Effect of Short-Term Desiccation, Recovery Time, and CAPA–PVK Neuropeptide on the Immune System of the Burying Beetle Nicrophorus vespilloides

Environmental conditions, especially related to winter, are crucial for shaping activity of insect immune system. However, our previous research clearly indicates differences in the immune system functioning when the cold stress was induced in the laboratory conditions and when the beetles were collected from natural environment during winter. This is probably related to the multiplication of observed effects by simultaneous presence of different stress factors characteristic of winter, including desiccation. For these reasons, our next step was analysis of the effects of short-term desiccation and recovery time on the functioning of immune system of burying beetle Nicrophorus vespilloides. Also, the effect of Tenmo–PVK-2 (tenebrionid periviscerokinin), member of the CAPA–PVK neuropeptide family, was investigated to better understand observed changes. Short-term desiccation decreases the phagocytic activity of burying beetle haemocytes, which is correlated with a reduction in their adhesive ability. On the other hand, there was a significant increase in phenoloxidase (PO) activity and the level of proPO expression, which may suggest sealing the cuticula by melanin deposition and prevention of water loss. Additionally, the elevated level of defensin expression may be associated with the cross-talk between mechanisms, which participate in insect response to environmental stress, including pathogen infection. After 1 h of recovery time, the activity of tested cellular and humoral mechanisms was mostly back to the control level. However, inhibition of the activity of PO and down-regulation of proPO were noted. These results also indicate importance of melanin deposition during water loss. Moreover, it suggests that some changes in immune system functioning during stress conditions do not have an immune function. Interestingly, part of the effects characteristic of recovery time were also observed after the application of Tenmo–PVK-2, mainly related to haemocyte morphology. These results indicate that CAPA–PVK neuropeptides may also influence on activity of burying beetle immune system. It should be also highlighted that, because of the study of the effects of CAPA–PVK neuropeptides, homologs of vertebrate neuromedin U, the results may be interesting for search evolutionary similarities in the functioning of the neuroendocrine system of insects and vertebrates

Solanum nigrum Fruit Extract Modulates Immune System Activity of Mealworm Beetle, Tenebrio molitor L

: Here, we report the first evidence concerning the modulation of insect immune system activity after applying Solanum nigrum fruit extract (EXT). We focused on two main issues: (1) is EXT cytotoxic for Tenebrio molitor haemocytes? and (2) how EXT affects the basic immune mechanisms of T. molitor. The results indicate cytotoxic action of 0.01 and 0.1% EXT on beetle haemocytes. Both the injection of EXT and incubating haemocytes with the EXT solution on microscopic slides significantly increased the number of apoptotic cells. However, 24 h after injection of 0.1% EXT cytotoxic effect of the tested extract probably was masked by the increased number of circulating haemocytes. Application of 0.01 and 0.1% EXT led to impairment of the activity of basic immune mechanisms such as phenoloxidase activity and the lysozyme-like antimicrobial activity of T. molitor haemolymph. Moreover, the EXT elicited significant changes in the expression level of selected immune genes. However, some of the immunomodulatory effects of EXT were different in beetles with and without an activated immune system. The obtained results are an essential step toward a complete understanding of the EXT mode of action on the T. molitor physiology and its potential usage in pest control

Tachykinin-related peptides modulate immune-gene expression in the mealworm beetle Tenebrio molitor L

