Survival at low temperature of larvae of the pine processionary moth Thaumetopoea pityocampa from an area of range expansion

1 Larvae of Thaumetopoea pityocampa (Lepidoptera: Notodontidae) develop throughout the winter, although their feeding activity and survival can be impaired by adverse climatic factors. The present study investigated the survival at low temperature of larvae originating from a population with range expansion in an alpine valley in Northern Italy.
2 The supercooling point of individually analysed larvae averaged at −7 °C. This value insufficiently described the cold hardiness of the larvae; 39% of the tested larvae were alive when returned to room temperature immediately after freezing. When larval colonies inside their nest were exposed to −17 °C for 1 h after gradual temperature decrease, survival was 70.4%.
3 Rearing of larvae in the laboratory at different day/night temperatures indicated an effect of cumulative chill injury on larvae. A logistic regression explained the relationship between negative thermal sum (h°C below 0 °C) received in the laboratory experiment and larval survival. A similar relationship was demonstrated between negative thermal sum and survival of larval colonies in the field.
4 In the laboratory experiment, some tested larvae were able to survive for up to 8 weeks without feeding depending on rearing temperature. As expected, feeding occurred only when larvae were reared at temperatures of 9 °C day/0 °C night.
5 We classify the larvae of T. pityocampa as being moderate freezing tolerant. The winter behaviour allows this species to track climate warming by a rapid expansion into those areas that become compatible with the insect's development.     

Digestive processes during the development of the roach, Rutilus rutilus L.

During development of the roach from larvae to adults relative gut length increases from 44 to 104% of the body length. Gut passage rate correlates with gut length, ranging from 2.25 h in larvae to 6.2 h in adults (at 20 ± 1°C). In controlled experiments (20°C, feeding on carp diet) tryptic activity of the gut content increases with the age of the fish. In larvae artificial diet leads to considerable increase of tryptic activity, but to low growth rate and finally to total mortality. The reabsorption of digestive enzymes in the hindgut is efficient only in adults in which tryptic activity in the second half of the intestine is reduced to about 12% of total activity.     

Consumption and gut evacuation rate of laboratory‐reared spotted seatrout (Sciaenidae) larvae and juveniles

The temperature and mass dependence of maximum consumption rate was measured for larval and early juvenile spotted seatrout Cynoscion nebulosus . Maximum consumption ( C _MAX) estimates were obtained from feeding and gut evacuation experiments on larvae (3·8–19 mm standard length, L _S) at three temperatures (24, 28 and 32° C), and maximum consumption experiments on juveniles at three temperatures (20, 26 and 32° C). Feeding levels were determined for larvae fed live prey ( Brachionus plicatilis and Artemia salina ) ad libitum . The midgut and total evacuation times were estimated for fish feeding continuously and discontinuously using alternate meals of tagged and untagged live prey. Temperature and fish size had significant effects on gut evacuation and consumption. The gut evacuation time increased with increasing fish size, and decreased with increasing temperatures. Mass‐specific midgut contents increased for small larvae <0·156 mg dry mass ( M _D)( c . 4 mm L _S), and decreased for larger larvae and juveniles. Maximum consumption was modelled by fitting a polynomial function to a reduced dataset of individuals feeding at high levels. The C _MAX model predicted an initial increase in specific feeding rate from 70 to 155% M _D day⁻¹ for small larvae, before declining for larger larvae and juveniles.     

Bimodal life cycle of the burying beetle Nicrophorus quadripunctatus in relation to its summer reproductive diapause

