JHR 96: 967-982 (2023) ieee, JOURNAL OF Seetetenseationt

doi: 10.3897/jhr.96.1 11382 RESEARCH ARTICLE () I Tymenopter a
9

https://jhr.pensoft.net Theleternaonl Society of Hymenopexriss, RESEARCH

The wing interference patterns (WIPs) of Parapanteles
(Braconidae, Microgastrinae): demonstrating
a powerful and accessible tool for species-level
identification of small and clear winged insects

Shuyang Jin', Kyle S. Parks’, Daniel H. Janzen’,
Winnie Hallwachs?, Lee A. Dyer*, James B. Whitfield?

| Department of Neurobiology, Duke University, Durham NC 27708, USA 2. Department of Physical and
Biological Sciences, Western New England University, Springfield MA 01119, USA 3 Department of Biology,
University of Pennsylvania, Philadelphia, PA 19104, USA 4 Department of Biology, University of Nevada,
Reno NV 89557, Nevada, USA 5 Department of Entomology, University of Illinois Urbana-Champaign,
Urbana IL 61801, USA

Corresponding author: Kyle S. Parks (kyle.parks@wne.edu)

Academiceditor:J.M. Jasso-Martinez | Received 19 September 2023 | Accepted25 October2023 | Published 13 November2023
https://zoobank. org/5D12BB28-CFEF-4360-BF27-B0C761D938A0

Citation: Jin S, Parks KS, Janzen DH, Hallwachs W, Dyer LA, Whitfield JB (2023) The wing interference patterns
(WIPs) of Parapanteles (Braconidae, Microgastrinae): demonstrating a powerful and accessible tool for species-
level identification of small and clear winged insects. Journal of Hymenoptera Research 96: 967-982. https://doi.
org/10.3897/jhr.96.111382

Abstract

Wing interference patterns (WIPs) are color patterns of insect wings caused by thin film interference.
Thin film interference is the same phenomenon responsible for the refracted spectral colors sometimes
visible on soap bubbles. Insect WIPs are static patterns due to the variable thickness of wing membranes
and the colors produced depend on the thicknesses of wing membranes. While WIPs have been studied
in several taxa of small insects, they have not been broadly adopted by insect taxonomists. We surveyed
WIPs in one moderate-sized genus of parasitoid wasps, Parapanteles (Braconidae: Microgastrinae). Using
an inexpensive microscope camera set-up and free imaging and analysis software, we detected consistent
WIP differences between Parapanteles species. In some cases, WIPs can be used to diagnose sibling species
that would otherwise require SEM images to differentiate or DNA barcodes. Wing interference patters are

an underemployed character that may be similarly useful in many other taxa of small clear-winged insects.

Keywords

Braconidae, color patterns, Microgastrinae, Parapanteles, WIP, Wing interference patterns

Copyright Shuyang Jin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

968 Shuyang Jin et al. / Journal of Hymenoptera Research 96: 967-982 (2023)

Introduction

Wing interference patterns (WIPs), the rainbow colors that can appear on clear in-
sect wings against dark background, have not been broadly adopted by insect tax-
onomists as morphological characters. Shevtsova et al. (2011) comprehensively in-
vestigated and called attention to these patterns, discovering that they are stable
non-iridescent color patterns produced by thin film interference, where light that
is reflected off of the upper or lower surface of a clear membrane constructively or
destructively interferes with light approaching the membrane. The perceived color
pattern is primarily caused by the varying thickness of the wing itself, and are, un-
like iridescent colors from butterfly wing scales, static at a range of viewing angles
(Shevtsova et al. 2011).

