Like most other animals, the fruit fly Drosophila melanogaster has blood cells patrolling all parts of the organism (Rizki, 1978; Rizki, 1984; Meister and Lagueux, 2003). These cells, the hemocytes, attack pathogens, participate in blood clotting and wound healing, and mediate the remodeling of tissues during development. Our understanding of how hemocytes carry out these tasks is surprisingly limited, at least on the molecular level. This may seem surprising, considering the popularity of this model organism. However, important steps forward have been taken during the past two decades. Antibodies and genetic markers have been developed to follow the hemocytes in vivo and manipulate their activities (Evans et al., 2014), and the hematopoietic events that control the production of these cells have now been characterized in considerable detail, as summarized in excellent reviews (Evans et al., 2003; Martinez-Agosto et al., 2007; Honti et al., 2014; Ramond et al., 2015; Gold and Brückner, 2015; Letourneau et al., 2016; Csordás et al., 2021). Our present knowledge about the role of hemocytes in immunity has been reviewed by Carton et al., 2008; Williams, 2007; Theopold and Dushay, 2007; Wang et al., 2014; and Yang et al., 2020, and their involvement in embryology and wound healing by Fauvarque and Williams, 2011; Evans and Wood, 2014; Theopold et al., 2014; and Ratheesh et al., 2015. For a recent comprehensive review of the entire field, with an emphasis on hematopoiesis, see Banerjee et al., 2019.

In spite of this progress, many central questions remain to be answered, but the development of single-cell sequencing technology may change the picture. During the past years, this technique has generated a wealth of data that may look incoherent, but that could potentially change the field. This review is an attempt to summarize and critically analyze this new information in the light of what we knew before.

Recently, several groups have used single-cell RNA sequencing technology to study the hemocyte diversity in Drosophila larvae and the relationship between the different hemocyte classes. Four published studies deal with the peripheral hemocytes, that is, the sessile and circulating hemocytes of the first hematopoietic wave (Cattenoz et al., 2020; Tattikota et al., 2020; Fu et al., 2020; Leitão et al., 2020), and two focus on the lymph glands (Cho et al., 2020; Girard et al., 2021). The Fly Cell Atlas, which appeared recently (Li et al., 2022), presents additional data on all cells in the adult fly, including hemocytes. These studies have confirmed the existence of unique hemocyte ‘clusters,’ corresponding to the classically defined crystal cell and lamellocyte classes, and in some cases also to their precursors. Not surprisingly, however, the plasmatocytes turned out to be heterogenous, and they were split by the different authors into several different clusters. Each cluster was defined by a unique pattern of gene expression, but these patterns were not entirely congruent between the different studies. The six studies on larval hemocytes identified between 2 and 13 plasmatocyte clusters, and in addition between 3 and 6 prohemocyte clusters in the lymph glands. A consensus view of the situation was recently published (Cattenoz et al., 2021), drawing general conclusions from three of the studies (Cattenoz et al., 2020; Tattikota et al., 2020; Fu et al., 2020). To bring further clarity to this issue, we have now compiled lists of the genes that are specifically expressed in each cluster and compared these lists from the different studies (Figure 1—source data 1). To reduce noise, we set a threshold of at least 1.4-fold enrichment (2-fold for the data from Girard et al., 2021, where the relative enrichment values were generally higher). As Fu et al., 2020. did not provide comprehensive lists of specifically expressed genes, we have only listed genes mentioned in the text and figures. The most characteristic lamellocyte and crystal cell markers are listed in Figure 1, and primocyte and plasmatocyte markers in Figure 2.

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Figure 1—source data 1

Genes with enhanced expression in specific cell classes.

Complete lists of differentially expressed genes.

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Figure 1—source data 2

Gene Ontology (GO) terms.

Significantly enriched GO terms in differentially expressed genes in lamellocyte, crystal cell, and plasmatocyte clusters in at least two of the published studies.

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Crystal cells

The crystal cell clusters also form a well-defined class, in this case with a high degree of overlap between all six studies (Figure 3). 137 genes were preferentially expressed in crystal cell clusters in at least two of the studies and 35 genes in at least five of them (Figure 1, Figure 1—source data 1). However, the level of enrichment varied enormously between the studies, with particularly high values reported by Girard et al., 2021. The putative precursors in the CC1 clusters (Tattikota et al., 2020; Cho et al., 2020) overlap less with the major crystal cell clusters.

