Effect of temperature on reproduction and embryonic development of the cabbage stem flea beetle,Psylliodes chrysocephala L., (Coleoptera: Chrysomelidae)

The cabbage stem flea beetle, Psylliodes chrysocephala (L.) (Coleoptera: Chrysomelidae), is a major pest of winter oilseed rape. Despite the importance of this pest, detailed information on reproduction to predict risk of crop damage is lacking. This study investigates the effect of temperature on parameters of reproduction, egg development and viability at five constant temperatures. Significant temperature effects were found on the pre‐oviposition period, total number of eggs laid, daily oviposition rate, female longevity, egg‐development rate and viability. The mean length of the pre‐oviposition period ranged from 93.1 days at 4°C to 14.6 days at 20°C. Analysis of total number of eggs laid and daily oviposition rate during female lifespan estimated the highest total number of eggs laid (696 eggs/female) at 16°C and the highest oviposition rate (6.8 eggs/female and day) at 20°C. The daily oviposition rate at 20°C was not significantly higher than 5.4 eggs/female and day at 16°C. Female longevity was significantly longer at 4°C, shorter at 20°C and not significantly different between 8, 12 and 16°C. Estimated 50% survival time of females was 239, 153, 195, 186 and 78 days at 4, 8, 12, 16 and 20°C, respectively. A linear model of egg development at 8–20°C estimated the lower developmental threshold to be 5.1°C and the thermal constant for development 184.9 degree‐days. The percentage of eggs hatching was significantly lower at 4°C than at all other temperatures tested. The estimated mean hatching percentages were 47.3%, 70.0%, 72.4%, 66.2% and 67.9% at 4, 8, 12, 16 and 20°C, respectively. These results can be used to predict the start and intensity of egg‐laying in the autumn and the appearance of larvae in the field from knowledge about time of field invasion and from monitoring the weather.     

Oviposition behavior and progeny allocation of the polyembryonic waspCopidosoma floridanum (Hymenoptera: Encyrtidae)

Oviposition behavior was used to determine the primary clutch size and sex ratio of the polyembryonic wasp Copidosoma floridanumAshmead (Hymenoptera: Encyrtidae) parasitizing Pseudoplusia includens(Walker) (Lepidoptera: Noctuidae). The laying of a female egg was associated with a pause in abdominal contractions during oviposition, while the laying of a male egg was associated with uninterrupted abdominal contractions. Although unmated females produced only male broods, they also displayed male and female egg oviposition movements. Wasps always laid a primary clutch of one or two eggs. For mated females if only one egg was laid, the emerging secondary clutch was all male or female, but if two eggs were laid a mixed brood of males and females was almost always produced. The secondary clutch of single sex broods was usually between 1000 and 1200 individuals, but the secondary clutch of mixed broods averaged 1143 females and 49 males. Thus, the primary sex ratio for mixed broods was 0.5 (frequency males), but the secondary sex ratio was 0.042. Manipulation of the sequence of male and female egg oviposition or of the primary clutch did not produce major alterations in the secondary clutch size or sex ratio.     

Harem size and oviposition behaviour in a polygynous bark beetle

Abstract. 1. Harem polygyny can have fitness benefits and costs on females. In bark beetles of the genus Ips the latter may include within‐harem competition between larvae. However, earlier competition between females for male care and mating opportunities may also influence oviposition behaviour. There has been relatively little investigation into the relationship between harem size and initial egg output. The present study investigated this relationship in the bark beetle Ips grandicollis. 2. The measure of egg output used was the number of eggs in the gallery with the most eggs in each harem. Mean (±SE) harem size of 242 observed harems was 3.25 ± 0.10. A curvilinear relationship was found between egg output and harem size, with females in smaller harems (one to four females) laying more eggs with increased harem size. However, females in larger harems (five to seven females) laid fewer eggs as harem size increased. The optimal harem size (in terms of number of eggs laid) was close to four females. 3. We found no evidence from a behavioural assay that females could preferentially choose unmated males over mated males with harems of two females. Additionally, the distribution of harem sizes suggests that females distribute themselves among males randomly. 4. The results suggest that harem size has effects on female reproduction that extend beyond larval competition and influence patterns of oviposition. The mechanism that determines why egg laying is greatest at intermediate levels is unknown. There is no evidence that smaller harems belong to lower quality males, but females may adjust egg‐laying behaviour in large harems as a result of reduced male attendance or anticipated larval competition.     

