Insectes Sociaux (2019) 66:91–105 https://doi.org/10.1007/s00040-018-0668-x Insectes Sociaux

RESEARCH ARTICLE

An improved method for testing invertebrate encapsulation response as shown in the honey bee

N. Wilson‑Rich1,2 · R. E. Bonoan1 · E. Taylor1 · L. Lwanga1 · P. T. Starks1

Received: 26 February 2018 / Revised: 26 September 2018 / Accepted: 6 October 2018 / Published online: 25 October 2018 © International Union for the Study of Social Insects (IUSSI) 2018

Abstract In 1882, Metchnikoff documented the encapsulation response (ER) of the invertebrate immune system. Since then, researchers have used Metchnikoff’s method to quantify immune function—and examine its relationship with ecological and behavio- ral factors—across various insect taxa. While scientists continue to uncover information regarding invertebrate immunity, behavioral ecology, and ecological immunology, the basics of Metchnikoff’s method have remained unchanged. All but two previous studies investigating insect immunity have used sterile or PBS-coated inducers, although we know that the immune system recognizes specific pathogens. To account for the specificity of the immune system, we modified Metchnikoff’s method and coated nylon monofilaments with pathogen-associated molecular patterns (PAMPs). Using honey bees (Apis mellifera), we examined ER using implants coated with PAMPs (“PAMPlants”) found on known honey bee parasites and pathogens. Lipopolysaccharide (LPS), peptidoglycan (PGN), and β-1, 3-glucan (B13G) PAMPlants mimicked an infection with Gram-negative bacteria, Gram-positive bacteria, and fungi, respectively. Our PAMPlants induced stronger responses than the control implants in both singly- (one PBS-coated or PAMP-coated implant) and doubly- (internal control; one PBS- coated and one PAMP-coated implant) implanted animals. In doubly-implanted individuals, there was a significant increase in response to B13G and LPS when compared with internal controls. The PGN and BSA did not differ from the internal controls in the doubly implanted individuals. These methods provide an improvement when exploring responses to specific pathogens and exploring topics within the field of invertebrate ecological immunity. When applied to social systems, these methods can be used to examine the evolution of disease resistance in societies.

Keywords Disease resistance · Innate immunity · Insect societies · Invertebrate immunity · Sociality

Introduction for disease to spread. Furthermore, many related individuals in one place lead to lower genetic diversity which can facili- A common cost of social living is the spread of disease (Wil- tate the spread of disease. Despite these threats, insect socie- son 1975). Many individuals living in close association lead ties are wildly successful (Wilson 1971). For many insect to frequent physical interaction and thus many opportunities societies, the first line of defense against invaders is behav- ioral. For example, when a honey bee colony is infected with a heat-sensitive pathogen, workers collectively raise the Noah Wilson-Rich and Rachael E. Bonoan contributed equally. temperature of the hive to generate a preventative colony- Electronic supplementary material The online version of this level fever (Starks et al. 2000). article (https​://doi.org/10.1007/s0004​0-018-0668-x) contains One hypothesis that may explain the success of insect supplementary material, which is available to authorized users. societies in spite of disease threats is the relaxed selection hypothesis, which postulates that social species have evolved * P. T. Starks [email protected] behavioral immune responses, such as honey bee fever, that decrease both disease risk to the group as well as the need 1 Department of Biology, Tufts University, Medford, MA, for strong individual immunity. When behavioral defenses USA are not successful, however, individuals must turn to physi- 2 Present Address: Urban Beekeeping Laboratory and Bee ological defenses. The social group hypothesis postulates Sanctuary, Inc., Best Bees Company, Llc, 839 Albany Street, that due to the increased risk of disease spread in societies, Boston, Ma 02119, USA

Vol.:(0123456789)1 3 92 N. Wilson‑Rich et al. sociality may lead to stronger physiological immunity in coast in 1882, Metchnikoff implanted a rose thorn into a individuals. In support of the relaxed selection hypothesis, starfish larva, observed an ER, and thus invented the first Lopez-Uribe et al. (2016) have shown reduced physiological method of studying the invertebrate ER (Beck and Habicht response to lipopolysaccharide, a protein on Gram-negative 1996; Tauber 2003). pathogens, in social insect lineages. Here, we describe a Nearly a century later, Metchnikoff’s method for measur- method to investigate physiological immunity, and tease ing invertebrate immune function was resurrected and used apart the two hypotheses, in response to various classes of to answer questions relating to ecological immunity—the pathogens. study of how abiotic and biotic factors influence variation Like vertebrates, an invertebrate’s first line of defense to in immune traits. As the immune system of invertebrates a physiological assault is a physical barrier (Antunez et al. is simpler than that of vertebrates, and is phylogenetically 2009). For insects, the exoskeleton’s cuticle and the diges- conserved, an understanding of invertebrate ecological tive tract’s peritrophic membranes serve as protection. When immunity provides insights in the evolution of immunity an infection evades such barriers, the host must recognize overall (Beck and Habicht 1996). Recent empirical ER the invader as both foreign and dangerous to launch the studies (Table 1) using Metchnikoffian methods of Konig appropriate immune response. In invertebrates, this process and Schmid-Hempel (1995) have elucidated relationships is carried out via proteins called pattern recognition recep- between immune function and many ecological and behav- tors (PRRs), and other recognition molecules, that recognize ioral factors in insects, including: sex, behavioral role, dom- specific, highly conserved pathogen-associated molecular inance, female mate choice, foraging ability, diet, habitat patterns (PAMPs) found on the microbial cell wall (reviewed type, infection risk, co-infection, energy cost, individual and in Nurnberger et al. (2004); Zipfel and Felix (2005)). For colony condition, ontogeny, and invasion biology (Table 1 example, peptidoglyan (PGN) is an essential component and references therein). In addition, as exhibited by Lopez- of the bacterial cell wall in both Gram-positive and Gram- Uribe et al. (2016), studying ecological immunity in insect negative bacteria and thus serves as a PAMP recognized by societies provides insight into the evolution of sociality and the invertebrate immune system. In addition to PGN, the cell how individuals deal with the spread of disease in close wall of Gram-negative bacteria also contains the endotoxin quarters. lipopolysaccharide (LPS) that is recognized by host PRRs. In this paper, we report results from an improved PAMPs such as β-1, 3-glucans (B13G), mannoproteins, and approach to the classic method for testing immune function phospholipomannan are found on fungi. using foreign bodies coated with PAMPs. All but two prior Upon recognition of a foreign body, insects can employ insect ER studies (Table 1) employed implants (monofila- one or more of three general innate responses: (1) synthesis ments or beads) that were either sterile and uncoated or ster- of antimicrobial peptides; (2) initiation of proteolytic cas- ile and coated with PBS. The two studies (Appler et al. 2015; cades, which lead to clotting and melanization; (3) phagocy- Lopez-Uribe et al. 2016) that used PAMP-coated implants tosis or encapsulation by hemocytes (Kimbrell and Beutler only used one type of PAMP: LPS. Our comparative method 2001). For the purposes of this study, we focused on the allows for further investigation of the ability of a host to encapsulation response (ER) by which the host immune identify and respond to a specific class of pathogen. Fur- system surrounds a foreign body with multiple hemocytes. thermore, of the studies listed in Table 1, only 15 (about The bound hemocytes then isolate and neutralize foreign 20%) focus on a social study system. Investigating physi- bodies via anoxia, toxic reactive oxygen species, or starva- ological immunity in response to various classes of patho- tion (reviewed in Moreno-Garcia et al. (2013)). In inver- gens in social insects could distinguish between, or support tebrates, the encapsulation response is highly effective at both, the social group hypothesis and the relaxed selection clearing small foreign bodies or bacteria (Smith 2016). The hypothesis. It is possible, for example, that while the relaxed first observation of the ER, and the creation of the assay to selection hypothesis holds true for Gram-negative pathogens measure ER, is attributed to Élie Metchnikoff (Beck and (see Lopez-Uribe et al. (2016)), the social group hypothesis Habicht 1996; Chernyak and Tauber 1988; Gordon 2008; holds true for Gram-positive and/or fungal pathogens. Tauber 2003). Since much is known concerning both behavioral and Although he began as a zoologist and embryologist, physiological immune mechanisms of honey bees (Apis mel- Metchnikoff is now referred to as “The Father of Natural lifera), this social insect served as our study system (Wilson- Immunity” (Beck and Habicht 1996; Gordon 2008). In 1908, Rich et al. 2008; Wilson-Rich et al. 2014; Wilson-Rich et al. Metchnikoff won the Nobel Prize in medicine for research 2009). Aside from immune mechanisms—including ER— stemming from his novel methods of measuring immune being well documented, further understanding immunity in strength in invertebrates (Metchnikoff 1884, as translated the honey bee is of ecological and economic importance. As and reprinted in Beck and Habicht (1996) and references the honey bee is naturally challenged by various pathogens therein). While vacationing with his family on the Sicilian and parasites, and its genome is fully sequenced (Honeybee