Abstract 1. Under natural conditions in Kyoto, Japan, the reproductive activities of Nicrophorus quadripunctatus Kraatz (Coleoptera: Silphidae) decreased in summer and the species showed a bimodal life cycle.
2. In the laboratory, most adult pairs raised at 20 °C under a LD 12:12 h regime reproduced when provided with a piece of chicken. In adults raised at 20 °C under a LD 16:8 h regime, however, both reproductive behaviour and ovarian development were reduced. It is concluded that these adults entered a reproductive summer diapause.
3. High temperature (25 °C) also suppressed the reproductive behaviour even under a favourable LD 12:12 h regime. In the field, therefore, adults reduce their reproductive activity in summer because of diapause induced by long-day photoperiods and direct inhibition of reproduction by high temperatures.
4. When the temperature was changed from 20 °C to 25 °C immediately after hatching of larvae, they reached the wandering stage in 95% of adult pairs. When the temperature was changed from 20 °C to 25 °C immediately after oviposition, however, no larvae hatched in 85% of pairs. Egg mortality was significantly higher at 25 °C than at 20 and 22.5 °C; no eggs hatched at 27.5 °C. The physiological mechanisms for reducing reproduction probably prevent the beetles from inefficient oviposition in summer.     

Thermal conditioning of fifth-instar Cydia pomonella (Lepidoptera: Tortricidae) affects HSP70 accumulation and insect mortality

Abstract.  Levels of HSP70 protein of fifth-instar codling moth [ Cydia pomonella (L.) (Lepidoptera: Tortricidae)] are determined after conditioning at 35 °C for different times and also after recovery at 22 °C. Protein samples from larvae conditioned for different times are separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis electrophoresis. Sub-lethal thermal conditioning at 35 °C for 40 min, 2, 6 and 18 h induces new protein bands in the extracts from treated codling moth larvae. Immunodetection with an antibody to a heat-inducible HSP70 indicates a stronger reaction after 35 °C for 2, 6 and 18 h than after 35 °C for 40 min or control and, during the recovery period at 22 °C, the level of heat shock protein decreases. Conditioning of fifth-instar codling moths at 35 °C also induces thermotolerance in the insects and necessitates longer times at a lethal temperature to ensure mortality. Thermotolerance is correlated with the accumulation of heat inducible HSP70 protein.     

Cold shock and cold tolerance in larvae and pupae of the blow fly, Lucilia sericata

Abstract.  Understanding the effects of low winter temperatures on mortality is essential in the development of a full understanding of the long-term population dynamics of any insect. The present study aims to examine the survival of pupae and larvae of the blow fly, Lucilia sericata , at overwintering temperatures. Groups of pupae and diapausing and nondiapausing third-stage larvae of L. sericata are maintained in cooled incubators at either 3 °C and 6 °C. Groups are removed from the incubators at 3–4-day intervals and transferred either to−8 °C or to 25 °C. After 1 h in the freezer, the larvae and pupae exposed to this cold-shock are also transferred to 25 °C. Larvae and pupae are then allowed to continue development and the number of adults emerging from each group is counted. The results demonstrate that survival decreases linearly with the period of exposure at both 3 °C and 6 °C. Mortality is higher at 3 °C than at 6 °C and, in groups that receive the cold shock, cold-shock reduces emergence by over 50%. However, there is no consistent tendency for diapausing larvae to survive prolonged cold or cold shock better than other life-cycle stages. The results suggest that the facultative development of an overwintering diapause stage in L. sericata does not appear to be an adaptation to enhance cold tolerance or resistance to cold shock. It is concluded that the survival of overwintering L. sericata is likely to be relatively less affected by low temperatures than it is by, for example, biotic factors, particularly given the buffered soil environment and short time-scales over which periods of cold act.     

Temperature and neuromuscular development in the tambaqui

The development of muscle innervation pattern was investigated in larvae of the Amazonian fish, the tambaqui Colossoma macropomum. The time to hatching decreased from 28–29 h at 23.5° C to 11–12 h at 31° C. The larvae hatched after the completion of somitogenesis (38-somite stage) at 23.5° C but only at the 33-somite stage at 28–31° C. Embryos were stained for acetylcholinesterase activity and with an acetylated tubulin antibody in order to visualize neural processes. All muscle fibre types were initially innervated at their myoseptal ends. The development of motor innervation to the trunk muscle was delayed with respect to hatching at higher temperatures. At hatching, muscle fibres were innervated only to somites 16–17 at 28–31° C and somite 23–26 at 23.5–25° C (counting from the head), although the larvae swam vigorously to avoid sinking. In contrast, in newly hatched larvae myofibrils were present right along the trunk at all temperatures in both the superficial and inner muscle fibres. At hatching numerous multi-layered membrane contacts with the ultrastructural characteristics of gap junctions, were found between muscle fibres and at the inter-somite junctions, suggesting the somites were initially electrically coupled. These structures disappeared concomitant with the development of muscle endplates right down the trunk. The larvae started feeding 5 days post-hatch at 28° C. First feeding was associated with a dramatic decrease in the volume density of mitochondria and an increase in the volume density of myofibrils in the inner muscle fibres. The polyneuronal and multi-terminal pattern of innervation characteristic of adult slow-muscle fibres also developed around the time of first feeding.     

