PLOS ONE
RESEARCH ARTICLE
First evidence for an aposematic function of a
very common color pattern in small insects
Rebeca Mora-Castro 1,2,3ID *, Marcela Alfaro-Córdoba 4,5 2,6ID , Marcela Hernández-Jiménez ,
Mauricio Fernández Otárola3,7, Michael Méndez-Rivera8, Didier Ramı́rez-Morales8, Carlos
E. Rodrı́guez-Rodrı́guez 8ID , Andrés Durán-Rodrı́guez
9, Paul E. Hanson3
1 Centro de Investigación en Biologı́a Celular y Molecular, University of Costa Rica, San José, Costa Rica,
2 Centro de Investigación en Ciencia e Ingenierı́a de Materiales, University of Costa Rica, San José, Costa
Rica, 3 Escuela de Biologı́a, University of Costa Rica, San José, Costa Rica, 4 Centro de Investigación en
Matemática Pura y Aplicada, University of Costa Rica, San José, Costa Rica, 5 Escuela de Estadı́stica,
a1111111111 University of Costa Rica, San José, Costa Rica, 6 Escuela de Fı́sica, University of Costa Rica, San José,
a1111111111 Costa Rica, 7 Centro de Investigación en Biodiversidad y Ecologı́a Tropical (CIBET), University of Costa
a1111111111 Rica, San José, Costa Rica, 8 Centro de Investigación en Contaminación Ambiental (CICA), University of
a1111111111 Costa Rica, San José, Costa Rica, 9 Protolab, University of Costa Rica, San José, Costa Rica
a1111111111
* rebeca.mora@ucr.ac.cr
Abstract
OPEN ACCESS
Many small parasitoid wasps have a black head, an orange mesosoma and a black meta-
Citation: Mora-Castro R, Alfaro-Córdoba M,
Hernández-Jiménez M, Fernández Otárola M, soma (BOB color pattern), which is usually present in both sexes. A likely function of this
Méndez-Rivera M, Ramı́rez-Morales D, et al. widespread pattern is aposematic (warning) coloration, but this has never been investi-
(2021) First evidence for an aposematic function of gated. To test this hypothesis, we presented spider predators (Lyssomanes jemineus), both
a very common color pattern in small insects.
field-captured and bred in captivity from eggs, to four wasp genera (Baryconus, Chromote-
PLoS ONE 16(2): e0237288. https://doi.org/
10.1371/journal.pone.0237288 leia, Macroteleia and Scelio), each genus being represented by a BOB morphospecies and
black morphospecies. We also used false prey, consisting of lures made of painted rice
Editor: Phillip Barden, New Jersey Institute of
Technology, UNITED STATES grains. Behavioral responses were analyzed with respect to presence or absence of the
BOB pattern. In order to better understand the results obtained, two additional studies were
Received: July 17, 2020
performed. First, the reflection spectrum of the cuticle of the wasp and a theoretical visual
Accepted: January 27, 2021
sensibility of the spider were used to calculate a parameter we called “absorption contrast”
Published: February 11, 2021 that allows comparing the perception contrast between black and orange in each wasp
Peer Review History: PLOS recognizes the genus as viewed by the spider. Second, acute toxicity trials with the water flea, Daphnia
benefits of transparency in the peer review magna, were performed to determine toxicity differences between BOB and non-BOB
process; therefore, we enable the publication of
wasps. At least some of the results suggest that the BOB color pattern may possibly play an
all of the content of peer review and author
responses alongside final, published articles. The aposematic role.
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0237288
Copyright: © 2021 Mora-Castro et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which Introduction
permits unrestricted use, distribution, and
reproduction in any medium, provided the original Many small (<10 mm) parasitoid wasps have a black head, an orange (or reddish orange)
author and source are credited. mesosoma and a black metasoma (BOB color pattern). This color pattern has been found in
species belonging to 23 families of Hymenoptera (including phytophagous sawflies), and is
Data Availability Statement: All data and code files
are available from the public repository: https:// especially common in neotropical scelionid wasps (Platygastridae; formerly Scelionidae), but
malfaro2.github.io/Mora_et_al2/. is also common in evaniid wasps from diverse biogeographic regions; moreover, this color
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 1 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Funding: This work was funded by the University pattern is usually present in both sexes [1]. In previous research it was found that the spectral
of Costa Rica, project 801A5B50. The funders had blue components of the orange and black color in scelionid wasps are almost identical, sug-
no role in study design, data collection and
gesting that there is a common compound for the pigments [2], but the identity of the pigment
analysis, decision to publish, or preparation of the
remains unknown.
manuscript.
Conspicuous coloration such as the BOB pattern is often, but not always, indicative of
Competing interests: The authors have declared
aposematism [3], whereby predators learn to associate particular color patterns with noxious
that no competing interests exist.
chemical defenses, although this learning process is much more complex than simply develop-
ing an aversion to specific types of prey [4]. In some larger insects contrasting black and
orange color patterns are known to serve as aposematic (warning) coloration for potential
predators, mostly vertebrates [5], and it is possible that the BOB pattern serves as a warning
pattern for smaller (invertebrate) predators, although this has not yet been tested.
To test whether the BOB pattern also serves as aposematic coloration for predatory inverte-
brates, we chose a common (but understudied) jumping spider (Salticidae) for our experimen-
tal trials. Molecular and electrophysiological data suggest that color vision in the principal eyes
of most jumping spiders is based on only two types of photosensitive pigments, one sensitive
to ultraviolet (UV) light, the other to green light, although a few of them also exhibit filter-
based trichromacy [6, 7]. Jumping spiders exhibit the following pairs of eyes: anterior median
eyes (spatial acuity and depth of field), posterior lateral eyes (lower spatial acuity and wide
field of view), anterior lateral eyes (high-resolution spatial vision due to densely packed recep-
tors with forward-facing acute zones) and side-facing posterior median eyes (reduced in most
genera). These characteristics of salticid spiders have allowed them to achieve the highest visual
acuity thus far measured in any arthropod, with the ability to classify prey into categories [8–
10].
Among the various jumping spiders present in our collecting sites, we chose to use Lysso-
manes jemineus Peckham & Wheeler, because it was one of the most common species. Lysso-
manes, like most salticids, are hunting spiders, primarily insectivorous, and behavioral
observations suggest a strong role for vision in predation, although visual acuity in Lyssomanes
may be somewhat lower than that of other salticids, such as Portia [11]. Lyssomanes species are
diurnal foliage dwellers and two hunting behaviors predominate: a sit-wait strategy followed
by springing from the underside of the leaf to the upper surface (they usually sit on leaves that
are exposed to the sun waiting to ambush prey on the upper surface), and a hunting behavior
that consists of exploring both sides of the leaf, actively searching for passing insects [12].
Thus, Lyssomanes, like most salticids, can be characterized as a hunting spider that does not
use webs, and which executes behavioral responses such as: prey detection by sight, stalking,
and attacking by jumping towards the prey [13, 14]. Penultimate juveniles and adults of Lysso-
manes usually hunt and feed on soft-bodied insects such as dipterans, small hemipterans, and
small wasps [12].