Wing interference patterns are under-used in species descriptions and as a tool
for species-level identification of small clear-winged insects. Since their discovery as
stable color patterns, they have rarely been reported in taxonomic works and even
less frequently been used in species diagnoses or identification keys. In addition to
discovering them, Shevtsova et al. (2011) comprehensively described the physical phe-
nomenon that causes them and documented examples of WIPs in several Diptera and
Hymenoptera taxa. Since then, WIPs have been documented in just 20 taxonomic
or descriptive works (Hansson 2011; Shevtsova and Hansson 2011; Buffington and
Sandler 2012; Hansson 2012; Hansson and Shevtsova 2012; Hernandez-Lépez et al.
2012; Simon 2012; Stigenberg 2012; Buffington and Condon 2013; Mitroiu 2013;
Buffington and Forshage 2014; Drohojowska and Szwedo 2015; Zhang et al. 2014a,
2014b, 2016; Hosseini et al. 2019, 2020, 2021 2021; Pielowska-Ceranowska and
Szwedo 2020; Butterworth et al. 2021; Conrow and Gelhaus 2022) and five experi-
mental studies (Katayama et al. 2014; Brydegaard et al. 2018; Hawkes et al. 2019;
Dong et al. 2020; White et al. 2021). Most of these studies focus on Hymenoptera
(161 species), followed by Diptera (58 species), Hemiptera (8 species), and Odonata
(1 species) (Suppl. material 1).

Because WIPs are a function of the varying thickness of wings, some authors have
speculated that color may vary intraspecifically because overall wing thickness may
be correlated to individual size (Shevtsova and Hansson 2011; Hernandez-Lopez et
al. 2012). Therefore, they concluded that the colors of WIPs are less important than
the patterns they form. Despite this, the majority of taxonomic works that document
WIPs describe them in terms of qualitative colors and the relative portion of the wing
those colors occupy (e.g., distal 1/3 magenta). Wing interference patterns have been
used as characters in species diagnoses in only three publications to date (Hansson
2011; Shevtsova and Hansson 2011; Hansson and Shevtsova 2012), and have been
used in a taxonomic key only three times (Mitroiu 2013; Zhang et al. 2014b; Hos-
seini et al. 2021). Efforts to quantify and compare WIPs have generally found them to
be species-specific but rarely sexually dimorphic (Hawkes et al. 2019; Hosseini et al.
2019; Hosseini et al. 2020; Butterworth et al. 2021; White et al. 2021). To-date, WIPs
have not been broadly adopted by taxonomists of small insects.

The wing interference patterns of Parapanteles (Braconidae, Microgastrinae) 969

Microgastrinae (Hymenoptera: Braconidae) is a hyper-diverse subfamily of small
parasitoid wasps that attack Lepidoptera (Mardulyn and Whitfield 1999). Microgastri-
nae currently has 2,999 described species (Fernandez-Triana et al. 2020), representing
roughly 5—10% of the estimated worldwide diversity of this group (Rodriguez et al.
2012; Fernandez-Triana and Ward 2015). Their diminutive adult size and small num-
ber of morphological characters have made the generic-level taxonomy of this group
difficult, and species-level diagnoses, absent DNA barcoding, often rely on subtly vari-
able or minute characters that often require SEM imaging to observe (e.g., Valerio et
al. 2009). Wing interference patterns have never been reported for microgastrines but
are readily visible in living wasps in a container (DHJ, WH, personal communication).

Parapanteles Ashmead is a small genus of Microgastrinae with several species that are
morphologically very similar to other genera (Dolichogenidea and Glyptapanteles) and
frequently misdiagnosed (Valerio et al. 2009; Parks et al. 2020). Here, we document
the WIPs of 7 described and 12 putative undescribed Parapanteles species from Costa
Rica and Ecuador and present a simple and inexpensive method for quantifying and
comparing WIPs that can contribute to identification keys and rapid species diagnosis.

Here we present the first study of WIPs in Microgastrinae (Hymenoptera: Braco-
nidae), and an attempt to quantitatively compare the WIPs of closely related species
using materials and methods already common in or freely available to most taxonomic
laboratories that focus on small clear-winged insects.

Methods

Adult wasps used in this study were collected by two long-term Lepidoptera/parasi-
toid rearing projects: Area de Conservacién Guanacaste (ACG) in Costa Rica (Janzen
and Hallwachs 2009, 2016) and Yanayacu Biological Station in Ecuador (Dyer et al.
2017). A list of specimens used in this study is available in Table 1.