The effect of temperature on the size and population density of dinoflagellates in larvae of the reef coral Porites astreoides

Abstract. Pre-settlement events play an important role in determining larval success in marine invertebrates with bentho-pelagic life histories, yet the consequences of these events typically are not well understood. The purpose of this study was to examine the pre-settlement impacts of different seawater temperatures on the size and population density of dinoflagellate symbionts in brooded larvae of the Caribbean coral Porites astreoides. Larvae were collected from P. astreoides at 14–20 m depth on Conch Reef (Florida) in June 2002, and incubated for 24 h at 15 temperatures spanning the range 25.1°–30.0°C in mean increments of 0.4±0.1°C (±SD). The most striking feature of the larval responses was the magnitude of change in both parameters across this 5°C temperature range within 24 h. In general, larvae were largest and had the highest population densities of Symbiodinium sp. between 26.4°–27.7°C, and were smallest and had the lowest population densities at 25.8°C and 28.8°C. Larval size and symbiont population density were elevated slightly (relative to the minimal values) at the temperature extremes of 25.1°C and 30°C. These data demonstrate that coral larvae are highly sensitive to seawater temperature during their pelagic phase, and respond through changes in size and the population densities of Symbiodinium sp. to ecologically relevant temperature signals within 24 h. The extent to which these changes are biologically meaningful will depend on the duration and frequency of exposure of coral larvae to spatio-temporal variability in seawater temperature, and whether the responses have cascading effects on larval success and their entry to the post-settlement and recruitment phase.     

Gut evacuation and absorption efficiency of walleye larvae

Gut evacuation and absorption efficiency in zooplanktivorous walleye Stizostedion vitreum larvae (10–19 mm; 0.53–6.8 mg dry weight) were examined in the laboratory. The decline in dry weight of whole digestive tract contents was examined at 15, 20, and 25) C in discontinuous feeding larvae. The observed relationship between short-term evacuation rate and gut fullness indicated that evacuation approximated most closely an exponential decline. When gut evacuation rate, R , was calculated as the slope of loge gut fullness v . time there were significant effects of temperature and walleye size on R . Gut evacuation rate was higher at 20° C than at 15 or 25° C and declined with increasing walleye dry weight. Absorption efficiency at 20° C was examined by qualitative analysis of food and faeces. Apparent digestibility, D _a, of crustacean zooplankton increased with gut retention time and declined with walleye dry weight. Maximum D _a was estimated to be 79% of organic matter from food retained in the gut for 6 h.     

Effects of temperature on the development and survival of an endangered butterfly, Shijimiaeoides divinus barine (Leech) (Lepidoptera: Lycaenidae)

The effects of temperature on the development and survival of Shijimiaeoides divinus barine were examined in the laboratory in 2008. The eggs and larvae were reared at temperatures of 15, 17.5, 20, 25, 30 and 35°C with a long-day photoperiod of 16 h light : 8 h dark (16L : 8D). The highest hatchability of eggs was 88.0% at 20°C, but hatchability at high temperatures of 30 and 35°C was 30 and 0%, respectively. The lowest and highest survival rates from the first to third instar were 18.8% at 15°C and 76.9% at 20°C. Few deaths were observed after the fourth instar. The shortest developmental periods of the eggs and larvae were 4.0 and 15.8 days at 30°C, and the durations of the egg and larval stages increased significantly as the temperature decreased. The developmental zero and thermal constants were 9.6°C and 82.6 degree–days for the egg stage, and 10.7°C and 306.8 degree–days for the larval stage. The developmental period of the natural population of S. divinus barine in Azumino City, Nagano Prefecture was calculated using the developmental zero, thermal constants and Azumino City temperature data.     

