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Please do not remove this page Ontogenetic changes in Phoneutria nigriventer (Araneae, Ctenidae) spider venom Herzig, Volker; Ward, R J; Dos Santos, W F https://research.usc.edu.au/discovery/delivery/61USC_INST:ResearchRepository/12126673790002621?l#13127275900002621 Herzig, V., Ward, R. J., & Dos Santos, W. F. (2004). Ontogenetic changes in Phoneutria nigriventer (Araneae, Ctenidae) spider venom. Toxicon, 44(6), 635–640. https://doi.org/10.1016/j.toxicon.2004.07.020 Link to Published Version: https://dx.doi.org/10.1016/j.toxicon.2004.07.020 Document Type: Published Version USC Research Bank: https://research.usc.edu.au research-repository@usc.edu.au Copyright © 2004 Elsevier Science Ltd. All rights reserved. Reproduced here with kind permission of the publisher. Downloaded On 2021/01/26 06:03:26 +1000 Please do not remove this page
Toxicon 44 (2004) 635–640
www.elsevier.com/locate/toxicon
Ontogenetic changes in Phoneutria nigriventer
(Araneae, Ctenidae) spider venom
Volker Herziga, Richard John Wardb, Wagner Ferreira dos Santosc,*
a
Department of Neuropharmacology, Zoological Institute, Faculty of Biology, University of Tübingen, Germany
b
Departamento de Quimica, FFCLRP-USP, Ribeirão Preto, Brazil
c
Laboratorio de Neurobiologia e Peçonhas, Departamento de Biologia, FFCLRP-USP, Ribeirão Preto,
Av. Bandeirantes, 3900, 140140-901, Brazil
Received 21 January 2004; revised 28 April 2004; accepted 26 July 2004
Available online 30 September 2004
Abstract
Venom-yield and composition of differently sized individuals of the medically most important Brazilian spider Phoneutria
nigriventer (Keyserling, 1891) was analysed. During growth the venom-mass increases according to a fourth order function of
the prosoma size, which mainly reflects an increase of the venom gland volume. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis revealed increasing percentages of proteins %17 kDa from 4.1% in the smallest analysed spiders (2–3 months-
old) to 79.1% in adult female venom. Additionally, high-pressure liquid-chromatography showed an increase of a single
(‘main’) peak from 4.6 to 64.9%, while the overall number of other major-peaks decreased. Venom from young instars
completely lacked lethality in mice up to a dose of 3.28 mg/kg i.v. as compared to a LD50 of 0.63 mg/kg for adult female or
1.57 mg/kg for adult male venom that we reported previously. In conclusion, ontogenetic changes in venom protein-
composition of growing P. nigriventer are suggested to produce increasing lethality in vertebrates.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Phoneutria nigriventer; Ontogenetic changes; Venom yield; Protein composition; Vertebrate-lethality (LD50)
1. Introduction (Ramos et al., 1998) and females with egg-sacs appear at the
middle of July. After copulation females build up to four
In Brazil the genus Phoneutria Perty, 1833 (family: white oval egg-sacs, and from the time the young spiders
Ctenidae) is responsible for about 60% of all spiderbite- leave the egg-sac, they are able to move and capture prey.
accidents (Lucas, 1988). According to the latest revision Because spiders have an exoskeleton they have to shed it at
(Simó and Brescovit, 2001), only five valid species of regular intervals (moult) during growth. Depending on the
Phoneutria have been described (fera, nigriventer, boli- availability of food, young Phoneutria moult 5–10 times in
viensis, reidyi, bahiensis) and in Brazil P. nigriventer the first, 3–7 times in the second and 1–3 times in the third
(Keyserling, 1891) accounts for the majority of accidents year (Lucas, 1969, 1988; Bücherl, 1969). They become
(Bücherl, 1969). The length (prosomaCopisthosoma) of adult in the third year and the maximum age that can be
adult P. nigriventer female can reach 42 mm (Vital-Brazil reached is 6 years (Bücherl, 1969).