The objective of this study was to determine if the BOB color pattern in small parasitoid
wasps is aposematic, through toxicity tests and behavioral analysis of invertebrate predators
that depend on vision to capture prey. Our hypothesis is that the BOB pattern in scelionids
and other small wasps is aposematic, acting as a signal for vision-dependent invertebrate pred-
ators. We had four predictions: 1) all-black prey would be preferred over prey with the BOB
pattern; 2) experienced predators (collected in the field) would avoid the BOB pattern more
than inexperienced adults reared in captivity; 3) greater perceived contrast between black and
orange colors would trigger fewer attacks from the predator, since this would amplify the apo-
sematic BOB pattern; 4) when comparing wasp species with the BOB color pattern with all
black species, the former would show greater toxicity.
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 2 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Materials and methods
Scelionid collection
The collection site with the greatest number of scelionid wasps of interest was in the Central
Valley of Costa Rica, in a forest patch within the Protected Zone of El Rodeo, which is located
near Ciudad Colón, San José (9˚52’–9˚56’N, 84˚14’–84˚20’W, 650 m.a.s.l.). The area is com-
posed of secondary forest and a remnant of primary forest (approximately 200 ha). A collect-
ing permit was obtained from the National System of Conservation Areas (Ministry of
Environment and Energy), document N˚ ACTo-050-18, and all experimental protocols were
carried out in accordance with our institution’s guidelines and regulations. Because the hosts
of scelionid wasps (insect eggs) are very difficult to locate, live wasps were obtained via sweep-
ing with insect nets which requires considerable time and effort. Between March 2017 and
November 2018 thirty trips were made to the site, and on each visit three people working for
about six hours obtained four to six scelionid wasps. The latter were promptly used in trials
(see below) carried out in a makeshift laboratory at the same site. Within the wasp genera
showing the black-orange-black (BOB) pattern, only some species have this pattern, the other
species usually being totally black (Fig 1). The wasps were identified by R.M. and P.H. using
keys to scelionid genera [15]; voucher specimens were deposited in the Museum of Zoology at
the University of Costa Rica. Four genera were collected from this site and used in the experi-
ments: Baryconus Foerster, Chromoteleia Ashmead, Macroteleia Westwood and Scelio
Latreille. All four genera were represented by a BOB morphospecies and an all black morphos-
pecies, providing a total of eight morphospecies. Due to the lack of taxonomic studies for three
of the four genera, species level identifications were not possible. While there is a chance that
some of the morphospecies included a mixture of more than one species, this problem was
minimized (though not totally eliminated) by always collecting from the same site and care-
fully examining the specimens under a stereomicroscope.
Salticid collection
Lyssomanes jemineus egg sacs (from which adults were reared in captivity; see S1 Appendix in
S1 File), female adults, and juveniles were collected weekly from January 2017 to January 2019.
Collecting was done in Finca 3 of the University of Costa Rica, San Pedro de Montes de Oca,
San José, Costa Rica (9˚56’07”N, 84˚03’04”W, 1300 m.a.s.l.). This site is located in a disturbed
urban environment which exhibits mainly semi-woodland vegetation of native and introduced
herbaceous plants [16]. This locality has an average annual precipitation of 1200–1500 mm
and an average temperature of approximately 21˚C. Spiders and egg sacs were collected from 0
to 1.5 m above ground level. Since many of the plants were quite widely spaced, the transect
consisted of 25 to 30 locations where diverse herbaceous plants were haphazardly searched.
For the collection of egg sacs weekly samplings of approximately three hours each were carried
out from May to September, according to the reported phenology for this species [12]. Each
egg sac (usually located on the underside of the leaf) was collected manually without being
detached from the leaf and was quickly transferred to the laboratory using 15 ml sterile Falcon
tubes with a humid cotton ball. For the collection of adults, the same methodology was applied,
but mainly from April to October. Specimens were identified by G.B. Edwards.
Observations of spiders with live prey
Field-collected juvenile and adult spiders, as well as adults reared from egg sacs in the labora-
tory (for about a year), were observed one at a time in an experimental arena (see below).
Adult spiders reared in the laboratory had no prior contact with the wasps. Six types of
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 3 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Fig 1. Two scelionid wasp color patterns within the same genus. Color patterns in the genus Scelio: black (A), and BOB (B). The focus-stacked macro
photograph was obtained with a Reflex 850 camera coupled to a 20x microscope lens.
https://doi.org/10.1371/journal.pone.0237288.g001
behavior were recorded: 1) prey detection as indicated by swivelling the cephalothorax; 2) fol-
lowing or stalking prey; 3) crouching, jumping and contacting the prey; 4) piercing and ingest-
ing the prey; 5) prey detection followed by withdrawal from the prey; 6) prey undetected or
ignored. In the results the first two were combined as “detect” (although detection was not
always followed by stalking), three and four were combined as “attack” (although jumping was
rarely followed by ingestion), and five and six were combined as “avoid”.
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 4 / 19
PLOS ONE Aposematic black orange black color pattern in insects
To facilitate observations of such small specimens we used a closed transparent acrylic
box with an internal volume of 1550 cm3. The acrylic plates were made in a CNC cutting
machine, model Redsail CM1690, with a red laser tube with 100 watts of power. To provide
greater structural complexity and additional routes for approaching the prey, an acrylic, tree-
like structure was placed in the center of the floor [17]. We also incorporated a white back-
ground on the floor and natural light was allowed to enter the remaining walls of the acrylic
box. In order to emulate the natural environment as much as possible, all observations were
carried out in a makeshift kiosk embedded in the forest in which the wasps were collected.
Behavior was filmed with a Sony RX100 IV camera and was done in FullHD 1080 x 1920 at
60fps.
In order to minimize potential confounding factors, we used the following standardized
conditions for all experiments with spiders.
1. We standardized spider sex (females only), age (more than 160 days old) and body size
(considered in the statistical analysis).
2. An 8-day pre-test fast ensured that the test spiders would be motivated to feed during
testing.
3. We used each prey and predator for just a single trial.
4. Each spider was allowed 60 s of acclimatization in the chamber, before introducing the
prey.
5. Between tests, the chamber was washed with 80% ethanol, followed by distilled water, and
then dried in order to eliminate any chemical trail of predator or prey [18–20].
6. Each trial finished when the prey was eaten or after 40 minutes of observation.
Observations of spiders with false prey (rice lures) in the automated cage
Most of this research was based on live predators (salticids) and prey (scelionids). However,
false prey consisting of painted rice lures were also used, since some previous studies have
examined aposematic signals in insects by utilizing painted models as prey in order to achieve
greater standardization with respect to potentially confounding factors such as posture, size
and shape [21, 22].
