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Plasmodium sporozoites, the infective stage of malaria, must invade the mosquito salivary glands (SGs) before being transmitted to a vertebrate host. However, the physiological and biochemical effects of this invasion remain largely unexplored. We examined the impact of Plasmodium infection on Anopheles gambiae salivary glands using high-resolution proteomics, gene expression, and morphological analysis. The data reveal differential expression of various proteins, including the enrichment of hemolymph-derived humoral proteins in infected salivary glands. These proteins diffuse into the SGs due to structural damage caused by the sporozoites during invasion, while saliva proteins diffuse out into the circulation. Moreover, proteomic analysis of saliva from P. berghei – or P. falciparum –infected mosquitoes revealed changes in composition, with a pronounced reduction of immune proteins relative to uninfected mosquitoes. This reduction is likely due to the association of these proteins with the surface of sporozoites and/or changes in the saliva’s physical properties within the invaded salivary secretory cavities. The saliva protein profiles from mosquitoes infected with both Plasmodium species are remarkably similar, suggesting a conserved interaction between sporozoites and salivary glands. Our results provide a foundation for understanding the molecular interactions between Plasmodium sporozoites and mosquito salivary glands. Plasmodium sporozoites must invade mosquito salivary glands to facilitate malaria transmission, yet the effects of this invasion are not well understood. Here, the authors use high-resolution proteomics and show that malaria parasite invasion damages mosquito salivary glands, alters saliva protein composition, and attracts immune cells, potentially increasing parasite transmission.

Metarhizium anisopliae infects mosquitoes through the cuticle and proliferates in the hemolymph. To allow M. anisopliae to combat malaria in mosquitoes with advanced malaria infections, we produced recombinant strains expressing molecules that target sporozoites as they travel through the hemolymph to the salivary glands. Eleven days after a Plasmodium-infected blood meal, mosquitoes were treated with M. anisopliae expressing salivary gland and midgut peptide 1 (SM1), which blocks attachment of sporozoites to salivary glands; a single-chain antibody that agglutinates sporozoites; or scorpine, which is an antimicrobial toxin. These reduced sporozoite counts by 71%, 85%, and 90%, respectively. M. anisopliae expressing scorpine and an [SM1]₈:scorpine fusion protein reduced sporozoite counts by 98%, suggesting that Metarhizium-mediated inhibition of Plasmodium development could be a powerful weapon for combating malaria.

Invasive species that proliferate after colonizing new habitats have a negative environmental and economic impact. The reason why some species become successful invaders, whereas others, even closely related species, remain noninvasive is often unclear. The harlequin ladybird Harmonia axyridis, introduced for biological pest control, has become an invader that is outcompeting indigenous ladybird species in many countries. Here, we show that Harmonia carries abundant spores of obligate parasitic microsporidia closely related to Nosema thompsoni. These microsporidia, while not harming the carrier Harmonia, are lethal pathogens for the native ladybird Coccinella septempunctata. We propose that intraguild predation, representing a major selective force among competing ladybird species, causes the infection and ultimate death of native ladybirds when they feed on microsporidia-contaminated Harmonia eggs or larvae.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

Background The parasitic dinoflagellates of the genus Hematodinium represent the causative agent of so-called bitter or pink crab disease in a broad range of shellfish taxa. Outbreaks of Hematodinium- associated disease can devastate local fishing and aquaculture efforts. The goal of our study was to examine the potential role of the common shore (green) crab Carcinus maenas as a reservoir for Hematodinium . Carcinus maenas is native to all shores of the UK and Ireland and the North East Atlantic but has been introduced to, and subsequently invaded waters of, the USA, South Africa and Australia. This species is notable for its capacity to harbour a range of micro- and macro-parasites, and therefore may act as a vector for disease transfer. Methods Over a 12-month period, we interrogated 1191 crabs across two distinct locations (intertidal pier, semi-closed dock) in Swansea Bay (Wales, UK) for the presence and severity of Hematodinium in the haemolymph, gills, hepatopancreas and surrounding waters (eDNA) using PCR-based methods, haemolymph preparations and histopathology. Results Overall, 13.6% were Hematodinium -positive via PCR and confirmed via tissue examination. Only a small difference was observed between locations with 14.4% and 12.8% infected crabs in the Dock and Pier, respectively. Binomial logistic regression models revealed seasonality ( P  < 0.002) and sex ( P  < 0.001) to be significant factors in Hematodinium detection with peak infection recorded in spring (March to May). Male crabs overall were more likely to be infected. Phylogenetic analyses of the partial ITS and 18S rRNA gene regions of Hematodinium amplified from crabs determined the causative agent to be the host generalist Hematodinium sp., which blights several valuable crustaceans in the UK alone, including edible crabs ( Cancer pagurus ) and langoustines ( Nephrops norvegicus ). Conclusions Shore crabs were infected with the host generalist parasite Hematodinium sp. in each location tested, thereby enabling the parasite to persist in an environment shared with commercially important shellfish.