Field oviposition rates and egg load dynamics of pollen beetles (Meligethes aeneus Fab.) (Colepotera: Nitidulidae)

Abstract 1 Egg loads from field collected pollen beetles (Meligethes aeneus Fab., Coleoptera: Nitidulidae) were determined by dissecting beetles caught at the beginning and end of the putative daily oviposition period. Field collected beetles were offered Brassica napus (L.) plants in cages for 8 (morning and early afternoon), 16 (overnight), and 24 h to ascertain the number of eggs laid during these time periods. 2 Most eggs were laid in the morning and early afternoon. The proportion of gravid females was higher at the beginning of the oviposition period than at the end. Most females in the morning carried two eggs, whereas one egg was more common in the afternoon. 3 We hypothesized that the number of eggs laid during the oviposition period would be equivalent to the difference between egg loads at the beginning and end of oviposition. This was not the case; differences in egg loads were significantly lower than number of eggs laid. However, the number of eggs laid was equivalent to the egg load at the beginning of the oviposition period, suggesting that eggs available in the morning are laid during the following day. 4 Population estimates of daily oviposition rates, approximately 0.7 eggs per beetle and day, were close to estimates from laboratory studies when the proportion of gravid females was taken into consideration.     

Egg Deposition Behavior in the Haplodiploid Sawfly Athalia rosae ruficornis Jakovlev (Hymenoptera: Symphyta: Tenthredinidae)

The egg deposition behavior of the turnip sawfly, Athalia rosae (Hymenoptera: Symphyta), is described. Both unmated and mated females lay eggs individually inside of fresh young leaves of cruciferous plants. During an oviposition event, females exhibit a distinct pause in abdominal contractions just before the actual egg deposition act. Unmated females show a longer pause (11.31 s on average) than mated females (4.38 s on overall average). By employing an eye color mutation, the sex of the eggs laid by females was ascertained. Females mated once lay mostly fertilized (diploid female) eggs initially but begin to lay a considerable number of unfertilized (haploid male) eggs later in life. The laying of an unfertilized egg is associated with a longer pause (6.98 s on average) than the laying of a fertilized egg (3.76 s on average). These results are in contrast to previous reports on apocritan Hymenoptera, where the presence of a pause or a longer pause during oviposition was associated with the deposition of fertilized eggs rather than unfertilized eggs. The possibility that mated Athalia rosae females control fertilization and its implications for sex allocation strategies are discussed.     

两种寄生蜂的卵巢结构与卵成熟特点初步观察

对斜纹夜蛾的内寄生蜂双斑侧沟茧蜂MicroplitisbicoloratusChen和南美斑潜蝇的外寄生蜂潜蝇姬小蜂Diglyhusisaea(Walker)的卵巢结构进行了研究。双斑侧沟茧蜂的2个卵巢管底部膨大有大量的卵,通过凹陷的颈部与萼区相连接,在萼区里大量成熟待产的卵浸泡在萼液中。产卵量随产卵时间延长而降低,在日产卵实验中,日产卵量202(±114SE),从1000至1800每2h的产卵量分别占日产卵量的38,32,17和13%;在一生产卵量实验中,9d产卵期,每头雌峰一生平均产卵量为502(±78SE)粒,第1d产卵量占一生总产卵量的42%。潜蝇姬小蜂的每个卵巢由3条卵巢管组成,每条输卵管中仅有1粒成熟卵,整个卵巢中只有6粒成熟卵,每次产卵数不超过6粒。结果表明,M.bicoloratus是早熟卵(proovigenic)类昆虫;D.isaea是同步卵(synovigenic)类昆虫。     

Effect of temperature on development and reproduction of Neoseiulus barkeri (Acari: Phytoseiidae) fed on Aleuroglyphus ovatus