1 3 93 An improved method for testing invertebrate encapsulation response as shown in the honey bee Calleri ( 2006 ) et al. Calleri ( 2007 ) et al. Sorvari ( 2007 ) et al. Vainio et al. ( 2004 ) et al. Vainio Konig and Schmid-Hempel ( 1995 ) and Schmid-Hempel Konig Baer and Schmid-Hempel ( 2011 ) Baer and Schmid-Hempel Schmid-Hempel and Schmid-Hempel ( 1998 ) and Schmid-Hempel Schmid-Hempel Allander and Schmid-Hempel ( 2000 ) Allander and Schmid-Hempel Appler et al. ( 2015 ) Appler et al. Wilson-Rich et al. ( 2008 ) et al. Wilson-Rich Negri ( 2014 ) et al. Brandt et al. ( 2016 ) et al. Brandt Lopez-Uribe ( 2016 ) et al. References - termites significantly influenced ER, however, mass however, influenced ER, significantly affect ER and mated/unmated status did not environments and translocated groups; and translocated environments whereas in moderate, elevated ER was sug - metal levels in high heavy suppressed in heavily infections higher risk for gesting polluted areas males, micraners had lower ER than had lower males, micraners had nests larger and males from macraners no relationship ER; among queens, stronger ER and body size or colony between than those prevented from foraging; strong strong foraging; from than those prevented effect on ER colony ers; ER was lower in late-produced and in late-produced lower ers; ER was cohortslate-reproducing early to compared cohorts by C. bombi co-infection landscapes than in urban landscapes; no dif - and managed feral in ER between ference bees adults (nurses), and old (foragers) enhanced nitric oxide production and the production enhanced nitric oxide of hemocytes spread nificantly reduced compared to control bees to control reduced compared nificantly effect of colony size on ER effect of colony ER did not differ between inbred and outbred and outbred inbred differ between ER did not Sex, implant period, and colony of origin period, implant and colony Sex, ER was similar between ants in heavy metal ants in heavy similar between ER was Males have lower ER than queens; among ER than queens; lower Males have Workers allowed to forage had lower ER had lower forage to allowed Workers ER was not affected by diet or body mass by diet affected not ER was ER was not affected by or the size of implant affected not ER was Males had lower ER compared to sister work sister to ER compared Males had lower More variation in ER for bees living in rural in ER for variation More No change in ER across larvae, pupae, young larvae, pupae, young in ER across change No Challenging larvae with nylon implants implants Challenging larvae with nylon ER of bees treated withis sig - ER of bees treated neonicotinoids Within social insects, there is a negative social insects, thereWithin is a negative Major findings Major paper length Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated rubbed with sand - Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament of 1 mm or 2 mm Uncoated Uncoated monofilament Uncoated LPS-coated monofilament Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated LPS-coated monofilament Method used Method Findings uncovered using various Metchnikoffian methods with different insect study systems methods study with insect different Metchnikoffian using various uncovered Findings termite) wasps, termites) wasps, (dampwood (dampwood Zootermopsis angusticollis (forest dwelling ant) dwelling (forest aquilonia Formica (wood ant) (wood exsecta Formica (bumble bee) Bombus terresteris (honey bee) (honey Apis mellifera Insects of varying sociality (bees, ants, Study system Study 13 12 11 7 9 8 10 3 4 5 6 Eusocial insects 2 1 1 Table

1 3 94 N. Wilson‑Rich et al. Rantala et al. ( 2000 ) Rantala et al. Siva-Jothy ( 2000 ) Siva-Jothy Krams et al. ( 2015 b) et al. Krams Srygley et al. ( 2009 ) et al. Srygley ( 2011 ) and Lorch Srygley Srygley and Jaronski ( 2018 ) and Jaronski Srygley Silva and Elliot ( 2016 ) and Elliot Silva González et al. ( 2015 ) González et al. Boete et al. ( 2002 ) et al. Boete Ryder and Siva-Jothy ( 2000 ) and Siva-Jothy Ryder Wilson-Rich and Starks ( 2010 ) and Starks Wilson-Rich Wilson-Rich et al. ( 2014 ) et al. Wilson-Rich References - wingspots size and hemocyte number, but size and hemocyte number, wingspots correlated asym - with wingspot negatively metry ment heterogeneity; no effect of pre- or no effect of pre- ment heterogeneity; in PO activity levels post-implantation males unaffected by the dietaryunaffected in treatments the short term ets fed a carbohydrate diet had significantly had significantly diet a carbohydrate fed ets males proteins, greater ER than those fed greater encapsulation response had slightly than females slower ER than those fed high protein diets high protein ER than those fed slower perature or population density, however, however, or population density, perature caterpillars of the intermediate color pheno - greatest ER type showed than those prevented from constructing from than those prevented ER than females males had lower webs; had reduced ER versus control injections control ER versus had reduced hypothesis by showing male crickets with male crickets showing by hypothesis females); by (preferred song syllables more also had higher ER than the native, sympatric, congener, P. sympatric,than the congener, native, fuscatus early season workers early ER positively correlated with both male ER positively No relationship between ER and wing pig - between relationship No No effect of copulatoryNo activity on ER ER directly proportional body mass, but to ER directly ER directly correlatedER directly with body mass, crick a exhibited diets protein low Individuals fed ER was not affected by development tem - by development affected not ER was Females constructing webs had higher ER constructingFemales webs Mosquitoes injected with malariaMosquitoes parasites Supports handicap immunocompetence a had lower ER dominulus a had lower P. The invasive Late season workers exhibit lower ER than lower exhibit Late season workers Major findings Major Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated glass rod glass Uncoated Uncoated glass rod glass Uncoated rod glass Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Injected, uncoated beads Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Method used Method (continued) lar) name) Calopteryx splendens (damselfly) Anabrus (Mormon simplex cricket) (velvetbean caterpil - (velvetbean gemmatalis Anticarsia (spider, no common (spider, lagotis Aglaoctenus (yellow fever mosquito) fever (yellow aegypti Aedes (house cricket) domesticus Acheta Polistes dominulus, P. fuscatus (paper wasps) dominulus, P. Polistes Study system Study 24 25 21 22 23 20 19 18 17 15 Non-eusocial insects Non-eusocial 16 Primitively eusocial insects Primitively 14 1 Table

1 3 95 An improved method for testing invertebrate encapsulation response as shown in the honey bee Wu et al. ( 2014 ) et al. Wu Krams et al. ( 2015a ) et al. Krams Krams et al. ( 2017 ) et al. Krams Pomfret and Knell ( 2006 ) Pomfret Sorvari ( 2008 ) et al. Kapari ( 2006 ) et al. Haviola et al. ( 2007 ) et al. Haviola Klemola et al. ( 2007 ) Klemola et al. Kelly ( 2016 ) Kelly Pereira et al. ( 2017 ) et al. Pereira Debecker et al. ( 2015 ) et al. Debecker Wootton et al. ( 2006 ) et al. Wootton de Souza et al. ( 2008 ) de Souza et al. Koskimaki et al. ( 2004 ) et al. Koskimaki References dose-dependent elevation in ER dose-dependent elevation food of average nutritional when quality of average food withcompared on a high- larvae raised diet energy elytra length; no difference between sexes length; between no difference elytra type affected ER differently between castes between type affected ER differently defenses during a moth population increase defenses negatively associated with ER; flavenoid associated with ER; flavenoid negatively with had no relationship ER glycosides ER as pupae than on high- larvae reared had higher ER than females food; quality males females; fed individuals tend to have higher have individuals tend to fed females; ER than individuals starved Palmistichus elaisis the Palmistichus parasitoid and metamorphosed later than control larvae vigorous ER; non-specific electrostatic ER; non-specific electrostatic vigorous a have plasma factors and humoral forces on ER role synergistic compared to ringer injection; workers did ringer to injection; workers compared of self-grooming or the decrease level not allogrooming ER and higher post-fight fat reserves reserves fat than ER and higher post-fight losing males Priming with heat-killed bacteria causes ER response was stronger in larvae raised on in larvae raised stronger was ER response Larval diet and gut microbiome affect ER and gut microbiome Larval diet ER positively correlated and with age both ER positively Gynes had stronger ER than workers; habitat ER than workers; Gynes had stronger ER is positively correlated with plant ER is positively Several individual hydrolysable tannins were tannins were individual hydrolysable Several Larvae reared on low-quality food had higher food on low-quality Larvae reared On average, males have higher ER than males have On average, ER negatively affected by superparasitismby affected ER negatively Larvae exposed to UV exhibited higher ER UV exhibited to Larvae exposed Positively charged targets stimulated the most the stimulated most targets charged Positively Workers injected with ER PGN had reduced Workers Males winning male–male fights had higher Major findings Major paper paper paper paper monofilament PGN Injected, Congo red-stained beads Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated rubbed with sand - Uncoated monofilament Uncoated rubbed with sand - Uncoated monofilament Uncoated rubbed with sand - Uncoated monofilament Uncoated rubbed with sand - Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Injected, uncoated beads and Uncoated monofilament and injection with Uncoated Uncoated monofilament Uncoated Method used Method (continued) (greater wax moth) Galleria mellonella (greater wax Euoniticellus intermedius (dung beetle) Epirrita (autumnal moth) autumnata Deinacrida giant weta) rugosa (Cook Strait (Melonworm) Diaphania hyalinata (azure damselfly) puella (azure Coenagrion Cerastoderma edule (edible cockle) Cerastoderma (ant, no common name) fellah Camponotus (damselfly) Calopteryx virgo Study system Study 38 39 36 37 33 34 35 32 31 30 29 28 27 26 1 Table