Background The in vitro cultivation of individual stages of the Plasmodium falciparum mosquito life cycle is notably challenging. The main difficulty is replicating the intricate nutrient and metabolite exchanges necessary for oocyst development and sporozoite (SPZ) formation in the three-dimensional environment of the mosquito midgut. Replicating these conditions is essential for understanding the biological interactions between mosquito and parasite, as well as advancing malaria vaccine development. Methods An in vitro three-dimensional system was developed that closely mimics the mosquito midgut epithelium, basal lamina, and haemolymph, facilitating the production of substantial quantities of haemolymph-like Pf SPZ. Results Use of an extracellular matrix-coated Alvetex ® Strata scaffold, combined with optimized culture medium, supports efficient oocyst attachment and provides the necessary nutrients for robust production of haemolymph-like SPZ. This system enables full maturation of oocysts, as shown by immunofluorescence assays (IFA), and allows timely release of in vitro SPZ (IVS) between days 11 and 15, comparable to the in vivo mosquito timeline. Haemolymph-like SPZ generated were found to be infectious, as evidenced by successful HC04 infection in in vitro and in vivo studies using FRG-huHep mice. Similar outcomes were observed across different P. falciparum strains. Conclusions Implementation of the Alvetex Strata scaffold, optimized medium, and improved ookinete production consistently enables in vitro generation of large quantities of haemolymph-like SPZ.

Background Metabolic responses to infection differ based on arthropod and pathogen. Increased metabolic rates can result in faster depletion of energetic resources, and decreases may allow for energy conservation. Babesia bovis is a protozoan pathogen transmitted by the cattle fever tick, Rhipicephalus microplus . Adult female ticks acquire B. bovis by feeding on an infected animal. Babesia bovis undergoes development and invades the ovaries where it is transmitted transovarially to tick offspring. The effects of infection on R. microplus metabolic rate are not well studied. Methods We tested the hypothesis that R. microplus infected with B. bovis would have altered metabolic rates (volume of carbon dioxide [VCO 2 ]) across life stages using flow-through respirometry. Replete females from either an infected or naïve calf were measured across 3 days to determine differences in VCO 2 . Hemolymph smears were used to categorize the number of B. bovis kinetes present in the hemolymph of replete females during egg oviposition. The VCO 2 for groups of their offspring were measured twice as eggs and once as larvae. The number of individuals and successfully hatched larvae in each group were enumerated at the end of the experiment to determine the average VCO 2 per individual. Results Infected replete females have decreased VCO 2 while their offspring have increased VCO 2 at the egg and larval stages. Interestingly, replete females had a 25% reduction in body mass compared to uninfected female tick controls. Uninfected larvae were twice as likely to hatch than larvae from infected replete female ticks. Conclusions VCO 2 varied between control and infected ticks depending on life stage. Infected replete females had lower VCO 2 and body mass while their offspring had higher VCO 2 than their control counterparts. Higher larval VCO 2 may promote earlier questing and a shorter lifespan. Changes in metabolic and hatch rates have implications that may promote disease spread. Graphical Abstract

