Dissertations in Forestry and Natural Sciences
DISSERTATIONS | BADEJO OLUWATOBI ABAYOMI | CLIMATIC AND ENVIRONMENTAL EFFECTS ON PHENOTYPIC VARIATION... | No 449
BADEJO OLUWATOBI ABAYOMI
Climatic and
environmental
effects on Phenotypic
variation in common
wasp Vespula vulgaris
CLIMATIC AND ENVIRONMENTAL EFFECTS ON PHENOTYPIC VARIATION IN COMMON WASP VESPULA
VULGARIS
Badejo Oluwatobi Abayomi
CLIMATIC AND ENVIRONMENTAL EFFECTS ON PHENOTYPIC VARIATION IN COMMON WASP VESPULA
VULGARIS
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 449
University of Eastern Finland Kuopio
2021
Academic dissertation
To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium CA101 in the Canthia Building at the University of Eastern Finland, Kuopio, on
December, 8, 2021, at 12 o’clock noon
Punamusta Oy Joensuu, 2021 Editor: Pertti Pasanen
Distribution: University of Eastern Finland / Sales of publications www.uef.fi/kirjasto ISBN: 978-952-61-4398-9 (nid.)
ISBN: 978-952-61-4399-6 (PDF) ISSNL: 1798-5668
ISSN: 1798-5668 ISSN: 1798-5676 (PDF)
Author’s address: Oluwatobi Badejo
University of Eastern Finland
Department of Environmental and Biological Sciences P.O. Box 1627
70211 KUOPIO, FINLAND.
email: oluwatobi.badejo@uef.fi
Supervisors: Adjunct Professor Jouni Sorvari, Ph.D.
University of Turku Department of Biology 20014 TURKU, FINLAND.
email: jouni.sorvari@utu.fi
Research Director Arto Koistinen, Ph.D.
University of Eastern Finland SIB Labs
P.O. Box 1627
70211 KUOPIO, FINLAND.
email: arto.koistinen@uef.fi
Dr. Oksana Skaldina, Ph.D.
University of Eastern Finland
Department of Environmental and Biological Science P.O. Box 1627
70211 KUOPIO, FINLAND.
email: oksana.skaldina@uef.fi
Reviewers: Dr. Jukka Suhonen, Docent.
University of Turku Department of Biology 20014 TURKU, FINLAND.
email: jukka.suhonen@utu,fi Dr. Marko Mutanen, Docent.
University of Oulu
Ecology and Genetics Research Unit P.O. Box 8000
90014 OULU, FINLAND.
email: marko.mutanen@oulu.fi
Opponent: Professor Toomas Tammaru, Ph.D.
University of Tartu
Institute of Ecology and Earth Sciences 51014 TARTU, ESTONIA.
email: toomas.tammaru@ut.ee
Badejo, Oluwatobi A.
Climatic and environmental effects on phenotypic variation in common wasp Vespula vulgaris.
Kuopio: University of Eastern Finland, 2021 Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2021 ISBN: 978-952-61-4398-9 (print)
ISSNL: 1798-5668 ISSN: 1798-5668
ISBN: 978-952-61-4399-6 (PDF) ISSN: 1798-5676 (PDF)
ABSTRACT
Insects are ecologically important organisms as they regulate different processes within an environment. Social hymenopterans are specifically important because of their ability to control population of agricultural pests, pollination activities, and improvement of soil properties. The common wasp Vespula vulgaris is a social hymenopteran that is native to Finland and Europe but invasive in other parts of the world (e.g. Australia, New Zealand, and North America). The ability of this insect species to survive outside the native range involves modification of life cycle, morphology, and phenotypic properties. In the face of climate change and environmental alteration due to urbanization, industrial pollution, and other anthropogenic impacts, phenotypic changes and other modifications are expected to arise in this species within the native range.
Therefore, in this thesis I studied the effect of climate change using variation in latitude and weather condition of different cities on the growth (body size) and colouration (melanisation degree) in V. vulgaris. The pigmentation in insects is an important property that can be modified to improve thermoregulation and immunity. Secondly, I assessed the impact of urban heat island (UHI) on the expression of melanic pigmentation, the relative proportion of black and yellow pigmentation on the 2nd abdominal tergite, morph selection and structure of the cuticle using scanning electron microscopy (SEM). Finally, the impact of pollution on growth and morph selection was studied, and the internal mechanism of encapsulation of ingested pollutants (heavy metals) was assessed using transmission electron microscopy (TEM).
The result showed that the growth of the common wasps is location-specific and variation in body size within a geographical location (i.e. city) was only observed in a city exposed to metal pollution. The melanisation degree differed spatially across the different locations following
thermal melanism theory (in colder environments) and desiccation avoidance theory (in warmer environments). The SEM analysis of the yellow portion showed xanthopterin granules which are expected to assist the wasps in storing heat energy and aid thermoregulation of wasps in cold weather conditions. This showed the importance of the relative proportion of area of yellow and black pigmentation in thermoregulatory function in wasps, the morphs were grouped based on the proportion of both pigments on the 2nd tergite. The morph selection across the urbanization and pollution gradient showed relative association with the thermoregulatory needs for the location. The TEM analysis showed evidence of deployment of melanin for encapsulation of ingested metal pollutants, iron and nickel were identified within the midgut of wasps collected from the polluted zone. The overall results of the thesis demonstrate the importance of the complex assessment of wasps’ phenotypic plasticity during environmental surveys.
Universal Decimal Classification: 504.5, 504.7, 574.9, 591.128, 591.157, 595.798
CAB Thesaurus: climate change; pollution; pollutants; heavy metals; social insects;
Hymenoptera; Vespula vulgaris; growth; size; colour; colour patterns; pigmentation;
melanism; animal cuticle; thermoregulation; geographical distribution; latitude; cities;
weather; electron microscopy
Yleinen suomalainen ontologia: ilmastonmuutokset; saastuminen; saasteet; raskasmetallit;
yhteiskuntahyönteiset; ampiaiset; kasvu; koko; väri; pigmentti (biologia); lämmönsäätely;
paikka; leveyspiirit; kaupungit; sää; elektronimikroskopia
ACKNOWLEDGEMENTS
I am grateful to God for the gift of life and the opportunity to complete this thesis. I am particularly grateful to my main supervisor Dr. Jouni Sorvari (Adjunct Prof.) for his guidance and commitment in the past five years of working on this thesis. I am also eternally grateful to my other supervisors; Dr. Arto Koistinen and Dr. Oksana Skaldina, their encouragement and support is immeasurable.
