Induction of adult diapause in Pyrrhocoris apterus L. by short cold exposure

Summary The ripening of ovaries in the females of Pyrrhocoris apterus can be inhibited not only by short-day photoperiod but also by cold shocks during the first week of the adult life.Ovaries in a part of the females are prevented from ripening by transferring the bugs from 25° C and long-day photoperiod to substantially lower temperatures for 2 (or 4) days. The oviposition is inhibited at least for 3 weeks after the return to original conditions favourable for reproduction. This effect is achieved when cold treatment is combined either with darkness or with long day. A difference between 2 and 4 days of chilling was not recorded. The effect of cold exposure (evaluated in our experiments by the proportion of mature and immature females) depends on the temperature of the cold exposure and on the age of females at the time of transfer to cold. The incidence of immature females was higher when lower temperatures (+2 to +6° C) were used and when females were transferred to cold early (0–2 days) after the adult moult.     

Effects of photoperiod and temperature on blood feeding,oögenesis and fat body development in the mosquito, Culiseta inornata

In the laboratory, blood feeding rates, oögenesis and fat body development were utilized to assess the effects of photoperiod and temperature in the conditioning of adult female Culiseta inornata for aestivation. Long-day (16L:8D) and short-day (8L:16D) photoperiods were examined in combination with temperatures of 15°C, 20°C, and 25°C. All tested individuals were reared from egg to adult under one of six possible regimes.Blood feeding occurred in approximately 60% of the females subjected to short-day conditions. Females subjected to long-day regimes exhibited gonotrophic concordance (the inhibition of blood feeding), evidenced by a decrease in overall blood-feeding rate to 20%. Inhibition of blood feeding by long-day females decreased in a linear fashion with increasing temperature. No interaction of the effects of photoperiod and temperature upon blood feeding was apparent. Examination of the ovarioles of post-blood feeding females, reared under each of the above conditions, revealed no evidence of gonotrophic dissociation (the inhibition of oögenesis despite continued blood feeding).Fat body hypertrophy occurred in females reared under long-day conditions, whereas hypotrophy of this tissue was apparent in short-day females. No significant difference in fat body development occurred between parous and nulliparous females reared under long-day photoperiod conditions. Short-day blood-fed females reared at 15°C deposited a significantly greater amount of fat than short-day blood fed females reared at 20° and 25°C, and short-day nonbloodfed females reared at 15°, 20°, and 25°C. The primary stimulus for fat body hypertrophy appears to be long-day photoperiod conditions. Temperature exerted little discernible effect upon this process, and there was no interaction between the effects of photoperiod and temperature upon the degree of fat body development.     

Summer dormancy ensures univoltinism in the predatory bug Picromerus bidens

The seasonal cycle of Picromerus bidens L. (Heteroptera: Pentatomidae) is usually considered to be univoltine with an obligatory winter egg diapause. Seasonal adaptations of the species were studied in the laboratory and in field experiments. When reared under short-day photoperiodic conditions (L12:D12 and L14:D10), all females began to lay eggs synchronously soon after their emergence. However, in the females reared under long-day conditions (L18:D6 and L20:D4) and outdoors in June–July, oviposition was significantly delayed. This delay in reproduction induced by photoperiodic conditions and then spontaneously terminated was considered to be aestivation. Egg batches laid by females in the laboratory and in the field were kept at 25 °C for two months. From 30.8 to 93.8% of batches contained eggs which hatched without cold treatment between day 14 and 60 after oviposition. The proportion of eggs hatched was 17.7 to 20.9% in the short-day regimes, while it was significantly less (5.7 to 6.0%) under long-day conditions. It is concluded that in some eggs diapause is of low intensity and that if under natural conditions the first batches had been laid at the end of June, nymphs would have hatched at least from some eggs during the same season even without cold treatment. Such untimely hatching would have resulted in the death of nymphs and adults unprepared for overwintering. A photoperiodic response which induces aestivation in the early emerging adults in June–August may prevent early oviposition and occurrence of a second generation and thus maintains univoltinism in P. bidens.     