The effect of five constant temperatures (16, 20, 24, 28 and 32°C) on the development, survival and reproduction of Neoseiulus barkeri Hughes fed on Aleuroglyphus ovatus Toupeau (Acari: Acaridae) was examined in the laboratory at 85% relative humidity. Development time of different immature stages decreased with increasing temperature, total egg-to-adult development time varied from 5.0 ± 0.13 to 17.5 ± 0.29 days. The lower thermal threshold for development was 9.7 ± 2.48°C and the thermal constant from egg to adult was 111.1 ± 12.34 degree-days. Pre- and post-oviposition period and female longevity all shortened as temperature increased. The longest oviposition period was observed at 24°C with 20.4 ± 1.13 days. At 20, 24, 28 and 32°C, mated females laid on average 0.7 ± 0.08, 1.5 ± 0.04, 1.6 ± 0.11 and 1.5 ± 0.11 eggs per day, respectively, but no eggs were laid at 16°C. Both the maximum fecundity (30.9 eggs per female) and the highest intrinsic rate of increase (r _m = 0.166) were obtained at 28°C. The results of this study indicated that a mass rearing of N. barkeri with A. ovatus as prey is feasible at the appropriate temperature.     

Distribution ofChilo partellus (Lepidoptera: Pyralidae) egg batches on maize

Distribution ofChilo partellus egg masses was studied in field, greenhouse, and laboratory experiments. The eggs were laid in batches mainly on the lower side and the lower leaves of the plant. The egg batch size ranged from 1 to 169 eggs, with a median of 33.5 eggs per batch (average, 40.5). Oviposition ofC. partellus is described at two levels. The first level, choice of oviposition plants, followed a random distribution. The second level, number of egg batches per plant, followed an aggregated distribution in the field, where more than one egg batch was deposited on the same plant by the same female, which was found on 25% of the oviposition plants. A mechanism for egg-layingC. partellus females to perceive preceding oviposition or injured plants could not be detected. Oviposition site choice seemed to be mediated by tactile stimuli.     

Effect of host deprivation on egg quality,egg load,and oviposition in a solitary parasitoid,Chetogena edwardsii (Diptera: Tachinidae)

Solitary parasitoids are limited to laying one egg per host because larvae compete within hosts. If host encounter rate is low, females should not increase the number of eggs/host in response. The tachinid fly, Chetogena edwardsii,was used to evaluate the effect of host deprivation on egg accumulation, oviposition behavior, and egg quality in a solitary parasitoid. Females deprived of hosts for 2– 7 days accumulate about 1 day's supply of eggs. Egg output of deprived females once hosts are restored does not differ from that of control females. Deprived females retain one egg in the uterus where it undergoes embryogenesis. Maggots emerging from retained eggs are more likely to survive in hosts molting in 40 h or less after receipt of an egg than are maggots emerging from eggs fertilized shortly before oviposition. Egg retention is a consequence of host deprivation that permits females to broaden the range of hosts they can exploit to include soon-to-molt hosts and possibly multiply parasitized hosts.     

Geographical variation in reproductive traits and trade‐offs between size and number of eggs in the king ratsnake,Elaphe carinata

We collected gravid king ratsnakes (Elaphe carinata) from three geographically separated populations in Chenzhou (CZ), Lishui (LS) and Dinghai (DH) of China to study the geographical variation in female reproductive traits and trade‐offs between the size and number of eggs. Not all reproductive traits varied among the three populations. Of the traits examined, five (egg‐laying date, post‐oviposition body mass, clutch size, egg mass and egg width) differed among the three populations. The egg‐laying date, ranging from late June to early August, varied among populations in a geographically continuous trend, with females at the most northern latitude (DH) laying eggs latest, and females at the most southern latitude (CZ) laying eggs earliest. Such a trend was less evident or even absent in the other traits that differed among the three populations. CZ and DH females, although separated by a distance of approximately 1100 km as the crow flies, were similar to each other in most traits examined. LS females were distinguished from CZ and DH females by the fact that they laid a greater number of eggs, but these were smaller. The egg size–number trade‐off was evident in each of the three populations and, at a given level of relative fecundity, egg mass was significantly greater in the DH population than in the LS population. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 701–709.     