Figure 3

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As expected, well-established crystal cell markers like PPO1, PPO2 (Binggeli et al., 2014), and lozenge (lz) (Ferjoux et al., 2007) were represented among the preferentially expressed genes, although high enrichment of lozenge (53-fold) was reported in one study only. In the other studies, lozenge was enriched threefold at best, if at all (Figure 1, Figure 1—source data 1). By contrast, the phenoloxidase genes PPO1 and PPO2 were very highly enriched (up to 836-fold) in all six studies. PPO3 has also been used as a crystal cell marker, but only in the embryo (Waltzer et al., 2003; Bataillé et al., 2005; Ferjoux et al., 2007), but that gene was exclusively lamellocyte-specific in the studies discussed here. More rarely used crystal cell markers like peb, klu (Terriente-Felix et al., 2013) and Jafrac1 (Waltzer et al., 2003) were also overrepresented in the crystal cell clusters, as were 19 of 31 embryonic crystal cell markers listed by Ferjoux et al., 2007: PPO1, PPO2, CG10467, Pde1c, CG17109, Gip, fbp, CG17065, Cndp2, CG31431, Jafrac1, Fatp3, Kaz-m1, CG1847, ApepP, pfrx, CG15739, Rift, and CG8112 (Figure 1, Figure 1—source data 1). A Gene Ontology (GO) term analysis of genes specifically enriched in crystal cells in at least two of the studies shows that genes involved in basal metabolism are highly enriched (Figure 1—source data 2), suggesting that crystal cells are metabolically very active.

Crystal cells are best known for their role in the melanization of wounds and encapsulated parasites, as reflected in the list of genes that are preferentially expressed in these cells (Figure 1). PPO1 and PPO2 encode phenoloxidases, key enzymes in the melanization reaction. They are copper enzymes that catalyze the oxidation of tyrosine and other phenols, leading to their polymerization into melanin (Carton et al., 2008). PPO1 is produced in an inactive form, prophenoloxidase, which is possibly secreted directly into the hemolymph. By contrast, PPO2 is kept sequestered in the crystals and released only when the activated crystal cells rupture and the crystals dissolve (Binggeli et al., 2014; Schmid et al., 2019). The proenzymes are proteolytically processed in the hemolymph into active phenoloxidase forms, in a process that involves several serine proteases (Nam et al., 2012; Dudzic et al., 2019). Together, PPO1 and PPO2 mediate the melanization of wound sites and of infecting bacteria (Binggeli et al., 2014; Dudzic et al., 2019). PPO2 from crystal cells also acts together with PPO3 in lamellocytes to melanize encapsulated parasites (Dudzic et al., 2015). Related to the production of these copper enzymes, the transcripts for two copper-transporting and -concentrating proteins, encoded by the Ctr1A and Atox1 genes, are overrepresented in the crystal cells. Four metallothionein genes are also specifically transcribed in the crystal cells: MtnA, MtnB, MtnD, and MtnE (Figure 1, Figure 1—source data 1). Their role may be to deal with copper toxicity.

The capacity of crystal cells to burst and release their contents, via a pyroptosis-like mechanism (Dziedziech and Theopold, 2021), in response to infection and other challenges, may also be reflected in the single-cell transcriptome data. In all six studies, Ninjurin B (NijB) transcripts were enriched in the crystal cell clusters (Figure 1—source data 1). NijB encodes a homolog of the human Ninjurin-1, which is described as a cell adhesion protein. The corresponding mouse protein, NINJ1, was recently found to mediate plasma membrane rupture in macrophages, in response to bacterial infection, and thereby the release of pro-inflammatory cytokine IL-1β and other danger signals (Kayagaki et al., 2021). NijB may play a similar role in the infection-induced rupture of crystal cells.