One set of fore and hind wings were removed from each adult wasp from samples
stored in ethanol. Where available, wings from one male and one female per brood
were removed and slide mounted on temporary slides. All species sampled are gregari-
ous (i.e. the female lays multiple eggs in a single host) except Parapanteles sp. J and
Parapanteles sp. K, which are solitary (i.e. females lay a single egg per host). We assume
that all wasps eclosing from the cocoons from one caterpillar are siblings. Wings were
sandwiched between two microscope slides which were taped together at the ends.
This flattens wings more reliably than using a standard slide cover. As in Shevtsova and
Hansson 2011, a drop of black India ink was spread on one slide to create a uniform
black background behind the wings.

Wings were photographed at 50x magnification using a Cannon Rebel Xsi camera
and an Amscope LED-144A-YK 144 LED ring light at maximum brightness. Wing
images were not visually adjusted. Materials examined and qualitative descriptions of
WIPs are available in Suppl. material 2. Images used in our analyses are available in

Suppl. material 3.

970 Shuyang Jin et al. / Journal of Hymenoptera Research 96: 967-982 (2023)

Table |. Materials examined for Parapanteles species included in this study from Area de Conservacién Gua-
nacaste (ACG), Costa Rica and Yanayacu Biological Station (YBS), Ecuador. Identification numbers for ACG
specimens reflect voucher codes for COI DNA barcoding sequences on the Barcode of Life Database (BOLD).

Specie IDF

Parapanteles continua ACG, Costa Rica DHJPAR0013724, DHJPAR0013810, DHJPAR0013718,
DHJPARO0013733, DHJPAR0020230, DHJPAR0013716,
DHJPARO0013725, DHJPAR0020228, DHJPARO0013723,
DHJPARO0013717, DHJPAR0020236, DHJPAR0004196,
DHJPAR0004192, DHJPAR0004189, DHJPAR0004190,
DHJPARO0002808, DHJPAR0004798, DHJPAR0005102,
DHJPAR0020859, DHJPAR0020911, DHJPAR0030974,
DHJPAR0020231
Parapanteles em ACG, Costa Rica DHJPARO0004212, DHJPAR0004543, DHJPAR0004535,
DHJPAR0004539, DHJPAR0002757, DHJPAR0020573,
DHJPAR0020466, DHJPAR0020785, DHJPAR0020788,
DHJPARO0020261, DHJPARO0002802
Parapanteles paradoxus ACG, Costa Rica DHJPARO0000248, DHJPAR0012335, DHJPAR0030924,
DHJPAR0004544, DHJPAR0004209, DHJPAR0004534,
DHJPAR0000246, DHJPAR0004194, DHJPAR0004541,
DHJPARO0005103, DHJPAR0004796, DHJPAR0004800
Parapanteles sicpolus ACG, Costa Rica DHJPARO0004542, DHJPAR0000204, DHJPAR0000199,
DHJPARO0004201, DHJPAR0004200, DHJPAR0004537,
DHJPARO0004198, DHJPAR0004187
Parapanteles tessares ACG, Costa Rica DHJPAR0030744, DHJPAR0020916, DHJPAR0030733,
DHJPARO0030762, DHJPAR0020905, DHJPAR0020850,
DHJPAR0020849, DHJPAR0030752, DHJPAR0020904,
DHJPAR0020852, DHJPAR0020857, DHJPAR0030773,
DHJPARO0030975
Parapanteles tlinea ACG, Costa Rica DHJPARO0004188
DHJPAR0020792, DHJPAR0012000, DHJPAR0020574,
DHJPAR0020570, DHJPAR0020568, DHJPAR0020569,

Parapanteles sp. “valerio05” | ACG, Costa Rica

DHJPAR0031011
Parapanteles sp. “B” 45714, 26049, 37474, 20919, 24670
Parapanteles sp. “C” 12105, 45981, 48054
Parapanteles sp. “D” 8275, 35934, 37263, 37275, 37791, 44117
Parapanteles sp. “E” YBS, Ecuador 36197, 36198, 36520
Parapanteles sp. “H” YBS, Ecuador 2365, 2366, 2466, 4503
Parapanteles sp. “1” YBS, Ecuador 42069, 43211, 46466, 66971
Parapanteles sp. “J” YBS, Ecuador 27850, 27851, 34403, 34413, 36533
Parapanteles sp. “K” 28620, 32234, 36406, 36534, 38844