I wish to thank members of the Department of Environmental and Biological Sciences of the University of Eastern Finland (Kuopio), SIB Labs and the Faculty of Science and Forestry for the opportunity to use their facility during the research project. My appreciation also goes to the members of our research group in University of Eastern Finland and University of Turku.
I express my most sincere thanks to my extended family; especially my mother, my aunt (Toyin Ogundeji) and my siblings. My special gratitude goes to my wife Ozioma Badejo and my daughter Diekololaoluwa Chikaima Badejo for their support and tolerance throughout the period of making this thesis. Thanks to other friends and family that provided moral support during this period.
Finally, I thank Finnish Entomological Society, Societas pro Fauna et Flora Fennica, KLYY/Betty Väänänen fund and Alfred Kordelin Foundation for supporting this thesis with funds.
LIST OF ABBREVIATION HW Head width
ITD Intertegular distance MD Melanisation degree
MAf Melanised area on the Clypeus/wasp face MAb Melanised area on the 2nd abdominal tergite SEM Scanning electron microscope/microscopy TEM Transmission electron microscope/microscopy TW Thorax width
UHI Urban heat island UV Ultra-violet radiation
LIST OF ORIGINAL PUBLICATIONS
The thesis is a summary of four published papers which are referred to in the text as I – IV, the articles I - IV are reproduced with the permission of the publishers.
I. Badejo O., Skaldina O., Gilev A., Sorvari J. (2020) Benefit of insect colours: a review from social insect studies. Oecologia 194: 27–40. doi: 10.1007/s00442-020- 04738-1.
II. Badejo O., Skaldina O., Sorvari J. (2018) Spatial and temporal variation in thermal melanism in the aposematic common wasp (Vespula vulgaris) in northern Europe.
Annales Zoologici Fennici 55: 67-78.doi: 10.5735/086.055.0107.
III. Badejo O., Leskinen J.T.T., Koistinen A., Sorvari J. (2020) Urban environment and climate condition-related phenotypic plasticity of the common wasp Vespula vulgaris. Bulletin of Insectology 73 (2): 285-294.
IV. Badejo O., Skaldina O.,Peräniemi S., Navarro V.C., Sorvari J. (2021)Phenotypic plasticity of common wasps in an industrially polluted environment in southwestern Finland. Insects 12 (10): 888. doi: 10.3390/insects12100888.
AUTHOR’S CONTRIBUTION
The thesis has one review article I and three original research articles (II, III and IV). I contributed to the writing of the article I and all modifications during the review process. I was involved in the processing of all wasp samples and data collections for articles (II – IV), I took the pictures of all assessed parts of the samples, measured all parameters used in the thesis and took part in the data analysis.
I was involved in sample preparation for SEM analysis and microscopy sessions (III) and for IV, I was involved in the TEM imaging sessions. All authors contributed to the drafting and editing of the manuscripts and the major sample collection was done by JS while OS collected samples for TEM.
CONTENTS
ABSTRACT ... 5
ACKNOWLEDGEMENTS ... 7
LIST OF ABBREVIATION... 8
LIST OF ORIGINAL PUBLICATIONS ... 9
AUTHOR’S CONTRIBUTION ... 10
1 INTRODUCTION... 12
1.1 Background ... 12
1.2 Phenotypic variation in insects ... 14
1.3 Climatic variation and insects ... 15
1.4 Ecological functions of eusocial insects ... 16
1.5 Pigmentation of Vespula vulgaris ... 17
1.6 Aim of the study ... 18
2 MATERIAL AND METHODS ... 19
2.1 Study area and sample collection ... 19
2.2 Digital photography, SEM and TEM imaging ... 20
2.3 Image analysis ... 21
2.4 Data analysis ... 22
3 MAIN RESULTS AND DISCUSSION ... 23
3.1 Body size (ITD) variation across regions, urbanization gradient and pollution gradient. ... 23
3.2 Variation in melanisation degree across regions ... 24
3.3 Xanthopterin layer and colour morphs ... 25
3.4 Yearly variation in wasps colour morphs ... 29
3.5 Pollution mediated melanisation and phenotypic variation ... 30
4 CONCLUSIONS ... 33
5 REFERENCE ... 34
ARTICLES……… 45
1 INTRODUCTION 1.1 Background
Environmental modification occurs through natural and anthropogenic means. Insects are mostly affected by such modifications because of their reliance on the environment for food and coordination of physiological activities. One of the major physiological activities in insect is thermoregulation, insects are mostly ectothermic organisms while few exhibits some level of endothermy. Endothermy is the ability to regulate body temperature irrespective of the environmental temperature, and this type of thermoregulation has been reported in some hymenopteran insect species (Weiner et al. 2010; Kovac and Stabentheiner 2012; Giménez Gómez et al 2020). Ectothermic insects depend on environmental temperature for their thermoregulation and changes in the ambient temperature can prompt different physiological response in such insect species. Insects can respond through change in cycle stages, population growth or metabolism (Khaliq et al. 2014).
Climate change is a long-term consequence of environmental alteration, it occurs mainly due to unsustainable utilization and consumption of natural resources (Warner et al. 2009; Ahmed et al. 2018). Different activities that contribute to changes in climatic conditions include different land use activities like logging, agricultural operations, industrialization, urbanization and so on. Gaseous emissions from different industrial activities have strong links to ozone layer depletion and this increases the intensity of radiation from the sun on the earth surface.
Uncontrolled logging has also reduced the ecological function of trees in trapping carbon emitted from various industrial activities, this provides more catalyst for ozone depletion and disruption of the hydrology of different locations. Thus, causing changes in rainfall pattern and ambient temperature (Mgbemene et al. 2016) of different locations across the world.