The membrane receptor Notch plays a central role in crystal cell fate determination and maintenance, together with the Runt domain transcription factor Lozenge, which acts downstream and together with Notch. Consequently, Notch (N) and lozenge (lz) transcripts were picked up in most of the studies as preferentially expressed in the crystal cells, as were several Notch and Lozenge transcriptional targets: the early-onset Notch targets E(spl)m3-HLH and E(spl)mβ-HLH (Couturier et al., 2019), which encode basic helix–loop–helix transcription factors; and the Notch/Lozenge target genes pebbled (peb = hindsight); klumpfuss (klu); and CG32369 (Terriente-Felix et al., 2013). Transcripts of the numb gene, which encodes a membrane-associated inhibitor of signaling from Notch (Wu and Li, 2015), were also found to be enriched, but only in the lymph gland studies.

The interpretation of the crystal cell transcriptome data is complicated by the fact that the recovery of crystal cells was in some cases very low. While normally about 5–10% of the hemocyte population are crystal cells in uninfected third-instar larvae, only 0.6% (Cattenoz et al., 2020) or 0.35% (Fu et al., 2020) of all counted hemocytes were assigned to the crystal cell clusters. Although other studies found higher numbers, many cells may have been lost due to the sensitive nature of crystal cells, which tend to burst after bleeding.

Armed with the new markers for specific cell classes in D. melanogaster, we can begin to look for homologous cell types in other species. Beginning with the crystal cells, where the relationships are more clear, we will here discuss the results from three single-cell transcriptomic studies of hemocytes from the malaria mosquito, Anopheles gambiae, and one from the silkworm, B. mori (Severo et al., 2018; Raddi et al., 2020; Kwon et al., 2021b; Feng et al., 2021). We will also discuss transcriptomic and genomic information available for drosophilid flies other than D. melanogaster.

Crystal cells/oenocytoids

Crystal cells are generally considered equivalent to the cells called oenocytoids in other insects (Lavine and Strand, 2002; Ribeiro and Brehélin, 2006; Hillyer, 2016; Eleftherianos et al., 2021), and there is plenty of evidence supporting that view. Crystal cells and oenocytoids have similar cytology, and neither cell type is known to undergo mitosis. Like crystal cells, oenocytoids are the main or sole source of phenoloxidases (Iwama and Ashida, 1986; Ashida et al., 1988), which are required for melanin deposition around parasites, at wound sites, and in pigmented cuticle. Lepidopteran oenocytoids can release their phenoloxidases in a lytic reaction, in which the cells burst and release their entire contents (Strand and Noda, 1991; Ribeiro and Brehélin, 2006). This response is triggered by prostaglandin (Shrestha and Kim, 2008; Shrestha et al., 2011; Park and Kim, 2012), and a similar prostaglandin-dependent response has also been reported from mosquitoes (Kwon et al., 2021a). Likewise, Drosophila crystal cells are triggered to burst at wound sites and in response to parasitization (Rizki, 1957; Rizki and Rizki, 1959; Rizki, 1978; Bidla et al., 2007; Schmid et al., 2019). One difference is that phenoloxidase is stored in regular polyhedral (‘pseudocrystalline’) inclusion bodies in the crystal cells of D. melanogaster, while phenoloxidases are found in the cytoplasm or in various non-crystalline inclusions in lepidopteran oenocytoids (Iwama and Ashida, 1986; Ashida et al., 1988). Mosquito oenocytoids lack cytoplasmic inclusions, and they stain homogenously for phenoloxidase in the cytoplasm (Hillyer et al., 2003). However, most drosophilids have their phenoloxidases stored in amorphous granules, as summarized in Figure 4 (Rizki and Rizki, 1980; Rizki, 1984). Crystal cells with well-ordered crystalline inclusions have only been observed in the closest relatives of D. melanogaster. Even within the melanogaster species subgroup, D. yakuba and D. teissieri have less regular inclusion bodies. Thus, there are good reasons to conclude that crystal cells are indeed oenocytoids. The term ‘crystal cell’ should either be dropped altogether (Ribeiro and Brehélin, 2006) or at least be restricted to the few species that have oenocytoids with crystalline inclusions.

Figure 4

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Mosquitoes

We can therefore expect that the relatedness between oenocytoids and crystal cells should also be reflected by the genes they express. Three studies on hemocytes from the malaria mosquito (A. gambiae) have been published, but the data do not give an entirely coherent picture (Severo et al., 2018; Raddi et al., 2020; Kwon et al., 2021b). In Figure 5, we have summarized mosquito clusters that express orthologs of D. melanogaster crystal cell-specific markers.