Emergence and mating behavior of the oriental fruit moth Cydia molesta (Lepidoptera: Tortricidae) and its potential for reproduction

The Oriental fruit moth, Cydia molesta (Busck, 1916) (Lepidoptera: Tortricidae), is a key pest of fruit and is widely distributed around the world. There are important connections between its behavior and biology and its management in agriculture, but few studies have investigated the associations between adult behaviors and oviposition. In this study, adult emergence, mating, and reproduction were investigated under laboratory and field conditions. The ratio of females to males at eclosion was approximately 1:1. When one virgin female had access to one virgin male, 66% and 34% of the couples copulated just once and twice, respectively; and the infertility rate of eggs (21.39 ± 1.25%) did not vary daily. Males, given access to one new female daily, could copulate multiple times, whereas females seldom mated more than once, indicating a male-biased operational sex ratio, but mating status of the male parent had no effect on progeny egg reproduction. Also, the number of eggs that hatched by all female partners of a male was inversely proportional to copulation duration for the female laying the eggs for total female reproductive success; and the number of eggs laid by all female partners of a male was proportional to their number of matings for total male reproductive success. However, the total number of eggs that hatched did not significantly differ for eggs laid by a female given new virgin males daily for mating (17.75 ± 4.28) versus eggs laid by virgin females (19.17 ± 7.51) presented daily with a male that re-mated daily with the series of females. Therefore, our results showed that females engaged in mate choice and males engaged in mate competition, affecting egg production, a factor that may be used to enhance mating disruption technology against Cydia molesta.     

Diamondback moth egg susceptibility to rainfall: effects of host plant and oviposition behavior

Oviposition patterns of the diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), differ between common cabbage (Brassica oleracea L. var. capitata) and Chinese cabbage (Brassica rapa L. var. pekinensis) (Brassicaceae) host plants. This study shows that the moth prefers to oviposit on adaxial rather than abaxial leaf surfaces and petioles of both host plants. More eggs were laid in leaf veins than on leaf laminas of both host plants, especially in Chinese cabbage, where 94.6% of eggs were laid in veins. On Chinese cabbage, very few eggs were laid in clusters (≥2 eggs), whereas on common cabbage approximately 30% of eggs were laid in groups of 2 or more eggs. Removal of wax from common cabbage leaves dramatically increased the number of eggs laid singly on the leaf lamina of treated plants, suggesting that leaf waxes affect how eggs are distributed by ovipositing DBM. Eggs were most susceptible to removal by rainfall from the plant surface immediately (<1 h) after oviposition and when close to hatching (>72h old) whereas they were least susceptible 24 h after oviposition. Eggs laid on common cabbage plants were more susceptible to simulated rainfall than eggs laid on Chinese cabbage plants. On common cabbage plants, egg susceptibility to rainfall on different plant parts ranked adaxial leaf surfaces>petioles = abaxial leaf surfaces>stem, but there was no difference in egg susceptibility to rainfall on the various plant parts of Chinese cabbage. Furthermore, on common cabbage plants, eggs laid on both adaxial and abaxial leaf surfaces were afforded significant protection from the effects of rainfall by leaves higher in the plant canopy. On common cabbage plants, oviposition patterns reduce the potential impact of rainfall on eggs, possibly reducing the effect of this important abiotic mortality factor in the field.     

Do Golden Egg Bugs (Phyllomorpha laciniata: Heteroptera, Coreidae) Require Conspecifics for Oviposition?

Golden egg bug (Phyllomorpha laciniata) females lay eggs on the bodies of conspecifics of both sexes. We investigated to what extent reproduction depended on the availability of conspecifics as oviposition substrate and the acceptability of the host plant as an alternative oviposition substrate in the absence of conspecifics. Mated females were placed in experimental enclosures each containing a sprig of fresh host plant. Each experimental female was subjected to one of three treatments: isolated from conspecifics (solitary), paired with another female, or paired with a male. Solitary females laid a few eggs on the host plant but then stopped laying eggs, and solitary females laid significantly fewer eggs than those enclosed with another female or a male. Females enclosed with a male laid no more eggs than those enclosed with a female. When two previously isolated females were later enclosed together, they soon renewed oviposition. Females in nature contained significantly more oviducal eggs than did females that were enclosed with other females for a short period. Thus the availability of suitable conspecifics as oviposition substrate stimulates the deposition of mature eggs, and reproduction depends on the presence of conspecifics of either sex as oviposition substrate.     