Diapause termination, post-diapause development and reproduction in the beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae)

Effects of photoperiod and cold exposure on diapause termination, post-diapause development and reproduction in Loxostege sticticalis were examined. Larvae were reared at diapause inducing condition (22 °C, L:D 12:12) consistently or transferred to long day photoperiod (L:D 16:8) and darkness (L:D 0:24) respectively, after entering into diapause. Diapause was terminated in approximately 40% of the larvae after 36 days, and no significant differences were observed between photoperiods, suggesting larval diapause was terminated spontaneously without being induced by photoperiods. Cold exposure significantly hastened diapause termination. The diapause termination incidence increased significantly with peaks of 98% at both 5 °C and 0 °C exposure for 30 days, as compared to 42% in controls not exposed to cold, while the mortality and number of days required for diapause termination decreased dramatically. The optimal low temperature exposure periods under 5 °C or 0 °C were 20 days and 30 days, showing a higher termination incidence and shorter time for diapause termination. This suggests that the low temperatures in winter play an important role in diapause termination under natural conditions. The threshold temperatures for post-diapause development in prepupae and pupae were 9.13 °C and 10.60 °C respectively, with corresponding accumulations of 125 and 200 degree-days. Adults that experienced larval diapause significantly delayed their first oviposition, oviposition period was prolonged, and the lifetime number of eggs laid was decreased, however both males and females have significantly longer longevity. The field validation of diapause termination, the degree-days model, and the relationship between diapause and migration in L. sticticalis were also discussed.     

Induction of adult diapause and of low and high reproductive states in a parasitoid wasp,Ooencyrtus nezarae, by photoperiod and temperature

Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) was reared on eggs ofRiptortus clavatus (Thunberg) (Heteroptera: Alydidae) at various temperatures under long-day (L16:D8) or short-day (L10:D14) conditions. There was no diapause during egg, larval or pupal stages under any set of conditions examined. However, at 15°C under short-day conditions, vitellogenesis was arrested in all adult females and they entered diapause. At 15°C under long-day conditions, or at 20°C under short-day conditions, some adult females entered diapause. Under the latter set of conditions, the adult females laid eggs but they laid fewer eggs than under long-day conditions, Even at 25°C, under short-day conditions, adult females laid fewer eggs than under long-day conditions, and this low rate of oviposition was attributed to the retarded development of ovaries. Diapause adults reared at 15°C were more resistant to low temperature than nondiapause adults reared at 25°C.     

Diapause induction in the codling moth, Cydia pomonella: effect of prediapause temperatures

The effect of four prediapause temperatures (18, 22, 26 and 30°C) on the photoperiodic response of the codling moth, Cydia pomonella (L.), was studied under controlled conditions. The highest rates of diapause were recorded, for all day-lengths, at temperatures of 22 and 26°C while relatively lower rates of diapause were elicited at 18 and 30°C. The same trend was demonstrated by projecting the values of the critical photoperiod which induces 50% diapause (=CPhP₅₀) over the prediapause temperature. The change in diapause incidence as a function of photoperiod, at all prediapause temperatures, exhibited a response characteristic of long-day insects, i.e. high rates of diapause at short days (12–13.5 h) and a decrease in diapause incidence at long days (14–15 h). The results for temperatures 22, 26 and 30°C support the view that lower prediapause temperatures enhance diapause induction, at a give photoperiod, while higher temperatures tend to avert or diminish the process. On the other hand, the low rates of diapause obtained at 18°C contradict this view. Nevertheless, high correlation was found between the laboratory evidence and field data, indicating the adaptability of the Israeli codling moth to subtropical climate.     

Egg production in response to combined alternative foods by the predator Coccinella transversalis

The peformance of the parasitoid Anagyrus kamali Moursi [Hymenoptera: Encyrtidae], as a function of host density, temperature, and photoperiod was investigated with the objective to optimize a mass-rearing system in the context of a biological control program. The number of hosts parasitized at densities varying from 2–100 hibiscus mealybug (HMB), Maconellicoccus hirsutus Green [Homoptera: Pseudococcidae], corresponded to a type II-III functional response in fixed-time conditions and a type III in variable-time conditions. Twenty-six percent of the oviposited eggs led to progeny emergence with a sex ratio of 0.49±0.102 (M/F), regardless of host density. Fecundity and oviposition period under six abiotic combinations (i.e., two temperatures (26±2 °C and 32±2 °C) and three photoperiods (L0:D24, L12:D12, L24:D0)) were measured. Lifetime fecundity and reproductive life were significantly affected by temperature and photoperiod conditions. Optimum female parasitoid lifetime fecundity was attained at 26±2 °C, L0:D24 with an average of 116.1±17.43 eggs. At 32±2 °C, L24:D0 and L12:D12, an average of 79.4±34.57 and 85.8±35.81 eggs were laid, respectively. Reproductive longevity was maximal at 26±2 °C, L0:D24 with 12±4.85 days of oviposition. Because the parasite A. kamali can be reared optimally without light, this may save tremendous energy costs.     