Characterization of pyrolysin, a hyperthermoactive serine protease from the archaebacterium Pyrococcus furiosus

Abstract From the hyperthermophilic archaebacterium Pyrococcus furiosus an oxygen-stable, extremely thermostable protease activity, which we designate pyrolysin, has been identified and characterized. Pyrolysin is a cell-envelope associated protease activity high thermo-activity and stability. The temperature optimum is 115°C and half-life values in the absence of substrate are: at least 96 h at 80°C, 9 h at 95°C, 4h at 100°C, 20 min at 105°C and 3 min at 110°C. Pyrolysin is active at a broad pH range between 6.5 and 10.5, and was classified as a serine-type protease activity. Zymogram staining showed the presence of multiple protease bands of about 140, 130, 115, 100 and 65 kDa.     

Diapause induction in Trichogramma embryophagum Htg. (Hym., Trichogrammatidae): the dynamics of thermosensitivity

It is known that the low temperature is the most important factor inducing the pre-pupal diapause in Trichogramma species. The position of the thermosensitive period over the life cycle and temporal variation of the degree of responsiveness were investigated in T. embryophagum Htg. by transferring pre-imaginal stages between 'neutral' temperature of 15°C and 'diapause-inducing' temperature of 10°C. Our experiments showed that 6 days long exposure at 10°C significantly increased the percentage of diapausing pre-pupae when started during rather large part of development: from embryo up to early pre-pupa. The highest thermosensitivity was recorded during the embryo and the larval stages, with some decrease during the hatching period. Treatments with shorter cold exposures (2–3 days) gave similar results. Even 24 h long exposure at 10°C increased the percentage of diapausing pre-pupae when applied during egg or early larval stage. Being started at the same stage of development, longer cold exposures caused stronger increase in the percentage of diapausing individuals. The experiments did not reveal any significant daily changes in thermosensitivity: at 12 : 12 h light : dark, larvae subjected to the low temperature during six photophases showed practically the same percentage of diapausing individuals as those subjected to the low temperature during six scotophases, and as those subjected to the 3 days long uninterrupted cold exposure. Hence, in natural conditions even occasional short-term cold periods could be accumulated.     

Insecticidal and antifeedant activities of proteins secreted by entomopathogenic fungi against Spodoptera littoralis (Lep., Noctuidae)

Abstract:  Entomopathogenic fungi are a poorly exploited source of insecticidal proteins, which may be used for the development of new natural insecticides and as alternative molecules for transgenic deployment. The crude soluble protein extracts in Adamek's liquid medium of 25 fungal isolates belonging to the fungal species Metarhizum anisopliae , Beauveria bassiana , Beauveria brongniartii and Scopulariopsis brevicaulis were screened by per os toxicity on Spodoptera littoralis larvae. Whilst extracts from two M. anisopliae and two B. bassiana isolates gave significant mortalities when applied either on alfalfa leaf discs or incorporated into artificial diet, the one from M. anisopliae 01/58-Su isolate was the only most toxic that showed promise for S. littoralis control. In leaf disc assays, this extract exhibited strong dose-related toxic and antifeedant activity against the larvae. At 10, 20 and 40  μ g protein/insect, the extract gave 61.3%, 96.6% and 100.0% mortality, respectively, and average survival time of 5.7, 4.3 and 3.1 days respectively. Not only the antifeeding index was dose-related, but it significantly increased over time in a dose-related manner. Longer exposure times led to a dose-related significant increase in larval mortality. The exposure times for 50% mortality were 91.3 h and 62.1 h for 20 and 40  μ g protein/insect respectively. The crude extract when exposed to higher temperature or protease treatment lost toxicity, indicating that toxicity was protein mediated. In addition, the liquid medium composition did not influence its insecticidal activity. The effects of the protein extract on midgut cells of second instar larvae of S. littoralis were investigated by using both light and electron microscopy. A progressive bleeding of the midgut epithelium into the gut lumen was observed along with lysis of the epithelium and deterioration of the microvilli.     