and Vellard, 1925). The mating season is from April to July More than 25 neurotoxic peptides and proteins in the
range from 2–15 kDa have been purified from the venom of
P. nigriventer, and to date the complete amino
* Corresponding author. Tel.: C55 16 6023657; fax: C55 16 acid sequence of 15 of these polypeptides is known
6331758. (Diniz et al., 1993; Cordeiro et al., 1995). For intravenous
E-mail address: wagnerf@usp.br (W.F. dos Santos). (i.v.) injections in mice, the median lethal dose (LD50)
0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2004.07.020636 V. Herzig et al. / Toxicon 44 (2004) 635–640
was determined as 0.33 mg/kg mice (Bücherl, 1956; After capturing the spiders were maintained in the laboratory
Schenberg and Pereira-Lima, 1966). The venom causes for a maximum of 8 months, where they were fed on crickets
dyspnea, prostration, paralysis of hind legs and tail and and cockroaches (see also Herzig et al., 2002). During this
finally death by paralysis of the respiratory system. period, the spiders were allowed to reproduce in order to have
However, individual venom quantity varies considerably, enough young spiders for venom extraction. However,
ranging from 0.3–8.0 mg dried weight per spider (Lucas, venom-extraction from neonate spiders was not possible
1988). Furthermore, P. nigriventer venom quantity can vary therefore they were kept until they reached a size that allowed
seasonally in summer (with 1.8 mg average) and venom-extraction (after 2–3 months). The size and (if
winter (2.5 mg) (Schenberg and Pereira-Lima, 1966) and possible) sex of all spiders was determined before venom
intersexual differences can also account for variations extraction and the length of the prosoma was measured. Since
(Herzig et al., 2002). Variations in venom quantity, caused P. nigriventer requires 3 years from leaving the egg-sac to
by different factors, have also been documented in other adulthood and since all spiders were collected only over a
spider species. In electrically extracted venom from period of 6 months, a correlation of the spiders sizes with the
Loxosceles reclusa, smaller spiders yielded less venom instar-stages (as done by Malli et al., 1993) was not possible.
than larger ones (Morgan, 1969). Kuhn-Nentwig and Thus, all spiders were organized into seven size-classes
Nentwig (1997) reported that venom production in adult according to their prosoma-length, i.e. size-class 1
Cupiennius salei (Ctenidae) can depend on age, gender, (adult females); size-class 2 (sub-adult females) and
degree of hunger and probably on the reproductive status. size-classes 3–7 (instars in order of decreasing size). The
Periods of high or low temperature may also influence the size-classes 1–3 contain only female P. nigriventer, whereas
venom yield (Jong et al., 1979). Therefore, variations in the size-classes 4–7 include spiders of both sexes.
the venom quantity can be an important aspect regarding the
danger of a spiderbite. Individual P. nigriventer with high 2.2. Method of venom extraction and treatment
venom quantities may be dangerous for physically weak or
diseased adult humans. The spiders were anesthetized with CO2 and venom was
Not only the venom quantity, but also its composition, extracted by applying a few times electric shocks of 13–18 V
can show intraspecific variations. In the brown recluse (depending on the size of the spider) that lasted about 1 s
spider L. intermedia, the ontogenetic development of a each. To avoid contamination with saliva, venom was only
single 35 kDa protein (denominated F35) was demonstrated collected from the tips of the fangs. Venom was extracted
(De Andrade et al., 1999). F35 has dermonecrotic and about once per month over a period of 8 months. Other
haemolytic activity and is responsible for the main toxic procedures are described in detail by Herzig et al. (2002).
effects of the venom. Eggs and spiderlings of the first and
second instar contained no F35. It was first detected in 2.3. Gel electrophoresis
instars of the third stage and its amount increased until
adulthood. The authors conclude that F35 is an important Sodium dodecyl sulfate-polyacrylamide gel electrophor-
factor for the spiders’ survival, because it precisely occurred esis (SDS-PAGE) was performed in a running gel with a
at the point of development when the spider is able to hunt, linear gradient of 5–20% acrylamide at a voltage of 120 V
and defend itself. For the ctenid spider Cupiennius salei, and a current of 20 mA (Laemmli, 1970). Venom samples
Malli et al. (1993) showed that the insect-lethality is highest were dissolved in 20 ml of water (milli-Q) and 5 ml sample
in the sixth instar (the first instar stage that was tested) and in buffer (25% b-mercaptoethanol and 75% of 0.313 M
adult spiders (without intersexual differences). Tris–HCl; 10% glycerol and 0.001% bromphenol blue,
The present study was designed to determine if and how pH 6.8) was added. Samples were boiled for 2 min, vortexed
ontogenetic development affects venom yield or venom for 30 s and applied to the gel. After electrophoresis, gels
composition in the ctenid spider P. nigriventer. Further- were stained in a 0.25% Coomassie Brilliant Blue R250
more, the effect of such developmental changes in venom solution and destained by using a solution containing 10%
composition on the lethality was examined in mice. methanol and 10% acetic acid. The molecular weights of the
venom proteins were estimated by using known molecular
weight standards (Sigma 6H standard). The gel was scanned
2. Materials and methods and analysed using the program Easy Win 32 (Herolab). The
total intensity of the protein bands in each size-class was set
2.1. Spiders to 100% and the intensities of the individual protein bands
were normalized to this value.