Figure 5

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First, in a small study specifically focusing on oenocytoids, Severo et al., 2018 purified a population of hemocytes that express an oenocytoid-specific fluorescent marker, driven by the prophenoloxidase 6 (PPO6) gene promoter. Unexpectedly, single-cell RNA sequencing of that population identified two different kinds of cells, expressing either high or low levels of the PPO6 marker, respectively. The PPO6 gene encodes one of the nine different phenoloxidase genes of Anopheles, PPO1-PPO9. Anopheles PPO1 corresponds to the PPO2 gene in Drosophila, while Anopheles PPO2-9 are all related to Drosophila PPO1 (Figure 6). Five of them, PPO2, 4, 5, 6, and 9, were more than 1000-fold enriched in the PPO6^high population compared to PPO6^low (Figure 5; Severo et al., 2018). The remaining four genes, PPO1, 3, 7, and 8, were only expressed in a few scattered cells, but these cells also belonged to the PPO6^high cluster. The high expression of phenoloxidase genes confirms the expectation that the PPO6^high cells are oenocytoids or a subpopulation of oenocytoids. By contrast, the expression pattern of the other cluster, the PPO6^low cells, corresponded to what might be expected in granular cells, with an enrichment of orthologs of Drosophila plasmatocyte markers as discussed below, although no morphological differences were found between PPO6^high and PPO6^low cells (Severo et al., 2018). The homology between the Anopheles PPO6^high cells and Drosophila crystal cells is further supported by the fact that orthologs of several other crystal cell marker genes are enriched in the PPO6^high and none in the PPO6^low cluster (Figure 5). Besides the phenoloxidase genes, homologs of CG9119, meep, Fkbp59, and CG17109 were all more than 1000-fold enriched in the PPO6^high population (Severo et al., 2018). Homologs of CG17065, Ctr1A, CG10467, and pathetic were also enriched, but to a lesser extent. However, homologs of the classical crystal cell markers, lozenge, Notch, or pebbled, were not detected, perhaps due to low expression levels of these genes, and because very few cells were analyzed in this study. It should be noted that the recovery of oenocytoids was very low in this study. It may be that the lytic program of these cells was activated during the handling of the samples. In that case, the surviving cells may not be entirely representative of oenocytoids in general. It was suggested that the detection of PPO6 marker in PPO6^low cells was due to uptake of RNA-laden microvesicles, shed by the PPO6^high cells (Severo et al., 2018). Alternatively, phagocytic PPO6^low granular cells may have taken up fragments of disrupted oenocytoids.

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In a larger study, Raddi et al., 2020 identified a likely oenocytoid cluster with enhanced transcription of the five phenoloxidase genes PPO2, 4, 5, 6, and 9. Furthermore, this cluster, called HC1, expressed two additional homologs of the Drosophila crystal cell markers (Figure 5), giving further support for the homology between Drosophila crystal cells and Anopheles oenocytoids. However, the enrichment of these or other transcripts was in general much lower than in the data from Severo et al., 2018. Again, none of the crystal cell markers lozenge, Notch, or pebbled were detected.

Unlike the other single-cell transcriptomic studies, Kwon et al., 2021b did not find statistically significant enrichment of the phenoloxidase genes in any specific hemocyte cluster. The primary markers for oenocytoids, PPO2, 4, 5, 6, and 9, were expressed at moderate levels, and relatively evenly distributed between seven different hemocyte clusters (Kwon et al., 2021b). One possible explanation is that the oenocytoids were completely lysed in this experiment, and that the remnants were taken up by other hemocytes. Interestingly, however, PPO1, 3, 7, and 8 transcripts were primarily found in two clusters, cluster 7 and 8, albeit at low levels. Incidentally, the latter four genes are exactly the ones that have been linked to prostaglandin-dependent induction in oenocytes of Plasmodium-infected mosquitoes (Kwon et al., 2021a). The same clusters were also reported to express the crystal cell markers peb, DnaJ-1, Mlf, klu, and lozenge, although not to levels that reached statistical significance. The authors conclude that clusters 7 and 8 correspond to the oenocytoid class, but that assignment may have to be revised, as cells in cluster 8 also express primocyte markers (see below).