Urbanization and habitat fragmentation are important products of civilization that has terrible immediate consequence on species distribution and abundance. Extinction of valuable insect species have been attributed to urbanization (Fattorini 2011), disappearance of agricultural activities and replacement of green areas with urban structures have adverse effect on survival of insect species. Insect species that survive and cope significantly in urbanized ecosystems are typically generalist species while species associated with specific habitats rapidly decline in abundance in urban cores (Crowther et al. 2014). Spatial isolation of suitable biotopes contributes greatly to changes in life cycle and growth of insects (Fattorini 2011). Insects that survive in urban zones modify their life cycle to grow in different patches of biotopes that can
support their growth at each stage of their life cycle. This kind of life cycle modification involves internal migration within urban zones which can expose the insect to fatal collision with urban structures and activities. Also, reproductive isolation within species in ecosystems that are highly modified and fragmented can result in production of unfit offspring due to continuous inbreeding within urban population.
Insect predator density increase is a major attribute of urban centers (McCabe et al. 2018) due to disruption of natural communities and reduction in abundance of top predators. Birds and other insectivorous animals increase in urban areas because of easy availability of food, nesting location and adoption as pets by urban dwellers (personal observation). Birds like Mississippi Kite (Ictinia mississippiensis), Northern Goshawks (Accipiter gentilis) and peregrine falcon (Falco peregrinus) have shown high nesting success and abundance in urban environments (Welch and Boal 2015; Kettel et al. 2019; Solonen et al. 2019). The presence of such predators alters the foraging behavior and distribution of insect species within urban environment.
Pollution is also associated with urbanization; light, noise, chemical and other forms of pollution actively exist in urban centers and anthropogenically altered ecosystems. Artificial light at night (ALAN) affects the foraging orientation of nocturnal insects and expose them to predators at night in urban areas, such opportunistic predators include orb‐web spiders (Willmott et al. 2019) and bats (Minnaar et al. 2015). Noise pollution have been indicted in reduction in signaling and development of sexual properties in insects (Bowen et al. 2020), noise also interfere with intraspecific communication in insects (Lampe et al. 2012; Gallego- Abenza et al. 2020). Chemical pollution in urban environment comes from industrial and mechanical operations associated with urban life. Industrial effluents and gaseous emission can contaminate food sources (Shi et al. 2020) and penetrate the insect community through uptake with food or gaseous exchange processes. This leads to bioaccumulation of chemical pollutants in insects with adverse effect on their growth and other physiological activities, e.g., heavy metals like Zinc (Zn) affects fertility in female ladybird (Shi et al. 2020) and Cadmium (Cd) causes growth retardation in moths and other insects (Jiang et al. 2020; Yu et al 2020). Other metals that can potentially bioaccumulate in insects includes arsenic (As), cobalt (Co), copper (Cu), iron (Fe), lead (Pb) and mercury (Hg) (Eeva et al. 2004; Butt et al. 2018; Skaldina et al.
2020).
Nano- and micro-plastics are also emerging as a major threat to animals in both terrestrial and aquatic ecosystems, insects being widely distributed across both environments are exposed to
these pollutants. These plastic residues have been identified in midguts of insects (Parenti et al. 2020) and associated with erratic movements and fitness defects. The combination of environmental modification and changing climatic conditions in urban centers can prompt various adaptations in insect species.
1.2 Phenotypic variation in insects
Phenotypic variations occur when there are intraspecific differences in appearance and morphology in animal species. Climate change has been identified as an important driver of phenotypic variation in different insects (Zeuss et al. 2014; Juhász et al. 2016). Such variations occur mostly with expression of colouration, variation in size and body parts in insects. Colour variation have been used to explain different thermoregulatory and adaptive theories in response to climate change. The major theories are thermal melanism theory and melanin- desiccation theory. Thermal melanism theory proposes higher degree of melanisation in insects from colder environment (Clusella-Trullas et al. 2007) because insects with higher expression of melanin pigmentation heat up faster and can survive under lower environmental temperatures. However, as the temperature is predicted to get warmer globally, paler phenotypic expression is expected to increase within insect species (Zeuss et al. 2014). The paler or brighter phenotypes will reduce possibility of overheating in the face of climate change but at high cost of increased predation.
On the other hand, melanin desiccation theory attributes selection of melanic pigmentation in insects to the need to prevent water loss through evaporation in response to elevated environmental temperature (Rajpurohit et al. 2016; Law et al., 2019). Therefore, different selective pressure can prompt phenotypic variation in different environments. Other environmental challenges that can impose phenotypic selection in insects are the need for protection against predator through cryptic and aposematic colouration, as well as disease and parasitic resistance (Armitage and SivaJothy 2005; Stevens and Merilaita 2009; Dubovsiy et al. 2013; Evison et al. 2017). Aposematic colouration involves the use of bright colours in advertisement of unpalatability to potential predators, this property has been reported in different species of insects (Prudic et al. 2007; Svádová et al. 2009; Fabricant and Heberstein 2015; Rojas et al. 2018) and can also be mimicked by other animals to benefit from the defense advantage.
Genetics can also drive phenotypic selection within insect population. In Drosophila melanogaster, chromosomal inversion has been attributed to intraspecific variation in eye size
and wing size (Rako et al. 2006; Reis et al. 2020) forming different phenotypes based on eye and wing morphology. Heritability of different phenotypic colouration occur in club-legged grasshopper Gomphocerus sibiricus, the green-brown polymorphism in which the green allele is mostly dominant over the brown allele when there is crossbreeding between the two morphs (Schielzeth and Dieker 2020). The authors acknowledged the action of genetic modifiers in the expression of dominance of the green allele and this also points to the possibility of brown morphs carrying green alleles recessively which now becomes dominant in their offspring.
Most grasshoppers across the world also have a green-brown polymorphism system, studies have linked this phenotypic variation to its ecological relevance in protection of this group of insects from predators within the environment (Bond 2007).
1.3 Climatic variation and insects
Different locations experience changes in climatic conditions, these changes are mostly observed as unpredictable pattern of weather condition over time. The most important weather parameters that are used to monitor climate of a place are rainfall and temperature. As stated earlier, temperature is important for thermoregulation in insects while prevailing rainfall pattern has also been linked to nesting and foraging success in some insect species (Dejean et al. 2010, 2011).
Insects’ response to climate change have been reported and discussed by different researchers (Pawlikowski and Pawlikowski 2009; Robinet and Roques 2010; Kellermann and van Heerwaarden 2019) and the mechanisms deployed into adaptation varies amongst insect species. Some wasp species modify their phenology to cope with climate change, reduction in the flight and foraging period have been identified as an adaptation to changing climate in Vespula and Dolichovespula wasp species (Pawlikowski and Pawlikowski 2009). In Polistes paper wasps, expression of high melanic pigmentation was discovered in association to cold climatic conditions (De Souza et al. 2016). Climatic variation is also a major attribute of locations with different levels of elevation, Agelaia pallipes paper wasp showed higher degree of melanisation of the thorax at locations with higher elevation (De Souza et al. 2020).