The spiders for the present study were all collected
between March and August 1999 on the campus of the 2.4. Reverse-phase liquid chromatography
University of São Paulo (USP) in Ribeirão Preto (Brazil). The
spiders were identified as P. nigriventer by High-pressure liquid-chromatography (HPLC) was
Antonio Brescovit (Butantan Institute, São Paulo, Brazil). performed using a dual pump solvent delivery systemV. Herzig et al. / Toxicon 44 (2004) 635–640 637
(LC-10Ai, Shimadzu, Japan), coupled to a fraction collector 3. Results
(FRC-10A, Shimadzu, Japan). The flow rate was 1 ml/min
and a linear gradient (2.5%/min) of solvent A (0.1% The spiders of size-class 7 (Table 1) were the smallest
aqueous solution of trifluoracetic acid (TFA, Nuclear)) spiders that yielded sufficient venom for analysis, however
and solvent B [60% acetonitrile (chrom HR HPLC, the quantity was to low for weight-determination. Instead,
Mallinckroft)C40% solution A] was used according to the venom was used for a single SDS-PAGE run and no
Rego et al. (1996). The lyophilized venom samples HPLC analysis was performed. The next size-class analysed
were dissolved in 120 ml water and applied to a reverse- (size-class 6) was approximately 6–7 months old and
phase C-18 column (Waters Spherisorb ODS2 5 mm, yielded enough venom for both HPLC and SDS-PAGE
250!4.6 mm), previously equilibrated with a 0.1% aqueous analysis. Data for size-classes 1–3 have already been
TFA. The absorbance of the eluate was monitored published (Herzig et al., 2002) and are included for reasons
continuously at 220 nm. Based on the total peak area per of comparison. Table 1 further shows an increase of the
run, a percentage value for each peak area was calculated venom-mass yielded per spider during growth, with the
(using the program CLASS-LC10, version 1.63, Shimadzu) exception that size-class 6 yielded more venom than size-
and compared with peaks from different size-classes that class 5. The dried venom yield (y, in mg) increased
showed the same retention times. exponentially during the spider’s growth according to the
equation yZ0.01 * x4.21, where x is the prosoma length in
millimeter. This equation shows a r2 of 0.998 and was
2.5. Lethality test in mice iterated by using the ‘solver’ tool in Microsoft Excel 2002.
According to Fig. 1, each of the size-classes 1–5 showed
The lethality test in mice was performed to determine nine protein bands of the same molecular weights or weight-
any potential dangers for vertebrates caused by venom ranges (i.e. 7–12, 13, 16, 19, 20, 22–27, 32, 35 and
from young P. nigriventer. Assuming varying proportions 41–47 kDa), however their percentages varied among
of different toxins in different size-classes, a bioassay different size-classes. In size-class 6, 10 bands were
using invertebrates could therefore not be used, since detected, which is due to an additional protein of 17 kDa.
different toxins in the venom are responsible for effects in The venom of the youngest spiders (size-class 7) consisted
vertebrates and invertebrates. Venom from size-class 6 mainly (82%) of a 45 kDa protein and very low quantities of
(about 6–7 month-old spiders) was dissolved in 100 ml of some of the proteins that have been detected in size-classes
0.9% NaCl solution and injected into the tail-vein (i.v.) of 1–6. By delineating two molecular-weight classes (Table 1),
male Swiss white mice. The mice were supplied from the a general variation in the venom protein composition was
main animal house, USP Ribeirão Preto, São Paulo, Brazil revealed. The quantity of proteins with more than 17 kDa
and had a body-weight of 22–26 g. Three increasing molecular weight decreased during the spiders’ growth from
venom concentrations were injected into 5 mice at each about 96% in size-class 7 to about 21% in adult females. On
concentration and 15 mice were used in total. All animal the other hand, the quantity of proteins %17 kDa increased
experiments comply with the guidelines of the Brazilian from 4% in size-class 7 to 79% in size-class 1.