1 3 96 N. Wilson‑Rich et al. Smilanich et al. ( 2009 ) et al. Smilanich Ahtiainen et al. ( 2004 ) Ahtiainen et al. Contreras-Garduno et al. ( 2006 ) et al. Contreras-Garduno Robb et al. ( 2003 ) et al. Robb Kelly and Jennions ( 2009 ) Kelly Kelly ( 2017 ) Kelly Wang et al. ( 2017 ) et al. Wang Zhong et al. ( 2017 ) Zhong et al. Zhuo et al. ( 2018 ) Zhuo et al. Hu et al. ( 2017 ) Hu et al. Gershman ( 2008 ) Kirschman et al. ( 2017 ) et al. Kirschman Rantala ( 2004 ) and Kortet Rantala and Roff ( 2005 ) Rantala and Roff Rantala ( 2003 ) and Kortet References - ism incidence drumming rates, but not affected by mobil - drumming affected but not rates, ity or body mass mentation, while fat reserves and immune reserves mentation, while fat constant during were territorialdefense tenure and the number of beads melanized was and the number of beads melanized was combi - morphs sex by all four for positive nations females blocking peptide genes HaGBPB1 and genes peptide blocking plasma HaGBPB2 into in ER females showed higher ER than males; the showed females 10) had no number of matings (5 versus effect on ER short-winged morphs - subordi had higher ER than size-matched and weight nates; no correlation between ER with body size, development time and lytic time and lytic with body size, development activity from males withcor from a high ER; ER did not relate withrelate male body weight - ER and parasit correlation between Negative ER was higher among males with higher ER was Territorial males had higher levels of pig - Territorial males had higher levels The relationship between beads recovered beads recovered between The relationship Males exhibited significantly ER greater than significantly Males exhibited Mated females showed an increase in ER an increase showed Mated females HaCTL3, a C-type lectin, enhances ER Fungal infection reduced ER reduced infection Fungal bead challenge stimulated release of growth of growth release stimulated bead challenge Immune-related gene Ha-DFP1 is involved Ha-DFP1 is involved gene Immune-related Following experimentally assigned matings, experimentally Following Long-winged morphsLong-winged had a higher ER than Dominant males obtained mates and more In both sexes, ER was negatively correlated negatively ER was In both sexes, Female crickets preferred courtship preferred songs crickets Female Major findings Major paper paper Uncoated monofilament Uncoated Injected, uncoated beads Uncoated monofilament Uncoated Uncoated monofilament Uncoated Injected, uncoated beads Uncoated monofilament Uncoated Uncoated monofilament Uncoated Injected, uncoated beads Injected, uncoated beads Injected, uncoated beads and PBS In vitro assay with PBS-coated beads assay In vitro Uncoated monofilament Uncoated rubbed with sand - Injected, uncoated beads Uncoated monofilament Uncoated Uncoated monofilament Uncoated rubbed with sand - Method used Method (continued) damselfly) spp. (caterpillars) Lepidopteran (wolf spider) (wolf rubrofasciata Hygrolycosa Hetaerina americana (American Hetaerina rubyspot Hemideina maori weta) (mountain stone (tree weta) (tree Hemideina crassidens Helicoverpa armigera (cotton bollworm) armigera (cotton Helicoverpa Gryllus (field cricket) vocalis Gryllus firmus (field cricket) Gryllus bimaculatus (field cricket) Study system Study 54 53 52 50 51 46 47 48 49 45 44 41 42 43 40 1 Table

1 3 97 An improved method for testing invertebrate encapsulation response as shown in the honey bee Rantala et al. ( 2003 ) Rantala et al. Vainikka et al. ( 2007 ) et al. Vainikka Kivleniece et al. ( 2010 ) et al. Kivleniece Lee et al. ( 2006 ) Lee et al. Di Lelio et al. ( 2014 ) Di Lelio et al. Gilbert ( 2016 ) and Uetz Wilson et al. ( 2002 ) et al. Wilson Duong and McCauley ( 2016 ) Duong and McCauley Polkki et al. ( 2014 ) et al. Polkki Yaroslavtseva et al. ( 2017 ) et al. Yaroslavtseva Freitak et al. ( 2003 ) Freitak et al. References Teng et al. ( 2016 ) et al. Teng Gherlenda et al. ( 2016 ) et al. Gherlenda

2 CO (JH) treatment group exhibited lower ER lower group(JH) treatment exhibited and PO activity than the non-JH treated group control implanted with one, two, or no implants or no implants withimplanted one, two, males lost starved; when the males were less body mass when immune challenged an opportunity feed to and given exhibited significantly ER greater than significantly that exhibited of the males repeat-treatment protein-to-carbohydrate ratio diet than those diet ratio protein-to-carbohydrate diet ratio on a low size (ornamentation) and ER; males that ER performed 5 min of courtship had lower than those perform that courtship did not solitary phases of living higher ER than those not exposed to caged caged to higher ER than thoseexposed not predators immune defense; males had a stronger ER males had a stronger immune defense; with when compared at higher temperatures females affect ER in larvae, primary inoculation with bacteria ER in larvae decreased ard metabolic rate by approximately 8% approximately by metabolic rate ard hence an energetic controls, to compared ER to cost exhibited a lower ER than unparasitized a lower exhibited larvae exhibited a higher ER than those on raised exhibited plants control Individuals treated with juvenile hormone withIndividuals treated juvenile No difference in difference of males the survival curves No ER of males in the group single-treatment ER was great in individuals on a high ER was ER depends on an immune gene, 102 SI ER depends on an immune gene, Significant positive correlation between tuft correlation Significant positive between Larvae raised on plants under elevated ­ on plants under elevated Larvae raised No difference in ER between social and in ER between difference No Larvae exposed to caged predators had a predators caged to Larvae exposed Temperature influenced sex differences in differences influenced sex Temperature Primary inoculation with fungus did not Implanted individuals increased their individuals increased Implanted stand - Major findings Major Within 24 h of inoculation, parasitized larvae 24 h of inoculation, parasitized Within Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Injected, PBS-coated beads Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Method used Method Injected, PBS-coated beads (continued) caterpillar) spider) dragonfly) beetle) (mealworm beetle) (mealworm molitor Tenebrio (cotton leafworm leafworm (cotton littoralis Spodoptera (brush-legged wolf (brush-legged wolf ocreata Schizocosa Paropsis atomaria (common leaf beetle) Paropsis Schistocerca gregaria (desertSchistocerca locust) (dot-tailed whiteface (dot-tailed whiteface intacta Leucorrhinia (blow fly) (blow terraenovae Protophormia (Colorado potato potato (Colorado decemlineata Leptinotarsa (white cabbage butterfly) (white cabbage Pieris brassicae Study system Study 66 67 64 65 63 62 58 61 57 60 55 56 1 Table 59