The research was partially supported by Grant No. 2021/41/B/NZ9/01054 from the National Science Centre (Poland). AU was supported by a scholarship from the Polish National Agency for Academic Exchange (NAWA) within the Bekker Programme, 2019 (personal stipend, PPN/BEK/2019/1/00167), a scholarship from the Deutscher Akademischer Austauschdienst (DAAD) within the program for Research Stays for University Academics and Scientists, 2018 (personal stipend, 91696887), and a scholarship from the Initiative of Excellence, Research University (ID-UB Project), within the International Junior and Senior Exchange, 2021 (personal stipend, 018/07/POB2/0001). The publication fee was supported by the ID-UB Project (040/08/POB2/0010). JR was funded by DFG FOR 5026.Tachykinins (TKs) are a group of conserved neuropeptides. In insects, tachykinin-related peptides (TRPs) are important modulators of several functions such as nociception and lipid metabolism. Recently, it has become clear that TRPs also play a role in regulating the insect immune system. Here, we report a transcriptomic analysis of changes in the expression levels of immune-related genes in the storage pest Tenebrio molitor after treatment with Tenmo-TRP-7. We tested two concentrations (10-8 and 10-6 M) at two time points, 6 and 24 h post-injection. We found significant changes in the transcript levels of a wide spectrum of immune-related genes. Some changes were observed 6 h after the injection of Tenmo-TRP-7, especially in relation to its putative anti-apoptotic action. Interestingly, 24 h after the injection of 10-8 M Tenmo-TRP-7, most changes were related to the regulation of the cellular response. Applying 10-6 M Tenmo-TRP-7 resulted in the downregulation of genes associated with humoral responses. Injecting Tenmo-TRP-7 did not affect beetle survival but led to a reduction in haemolymph lysozyme-like antibacterial activity, consistent with the transcriptomic data. The results confirmed the immunomodulatory role of TRP and shed new light on the functional homology between TRPs and TKs.Peer reviewe

Tachykinin-related peptides modulate immune-gene expression in the mealworm beetle Tenebrio molitor L.

Tachykinins (TKs) are a group of conserved neuropeptides. In insects, tachykinin-related peptides (TRPs) are important modulators of several functions such as nociception and lipid metabolism. Recently, it has become clear that TRPs also play a role in regulating the insect immune system. Here, we report a transcriptomic analysis of changes in the expression levels of immune-related genes in the storage pest Tenebrio molitor after treatment with Tenmo-TRP-7. We tested two concentrations (10–8 and 10–6 M) at two time points, 6 and 24 h post-injection. We found significant changes in the transcript levels of a wide spectrum of immune-related genes. Some changes were observed 6 h after the injection of Tenmo-TRP-7, especially in relation to its putative anti-apoptotic action. Interestingly, 24 h after the injection of 10–8 M Tenmo-TRP-7, most changes were related to the regulation of the cellular response. Applying 10–6 M Tenmo-TRP-7 resulted in the downregulation of genes associated with humoral responses. Injecting Tenmo-TRP-7 did not affect beetle survival but led to a reduction in haemolymph lysozyme-like antibacterial activity, consistent with the transcriptomic data. The results confirmed the immunomodulatory role of TRP and shed new light on the functional homology between TRPs and TKs

Impact of environmental factors on the immune system and early seasonal activity of beetles in the genus Nicrophorus (Coleoptera: Silphidae)

Wydział Biologii: Instytut Biologii ŚrodowiskaPomimo dużej wiedzy na temat chrząszczy z rodzaju Nicrophorus (grabarz) nadal wiele aspektów fizjologicznych i ekologicznych nie zostało w pełni poznanych. Dlatego celem rozprawy doktorskiej było określenie rozwojowo-zależnych zmian w aktywności układu odpornościowego Nicrophorus vespilloides i wpływu niskiej temperatury na funkcjonowanie tego układu oraz analiza wczesno-sezonowej aktywność lokalnych populacji grabarzy. Przeprowadzone badania wykazały różnice w aktywności układu odpornościowego N. vespilloides w poszczególnych fazach jego rozwoju. W szczególności widoczne było to u osobników larwalnych. W stadium tym obserwowano m.in. wzrost aktywności oksydazy polifenolowej i liczby fagocytujących hemocytów. Podczas realizacji drugiego celu wykazano, po raz pierwszy, wpływ zimna na funkcjonowanie układu odpornościowego chrząszczy. Układ odpornościowy grabarzy reagował odmiennie na niską temperaturę gdy warunki stresowe były indukowane w warunkach laboratoryjnych oraz gdy osobniki do badań pobierane były ze środowiska naturalnego w trakcie okresu zimowego. Z tematem zimowania nierozerwalnie związana jest fenologia owadów. Obserwacje wykazały różnice we wczesno-sezonowej aktywności grabarzy. Jednakże uzyskane wyniki sugerują, że w początkowej fazie ich aktywności kluczowa nie jest obecność innych gatunków grabarzy a dynamika preferowanych siedlisk po okresie zimowym.Despite large knowledge about beetles in the genus Nicrophorus (burying beetles) still little is known about their physiology and ecology. For this reason the aim of PhD thesis was to analyse the developmental changes of immune system activity and impact of cold on immune system functioning in Nicrophorus vespilloides and determination of differences in early-seasonal activity of local population of burying beetles. The results showed differences in immune system activity in different phase of N. vespilloides development. Especially this was visible in case of larvae. In this stage inter alia increase of phenoloxidase activity and number of phagocytic haemocytes were observed. During the realization of the second aim, I have shown, for the first time, the impact of cold on the beetles immune system. However, burying beetles immune system reacted differently on the low temperature when the stress factor was induced in laboratory conditions, and when the individuals for analysis were obtained from natural habitats during winter. Aspect which is inseparably connected with insect overwintering is their phenology. The results of my observation showed differences in early-seasonal activity of burying beetle. However, the obtained results suggested that, for their activity, crucial is post-winter dynamics of preferred habitat rather than presence of other burying beetles species

