Age-related mating rates among ecologically distinct lineages of bedbugs, Cimex lectularius.

Understanding how many mates an animal has in its lifetime is a critical factor in sexual selection. At the same time, differences in an organism's ecology, such as the quantity and quality of food, could be reflected in different mating rates. Mating rate had a significant effect on female net fitness (i.e., lifetime offspring production), however, laboratory measurements cannot well mirror the situation in wild. The common bedbug (Cimex lectularius) is a well-established model for studying traumatic insemination and sexual conflict. The species comprises two host lineages that feed on bats (BL) or humans (HL). HL can constantly feed on human hosts throughout the year, while BLs feed only during summer months when their bat hosts occupy the roosts. Because mating in female bedbugs is closely linked to foraging, this system provides a valuable model to study mating variation in the field. We established a new method for estimating age-dependent mating rates of females in the wild by relating the fluorescent pigment accumulation in the eyes of females to the number of mating scars that manifest as melanized spots caused by the injection of sperm through the wall of the female abdomen by the male into the spermalege. In addition, using laboratory bedbugs we found that three and a half observed matings on average lead to one observed melanized mating scar. Although young BL and HL females (with low pteridine concentrations) did not differ in the number of matings, the mating rate increased with age only in HL but not in BL females. We sampled on average older BL than HL females. The lack of access to food (bat blood) during winter could explain the lack of increase in the number of scars with age in BL. In species where mating leaves visible marks, using fluorescent pigments to determine female age (applicable to most arthropods) could be an important tool to study sexual selection and mating rate in the wild. The method can help formulate sustainable and biologically lucid approaches for their control.

Despite genetic isolation in sympatry, post-copulatory reproductive barriers have not evolved between bat- and human-associated common bedbugs (Cimex lectularius L.).

BACKGROUND: The common bedbug Cimex lectularius is a widespread ectoparasite on humans and bats. Two genetically isolated lineages, parasitizing either human (HL) or bat (BL) hosts, have been suggested to differentiate because of their distinct ecology. The distribution range of BL is within that of HL and bedbugs live mostly on synanthropic bat hosts. This sympatric co-occurrence predicts strong reproductive isolation at the post-copulatory level. RESULTS: We tested the post-copulatory barrier in three BL and three HL populations in reciprocal crosses, using a common-garden blood diet that was novel to both lineages. We excluded pre-copulation isolation mechanisms and studied egg-laying rates after a single mating until the depletion of sperm, and the fitness of the resulting offspring. We found a higher sperm storage capability in BL, likely reflecting the different seasonal availability of HL and BL hosts. We also observed a notable variation in sperm function at the population level within lineages and significant differences in fecundity and offspring fitness between lineages. However, no difference in egg numbers or offspring fitness was observed between within- and between-lineage crosses. CONCLUSIONS: Differences in sperm storage or egg-laying rates between HL and BL that we found did not affect reproductive isolation. Neither did the population-specific variation in sperm function. Overall, our results show no post-copulatory reproductive isolation between the lineages. How genetic differentiation in sympatry is maintained in the absence of a post-copulatory barrier between BL and HL remains to be investigated.

Cold arousal - A mechanism used by hibernating bats to reduce the energetic costs of disturbance.

During the season of hibernation, temperate bats alternate between prolonged bouts of torpor with reduced body temperature and short arousals with a return to normothermy. Hibernating bats are sensitive to non-tactile stimuli and arouse following changes in microclimatic conditions or disturbance from other bats, potential predators, or humans. Here, we used temperature data loggers to register the skin temperature of 38 Myotis myotis bats over two winters (between January and March), during which regular visits were made to the hibernaculum. Two kinds of arousal were observed, normothermic (Tsk > 25 °C) and cold (Tsk < 15 °C). Although bats responded to the presence of a researcher by arousals of both kinds, cold arousals were more frequent (63.8%). We found that mass loss was not affected by the number of disturbances, however it was in positive relationship with the mass at the beginning of the observation and differed between sex and age categories. Furthermore normothermic bats crawling among cluster-mates initiated arousal cascades, which mainly consisted of cold arousals. We failed to detect any effect of age or sex on the number of arousals initiated by normothermic individuals. Warming by only a few degrees requires less energy than a normothermic arousal and we propose it is sufficient to activate the sensory system in order to assess the relevance of external stimuli. Our results indicate that cold arousals reflect a physiological and behavioural adaptation aimed at avoiding the energetic costs of disturbance that can lead to depletion of fat reserves.