1 3 98 N. Wilson‑Rich et al. Civantos et al. ( 2005 ) et al. Civantos References Rantala et al. ( 2011 ) Rantala et al. Daukste et al. ( 2012 ) et al. Daukste Krams et al. ( 2013 ) et al. Krams Prokkola et al. ( 2013 ) et al. Prokkola Sadd and Siva-Jothy ( 2006 ) Sadd and Siva-Jothy Rantala et al. ( 2002 ) Rantala et al. ); Krams et al. ( 2016 ) et al. ( 2016 ); Krams et al. Kangassalo Li et al. ( 2018 ) Li et al. size - resist reduce it did significantly however, bassiana fungi Beauveria ance against and second implantations in males activation by a nylon implant previous to to previous implant a nylon by activation higher than survival was exposure fungal fungal subjected to were which of beetles only infection rise in temperature pighian tubule; physiology in the proximate in the proximate pighian tubule; physiology implant the to area nylon from males withfrom high ER; ER correlated with body PO enzyme activity and fresh weight higher ER hemocytes, but not in a dose-dependent hemocytes, but not manner No dependence of ER on individual body No Major findings Major Inbreeding did not significantly affect ER; significantly did not Inbreeding Significant increase in ER between the first in ER between Significant increase Survival of beetles subjected to immune subjected to Survival of beetles ER and development time decreased with time decreased a ER and development ER results in significant damage to Mal - in significant damage ER results Females were more attracted to pheromones pheromones to attracted more were Females Individuals with a darker cuticle exhibited a cuticle exhibited Individuals with a darker Ectoparasitoid venom inhibits ER of host inhibits ER of host venom Ectoparasitoid attached to a Malpighian tubule tissue graft to attached paper paper ectoparasitoid venom ectoparasitoid Uncoated monofilament Uncoated Method used Method Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament Uncoated Uncoated monofilament and Uncoated Uncoated monofilament Uncoated rubbed with sand - Uncoated monofilament Uncoated rubbed with sand - In vitro assay with uncoated beads and assay In vitro (continued) Tetrix undulate (pygmy grasshopper) undulate (pygmy Tetrix Study system Study Appler et al. and Lopez-Uribe et al. to be highlighted in gray, used sterile and/or PBS-coated implants. Only 15 of the 76 studies examine ER in social insects 15 of theexamine 76 studies used sterile Only and/or PBS-coated implants. and Lopez-Uribe be highlighted in gray, to Appler et al. et al. 68 69 70 71 72 73 74 75 76 1 Table

1 3 99 An improved method for testing invertebrate encapsulation response as shown in the honey bee

Genome Sequencing Consortium 2006), immune function ER. Implants were allowed to dry overnight in sterile petri can be studied under near-natural conditions and linked to dishes. specific immune genes for phylogenetic analysis (e.g. Lopez- To quantify the ER of individuals to the PAMPlants, we Uribe et al. (2016)). performed a series of modified, standard ER assays (Konig While honey bees are affected by many pests and path- and Schmid-Hempel 1995; Wilson-Rich et al. 2008) over ogens, the most notorious is the parasitic mite, Varroa 2 years (2008, 2009). Each year, we collected foraging destructor. In adult honey bees, the Varroa mite implants honey bees—which are all similar age (Winston 1987)— itself into the host cuticle (Martin 2001; Spivak 1996). Even at the entrance of a single hive maintained at Tufts Uni- if the mite itself does not induce ER, it creates a structural versity in Medford, MA. This allowed us to control for insult for other pathogens and parasites to pass through, and genetic variation and health status. Individuals were stored is the main cause of disease spread in a hive (Kang et al. collectively and immediately brought to the laboratory for 2016). Honey bee colonies affected by large numbers of experimentation. mites suffer from Parasitic Mite Syndrome, which causes Collected foragers were ice-anesthetized and the nylon the population to dwindle and the colony to eventually die monofilaments were implanted between the third and fourth out (Sammataro et al. 2000). Our PAMPplants mimic this ventral intersegmental membrane (N = 74 singly implanted, natural process while focusing on the immune response of and N = 102 doubly implanted, individuals). All implants the host to the bacterial and fungal pathogens commonly remained in situ for 4 h [see Wilson-Rich et al. (2008) transmitted by Varroa (Mariani et al. 2012; Vanikova et al. and references therein]. Experimental individuals in 2008 2015). We did this using three different PAMPs to coat the received a single implant, either an experimental PAMPlant implants: PGN, LPS, and B13G (reviewed in Sammataro or a control implant coated with PBS. Experimental individ- et al. (2000)). Due to the historical relationship between uals in 2009 received double implants, with one PAMPlant pathogens and host immune system (van Engelsdorp and matched with an internal PSB-coated control implant for a Meixner 2010), each PAMP coating was predicted to elicit self-paired design. To control for side, side was switched a more specific ER relative to control implants. for PAMPlant and control implants, and the left side was Each study listed in Table 1 added valuable information always inserted first, thus controlling for which condition to the field of insect immunity. By incorporating pathogen- (PAMPlant or control implant) was performed first. In com- specific molecules on experimental implants, we hope to parison with PBS, an additional control—an insert coated better mimic the natural host–pathogen relationship. In addi- with BSA—was used, because unlike LPS, PGN, and β13G, tion, in doing so, provide an improved method to researchers it is predicted to elicit a baseline immune response. to create a clear picture of ecological immunity in insects After 4 h, experimental individuals were re-anesthetized and invertebrates in general. Applied across various levels on ice and the implants were removed with fine forceps. of sociality, our method could be used to better understand The removed implants were then analyzed for mean gray the interplay of the social group hypothesis and the relaxed value using a fluorescence-detecting microscope (Olympus selection hypothesis in the evolution of disease resistance VX40), and image capturing (Optronics Magna Fire-SP, and sociality in insect societies. v1.0_5) and analysis [ImageJ (NIH)] software [see Wilson- Rich et al. (2008) and references therein]. Darker implants indicated more melanization, which served as a proxy for a Materials and methods stronger ER (Appler et al. 2015; Lopez-Uribe et al. 2016; Wilson-Rich et al. 2008), and thus greater immune func- To create the PAMPlants, sterile (rinsed with 70% ethanol) tion according to this measure, see Online Appendix 1 for a cuts of nylon monofilament (2 mm long, 0.4 mm diameter, detailed description of the methods. Scientific Anglers Tippet, 3M, diameter previously incor- rectly reported in Wilson-Rich et al. (2008)) were dipped through one of the five different solutions with varying con- Statistical methods centrations: (1) phosphate-buffered saline (PBS, our con- trol); (2) lipopolysaccharide in PBS (LPS; 0.1 mg/ml, 1 mg/ Singly implanted mean ER values were compared using ml, and 10 mg/ml); (3) peptidoglycan in PBS (PGN; 1 mg/ ANOVA followed by post-hoc Tukey HSD pairwise com- ml, 10 mg/ml, and 100 mg/ml); (4) β-1,3-glucans in PBS parisons. Doubly implanted mean values within PAMP treat- (β13G; 1 mg/ml, 10 mg/ml, and 100 mg/ml); and (5) bovine ments (i.e., between PAMP-coated and PBS-coated implants serum albumin in PBS (BSA; 1 mg/ml, another control). or PBS-coated and BSA-coated implants) were compared As such, there was a proportional fold increased in PAMP using paired t tests within individuals. The ER to different concentrations across treatments. PBS and BSA mono- PAMPlants in doubly implanted individuals was compared filaments were controls, and expected to induce a baseline using ANOVA followed by post-hoc Tukey HSD pairwise

1 3 100 N. Wilson‑Rich et al. comparisons. All statistics were calculated using SPSS for Windows v. 11.0.

Results

There were no significant effects of PAMP concentration on ER (Two-way ANOVA, F = 1.12, df = 3, p = 0.35) (Supp. Fig. 1). As such, data from all samples were combined within the singly- and doubly implanted groups. Overall, the PAMPlants induced higher ERs than the control (PBS, BSA) implants (Figs. 1, 2). In 2008, honey bees with a single implant exhibited a significantly upregulated ER in response to each of the three PAMPs when compared with the control (PBS-coated) implants (One-way ANOVA, F = 8.71, df = 3.70, p < 0.001). There were no significant differences in ER across the three PAMP treatments (Fig. 1). Fig. 2 Mean encapsulation response (as measured via optical density, In 2009, within doubly implanted individuals, PAMPlants OD) of doubly implanted honey bees (Apis mellifera) ± 1 SE. Adult bees were doubly implanted with two nylon monofilaments. One induced stronger responses than PBS-coated implants. Com- monofilament was PBS-coated, while the other was coated in one of pared with the PBS-coated control, this relationship was sig- the four types of molecules: β-1,3-glucan (B13G), lipopolysaccha- nificant for two PAMPs: B13G (paired t test, t = 3.30, df = 22, ride (LPS), peptidoglycan (PGN), or bovine serum albumin (BSA). p = 0.02), and LPS (paired t test, t = 1.85, df = 25, p = 0.04) The optical density (OD) of implants was quantified using fluores- cence microscopy and imageJ software to measure the encapsulation (Fig. 2). PGN and BSA showed no overall difference from response around the implants. Asterisks indicate p < 0.05 between the the internal PBS control. There was no effect of side (left PAMPlant and the internal control using paired t tests. Sample sizes (N) indicate the number of bees doubly implanted

versus right) on ER. Across all treatments, doubly implanted individuals had the strongest ER to B13G (Tukey’s HSD, pB13G−LPS = 0.03, pB13G−PGN < 0.001, pB13G−BSA < 0.001). There was no difference in ER between LPS, PGN, or BSA within doubly implanted treatments.