Age related changes in the calling behaviour and the attractiveness of obliquebanded leafroller virgin females, Choristoneura rosaceana, under different constant and fluctuating temperature conditions

C. rosaceana (Lepidoptera: Tortricidae) virgin females initiated calling in the first scotophase following emergence at both 15° and 20°C constant temperature under a 16L:8D photoperiod. As females aged, the onset of calling occurred much earlier, and the time spent calling increased significantly at both temperature conditions. For a given age, the onset of calling occurred significantly earlier at 15°C than at 20°C, but the duration of calling did not vary with temperature. Similar results were obtained under warm and cold thermocycles that simulated natural conditions prevailing during the summer and fall flight periods. A comparison of the degree of attractancy of different-aged females (0, 3, and 5 days old) in the field showed that the attractiveness of 0-day-old females relative to that of 3-day old females did not vary between flights. However, compared to day-5 females, the relative attractiveness of 0- and 3-day-old females was greater in the summer than in the fall flights. The potential adaptative value of these changes for seasonal mating success are discussed.     

Photoperiodic responses during larval development and diapause of two geographic ecotypes of the rice stem maggot, Chlorops oryzae

Chlorops oryzae is bivoltine in northern Japan but trivoltine in the southern part of the country. In the bivoltine strain, both the egg and larval stages were found to be sensitive to photoperiod. When the egg stage was exposed to a long-day photoperiod (16L:8D), larval development showed a short-day type response, and mature third-instar larvae entered a summer diapause under a long-day photoperiod (15L:9D). When eggs experienced short days, the first-instar larvae entered a winter diapause under short-day conditions, and the critical photoperiod in the larval stage ranged from about 14L:10D to about 12L:12D as the photoperiod experienced by the eggs increased from 12L:12D to 14L:10D. However, the development of the larvae after overwintering was not influenced by the photoperiod. In the trivoltine strain, larval development was retarded under a 14L:10D photoperiod but not under either shorter or longer photoperiods, when larvae had spent the egg stage under a 16L:8D photoperiod. The critical photoperiod of the larval stage for the induction of a winter diapause in the first instar was about 12L:12D, though it varied to some extent with the photoperiod during the egg stage. Thus, Chlorops oryzae was able to adapt itself to the local climatic conditions by the development of variable and complicated photoperiodic responses.     

Photoperiod- and temperature-dependent induction of larval diapause in a multivoltine bruchid, Bruchidius dorsalis

The wild bruchid beetle, Bruchidius dorsalis Fahraeus (Coleoptera: Bruchidae), has a multivoltine life cycle and overwinters in several developmental stages in the middle part of Japan. We investigated the incidence of diapause under different conditions of photoperiod (from L8:D16 to L16:D8) and temperature (at 20 °C and 24 °C). Our experiments revealed the following results: (1) B. dorsalis entered diapause at the final (late fourth) instar larva under short photoperiods, (2) the larval diapause incidence was dependent on temperature (critical photoperiods were 12.5 h at 20 °C and 12 h at 24 °C), (3) some individuals did not enter diapause under short-photoperiod conditions at 24 °C, and (4) the sensitive stages to the photoperiod were from the late egg stage to the early first instar larva. Based on these results, we discuss not only the evolution of a complex overwintering strategy inB. dorsalis but also the domestication process of stored-bean pests.     