Age- and density-related oviposition behavior of the European corn borer,Ostrinia nubilalis (Lepidoptera: Pyralidae)

Age-related oviposition patterns ofOstrinia nubilalis were studied at three population densities in the laboratory by releasing newly eclosed adults in wire-screened cages and analyzing their oviposition throughout the adult stage with digital analysis. Oviposition sequences of individual females depositing egg masses were documented on the third and seventh nights after eclosion with a video camcorder. During a sequence, a female produced an egg in an average time of 15 or 26 s on the third and seventh nights, respectively, and completed depositing an egg mass the size of 20–39 eggs in an average time of 316 and 525 s, respectively. Females were not easily disturbed during egg mass deposition and pulsated their abdomen before deposition of each egg. Females produced few egg masses the first night after eclosion. Oviposition increased on nights 2 and 3 but declined steadily thereafter as females matured. Females older than 6 nights produced fewer egg masses; the proportion of egg masses with fewer than 20 eggs increased gradually. By the end of the adults' lifetime, nearly 100% of the egg masses had fewer than 20 eggs. The data are fundamental to our research to define the role of phytochemicals in modifying oviposition behavior of the European corn borer.     

Effects of temperature on the fecundity and longevity of Zeugodacus cucurbitae (Coquillet) (Diptera: Tephritidae) on artificial diet

The melon fruit fly, Zeugodacus cucurbitae (Coquillett), is an important polyphagous pest that damages to various agricultural crops, whose distribution has become global as a result of human activity. In this study, we investigated the fecundity, pre-oviposition and oviposition periods, and the longevity of adult Z. cucurbitae at various constant temperatures ranging from 15 to 35.2°C. One newly emerged one virgin female and two males of Z. cucurbitae were used, and the longevity of both sexes and the fecundity of the females were examined daily. The longevity of female Z. cucurbitae ranged from 183.8 days at 15.0°C to 30.8 days at 35.2°C, and the maximum fecundity per female was 1204 eggs at 24.5°C. The lower development threshold (LDT) and thermal constant (K) of females were estimated as 14.8°C and 781.13 degree-days, respectively. Female reproduction was modeled using a two-phase oviposition model. Oviposition was estimated using females in the oviposition phase, which had a complete pre-oviposition phase. The oviposition model consisted of two reproductive components (total fecundity and age-specific cumulative oviposition rate during oviposition) and a survival component (age-specific survival rate). The daily egg production of Z. cucurbitae was estimated in relation to adult age and temperature.     

Nesting Phenology and Clutch Characteristics of Captive Siamese Crocodiles (Crocodylus siamensis) in Cambodia

The Siamese crocodile (Crocodylus siamensis) is considered one of the least studied and most critically endangered crocodilians in the world. Although few wild populations remain, more than 700,000 C. siamensis are held on commercial crocodile farms in Southeast Asia. Despite conservation concerns, many aspects of C. siamensis life history remain poorly known, particularly with regards to its reproductive biology. We studied nesting phenology, clutch characteristics, and other aspects of C. siamensis reproductive biology on crocodile farms in Cambodia during 2000 and 2001. Oviposition among captive crocodiles began in February and continued into early June. The mean (±1 SD) oviposition date based on pooled data from 2000 and 2001 was 5 April ± 24 days. Mean oviposition date differed significantly between 2000 and 2001, possibly as a result of annual variability among nesting cues. The mean incubation period was 72 ± 3 days and eggs hatched from 5 May to 18 August. Mean clutch size (25.0 ± 8.8 eggs; n = 183) differed significantly between years, possibly resulting from the >2.5‐fold increase in sample size during 2001. There was no correlation between clutch size and oviposition date during either 2000 or 2001. A single female produced two clutches during 2001, complimenting previous reports of double‐clutching among C. siamensis. The mean length and width of 515 eggs were 78.2 ± 4.9 and 48.1 ± 2.5 mm, respectively; mean egg mass was 90.8 ± 16.5 g (n = 471). One unpipped egg contained a set of twins. Zoo Biol 31:534‐545, 2012. © 2011 Wiley Periodicals, Inc.     