The average RGB (red, green, and blue) values of pixels in each fore wing image
were measured using the “RGB Measure” feature in ImageJ v1.49 (Schneider et al.
2012). The value for each color component was divided by the average of all three
average color values to calculate the relative “redness,” “greenness,” and “blueness” of
each fore wing image (e.g., redness=R/((R+G+B)/3)). This averages out the contribu-
tion of black (R/G/B=0/0/0), white (R/G/B=255/255/255), and grey (R/G/B are all
equal) pixels.

Arrays of relative redness, greenness, and blueness for each species were tested for
normality in R v4.2.2 (R Core Team 2017) using the ‘agricolae’ and ‘nortest’ packages

The wing interference patterns of Parapanteles (Braconidae, Microgastrinae) 971

(Gross and Ligges 2015; de Mendiburu and Yaseen 2020) via the Shapiro-Wilk test and
for skewness, and then compared across species via ANOVA and Tukey’s HSD test and
visualized with geplot2 (Beck 2017). Species with sample size lower than 3 were exclud-
ed from our statistical analysis. Data files and R code are available in Suppl. material 4.

Several metrics of fore wing size were measured to test whether they correlated with
WIP patterns, because if they do then species-level differences in WIPs may simply be
caused by some species being larger than others. Fore wing length (measured from the
junction of C+Sc+R and M+Cu to the distal end of 3/M) and area were compared to
each color array. In addition, overall fore wing shape was measured by dividing length
by width (measured from the junction of r-rs and the stigma to the distal end of the
anal lobe) to test if wing narrowness has any effect on wing thickness. Measurements
were done in Image] v1.49 (Schneider et al. 2012) and tested for correlation via the
Pearson Correlation test in R v4.2.2 (R Core Team 2017) using the ‘hmisc’ package
(Harrell and Dupont 2019).

Linear discriminate function analyses were used to test how useful our quantifica-
tion of microgastrine WIPs were by themselves for identifying species. Linear discrimi-
nation analyses were done in R v4.2.2 (R Core Team 2017). Several subsets of models
were tested, and variables included the relative redness/greenness/blueness values for
both fore wing and hind wing for all species, fore wing only for all species, hind wing
only for all species, fore wing and hind wing data for each subclade containing two
or more taxa, fore wing and hind wing data for species collected in the same country
(Costa Rica or Ecuador), and fore wing and hind wing data for species that attack the
same host family (Erebidae, Geometridae, Notodontidae, or Saturniidae). In each case
50% of the dataset was used to train the model and 50% of the dataset was used for
validation. R code and data files are available in Suppl. material 4.

Results

Inter- and intraspecific variation in WIPs

The wing interference patterns of the species surveyed are generally consistent within
species, although intraspecific consistency does vary. Both qualitatively (Suppl. mate-
rial 3) and in terms of relative redness, greenness, and blueness (R.RGBs) (Table 2,
Fig. 1), the species with purplish WIPs (Parapanteles tessares, P. continua, P. sicpolus, and
P sp. H) have the most consistent WIPs, while species with reddish or yellowish WIPs
are more variable, especially Parapanteles sp. J and Parapanteles sp. K.

All R.RGB arrays were normally distributed except two P continua arrays, one
Parapanteles sp. E, one P paradoxus, one P sicpolus, and four P tessares arrays (Table 2).
The distributions of fore wing and hind wing R.RGBs among closely related species are
often similar with one or two parameters significantly different (Fig. 1). For example,
the R.RGBs of the sister species P tessares and P continua are not significantly differ-
ent except for fore wing relative redness (higher in P continua) and relative blueness

Shuyang Jin et al. / Journal of Hymenoptera Research 96: 967-982 (2023)

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The wing interference patterns of Parapanteles (Braconidae, Microgastrinae) O73

P. tessares P. continua P. sicpolus P.sp.H P.sp.D P.em _ P.sp.valerio05 P. paradoxus_P. sp. | P.sp. J P.sp.K P.sp.E P.tlinea P.sp.B P.sp.C