Influence of temperature and photoperiod on the re-attachment process ofGelidium sesquipedale (Clem.) Born. et Thur. (Gelidiales: Rhodophyta)

Thalli of the economically important rhodophyteGelidium sesquipedale were cultured for 8 weeks under laboratory conditions, and the influence of temperature and photoperiod on the re-attachment process were studied. Four different temperatures (16, 18, 20, 22 °C) and four different photoperiods (6:18, 12:12, 14:10, 16:8) were used and the results obtained in the thalli responses such as apical growth (measured as elongation of principal apex), rhizoidal cluster production and number of necrotic patches were tested.During the re-attachment process, the best results were obtained at temperatures of 16–18 °C, when rhizoidal cluster production was high and necrotic patch development was low (18 °C) or absent (16 °C). Temperatures of 20 and 22 °C favoured high rhizoidal cluster production, but also a high production of necrotic patches that finally led to death. The results suggest that long-day photoperiods (14:10, 16:8) produce a higher number of rhizoidal cluster bands than short-day photoperiods (6:18) at the same temperature.     

Causes of Flowering of Long-Day Potato Species under Short-Day and Cold-Night Conditions

Flowering and tuber formation in high-mountain potato species Solanum sparsipilum Bitt., S. acaule Bitt., S. punae Juz., S. demissum Lindl., and a tuber crop Ullucus tuberosus Caldas. were investigated. All these species are characterized by absolute requirement of long day-length for flowering and short day-length for tuberization. Plants were grown under the following conditions: natural day-length with a photoperiod of 17 h or longer (treatment 1), short days with a photoperiod of 12 h and warm nights (15–20°C) (treatment 2), and short days with cold nights (5–6°C) (treatment 3). In the first treatment, plants produced flowers but no tubers. In the second treatment, plants produced tubers but no flowers. In the third treatment, plants produced both flowers and tubers. In leaves of S. acaule and U. tuberosus, the levels of gibberellins and ABA were determined. A high activity of gibberellins in the third treatment was similar to that in the first treatment, whereas high ABA activity in the third treatment was similar to that in the second treatment. It is supposed that cold nights retard the destruction of GA in plants during the dark period of diurnal cycle and ensure a permanently high level of gibberellins, which facilitates flowering of long-day species under short-day conditions. The high level of ABA is considered a plant response to short-day conditions, which is favorable for tuberization.     

Alternative life cycles controlled by temperature and photoperiod in the oligophagous bug, Dybowskyia reticulata

Abstract. Effects of temperature and photoperiod on the induction and re-induction of adult diapause were examined in Dybowskyia reticulata (Dallas) (Heteroptera: Pentatomidae). Adults collected from the field after overwintering in early summer continued oviposition under long-day conditions of LD 16:8 h at 20 or 25°C, while they re-entered diapause under short-day conditions of LD 12:12 h at 25, 27.5 or 30°C. By contrast, adults reared in the laboratory from eggs at 20 or 25°C entered diapause under both long-day and short-day conditions, whereas those reared at 27.5 and 30°C entered diapause only under short-day conditions. Under quasi-natural conditions in 1993, when summer temperature was low, most adults of the first generation entered diapause in late July. However, in the warmer summer of 1996, oviposition was recorded in many females that ecdysed into adults from July to early August. Even though the seeds of the host plants occur in a restricted period from early summer to early autumn, in warmer years D. reticulata may produce a second generation. The response to temperature with a threshold between 25 and 27.5°C in D. reticulata brings about a switch between the univoltine and bivoltine life cycles.     

Cold-hardiness in the predatory miteAmblyseius potentillae (Acari: Phytoseiidae)

Cold-hardiness was studied in the predatory miteAmblyseius potentillae (Garman) by determining the mortality after exposure to –5°C. Predators reared under diapause-inducing conditions were more cold-resistant than mites that had been kept under long-day conditions. An acclimatization period at 4°C prior to exposure to sub-zero temperatures had a positive effect on the cold-hardiness of the predator, and was remarkable in diapausing mites.Lower temperatures during diapause induction had a positive effect on cold resistance in this predator; mites in which diapause was induced at 15°C were more cold-resistant than mites that had been subjected to short-day conditions at 18 and 20°C.A day/night temperature rhythm did not increase the cold-hardiness of the mite when grown under long-day conditions. Such a rhythm did increase the cold-hardiness of the diapausing predator when given a short acclimatization period, but this effect disappeared after longer acclimatization periods.     