The experimental arena, consisting of an automated cage for observing spiders with false
movable prey (rice lures, explained below), was made of a closed transparent acrylic box with
the same characteristics as the ones used for live prey. In order to study whether background
colour affected the detectability of BOB or black rice lures, two types of background were used:
one acrylic box had white-colored walls and the other had black-colored walls. All observations
were carried out between 07:00 h and 11:00 h in a laboratory with ambient lighting from fluo-
rescent ceiling lamps (photoperiod 12h:12h L:D).
In order to generate movement in the lures, the acrylic box was placed inside a frame that
contained an automated mechanism (Fig 2). The rice lures were moved by a motor mechanism
that allowed for two-dimensional movements. Each lure was connected to a moving part
through a nylon thread, generating movement along two axes. This mobile part consists of a
magnet on the outside of the acrylic box which in turn moves a magnet on the inside of the
box. The control unit has programming functions that allow one to execute different patterns
of movement and comprises an interaction interface with the user; for this study, we used only
horizontal movements along the side walls of the box (attempting to simulate the movements
of live scelionid wasps that we previously observed to be attractive to the spiders). For the pro-
gramming of this movement pattern the Arduino platform was used.
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 5 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Fig 2. Components of the automated cage. Aperture for introducing the spider (A). Aperture for introducing live
prey (B). Buttons controlling the moving lure (C). Motor mechanism consisting of 4 guide blocks, 4 rails and 2 means
of movement that use two pairs of independent rails; each electric motor is connected by a toothed belt to a guide
block, which in turn is connected to a busbar that moves the remaining guide block located in parallel; each pair of
guide blocks located in parallel is connected to a moving part through a nylon thread, generating movement along an
axis (D). Upper magnet (E). Aperture for introducing false prey (lure) (F). Lower magnet from which the lure is
suspended by a transparent thread inside the acrylic box (G).
https://doi.org/10.1371/journal.pone.0237288.g002
The interaction between live spiders, lures (made of painted rice) and the experimental
arena consisted of the following:
a. Spiders and lures: during each trial an adult spider had simultaneous access to two types of
lures, one painted in the BOB pattern and the other painted all black.
b. Lures and automated cage: The BOB and black lure moved continuously and simulta-
neously, one on each lateral wall of the arena, emulating live scelionid wasp movement. All
lures were 4 mm in length, similar to that of the scelionid wasps. In order to objectively
paint the grains, not based on the colors detected from our human visual space, but on the
spectral characteristics of the BOB colors in wasps, the reflectance spectra of both wasps
and painted rice grains were considered (S2 Appendix in S1 File). The spectral characteris-
tics of BOB colors were compared with those of paints in order to choose the lures having
spectral features most likely to interact with the visual system of the jumping spiders (S1 Fig
in S1 File). With respect to the four scelionid genera included here, we chose to use the pre-
viously reported mean reflectance curves for Baryconus because this data set showed low
dispersion compared with those for other scelionid genera [2].
c. Data collection: Each trial was carried out for 40 minutes. We recorded the following behav-
iors: a) spider detects prey, as indicated by swiveling its cephalothorax; b) prey stalked and/
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 6 / 19
PLOS ONE Aposematic black orange black color pattern in insects
or contacted; c) prey undetected or ignored; d) prey detected and spider withdraws; e) the
color pattern (BOB or black) that is detected first. We also measured the body length of
each spider, and noted whether a noticeable silk dragline was produced while jumping
toward the prey (small amounts of silk produced while walking were not included).
Absorption contrast parameter for the green spectral components of the
BOB pattern
In order to elucidate whether the spider can distinguish between orange (BOB) and black, and
to determine whether the orange color varies among genera, the method described in S2
Appendix in S1 File was also used to obtain spectral green components of the reflectance spec-
tra of the black and orange color of the BOB pattern in the four wasp genera, as previously
reported [2]. Since preliminary studies of other salticid spiders suggest the presence of photo-
pigments with absorption bands in the UV (ca. 360nm) and the green (ca. 520nm) [23], we
used a Govardovskii nomogram [24] for the normalized absorption spectrum of the α band of
A1 type pigment with λ max = 520nm. Nevertheless, this description is not complete since it
lacks information about the UV visual sensibility.
After calculating the spectral green components, the areas under each curve were obtained
numerically. This quantity represents how much of the light reflected by the cuticle of the
wasp can be absorbed by the photosensitive pigment of the spider and therefore can be pro-
cessed by its visual system. According to this criterion, the absolute value of the arithmetic dif-
ference between areas should be proportional to a higher or lower capability of detecting a
difference between colors. The difference was calculated as the area for the green component
of the black color minus the area of the green component of the orange color. Negative values
therefore indicate a major absorption of electromagnetic input corresponding to orange,
whereas positive values indicate a major contribution to the absorbed light coming from the
black color. We refer to these differences as “absorption contrasts”, in the sense that the only
assumption we are making about the physiology of the visual system of the spider is the type of
photosensitive pigment present and its maximum absorption wavelength (as described above).
The calculations described here were performed using the reflectance curves reported in refer-
ence [2]. The standard deviation of the areas under curve from the green components were
used for estimating an error for the calculation of the absorption contrast.
Statistical analysis
There were three different statistical analyses in this work: (1) an experimental design with spi-
ders and live prey, that uses a multinomial model to describe 136 trials, with each trial as the
statistical unit, and the most common spider activity per trial as a response, (2) a statistical
description of the timeline for the same trials, taking each spider action as the statistical unit,
and (3) an experimental design with spiders and false prey in the automated cage, with 30 sepa-
rate trials.
The first analysis consisted of a total of 136 trials, with three groups of spiders (68 field juve-
niles, 51 field adults and 17 captive adults) and live prey during which the following data were
recorded: spider type (juvenile, field adult, captive adult), wasp color (BOB or black), spider
behavior (behavioural scale explained before), time when the spider reacted (0–5, 5–10, 10–20,
20–30, 30–40 minutes), the distance between the spider and the prey during each behavior,
presence/absence of a noticeable silk dragline, and wasp and spider body length. For each of
the 136 trials consisting of a wasp with a spider, the size of each was measured (S2 Fig in S1
File). Although there is size variation within each group, predator (spiders) and prey (wasps)
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 7 / 19
PLOS ONE Aposematic black orange black color pattern in insects
fall within the same general range, with field-captured adult spiders being the largest (S2 Fig in
S1 File).
A multinomial logistic regression [25] was fitted to a simplified response: the most common
activity for each spider was classified as “detect”, “attack” or “avoid”, in order to correct for
zero or near zero counts in each response category. The full model includes the following
covariates: wasp color, spider type, wasp size, spider size, wasp genus and presence/absence of
a noticeable silk dragline during jumping. This model was compared with simplified versions,
with the result being that the model with wasp color and spider type was the model with the
best fit, according to the AIC statistic.
Additionally, a second analysis was carried out with the same trial, where the number of
individual actions for each spider was recorded during each of the four 10-minute time slots,
and distinguished between the wasp colors they were acting on, in order to define a clear time-
line of spider behavior in the controlled experiments. In this case we had 954 individual
actions included in the 136 trials.