Figure |. Box-and-whiskers plots of forewing and hind wing wing interference pattern relative red-
nesses (RR), greennesses (RG), and bluenesses (RB) shown in phylogenetic order. The cladogram above
the figure is based on results from Parks et al. 2020. RR box-and-whiskers are shown in red, RB in blue,
and RG in green (for colorblind: all RR values are greater than their corresponding RG values, so all red
box-and-whisker plots are above green box-and-whisker plots in the figure). Results of Tukey’s HSD test
are displayed above or below each box-and-whisker. The white horizontal bar below each wing image

represents 2 mm.

(higher in P tessares), which corroborates the more uniformly purple appearance of
P tessaress WIP.

We did not find evidence of sexual dimorphism in Parapanteles WIPs. Males and
females of most species have similar WIPs, although in Parapanteles sp. D and P em
male WIPs are slightly more yellowish (Suppl. material 3: f and g.). Sexual dimorphism
could not be assessed for 6 species: only females were available for Parapanteles sp. C, sp.
J, sp. K, and sp. Valerio05, and only males were available for Parapanteles sp. land sp. E.

Relative redness, greenness, and blueness and wing size

The majority of R.RGB arrays were not significantly correlated with wing length,
area, or shape. Eleven of the 33 R.RGB tested were significantly correlated with wing
length and 8 of 33 were significantly correlated with wing area. In each case the slope
of the line of regression was slight and no R.RGB arrays were correlated with wing

shape (Table 3).

974 Shuyang Jin et al. / Journal of Hymenoptera Research 96: 967-982 (2023)

Table 3. Average length, area, and shape (length/height) of the fore wings of fifteen Parapanteles species
plus or minus one standard deviation, with coefficient of determination and the p-value of Pearson cor-

relation tests of each measurement for each fore wing color array (relative redness (RR), greenness (RG),

and blueness (RB)).