Factors limiting competition of the burying beetles (Coleoptera: Nicrophorus)

Nekrofagiczne chrząszcze z rodzaju Nicrophorus (grabarz) w wyniku intensywnej konkurencji o zasoby, wykształciły szereg mechanizmów redukujących negatywne oddziaływanie rywali. Najważniejszym z nich jest opieka samca i samicy nad potomstwem. Grabarze zakopują oraz przygotowują padlinę tak, aby stanowiła bazę pokarmową dla potomstwa. Osobniki rodzicielskie zostają w dole rozrodczym, aż do momentu przemiany larw w poczwarki. Jednak nie mniej ważny element stanowi fenologia omawianej grupy. Preferencje siedliskowe oraz aktywność sezonowa poszczególnych gatunków skorelowane są z występowaniem konkurentów. Grabarze występujące w tym samym czasie zwykle zasiedlają odmienne siedliska. W okresie jesiennym może dochodzić do zmiany preferencji siedliskowych. Dzieje się tak, ponieważ grabarze dążą do zmagazynowania dużej ilości materiałów zapasowych przed hibernacją. Aktywność chrząszczy z rodzaju Nicrophorus przypada głównie na okres od godzin popołudniowych do zmierzchu. Jednakże, i w tym przypadku duży wpływ ma konkurencja ze strony innych owadów. Aktywne w tym samym czasie grabarze zamieszkują różne środowiska. Natomiast u chrząszczy zamieszkujących podobne siedliska obserwuje się przesunięcie szczytów ich aktywności dobowej względem siebie. Innym mechanizmem mającym redukować konkurencję są preferencje odnośnie wielkości wykorzystywanych zasobów pokarmowych.Beetles of the Nicrophorus species (burying beetles) under the pressure from scavengers developed a number of mechanisms that reduce negative impact of rivals. The most important mechanism is extensive parental care provided by the male and female. Burying beetles conceal carcasses underground and prepare them for the consumption by their young. Parents stay in the resources until the offspring transforms to the pupa. But equally important part of reduced competition strategies is phenology of burying beetles. Habitat preferences and seasonal activity of the Nicrophorus species are correlated with the presence of competitors. Usually burying beetles occurring at the same time, occupy different habitat. During the autumn period niche releases are observed, because species only need feeding before hibernation. Beetles of Nicrophorus species mostly are active from late afternoon to the nightfall. But diurnal activity of burying beetles depends also of competition from other insects. Beetles active at the same time, prefer different habitats. However, the beetles occupying the same habitat have their peaks of diurnal activity shifted relative to each other. Another mechanism which might reduce competition from other carrion beetles is carcasses preferences

Behavior of burying beetles (Coleoptera: Nicrophorus)

