bartlett test) and, when necessary, were appropriately transformed (Statistica 6.0, Statsoft Inc., 2001;Zar, 1999).

RESULTS
The stage-specific survival of the N1, N2, N4 and N5 instars of the first generation (F1) was significantly higher for nymphs reared on beans and beans + pupae, compared to those kept on the artificial diet, although in N3 instar the differences do not emerge in the internal comparisons (Table 1).Consequently, total nymphal survival (N1-N5) was higher for individuals maintained on the beans and beans + pupae diets than on the artificial diet (Table 1).
Larger differences were recorded in the second generation (F2) (Table 1).For the N1 instar, there were significant differences among the three diets, with Lygus on beans surviving better than those reared on beans + pupae, or artificial diet (Table 1).Moreover, the survival of the N2 instar kept on the artificial diet was 0.0%,  3. Effects of diet (beans, beans + pupae, artificial diet; F2 nymphs did not survive on the artificial diet), sex and their interaction on the stage dependent duration of post-embryonic development of F1 and F2 L. rugulipennis generations (25 ± 1°C, 50-60% r.h., 14L : 10D) (2-way ANOVA) (see also Fig. 3).resulting in the extinction of the F2 generation reared on this diet (Table 1).Total nymphal survival (N1-N5) was significantly different among the three diets, highest on beans, followed by beans + pupae and lowest on the artificial diet (Table 1), on which as already stated, no individuals survived.
The duration of embryonic development was evaluated only for the F2 generation, because it could have been influenced by the type of diet.Differences among diets were significant, with the shortest embryonic development recorded for the L. rugulipennis eggs produced by females reared and fed on beans, followed by beans + pupae and longest for those on artificial diet (Fig. 1).
The total duration of the post-embryonic development was evaluated both for the F1 and the F2 generations.In F1 the effects of diet, sex and their interaction were all significant (Table 2).Specifically, while the total devel-opmental time of females did not differ significantly among diet types, the males took longer to develop on beans + pupae than on the artificial diet, and an intermediate time to develop on beans (Fig. 2).In F2 none of the effects were significant (16.03 ± 0.09; mean ±SE) (Table 2).
The duration of individual nymphal stages was also evaluated for the F1 and F2 generations.The effects of diet, sex and their interaction during the different nymphal instars were not significant, except for a diet effect on the N2 instar of F2 (beans: 2.77 ± 0.07; beans + pupae: 3.08 ± 0.14; mean ± SE) (Table 3 and Fig. 3).
In the case of adult longevity, diet only significantly affected the F1, whereas both diet and sex effects were significant in F2, but not their interaction (Table 4).
F1 adults lived longer on beans and beans + pupae than on artificial diet (Figs 4 and 5).F2 adults lived longer when maintained on beans compared to beans + pupae and females lived longer than males (Figs 4 and 5).
Female fecundity was also influenced by the diet.F1 females laid significantly more eggs when maintained on bean and bean + pupae compared with those on the artifi-cial diet, and egg production in the F2 was higher for females on bean compared to bean + pupae (Fig. 7).
The diet did not influence the morphological parameters (i.e., weight, width of cephalic capsule, and tibia and hemelytron length) of males and females.However, sex of the bugs significantly affected the weight in the F1, in which females were heavier than males (female = 0.008 g ± 0.0001; male = 0.007 g ± 0.0001.Mean ± SE), and the width of cephalic capsule in the F2, with males having smaller heads (female = 1128.37µm ± 4.05; male = 1108.04µm ± 3.5.Mean ± SE) (Table 5).

DISCUSSION
In our laboratory experiments, the artificial diet developed for rearing L. hesperus (Debolt, 1982;Patana, 1982;Debolt & Patana, 1985;Cohen, 2000;Habibi et al., 2001;Zeng & Cohen, 2001) was not as good as the vegetable     5. Effects of diet, sex and their interaction on the morphological parameters of F1 and F2 L. rugulipennis adults (25 ± 1°C, 50-60% r.h., 14L : 10D) (2-way ANOVA).diet for L. rugulipennis, as indicated by reduced survival and longevity in the F1 generation, and extinction in the F2.L. rugulipennis fecundity, evaluated as number of eggs laid, was also lower in females reared on the artificial diet compared to those reared on fresh green beans.This may be because of the diet or the lower suitability of the artificial oviposition substrate (agar-parafilm cylinders) compared to beans, as indicated by preliminary tests.In laboratory choice tests L. rugulipennis females preferred to oviposit into fresh green beans compared to artificial substrates, even when these were treated with bean extracts (data not shown), as suggested by Whitbey (1999) for L. hesperus.
Rearing on the artificial diet also appears to affect embryonic development, suggesting a poor yolk quality.However, other effects of the artificial oviposition substrate on embryonic development should be evaluated.
In our experiments it was difficult to separate the effects of the artificial diet and artificial oviposition substrate.It was not possible to combine a diet with an artificial oviposition substrate and vice-versa, because oviposition behaviour in Lygus species is always linked with stylet probing (Romani et al., 2005).
Lygus spp., as many mirid bugs and other Heteroptera, often show a partial zoophagy (Wagner & Weber, 1964;Alomar & Wiedenmann, 1996;Wheeler, 2001;Boyd et al., 2002;Boyd, 2003), which in several cases is confirmed by the enzyme profiles of the saliva.For example, there is protease in addition to pectinase and amylase in the saliva of L. hesperus, L. lineolaris and L. rugulipennis, suggesting that during extra-oral digestion they are capable of ingesting structural proteins from animals (Laurema et al., 1985;Agusti & Cohen, 2000).A partial zoophagy was, in fact, also noticed in the case of L. rugulipennis fed on Aphis fabae and Tenebrio molitor pupae (personal observations).However, in our experiments this species developed better on a diet of fresh green beans than on a combined diet composed of green beans and T. molitor pupae.These results may suggest that the nutritional quality of these pupae is not suitable for L. rugulipennis, although they are commonly used as protein source in mass rearing of many heteropteran predators (Oliveira et al., 1999(Oliveira et al., , 2004;;Jusselino et al., 2001;Zanuncio et al., 2001).Therefore, future research should also consider alternative animal sources of protein.
Because the lack of positive results in the use of an artificial diet to rear L. rugulipennis, further efforts are needed to improve this diet and in particular to find a suitable oviposition substrate for the mass rearing of this species.In addition, a suitable oviposition substrate will allow the mass rearing of the specific egg parasitod, A. fuscipennis, for use as a biological control agent (Conti et al., 1994;Coutinot & Hoelmer, 1999) in IPM programmes.In fact, maintaining this parasitoid on host eggs embedded in green beans is very difficult (personal observations) as the beans tend to deteriorate rapidly under standard rearing conditions.