Species n Fore wing Average */RRr p */RGr p */RBr p
measurement
Parapanteles continua 41 Length* (mm) 2.5 £0.18 0.09 0.06 0.16 0.01 0.29 0.00
Height (mm) 0.67 + 0.05 _ - - - - -
Area* (mm7) 0.67 + 0.05 0.06 0.13 0.15 0.01 0.24 0.00
Shape* (L/H) 3.76 + 0.13 0.01 0.56 0.01 0.51 0.03 0.33
Parapanteles em 16 Length* (mm) 2.36 + 0.21 0.45 0.00 0.79 0.00 0.29 0.03
Height (mm) 0.64 + 0.06 _ - - - - -
Area* (mm7) 0.64 + 0.06 0.42 0.01 0.85 0.00 0.35 0.02
Shape* (L/H) 3.71 £0.14 0.00 0.84 0.01 0.71 0.03 0.55
Parapanteles paradoxus 16 Length* (mm) 2.36 + 0.21 0.08 0.28 0.04 0.49 0.01 0.76
Height (mm) 0.62 + 0.05 - - - - - -
Area* (mm7) 0.62 + 0.05 0.08 0.28 0.12 0.19 0.00 0.92
Shape* (L/H) 3.81 + 0.23 0.00 0.88 0.01 0.73 0.01 0.73
Parapanteles sicpulus 14 Length* (mm) 2.74 = 0.13 0.24 0.08 0.07 0.34 0.24 0.07
Height (mm) 0.74 = 0.04 _ - - _ — -
Area* (mm?) 0.74 = 0.04 0.25 0.07 0.04 0.47 0.23 0.08
Shape* (L/H) 3.7 +£0.18 0.03 0.53 0.03 0.53 0.04 0.47
Parapanteles sp. B 8 Length* (mm) 2.11 240.1 0.00 0.87 0.86 0.00 0.49 0.05
Height (mm) 0.51 + 0.03 ~ - - _ - —
Area* (mm7) 0.51 + 0.03 0.08 0.49 0.69 0.01 0.28 0.18
Shape* (L/H) 4.16+0.15 0.37 0.11 0.04 0.65 0.18 0.30
Parapanteles sp. D 10 Length* (mm) 3.59 + 0.15 0.00 0.95 0.66 0.00 0.48 0.03
Height (mm) 0.93 + 0.06 = = = = =
Area* (mm?) 0.93 + 0.06 0.01 0.79 0.76 0.00 0.49 0.02
Shape* (L/H) 3.88 + 0.14 0.00 0.98 0.13 0.30 0.10 0.36
Parapanteles sp. H 9 Length* (mm) 3.11 + 0.47 0.06 0.53 0.00 0.88 0.01 0.82
Height (mm) 0.82 + 0.13 — - — — — —
Area* (mm7) 0.82 + 0.13 0.06 0.51 0.00 0.00 0.02 0.72
Shape* (L/H) 3.81 + 0.08 0.10 042 0.37 0.08 0.34 0.10
Parapanteles sp. J 5 Length* (mm) 2.96 = 0.21 0.02 0.82 0.02 0.83 0.00 0.95
Height (mm) 0.78 = 0.07 - - - - - -
Area* (mm7) 0.78 + 0.07 0.03. 0.77 0.03 0.77 0.00 0.93
Shape* (L/H) 3.81 + 0.12 0.62 0.11 0.01 0.85 0.16 0.50
Parapanteles sp. K 5 Length* (mm) 2.66 = 0.47 0.62 0.11 0.02 0.83 0.74 0.06
Height (mm) 0.7 £0.12 - - - - - -
Area* (mm?) 0.7 £0.12 0.09 0.16 0.02 0.82 0.61 0.12
Shape* (L/H) 3.81 + 0.17 016 O57 0:05 0.77 0.29: _0.35
Parapanteles sp. valerio05 7 Length* (mm) 2.4 +£0.13 0.07 0.58 0.48 0.09 0.10 0.48
Height (mm) 0.62 + 0.04 - - — — — -
Area* (mm7) 0.62 + 0.04 0.02 0.77 055 0:06 0.18 0.35
Shape* (L/H) 3.87 + 0.2 0.30 0.20 0.10 049 0.25 0.25
Parapanteles tessares 25 Length* (mm) 2.33 = 0.09 0.04 0.37 0.18 0.03 0.18 0.03
Height (mm) 0.61 + 0.04 — - _ - — —
Area* (mm7”) 0.61 + 0.04 0.00 0.89 0.13 0.08 0.07 0.19
Shape* (L/H) 3.83 + 0.15 0.00 0.91 0.00 0.73 0.00 0.78

The wing interference patterns of Parapanteles (Braconidae, Microgastrinae) 975

Linear discriminate function analysis

Results for linear discriminate function analyses varied widely and are available in
Suppl. material 4. Linear discriminate function analysis using our complete dataset
predicted species accurately only 34% of the time, but was more accurate with some
subsets of species separated by subclade, geography, or host use (e.g., species prediction
of species found in Costa Rica was 83% and species parasitizing saturniids was 75%).

Discussion

‘The wing interference patterns of Parapanteles are consistent within species and distinct
between species, often enough to be diagnostic by themselves. Among the species sur-
veyed, the WIPs of Parapanteles tessares, P continua, P sicpolus, P sp. H, and P sp. C
were the most distinct. These species tended to have more green and purple in their
WIPs, while the remaining species’ WIPs were predominantly red and/or yellow.
Wing interference patterns are directly related to the thickness of wing membranes,
and previous publications have speculated that WIP colors should change as individu-
als get larger because cuticle thickness may increase with body size (Shevtsova and
Hansson 2011; Hernandez-Lépez et al. 2012). We are not aware of any studies investi-
gating the allometry of body or wing cuticle thickness. Among the species we surveyed,
some relative redness, greenness, and/or blueness arrays were significantly correlated
with wing size and/or area in some species, but in each of these cases the slope of the
corresponding linear regression was very slight (Table 3). Correlation with wing size
(as a proxy for body size) alone does not account for the differences between the WIPs
of closely related Parapanteles species. We were not able to use WIPs alone to reliably
predict the identity of an unknown specimen from a large number of species, but were
able to discriminate between species in some subclades or subsets of species defined by
location or host use (Suppl. material 4). Wing interference patterns are not likely to
be useful for automated species identification for many taxa, but are useful as an ad-
ditional and generally overlooked morphological character to be used in conjunction
with other characters for species diagnosis, as any morphological character tradition-
ally would be. When viewed this way they are often one of the most conspicuous and
accessible morphological characters of the physically small taxa on which they appear.
Wing interference patterns are directly related to the wavelength of the light pass-
ing through the wing membrane, which is a major weakness for using any measure-
ment derived from RGB values for diagnostic purposes. The relative RGB values we
measured in this study were not consistent if the wing was illuminated with a different
light source. This limitation can be solved by using a consistent light source, and the
light source which we used for all WIP photographs in this study, an Amscope LED-
144A-YK 144 LED ring light, is widely available and relatively inexpensive. Using one