Although the authors suggested that increased exposure to UV radiation at higher elevation might prompt increased melanisation for photoprotection, but they provided superior arguments for climate induced phenotypic variation.
Other insect species respond to climate change induced elevated temperature with reduction in body size (Polidori et al. 2020). The authors have associated elevated temperature with
reduction in developmental time and quick emergence of insects which can be responsible for the reduction in adult size. These can cause different fitness problems in the insects ranging from deficiency in competitive interactions to decline in foraging efficiency (Arendt 2007;
Jourdan et al. 2019; Polidori et al. 2020).
Climate change increase success of biological invasion, this can happen through natural range expansion or deliberate introduction of exotic species. Increased temperature and changes in hydrology of locations can cause range shift of insect species into new favorable ecosystems bringing new species or exit of native species. Such migration destabilizes the community structure of invaded ecosystem prompting competition with native species, causing introduction of new pests and predators. Most invasive alien species have higher tolerance range of environmental parameters and are generalist species that can easily become dominant and suppress the population of native species. This thesis study insect (Vespula vulgaris) has been classified as an invasive species in different parts of the world (Donovan 1984; Beggs et al. 1998; Mathews et al. 2000; Lester and Beggs 2019) but native to Europe and Asia (Archer 1989; Dvorak 2007; Lester et al 2014). Other known invasive insect species include – German wasps Vespula germanica, little fire ant Wasmania auropunctata, cabinet beetle Trogoderma granarium, Gypsy moth Lymantria dispar and silver leaf whitefly Bemisia tabaci (McLaughlin and Dearden 2019).
1.4 Ecological functions of eusocial insects
Insects perform many ecosystem services as pollinators, ecological engineers, plant pests, vectors of disease, predators and so on. Eusocial insects are particularly important because of the specialization of duties within their population and differentiation into castes to work for the collective success of their colony (Mateus et al. 2019; Sun et al. 2020; West and Purcell 2020). Eusociality occur mostly in Hymenopterans (ants, bees, and wasps) but have been reported in Isoptera (termites). The castes are divided into the reproductive and workers (Yoshimura et al. 2019), the workers are divided based on the task performed ranging from foraging, nest defense to brood care.
Ants are environmental cleaners, that acts as decomposers of organic wastes and dead animals.
Ants have been identified as an important agent in the regeneration of forest through effective dispersal of tree seed (Gallegos et al. 2014; Magalhães et al. 2018; Silva et al. 2019; Anjos et al. 2020), the ants remove all existing appendages on the seeds while foraging for pulp or aril and this prevent pathogenic infection through secretions from the ants’ mandibles that have
antibiotic and antifungal properties (Ohkawara and Akino 2005; Silva et al 2019). This group of Hymenoptera can also be used to monitor environmental health (Ribas et al. 2012; Bharti et al. 2016) through the level of abundance and distribution of different species within an ecosystem. Also, ants contribute to agricultural success through improvement of soil structure by enhancing good water infiltration (Leite et al. 2018) and predation on plant pests (Ennis and Philpott 2019).
Another important group of plant pest predator are wasps. Some wasp species also attack beneficial insects such as Vespa velutina (Chen et al. 2020) known for hunting pollinators, especially honeybees. Other ecologically beneficial wasp species include V. vulgaris (Jules 1996; Brodmann et al. 2008), the species perform similar functions as the ants. Although V.
vulgaris regulates plant pest population and prey on other insects (Richter 2000), mutualism in seed dispersal have been reported between the species and ants (Bale et al. 2003). Other social insects perform similar ecological functions of ants and wasps like pollination and seed dispersal in bees. Termites are important environmental engineers through decomposition of biological matters and modification of soil structure.
1.5 Pigmentation of Vespula vulgaris
V. vulgaris is an important species whose high level of adaptation have established the species in different regions of the world where it is classified as invasive alien species (see Donovan 1984; Beggs et al., 1998; Mathews et al., 2000; Lester and Beggs, 2019). V. vulgaris is expected to possess majority of the general characteristic of invasive species. However, specific adaptative mechanisms reported for the species includes phenology modification (Pawlikowski and Pawlikowski 2009) as well as possible variation in size with latitude and pigmentation.
The common wasps V. vulgaris has an aposematic pigmentation of alternating black and yellow colouration across both ventral and dorsal parts of the body. The body is divided into three parts (the head, thorax, and the abdomen), all the body parts have great proportion of both black and yellow pigmentation. Similar yellow-black pigmentation in other wasp species have been identified with different functions like nest mate recognition (Injaian and Tibbetts 2014;
Baracchi et al. 2016), social dominance (Tibbetts and Sheehan 2011) and sexual selection (De Souza et al. 2014; Izzo and Tibbetts 2012). However, such intraspecific mediation has not been reported in the common wasps, the only information available indicates the importance of the yellow-black pigmentation in defense and thermoregulation (Vidal-Cordero et al. 2012).
1.6 Aim of the study
This thesis studied the environmental drivers of variation in pigmentation in an ecologically important insect species V. vulgaris. Only little information is available on the usefulness of the yellow and black pigmentation of this species, thus this study. The study assessed the effect of different weather conditions (temperature and rainfall) on the melanisation degree (MD) and body size of V. vulgaris across different regions of Finland. The study explored the melanic variation across rural-urban gradient across different regions of Finland and across a pollution gradient in an industrial zone of a Finnish city. The study aims were:
To review existing information on importance and function of colour in social insect species (I).
To reveal how local climatic conditions can affect phenotypes of V. vulgaris within its native ecosystem (II).
To determine if difference in climatic condition between years affect phenotypic pigmentation of the common wasps from same location (II).
To determine the presence of xanthopterin granules in the yellow pigment layer of the abdominal tergites and show the importance of relative proportion of yellow and black pigmentation in thermoregulation of wasps (III).
To provide information on the possible urban heat island (UHI) effect on phenotypic variation across rural-urban gradient (III).
To determine the variation in phenotypic morphs and tolerance ability of V. vulgaris in heavy- metal polluted environment (IV).