A third analysis of a separate experiment with lures in the automated cage included 30 tri-
als, during which the following variables were recorded: presence of noticeable silk/absence of
noticeable silk, spider size, background experimental arena color (black or white) and color of
false prey (BOB vs black) that first attracted the spider.
The response variable in this case was constructed by registering whether L. jemineus
responded in the same way to black and BOB lures, i.e. if it attacked, detected or avoided both
the black and BOB lures, coded as 1, and whether L. jemineus responded in a different way to
black and BOB, i.e. if it attacked black and detected the BOB, or if it detected the BOB and
avoided the black lures, coded as 2. Contingency tables were calculated, along with a log likeli-
hood ratio (G-test) independence test for the response versus each of the variables: presence/
absence of a noticeable silk dragline, background color and prey color that first attracted the
spider.
Statistical analyses were performed using R (version 3.6.2, R Core Team, 2019). Data for-
matting and figures were prepared using the Tidyverse packages [26]. Multinomial logistic
regression was done using the nnet package [25].
Wasp extract preparation for toxicity tests
To obtain the wasp extracts some modifications of Arenas research [5] were implemented.
Pools of three organisms from the same genus and with the same color were placed in 1.5 ml
centrifuge tubes; the initial weight of each pool was taken with a Mettler Toledo XP 205 analyt-
ical balance. Extraction was done by adding 0.5 ml of methanol (99.8% purity) to each pool of
individuals and subsequent maceration was done with a glass pestle for 5 minutes. After mac-
eration, the tubes were centrifuged in a Minispin plus Eppendorf centrifuge at 14500 rpm for
10 minutes. The supernatant was transferred into a 6 ml glass vial and the pellet was discarded.
The methanol from the supernatant was evaporated to dryness with an Organomation brand
nitrogen concentrator, model MULTIVAPTM (flow of 7 l/min at 28˚C). For testing, 1400 μl of
reconstituted water was added to the concentrate and homogenized using a vortex; this extract
was considered as 100%, then dilutions of 75%, 50% and 25% from the original extract were
prepared with reconstituted water to a final volume of 0.5 ml. A negative control of the extract,
lacking the wasp pool, was prepared following the same procedure.
Acute toxicity test with Daphnia magna
The toxicity of seven wasp extracts corresponding to four scelionid genera and the two color
patterns of interest was measured by an acute toxicity test with the water flea Daphnia magna
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 8 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Straus, based on a similar method described by [5]. This organism is frequently used for toxic-
ity tests due to its sensitivity to xenobiotics, wide distribution, short life cycle and ease of cul-
turing in the laboratory. The water fleas were kept in reconstituted water (MgSO4, NaHCO3,
KCl, CaSO4 x 2 H2O) [28], at 21 ± 2˚C, with a photoperiod of 12:12, and fed with the green
algae, Raphidocelis subcapitata. The culture medium was changed every week, and after five-
weeks adult individuals were discarded and a new culture started from neonates. For the
adapted acute toxicity test [27], ten 24-hour neonates were exposed to 0.5 ml of each different
dilution (triplicates) of the wasp extract during a 48-hour period in a dark room at a constant
temperature of 22˚C. The test end point was mortality (immobilization), which was recorded
at 24 h and 48 h. The data were used to calculate the mean lethal concentration (LC50), using
the R software, version 3.5.3 (R Development Core Team 2014) and using the “drc” package
[28]; LC50 was defined as the relative concentration of the extract (considering the pure
extract as 100%) that causes a mortality of 50% in the water fleas.
Acute toxicity test with Vibrio fischeri
The Microtox test detects inhibition of bioluminescence in the bacterium, Vibrio fischeri,
using the Microtox1 500 Analyzer. For the analysis, the 2% Basic Test method recommended
by the Microtox1 software was employed. Briefly, the lyophilized bacteria (Microtox Acute
Reagent) was reconstituted and exposed to four dilutions from the original 100% wasp extract,
prepared with Modern Water Microtox Diluent1. Light emission by the bacteria in contact
with the extract dilutions was measured at 0, 5, and 15 minutes; data analysis by the Micro-
tox1 software was employed to determine the EC50 value, defined as the relative concentra-
tion of the wasp extract (considering the pure extract as 100%) that causes 50%
bioluminescence inhibition.
Results
Experiment of spiders with live prey
A summary of the most common action per each of the 136 trials included in the final multi-
nomial model is presented in Fig 3. The number of spiders differs between groups, which is
why the bars for captive adults are smaller than the ones for field adults and field juveniles.
The aforementioned model suggests that not producing a noticeable silk dragline is associ-
ated with an increased chance of detecting versus attacking (p = 0.0160), which makes sense
Fig 3. Most common behaviours of L. jemineus in trials with different combinations of wasp color (BOB vs black)
and spider type. Each bar represents the proportion of spiders per type and wasp color (orange–BOB, gray–black),
according to the most common action taken by the spider in each of the 136 trials.
https://doi.org/10.1371/journal.pone.0237288.g003
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 9 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Fig 4. Frequency of individual behavioral responses experienced by L. jemineus when confronted with live wasps.
Behaviors are: detection (A), attacking (B), avoiding (C) All bar plots are represented according to behavior per time,
spider type and prey color (black and BOB). Time slots without bars indicate that none of the spiders performed that
action during that time slot. The total individual actions is n = 954.
https://doi.org/10.1371/journal.pone.0237288.g004
since in many cases silk was more evident before attacking the prey (R. Mora-Castro, unpub-
lished), an observation that warrants further research. Being a field-caught juvenile versus a
captive adult did not make a difference in the most common spider actions. Other factors such
as wasp color, wasp size, spider size, and wasp genus did not make a significant difference
when included in the full model.
When the behavior of all spiders is analyzed individually and during the 40 min and in the
different time slots a greater complexity of behaviors is observed, specifically in that the field
adults usually differed from the captive adults. Fig 4 summarizes the 954 actions taken by all
spiders throughout the timeline of the experiment, and data in Table 1 shows the detailed
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 10 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Table 1. L. jemineus individual behavioral responses.
Behavioral responses Black BOB TOTAL
1. Alert and swivel 160 192 352
2. Follow or stalk 99 111 210
3. Crouch, jump and contact prey 99 101 200
4. Pierce and ingest 8 0 8
5. Prey undetected or ignored 83 48 131
6. Detect visually and withdraw 14 37 51
7. None 2 0 2
TOTAL 465 489 954
Frequency of individual behavioral responses experienced by L. jemineus when confronted with live wasps, with
seven ungrouped behaviors, per wasp color. The total individual actions is n = 954.
https://doi.org/10.1371/journal.pone.0237288.t001
count with the original individual actions. There are some time slots without observations
such as 0–10 mins for captive adults in plot B. Thus, the number of spiders for each category
changes according to each combination. It can be seen that captive adults detect/stalk wasps of
both color patterns, but during certain time periods their behaviour is directed slightly more
toward the black wasps than the BOB wasps (10–20 min and 30–40 min), a behavior not
observed in field adults or field juveniles (Fig 4A). It is worth noting that captive adults attack
only black wasps during the last time period, unlike the first 30 min during which both colors
are attacked, as in field juveniles and adults (Fig 4B). Captive adults avoid wasps with the BOB
pattern during most time periods, which differs from the behavior of field-caught spiders,
which showed equal avoidance of both colors of wasps (Fig 4C).