Evaluation of entomopathogenic nematodes for control of the beetle, Luperomorpha suturalis Chen (Col., Chrysomelidae)

Abstract: The strains Steinernema feltiae Otio and A54, Steinernema ceratophorum D43 and Steinernema carpocapsae BJ were tested for their infectivity to the larvae and pupae of beetle ( Luperomorpha suturalis Chen) at 25 ± 0.5°C and 15 ± 0.5°C in laboratory conditions. The results, based on comparison of the insect mortalities and nematode penetration rates among four nematode strains, showed that S. feltiae Otio was a potential biocontrol agent of the larvae and pupae of L. suturalis . The mortalities of the larvae and pupae exposed to S. feltiae Otio strain were 95.8 and 97.1% at 25 ± 0.5°C and 78.0 and 83.0% at 15 ± 0.5°C, respectively. The nematode penetration rates of S. feltiae Otio of the larvae and pupae were 15.6 and 19.0% at 25 ± 0.5°C, 2.6 and 6.3% at 15 ± 0.5°C, respectively. Field efficacy of S. feltiae Otio strain was examined against beetle larvae in Hebei province, northern China. The population reduction of insect larvae was 77.8 and 13.9% at doses of 30 and 15 infective juveniles (IJs)/cm² of S. feltiae Otio after 38 days of treatment and 90.2 and 92.4% after 100 days of treatment. However, the population of the insect larvae was reduced only to 15.5 and 15.7% when treated with pesticide after 38 and 100 days, respectively. The efficiency between the two nematode doses was not significantly different but it was remarkably higher than that of the pesticide after 100 days of application. The results suggest that S. feltiae Otio strain could be an alternative to pesticide for beetle control.     

Gut evacuation of walleye pollock larvae in response to feeding conditions

Gut residence times of first-feeding walleye pollock larvae were measured at 6°C in continuous and discontinuous feeding regimes. Larvae fed tagged copepod prey evacuated their guts more quickly in continuous feeding as compared to the discontinuous feeding treatment. The mean gut residence time was estimated to be 5.0 h for larvae feeding continuously. Gut evacuation by larvae fed tagged prey and then isolated without food (discontinuous treatment) was slower and more variable, with an estimated gut residence time exceeding 8.0 h. Field and laboratory observations suggest that the larval fish gut may be modeled as an intermittent plug-flow reactor (PFR) in response to diel feeding patterns. The cyclical nature of gut dynamics has implications for gut content analyses and the estimation of daily food rations.     

Effects of temperature and delayed initial feeding on the survival and growth of Japanese flounder larvae

The effects of the timing of initial feeding (0, 1, 2, 3 and 4 days after yolk exhaustion) and temperature (15, 18 and 21° C) on the point‐of‐no‐return (PNR), survival and growth of laboratory‐reared Japanese flounder Paralichthys olivaceus larvae were studied under controlled conditions. The larvae reached PNR on 7·7, 5·2 and 4·2 days‐post‐hatching (dph) at 15, 18 and 21° C, respectively. At each temperature, larval growth did not differ significantly among the delayed initial feedings 1 day before PNR but decreased significantly in larvae first fed after that. In the treatments where initial feeding was equally delayed, larvae grew significantly faster at 18 and 21° C than at 15° C. The larvae survived apparently better at 15 and 18° C than at 21° C when initial feeding was equally delayed. At each temperature, survival of the larvae first fed before PNR did not differ noticeably, while delayed initial feeding after that apparently reduced their survival. These results indicated that there existed a negatively temperature‐dependent PNR in the Japanese flounder larvae. Survival and growth of the larvae strongly depended on temperature as well as the timing of initial feeding. High temperature accelerated the yolk exhaustion and growth of the larvae and thus reduced their starvation tolerance and survival. To avoid potential starvation mortality and obtain good growth, the Japanese flounder larvae must establish successful initial feeding within 2 days after yolk exhaustion at 15° C and within 1 day at both 18 and 21° C.     