Discussion

Using a simple technique, Metchnikoff discovered the pro- cess of ER in invertebrates resulting in a better understand- ing of cellular immunity and paving the way for modern advancements in assay methods (see Table 1). Building upon these approaches, we report results from a method for test- ing more specific immune function in a social insect. Our Fig. 1 Mean encapsulation response (as measured via optical den- results suggest that the evolution of physiological immu- sity, OD) of singly implanted honey bees (Apis mellifera) ± 1 SE. nity in insect societies is likely context dependent: both the Adult bees were implanted with one PAMP-coated or PBS-coated relaxed selection hypothesis and the social group hypothesis monofilament. OD of each implant was quantified using fluores- could hold true, depending on the invader. cence microscopy and imageJ software to measure the encapsulation response around the implants. Asterisks indicate p < 0.05 between Our experimental honey bees exhibited a stronger ER coated implants (“PAMPlants” and control PBS-coated implants when exposed to foreign bodies coated with molecules using Tukey HSD pairwise comparisons). All coated implants nearly found on pathogens (i.e., PAMPs). As these PAMPs are doubled the immune response compared to the control. Sample sizes conserved molecules found on and in the cell wall of bac- (N) indicate the number of bees singly implanted. B13G β-1,3-glucan (fungal PAMP); LPS lipopolysaccharide (Gram-negative bacteria teria and fungi (see Postel and Kemmerling (2009)), it is PAMP); PGN peptidoglycan (Gram-positive bacteria PAMP) not surprising that hosts evolved to recognize PAMPs as

1 3 101 An improved method for testing invertebrate encapsulation response as shown in the honey bee foreign cues, and induce an appropriate immune response on the study system and the question being asked, this to clear them. Uncoated implants are foreign and elicit an simultaneous internal control may be more ecologically immune response (Table 1); however, our findings suggest relevant: honey bees can be plagued by multiple parasites that results gathered from uncoated or PBS-coated implants and pathogens at the same time. may not account for the specificity and/or degree of insect In contrast to the single implant trials, the data in our immune response. paired implant trials were not uniform. In the paired When compared with control treatments, singly implanted implant trials, honey bees showed a stronger response to individuals showed a greater immune response to all PAM- fungi (B13G) over Gram-negative bacteria (LPS) and even Plants. The doubly implanted individuals exhibited a signifi- more so over Gram-positive bacteria (PGN). We speculate cantly stronger immune response to B13G and LPS when that this makes sense both evolutionarily and ecologically; compared with internal controls. These data suggest that as mentioned earlier, colonies are commonly plagued by while Lopez-Uribe et al. (2016) found reduced ER to LPS in microsporidian Apsergillus and Nosema spp. (Glinski and social insect lineages, physiological immunity is likely still Jaroz 2001; Li et al. 2017; Paxton et al. 2007). important for these two classes of pathogens. B13G PAM- Although our focus is on ER, the immune response Plants are a good candidate to build upon results found by is multi-faceted and insect societies have myriad weap- Lopez-Uribe et al. (2016) as honey bees use both behavio- ons for thwarting disease. The ER mode of immunity is ral and physiological immunity to combat fungal invaders. particularly important and is associated with physiologi- Behavioral immunity is used to combat a common fungal cal disease resistance to virally infected cells (Trudeau pathogen, Ascosphaera apis (chalkbrood) (Starks et al. et al. 2001; Washburn et al. 1996), parasitoids (Carton 2000), while physiological immunity is used to combat more and David 1983; Kraaijeveld et al. 2001), and parasites virulent fungal diseases caused by Aspergillus and Nosema (Doums and Schmid-Hempel 2000). Indeed, the phenolox- spp. (Glinski and Jaroz 2001; Li et al. 2017; Paxton et al. idase (PO) cascade is similarly important, as it plays a 2007). complementary role in the ER process by contributing to There was no difference in ER for individuals doubly resistance to bacteria (Ashida and Brey 1997; Pye 1974), implanted with PGN and the PBS-coated implant. In accord- fungi (Ochiai and Ashida 1988), viruses (Beck and Strand ance with our results, it has been shown that honey bees 2007; Wilson et al. 2001), parasites (Gorman et al. 1996; protect the colony from Gram-positive infections, such as Leonard et al. 1985; Paskewitz and Riehle 1994), and Paenibacillus larvae (American foulbrood), via hygienic parasitoids (Wilson et al. 2001). The single measure of behavior (Spivak and Reuter 2001). Hence, in honey bees, immune function reported here (ER) has provided impor- as predicted by the social group hypothesis, social immu- tant information to researchers interested the evolutionary nity may be more important than individual immunity when dynamics of host/pathogen relationships (Table 1). it comes to defending against Gram-positive pathogens. Our PAMPlant technique enables researchers to explore Alternatively, the lack of ER in response to PGN in dou- the mechanism associated with defense against specific bly implanted individuals could stem from the evolutionary classes of microbes on host disease resistance. Data pre- relationship between honey bees and their microbial sym- sented here confirm the specificity of the invertebrate bionts. Worldwide, Lactobacillus and Bifidobacterium spp. immune system. Accordingly, understanding immune are found in the gut microbiome of adult bees; both species responses to historically common pathogens will be facili- are likely important for metabolism and immune function tated using biologically inspired techniques. Each study [Raymann and Moran (2018) and references therein]. in Table 1 documents an opportunity to dig deeper and Interestingly, the immune response to single implants understand the degree to which immune function inter- was nearly double that of doubly implanted individu- acts with sex, behavior, dominance, mate choice, forag- als. One possible explanation for this finding is that the ing ability, energetics, diet, habitat type, infection risk, immune response was divided between the two foreign co-infection, individual and colony condition, ontogeny, bodies, albeit unevenly. Although these data were col- and invasion biology. Moreover, comparative studies using lected over two different years, we feel confident in this social species as a study system will help us better under- explanation as this uneven response was also observed by stand the evolution of disease resistance in insect societies, Allander and Schmid-Hempel (2000) in bumble bees. The as well as the evolution of sociality. bumble bee immune response to a primary immune chal- lenge (either an implant or infection with Crithida bombi) Acknowledgements We are grateful for the helpful comments from Kelsey K. Graham, Stephanie Clarke, Rebecca Czaja. ET and LL were was higher than the immune response to a second implant funded through NSF DBI 0649190 granted to PTS. We also thank two in the same individual. Our study is the first to show the anonymous reviewers and the editor for their helpful comments and possible division of the immune response in an individual suggestions. with multiple, simultaneous, physical insults. Depending

1 3 102 N. Wilson‑Rich et al.