Society of Neurosciences that follow the guidelines By HPLC, a total number of 31 different peaks
for animal care of the Committee on Care and Use was detected in the venoms of all size-classes. The number
of Laboratory Animal Resources, National Research of peaks varied between 25 in size-class 2 and 20 in size-
Council (USA). class 4 (Table 2). A decrease in the number of ‘major-peaks’
Table 1
Ontogenetic changes in prosoma-length, venom yield and venom protein sizes: venom data of different size-classes of Phoneutria nigriventer
are shown by decreasing size
Size-class Sex of Length of prosoma Number of Venom-yield Protein content (%) Protein content (%)
spiders (mm)GSD spiders per spider (mg) with MW O17 kDa with MW %17 kDa
1 Adult female 15.8G0.6 21 1079 20.9 79.1
2 Subadult female 13.5G0.6 17 548 not det. not det.
3 Instar female 11.5G0.7 16 296 21.6 78.4
4 Instars, sex not det. 9.5G0.6 7 117 29.9 70.1
5 7.5G0.5 33 36 37.2 62.8
6 5.5G1.7 36 50 44.1 55.9
7 1.9G0.4 9 not det. 95.9 4.1
The data of size-classes 1–3 (in italics), have already been published (Herzig et al., 2002) and are only provided for ease of comparison.
SDS-PAGE revealed increasing percentages of proteins %17 kDa and decreasing percentages of proteins O17 kDa during the spiders growth
(MW, molecular weight; not det., not determined).638 V. Herzig et al. / Toxicon 44 (2004) 635–640
of size-classes 2–5 are not presented, since they are very
similar to the chromatogram of size-class 1.
The venom of small spiders (size-class 6) showed no
lethality in Swiss white mice (i.e. no mice died) even after
injection of the highest dose (3.28 mg/kg) and also no signs
of toxicity (e.g. disturbance of respiration or paralysis of the
extremities) were observed.
4. Discussion
The dried venom yield (y) is a fourth order function (x4)
of the prosoma-length (x). A third order function would have
been easily explained by the increase of the venom gland
volume (and therefore its venom content) within three
dimensions, whereas the length of the prosoma increases
only in one dimension. The observation of a fourth order
function is difficult to interpret but it is likely that the
Fig. 1. SDS-PAGE gel. Coomassie-blue stained SDS-PAGE gel of increase in the venom gland volume accounts for the main
venom from six different size-classes (for details about the size-
increase in the venom yield. However, other (unknown)
classes see Table 1) of Phoneutria nigriventer (with exception of
factors also play an additional role in this phenomenon
size-class 2 that was not analysed). The data of size-classes 1 and 3
were already published (comply with venom pools 3 and 4 in Herzig (e.g. state of nutrition, time since the last feeding or ecdysis,
et al., 2002) and are only provided for ease of comparison. The dried etc.). Interestingly, the observed exponential increase in
venom quantities applied to the gel are indicated below. The venom yield is consistent with the results obtained by Malli
standard marker proteins (‘M’Z6H, Sigma) and the two molecular et al. (1993) on electrically extracted venom from instars of
weight classes (! or R17 kDa, corresponding to Table 1) are also Cupiennius salei. In summary, the observed increase of the
indicated. dried venom yield mainly reflects the increase of the venom
gland volume during growth of the spiders.