Photoperiodic regulation of the phospholipid molecular species composition in thoracic muscles and fat body of Pyrrhocoris apterus (Heteroptera) via an endocrine gland,corpus allatum

In the conventional view, the winter adaptation of membrane lipids is induced by temperature decrease. We propose that winter remodelling of membranes in Pyrrhocoris apterus is triggered by short-day photoperiod before the temperature decrease and changes caused by cold temperature represent the later phase of adaptation. The induction of diapause by short-day photoperiod results in an accumulation of phosphatidylethanolamine (PE) molecular species with C_16:0/C_18:2 acyl chains esterified to sn-1/sn-2 positions of glycerol at the expense of C_18:0/C_18:2. Proportions of C_16:0/C_18:2-PE are enhanced in short-day compared to long-day insects in both thoracic muscles (TM, 15.0 vs. 8.2%) and fat bodies (FB, 24.9 vs. 13.6 %). Proportions of C_16:0/C_18:2-PE are further enhanced during cold acclimation (to 26.5% in TM, 33.6 % in FB) at the expense of a more saturated species, C_18:0/C_18:1-PE. These changes are less prominent in phosphatidylcholines (PC). The effect of photoperiod seems to be mediated via the corpus allatum. Long-day non-diapause females deprived of their corpus allatum have the phospholipid molecular species profile similar to that found in short-day diapausing females. While the acyl chain remodelling is regulated by both photoperiod and temperature, the head group composition is regulated by temperature only. Similar to most other organisms, the level of PE is higher (50.3 vs. 43.5% in TM, 44.3 vs. 37.8% in FB) and that of PC is lower (35.9 vs. 40.2% in TM, 41.6 vs. 46.1 % in FB) at cold temperatures (≤1°C) compared to warm temperatures (≥16°C). In contrast to a general rule, the PE is less unsaturated than PC. In both TM and FB, proportions of unsaturated/unsaturated molecular species are consistently high in PC (56.3-67.5% in TM, 59.2-66.6% in FB), while they are consistently low in PE (19.1-26.7% in TM, 12.1-15.1% in FB). An adaptive significance of changes in the phospholipid composition for the low temperature and/or dehydration stress is discussed in relation to known physical properties of phospholipids.     

Photoperiodic and Thermal Regulation of Development and Cold Hardiness in Larvae of the Clover Leaf Weevil, Hypera punctata

Effects of photoperiod and temperature on the development and cold hardiness were investigated in larvae of Hypera punctata. At a relatively low temperature (15°C), the larvae fed less and developed more slowly under a 12L:12D (SD) photoperiod than under a 16L:8D photoperiod (LD). SD larvae had lower gut weight against the whole body weight and lower supercooling point (SCP) than the LD counterparts for the same instar and same body weight. This was because the larval SCP is markedly affected by the quantity of the gut content. Laboratory experiments indicated that the low temperature mortality of this larvae occurred mainly due to freezing irrespective of the photoperiod and temperature, suggesting that the lower lethal temperature (LLT) depends on the supercooling ability of larvae. The SD larvae tended to have a lower SCP and hence a lower LLT than the LD counterparts at 15 or 10°C, unlike at 20°C. Thus, the slower larval development under SD conditions at relatively low temperatures may prevent larvae from reaching the later instar, which have a higher SCP and thus less cold tolerance, during the coldest season. The suppressed feeding activity under SD conditions would lower the SCP, thereby reducing the possibility of lethal tissue freezing. Such a photoperiodic and thermal regulation of the larval development and the supercooling ability appear to represent adaptive mechanisms for winter survival in this beetle.     

Effect of temperature on photoperiodic response in a selected 'non-diapause' strain of Pyrrhocoris apterus (Heteroptera)

Abstract. The artificially selected 'non-diapause' strain of Pyrrhocoris apterus (L.) (Heteroptera) showed no diapause response to photoperiod at 26°C (Socha & Hodkova, 1994). However, the diapause response to short-day photoperiod (LD 12:12 h) became apparent at lower temperatures of 17°C (70% diapause) or 20°C (41% diapause). Diapause was induced in 60% females by short-day photoperiod combined with thermoperiod of 26/16°C, whereas only 20% diapause was induced by the same thermoperiod under continuous darkness. Thus the time-measuring system was not removed by artificial selection but the diapause response was shifted to lower temperatures. The diapause response to short days seems to be favoured rather by low temperature during scotophase than by low temperature throughout the whole light/dark cycle. If the percentage of diapause at 26°C is compared in F₁ hybrids and in wild and selected parental strains the diapause appears to be dominant at LD 13:11 h but recessive at LD 11:13 h and LD 10:14 h. A hypothesis is proposed that the inheritance of the percentage of diapause in F1 hybrids is determined by interactions of genes controlling the temperature dependence of photoperiodic response.     