2 MATERIAL AND METHODS 2.1 Study area and sample collection
Wasp samples were collected from different geographical zones across Finland (Fig. 1a), the sampling locations represent different habitats with varying microclimatic conditions. The samples were collected using a beer-trap type developed by Sorvari (2013, Fig. 1b), the trap was made of transparent plastic beer pint glasses (0.5 L) covered with a transparent plastic petri-dish top and with a circular 2.5 cm hole on the upper part of the plastic glass to allow wasps to enter. The traps were hung on trees and bushes (at a height of 1-4 m) with a steel wire.
The trapping method was used across all sampling locations with an average of 200m between the closest set of traps. The traps were left in the field for a period of seven days, after which traps were retrieved and nontarget insect species were removed from the trap and V. vulgaris samples collected. In 2019, sweep-netting method was used for samples used in TEM analysis.
Figure 1. Map showing distribution of sampling location across Finland (a) and (b) shows the beer trap method used for sampling. Map was downloaded from National Land Survey of Finland website.
The samples were collected in Harjavalta, Helsinki, Kuopio, Oulu, Tampere, and Turku.
Harjavalta is located in the southwestern part of Finland associated with high heavy metal pollution, Helsinki is on the southern peninsula by the Gulf of Finland with humid continental climate, Kuopio belongs to the southern boreal zone, with a continental climate, Ouluis located in the central boreal zone, close to the northern tip of the Baltic Sea with a humid continental climate, Tampere is located in the western part of Finland with a continental climate and Turku
is located in a hemiboreal zone (a transitional zone between a boreal and temperate zone) on the southwestern coast of Finland with humid continental climate. The samples were collected in August of the respective years of collection, the month was chosen based on the phenology of the insect – colonies are established in spring and grow through summer, in August the population would be at peak before it gradually dies off during autumn leaving mated queens that will overwinter to start new colonies the next spring (e.g. Pawlikowski and Pawlikowski, 2009). The number of samples collected varied across locations (Table 1) with varying environmental condition across the sampling years. A total of 1049 samples were used for these studies, the samples were preserved in 95% alcohol immediately after collection from traps and stored at +5 °C.
Table 1. Samples assessed from different locations in Finland with (article) and the corresponding year of sampling.
Location year
Harjavalta Helsinki Kuopio Oulu Tampere Turku
2011 73 (II, III)
2012 150 (II) 189 (II, III)
2013 62 (II)
2014 150 (IV) 148 (III) 49 (II) 108 (III) 105 (III)
2015 6 (II)
2019 9 (IV)
2.2 Digital photography, SEM and TEM imaging
For climate related melanisation and phenotype studies, digital photographs of the thorax and abdomen of individual specimens were obtained under constant lighting in a room without natural light penetration, using a binocular microscope camera setting with a Nikon DS-Fi1 microscope camera attached to an Olympus SZX9 microscope and using Nikon NIS-Elements BR version 3.2 software. The camera setting was set at 63X (for II) and 80X (for III) magnification, the exposure was 125ms for both studies. The photos were saved as 8-bit RGB colour JPG images. Image of wasps from Harjavalta (for IV) were obtained using the same method.
The structure of the wasp chitin specimen was studied using scanning electron microscope (SEM) Zeiss Sigma HD|VP (Carl Zeiss NTS, Cambridge, UK). The cross-section of the black
and yellow chitin coat was obtained by fracturing the wasp cuticle in two pieces with a help of freezing with liquid nitrogen (LN2). Thereafter, the frozen specimen was mounted onto a custom-made cryoholder with 10 μl silicon vacuum grease (Wacker Hochvakuumfett Schwer, Wacker Chemie AG, Burghausen, Nünchritz, Germany). The cryoholder consisted of a 0.5 kg LN2 cooled cool reserve made of copper and a heat insulator made of PTFE to insulate the heat conduction from the SEM framework. The electron acceleration voltage of 0.5 kV was applied for surface-sensitive structural imaging. The specimens were kept stable in high vacuum (P <
0.01 Pa) condition using cryogenic fixation, SEM micrographs were recorded using Everhart- Thornley type II secondary electron detector with +300 V grid bias and working distance of 3.5 mm.
Transmission electron microscopy (TEM) was used to analyze sections of the midgut of wasp samples from the polluted, intermediate and low polluted zones (for IV) to identify melanin encapsulation of metal pollutants. Energy-dispersive X-ray analyses (EDX) was then used to identify the metals present in the midgut. Detailed description of the sample processing and analysis procedure is provided in the article (IV).
To quantify metal elements, we used an inductively coupled plasma mass spectrometer (ICP- MS). The method enabled the identification of the following elements: arsenic (As), cadmium (Cd), cobalt (Co), copper (Cu), iron (Fe), mercury (Hg), nickel (Ni), lead (Pb) and zinc (Zn).
Individual wasp samples (0.015–0.020 g) were dissolved using a microwave digestion system (MARSTM 6 iWave instrument CEM Corporation, USA). The determination of metal concentrations was performed with a NexION 350D ICP-MS (PerkinElmer, USA) equipped with an ESI prepFAST autosampler (Elemental Scientific, USA). Each metal element was detected at the approximately 0.0001 μg/g detection limit. Further details are provided in the article (IV).
2.3 Image analysis
The digital photographs were processed with Adobe® Photoshop CS5 and then analyzed using ImageJ software. For (II), the photos of the abdomen were converted into black and white, inverted and the images were cropped using the peaks of dark pigment on both sides of the central peak as landmarks to only display the second tergites. Cuticular melanisation was measured as a degree of darkness (melanisation degree [MD]) of the cuticle.The value of darkness was measured using ImageJ by scaling from zero (totally white) to 255 (totally black).
Thorax width was used as an estimate of individual body size, and this was measured as the distance between the bases of the wings - the tegula (intertegular distance [ITD]).
For (III), the processing and analysis were like (II) but the first and second tergites were cropped from the photographs of the wasps’ abdomen. We visually observed and grouped the first and second tergite into morphs using similar method used by Clapperton et al. (1989) with some modifications. Clapperton et al. (1989) classified the fusion (the side peaks) on the second tergite separately, while we classified the morphs using the pattern of the entire melanised part of the tergite.The ITD was measured from the thorax photographs and proportion of black and yellow pigments on second tergite was measured.