Silkworms

A single-cell transcriptomic study of hemocytes from silkworm, B. mori, gives further support for a relationship between crystal cells and oenocytoids (Feng et al., 2021). In that study, no less than 20 different hemocyte clusters were identified. Four of them, numbers 5, 8, 12, and 16, were assigned to the oenocytoid class, by the criterion that they expressed the paralytic peptide-binding protein genes 1 and 2, PPBP1 and PPBP2, which lack orthologs in Drosophila and Anopheles. These clusters are also highly enriched for the homologs of several crystal cell markers from Drosophila (Figure 5). Notably, the silkworm has three different phenoloxidase genes (Figure 6). By yet another unfortunate twist of nomenclature, the gene related to Drosophila PPO1 is called PO2 (or PPO2), and two genes related to Drosophila PPO2 are called PO1 and PO1-like (or PPO1 and PPO1-like). Only PO1 and PO2 were annotated in the database used by Feng et al., and both of them were found to be highly expressed in all four oenocytoid clusters (Figure 5). Several genes involved in general metabolism and one copper ion transporter were also upregulated, presumably to meet the needs of the copper enzyme phenoloxidase. Importantly, lozenge transcripts were significantly enriched in all four oenocytoid clusters and Notch in two of them. As lozenge and Notch are characteristic markers for the crystal cell fate in D. melanogaster and directly involved in their hematopoiesis, this is strong evidence that crystal cells are indeed oenocytoids.

Lamellocytes

Drosophilids other than D. melanogaster

Of particular interest are the lamellocytes, a cell type that is uniquely found only among the drosophilid flies. According to most authors, typical lamellocytes do not occur outside the genus Drosophila, or even outside the melanogaster and suzuki subgroups (Eslin and Doury, 2006; Eslin et al., 2009; Havard et al., 2009; Salazar-Jaramillo et al., 2014; Wan et al., 2019; Cinege et al., 2020; Figure 4). In apparent contradiction to that view, a master’s thesis by Kacsoh, 2012 documents a type of large lamellocyte-like cells in wasp-infected larvae of several other more distantly related drosophilid flies (see asterisks in Figure 4), and similar cells have also been reported from Zaprionus indianus and Drosophila willistoni (Kacsoh et al., 2014; Salazar-Jaramillo et al., 2014). They were described as large cells that flatten out on a dissection slide (Kacsoh, 2012), though not as large or flat as lamellocytes (Salazar-Jaramillo et al., 2014). It is possible that these lamellocyte-like cells correspond to the ‘activated plasmatocytes’ or to the ‘lamellocytes type II’ that have been observed in wasp-infected animals (Anderl et al., 2016; Cinege et al., 2021).

Regardless of the status of such lamellocyte-like cells, two of the more prominent lamellocyte-specific marker genes, PPO3 and ItgaPS4, are uniquely present only in the genomes of the ‘oriental’ subgroups of the melanogaster species group (Figure 4, Figure 6, Figure 7). These are also the only species where typical lamellocytes have been found. PPO3 and ItgaPS4 both originate from gene duplication events in the ancestors of the ‘oriental’ species groups. It has been proposed that PPO3 originates from a duplication of an ancestral PPO2-like gene (Salazar-Jaramillo et al., 2014; Dudzic et al., 2015) (node 1 in Figure 6). A more detailed phylogenetic analysis supports this idea and suggests that the duplication happened before the split between the melanogaster and obscura species groups (Figure 6). The exact branching order is uncertain, and a likely scenario is that the duplication actually happened even later, in the immediate ancestors of the ‘oriental’ subgroups (node 2 in Figure 6) about 20 million years ago (Figure 4). The resulting tree (Figure 6—figure supplement 1) would fit with the present distribution of the PPO3 gene.

Figure 7

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Similar to the PPO3 gene, the lamellocyte marker ItgaPS4 originates from a series of gene duplications at about time when lamellocytes first appeared on the scene. The ItgaPS4 gene encodes an integrin alpha subunit that is expressed on the surface of lamellocytes. It is closely related to ItgaPS5, which is also highly enriched in hemocytes, though primarily in plasmatocytes (Leitão and Sucena, 2015). A phylogenetic analysis (Figure 7) suggests that ItgaPS4 and ItgaPS5 originate from the duplication of a common ItgaPS4/5 precursor around 20 million years ago (Figure 4). Interestingly, the ancestral ItgaPS4/5 gene in turn comes from a prior duplication of an ItgaPS3-like gene, maybe 10 million years earlier. Species in the montium subgroup still have separate ItgaPS4/5-like and ItgaPS3-like genes (Figure 4, Figure 7).