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Active surveillance for antibodies confirms circulation of lyssaviruses in Palearctic bats.

BACKGROUND: Palearctic bats host a diversity of lyssaviruses, though not the classical rabies virus (RABV). As surveillance for bat rabies over the Palearctic area covering Central and Eastern Europe and Siberian regions of Russia has been irregular, we lack data on geographic and seasonal patterns of the infection. RESULTS: To address this, we undertook serological testing, using non-lethally sampled blood, on 1027 bats of 25 species in Bulgaria, the Czech Republic, Poland, Russia and Slovenia between 2014 and 2018. The indirect enzyme-linked immunosorbent assay (ELISA) detected rabies virus anti-glycoprotein antibodies in 33 bats, giving an overall seroprevalence of 3.2%. Bat species exceeding the seroconversion threshold included Myotis blythii, Myotis gracilis, Myotis petax, Myotis myotis, Murina hilgendorfi, Rhinolophus ferrumequinum and Vespertilio murinus. While Myotis species (84.8%) and adult females (48.5%) dominated in seropositive bats, juveniles of both sexes showed no difference in seroprevalence. Higher numbers tested positive when sampled during the active season (10.5%), as compared with the hibernation period (0.9%). Bat rabies seroprevalence was significantly higher in natural habitats (4.0%) compared with synanthropic roosts (1.2%). Importantly, in 2018, we recorded 73.1% seroprevalence in a cave containing a M. blythii maternity colony in the Altai Krai of Russia. CONCLUSIONS: Identification of such "hotspots" of non-RABV lyssavirus circulation not only provides important information for public health protection, it can also guide research activities aimed at more in-depth bat rabies studies.

Resistance is futile: RNA-sequencing reveals differing responses to bat fungal pathogen in Nearctic Myotis lucifugus and Palearctic Myotis myotis.

Resistance and tolerance allow organisms to cope with potentially life-threatening pathogens. Recently introduced pathogens initially induce resistance responses, but natural selection favors the development of tolerance, allowing for a commensal relationship to evolve. Mycosis by Pseudogymnoascus destructans, causing white-nose syndrome (WNS) in Nearctic hibernating bats, has resulted in population declines since 2006. The pathogen, which spread from Europe, has infected species of Palearctic Myotis for a longer period. We compared ecologically relevant responses to the fungal infection in the susceptible Nearctic M. lucifugus and less susceptible Palearctic M. myotis, to uncover factors contributing to survival differences in the two species. Samples were collected from euthermic bats during arousal from hibernation, a naturally occurring phenomenon, during which transcriptional responses are activated. We compared the whole-transcriptome responses in wild bats infected with P. destructans hibernating in their natural habitat. Our results show dramatically different local transcriptional responses to the pathogen between uninfected and infected samples from the two species. Whereas we found 1526 significantly upregulated or downregulated transcripts in infected M. lucifugus, only one transcript was downregulated in M. myotis. The upregulated response pathways in M. lucifugus include immune cell activation and migration, and inflammatory pathways, indicative of an unsuccessful attempt to resist the infection. In contrast, M. myotis appears to tolerate P. destructans infection by not activating a transcriptional response. These host-microbe interactions determine pathology, contributing to WNS susceptibility, or commensalism, promoting tolerance to fungal colonization during hibernation that favors survival.

Numerous cold arousals and rare arousal cascades as a hibernation strategy in European Myotis bats.