For (IV), the image processing was like (II) and (III) but the morph categorization of the sternite was included. Morphological measurements assessed include head width (HW), thorax width/ITD, dry body weight and colouration area on the face and abdomen. The measurements were performed using ImageJ and WaspFacer softwares.
2.4 Data analysis
The data collected for (II) was used to analyze variation in melanisation degree between regions and between years, (III) analyzed morph variation across urbanization gradient in 3 different cities in Finland and between years at Turku while (IV) analyzed melanisation and morph selection across pollution gradient in Harjavalta. The size variation was assessed in all studies.
All statistical tests were performed using SAS 9.4 statistical software (SAS Inc., Cary, IL, USA)and detailed description of analysis are provided in the articles.
3 MAIN RESULTS AND DISCUSSION
3.1 Body size (ITD) variation across regions, urbanization gradient and pollution gradient.
The body size was measured mostly using the ITD as an estimate but in the last article (IV), the ITD was represented with the thorax width (TW) and other parameters such as the head width (HW) and the body weight was measured. The measurements were aimed at comparing differences across the sampling locations and zones. The ITD/TW of all samples used varied between 1.68 – 2.86mm. In the study between regions, the body size was significantly associated with location while in the study between urbanization gradient, there was no significant difference in the body size between zones but there was a marginally nonsignificant difference among the three studied regions. In both studies that required sampling in different cities and across climatic zones, there was obvious differences in the body size across the different locations and this is an indication that major drivers of growth and size variation are location specific. This is because condition of growth is an important factor that determines the adult size in most insects and environmental condition within a geographical location is expected to be similar except in polluted sections of a location. Other important factors that can affect growth and development of wasps includes climatic change (Sambaraju et al. 2012;
Polidori et al. 2020), availability of food and nesting location (Chown and Gaston 2010;
Jiménez-Cortés et al. 2012) and pathogens (Páez et al. 2015).
In contrast to the two earlier studies, the body size (TW) revealed significant difference within the compromised environment and the reference site in the same geographical location.
Samples from Harjavalta had wider TW from polluted zones while the wasps from the low polluted environment were narrower, other measured morphometric parameters were similar across pollution gradient. The presence of contaminants within the polluted zone might prompt adaptive response for survival and to minimize intake of the contaminants, the increase in TW might be to accommodate more flight muscles to be able to forage far away from the polluted zone. Although the foraging distance of the wasps from the polluted zone were not studied, Hernández et al. (2015) has described the ability of insects to modify their morphology to improve their dispersal ability by flight and this is possibly the case here.
3.2 Variation in melanisation degree across regions
Melanisation degree (MD) was studied across different zones in Finland with different climatic conditions and there was significant different in the expression of melanin across the locations (Fig. 2). The darkest individual wasps were from Oulu, followed by Turku while the lightest wasps were from Kuopio. The spatial variation in darkness follows basically the thermal melanisation theory (Clusella-Trullas et al. 2007) which attributes darker individuals to colder environmental temperature and climate, especially in Oulu. Although Turku is warmer than the other two locations, studies have also linked melanisation degree to desiccation avoidance properties (Law et al. 2019) in warmer climates and this might be an adaptation to avoid dehydration and water loss. However, Oulu and Turku are both coastal towns, melanisation pattern variation might be more linked to the coastal–continental differences than latitudinal differences. The increase in MD in Oulu and Turku is possibly for thermoregulation in cold environment and desiccation resistance in warmer climate, respectively. This reduces the aposematic properties of wasps in these locations and ability to protect themselves from predators, studies have associated expression of bright aposematic colouration with poison gland size in wasps (Vidal-Cardero et al. 2012) and this property reduces with increase in MD.
Figure 2. Estimated marginal means with 95% confidence intervals of melanisation degree in the studied locations in Finland. Different letters denote the significant differences (Tukey’s test: P < 0.05). The melanisation degree was marginally non-significant between wasps from Oulu and Turku (Tukey P = 0.066).
The result obtained in Kuopio shows that connection between climate and colouration is not always direct, and this brings up the importance of microclimatic properties of the locations in
expression of pigmentation. Also, the duration of the study might not be sufficient to generalize our conclusion as longer monitoring can show different trends in environmental conditions and subsequent phenotypic adaptation in the species.
3.3 Xanthopterin layer and colour morphs
The structure of the cuticle of wasps across rural-urban gradient was studied using SEM. A layer of xanthopterin was discovered in the SEM analysis of the yellow strip on the cuticle of V. vulgaris. The pigments were arranged below the layers of chitin lamellae (Fig. 3), similar in size and shape, like the barrel shaped cylindrical yellow granules identified in the oriental hornet by Ishay et al. (2004). The method used was not entirely the same as in Ishay et al.
(2004) but it was sufficient to show the presence and features of xanthopterin pigment granules.
Figure 3. SEM image showing xanthopterin pigment granules in the yellow stripe of the common wasp V. vulgaris cuticle. The pigment granules pointed by an arrow are located below the cuticle layer.
Ishay and Pertsis (2002) described the xanthopterin granules as a battery which stores excess heat absorbed by the melanin pigments. The authors described the wasp cuticle as an electric capacitor where the melanin stripe acts as n-type semiconductor which donates extra electrons to the p-type semiconductor (yellow xanthopterin stripe) for energy storage. Our study presents information of structural similarity in xanthopterin pigments described in oriental hornet within the yellow stripe of the cuticle of V. vulgaris. Therefore, the xanthopterin pigmentation in V.
vulgaris is expected to perform similar photoreceptive function. Thus, the results suggest that
a balance between the proportion of black and yellow pigmentation might be advantageous for effective location-specific thermoregulation in common wasps.
The proportion of black and yellow pigmentation on the wasps’ abdominal cuticle was used to group the wasps into different morphs. The first tergite had three morphs (Fig. 4a) and six morphs was identified for the second tergite (Fig. 4b). The second tergite showed more variability across the study zones and it presents the most conspicuous aposematic display of pigmentation on the abdomen of the wasps. When the city regions were pooled together, there was no difference between zones in morph frequencies in the first tergite nor in the second tergite. However, the cities were analyzed separately because the size and properties of the cities were different. Helsinki being the largest city in the study showed some differences in morph frequencies on the 2nd tergite (Figure 5; Table 2). The difference was mostly due to opposite trends between morphs B and C, as morph B was the most common in urban areas and C in rural areas. The morph frequencies of the first and second tergite between urban and rural samples did not differ in Tampere and Turku (Table 2).
a b
Figure 4. Image showing morph of Finnish samples of V. vulgaris on the first tergite (a) and (b) second tergite.