There is a clear effect of experience, with captive adults showing a lower attack capacity
than field spiders (including juveniles). This may be due to the fact that captive adults were
reared on a simple diet and were thus responding to unfamiliar prey. However, there is some
evidence for an innate response by captive adults, in avoiding attack responses and aversion to
BOB wasps (Fig 4C).
Green spectral components of the BOB pattern
The mean green spectral components of the black and orange colors of the BOB pattern in the
four wasp genera are shown in Fig 5. In general, the spectral components for black and orange
colors differ in the position of the maximum wavelength and the height of the peak, indicating
different interaction of the reflected light with the photosensitive pigment. The exception is
Baryconus, for which the spectral components of black and orange colors are similar. The
absorption contrast parameters, calculated as described in the Methods section, together with
the results of the predation experiments, are presented in Fig 6. The four genera were ranked
from the highest negative value to the highest positive value of absorption contrast between
black and orange spectral components. Also, the mean green spectral components of the black
and orange colors of the BOB pattern from Fig 5, used for the calculation of the absorption
contrast, were included as a visual aid. The standard deviation for the areas under the curve,
used for estimating the error in the calculation, are shown as percentages inside square brack-
ets. The observed events, classified in the categories “detect”, “avoid” and “attack”, as explained
in the Methods section, are presented as percentages calculated with respect to total number of
trials for each genus in the case of BOB specimens, and the total number of trials for black
specimens of all genera. This choice is based on a conclusion of previous work of our group
[2], which suggests that black color is spectrally similar between genera.
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 11 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Fig 5. Mean green component of the reflection spectra of both black and orange colors for both the cuticle of each
genus and the blend chosen for painting the false prey (painted rice grain). Green component, in arbitrary units (a.
u.), of both black (black line) and orange (orange line) colors for the BOB color pattern of four genera of scelionid
wasps (solid lines) and the paint chosen for the false prey (dashed lines).
https://doi.org/10.1371/journal.pone.0237288.g005
The information in Fig 6 shows that for the wasps with a BOB color pattern, most of the
“attack” events occurred with BOB-colored Baryconus, which also has the lowest absorption
contrast. On the other hand, the greatest number of “detect” events occurred with BOB-col-
ored Macroteleia, which has the highest absorption contrast and the highest contribution of
orange, followed by Chromoteleia which has the second highest contrast value in absolute
numbers, while lower values of black contribution, as in Scelio and Baryconus, yielded a lower
percentage of “detect” events. The greatest number of “avoid” events occurred with BOB-col-
ored Scelio, which has the second highest absolute absorption contrast.
Experiment of spiders with false prey in the automated cage
Table 2 presents the results from the experiment with false prey. Each contingency table was
tested for independence, and there was no evidence of dependence for background
(G = 2.0879, p-value = 0.1485), color (G = 0.06728, p-value = 0.7953), or presence of noticeable
silk (G = 0.0089311, p-value = 0.9247). In other words, spider behaviors are not associated
with background color (black or white walls in the arena), the color of lure that was detected
first, or the presence or absence of a noticeable silk dragline.
Acute toxicity tests with Daphnia magna and Vibrio fischeri
During the exposure period, acute toxicity trials with D. magna resulted in a higher mortality
(LC50< 65.2%) of water fleas when exposed to the extracts from the BOB wasps, which
employed Chromoteleia and Baryconus (Table 3). In the case of the black wasps, three out of
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 12 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Fig 6. Relationship between the green components of the BOB colors of each scelionid genus and the percentage of behavioral responses by L.
jemineus. Summary of information about the relationship between the green components of the BOB colors of each scelionid genus and the percentage
of behavioral responses by L. jemineus observed and grouped in the categories “detect”, “avoid” and “attack”. The mean BOB color pattern areas under
the curve, in arbitrary units, were calculated and compared for the green component of the black and orange colors, respectively (see also Fig 5). Values
within square brackets represent the standard deviation of the areas.
https://doi.org/10.1371/journal.pone.0237288.g006
four extract samples did not show toxic effects (LC50 > 100%) on D. magna; the one sample of
black Macroteleia that showed toxicity had a lower mortality than with the BOB wasps. No
mortality of water fleas was observed with the negative control, thus suggesting that the toxic
effects are due to the components of the wasps. In the toxicity tests with V. fischeri only black
Macroteleia and BOB Chromoteleia resulted in high bioluminescence inhibition. The results
for the two genera have overlapping confidence intervals, so no difference can be inferred; on
the contrary, there were no inhibitory effects with BOB Baryconus or black Scelio (Table 3).
Table 2. Results from experiment with false prey in three contingency tables.
Behavioural responses (A) (B) (C)
Background Color Presence of a silk dragline
White Black Black BOB No Yes
L. jemineus took the same actions with both black and BOB wasps 13 8 15 6 16 5
43.3% 26.6% 50.0% 20.0% 53.3% 16.6%
L. jemineus took different actions with black and BOB 3 6 6 3 7 2
10.0% 20.0% 20.0% 10.0% 23.3% 6.6%
TOTAL (30) 16 14 21 9 23 7
A total of 30 observations were aggregated according to the response variable (same or different actions) and three variables: (A) background color (white or black), (B)
color (black or BOB) that first attracted the spider, (C) presence of a noticeable silk dragline while jumping toward the prey (yes or no). The counts and the percentage
of total cases are indicated.
https://doi.org/10.1371/journal.pone.0237288.t002
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 13 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Table 3. Toxicity (± C.I.) of extracts of scelionid wasps as determined in acute tests in the bioindicators Daphnia
magna and Vibrio fischeri.
Wasp genus and color (pools of three Total mass LC50a (%c) (Daphnia EC50b (%c) (Vibrio
specimens) (mg) magna) fischeri)
Baryconus (BOBd) 3,41 59.6% (50.2, 68.9) > 100%
Chromoteleia (BOB) 3,46 60.6% (51.5, 69.7) 1.7% (0.9, 3.0)
Chromoteleia (BOB) 3,75 65.2% (54.4, 75.9) 5.2% (0.8, 32)
Macroteleia (blacke) 1,34 85.9% (73.5, 98.3) 1.0% (0.4, 2.0)
Macroteleia (black) 1,42 > 100% 2.1% (1.1, 4.1)
Scelio (black) 0,66 >100% > 100%
Scelio (black) 0,86 >100% > 100%
a LC50: mean lethal concentration
b EC50: mean effect concentration
c %: Refers to the % of original extract dilution; 100% corresponds to the pure extract.
d BOB: black-orange-black color pattern
e black color pattern
https://doi.org/10.1371/journal.pone.0237288.t003
Overall, the wasp extracts showed a much higher toxicity for the bacteria bioindicator (V.
fischeri) than for the arthropods (D. magna).