References de Souza DJ, van Vlaenderen J, Moret Y, Lenoir A (2008) Immune response affects ant trophallactic behaviour. J Insect Physiol 54:828–832. https​://doi.org/10.1016/j.jinsp​hys.2008.03.001 Ahtiainen JJ, Alatalo RV, Kortet R, Rantala MJ (2004) Sexual adver- Debecker S, Sommaruga R, Maes T, Stoks R (2015) Larval UV expo- tisement and immune function in an arachnid species (Lycosi- sure impairs adult immune function through a trade-off with dae). Behav Ecol 15:602–606. https​://doi.org/10.1093/behec​ larval investment in cuticular melanin. Funct Ecol 29:1292– o/arh06​2 1299. https​://doi.org/10.1111/1365-2435.12435​ Allander K, Schmid-Hempel P (2000) Immune defence reaction in Di Lelio I et al (2014) Functional analysis of an immune gene of bumble-bee workers after a previous challenge and parasitic Spodoptera littoralis by RNAi. J Insect Physiol 64:90–97. https​ coinfection. Funct Ecol 14:711–717. https​://doi.org/10.104 ://doi.org/10.1016/j.jinsp​hys.2014.03.008 6/j.1365-2435.2000.00476​.x Doums C, Schmid-Hempel P (2000) Immunocompetence in workers Antunez K, Martin-Hernandez R, Prieto L, Meana A, Zunino P, of a social insect, Bombus terrestris L., in relation to foraging Higes M (2009) Immune suppression in the honey bee (Apis activity and parasitic infection. Can J Zool 78:1060–1066 mellifera) following infection by Nosema ceranae (Micro- Duong TM, McCauley SJ (2016) Predation risk increases immune sporidia). Envrion Microbiol 11:2284–2290 response in a larval dragonfly (Leucorrhinia intacta). Ecology Appler RH, Frank SD, Tarpy DR (2015) Within-colony variation in 97:1605–1610 the immunocompetency of managed and feral honey bees (Apis Freitak D, Ots I, Vanatoa A, Horak P (2003) Immune response is mellifera L.) in different urban landscapes. Insects 6:912–925. energetically costly in white cabbage butterfly pupae. Proc R https​://doi.org/10.3390/insec​ts604​0912 Soc B 270:S220–S222. https://doi.org/10.1098/rsbl.2003.0069​ Ashida M, Brey P (1997) Recent advances in research on the insect Gershman SN (2008) Sex-specific differences in immunological costs phenoloxidase cascade. In: Brey P, Hultmark D (eds) Molecu- of multiple mating in Gryllus vocalis field crickets. Behav Ecol lar mechanisms of immune responses in insects. Chapman and 19:810–815. https​://doi.org/10.1093/behec​o/arn04​0 Hall, London, pp 135–172 Gherlenda AN, Haigh AM, Moore BD, Johnson SN, Riegler M Baer B, Schmid-Hempel P (2011) Unexpected consequences of poly- (2016) Climate change, nutrition and immunity: effects of andry for parasitism and fitness in the bumble bee, Bombus elevated ­CO and temperature on the immune function of terrestris. Evolution 55:1639–1643 2 an insect herbivore. J Insect Physiol 85:57–64. https​://doi. Beck G, Habicht GS (1996) Immunity and the invertebrates. Sci org/10.1016/j.jinsp​hys.2015.12.002 Am 275:60–66 Gilbert R, Uetz GW (2016) Courtship and male ornaments as honest Beck MH, Strand MR (2007) A novel polydnavirus protein inhib- indicators of immune function. Anim Behav 117:97–103. https​ its the insect prophenoloxidase activation pathway. Proc Natl ://doi.org/10.1016/j.anbeh​av.2016.04.013 Acad Sci USA 104:19267–19272 Glinski Z, Jaroz J (2001) Infection and immunity in the honey bee, Boete C, Paul REL, Koella JC (2002) Reduced efficacy of the Apis mellifera. Apiacta 36:12–20 immune melanization response in mosquitoes infected González M, Cost FG, Peretti AV (2015) Funnel-web construction by malaria parasites. Parasitology 125:93–98. https​://doi. and estimated immune costs in Aglaoctenus lagotis (Araneae: org/10.1017/s0031​18200​20019​44 Lycosidae). J Arachnol 43:158–167 Brandt A, Gorenflo A, Siede R, Meixner M, Buchler R (2016) The Gordon SM (2008) Elie Metchnikoff: father of natural immunity. J neonicotinoids thiacloprid, imidacloprid, and clothianidin Eur Immunol 38:3257–3264 affect the immunocompetence of honey bees (Apis mellifera Gorman MJ, Cornel AJ, Collins FH, Paskewitz SM (1996) A shared L.). J Insect Physiol 86:40–47. https​://doi.org/10.1016/j.jinsp​ genetic mechanism for melanotic encapsulation of CM-Sepha- hys.2016.01.001 dex beads and a malaria parasite, Plasmodium cynomolgi B, in Calleri DV, Reid EM, Rosengaus RB, Vargo EL, Traniello JFA the mosquito, Anopheles gambiae. Exp Parasitol 84:380–386 (2006) Inbreeding and disease resistance in a social insect: Haviola S, Kapari L, Ossipov V, Rantala MJ, Ruuhola T, Haukioja effects of heterozygosity on immnnocompetence in the termite E (2007) Foliar phenolics are differently associated with Epir- Zootermopsis angusticollis. Proc R Soc B 273:2633–2640. rita autumnata growth and immunocompetence. J Chem Ecol https​://doi.org/10.1098/rspb.2006.3622 33:1013–1023. https​://doi.org/10.1007/s1088​6-007-9271-8 Calleri DV, Rosengaus RB, Traniello JFA (2007) Immunity and Honeybee Genome Sequencing Consortium (2006) Insights into reproduction during colony foundation in the dampwood ter- social insects from the genome of the honeybee Apis mellifera. mite Zootermopsis angusticollis. Physiol Entomol 32:136–142. Nature 443:931–949 https​://doi.org/10.1111/j.1365-3032.2007.00559​.x Hu QQ, Wei XH, Li YP, Wang JL, Liu XS (2017) Identification Carton Y, David JR (1983) Reduction of fitness in Drosophila and characterization of a gene involved in the encapsulation adults surviving parasitization by a cynipid wasp. Experientia response of Helicoverpa armigera haemocytes. Insect Mol Biol 39:231–233 26:752–762. https​://doi.org/10.1111/imb.12336​ Chernyak L, Tauber AI (1988) The birth of immunology: Metch- Kang Y, Blanco K, Davis T, Wang Y, DeGrandi-Hoffman G (2016) nikoff, the embryologist. Cell Immunol 117:218–233 Disease dynamics of honeybees with Varroa destructor as Civantos E, Ahnesjo J, Forsman A (2005) Immune function, parasiti- parasite and virus vector. Math Biosci 275:71–92 zation and extended phenotypes in colour polymorphic pygmy Kangassalo K, Kosonen K, Pölkki M, Sorvari J, Krams I, Rantala grasshoppers. Biol J Lin Soc 85:373–383. https://doi.org/10.1​ MJ (2016) Immune challenge has a negative effect on cuticular 111/j.1095-8312.2005.00494​.x darkness in the mealworm beetle, Tenebrio molitor. Annales Contreras-Garduno J, Canales-Lazcano J, Cordoba-Aguilar A (2006) Zoologici Fennici 53:255–262 Wing pigmentation, immune ability, fat reserves and territorial Kapari L, Haukioja E, Rantala MJ, Ruuhola T (2006) Defoliating status in males of the rubyspot damselfly, Hetaerina americana. insect immune defense interacts with induced plant defense J Ethol 24:165–173. https://doi.org/10.1007/s1016​ 4-005-0177-z​ during a population outbreak. Ecology 87:291–296. https​:// Daukste J, Kivleniece I, Krama T, Rantala MJ, Krams I (2012) doi.org/10.1890/05-0362 Senescence in immune priming and attractiveness in a bee- Kelly CD (2016) Effect of nutritional stress and sex on melanotic tle. J Evolut Biol 25:1298–1304. https​://doi.org/10.111 encapsulation rate in the sexually size dimorphic Cook Strait 1/j.1420-9101.2012.02516​.x

1 3 103 An improved method for testing invertebrate encapsulation response as shown in the honey bee