(i.e. peaks with more than 5% of the total peak area) was The observed increase in the relative amounts of proteins
observed during the spiders growth (Table 2). Venom of %17 kDa (up to 79%) during the development of the spiders
small spiders from size-class 6 showed nine major-peaks, could indicate that in young spiders the venom gland and
while only two major-peaks were observed in venom of size- especially the venom secreting cells are limited in their
class 1. A comparison of HPLC data from size-class 1 and capacity to synthesize these proteins. Another possible
size-class 6 venoms (Fig. 2) showed various differences in the explanation could be that young spiders already have the
venom composition of both size-classes, but a clear tendency ability to synthesize these proteins, but are limited in their
towards an increase of a single peak (eluting at 19.2 min). In ability to posttranslationally process the prepropeptide
venom of size-class 1, this peak (the ‘main peak’) accounted precursors of the mature toxins. Similar changes in venom
for about 65% the total protein content, while in size-class 6 composition during ontogenetic development have been
the content of this peak was only 4.6%. The chromatograms detected by HPLC, where an increase of the ‘main’-peak
Table 2
Ontogenetic changes in P. nigriventer venom protein composition: percentage content of ‘major’ protein peaks (peaks with more than 5% peak
area) and their retention times according to HPLC of all analysed size-classes
HPLC major protein peaks Total number
of peaks
Retention 2.3 2.8 3.0 3.2 3.5 3.7 4.3 8.1 11.1 11.5 16.9 19.2 ’main’ peak 25.6 29.3
time (min)
Size-class % of total protein content
1 5.6 64.9 21
2 8.5 6.1 56.3 25
3 5.4 65.4 22
4 5.8 8.0 38.5 12.0 20
5 11.5 6.2 5.5 43.1 6.4 23
6 8.3 7.5 7.1 6.1 6.5 8.9 6.2 4.6 10.3 8.2 23
The total number of all peaks (incl. peaks !5% peak area) is also presented. The percentage of the ‘main’ peak increased with the size of the
spiders. Venom of size-class 7 was not analysed due to a lack of sufficient venom quantity.V. Herzig et al. / Toxicon 44 (2004) 635–640 639
venom from larger specimens of P. nigriventer has a higher
lethality in mice, since toxins with lethal effects in vertebrates
(PhTx 1, PhTx 2–5 and PhTx 2–6) also show molecular
weights in that range (Diniz et al., 1990; Cordeiro et al.,
1992). This hypothesis is strongly supported by the lack of
lethality in venom from 6–7 month-old spiders (up to
3.28 mg/kg), whereas adult spiders showed lethal effects
(LD50 of 0.63 mg/kg for female venom and 1.57 mg/kg for
male venom according to Herzig et al., 2002). It furthermore
strengthens the assumption that venom-secreting cells
undergo ontogenetic development, showing full capacity
for toxin-synthesis only in larger instars. On the other hand,
the insect-lethality of venom from several stages of C. salei
was highest in young instars (sixth instar) and in adults,
suggesting a high content of invertebrate-specific toxins
especially in young spiders (Malli et al., 1993). The present
Fig. 2. HPLC results. HPLC venom chromatograms of size-class 1 findings do not exclude the possibility that venom from
(adult females, 700 mg venom applied) compared to size-class 6
young P. nigriventer also has a higher invertebrate-lethality
(instars, about 6–7 months-old, 741 mg venom applied). The
absorbance of the eluates was monitored at 220 nm for 30 min.
compared to venom from larger instars. Biologically it would
The peak showing the largest percental variation during ontogenetic even make sense that young spiders show a higher
development was termed as ‘main-peak’. The chromatograms of invertebrate-lethality, since this forms their main prey-type.
size-classes 2–5 are not presented, since they are very similar to the Furthermore, our study was not designed to identify specific
chromatogram of size-class 1. Due to the low quantity, venom of proteins and due to the lack of sufficient venom quantity
size-class 7 was not analysed by HPLC. especially from the smallest instars, the isolation and
characterisation of single venom proteins was not possible.