Discussion
Evidence for an aposematic function of BOB coloration
A previous study showed that the black-orange-black (BOB) color pattern is very widespread
among small parasitoid wasps and that it is usually present in both sexes [1], suggesting that
this color pattern is not related to sexual behavior. The principal hypothesis regarding the
function of this widespread color pattern is aposematism and to the best of our knowledge, the
present research is the first to test this hypothesis. Although the results were mixed, at least
two of our findings provide evidence that BOB coloration is indeed aposematic. First, although
the behavioral responses were quite variable, we never observed a spider consuming a wasp
with BOB coloration (Table 1), but we did observe a few instances of black wasps being
ingested. Second, toxicity, a well-studied and common attribute utilized as a defense by apose-
matic prey [29], was determined in acute toxicity tests with Daphnia magna and showed that
wasps with BOB coloration caused greater mortality than did the black wasps. This suggests
that BOB wasps contain defensive compounds unlike black wasps, which would explain why
only black wasps were consumed, but this requires further investigation. It is also possible that
the BOB coloration acts synergistically with repellent odors, which has been shown to affect
foraging behavior in another jumping spider [30].
It was also observed that the wasp extracts were more toxic for the bacteria (V. fischeri) than
for the crustacean (D. magna). Unfortunately, although in the same toxicity range, no clear dif-
ferences were observed for the extract samples that showed toxicity in the V. fischeri assays,
due to overlapping confidence intervals. Nonetheless, results from this test should be taken
into account when considering the potential presence of toxic compounds in the wasp extracts.
A consistent result for both bioindicators is that the black Scelio did not show toxicity, and
might be considered the least toxic of the four genera. The results for D. magna are more rele-
vant for this study since differences between the wasp genera can be observed and because it is
an arthropod as is the spider. Moreover, the extracts from the BOB wasps all showed toxicity
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 14 / 19
PLOS ONE Aposematic black orange black color pattern in insects
while just one out of four black wasp extracts did so; moreover, the EC50 values for the BOB
wasp extracts were lower, i.e. had higher toxicity.
Predator responses towards BOB wasps
It should be emphasized that considerable effort is needed to obtain live scelionid wasps; a
total of about 540 hours were required to collect the wasps used in our experiments and even
with this effort there were insufficient numbers for some of the trials. This limitation was one
of the motivations for carrying out trials with lures. Despite considerable care taken in provid-
ing lures that closely matched the black and orange colors of the wasps, not subjectively
through our visual perception but through spectrophotometric measurements, and an arena
in which the movements of the lures were carefully controlled, the results were inconclusive.
The likely explanation is that the lures lacked additional visual cues (e.g. legs and eyes) and/or
chemical or behavioral cues necessary to elicit a predatory response, which has been shown in
other jumping spiders [6]. Also, the absorption contrast of the paint chosen is somewhere
between the contrast for Scelio and Baryconus, and might not be enough to trigger a clear
response from the spider. In future research it would be interesting to use lures simulating
additional wasp features, as well as dead scelionid wasps as lures.
In our experiments with predators, we used three types of Lyssomanes spiders: field-col-
lected adults, field-collected juveniles, and lab-reared (captive) adults. In order to consider
warning signals more generally, we integrated background contrast, predator vision, predator
life stage, predator and prey sizes, and prior opportunities for learning by the predator [3, 5].
Predator origin (being a field adult) was the only significant factor that increased the main
probability of detecting vs attacking and avoiding vs attacking. If learning plays a role in avoid-
ing aposematic prey, one would expect field-collected adults to show the greatest discrimina-
tion between black and BOB wasps, since the use of lab-reared specimens eliminates the role
of learned generalization [31], and field-collected juveniles have had less time to learn. The
results of our experiments therefore provide some evidence for learning, an unsurprising find-
ing since learning is common in arthropods [32]. Moreover, the only instances of spiders con-
suming wasps (non-aposematic, black wasps) were of Lyssomanes that had been captured in
the field.
A possible innate effect is evident in the clear aversion by captive adults to wasps with the
BOB coloration. Evidence for innate aversion has been observed in naive chickens presented
with hibiscus harlequin bugs [33], in naive mantises presented with ants [34] and even with
ant-mimicking spiders [35], and in jumping spiders presented with army ants [36] or with
prey of different colors [37]. Furthermore, it has been shown that many predators are very
conservative in their prey preferences, and avoid brightly colored prey even when they are edi-
ble [33]. Nonetheless, finding evidence for innate aversion of captive adult spiders to wasps
with the BOB coloration does not rule out the possibility that learning is important in this
predator prey interaction; indeed, aversions to particular prey colors can change with experi-
ence [38]. We found that spiders from natural conditions showed increased aversion over
time, and this aversion coincides with the toxicity analyses and the aposematic pattern of the
wasps.
Spectral components of the BOB pattern
Although orange is usually present in aposematic coloration and warning signaling [3, 34], our
results show that not all BOB specimens were treated similarly by the spider predators. The
orange coloration in BOB wasps varies between genera, despite appearing similar to the
human eye. It is possible that the difference between the two light absorbing elements (black
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 15 / 19
PLOS ONE Aposematic black orange black color pattern in insects
and orange pigments) within each genus of wasps is important in affecting predator responses.
Such differences could favor the increase of conspicuousness and consequently intervene in
initial avoidance [39, 40].
In general, the dispersion of the reflectance data taken from reference [2] prevent us from
making strong assertions, but some features or tendencies can be pointed out from the com-
parison between the contrast absorption parameter and the results of experiments with live
prey summarized in Fig 6. As discussed in reference [2], the dispersion in those measurements
derives from several factors, including the fact that for this study, species were grouped into
genera. As mentioned previously, the reflectance curves of Baryconus have the least dispersion.
Therefore, the estimated error in the contrast parameter is the lowest between the genera stud-
ied. Nevertheless, the contrast parameter resulted in a value that is within the error, meaning
that black and orange cuticle segments have the same absorption. This is consistent with the
fact that Baryconus presented the higher percentage of “attack” events, suggesting that if color
perception is the only variable considered, in this case black and orange are not conspicuous
from the point of view of the spider. Scelio and Macroteleia have higher absorption contrast,
and higher orange contribution, suggesting that in those cases the spider should be more likely
to differentiate between black and orange. In fact, no experiment involving Macroteleia wasps
resulted in an “attack” event, all of them fell into the category “detect”. The results for Chromo-
teleia are similar in the sense that all the events were distributed between the categories
“detect” and “avoid”, and the contrast parameter is the second highest in terms of absolute
value. Nevertheless, the standard deviation of reflectance measurements is the highest among
the genera considered, resulting in an absorption parameter that is within the error estimation.
Even though the relationship between the BOB pattern and the behavior of the predator
should be taken with caution because of the estimated error in the calculations, we hypothesize
that the contrast difference between black and orange from the point of view of the predator
visual capabilities is an important factor to be taken into account in behavioral experiments.