Effect of temperature on the susceptibility of Culiseta longiareolata (Macquart) (Dipt., Culicidae) to two standard strains of biocontrol bacteria

The susceptibility of fourth instar larvae of Culiseta longiareolata (Macq.) to two standard strains of biocontrol bacteria was investigated under laboratory conditions. Larvae were found to be more susceptible to Bacillus sphaericus (Bs-IPS88) than to Bacillus thuringiensis H14 (Bti-IPS82) at 25 ± 1°C at concentrations ranging from 0.003 to 0.004 ppm at 2 and 3 days post-treatment. Statistical regression analysis revealed a significant mortality–concentration relationship for the two bacteria. Larval mortality increased significantly as temperature increased from 20 ± 1°C to 28 ± 1°C.     

Effects of photoperiod and temperature on the development and diapause of the bark beetle Ips typographus

Abstract:  Diapause was induced in a Central European population of Ips typographus grown at 20°C when the day length decreased below 16 h [50% diapause incidence occurred in the 14.7:9.3 h L:D (light:dark) regime]. The non-diapausing adults fed on days 2–6 and 10–14 after the ecdysis and swarmed after the second feeding bout with chorionated eggs in the ovaries and sperm in the spermiducts. Neither gonads nor the flight muscles matured and no swarming occurred in the diapausing adults. The development from egg to adult took about 34 days in both 18:6 h (no diapause) and 12:12 h L:D (diapause) regimes, but it was extended by up to 30% without diapause induction when only larvae or pupae were exposed to L:D 12:12 h. Diapause was induced in insects reared at L:D 12:12 h through the last larval and the pupal instars and/or in the adult stage. Temperature ≥ 23°C prevented diapause induction at L:D 12:12 h but diapause occurred at L:D 14:10 h associated with 26:6°C thermoperiod. The effect of thermoperiods on the developmental rate requires further research. Exposure of the non-diapausing adults to 5°C for several days blocked feeding and evoked a diapause-like state, whereas diapausing adults fed and their gonads slowly developed at this temperature. Diapausing adults exposed in forest to low night temperatures and transferred in October to 20°C readily reproduced at 18:6, but not 12:12 h L:D photoperiods. After 2-months at 5°C and darkness, they became insensitive to the photoperiod, matured and most of them also swarmed at 20°C in the 12:12 h L:D regime. In a Scandinavian population, diapause occurred at 18:6 h L:D and was terminated either by exposure to 5°C or by very long photoperiod (L:D 20:4 h) combined with high temperature (23°C).     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.     

Thermal ecology of western tent caterpillars Malacosoma californicum pluviale and infection by nucleopolyhedrovirus

Abstract 1. Western tent caterpillars hatch in the early spring when temperatures are cool and variable. They compensate for sub-optimal air temperatures by basking in the sun.
2. Tent caterpillars have cyclic population dynamics and infection by nucleopolyhedrovirus (NPV) often occurs in populations at high density.
3. To determine whether climatic variation might influence viral infection, the environmental determinants of larval body temperature and the effects of temperature on growth and development rates and larval susceptibility to NPV were examined.
4. In the field, larval body temperature was determined by ambient temperature, irradiance, and larval stage. The relationship between larval body temperature and ambient temperature was curvilinear, a property consistent with, but not necessarily limited to, behaviourally thermoregulating organisms.
5. Larvae were reared at seven temperatures between 18 and 36 °C. Larval growth and development increased linearly with temperature to 30 °C, increased at a lower rate to 33 °C, then decreased to 36 °C. Pupal weights were highest for larvae reared between 27 and 30 °C.
6. The pathogenicity (LD₅₀) of NPV was not influenced by temperature, but the time to death of infected larvae declined asymptotically as temperature increased.
7. Taking into account larval growth, the theoretical yield of the virus increased significantly between 18 and 21 °C then decreased slightly as temperatures increased to 36 °C.
8. Control and infected larvae showed no difference in temperature preference on a thermal gradient. The modes of temperature preference were similar to those for optimal growth and asymptotic body temperatures measured in the field on sunny days.
9. Warmer temperatures attained by basking may increase the number of infection cycles in sunny springs but do not protect larvae from viral infection.