(up to 65%) was observed. Thus, it would be of interest to Therefore more detailed studies are necessary to confirm our
further analyse this ‘main-peak’ to confirm if it contains suggestion that the lack of lethality of venom from instars of
proteins %17 kDa. However, only based on the results P. nigriventer is due to the absence of exactly these known
vertebrate-specific toxins (see Diniz et al., 1990; Cordeiro
obtained in the present study and due to different physico-
et al., 1992) and to account for alterations in the distribution
chemical bases of the separation techniques, it is not
of invertebrate-specific toxins within differently sized
possible to establish a correlation of HPLC and SDS-PAGE
spiders.
data. This could be achieved in future experiments, using
The ‘male specific’ venom components, that have been
LC-MS techniques.
detected in our previous study of intersexual differences in
The only other report considering the distribution of a
P. nigriventer venom (Herzig et al., 2002), were completely
specific toxin during ontogenetic development showed that
absent in size-classes 1–6. This is interesting, since the size-
even instars of the third stage of Loxosceles intermedia
classes 4–6 include both male and female spiders. Therefore
(exactly the stage when young spiders leave their egg-sac) we suggest that these male specific components develop
contain F35, which is responsible for the main toxic effects fairly late, i.e. after their last moult. The function of these
of the venom (De Andrade et al., 1999). However, no report components is unknown, yet the late development could
about the distribution of other venom components of L. indicate that it may be for sexual purposes. Because adult
intermedia was given. In line with these findings, quanti- males roam around in search of females, another possible
tative but not qualitative differences in the venom explanation for these components may be defensive
components have been reported from adults of both sexes purposes, however, the lower vertebrate lethality of male
and immature juveniles of the theraphosid spider Poeci- P. nigriventer venom (Herzig et al., 2002) argues against this
lotheria rufilata (Escoubas et al., 2002). Another study hypothesis. A partial loss of the protein-producing or
failed to correlate the lethality for insects with the total processing capacity in male P. nigriventer is therefore
protein content in different growth-stages of the ctenid more likely. This explanation is supported by the fact that
spider C. salei (Malli et al., 1993). Thus it remains unclear traces of some ‘male specific’ components were detected by
whether venom secreting cells generally undergo SDS-PAGE (2.9% of the 66 kDa protein and 0.7% of the
ontogenetic development in young spiders, if this 80 kDa protein) in venom from the smallest instars
phenomenon can only be observed in some spider species (2–3 months-old, size-class 7) that are presumably also
(e.g. P. nigriventer), or if it’s restricted to the synthesis of limited in their protein-producing or processing capacity. A
some toxins. partial loss of toxicity of male P. nigriventer venom fits to
Based on the observed increase in the proportion of their reduced need of venom to overcome potential prey,
proteins %17 kDa during growth, it could be assumed that because they rarely eat and mainly roam in search of females.640 V. Herzig et al. / Toxicon 44 (2004) 635–640
Acknowledgements Escoubas, P., Corzo, G., Whiteley, B.J., Celerier, M.L.,
Nakajima, T., 2002. Matrix-assisted laser desorption/ionization
Volker Herzig was the recipient of a DAAD grant time-of-flight mass spectrometry and high-performance liquid
within the exchange program University Tübingen chromatography study of quantitative and qualitative variation
in tarantula spider venoms. Rapid Commun. Mass Spectrom. 16,
(Germany)/University of São Paulo (USP), campus Ribeirão
403–413.
Preto (Brazil). We wish to thank Profs. Norberto Cysne
Herzig, V., Ward, R.J., dos Santos, W.F., 2002. Intersexual
Coimbra, Roy Larson, Antunes-Rodriguez, Lewis Greene, variations in the venom of the Brazilian ‘armed’ spider
Jarbas Georgini and Ronaldo Zucchi (USP, Ribeirão Preto- Phoneutria nigriventer (Keyserling, 1891). Toxicon 40,
SP) for use of their laboratories; Renato Guizzo (USP, 1399–1406.
Ribeirão Preto-SP) for assisting in the mouse-assay; Jong, Y.S., Norment, B.R., Heitz, J.R., 1979. Separation and
Johannes Müller and Birgitt Schönfisch (University of characterization of venom components in the brown recluse
Tübingen, Germany) for statistical advice and Antonio spider (Loxosceles reclusa)—I. Preparative-disc electrophor-
Brescovit (Butantan Institute, Brazil) for identification of esis. Toxicon 17, 307–312.
the spiders. Furthermore, we wish to thank all the Kuhn-Nentwig, L., Nentwig, W., 1997. Venom of the hunting
anonymous reviewers for their helpful and constructive spider Cupiennius salei (Ctenidae). Toxicon 35 (6), 813
(abstract).
comments on the manuscript.
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assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lucas, S., 1969. Contribuição ao estudo da ooteca, dos ovos, e a
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