It has been reported that jumping spiders are less sensitive to the orange end of the spec-
trum and thus perceive orange cuticles as more achromatic than structures with relatively
more reflectance in the green portion of the spectrum [6]. The aforementioned, plus the
absence of a red receptor in many spiders, could cause orange objects to be perceived as mono-
chromatically "green" objects, difficult to distinguish chromatically from green foliage [40–42].
Our results seem to be consistent with observations that arthropods may be able to perceive
long wavelength difference via achromatic or luminance information [43], and that some
color patterns may function as multicomponent signals [44, 45].
Conclusion
Most studies of aposematism have used vertebrate predators and larger prey, whereas arthro-
pod predators of smaller prey have generally been neglected [37]. The BOB pattern is
extremely widespread in small parasitoid wasps (as well as in a few other insects) and has evi-
dently evolved independently on numerous occasions [1]. To the best of our knowledge, the
present study provides the first evidence that this common color pattern might have an apose-
matic function in these small insects, although further research is needed to confirm this.
There are still many unanswered questions regarding the black-orange-black color pattern
found in scelionids and other small wasps. For example, the chemical identity of the pigments
and of the putative noxious compounds are still unknown. How does learning interact with
variability in the orange tones and the putative toxins? Finally, it would be instructive to exam-
ine the spectral properties of other wasps with this color pattern, as well as the visual capacity
and responses of other potential predators.
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 16 / 19
PLOS ONE Aposematic black orange black color pattern in insects
Supporting information
S1 File.
(DOCX)
Acknowledgments
We give special thanks to Geovanna Rojas Malavasi, Natalia Jiménez Conejo, Paulina Morales
Vargas and Anthony Ulate for their support in field data collection, maintenance and rearing
of spiders. We also thank Mauricio Valverde Arce for his support in macrophotography and
G.B Edwards for his valuable help in the identification of the salticid spiders. We thank our
filming crew, Valeria Romero and Soren Pessoa of MANDUCA productions, for their crucial
collaboration. We thank two anonymous referees whose comments greatly improved the qual-
ity of the manuscript.
Author Contributions
Conceptualization: Rebeca Mora-Castro.
Formal analysis: Rebeca Mora-Castro, Marcela Alfaro-Córdoba, Marcela Hernández-
Jiménez.
Funding acquisition: Rebeca Mora-Castro.
Investigation: Rebeca Mora-Castro.
Methodology: Rebeca Mora-Castro, Marcela Alfaro-Córdoba, Marcela Hernández-Jiménez,
Mauricio Fernández Otárola, Michael Méndez-Rivera, Didier Ramı́rez-Morales, Carlos E.
Rodrı́guez-Rodrı́guez, Andrés Durán-Rodrı́guez, Paul E. Hanson.
Project administration: Rebeca Mora-Castro.
Resources: Rebeca Mora-Castro.
Software: Marcela Alfaro-Córdoba, Marcela Hernández-Jiménez.
Supervision: Rebeca Mora-Castro.
Validation: Rebeca Mora-Castro.
Visualization: Rebeca Mora-Castro, Marcela Alfaro-Córdoba, Marcela Hernández-Jiménez,
Mauricio Fernández Otárola, Michael Méndez-Rivera, Didier Ramı́rez-Morales, Carlos E.
Rodrı́guez-Rodrı́guez, Andrés Durán-Rodrı́guez, Paul E. Hanson.
Writing – original draft: Rebeca Mora-Castro, Marcela Alfaro-Córdoba, Marcela Hernández-
Jiménez, Mauricio Fernández Otárola, Michael Méndez-Rivera, Didier Ramı́rez-Morales,
Carlos E. Rodrı́guez-Rodrı́guez, Andrés Durán-Rodrı́guez, Paul E. Hanson.
Writing – review & editing: Marcela Alfaro-Córdoba, Marcela Hernández-Jiménez, Mauricio
Fernández Otárola, Michael Méndez-Rivera, Didier Ramı́rez-Morales, Carlos E. Rodrı́-
guez-Rodrı́guez, Paul E. Hanson.
References
1. Mora R, Hanson P E. Widespread occurrence of black-orange-black color pattern in Hymenoptera.
Journal of Insect Science. 2019; 19 (2). https://doi.org/10.1093/jisesa/iez021 PMID: 30851035
2. Mora-Castro R, Hernández-Jiménez M, Alfaro-Cordoba M, Avendano E, Hanson-Snortum P. Spectral
measure of color variation of black-orange-black (BOB) pattern in small parasitoid wasps (Hymenop-
tera: Scelionidae), a statistical approach. PLOS ONE. 2019b; 14 (10).
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 17 / 19
PLOS ONE Aposematic black orange black color pattern in insects
3. Stevens M, Ruxton GD. Linking the evolution and form of warning coloration in nature. Proceedings of
the Royal Society B: Biological Sciences.2012; 279 (1728): 417–426. https://doi.org/10.1098/rspb.
2011.1932 PMID: 22113031
4. Skelhorn J, Halpin CG, Rowe C. Learning about aposematic prey. Behavioral Ecology. 2016; 27 (4):
955–964.
5. Arenas LM, Walter D, Stevens M. Signal honesty and predation risk among a closely related group of
aposematic species. Scientific Reports. 2015; 5:11021. https://doi.org/10.1038/srep11021 PMID:
26046332
6. Harland DP, Jackson RR. Eight-legged cats and how they see: A review of recent research on jumping
spiders (Araneae: Salticidae). Cimbebasia. 2000; 16: 231–240.
7. Nagata T, Koyanagi M, Tsukamoto H, Saeki S, Isono K, Shichida Y., et al. Depth perception from image
defocus in a jumping spider. Science.2012; 335:469–471. https://doi.org/10.1126/science.1211667
PMID: 22282813
8. Harland DP, Li D, Jackson RR. How jumping spiders see the world. Cambridge, MA: the MIT Press;
2012.
9. Williams DS, McIntyre P. The principal eyes of a jumping spider have a telephoto component.
Nature.1980; 288 (5791): 578.
10. Nelson XJ, Jackson RR. The discerning predator: decision rules underlying prey classification by a mos-
quito-eating jumping spider. Journal of Experimental Biology 2012; 215 (13): 2255–2261. https://doi.
org/10.1242/jeb.069609 PMID: 22675186
11. Blest AD, Sigmund C. Retinal mosaics of the principal eyes of two primitive jumping spiders, Yaginuma-
nis and Lyssomanes: clues to the evolution of salticid vision. Proceedings of the Royal Society of Lon-
don. Series B. Biological sciences.1984; 221(1222): 111–125.
12. Mora-Castro R, Edwards GB, Hanson Snortum P. Phenology of an urban population of Lyssomanes
jemineus Peckham & Wheeler (Araneae: Salticidae) with a list of other jumping spiders from the same
Costa Rican site. Peckhamia. 2019a; 194:1.