or more lasers of specific wavelengths to illuminate WIPs could offer a more replicable

976 Shuyang Jin et al. / Journal of Hymenoptera Research 96: 967-982 (2023)

and standardizable method for documenting WIPs, although using one or a few wave-
lengths would result in less data than full spectrum white light. Wing interference pat-
terns can be observed in situ on pinned specimens, but these are of little use compared
to WIPs observed on slide-mounted wings. Including WIP slides (wing slides with
India Ink painted on the back) of at least a few paratype individuals with the type se-
ries of small winged insects would ameliorate most of the problem posed by variations
between light sources, and expand the usefulness of WIPs for future studies.
Experiments in Drosophila have repeatedly shown WIPs to be subject to sexual
selection (Katayama et al. 2014; Hawkes et al. 2019). While this has not been ex-
perimentally tested in other taxa, this and other studies have found that WIPs are fre-
quently species-specific (Shevtsova et al. 2011; Buffington and Sandler 2012; Zhang et
al. 2014b, 2016; Hosseini et al. 2019; Butterworth et al. 2021; Hosseini et al. 2021).
Similarly to the Drosophila species used in the sexual selection experiments, microgas-
trinae males also display their wings to females during courtship (Bredlau and Kester
2019). The colors of WIPs are visible in situ and in natural settings whenever insect
wings are displayed in front of a dark background (e.g. green leaves), and the colors
that compose them occur in spectra visible to most insects (Shevtsova et al. 2011;
Brydegaard et al. 2018; Butterworth et al. 2021). Anecdotally, we found that closely
related sympatric species tended to be more subjectively different (i.e. ((Parapanteles
tessares, P continua), P. sicpolus) and (P. em, P valerio05) from Costa Rica and (P sp.
B, P sp. C) from Ecuador), while closely related allopatric species tended to be less
distinct (i.e. (P paradoxus, P sp. I) and (P sp. E, P tlinea) (Fig. 2). This suggests that
WIPs may be used by microgastrines for conspecific recognition, but this is entirely
speculative and would require a broader survey of microgastrine WIPs to test. We only
included two solitary species (i.e. females oviposit a single egg into each host, P sp. J
and P sp. K) in our study. These two species had the most variable WIPs and wing
sizes. The relationship to host quality and adult wasp size may be more direct in solitary

Parapanteles tessares Parapanteles continua Parapanteles sicpolus Parapanteles paradoxus Parapanteles sp. | Parapanteles sp. | Parapanteles sp. K

Figure 2. Right wings of three different individuals from seven Parapanteles species showing wing inter-
ference patterns. A shows three gregarious sympatric sister species ((P tessares, P continua), P sicpolus) from
Area de Conservacién Guanacaste (ACG) in Costa Rica. B shows two gregarious allopatric sister species,
one from AVG (P paradoxus) and one from Yanayacu Biological Station in Ecuador (P sp. I). C shows two

solitary sister species from Yanayacu Biological Station.

The wing interference patterns of Parapanteles (Braconidae, Microgastrinae) Di,

species that use small host caterpillars than gregarious species attacking larger caterpil-
lars. In such solitary species, poor quality hosts may have less resources available for
parasitoids and result in smaller adults, while gregarious species can oviposit fewer eggs
to account for poor quality hosts which may result in more consistent adult wasp sizes.
Even so, P sp. J fore wings are significantly redder than P sp. K (Figs 1, 2).