Looking at Figure 6 and Figure 7, it is striking how the evolutionary rate accelerated in the PPO3 and ItgaPS4 genes, immediately after they split from their rather conservative sister genes, as illustrated by their long branch lengths and complex patterns of additional gene duplications. Obviously, they must have acquired new roles, showing that the typical lamellocyte is indeed an evolutionary novelty within the ‘oriental’ lineage.

While lamellocytes are in many ways unique, most drosophilids, like insects in general, have other specialized hemocyte types that participate in the encapsulation of parasites (Figure 8). It is an open question how these effector hemocytes are related to each other. Nematocytes, a characteristic class of very thin filamentous hemocytes, were first found by Rizki, 1953 in larvae of D. willistoni, and similar cells have later been found in D. hydei, Z. indianus, and many other drosophilids (Srdic and Gloor, 1893; Kacsoh et al., 2014; Bozler et al., 2017). Possibly related to the nematocytes are the multinucleated giant hemocytes (MGHs), first discovered in species of the ananassae subgroup (Márkus et al., 2015). MGHs have also been identified in Z. indianus, D. falleni, and D. phalerata, and perhaps D. grimshawi (Cinege et al., 2020; Bozler et al., 2017). The multinucleated giant hemocytes form huge and highly motile networks of fused hemocytes that ensnare parasite eggs. Together with activated plasmatocytes, they form a capsule that envelops the parasite (Cinege et al., 2021). The wide distribution of nematocytes and/or MGHs among both distant and close relatives of D. melanogaster (Figure 4) suggests that they must have been present already in the common ancestor of all drosophilids. Yet another type of effector cell, the pseudopodocyte, has been described from species in the obscura subgroup (Havard et al., 2009; Havard et al., 2012). Pseudopodocytes are large plasmatocyte-like cells equipped with numerous long pseudopods, and they participate in the encapsulation of parasites.

Figure 8

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As the PPO3 and ItgaPS4 genes are markers for typical lamellocytes only, they are not informative about the possible homology between lamellocytes and other effector cells that can be found in species that lack lamellocytes. We have no single-cell transcriptomic data yet of hemocytes from drosophilids other than D. melanogaster, but recently the bulk transcriptome of D. ananassae multinuclear giant hemocytes was compared to that of other hemocytes in infected and uninfected larvae (Cinege et al., 2021). Strikingly, transcripts of one potential lamellocyte marker, the atilla ortholog, were found to be 300-fold enriched in the giant cells of wasp-infected larvae, compared to the circulating activated plasmatocytes in these larvae. However, this difference is mainly due to a very low expression of this gene in the latter cells. The atilla gene is otherwise also highly expressed in the naïve hemocytes of uninfected larvae, perhaps due to the presence of giant cell precursors. On the other hand, some lamellocyte-specific gene homologs, like the integrin Itgbn, are induced by infection in multinuclear giant hemocytes, and yet others, like Trehalase, are induced both there and in activated plasmatocytes. A substantial number of homologs of lamellocyte-specific genes are even downregulated after infection in one or both hemocyte classes. In conclusion, it is still difficult to judge if the limited overlap between gene expression in lamellocytes and multinuclear giant hemocytes is evidence of true homology or if it merely reflects an active role of these hemocytes.

Mosquitoes

Similarly, none of the mosquito hemocyte clusters described in the recent single-cell transcriptomic analyses (Severo et al., 2018; Raddi et al., 2020; Kwon et al., 2021b) show obvious homologies to the lamellocytes of D. melanogaster. Transcripts of atilla and CG15347 homologs are, for instance, not enriched in any hemocyte cluster (Raddi et al., 2020; Figure 5). However, we only have information from adult mosquitoes about hemocyte clustering, while lamellocytes and hemocytes with similar functions in other insects are typically found only in larvae. Thus, it is too early to speculate about possible lamellocyte homologs in mosquitoes. Besides, parasitoid wasps are certainly less of a problem for aquatic larvae.