Hibernating bats optimise the duration of torpor bouts and arousals in relation to hibernaculum microclimatic conditions and fat reserves. Clustering has significant physiological and ecological benefits, promoting successful hibernation of individuals. Such aggregations may help maintain optimal temperatures, allowing better energy utilisation than in solitarily bats. However, aroused bats in a cluster could conceivably disturb those still hibernating, starting an energy-demanding arousal process. Our study was conducted over two winters in two different hibernacula (cave and mine) in the Czech Republic, where Greater mouse-eared bats (Myotis myotis) have previously been diagnosed with white-nose syndrome. In 118 arousal episodes we recorded 193 individual arousals in which a warming phase was observed, 135 (69.9%) being cold arousals, where bats ceased increasing their body temperatures at ≤ 10 °C. The remaining arousals were standard normothermic arousals, where body (fur) surface temperatures reached > 20 °C. Cold arousals occurred during the mid- and late hibernation periods, suggesting they were a response to disturbance by a neighbour in the same cluster. Arousal cascades, where bats aroused in series, were rare (12.7%) and reached a maximum in mid-January. Our data suggest that Myotis bats prolong their torpor bouts using numerous cold arousals but few arousal cascades. Upon arrival of a bat, the clustered bats show tolerance to disturbing by conspecifics.

An evaluation of odor repellent effectiveness in prevention of wildlife-vehicle collisions.

Wildlife-vehicle collisions (WVC) amount to 11% of all registered traffic crashes in the Czech Republic causing, apart from numerous deaths and serious injuries to animals, property damage and injuries to car passengers. Odor repellents have the potential to lower the overall number of WVC and allow animals to cross roads at the same time. We tested the effectiveness of odor repellent preparation in prevention of WVC. 18 places were selected on the Czech road network where WVC were concentrated on the basis of traffic crash data. Control sections on the same road segments were also delimited in order to keep the traffic intensities constant. We applied a Before-After-Control-Impact (BACI) study design to control not only the effect of the measures but also the expected natural variations in wildlife populations over time. Data were compared before and after odor repellent installations. Wildlife carcass gathering was carried out during the spring and autumn. We also used the police crash database to supplement carcass data when no field works were carried out. 201 killed mammals (roe deer and wild boars) were identified in total over 47 months. We applied a Bayesian approach as only a limited numbers of WVC were available. A WVC decrease between 26 - 43% can be expected on the treated road sections. These numbers are, however, up to three-times lower than those claimed by producers of odor preparations.

Discrimination between lineage-specific shelters by bat- and human-associated bed bugs does not constitute a stable reproductive barrier.

The common bed bug Cimex lectularius, has been recently shown to constitute two host races, which are likely in the course of incipient speciation. The human-associated lineage splits from the ancestral bat-associated species deep in the history of modern humans, likely even prior to the Neolithic Period and establishment of the first permanent human settlements. Hybridization experiments between these two lineages show that post-mating reproductive barriers are incomplete due to local variation. As mating takes place in off-host refugia marked by aggregation semiochemicals, the present investigation tested the hypothesis that bed bugs use these semiochemicals to differentiate between refugia marked by bat- and human-associated bed bugs; this would constitute a pre-copulation isolation mechanism. The preference for lineage-specific odors was tested using artificial shelters conditioned by a group of either male or female bed bugs. Adult males were assayed individually in four-choice assays that included two clean unconditioned control shelters. In most assays, bed bugs preferred to rest in conditioned shelters, with no apparent fidelity to shelters conditioned by their specific lineage. However, 51 % of the bat-associated males preferred unconditioned shelters over female-conditioned shelters of either lineage. Thus, bed bugs show no preferences for lineage-specific shelters, strongly suggesting that semiochemicals associated with shelters alone do not function in reproductive isolation.

Deeply torpid bats can change position without elevation of body temperature.

Because body temperature is tightly coupled to physiological function, hibernating animals entering deep torpor are typically immobile. We analysed thermal behaviour and locomotory activity of hibernating greater mouse-eared bats Myotis myotis and found two types of movement behaviour related to body temperature, i.e. movement at high fur temperature and at low fur temperatures (Tflow; <5°C). First Tflow movements appeared at the beginning of March and often occurred during long torpor bouts. In most cases, Tflow events represented slow displacements between clusters of bats. In several cases, however, departure or arrivals from and into clusters was also recorded without any elevation in body temperature. Distance travelled, flight duration and speed of locomotion during Tflow events was lower than in high fur temperature events. Such behaviour could allow bats to save energy long-term and prolong torpor bouts. Tflow movement in torpid bats significantly changes our understanding of basic hibernation principles and we strongly recommend further studies on the subject.