The observed variation in the morphs of the first and second tergite points to different microclimatic drivers within the different locations, temperature have been identified as an important factor that can affect morph selection and pigmentation expression in common wasps. Studies have shown that lower temperature of the environment can affect the selection of more melanised individuals in insect species (Fedorka et al. 2013; Sibilia et al. 2018), because highly melanised individuals heat up faster in cold environment than lighter ones.
However, in the wasp case, the balance between heating speed and heat storage may affect the thermal melanism process. Other studies have stated that melanised individuals are more resistant to parasites and pathogens (Armitage and Siva-Jothy, 2005; Bailey, 2011); this might be the reason why the morphs with the highest frequency were those with a larger melanised portion and relative proportion of yellow pigmenation..
Figure 5. Relative colour pattern morph frequencies and the observed numbers appear on the top of the bars of urban and rural samples of the common wasp V. vulgaris in Helsinki 2014.
The proportion of yellow-pigmented (xanthopterin) area on the dorsal surface of second gastral tergite was on average smaller in urban areas compared to that of rural areas in Helsinki, but the comparison only showed a trend. In Tampere and Turku there was no difference between urban and rural areas. The overall comparison between rural and urban areas across the three sampling cities on proportion of yellow pigmented area was not significant. The proportion of yellow pigmented area on second tergite did not differ between the studied city regions. The morphs with low frequency had a reduced melanised portion on the second tergite. The melanised portion on the second tergite might give thermoregulatory advantage to morphs with a higher melanised portion combined with conspicuous yellow portion, thus, increasing the survival rate of morphs B and C. This indicates that the total melanised portion of the insect tergite is important in thermoregulation, but not just the degree of melanisation.
Table 2. Test for the frequencies of colour pattern morphs in different towns and their surroundings. The P-values are probabilities from Fisher’s exact tests. † The likelihood ratio chi-square test χ2 was applied due to low frequencies in some morphs. ‡ One sample was omitted due to poor quality of the first tergite.
Tergite N urban N rural df χ2 P
Helsinki 1st 48 100 2 1.669 0.50
2nd 48 100 5 11.465† 0.045
Tampere 1st 26‡ 81 2 0.819 0.70
2nd 27 81 4 5.319† 0.36
Turku 1st 79 25‡ 2 0.908 0.71
2nd 79 26 3 4.400† 0.23
The temperature of urban areas is typically higher than that of the surrounding areas; studies have shown that some insect species establish their colonies first in urban areas in invaded environments because of the UHI effect (Mieneke et al., 2013; Branco et al., 2019). This scenario was suggested for the German wasp V. germanica in Finland (Sorvari, 2018). The city centre of Helsinki can be about 8 °C warmer than nearby rural areas during the daytime in August (Savijärvi, 1985), whereas in Turku the UHI effect can be about 3-4 °C (Suomi and Käyhkö, 2002). There is no such data from Tampere, but as it is slightly larger and more continental than Turku, the UHI effect might be at least the same as in Turku. In Helsinki, the second tergite morph B, which had reduced side peaks (fusion) and smaller proportion of yellow pigmentation, was more abundant in the urban areas, while morph C, with conspicuous fusions and larger proportion of yellow pigmentation, was predominant in the rural part of the city. The dominance of morph C in the rural area might link the occurrence of melanin fusion and larger yellow portion on the second tergite to thermoregulatory functions as the rural part of Helsinki is expected to be cooler than the city centre. Reduced fusion and yellow portion in morph B, might be due to the UHI effect and the need for the modification of morph C in order to reduce unnecessary heat storage and overheating in warmer environment. In other words, morph C may present a standard morph for V. vulgaris while morphs A and B are variants of
it which could be induced by the temperature regime of the location.The intensity of the UHI effect has a significant positive relationship with surface area of urban cover (Du et al., 2016).
Thus, the result from Turku and Tampere - with no significant difference in morph frequencies was expected. These cities have smaller urban cover compared to Helsinki and the UHI effect is likely less conspicuous. This suggests that there is a level of influence of the UHI effect in morph selection and more pronounced morph variation is expected in larger cities. Access to human originated food and grid plan habitat was seemingly similar among the cities; thus, the observed difference between cities points more towards the UHI effect.
3.4 Yearly variation in wasps colour morphs
The wasp samples from Turku were tested for variation in the degree of melanisation (MD) and morphs between two years with varying temperature and rainfall pattern (Table 3). There was significant difference in the MD between years in Turku, 2012 presented a colder weather condition and the wasps had low degree of MD compared with 2011. While the total area of melanin pigmentation on the abdominal tergite in relation to the total proportion of yellow pigmentation have been established in the previous section as an important factor in thermoregulation in V. vulgaris. It is important to note that other factors can also affect the expression of melanisation and proportion of different pigments on the cuticle. Rainfall is expected to affect morph selection because of its direct impact on temperature and climate, Dejean et al (2011) have shown the negative impact rainfall pattern has on wasps’ survival and there is high possibility of rainfall fluctuations affecting phenotypic properties in the process of adaptation for survival.
Table 3. Average daily temperature (°C), the number of rainy days (days with rainfall of over 1 mm) and average daily precipitation (mm) in May, June and July in Turku (2011 & 2012)
Temp., °C Rainy days, N Rainfall in mm 2011 2012 2011 2012 2011 2012
May 10.2 11.0 12 5 1.8 0.9
June 17.5 13.3 9 11 2.5 2.3
July 20.5 17.6 10 12 3.4 2.4
There were differences in morphs variation between the two years studied, this might be to compensate for the reduced degree of MD (Fig. 6). In both years, morph B and C were the most
abundant. Morph C were more abundant in the colder year 2012, this is expected because the relative area of both yellow and melanin pigments is needed to improve the thermoregulation and heat retention capacity of the wasps to survive with the weather condition.
Figure 6. The relative colour pattern morph frequencies of the common wasp Vespula vulgaris, collected in 2011 and 2012 in the park-like Aurajoki river valley in Turku, southwest Finland.