giant weta (Deinacrida rugosa). Can J Zool 94:787–792. https​ cellular immunity. Arch Insect Biochem Physiol 98 https://doi.​ ://doi.org/10.1139/cjz-2016-0108 org/10.1002/arch.21451​ Kelly CD (2017) Sexually dimorphic effect of mating on the mel- Lopez-Uribe MM, Sconiers WB, Frank SD, Dunn RR, Tarpy DR anotic encapsulation response in the harem-defending Wel- (2016) Reduced cellular immune response in social insect lington tree weta, Hemideina crassidens (Orthoptera: Tettigo- lineages. Biol Lett 12:20150984 https​://doi.org/10.1098/ nioidea: Anostostomatidae). Ethology 123:785–792. https​:// rsbl.2015.0984 doi.org/10.1111/eth.12650​ Mariani F et al (2012) Parasitic interactions between Nosema spp. Kelly CD, Jennions MD (2009) Sexually dimorphic immune and Varroa destructor in Apis mellifera colonies. Zoo Trop response in the harem polygynous Wellington tree weta Hemid- 30:81–90 eina crassidens. Physiol Entomol 34:177–179 Martin SJ (2001) The role of Varroa and viral pathogens in the col- Kirschman LJ, Quade AH, Zera AJ, Warne RW (2017) Immune func- lapse of honeybee colonies: a modelling approach. J Appl Ecol tion trade-offs in response to parasite threats. J Insect Physiol 38:1082–1093 98:199–204. https​://doi.org/10.1016/j.jinsp​hys.2017.01.009 Moreno-Garcia M, Cordoba-Aguilar A, Conde R, Lanz-Mendoza H Kivleniece I, Krams I, Daukste J, Krama T, Rantala MJ (2010) Sex- (2013) Current immunity markers in insect ecological immu- ual attractiveness of immune-challenged male mealworm bee- nology: assumed trade-offs and methodological issues. B Ento- tles suggests terminal investment in reproduction. Anim Behav mol Res 103:127–139 80:1015–1021. https://doi.org/10.1016/j.anbeh​ av.2010.09.004​ Negri P, Quintana S, Maggi M, Szawarski N, Lamattina L, Eguaras Klemola N, Klemola T, Rantala MJ, Ruuhola T (2007) Natural host- M (2014) Apis mellifera hemocytes generate increased plant quality affects immune defence of an insect herbivore. amounts of nitric oxide in response to wounding/encapsula- Entomologia Experimentalis Et Applicata 123:167–176. https​ tion. Apidologie 45:610–617. https​://doi.org/10.1007/s1359​ ://doi.org/10.1111/j.1570-7458.2007.00533​.x 2-014-0279-0 Konig C, Schmid-Hempel P (1995) Foraging activity and immu- Nurnberger T, Brunner F, Kemmerling B, Piater L (2004) Innate immu- nocompetence in workers of the bumble bee, Bombus ter- nity in plants and animals: striking similarities and obvious dif- restris L. Proc R Soc B 260:225–227. https://doi.org/10.1098/​ ferences. Immunol Rev 198:249–266 rspb.1995.0084 Ochiai M, Ashida M (1988) Purification of a B1,3-glucan recogni- Koskimaki J, Rantala MJ, Taskinen J, Tynkkynen K, Suhonen J (2004) tion protein in the prophenoloxidase activating system from Immunocompetence and resource holding potential in the dam- hemolymph of the silkworm, Bombyx mori. J Biol Chem selfly, Calopteryx virgo L. Behav Ecol 15:169–173. https​://doi. 263:12056–12062 org/10.1093/behec​o/arg08​8 Paskewitz S, Riehle MA (1994) Response of plasmodium refractory Kimbrell DA, Beutler B (2001) The evolution and genetics of innate and susceptible strains of Anopheles gambiae to inoculated immunity. Nat Rev Genet 2:256–267 sephadex beads. Dev Comp Immunol 18:369–375 Kraaijeveld AR, Limentani EC, Godfray HCJ (2001) Basis of the trade- Paxton RJ, Klee J, Korpela S, Fries I (2007) Nosema ceranae has off between parasitoid resistance and larval competitive ability in infected Apis mellifera in Europe since at least 1998 and may be Drosophila melanogaster. Proc R Soc B Biol Sci 268:259–261 more virulent than Nosema apis. Apidologie 38:558–565 Krams I, Daukste J, Kivleniece I, Krama T, Rantala MJ (2013) Pre- Pereira KD, Guedes NMP, Serrao JE, Zanuncio JC, Guedes RNC vious encapsulation response enhances within individual pro- (2017) Superparasitism, immune response and optimum prog- tection against fungal parasite in the mealworm beetle Ten- eny yield in the gregarious parasitoid Palmistichus elaeisis. Pest ebrio molitor. Insect Sci 20:771–777. https​://doi.org/10.111 Manag Sci 73:1101–1109. https​://doi.org/10.1002/ps.4534 1/j.1744-7917.2012.01574​.x Polkki M, Kangassalo K, Rantala MJ (2014) Effects of interaction Krams I et al (2015a) Effects of food quality on trade-offs among between temperature conditions and copper exposure on immune growth, immunity and survival in the greater wax moth defense and other life-history traits of the blow fly Protophormia Galleria mellonella. Insect Sci 22:431–439. https​://doi. terraenovae. Environ Sci Technol 48:8793–8799. https​://doi. org/10.1111/1744-7917.12132​ org/10.1021/es501​880b Krams IA, Krama T, Trakimas G, Kaasik A, Rantala MJ, Škute A Pomfret JC, Knell RJ (2006) Immunity and the expression of a second- (2015b) Reproduction is costly in an infected aquatic insect. ary sexual trait in a horned beetle. Behav Ecol 17:466–472. https​ Ethol Ecol Evolut 29:74–84. https​://doi.org/10.1080/03949​ ://doi.org/10.1093/behec​o/arj05​0 370.2015.10899​43 Postel S, Kemmerling B (2009) Plant systems for recognition of Krams I et al (2016) A dark cuticle allows higher investment in immu- pathogen-associated molecular patterns. Semin Cell Dev Biol nity, longevity and fecundity in a beetle upon a simulated para- 20:1025–1031 site attack. Oecologia 182:99–109. https://doi.org/10.1007/s0044​ ​ Prokkola J, Roff D, Karkkainen T, Krams I, Rantala MJ (2013) Genetic 2-016-3654-x and phenotypic relationships between immune defense, mela- Krams IA et al (2017) Microbiome symbionts and diet diversity nism and life-history traits at different temperatures and sexes in incur costs on the immune system of insect larvae. J Exp Biol Tenebrio molitor. Heredity 111:89–96. https​://doi.org/10.1038/ 220:4204–4212. https​://doi.org/10.1242/jeb.16922​7 hdy.2013.20 Lee KP, Cory JS, Wilson K, Raubenheimer D, Simpson SJ (2006) Flex- Pye AE (1974) Microbial activation of prophenoloxidase from immune ible diet choice offsets protein costs of pathogen resistance in a insect larvae. Nature 251:610–613 caterpillar. Proc R Soc B 273:823–829. https​://doi.org/10.1098/ Rantala MJ, Kortet R (2003) Courtship song and immune function in rspb.2005.3385 the field cricket Gryllus bimaculatus. Biol J Linn Soc 79:503– Leonard C, Ratcliffe NA, Rowley AF (1985) The role of prophenolox- 510. https​://doi.org/10.1046/j.1095-8312.2003.00202​.x idase activation in non-self recognition and phagocytosis by Rantala MJ, Kortet R (2004) Male dominance and immunocompe- insect blood cells. J Insect Physiol 31:789–799 tence in a field cricket. Behav Ecol 15:187–191. https​://doi. Li W, Evans JD, Li J, Su S, Hamilton M, Chen Y (2017) Spore load and org/10.1093/behec​o/arg10​3 immune response of honey bees naturally infected by Nosema Rantala MJ, Roff DA (2005) An analysis of trade-offs in immune func- ceranae. Parasitol Res 116:3265–3274 tion, body size and development time in the Mediterranean field Li LF, Xu ZW, Liu NY, Wu GX, Ren XM, Zhu JY (2018) Parasitism cricket Gryllus bimaculatus. Funct Ecol 19:323–330. https://doi.​ and venom of ectoparasitoid Scleroderma guani impairs host org/10.1111/j.1365-2435.2005.00979​.x

1 3 104 N. Wilson‑Rich et al.