BACKGROUND: Tick-borne pathogen (TBP) surveillance studies often use whole-tick homogenates when inferring tick-pathogen associations. However, localized TBP infections within tick tissues (saliva, hemolymph, salivary glands, and midgut) can inform pathogen transmission mechanisms and are key to disentangling pathogen detection from vector competence. METHODS: We screened 278 camel blood samples and 504 tick tissue samples derived from 126 camel ticks sampled in two Kenyan counties (Laikipia and Marsabit) for Anaplasma, Ehrlichia, Coxiella, Rickettsia, Theileria, and Babesia by PCR-HRM analysis. RESULTS: Candidatus Anaplasma camelii infections were common in camels (91%), but absent in all samples from Rhipicephalus pulchellus, Amblyomma gemma, Hyalomma dromedarii, and Hyalomma rufipes ticks. We detected Ehrlichia ruminantium in all tissues of the four tick species, but Rickettsia aeschlimannii was only found in Hy. rufipes (all tissues). Rickettsia africae was highest in Am. gemma (62.5%), mainly in the hemolymph (45%) and less frequently in the midgut (27.5%) and lowest in Rh. pulchellus (29.4%), where midgut and hemolymph detection rates were 17.6% and 11.8%, respectively. Similarly, in Hy. dromedarii, R. africae was mainly detected in the midgut (41.7%) but was absent in the hemolymph. Rickettsia africae was not detected in Hy. rufipes. No Coxiella, Theileria, or Babesia spp. were detected in this study. CONCLUSIONS: The tissue-specific localization of R. africae, found mainly in the hemolymph of Am. gemma, is congruent with the role of this tick species as its transmission vector. Thus, occurrence of TBPs in the hemolymph could serve as a predictor of vector competence of TBP transmission, especially in comparison to detection rates in the midgut, from which they must cross tissue barriers to effectively replicate and disseminate across tick tissues. Further studies should focus on exploring the distribution of TBPs within tick tissues to enhance knowledge of TBP epidemiology and to distinguish competent vectors from dead-end hosts.

Lake Baikal is one of the largest and oldest freshwater reservoirs on the planet with a huge endemic diversity of amphipods (Amphipoda, Crustacea). These crustaceans have various symbiotic relationships, including the rarely described phenomenon of leech parasitism on amphipods. It is known that leeches feeding on hemolymph of crustacean hosts can influence their physiology, especially under stressful conditions. Here we show that leeches Baicalobdella torquata (Grube, 1871) found on gills of Eulimnogammarus verrucosus (Gerstfeldt, 1858), one of the most abundant amphipods in the Baikal littoral zone, indeed feed on the hemolymph of their host. However, the leech infection had no effect on immune parameters such as hemocyte concentration or phenoloxidase activity and also did not affect glycogen content. The intensity of hemocyte reaction to foreign bodies in a primary culture was identical between leech-free and leech-infected animals. Artificial infection with leeches also had only a subtle effect on the course of a model microbial infection in terms of hemocyte concentration and composition. Despite we cannot fully exclude deleterious effects of the parasites, our study indicates a low influence of a few leeches on E. verrucosus and shows that leech-infected amphipods can be used at least for some types of ecophysiological experiments.

Despite the economic importance of Mizuhopecten yessoensis, little is known about their parasites and immunity. This study, the first to examine the prevalence and intensity of Perkinsus across three age groups of scallops from four locations in the Sea of Japan, revealed that the gills, mantle, and digestive glands of one-year-old specimens from mariculture farms are heavily colonized. The cases of infection were notably higher in older specimens (mostly hemolymph and shell), suggesting that they act as carriers of Perkinsus. An immunological analysis indicated that when the pathogen is found only in the hemolymph and mantle, there is an increase in plasma protein concentrations, which likely plays a crucial role in resisting infection. However, when hypnospores were present in the mantle and gills, a decrease in reactive oxygen species and granulocytes occurred, accompanied by an increase in hemoblasts and agranulocytes. Phagocytic activity increased only when the pathogen appeared in the digestive gland. This evidence highlights the heightened vulnerability of young scallops, emphasizing the necessity for preventive measures against infection. The current troubling epidemiological situation regarding scallop diseases in the region suggests a rise in epizootics, raising doubts about the sustainability of the scallop farming industry unless timely interventions are implemented.