Phenological studies on the wing dimorphism of a semi-aquatic bug,Microvelia douglasi (Heteroptera: Veliidae)

Seasonal changes in the percentage of macropterous individuals in natural population of Microvelia douglasi in permanent habitats were investigated in Shimane, western part of Japan. Macropterous forms of 5th instar nymphs occurred most frequently from late June to early July. However, the seasonal appearance of macropterous adults differed between habitats and years. There were significant effects of density, temperature, photoperiod and food availability on the percentage of macropters under laboratory conditions. The percentage of macropterous forms generally increased with density, but the mode of the response was modified by temperature, photoperiod and food availability. When insects were reared under long day photperiod (16L8D) with abundant food (0.5–1.0 g/container/day), the percentage of macroptery varied most among the different density treatments at the intermediate temperature (24°C) and the difference was reduced at higher temperature (28° and 32°C). Effects of temperature were small under the short day photoperiod (12L12D). Under 12L12D photoperiod sensitivity of density responses were higher at 32°C and lower at 24°C than those under 15L8D photoperiod. When the insects were supplied with food every other day, the density response became obscure. Ecological significance of environmental responses in the wing morph determination was discussed in relation to the heterogeneous environment of water surface.     

The role of photoperiod and temperature in determination of summer and winter diapause in the cabbage beetle,Colaphellus bowringi (Coleoptera: Chrysomelidae)

The cabbage beetle, Colaphellus bowringi, is a short-day species undergoing an imaginal summer and winter diapause. Its photoperiodic response highly depends on temperature. All adults entered diapause at ≤ 20 °C regardless of photoperiods. High temperatures strongly weakened the diapause-inducing effects of long daylengths. The diapause-averting influence of short daylengths was expressed only at high temperatures (above 20 °C). This indicates that the beetle has a cryptic ability to reproduce in summer. In fact, summer and winter diapause were induced principally by relatively low temperatures in the field, whereas photoperiod had less influence on diapause induction. The critical daylength for the autumnal population was between 12 h and 13 h. By transferring from a long day to a short day or vice versa at different times after hatching, it was shown that the sensitive stage with regard to photoperiod was the larva, whereas a long day was photoperiodically more potent than a short day. The sensitive stage to temperature encompassed the larval, pupal and adult stages. This different response pattern serves to ensure that the beetle enters summer and winter diapause in time. The selections for non-diapause trait under laboratory (at 25 °C) and natural conditions (at >24 °C) showed that the beetle could lose its sensitivity to photoperiod very rapidly.     

Androgenesis in isolated pollen cultures ofNicotiana tabacum: Dependence upon pollen development

Summary The influence of the stage of pollen development and of the growth conditions of donor plants on the performance of cultures of isolated pollen fromNicotiana tabacum, var. Badischer Burley has been studied. The method described includes cold treatment (4–5 °C for 3 days) and a pre-culture of the anthers for 7 days at 24 °C before the pollen is isolated. With this system reproducible results were obtained with pollen at the early binucleate stage collected from plants 11–13 weeks old. Another prerequisite for reproducibility is that the donor plants must have been grown for eight weeks in soil with an additional supply of mineral salts. Furthermore, the production of haploids by these pollen cultures was strongly influenced by the photoperiodic and temperature regime experienced by the donor plants; it was best (0.07%) with pollen from short-day plants (8 hours light per day at 18 °C) and rather weak (0.015%) with pollen from long-day plants (16 hours light per day at 24 °C). In contrast to other reports, haploid production from anther cultures was not influenced by the photoperiod or temperature.Cytological studies undertaken at the end of the pre-culture period showed that there were no differences in the percentage of potential embryos for the stages of the late uninucleate, 1. pollen mitosis and early binucleate pollen of long-day plants (1.5%). This value was considerably higher with pollen from short-day plants (7–9%), indicating that short-day conditions at 18 °C of the donor plants are favourable for the induction of androgenesis. However, only the potential embryos formed by the pollen at the initial binucleate stage were able to continue androgenetic development after isolation.