13. Forster L. Vision and prey-catching strategies in jumping spiders. American Scientist. 1982; 70(2):
165–175.
14. Herberstein ME. Spider behaviour: flexibility and versatility. Cambridge University Press; 2011.
15. Masner L. Revisionary notes and keys to world genera of Scelionidae (Hymenoptera: Proctotrupoidea).
Memoirs of the Entomological Society of Canada. 1976; 108 (S97):1–97.
16. Biamonte E, Sandoval L, Chacón E, Barrantes, G. Effect of urbanization on the avifauna in a tropical
metropolitan area. Landscape Ecology. 2011; 26 (2): 183–194.
17. Tarsitano MS, Andrew R. Scanning and route selection in the jumping spider Portia labiata. Animal
Behaviour. 1999; 58: 255–265. https://doi.org/10.1006/anbe.1999.1138 PMID: 10458876
18. Jackson RR, Cross FR. Olfaction-based mate-odor identification by jumping spiders from the genus
Portia. Journal of Arachnology. 2011; 39(3): 439–444.
19. Nelson XJ, Warui CM, Jackson RR. Widespread reliance on olfactory sex and species identification by
lyssomanine and spartaeine jumping spiders. Biological Journal of the Linnean Society. 2012; 107(3):
664–677.
20. Clark RJ, Jackson RR. Dragline-mediated sex recognition in two species of jumping spiders (Araneae
Salticidae), Portia labiata and P. fimbriata. Ethology Ecology & Evolution. 1995; 7(1): 73–77.
21. Brandley N, Johnson M, Johnsen S. Aposematic signals in North American black widows are more con-
spicuous to predators than to prey. Behavioral Ecology. 2016; 27 (4): 1104–1112.
22. Exnerova A, Svadova K, Stys P, Barcalova S, Landova E, Prokopova M, et al. Importance of colour in
the reaction of passerine predators to aposematic prey: experiments with mutants of Pyrrhocoris
apterus (Heteroptera). Biol J Linn Soc. 2006; 88:143–153
23. Kelber A, Vorobyev M, Osorio D. Animal colour vision—Behavioural tests and physiological concepts.
Biological Reviews of the Cambridge Philosophical Society. 2003; 78: 81–118. https://doi.org/10.1017/
s1464793102005985 PMID: 12620062
24. Govardovskii VI, Fyhrquist N, Reuter TOM, Kuzmin DG, Donner K. In search of the visual pigment tem-
plate. Visual Neuroscience. 2000; 17(4): 509–528. https://doi.org/10.1017/s0952523800174036 PMID:
11016572
25. Venables WN, Ripley BD. Modern Applied Statistics with S. Fourth Edition. Springer, New York, 2002.
26. Wickham H. tidyverse: Easily Install and Load the ’Tidyverse’. R package version 1.2.1. https://CRAN.
R-project.org/package=tidyverse, 2017.
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 18 / 19
PLOS ONE Aposematic black orange black color pattern in insects
27. EPA. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and
Marine Organisms Fifth Edition October 2002. Environmental Protection Agency (October): 266,
2002.
28. Ritz C, Baty F, Streibig JC, Gerhard D. Dose-response analysis using R. PLOS ONE. 2015; 10 (12):
e0146021. https://doi.org/10.1371/journal.pone.0146021 PMID: 26717316
29. Speed MP, Ruxton GD, Stephens PA, Blount JD. Warning displays may function as honest signals of
toxicity. Proceedings of the Royal Society B: Biological Sciences. 2009; 276(1658): 871–877. https://
doi.org/10.1098/rspb.2008.1407 PMID: 19019790
30. Vickers ME, Taylor LA. Odor alters color preference in a foraging jumping spider. Behavioral Ecology.
2018; 29(4):833–839. https://doi.org/10.1093/beheco/ary068 PMID: 30018487
31. Fabricant SA, Smith CL. Is the hibiscus harlequin bug aposematic? The importance of testing multiple
predators. 2014; Ecology and Evolution, 4(2): 113–120. https://doi.org/10.1002/ece3.914 PMID:
24558567
32. Dukas R. Evolutionary biology of insect learning. Annual review of entomology. 2008; 53. https://doi.
org/10.1146/annurev.ento.53.103106.093343 PMID: 17803459
33. Marples N, Kelly DJ, Thomas RJ. Perspective: the evolution of warning coloration is not paradoxical.
Evolution. 2005; 59, 933e940.
34. Nelson XJ, Jackson RR, Li D, Barrion A.T., Edwards GB, Innate aversion to ants (Hymenoptera: Formi-
cidae) and ant mimics: experimental findings from mantises (Mantodea). 2006. Biological Journal of the
Linnean Society. 88 (1): 23–32.
35. Thery M, Gomez D. Insect colours and visual appearance in the eyes of their predators. In Advances in
Insect Physiology. Vol. 38, pp. 267–353. Academic Press, 2010.
36. Nelson XJ, Aguilar-Arguello S, Jackson R.R. Widespread army ant aversion among East African jump-
ing spiders (Salticidae). Journal of Ethology. 2020; 38:185–194.
37. Taylor LA, Maier EB, Byrne KJ, Amin Z., Morehouse NI. Colour use by tiny predators: Jumping spiders
show colour biases during foraging. Animal Behaviour. 2014; 90: 149–157.
38. Powell EC, Cook C, Coco J, Brock M, Holian LA, Taylor LA. Prey colour biases in jumping spiders (Hab-
ronattus brunneus) differ across populations. Ethology. 2019; 125(6): 351–361.
39. Stevens M, Mappes J, Sandre SL. The effect of predator appetite, prey warning coloration and lumi-
nance on predator foraging decisions. Behaviour. 2010; 147(9): 1121–1143.
40. Fabricant SA, Herberstein ME. Hidden in plain orange: aposematic coloration is cryptic to a colorblind
insect predator. Behavioral Ecology. 2014; 26 (1): 38–44.
41. Chittka L, Waser NM. Why red flowers are not invisible to bees. Israel Journal of Plant Sciences. 1997;
45(2–3):169–183.
42. Briscoe AD, Chittka L. The evolution of color vision in insects. Annual Review of Entomology. 2001; 46
(1):471–510. https://doi.org/10.1146/annurev.ento.46.1.471 PMID: 11112177
43. Reisenman CE, Giurfa M. Chromatic and achromatic stimulus discrimination of long wavelength (red)
visual stimuli by the honeybee Apis mellifera. Arthropod-Plant Interactions. 2008; 2(3): 137.
44. Grether GF, Kolluru GR, Nersissian K. Individual colour patches as multicomponent signals. Biological
Reviews. 2004; 79(3): 583–610. https://doi.org/10.1017/s1464793103006390 PMID: 15366764
45. Lang A, Filser J, Henschel JR. Predation by ground beetles and wolf spiders on herbivorous insects in a
maize crop. Agriculture, Ecosystems & Environment. 1999; 72(2): 189–199.
PLOS ONE | https://doi.org/10.1371/journal.pone.0237288 February 11, 2021 19 / 19