Silkworms

In lepidopterans, such as B. mori, hemocytes called plasmatocytes (not to be confused with Drosophila plasmatocytes) play a similar role as the lamellocytes in Drosophila (Strand, 2008), and it is possible that these cell classes have a common origin in the ancestor of these insects. In line with this idea, a number of lamellocyte markers are in fact shared between Drosophila lamellocytes and the Bombyx plasmatocyte clusters 14 and 15 (Feng et al., 2021), for instance, the homologs of α-Tubulin at 85E, and β-Tubulin at 60D (Figure 5). However, the overlap is rather modest, and it could be due to convergent evolution. The Bombyx plasmatocyte cluster 14 also expresses mitosis markers, suggesting that unlike Drosophila lamellocytes these cells are mitotically active. Furthermore, a few crystal cell markers are also expressed in Bombyx plasmatocyte clusters 14 and 15 (Figure 5). Until we know the signaling pathways involved in their hematopoiesis, it will be difficult to judge if these cell types are related.

One additional hemocyte class has been described in lepidopterans, the spherule cells, which lack dipteran counterparts. Spherule cells constitute one cluster in the study of Feng et al., 2021, cluster 19. While many crystal cell orthologs are found in the silkworm oenocytoid clusters, a few are to some extent expressed in the spherule cell cluster 19 (Figure 5). These more promiscuous markers also tend to be expressed in the lepidopteran plasmatocyte clusters 14 and 15, making them less useful as indicators for a relationship to Drosophila lamellocytes or crystal cells.

Primocytes

Few of the primocyte markers are enriched in any particular hemocyte cluster in mosquitoes or silkworms, and there are no convincing candidates for a primocyte class in these species. Intriguingly, as indicated in Figure 9, homologs of the key markers of primocytes, knot and Antennapedia, are enriched in the Anopheles hemocyte cluster 8 of Kwon et al., 2021b, but there is no indication in the other studies that these genes are expressed in any of the hemocyte classes. Cluster 8 is otherwise the candidate of Kwon et al. for being oenocytoids.

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Some experimental difficulties will have to be dealt with in future studies. One is the fragility of the crystal cells, which is a likely cause of the low yields of these cells in some of the studies. Crystal cells are not easy to collect, and the problem may have been exacerbated by the violent pretreatment of the larvae, intended to force the release of sessile cells. The same is true for the oenocytoids in other insects, especially for mosquitoes that have to be transfused in order to get a reasonable yield.

Other artifacts may be caused by the habit of plasmatocytes and granular cells to phagocytose fragments of other cells. Such fragments are generated when crystal cells/oenocytoids release their contents. Cell fragments may also be generated in the turnover of superfluous lamellocytes and in the autolytic disruption of larval tissue in preparation for metamorphosis. When such fragments are internalized or attached to plasmatocytes, they will contaminate the transcriptional profile of these cells. This could explain the unexpected presence of markers for crystal cells, oenocytoids, lamellocytes, or fat body cells in the plasmatocyte or granular cell transcriptomes. Further experiments will be required to resolve these issues.

The lack of reproducibility in the subclustering of Drosophila plasmatocytes may be due to experimental details that were not common to all laboratories. The pooling of data from parasitized and unparasitized animals may have introduced further variability. The outcome of the subclustering may also be dependent on different parameters chosen for the clustering algorithms.

The yield is a problem of its own. Most of the Drosophila studies were done with 15,000–20,000 hemocytes or more (Figure 1—figure supplement 1), which seems sufficient. Fu et al. assayed a smaller number, 3424, but they tested fewer conditions. Similarly, Feng et al. analyzed over 20,000 cells from the silkworm. The mosquito studies have struggled with smaller numbers. Raddi et al. assayed over 5000 cells, but Kwon et al. and Severo et al. had to do with 262 and 26 cells, respectively. This means that stochastic errors become serious, and that rare hemocyte classes will be missed. These results must therefore be regarded as tentative.