Circum-Mediterranean phylogeography of a bat coupled with past environmental niche modeling: A new paradigm for the recolonization of Europe?

The isolation of populations in the Iberian, Italian and Balkan peninsulas during the ice ages define four main paradigms that explain much of the known distribution of intraspecific genetic diversity in Europe. In this study we investigated the phylogeography of a wide-spread bat species, the bent-winged bat, Miniopterus schreibersii around the Mediterranean basin and in the Caucasus. Environmental Niche Modeling (ENM) analysis was applied to predict both the current distribution of the species and its distribution during the last glacial maximum (LGM). The combination of genetics and ENM results suggest that the populations of M. schreibersii in Europe, the Caucasus and Anatolia went extinct during the LGM, and the refugium for the species was a relatively small area to the east of the Levantine Sea, corresponding to the Mediterranean coasts of present-day Syria, Lebanon, Israel, and northeastern and northwestern Egypt. Subsequently the species first repopulated Anatolia, diversified there, and afterwards expanded into the Caucasus, continental Europe and North Africa after the end of the LGM. The fossil record in Iberia and the ENM results indicate continuous presence of Miniopterus in this peninsula that most probably was related to the Maghrebian lineage during the LGM, which did not persist afterwards. Using our results combined with similar findings in previous studies, we propose a new paradigm explaining the general distribution of genetic diversity in Europe involving the recolonization of the continent, with the main contribution from refugial populations in Anatolia and the Middle East. The study shows how genetics and ENM approaches can complement each other in providing a more detailed picture of intraspecific evolution.

Reproduction barrier between two lineages of bed bug (Cimex lectularius) (Heteroptera: Cimicidae).

Populations of bed bugs, Cimex lectularius, have increased in recent years spreading into numerous urban areas across the Western world and making them an increasingly important pest of the twenty-first century. Research into hybridization within and between different lineages of bed bugs can help us to understand processes of micro- and macro-evolution in these ectoparasites and may inform the control of this pest species. Hybridization experiments between two host lineages of bed bug (C. lectularius) from Central Europe (Czech Republic), those associated with humans and those with bats, were conducted under laboratory conditions. Number of eggs and early instars were compared between crosses of mixed host lineages (interspecific mating) with pairs from the same host lineage, those from the same locality and same lineage from different localities (intraspecific mating). While crosses within host lineages resulted in egg production and later instars, crosses between different host lineages were unsuccessful, although of the mated females possessed sperm in their mesospermaleges and/or seminal conceptacles. These crosses did not even result in egg production. Moreover, in the mixed lineage crosses, mortality rates in adults were higher (51 and 50% higher in bat and human lineage, respectively) than in those animals from the same lineage. Survival of adults was in pairs from the same locality slightly higher than in pairs from different localities and differed statistically. These results support the existence of post-mating barriers and show reproductive isolation between two lineages of C. lectularius. Bat and human host adaptations can promote evolving of such barriers and can be product of alloxenic speciation.

Novel Cryptosporidium bat genotypes III and IV in bats from the USA and Czech Republic.

Bats from the families Rhinolophidae (n = 90) and Vespertilionidae (n = 191) in the USA and Czech Republic were screened for the presence of Cryptosporidium by microscopic and molecular analysis of faecal samples collected from rectum of dissected animals and from the ground beneath roosting sites. Cryptosporidium oocysts were not detected in any of the 281 faecal specimens examined using the aniline-carbol-methyl violet staining method. Nested PCR amplification, sequencing and phylogenetic analysis of the small ribosomal subunit rRNA and actin genes were used to identify isolates and infer evolutionary relationships. Cryptosporidium parvum was identified in a western small-footed bat (Myotis ciliolabrum) from the USA and a common pipistrelle bats (Pipistrellus pipistrellus) from the Czech Republic. Two novel genotypes were identified and named Cryptosporidium bat genotype III and IV. Bat genotype III was found in two big brown bats (Eptesicus fuscus) from the USA. Bat genotype IV was detected in two common pipistrelle bats from the Czech Republic.