The numbers above the bars represent the abundance of the morphs within samples collected.
3.5 Pollution mediated melanisation and phenotypic variation
The concentration of metal pollutants was higher in the areas around the smelter and significantly lower in the low polluted point, these metals included Co, Ni, Cu, As, Cd and Pb while Fe and Zn showed no difference across the zones (Table 4). These high concentration of metals across the pollution gradient is expected to affect the condition of growth of individual wasps within the respective environments. The compromised condition of growth might affect expression of pigmentation and phenotypic selection across the assessed zones. Morph frequencies of both the second tergite (Fig. 4b) and the second sternite (Fig. 7) did not differ between polluted, intermediate, and low polluted zones (Fig. 8a). Melanized marking on clypeus, MAf, differed significantly among zones. Wasps from low polluted (reference) zone had significantly larger MAf than those from intermediate and polluted zones (Fig. 8b). Also, melanised marking on the second abdominal tergite, MAb, differed among the zones. However, the association between MAb and zones was opposite compared to the MAf (Fig. 8b).
Table 4. The average heavy metal concentrations (µg g-1 ±95% confidence interval) of Vespula vulgaris specimens in the three studied zones in Harjavalta and the results of linear mixed models. Same letter symbol in metal levels denote statistical pair-wise similarity between sites (Tukey’s test P > 0.05).
Polluted zone (N=62)
Intermediate zone (N=36)
Reference zone (N=52)
Test result
Fe 194.04-233.18-280.16 170.84-202.63-240.33 169.75-186.77-205.49 F2,6.75=2.85, P=0.13 Co 1.12-1.41-1.76a 0.39-0.51-066b 0.31-0.39-0.49b F2,88.9=35.39, P<0.0001 Ni 6.32-11.25-20.03a 2.51-9.21-33.81a 2.17-3.43-5.15b F2,3.72=9.61, P=0.034 Cu 68.66-84.24-103.32a 45.00-56.34-70.52b 40.88-46.05-51.88b F2,6.49=16.82, P=0.0027 Zn 338.30-544.63-876.60 192.53-352.45-645.21 391.56-531.74-722.10 F2,6.54=1.52, P=0.29 As 4.80-6.98-10.15a 2.29-3.97-6.89a 1.22-1.63-2.17b F2,6.53=25.60, P=0.0008 Cd 1.24-1.60-2.06a 0.32-0.41-0.52b 0.28-0.34-0.41b F2,89.4=50.72, P<0.0001 Pb 0.70-1.26-2.29a 0.34-0.82-1.98a 0.12-0.20-0.32b F2,8.87=16.05, P=0.0011
Figure 7. Sternite morphs of the second tergite of the common wasps Vespula vulgaris.
The TEM analysis of the midgut of the wasps from the polluted zone showed evidence of metal encapsulation and further analysis identified the ingested metals as iron (Fe) and nickel (Ni), see further details in the article IV. From (Table 4), Ni was among the metals with significant concentration in the polluted environment compared to the low polluted zone. Although Fe was not significantly higher in the polluted zone, the concentration was obviously higher than in the low polluted zone. The observed capsule in the midgut is an adaptive mechanism to immobilize foreign and harmful objects from absorption into the tissue of insects (Polidori et al., 2018). The midgut is an important location in insect immune defense because it is the point of digestion and absorption of nutrients, and the epithelium provides a suitable point of penetration of foreign pathogens into the body tissue. Secondly, the midgut extends through the entire length of the abdomen and provides a considerable surface area for penetration. Also,
the foregut and the hindgut are protected with chitinous material, thus making the midgut the possible point of pathogen attack in the digestive track.
a b
Figure 8. (a)Relative frequencies of different colour morphs of 2nd tergites and 2nd sternites of Common wasps in three pollution zones in Harjavalta (b). Mean size (± 95 % CI) of melanised marking in clypeus and second tergite in the tree pollution zones. Different letter above bars indicates significant pairwise difference (Tukey’s test P < 0,05).
Although, the midgut has a chemical mechanism of killing pathogens, inorganic pollutants are effectively immobilized by melanization and subsequent encapsulation of the pollutants. The melanization process starts with the conversion of tyrosine to melanin precursors in the presence of phenoloxidase, the precursors are crosslinked to hemolymph protein to form a layer of melanin that surrounds and sequesters invading pollutants and pathogens (Hillyer 2016).
Therefore, the wasps in the polluted zone deployed melanin pigments to immobilize the pollutants and hinder absorption into body tissues. It is plausible that the encapsulation process of metal pollutants in wasps from the polluted zone is responsible for the reduction in melanin pigmentation on the face, because wasps from the low polluted zone had larger melanin pigmentation on their clypeus (MAf). The wasps in the low polluted zone showed lower area
of melanisation on the second tergite (MAb) while MAb increased towards the pollution zones, this suggest that expression of melanin pigmentation might follow different paths on parts of the wasps body. The MAf might provide better information on impact of pollution and allocation of melanin for immune response, other studies have shown importance of facial pigmentation in sex selection and dominance hierarchy (Cervo et al.2008; Tibbetts et al. 2011;
de Souza et al. 2014). Therefore, this result provides an addition to the importance of melanin pigmentation on the face of wasps, but more studies are needed to support and establish this assumption.
4 CONCLUSIONS
The results showed evidence that microclimatic conditions have major impact on expression of pigmentation and contribution of pigments to thermoregulatory functions in the common wasps. The variation in the MD across latitudinal zones in Finland provides an insight into the possible adaptation and modification of the species to climate change and the possibility of losing aposematic properties as the temperature regime changes. Climate change and urbanization impacted the morph selection, this impact is expected to be higher in larger urban centers and with increase in climate change. Therefore, urban development should be environmentally friendly, and alteration of the natural ecosystem should be reduced to the possible minimum to preserve native species.
The xanthopterin pigments in the yellow portion presents an adaptive advantage for the common wasps to improve thermoregulation but its usefulness depends on the corresponding proportion of melanin pigmentation. So, microclimatic conditions that affects morph selection in this species should be kept balanced to ensure survival of this species. Although, weather condition might be difficult to control, drivers of climate change and unnecessary disruption of environment should be discouraged. Pollution and urbanization showed a trend on the growth and morph selection in these studies, more industrialized and urbanized locations might have negative impact on the survival and competitive strength of the common wasps.
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