<Emphasis Type="Italic">Fopius arisanus</Emphasis> oviposition in four <Emphasis Type="Italic">Anastrepha</Emphasis> fruit fly species of economic importance in Mexico

We studied the oviposition performance of Fopius arisanus (Sonan) (Hymenoptera: Braconidae) attacking eggs of four fruit flies of the genus Anastrepha Schiner (Diptera: Tephritidae) under laboratory conditions. The complete process of oviposition on an individual egg of Anastrepha ludens (Loew) lasts in average 85.4 ± 2.9 s, including a tremor (25.8 ± 1.03 s) observed in the middle of this process related to the egg’s descent. The average parasitism of A. ludens egg was 60.9 ± 7.5%, with only 1.2% of superparasitized eggs. During individual acts of oviposition, we noted that F. arisanus possesses a highly flexible ovipositor that curves easily as it searches for additional suitable eggs, which may be of particular benefit when a female finds large clutches of eggs. The individual oviposition of F. arisanus in host fruits attacked by Anastrepha spp. varies with the egg clutch size of each fruit fly species: A. serpentina laid the biggest egg clutches (21.3 ± 1.4), followed by A. ludens (14.2 ± 0.9), and A. striata (1.0 ± 0.0) (=A. obliqua). The time spent by F. arisanus in individual ovipositions was parallel to these findings, reinforcing the idea that F. arisanus attacks several eggs in each individual insertion of its ovipositor. Neither formal oviposition acts, nor adult emergences of F. arisanus were registered in A. obliqua. We discuss the potential of F. arisanus as natural enemy of fruit flies of the genus Anastrepha, and explore the eventual developing of its mass rearing. Handling Editor: Torsten Meiners.     

Oviposition preferences of Maculinea alcon as influenced by aphid (Aphis gentianae) and fungal (Puccinia gentianae) infestation of larval host plants

Abstract 1. The influence of infestation of the larval host plant Gentiana cruciata on the egg‐laying preferences of the xerophilous ecotype of Alcon Blue butterfly (Maculinea alcon) was studied in a semi‐dry grassland area (Aggtelek Karst Region, Northern Hungary). 2. We examined whether oviposition patterns of females differed when G. cruciata stems were uninfested compared with when they were infested by an aphid (Aphis gentianae) or a rust (Puccinia gentianae) species. 3. Females laid more than 90% of their eggs on fertile, uninfested G. cruciata stems, although these stems comprised only ～ 50% of the total stems available. Stems infested by aphids were similar to uninfested ones in properties that had a strong correlation with egg numbers, and yet there were significantly fewer eggs on infested stems than on intact ones. 4. Females never laid eggs on parts of Gentiana stems infested by aphids, and the presence of Lasius paralienus ants, which have a mutualistic interaction with Aphis gentianae, did not increase the repulsive effect of aphids. Infection of Gentiana by Puccinia did not influence the egg‐laying behaviour of females, even though the flowers and buds of infested stems exhibited a delayed development. 5. Aphid infestation can influence butterfly oviposition patterns through both direct and indirect effects. The presence of aphids directly excluded oviposition, but our data also indicated the possibility of an indirect effect of aphid infestation. Stems that had no aphids at the last egg counting, but were infested prior to it, had significantly fewer eggs than those that were never infested.     

The life cycle of Hyalomma rufipes (Acari: Ixodidae) under laboratory conditions

Biological characteristics of Hyalomma rufipes parasitising on rabbits and sheep were compared under laboratory conditions in Gansu, China. Mature ticks could parasitize both rabbits and sheep, while immature ticks only fed on rabbits successfully. Adults sucked blood on sheep significantly longer than on rabbits (16 and 13 days, respectively). Other adult parasite characteristics fed on the two hosts were similar, including the weight of engorged adult, female daily oviposition, and the weight and amount of the egg mass laid. Those indicated that this tick species showed little host specificity between sheep and rabbits during its adult stage. In total, the life cycle of H. rufipes was completed in an average period of 179 days. The average developmental periods were 59 days for egg incubation, 3 and 21 days for immature tick prefeeding and feeding, 2, 12 and 40 days for adult prefeeding, female preoviposition and oviposition. The longer female fed for engorgement, the shorter preoviposition period of engorged female needed, although when the feeding period was less than 15 days, this relationship was not obvious. The results confirmed the correlation between the weight of the engorged female and the number of eggs laid (r = 0.909). The reproductive efficiency index (REI) and reproductive fitness index (RFI) in females was 10.63 and 7.22, respectively. Engorged nymphs moulting to females were significantly heavier (27.6 ± 0.89 mg) than those moulting to males (22.3 ± 0.52 mg). Males outnumbered females by 1.4:1.