Changes in body size and fertility due to artificial and natural feeding of laboratory common bed bugs (Heteroptera: Cimicidae).

Rearing common bed bugs (Cimex lectularius L.) and other hematophagous insects is essential for basic, medical, and pest-control research. Logistically, acquiring fresh blood can be a challenge, while biologically, the eventual effects of different rearing and blood preparation protocols on bed bug genotype and phenotype pose a risk of biased research results. Using bed bug populations that are either bat- (BL) or human-related (HL), we tested the short- and long-term effects of rearing bugs on live bats or human volunteers, or artificially on CPDA (citrate phosphate dextrose, adenine)-treated blood, measuring meal size, body size, and fertility. We found that artificial feeding did not affect meal size compared with feeding on natural hosts. Long-term rearing across many generations of HL on CPDA-preserved blood led to reduced body size and fertility compared with populations reared on human volunteers. Blood preservatives increased the proportion of sterile eggs even after a single feed. Finally, our results indicated that laboratory reared bed bugs were smaller, regardless of the blood source, than wild bugs. Similar effects of artificial feeding or laboratory rearing alone should be considered in future studies using bed bug cultures to choose an appropriate rearing protocol. With regard to switching between bat and human hosts, HL took smaller meals and BL had lower fertility when fed on bats than when fed on humans. We attribute these results to methodological constrains, specifically the inconsistency of bat feeding, rather than to host specialization. Nevertheless, BL can be easily reared using human blood and artificial feeding systems.

Brno loanvirus (BRNV) in bats inhabiting the urban area of Brno, Czech Republic.

Bats are known reservoirs of various emerging pathogens, and have recently been found to host a novel hantavirus, named Brno loanvirus (BRNV), from the Mammantavirinae subfamily (family Hantaviridae, order Bunyavirales). Here we report BRNV detection in bats from the urban area of Brno, Czech Republic in March 2022. Specifically, we uncovered a high prevalence of BRNV (8.8%, 5/57) among hibernating bats (Nyctalus noctula) in urban area, which poses a risk of human exposure. The positive bats included adult females (3/9 positive), a juvenile female (1/32 positive), and an adult male (1/6 positive). All 10 juvenile males were negative. We used RT-qPCR to quantify the BRNV RNA levels in various bat organs, which yielded positive results for viral RNA in organs, including the kidneys, heart, spleen, brain, liver, lung, and gut, and in body cavity fluid. Among all tested organs, the liver showed the highest levels of viral RNA in 4 out of 5 animals examined (average Ct value of 20.8 ± 7.4).

Two different lineages of bedbug (Cimex lectularius) reflected in host specificity.

Co-speciation between host-parasite species is generally thought to result in mirror-image congruent phylogenies. For the last several centuries, many bat species have been turning synanthropic, especially those that are hosted by bedbugs in Europe. There is evidence of only limited gene flow from the population of people to the population of bats. This study was focused on comparison of survival, development, and the reproduction rate based on cross-feeding experiments. In our research, we used two bedbugs groups of Cimex lectularius-bat- and human-associated and respectively as specific/non-specific host bat and commercial human blood. Both lineages show different behavior according to their host preferences. During the bat blood experiment, we found significant differences between both human- and bat-associated bedbugs (Log rank test fourth χ(2) = 9.93, p > 0.05; fifth χ(2) = 11.33, p < 0.05), while no differences occurred with the human blood experiment between the survival levels. In molting, differences between both groups were significant particularly in the case of the bat blood experiment (fourth χ(2) = 5.91, p < 0.05). In the case of the bat blood experiment, we found a higher probability of molting in bat-associated groups than in human-associated groups. In the case of the human blood experiment, molting probability was stable in both specific and non-specific, showing similar pattern in both cases for all stages. Our results indicate an occurrence of two ecotypes within the one species C. lectularius. These findings support earlier data about morphological and mitochondrial DNA differences. The differentiation of both lineages fits the concept of specific host choice.