Rantala MJ, Jokinen I, Kortet R, Vainikka A, Suhonen J (2002) Do Srygley RB, Lorch PD, Simpson SJ, Sword GA (2009) Immediate pheromones reveal male immunocompetence? Proc R Soc B protein dietary effects on movement and the generalised immu- 269:1681–1685. https​://doi.org/10.1098/rspb.2002.2056 nocompetence of migrating Mormon crickets Anabrus simplex Rantala MJ, Vainikka A, Kortet R (2003) The role of juvenile (Orthoptera: Tettigoniidae). Ecol Entomol 34:663–668. https​:// hormone in immune function and pheromone production doi.org/10.1111/j.1365-2311.2009.01117​.x trade-offs: a test of the immunocompetence handicap princi- Starks PT, Blackie CA, Seeley TD (2000) Fever in honey bee colonies. ple. Proc R Soc B 270:2257–2261. https​://doi.org/10.1098/ Naturwissenschaften 87:229–231 rspb.2003.2472 Tauber AI (2003) Metchnikoff and the phagocytosis theory. Nat Rev Rantala MJ, Viitaniemi H, Roff DA (2011) Effects of inbreeding on Mol Cell Biol 4:897–901 potential and realized immune responses in Tenebrio molitor. Teng ZW, Xu G, Gan SY, Chen X, Fang Q, Ye GY (2016) Effects Parasitology 138:906–912. https://doi.org/10.1017/S0031​ 18201​ ​ of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconi- 10004​73 dae) parasitism, venom, and calyx fluid on cellular and humoral Raymann K, Moran NA (2018) The role of the gut microbiome in immunity of its host Chilo suppressalis (Lepidoptera: Crambi- health and disease of adult honey bee workers. Curr Opin Insect dae) larvae. J Insect Physiol 85:46–56. https://doi.org/10.1016/j.​ Sci 26:97–104 jinsp​hys.2015.11.014 Robb T, Jamieson IG, Forbes MR (2003) Morphs and immunity of the Trudeau D, Washburn JO, Volkman LE (2001) Central role of hemo- alpine weta, Hemideina maora: influence of temperature, selec- cytes in Autographa californica M nucleopolyhedrovirus patho- tion, and associations with density and cuticular melanism. Ecol genesis in Heliothis virescens and Helicoverpa zea. J Virol Entomol 28:738–746 75:996–1003 Ryder JJ, Siva-Jothy MT (2000) Male calling song provides a reliable Vainikka A, Rantala MJ, Seppala O, Suhonen J (2007) Do male meal- signal of immune function in a cricket. Proc R Soc B 267:1171– worm beetles, Tenebrio molitor, sustain the honesty of phero- 1175. https​://doi.org/10.1098/rspb.2000.1125 mone signals under immune challenge? Acta Ethol 10:63–72. Sadd BM, Siva-Jothy MT (2006) Self-harm caused by an insect’s innate https​://doi.org/10.1007/s1021​1-007-0031-0 immunity. Proc R Soc B 273:2571–2574. https://doi.org/10.1098/​ Vainio L, Hakkarainen H, Rantala MJ, Sorvari J (2004) Individual rspb.2006.3574 variation in immune function in the ant Formica exsecta; effects Sammataro D, Gerson U, Needham G (2000) Parasitic mites of honey of the nest body size sex. Evolut Ecol 18:75–84. https​://doi. bees: life history, implications, and impact. Annu Rev Entomol org/10.1023/B:EVEC.00000​17726​.73906​.b2 45:519–548 van Engelsdorp D, Meixner MD (2010) A historical review of managed Schmid-Hempel R, Schmid-Hempel P (1998) Colony performance and honey bee populations in Europe and the United States and the immunocompetence of a social insect, Bombus terrestris in poor factors that may affect them. J Invertebr Pathol 103:S80–S95 and variable environments. Funct Ecol 12:22–30 Vanikova S, Noskova A, Pristas P, Judova J, Javorsky P (2015) Hetero- Silva FWS, Elliot SL (2016) Temperature and population density: inter- trophic bacteria associated with Varroa destructor mite. Apid- actional effects of environmental factors on phenotypic plasticity, ologie 46:369–379 immune defenses, and disease resistance in an insect pest. Ecol Wang P et al (2017) C-type lectin interacting with beta-integrin Evolut 6:3672–3683. https​://doi.org/10.1002/ece3.2158 enhances hemocytic encapsulation in the cotton bollworm, Siva-Jothy MT (2000) A mechanistic link between parasite resistance Helicoverpa armigera. Insect Biochemistry Molecular Biology and expression of a sexually selected trait in a damselfly. Proc 86:29–40. https​://doi.org/10.1016/j.ibmb.2017.05.005 Roy Soc B Biol Sci 267:2523–2527. https​://doi.org/10.1098/ Washburn JO, Kirkpatrick BA, Volkman LE (1996) Insect protection rspb.2000.1315 against viruses. Nature 383:767–767 Smilanich AM, Dyer LA, Gentry GL (2009) The insect immune Wilson EO (1971) The insect societies. Harvard University Press, response and other putative defenses as effective predic- Cambridge tors of parasitism. Ecology 90:1434–1440. https​://doi. Wilson EO (1975) Sociobiology: the new synthesis. Harvard Univer- org/10.1890/08-1906.1 sity Press, Cambridge Smith VJ (2016) Immunology of invertebrates: cellular. In: Delves P Wilson K, Cotter SC, Reeson AF, Pell JK (2001) Melanism and disease (ed) Encyclopedia of life science. Wiley, New York resistance in insects. Ecol Lett 4:637–649 Sorvari J, Hakkarainen H, Rantala MJ (2008) Immune defense of Wilson K, Thomas MB, Blanford S, Doggett M, Simpson SJ, Moore SL ants is associated with changes in habitat characteristics. (2002) Coping with crowds: density-dependent disease resistance Environ Entomol 37:51–56. https​://doi.org/10.1603/0046- in desert locusts. Proc Natl Acad Sci 99:5471–5475. https​://doi. 225x(2008)37%5B51:idoai​a%5D2.0.co;2 org/10.1073/pnas0​82461​999 Sorvari J, Rantala LM, Rantala MJ, Hakkarainen H, Eeva T (2007) Wilson-Rich N, Starks PT (2010) The Polistes war: weak immune func- Heavy metal pollution disturbs immune response in wild ant pop- tion in the invasive Polistes dominulus relative to the natie P. ulations. Environ Pollut 145:324–328. https://doi.org/10.1016/j.​ fuscatus. Insectes Soc 57:47–52 envpo​l.2006.03.004 Wootton EC, Dyrynda EA, Ratcliffe NA (2006) Interaction between Spivak M (1996) Honey bee hygienic behavior and defense against non-specific electrostatic forces and humoral factors in haemo- Varroa jacobsoni. Apidologie 27:245–260 cyte attachment and encapsulation in the edible cockle Cerasto- Spivak M, Reuter GS (2001) Resistance to American foulbrood disease derma edule. J Exp Biol 209:1326–1335. https://doi.org/10.1242/​ by honey bee colonies Apis mellifera bred for hygienic behavior. jeb.02118​ Apidologie 32:555–565 Wilson-Rich N, Dres ST, Starks PT (2008) The ontogeny of immu- Srygley RB, Jaronski ST (2018) Protein deficiency lowers resistance nity: development of innate immune strength in the honey bee of Mormon crickets to the pathogenic fungus Beauveria bassi- (Apis mellifera). J Insect Physiol 54:1392–1399. https​://doi. ana. J Insect Physiol 105:40–45. https​://doi.org/10.1016/j.jinsp​ org/10.1016/j.jinsp​hys.2008.07.016 hys.2018.01.005 Wilson-Rich N, Spivak M, Fefferman NH, Starks PT (2009) Genetic, Srygley RB, Lorch PD (2011) Weakness in the band: nutrient-medi- individual, and group facilitation of disease resistance in insect ated trade-offs between migration and immunity of Mormon societies. Annu Rev Entomol 54:405–423 crickets Anabrus simplex. Anim Behav 81:395–400. https​://doi. Wilson-Rich N, Hester F, Pilowsky JA, Foo B, Tien T, Starks PT org/10.1016/j.anbeh​av.2010.11.006 (2014) A test of the haploid susceptibility hypothesis using a

1 3 105 An improved method for testing invertebrate encapsulation response as shown in the honey bee

species with naturally occurring variation in ploidy. Insectes Soc larvae. J Insect Physiol 96:14–20. https://doi.org/10.1016/j.jinsp​ ​ 61:163–169 hys.2016.10.004 Winston ML (1987) The biology of the honey bee. Harvard University Zhong K, Liu ZC, Wang JL, Liu XS (2017) The entomopathogenic Press, Cambridge fungus Nomuraea rileyi impairs cellular immunity of its host Wu M et al (2014) Inhibitory effect of gut bacteria from the Japanese Helicoverpa armigera. Arch Insect Biochem Physiol. https://doi.​ honey bee, Apis cerana japonica, against Melissococcus pluto- org/10.1002/arch.21402​ nius, the causal agent of European foulbrood disease. J Insect Zhuo XR et al (2018) 20-Hydroxyecdysone promotes release of Sci 14:1–13 GBP-binding protein from oenocytoids to suppress hemocytic Yaroslavtseva ON, Dubovskiy IM, Khodyrev VP, Duisembekov BA, encapsulation. Insect Biochem Mol Biol 92:53–64. https​://doi. Kryukov VY, Glupov VV (2017) Immunological mechanisms org/10.1016/j.ibmb.2017.11.006 of synergy between fungus Metarhizium robertsii and bacteria Zipfel C, Felix G (2005) Plants and animals: a different taste for Bacillus thuringiensis ssp. morrisoni on Colorado potato beetle microbes? Curr Opin Plant Biol 8:353–360

1 3