Conclusions

In general, WIPs can be observed and documented with very little additional effort
for most taxonomists who work on small winged insects. We predict that they can
be a large source of new morphological characters for the taxonomy and systematics
of these tiny animals. The only materials required are a dissecting microscope with a
camera attachment, a ring light, glass slides, and India Ink. Wing interference patterns
are often species-specific and useful for Parapanteles wasps, and will likely be for most
other microgastrine wasps.

Acknowledgments

This work was supported in part by the National Science Foundation grant DEB
1146119 and DEB 1442103. We thank the National Institute of Biodiversity — Ec-
uador (INABIO) for support and the Ministry of the Environment of Ecuador for
providing permits under the genetic access contract MAE-DNB-CM-2016-0045
and the project “Interacciones entre plantas, orugas, y parasitoides de los Andes del
Ecuador.” All specimens were collected, exported and to be DNA barcoded under
Costa Rican government permits issued to BioAlfa (Janzen and Hallwachs 2019) (R-
054-2022-OT-CONAGEBIO; R-019-2019-CONAGEBIO; National Published De-
cree #41767), JICA-SAPI #0328497 (2014) and DHJ and WH (ACG-PI-036-2013;
R-SINAC-ACG-PI-061-2021; Resolucié6n N°001-2004 SINAC; PI-028-2021). We
thank the GDFCF/ACG parataxonomist team of Area de Conservacion Guanacaste,
northwestern Costa Rica for finding and rearing the caterpillar hosts of these parasites,
which are documented individually by their DHJPAR..... voucher codes at http://

janzen.sas.upenn.edu/caterpillars/database.lasso.

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The wing interference patterns of Parapanteles (Braconidae, Microgastrinae) 981

Supplementary material |

Taxonomic summary of published wing interference pattern images and/or

descriptions

Authors: Shuyang Jin, Kyle S. Parks, Daniel H. Janzen, Winnie Hallwachs, Lee A.

Dyer, James B. Whitfield

Data type: xlsx

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/jhr.96.111382.suppl1

Supplementary material 2

Qualitative descriptions and materials examined for Parapanteles species included

in this study

Authors: Shuyang Jin, Kyle S. Parks, Daniel H. Janzen, Winnie Hallwachs, Lee A.

Dyer, James B. Whitfield

Data type: docx

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/jhr.96.111382.suppl2

982 Shuyang Jin et al. / Journal of Hymenoptera Research 96: 967-982 (2023)

Supplementary material 3

Wing interference patterns

Authors: Shuyang Jin, Kyle S. Parks, Daniel H. Janzen, Winnie Hallwachs, Lee A.

Dyer, James B. Whitfield

Data type: zip

Explanation note: Wing interference patterns of Parapanteles tessares (a), PR continua
(b and c), P sicpolus (d), P sp. H (e), P sp. D (f), PB em (g), P sp. valerio05 (h),
P. paradoxus (i), P sp. I (j), P sp. J (k), P sp. K (1), 2 sp. E (m), P tinea (n),
P sp. B (0), and P sp. C (p). Female wings are shown to the left and males to the
right. Horizontal pairs of wing images are of sibling wasps from the same reared
brood while each vertical set is from a distinct brood.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/jhr.96.111382.suppl3

Supplementary material 4

Data files and R code

Authors: Shuyang Jin, Kyle S. Parks, Daniel H. Janzen, Winnie Hallwachs, Lee A.

Dyer, James B. Whitfield

Data type: zip

Explanation note: Data files and R code used to calculate mean, standard deviation,
ANOVA, Tukey’s HSD, Skewness, Shapiro-Wilks normality test, and linear dis-
criminate functions analysis of forewing and hindwing relative redness, greenness,
and blueness, and Pearson's correlation of forewing length, forewing area, and fore-
wing shape (H/L) to forewing relative redness, greenness, and blueness.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/jhr.96.111382.suppl4