Throughout, we were surprised by the relatively modest levels of enrichment (‘FC values’) reported for many purportedly cell-type-specific transcripts. Part of the explanation may be that the borders between clusters become blurred when cells gradually activate different programs or initiate transdifferentiation, or when too many subclusters are recognized. Standard bioinformatic algorithms also tend to underestimate differences in gene expression. In order to avoid zero denominators, a constant value (typically 1) is usually added to all standardized read counts (RPKM). This gives conservative and more reliable estimates of statistical significance, but the FC values will systematically be underestimated, and the problem will become larger when the total number of reads is small.

Our analysis of the recently published single-cell transcriptomic studies shows that Drosophila plasmatocyte heterogeneity is not due to the presence of distinct and reproducibly occurring cell classes. Rather, plasmatocytes are flexible and they have a capacity to engage in different tasks, such as production and reshaping of extracellular matrix, phagocytosis of cell debris and microbes, encapsulation of parasites, etc., (Mase et al., 2021), and to adjust their activity accordingly. The resulting heterogeneity is gradual, transient, and probably reversible, and it does not result in the formation of separate well-defined classes. A similar functional plasticity is also seen in vertebrate myeloid cells (Galli et al., 2011). In a broad sense, this capacity may be inherited from the phagocytes of early metazoans, but the more specific adaptations of these plastic cells have probably evolved independently, considering the over 600 million years of separate evolution of insects and mammals (Cunningham et al., 2017).

On the other hand, insect plasmatocytes/oenocytoids and vertebrate myeloid cells have several basal functions in common. They can phagocytize microbes and apoptotic cells, and they can detect and react to the presence of specific microbe-associated molecular patterns. These functions are probably very old, going back to the very first animals, and even to bacteria-eating protists (Gilmore and Wolenski, 2012; Franzenburg et al., 2012; Wenger et al., 2014; Menzel and Bigger, 2015; Emery et al., 2021). In Hydra, a jellyfish relative, these functions are carried out by phagocytic epithelial cells in the gut, while in corals like Swiftia, Pocillopora, and Nematostella, it is done by specialized motile immunocytes (Metchnikoff, 1892; Bosch et al., 2009; Franzenburg et al., 2012; Menzel and Bigger, 2015; Snyder et al., 2021).

In order to meet special needs of immunity and wound healing, plasmatocytes can terminally transdifferentiate to become crystal cells (oenocytoids) or lamellocytes. Oenocytoids were probably present already in the first insects, and a subclass of phenoloxidase-expressing mobile cells have even been described from the coral Swiftia exserta (Menzel and Bigger, 2015). By contrast, lamellocytes are products of recent and very rapid evolution. The arms race with parasites like the parasitoid wasps has brought forward a plethora of different types of highly specialized effector cells among the drosophilid flies, and typical lamellocytes can only be found in a subset of species in the genus Drosophila.

One novel and distinct class of hemocytes did come out of the transcriptomic studies, the primocytes. They populate the posterior signaling centers of the lymph glands, but they also appear to circulate freely in the hemolymph. It is possible that circulating or sessile primocytes play a similar role for the activation of peripheral hemocytes as the posterior signaling centers do for the cells in the lymph glands. The expression of Antennapedia suggests that primocytes may have an origin separate from that of other hemocytes.

A comparison of Drosophila crystal cell transcriptomes with oenocytoid data from Anopheles and Bombyx gives strong support for the long suspected homology of these cell types. Similarly, Drosophila plasmatocytes are most likely homologous to the granular cells of other insects. Unlike these well-conserved hemocyte classes, the designated effector cells of the immune defense seem to undergo very rapid evolution, generating formidable entities such as lamellocytes, multinucleated giant cells, and lepidopteran plasmatocytes.

The transcriptomic studies published so far provide a rich source of data, and further analysis can probably yield even more information. For instance, how are the changes in morphology and activity reflected in the transcriptomes of the ‘activated plasmatocytes’ in infected larvae? And, is it possible already from existing data to generate better catalogs of plasmatocyte and granular cell transcriptional markers?

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Author details

1. Dan Hultmark

   Department of Molecular Biology, Umeå University, Umeå, Sweden

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   dan.hultmark@ucmp.umu.se

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6506-5855

2. István Andó

   Biological Research Centre, Institute of Genetics, Innate Immunity Group, Eötvös Loránd Research Network, Szeged, Hungary

   Contribution

   Conceptualization, Data curation, Funding acquisition, Validation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4648-9396

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