Recolonization of bat roost by bat bugs (Cimex pipistrelli): could parasite load be a cause of bat roost switching?

Roost ectoparasites are believed to have a negative impact on fitness of their hosts as birds or mammals. Previous studies were mostly focussed on the synchronization between reproduction cycles of ectoparasites and hosts living in infested roosts. However, to date, it has not been examined how fast ectoparasites colonize new, non-infested roosts and thus increasing the impact on the local populations of hosts. The parasite-host model was studied, including bat bugs Cimex pipistrelli and soprano pipistrelles Pipistrellus pygmaeus, where bat behaviour was observed which tended to reduce the parasite load in bat roosts. We investigated (1) whether bats change their roosting behaviour when we discontinued synchronization of their reproduction and the life cycle of the bat bugs and (2) how fast and which stages of bat bugs reoccupy cleaned roosts. In a 3-year field experiment, we removed all bat bugs from six bat boxes in each spring. Pipistrelles bred young in all non-infested boxes during these 3 years. In addition, 8 years of regular observations before this experiment indicate that bats avoided breeding in the same bat boxes at all. Bat bugs were found again in clean boxes in mid-May. However, their densities did not maximise before the beginning of June, before parturition. A re-appearance of bugs was observed after 21-56 days after the first bat visit. Adult bugs, mainly females, colonised cleaned boxes first though at the same time there were a lot of younger and smaller instars in non-manipulated roosts in the vicinity.

Bat bugs (Cimex pipistrelli) and their impact on non-dwelling bats.

Bat bugs are often roost ectoparasites of bats. Previous studies have shown that bats shifting roosts within the growing season prevent the massive reproduction of these parasites. We postulated that there could be other antiparasitic strategies of philopatric bats roosting in non-dwelling spacious roosts. Unfortunately, there are no studies devoted to such a topic. For 3 years, two attics highly and less infested by bat bugs (Cimex pipistrelli) with breeding females of Myotis myotis were monitored. From April, after the arrival of the bats, to November, abundance of all instars and adult bugs was sampled in the attics by adhesive traps. We found different patterns in the bug abundances and dynamics in the two attics. In highly infested attic, bat bugs induced pregnant females to move from the infested site of the attic to the non-infested one. Internal temperature and relative humidity were similar in both infested and non-infested sites. Females roosted in the infested site till time before parturition and then moved to the non-infested site within attic. When bats were absent in their old site, the abundance of nymphal instars of bugs decreased by half. Although adult bats can survive under high parasite loads of bat bugs, reproducing females prevent parasite reproduction and simultaneously reduce parasite load in the young by shifting inside spacious roosts.

Ecological values of intermittent rivers for terrestrial vertebrate fauna.

Rivers are generally considered critical habitats for biodiversity; however, this often ignores the fact that many rivers may run dry and support terrestrial as well as aquatic fauna. Here, we investigated the ecological value of intermittent rivers for terrestrial vertebrates by installing camera traps along rivers subject to varying dry periods in two contrasting European climatic zones. We then analysed i) species presence and behaviours (as a proxy of ecological functions) on perennial and intermittent streams; ii) environmental (hydrological and geomorphological) and anthropogenic factors affecting the frequency of occurrence and number of species recorded; and iii) the importance of hydrological factors as regards ecological functioning. In both study areas, we recorded a higher number of species and individuals along intermittent streams than perennial streams, with highest values in intermittent reaches exhibiting shorter dry periods. Both abundance and species richness were strongly affected by hydrological factors in both study areas, including not only the occurrence but also the duration of the dry period. Dry channels played a key role as migration corridors and as a source of food, being used more frequently than riparian habitats when the river ran dry. Our findings indicate that terrestrial vertebrate fauna benefit from dry phases in rivers. Intermittent rivers, supporting a high abundance and diversity of fauna, should be considered as target ecosystems for wildlife conservation. Not doing so will jeopardise urgently needed conservation strategies in the face of accelerating global climate change.

Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging.

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.