Padlina mimo małej dostepności stanowi główną bazę pokarmową dla wielu gatunków owadów. Wszystkie grupy padlinożerców wykształciły szereg mechanizmów redukujących negatywny wpływ konkurentów. Jednak najciekawszym z nich jest opieka nad potomstwem wystepująca u chrząszczy z rodzaju Nicrophorus (grabarz). Nie mniej ciekawe u grabarzy są zachowania poprzedzające opiekę nad potomstwem. Dla zrozumienia roli złożonych taktyk życiowych grabarzy, przedstawione będą one na tle ich cyklu życiowego. Grabarze do detekcji padliny wykorzystują zmysł węchu. Chrząszcze te są w stanie zlokalizować żródło zasobów już po kilku godzinach od ustaniu czynności życiowych zwierzęcia. Umożliwiają im to oragny znajdujące się na ostatnich segmentach czułków. Ponadto grabarze wykorzystują zmysł węchu do komunikacji między osobnikami. Samce emitując feromony aby zwabić samicę. Po znalezieniu padliny oraz partnera do rozrodu, grabarze przystępują do preparacji padliny. Przy pomocy żuwaczek oczyszczają martwe zwierzę z futra lub piór i formują z niego kulę. Jednocześnie padlina jest zakopywany i "zaszczepiana" wydzielinami oralnymi i analnymi. Substancje te konserwują zasoby pokarmowe poprzez dekompozycję procesów rozkładu. Po spreparowaniu padliny samica składa jaja w ziemi. Opieka rodzicielska jest głównym czynnikiem wpyływającym na sukces reprodukcyjny grabarzy. W początkowym okresie przypomina bardziej zachowania obserwowane u ptaków i ssaków. Młode larwy, tuż po wykluciu wyciągaja się w kierunku osobników rodzicielskich, ruchem żuwaczek oraz dotykiem odnoży domagając się pożywienia. Larwy grabarzy w pierwszych stadiach rozwojowych nie są w stanie samodzielnie pobierać pokarmu. Zarówno samiec i samica prowadzą opiekę na potomstwem. Obecność samca, przede wszystkim gwarantuje lepszą ochronę gniazda. Osobniki rodzicielskie zostają wraz z potomstwem do czasu zamiany larw w poczwarki.Carcasses, despite their low availability, are base resources for many insects. Scavengers developed many mechanisms that reduce negative impact of rivals. But the most spectacular behavior is extensive biparental care occurring in burying beetles (Nicrophorus sp.). For better understanding of the complexity of behavior of all burying beetles their life cycle is presented. Burying beetles have perfectly developed sense of smell; used for searching of carrion. They are remarkably adapt to detect the odor of recently died animals using the organs of smell located on their antennae. Furthermore, sense of the smell is involved in the communication between individuals of the opposite sex. Male emits a pheromone that serves a female. After finding resources and mates, burying beetles, start carcasses preparation. Beetles of Nicrophorus species remove from dead animal's fur or feathers and form balls. Simultaneously, the resource is buried and inoculated with oral and anal secretions that preserve the carrion and modify the course of its decomposition. The female lays eggs in soil. Parental care is a most important factors affecting to reproductive success. At the beginning, parental care takes a form, resembling more behavior found among birds and mammals than insects. Larvae after hatching draw towards the parents demanding food. In early development stages, most burying beetles offspring are unable to feed. Both males and females provide extensive parental care, and the major benefit of male assistance is to help to defend the brood and carcass from competitors. Parents stay in the resources until the offspring transforms into the pupa

Differences in Early Seasonal Activity of Three Burying Beetle Species (Coleoptera: Silphidae:NicrophorusF.) in Poland

Urbański, Arkadiusz, Baraniak, Edward (2015): Differences in Early Seasonal Activity of Three Burying Beetle Species (Coleoptera: Silphidae:NicrophorusF.) in Poland. The Coleopterists Bulletin 69 (2): 283-292, DOI: 10.1649/0010-065x-69.2.283, URL: http://dx.doi.org/10.1649/0010-065x-69.2.28

Fig. 5 in Differences in Early Seasonal Activity of Three Burying Beetle Species (Coleoptera: Silphidae:NicrophorusF.) in Poland

Fig. 5. Day of carcass colonization by individuals of Nicrophorus vespillo. A) Differences among types of habitats, B) Differences among months.Published as part of <i>Urbański, Arkadiusz & Baraniak, Edward, 2015, Differences in Early Seasonal Activity of Three Burying Beetle Species (Coleoptera: Silphidae:NicrophorusF.) in Poland, pp. 283-292 in The Coleopterists Bulletin 69 (2)</i> on page 287, DOI: 10.1649/0010-065x-69.2.283, <a href="http://zenodo.org/record/10104818">http://zenodo.org/record/10104818</a&gt